The Role of the Wiskott-Aldrich Syndrome Protein in Regulating T

The Role of the Wiskott-Aldrich Syndrome Protein in Regulating T Cell Programmed Cell Death Mechanisms and Implications for Autoimmunity in the Wiskott-Aldrich Syndrome

by Sophia Y. Marjanovic

B.S. in Chemistry, June 1998, Arizona State University

A Dissertation submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

January 31, 2016

Dissertation directed by

Richard Siegel Senior Investigator, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases

David Leitenberg Associate Professor of Microbiology, Immunology & Tropical Medicine

The Columbian College of Arts and Sciences of The George Washington University certifies that Sophia Y. Marjanovic has passed the Final Examination for the degree of

Doctor of Philosophy as of June 3, 2015. This is the final and approved form of the dissertation.

The Role of the Wiskott-Aldrich Syndrome Protein in Regulating T Cell Programmed Cell Death Mechanisms and Implications for Autoimmunity in the Wiskott-Aldrich Syndrome

Sophia Y. Marjanovic

Dissertation Research Committee:

Richard Siegel, Senior Investigator, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Dissertation Co-Director

David Leitenberg, Associate Professor of Microbiology, Immunology & Tropical Medicine, Dissertation Co-Director

Pamela Schwartzberg, Chief & Senior Investigator, Genetic Disease Research Branch, National Human Genome Research Institute, Committee Member

Fabio Candotti, Head of Disorders of Immunity Section, National Human Genome Research Institute, Committee Member

iii Dedication

This Ph.D. thesis is dedicated to my son, Mahpiya Waablezya Marjanovic, my father, Lawrence J. Cleland, Sr., my mother, Mary G. Eder, my siblings, my unofficial adopted mothers, Charlotte Black Elk, Anna Fire Thunder-Fuhrman, my unofficial adopted aunt, Mara Cohen, my unofffical adopted father, Dr. Kim Winkelman (the first

American Indian to graduate from the Citadel), my friends/colleagues, and my Fort Peck

Lakota, Dakota, Nakota, Santa Ysabel Iipay, Mexican, Scottish, Irish and Jewish tribes and ancestors. Through my experiences with all of you and from your experiences, I have learned a lot about resilience. My father especially has taught me many profound lessons about resilience regarding his struggles of hunting and foraging as the primary way to access food on his Santa Ysabel Iipay reservation in California as a child. He was coerced off the reservation by the Bureau of Indian Affairs’ (BIA) Relocation

Program with the promise of a house and job in Los Angeles (LA). The BIA official dropped him off at a dilapidated hotel in a LA ghetto. Upon getting to his room, the hotel building was so dilapidated, he saw several stories below him through the huge hole in the floor of his room. He signed up to join the Marines, served several tours in Vietnam, used the GI Bill to attend a technical college as his professors singled him out for

“milking the system” as a baby killing Indian and graduated with an Associates degree in

Electrical Engineering. Hughes Aircraft hired him in California and he helped develop

Landsat. He went onto helping to develop several technological innovations for heart pacers, robotic toys, etc. at Medtronic Microrel in Arizona. As American Indians in the

USA now use Landsat based technology to solve problems within and outside of our tribal communities, I know that although the basic science is slow, tedious, requires

iv patience and tenacity, STEM education and innovation is the only long term solution for the many problems we face not only in our tribal communities, but that many communities around the world encounter. All human groups on this planet started off from some tribal origin before joining modern societies in cities. My upbringing in transitioning back and forth between tribal and urban life has influenced my perspective on STEM education and innovation. Securing this Ph.D. means that I can continue on in the tradition of my father in pushing the boundaries for STEM education and innovation for the long term solution of securing peace within our tribal communities as we interact with non-tribal communities.

It took a lot of resilience to get to this stage of completing and defending my

Ph.D. thesis. My journey of pursuing a Ph.D. thesis in immunology was not an easy road as the systematic problems of education for People of Color to help our underserved communities thrive in the USA persist. Since my advisor for my undergraduate research thesis was Dr. Therese Markow whose interactions with the Havasupai tribes lead to the famous legal battle Havasupai Tribe of the Havasupai Reservation v. Arizona Board of

Regents and Therese Ann Markow, I have sought to achieve a higher standard of ethics in working with tribal and marginalized communities for solving complex biomedical problems. As both an American Indian and a researcher, I understood the concerns of both sides of the issue. I have felt obliged to improve the ethical standards of working with tribal and marginalized communities in the USA.

I first set out on my journey to obtain a Ph.D. in genetics, but I was not convinced that the ethics of the field were optimal to do genetic research with tribal communities.

Before I could improve the ethical standards, it became clear to me that I needed a solid

v foundation in basic research since so few American Indians had the opportunity to get such training. I had a choice before I came to the DC area: 1) cease pursuing my dreams of getting the best training possible for solving health problems in our underserved communities by becoming a bioethicist; or 2) continue pursuing my dreams of getting the best training possible for solving health problems in our underserved communities so that

I had a better tool set to accomplish the changes. It is obvious that I chose to sacrifice my time and start over in another field, immunology, so that I may get the best training possible to reverse the brain drain from our underserved tribal communities.

Since all of the diverse tribes of American Indians in the USA are collectively

1.7% of the total population in the USA, finding a critical mass of American Indian colleagues in genetics and immunology was impossible. I often endured a lot of painful isolation being the only American Indian pursuing a Ph.D. in genetics or immunology that I knew for a significant portion of my journey. Tribal community members had no clue about how to advise me regarding the struggles I encountered as a Ph.D. student.

Other Ph.D. students and mentors had no clue about how to advise me about the struggles

I endured as a tribal member in the sciences. Somehow, I navigated a way forward, as tough as it was by finding small pockets of marginalized, ethnic people to take me in as an honorary member of their communities. I met a Serbian, who I eventually married through this difficult process and gave birth to a beautiful baby boy who gave me even more purpose in my life as a mother.

While writing this thesis, I struggled through several traumas and still managed to move forward in finishing the writing of this thesis as difficult as it was. I am grateful to the rabbis in the DC area and Maryland who have taken me into their congregations and

vi given me some tribal structure, community and spiritual practice that I miss so much being away from family and tribal kin. While the external forces of my life were often difficult to endure on a daily basis, at least every Friday and Saturday, I had a place in synagogues to release some of the pain by reminding myself to at least be grateful for what I have, to remember that I am not the only one who has had to be more resilient than one knew one could, to remember that grit matters through many role models of grit at the synagogues and to remember to have some pride in being obliged in the mitzvot of

Tikkun Olam (healing the world).

The advocacy regarding the concerns our tribal communities have for research in our communities became a challenge that I readily took on for changing before I switched to pursue this Ph.D. in immunology. Having realized in my previous research on the genetic basis for rapidly developing autoimmunity in American Indian communities that I could not change the systematic dynamics in the fields as a graduate student, I temporarily put that challenge aside as I focused on securing high quality training in completing my Ph.D. in immunology through the George Washington University and the

National Institutes of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Now that securing the title of Ph.D. in immunology is imminent, I plan on returning to overcoming the challenge of raising the standards of ethics in science regarding how tribal and other underserved communities are incorporated into biomedical solutions for this nation while creating a thriving economy within our tribal and other underserved communities of STEM innovators. As my father has taught me through his own example, technological innovation is the only solution throughout human history that assures long-term justice and peace for any community.

vii Acknowledgements

I am grateful to Dr. Linda Werling, Dr. David Leitenberg, Dr. Richard Siegel and

Dr. Sharon Milgram for giving me this opportunity to pursue the high quality training I have been able to access at the National Institutes of Health. I especially appreciate the mentoring that Dr. Richard Siegel provided to me throughout the years in challenging me to be more critical and more systematic in approaching hypotheses.

I am grateful to Dr. Pamela Schwartzberg and Dr. Fabio Candotti for serving on my committee over the years and for reading my thesis to give me feedback. I appreciate all of the time and energy you have put in to assuring that I stay up to the high standards you have for immunological research. I am grateful to Dr. Tim McCaffrey and Dr.

Imtiaz Khan for serving as the examiners for my Ph.D. thesis defense. Likewise, I appreciate all of the time and energy you will have put into assuring that I stay up to the high standards you have for research.

I also want to thank the Washington DC Comedy Writers Group, particularly

Wayne Manigo, Mandy Dalton, John Quinn, Kandyce August, Orlando Gaston Chandler

Aziz, Sean Coleman and the many members of my Indigenius Online Comedy Writers

Group, for not only providing me a space to vent about my frustrations regarding the absurd things in life, but for challenging me to laugh about the absurdities and figure out how to connect with other people in order to help others laugh about such absurdities.

The warmth I received from you at the meetings, at stand up comedy events throughout

DC and from your hugs meant a lot to me and kept me moving forward with a healthy outlet for dealing with the stress of not only this difficult process of writing and defending the Ph.D., but for the many other difficult things in my life. I smile and laugh

viii so much more because of your challenges to me. The lessons I have learned from you and will likely continue to learn from you have been extremely valuable.

I also want to thank the friends I have for checking in on me and helping me when things were tough. I hope to return the generosity you have given to me in my time of need. Finally, I want to thank the many teammates I have had in Brazilian Jiu Jitsu,

Muay Thai, Judo and Mixed Martial Arts throughout the years. Thank you for pushing me to be a physically and mentally stronger person in what started out as a male dominated sport. You beat me up really badly. It was embarrassing. However, I learned that the best things in life do not come easily because a hard work ethic and learning to fight smarter is what matters in overcoming obstacles that seem impossible. When you asked me to be your coach, it was an honor. There are too many of you to mention, but you know who you are.

ix Abstract of Dissertation

The Role of the Wiskott-Aldrich Syndrome Protein in Regulating T Cell Programmed Cell Death Mechanisms and Implications for Autoimmunity in the Wiskott-Aldrich Syndrome

The development of autoimmunity in the setting of immunodeficiency has been a paradoxical observation. Many primary immunodeficiency diseases (PIDDs) are associated with autoimmunity. Wiskott-Aldrich Syndrome (WAS), a PIDD caused by defects in the Wiskott-Aldrich Syndrome protein (WASp), has an extremely high percentage of autoimmunity associated among WAS patients (40%). We hypothesized that defects in cell death mechanisms underlie the autoimmunity in WAS. In T cells, since TCR activation involves WASp, and TCR activation is necessary for restimulation induced cell death (RICD), we investigated whether WASp regulates this mechanism of peripheral tolerance. We found that older WASp deficient mice develop autoimmmunity with immune complex nephritis. WASp deficient T cells have a cell-intrinsic defect in

RICD and have reduced secretion of secretory granules as well as high molecular weight

FasL (HMW FasL). WASp is also required for cytotoxicity of CD4+ T cells, but does not affect the killing of target cell by CD8+ T cells. This was not simply due defects in FasL secretion or CTL granule secretion because Rab27a or FasL deficiency resulted in different defects in target cell killing. The defects we have found in T cell death mechanisms may contribute to the development of autoimmunity in WASp deficient mice and patients with WAS.

x Table of Contents

Dedication……………………………………………………………………………...…iv

Acknowledgements……………………………………………………………………..viii

Abstract of Dissertation…………………………………………………………………...x

Table of Contents…………………………………………………………………………xi

List of Figures…………………………………………………………………………...xiii

Chapter 1: General Introduction………………………………………………………….1

WASp Deficiency in Platelets…………………………………………………...17

Platelet Interactions with T cells…………………………………………18

WASp Deficiency in Myeloid Cells……………………………………..18

WASp Deficiency in NK cells…………………………………………...19

WASp Deficiency in B cells……………………………………………..20

WASp Deficiency in T cells……………………………………………..20

Fas-FasL Deficiency Association with Autoimmunity…………………..23

Intrinsic vs Extrinsic Cell Death Mechanisms…………………………...25

Fas and FasL Function in Regulating Peripheral Tolerance………...... 31

Vesicle Trafficking……………………………………………………....35

Chapter 2: Specific Aims………………………………………………………………..45

Chapter 3: Materials and Methods…………………………………………………...... 47

Chapter 4. Systemic autoimmunity and defective Fas ligand secretion in the absence of the Wiskott-Aldrich syndrome protein………………………………..57

xi Introduction………………………………………………………………………57

Results……………………………………………………………………………59

Discussion………………………………………………………………………..78

Chapter 5. Distinct secretory pathways govern CD4+ and CD8+ T cell cytotoxicity and restimulation-induced cell death………………………………………….

Introduction………………………………………………………………………82

Results……………………………………………………………………………...

The Predominant Form of FasL in RICD is in High Molecular

Weight Complexes…………..…………………………………………..85

Distinct Roles of WASp vs Rab27a in CD4+ T Cell RICD……………..93

CD8+ T cell Cytotoxicity in Rab27a Dependent whereas CD4+

T cell Cytotoxicity Depends on WASp……………….…………………96

Cell Intrinsic Role for WASp in CD4+ T cell

Restimulation-Induced Cell Death………………...……………...... 115

FasL is Able to Induce Cell Death in Trans during RICD……………...118

Discussion……………………………………………………………………......

CD8+ T cells Rely on Rab27a Instead of WASp for Mediating Cell

Death…………………………………………………………………....126

CD4+ T cells Depend on WASp for Mediating Cell Death……………128

WASp Mediates a Cell Intrinsic Defect in CD4+ T cell RICD………...133

Chapter 6: Conclusions………………………………………………………………...135

References……………………………………………………………………………....144

xii List of Figures

Figure 1. WASp activation leading to branched actin polymerization…………………...9

Figure 2. WASp domains and associated functions of each domain……………………12

Figure 3. Immune cell defects from WASp deficiency…………………………………15

Figure 4. The Intrinsic vs the Extrinsic pathways for apoptosis………………………...28

Figure 5. Different trafficking mechanisms that may produce the different post- translational forms of FasL………………………………………………………………33

Figure 6. Examples of fluorescence images used for scoring glomerular Ig subclass and C3 deposition…………………………...………………………………….49

Figure 7. Autoantibody Production in WASp deficient mice…………………………...60

Figure 8: Immune Complex Deposition and Mesangial Cell Proliferation in WASp deficient mice……………………………………………………………………………64

Figure 9. Impaired TCR mediated apoptosis of activated WASp deficient CD4+ T lymphocytes……………………………………………………………………………...68

Figure 10. Normal up-regulation of FasL mRNA and surface expression in WASp- deficient T cells…………………………………………………………………………..71

Figure 11. Reduced bioactive FasL and granule secretion by WASp deficient T cell….75

Figure 12. RICD and the secretion of FasL in gld FasL mutant T cells………………...88

Figure 13. FasL secretion does not require caspase-dependent apoptosis………………91

Figure 14. RICD and FasL secretion by CD4+ T cells does not require Rab27a……….95

Figure 15. CD8+ T cell cytotoxicity is dependent on Rab27 but not WASp;

xiii partially dependent on FasL……………………………………..……………………...100

Figure 16. CD8+ T cell RICD in the absence of target cells…………………………..103

Figure 17. CD4+ T cells depend on WASp but not Rab27 and partially on FasL for target cells lysis…………………………………………………………………………107

Figure 18. High CD4+ T cell density during restimulation results in less cell death….110

Figure 19. Role for WASp in CD4+ T cell cytotoxicity and RICD in presence of APC presenting cognate antigen……………………………………………...……...113

Figure 20. WASp deficiency results in a cell autonomous defect in RICD…………...116

Figure 21. WT FasL can rescue gld/gld RICD defect in trans………………………….119

Figure 22. Secreted WT FasL can rescue gld/gld RICD defect in trans………………..124

Figure 23. Different cells that WASp deficiency could impact for the development of autoimmunity………………………………………………………….142

xiv Chapter 1: General Introduction

Autoimmunity and immunodeficiency are considered separate disorders, but insights from both common and rare Mendelian forms of these diseases have revealed common mechanisms that may underlie both diseases. While it may be paradoxical for immunodeficient patients to get autoimmune disorders, there are mechanisms that may account for the development of autoimmunity in immunodeficiency. In our studies that we describe here, we have addressed the question regarding how autoimmunity develops from immunodeficiency by examining in detail cellular immune functions in Wiskott-

Aldrich Syndrome protein deficient T cells, which may predispose to autoimmunity.

A primary immunodeficiency disorder (PIDD) is a rare disorder that results from a genetic defect and is associated with increased susceptibility to infection (Geha et al.,

2007; Notarangelo et al., 2009). Primary immunodeficiency disorders are a genetically heterogeneous group of over 250 disorders that affect distinct components of the innate and adaptive immune system (Geha et al., 2007; Notarangelo et al., 2009; Ochs and

Hagin, 2014). While genetic defects affecting distinct components of the immune system lead to increased susceptibility to infection in PIDDs, normal immune responses can acquire a decrease in the immune response due to some secondary factor, e.g. malnutrition, environmental exposure, infection, etc., in what are termed secondary immunodeficiencies (Chinen and Shearer, 2010). Determining the cellular mechanisms that the genetic defects of PIDDs cause helps in not only the understanding the many complex functions of cells in the immune system, but also helps resolve what

1 mechanisms contribute to the development of autoimmunity, which in turn can help to develop treatments for individuals with PIDDs.

Ogdeon Bruton described the first PID, agammaglobulinemia, in 1952 (Bruton,

1952). In 1970, the International Union of Immunological Societies Expert Committee on Primary Immunodeficiencies decided to meet every two years to classify and define the growing number of PIDs (Geha et al., 2007). The last meeting was in 2013 and over

250 primary immune deficiencies have been characterized (Ochs and Hagin, 2014).

About 20 new PIDs are characterized each year (Primary Immunodeficiency Expert

Committee website), but new generation sequencing has lead to more rapid increases in the characterizations of new PIDs. Because primary immunodeficiencies are due to genetic defects, establishing prevalence of each PIDD and making diagnoses for PIDDs are current challenges in combating the adverse affects of PIDDs (Ballow et al., 2009;

Boyle and Buckley, 2007).

While enhanced susceptibility to infection is the first most prevalent clinical phenotype associated with primary immunodeficiency (Geha et al., 2007), autoimmunity is the second most prevalent clinical phenotype associated with primary immunodeficiency (Coutinho and Carneiro-Sampaio, 2008). Despite the challenges in combating the adverse affects of PIDDs, the study of these disorders have lead to significant insights into how the immune system functions (Coutinho and Carneiro-

Sampaio, 2008; Geha et al., 2007). Thus, the study of PIDDs are useful for understanding autoimmunity (Coutinho and Carneiro-Sampaio, 2008) because cellular mechanisms can be resolved if the genetic defects are known for PIDDs.

2 Autoimmune diseases affect ~3% of the human population (Jacobson, 1997).

Most autoimmune diseases are thought to be due to a combination of mutations in several genes and various environmental triggers (Todd, 1995), although some rare autoimmune diseases are due to single gene defects. So far, clinicians have established two general categories of autoimmune diseases: organ-specific and systemic autoimmune diseases

(Davidson and Diamond, 2001) (Marrack et al., 2001). In organ-specific autoimmune diseases, there is a response to an antigen expressed only in a specific organ (Marrack et al., 2001). In systemic autoimmune diseases, there is a response to antigens that are widely expressed throughout the host (Marrack et al., 2001). Because autoimmunity could arise either due to an antigen-specific abnormality or a global abnormality in lymphocyte function, this division does not correspond to a difference in causation

(Davidson and Diamond, 2001). As genome wide association studies continue for autoimmune diseases, the classifications for autoimmune diseases will be further defined.

Autoimmune development occurs from various lymphocytes recognizing self-antigens

(Grossman and Paul, 1992). Since CD4+ T cells are required for helping B cell responses, and B cells are responsible for producing antibodies, it is likely that autoreactive CD4+ T cells and B cells exist against self-antigens. Because there are many subsets of lymphocytes, the exact mechanisms that result in autoimmunity, however, are not completely resolved. Therefore, the genetic studies, including the genome wide association studies for autoimmune diseases, will provide useful information about how autoimmune diseases develop.

Studying autoimmune diseases associated with single gene defects help to resolve the mechanisms contributing to the pathogenesis of autoimmunity (Surolia et al., 2010).

3 Examining the association of monogenic PID disorders with autoimmunity provides insight into which genes are involved in tolerance and provides insight into how molecular mechanisms progress into autoimmunity (Carneiro-Sampaio and Coutinho,

2007). Monogenic PIDs that are highly associated with autoimmunity are particularly useful in understanding which molecules are involved in maintaining tolerance.

The monogenic PIDDs, IPEX, Omenn Syndrome, and APECED, are ~100% associated with autoimmunity (Carneiro-Sampaio and Coutinho, 2007) suggesting that

FOXP3, RAG-1, RAG-2, Artemis, IL-7Ra, RMRP and AIRE are critical genes involved in tolerance (Geha et al., 2007). FoxP3 deficiency leads to defects in regulatory T cells and is associated with the development of IPEX (Curotto de Lafaille and Lafaille, 2002).

Defects in RAG-1, RAG-2, Artemis, IL-7Ra, RMRP genes lead to defects in V-D-J recombination. These decreases in antigen receptor repertoire likely lead to autoimmune complications and are associated with the development of Omenn Syndrome (Villa et al.,

2001) (Ege et al., 2005) (Roifman et al., 2006) (Giliani et al., 2006). Defects in AIRE lead to the development of autoimmunity as a result of defects in central tolerance. In the absence of AIRE, there is a defect in the negative selection/deletion of self-reactive T cells against the “tissue-specific” genes in thymic epithelial cells. These defects in AIRE lead to the development of APECED (Eisenbarth, 2004) (Notarangelo et al., 2004)

(Ulmanen et al., 2005). The monogenic primary immunodeficiencies, ALPS and C1q deficiency, are also more than 80% associated with autoimmunity suggesting that FAS,

FASL, CASP10, CASP8, NRAS and C1Q are critical genes involved in peripheral tolerance (Notarangelo et al., 2009) (Carneiro-Sampaio and Coutinho, 2007). Defects in

Fas, FasL, Caspase 10, Caspase 8, and NRAS lead to defects in the apoptotic functions of

4 immune cells such as T cells and are associated with the development of ALPS. Defects in C1q lead to defects in the Classical Complement Pathway, which is involved in the rapid clearance of immune complexes, dying cells and debris from damaged tissues, and are associated with the development of C1q deficiency which is characterized by recurrent skin lesions, chronic infection, systemic lupus erythematosus (SLE) or SLE-like diseases (Thompson et al., 1980) (Topaloglu et al., 1996). The monogenic primary immunodeficiencies, C4, C1r/C1s and Wiskott Aldrich Syndrome, are between 40-75% associated with autoimmunity among PIDs that are strongly associated with autoimmunity (between 20-80% autoimmunity) suggesting that C4A, C4B, C1R, C1S and WAS are critical genes involved in tolerance (Notarangelo et al., 2009) (Carneiro-

Sampaio and Coutinho, 2007). Defects in C4, C1r and C1s lead to defects in the

Classical Complement Pathway and are associated with the development of SLE or rheumatoid arthritis (RA) similar to C1q. Defects in WAS lead to defects in actin function within hematopoietic cells and are associated with the development of the

Wiskott Aldrich Syndrome. CVID, a disease of polygenic origin, is also associated with

~20% autoimmunity (Agarwal and Cunningham-Rundles, 2009). Over 90% of the individuals with CVID do not have an immune-deficient family member (Agarwal and

Cunningham-Rundles, 2009). Defects serum immmunoglobulins and/or defects in T cells contribute to the development of CVID, which include increased susceptibility to infections as well as autoimmunity, granulomata and tumors. Because CVID has such complex etiology, its complexity presents challenges in understanding the genes and gene functions involved in the pathogenesis of autoimmunity. Therefore, focusing on

5 pathogenesis of autoimmunity in monogenic PIDs is a logical step for providing more efficient therapy for autoimmunity.

The monogenic primary immunodeficiency, Wiskott-Aldrich Syndrome (WAS), is associated with defects in the Wiskott-Aldrich Syndrome protein (WASp) and is associated with 40-72% autoimmunity (Notarangelo et al., 2009) (Carneiro-Sampaio and

Coutinho, 2007). Premature termination and deletion mutations affect the expression of

Wiskott-Aldrich Syndrome protein and result in severe, also known as, classical Wiskott-

Aldrich Syndrome (Jin et al., 2004), that can lead to premature death if the patient is not treated with a bone marrow transplantation or gene therapy. The phenotypically milder diseases, X-linked thrombocytopenia (XLT) and attenuated Wiskott-Aldrich Syndrome result from hundreds of different missense mutations or activating mutations in X-linked neutropenia (XLN) (Imai et al., 2004). Generally, the complete absence of WASp expression results in WAS while low level expression of WASp results in a milder phenotype of “dysregulated immunity” associated with thrombocytopenia (Imai et al.,

2004).

WASp is only expressed in hematopoietic cells (Petrella et al., 1998) and functions in the branched actin polymerization that is necessary for the transduction of signals at the cell surface to the actin cytoskeleton (Mullins et al., 1998). Defects in

WASp result in cytoskeletal aberrations that affect the normal cellular activity of hematopoietic cells in mice and humans, including proliferation, phagocytosis, immune synapse formation, adhesion, directed migration, antigen receptor activation, Ca2+ mobilization and transcriptional activation (Ancliff et al., 2006) (Bouma et al., 2011)

(Ochs et al., 1980) (Zhang et al., 1999a) (Snapper et al., 1998) (Gallego et al., 1997)

6 (Molina et al., 1993) (Dupre et al., 2002) (Badour et al., 2003) (Tsuboi and Meerloo,

2007) (Burns et al., 2004b) (Ishihara et al., 2012) (Kumar et al., 2012) (Lorenzi et al.,

2000) (Leverrier et al., 2001) (Leverrier and Ridley, 2001) (Olivier et al., 2006) (Pulecio et al., 2008) (Borg et al., 2004). These defects in the normal cellular processes of hematopoietic cells result an X-linked recessive disease that first presents with the appearance of petechiae and abnormal bleeding. WAS is most characterized by thrombocytopenia (100% in patients) with small platelets and eczema (80% in patients)

(Sullivan et al., 1994) and recurrent infections due to mutations causing the premature termination or truncation of WASp. WAS patients and WASp deficient mice also have high autoantibody titers and compromised adaptive immunity (Dupuis-Girod et al.,

2003). The defects in WASp affect the cellular functions of many different hematopoietic cells. These defective hematopoietic cells can manifest in the development of autoimmunity.

In order to understand the mechanisms of WASp that contribute to immunodeficiency and autoimmunity, several strains of WASp-deficient mice have been produced. WASp comprises 502 aa and has five functional domains: N-terminal

Drosophila-enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, a basic region, a GTPase-binding domain (GBD), a polyproline-rich region (PRR) and a

C-terminal verprolin homology/central region/acidic region (VCA) domain (Burns et al.,

2004a). These different domains of WASp help regulate the polymerization of branched actin in the cytoskeleton of hematopoietic cells. Before WASp becomes activated to regulate branched actin polymerization, WASp remains in an auto-inhibited state due to binding between the VCA domain to the GBD domain. The interaction of the WH1

7 domain with WASp-interacting protein (WIP) stabilizes the auto-inhibited form of

WASp. Upon cell surface receptor activation, the Rho family GTPase cell division cycle

2 (CDC42) binds to the GBD, causing the release of the VCA. The WASp activators, proline-serine-threonine phosphatase-interacting protein (PSTPIP1) and phosphatidylinositol-4,5-biphosphate (PIP2) bind the PPP and B domains, respectively.

Src family tyrosine kinases and the tyrosine-protein phosphatase non-receptor type 12

(PTPN12) phosphorylate the tyrosine residue 291 (Y291) and stabilize the active form of

WASp. This active form of WASp binds the actin-related protein 2 and 3 (ARP 2/3) complex through the VCA and monomeric actin to produce a new actin branch in the cytoskeleton. WASp activation and degradation regulate how the cytoskeleton remodels actin (Figure 1).

8 Figure 1. WASp activation leading to branched actin polymerization.

9 Figure 1. WASp activation leading to branched actin polymerization. WASp comprises 502 aa and has five functional domains: N-terminal Drosophila- enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain, a basic region, a GTPase-binding domain (GBD), a polyproline-rich region (PRR) and a C- terminal verprolin homology/central region/acidic region (VCA) domain (Burns et al.,

WASp. Upon cell surface receptor activation, the Rho family GTPase cell division cycle

Src family tyrosine kinases and the tyrosine-protein phosphatase non-receptor type 12

(PTPN12) phosphorylate the tyrosine residue 291 (Y291) and stabilize the active form of

10 The expression of targeted mutations of WASp in T cells and in transgenic and knock-in mice have helped define the function of each WASp domain and have helped to resolve how each domain may contribute to the clinical manifestations of WAS (Figure

2). EVH1 domain deletion reduces the ability of WASp to bind to WIP, which binds to and stabilizes WASp (Konno et al., 2007). The majority of WAS patients have mutations in the N-terminal region of WASp, including many within the EVH1 domain suggesting that WIP binding is necessary for stabilizing WASp expression in vivo. GBD deletion of

WASp in mice results in a lack of Cdc42 binding, but T cells proliferate normally after activation suggesting that the GBD domain in WASp is not necessary for T cell activation (Badour et al., 2004). Phosphorylation of the tyrosine at position 291 in human (293 in mouse) GBD of WASp activates WASp to remain in its active conformation to polymerize actin and effectively form the immune synapse (Badour et al., 2004) (Blundell et al., 2009). Deletion of the WASp Proline Rich Region (PRR) reduces actin polymerization and the formation of T cell:APC conjugates suggesting the

TCR signaling depends on PRR (Badour et al., 2003). Deletion of the C terminal VCA domain in WASp leads to defective thymopoiesis (Zhang et al., 2002). This defective thymopoiesis is not observed in WASp deficient mice, which have a targeted deletion of intron 4 to exon 11, encompassing the deletion of GBD, PRR and V and C of the of the

VCA domain (Zhang et al., 1999a) suggesting that this mutant of WASp depends on the

C-terminal domain for actin polymerization and acts as a dominant negative for other N-

WASp and other NPF family proteins.

11 Figure 2. WASp domains and associated functions of each domain.

12 Figure 2. WASp domains and associated function of each domain (Cleland and

Siegel, 2011). The indicated domains of WASp are shown schematically, with proteins and other molecules binding WASp shown below the schematic, and functions shown above. EVH1/WH1 domain: enabled VASP (vasodilator-stimulated protein) homology

1)/WH1 (WASP Homology 1). B: Basic Domain. GBD: GTPase Binding Domain.

Proline Rich Domain (PRD). The PRD of WASp has been reported to bind numerous Src

Homology 3 (SH3) domain containing proteins, including Btk, Hck, Fyn, Lyn, Itk, Nck, c-Src. Grb2, p85a, PI-3K, PLC-g, PSTPIP1, PTP-PEST, profilin and VASP. PSTPIP1 binding has been implicated in trafficking of WASp to the immune synapse, and binding of Fyn and PTP-PEST has been implicated in regulating tyrosine 291 phosphorylation and the function of WASp in T cell activation. VCA domain: Verprolin, cofilin, acidic domain.

13 In 1998, Scott Snapper et al. generated WASp-deficient mice on the 129 background with a targeted insertion into the GBD (Snapper et al., 1998). These young

WASp deficient mice had mild thrombocytopenia, lymphopenia and defective T cell activation similar to WAS patients (Snapper et al., 1998). Unlike WAS patients, these

WASp deficient mice developed colitis, but did not develop eczema, hematopoietic malignancies or abnormal bleeding (Snapper et al., 1998). These WASp deficient mice did not have reduced sized platelets and did not have abnormal responses to antigens.

Because Snapper et al. examined mice that were 4 months old, the phenotype similar to

WAS patients may not have had the time to develop. Additionally, because mice are raised in pathogen-free conditions, the discrepancies between WAS patients and WASp- deficient mice may be due to the different in sterile conditions.

In 1999, the Siminovitch lab generated a WASp-deficient mouse with targeted deletion of intron 4 to exon 11, encompassing the deletion of GBD, PRR and V and C of the of the VCA domain (Zhang et al., 1999a). Additionally, Siminovitch et al. have generated several T cell-specific WASp-deficient mice that re-express WASp with targeted deletions specifically affecting the EVH1, GBD, PRR, or VCA domains of

WASp under the CD2 promoter. These mouse models of WASp have helped understanding of the cellular mechanisms that contribute to the development of WAS.

Studies of WASp deficient mice show that there are distinct defects in immune function in multiple cell types comparable to the defects observed in WAS patients

(Figure 3). While WASp deficient mice have thrombocytopenia and lymphopenia similar to WAS patients, as mentioned before, they do not develop eczema, hematopoietic

14 Figure 3. Immune cell defects from WASp deficiency.

15 Figure 3. Immune cell defects from WASp deficiency (Cleland and Siegel, 2011).

Green arrows represent processes with positive influence on immune responses and red arrows represent processes that reduce immune responses through cytotoxicity or other regulatory mechanisms. Immune processes influenced by WASp are numbered in the figure with green numbers denoting effects of WASp that enhance immune responses and red denoting effects of WASp which regulate immune responses. Specific functions of

WASp are numbered as follows: 1) WASp promotes dendritic cell (DC) podosome formation and migration, presentation of particulate antigens and T-DC contacts in the immune synapse in a DC intrinsic manner; 2) WASp in T cells can enhance the efficiency of immune synapse formation which contributes to T cell activation; 3) WASp also promotes cyokine secretion which also contributes to T cell activation and effector function; 4) WASp promotes restimulation-induced cell death and release of cytotoxic granules and FasL; 5) WASp promotes B cell adhesion, migration, and germinal center formation; 6) WASp promotes T reg homeostasis, Treg control of effector T cell proliferation and Treg killing of B cells; 7) WASp promotes cytotoxicity of NK cells; and

8) WASp may also promote uptake of apoptotic blebs by macrophages.

16 malignancies or abnormal bleeding that WAS patients often exhibit (Snapper et al., 1998)

(Zhang et al., 1999a). Spontaneous colitis developed in one colony of WASp deficient mice (Snapper et al., 1998), but colitis is not seen until after irradiation or other stresses suggesting that environmental factors contribute to the colitis development.

Because WASp affects the polymerization of branched actin filaments in hematopoietic cells, the cellular functions of hematopoietic cells are compromised.

Platelets, macrophages, neturophils, NK cells, B cells and T cells have cellular defects as a result of WASp deficiency.

WASp Deficiency in Platelets

The most penetrant phenotype of Wiskott-Aldrich Syndrome in patients is thrombocytopenia with small platelets (Sullivan et al., 1994), so the effect of WASp deficiency on platelets may provide clues about the molecular functions that may be affected in other immune cells. WASp localizes to the platelet membrane (Lutskiy et al.,

2007) and is phosphorylated in alphaIIb beta3 outside-in signaling. WASp deficiency results in defective outside-in signaling and leads to defects in platelet adhesiveness and platelet spreading (Shcherbina et al., 2010). WASp deficiency does not affect alphaIIb beta3 inside-out signaling in WAS patient or WASp deficient murine platelets

(Shcherbina et al., 2010). Both WAS patient and WASp deficient murine platelets have decreased spreading on fibrinogen (Shcherbina et al., 2010). WASp deficiency does not result in a decrease in degranulation by platelets in humans (Gross et al., 1999), although degranulation in murine T cells (Nikolov et al., 2010) and integrin-dependent

17 degranulation in murine neutrophils (Zhang et al., 2006b) is reduced when WASp is deficient.

Platelet Interactions with T cells

Platelets have been suggested to enhance CD4+ T cell lymphocyte adhesion to extracellular matrix under flow conditions (Solpov et al., 2006). When beta2 and beta1 integrins are blocked, there is a decrease in adhesion that is more pronounced by beta2 inhibition (Solpov et al., 2006). These authors did not examine the effect of beta3 integrin, so it is not known if WASp affects platelet interactions with T cells. NK cell lysis of tumor cells can be inhibited by platelets (Nieswandt et al., 1999). Tumor cells acquire platelet aggregates on their cell surfaces and aggregate with platelets (Nieswandt et al., 1999).

WASp Deficiency in Myeloid Cells

Macrophages, dendritic cells and neutrophils rely upon actin polymerization in order to adequately respond to stimuli. These cells must migrate through tissues after stimulation and then home to sites of inflammation or to secondary lymphoid tissues for initiating effector responses of other immune cells. WASp deficient macrophages have compromised responses to fMLP, MCP-1 and CSF-1 (Badolato et al., 1998) (Zicha et al.,

1998). WASp deficient dendritic cells do not migrate upon stimulation with fMLP or

RANTES and do not polarize or extend (Binks et al., 1998). Both WASp deficient macrophages and dendritic cells have reduced numbers of podosomes (Burns et al.,

2001). WASp deficient dendritic cells have reduced adherence to ICAM-1 and do not

18 localize integrins around podosomes (Burns et al., 2004b). WASp deficient macrophages have a defect in IgG mediated phagocytosis (Lorenzi et al., 2000) (Tsuboi and Meerloo,

2007). WASp-deficient Neutrophils do not aggregate integrins properly (Zhang et al.,

2006a) and have decreased migration in vivo and in vitro (Snapper et al., 2005).

Additionally, WASp deficient DCs prevent activation and polarization of T cells (Bouma et al., 2007; Bouma et al., 2011) (Pulecio et al., 2008). WASp deficient macrophages have a defect in engulfing apoptotic debris suggesting a mechanism by which WASp affects the maintenance of the immune system (Leverrier et al., 2001). Overall, WASp deficiency reduces myeloid cell migration.

WASp deficiency in NK cells

WASp deficiency results in an increase in NK cells in peripheral blood, defective intercellular interactions leading to defective activation by dendritic cells and cytotoxic activity of NK cells (Castellano et al., 2001; Soderling et al., 2002; Volkman et al., 2002).

This defective cytotoxic activity is a result of impaired immune synapse formation between NK cells and their target cells (Soderling et al., 2002). Impaired perforin localization at the immune synapse occurs from WASp deficiency as well as defective signaling downstream of CD16 (Soderling et al., 2002; Volkman et al., 2002).

Additionally, WASp deficiency results in delayed activation of nuclear factor of activated

T cells 2 (NFAT2) and nuclear factor-B (NF-B) in response to the NKp46 activating receptor (Scott et al., 2002d). This process is independent of actin polymerization suggesting that WASp is involved in NK signaling.

19 WASp deficiency results in NK-cell migration defects (Stabile et al., 2010) (Scott et al., 2002a). This reduction in migration was associated with a reduction in the up- regulation of CD18 and a subsequent inability to adhere to ICAM-1 or VCAM-1 following chemokine stimulation (Stabile et al., 2010) (Scott et al., 2002a). Cdc42 activation and WASp tyrosine phosphorylation occurred after NK cell the engagement of chemokine receptor or integrin (or ) (Stabile et al., 2010) (Scott et al., 2002a).

Additionally, WASp association with Fyn and Pyk-2 occurred after NK cell NK cell chemokine receptor or integrin (or ) engagement. These results suggest that the

Cdc42/WASp pathway is involved in inside-out signaling. Overall, WASp deficiency in

NK cells provides clues about the both innate and adaptive molecular and cellular mechanisms that may contribute to autoimmunity.

WASp deficiency in B cells

WASp deficiency may contribute to the development of autoimmunity through defective functions of B cells and/or the production of autoantibodies. WASp deficient mice have reduced B cell proliferation, reduced B cell migration and reduced germinal center formation (Westerberg et al., 2005). WASp deficiency impairs mature B cell survival and homeostasis in a B cell intrinsic fashion (Meyer-Bahlburg et al., 2008).

WASp deficient murine T regs also have a reduced ability to kill activated B cells

(Adriani et al., 2011). This inability of WASp deficient T regs to kill activated B cells may lead to the production of autoantibodies from potentially autoreactive B cells

(Adriani et al., 2011).

20 WASp Deficiency in T cells

WASp deficient T cells proliferate poorly after TCR stimulation (Zhang et al.,

1999a). IL-2 supplementation in vitro rescues this poor proliferation (Snapper et al.,

1998). The poor proliferation may be a result of an inefficient formation of an immune synapse between T cells and APCs, but WASp independently regulates the secretion of effector cytokines from T cells (Morales-Tirado et al., 2004). WASp deficiency results in varied T cell defects and the reason for this may be due to the partial redundancy of

WASp with other related proteins that promote actin polymerization. These related proteins, type I nucleation promoting factors (NPF) that may have redundant function to

WASp include N-WASp, WAVE, WHAMM and JMY. While WASp expression is restricted to hematopoietic cells, N-WASp is ubiquitously expressed in all cells. N-

WASp deficiency results in defective endocytosis, exocytosis and hair-follicle cycling

(Derivery and Gautreau, 2010). WASH mediates endosome trafficking, but functions at different times and in different vesicles than N-WASp (Duleh and Welch, 2010). WAVE mediates cell motility and membrane ruffling (Borm et al., 2005). WHAMM mediates

ER-Golgi transport and Golgi organization (Campellone et al., 2008). JMY mediates cell motility (Zuchero et al., 2009). Investigations into redundancy of WASp and N-WASp in lymphocytes by specifically ablating WASp, N-WASp or both proteins in T cells or in the lymphocytes of chimeras with RAG deficient blastocysts showed that N-WASp deficient only cells did not have defects (Cotta-de-Almeida et al., 2007). This lack of a defect in N-WASp deficient only cells indicates that N-WASp is redundant for many functions of lymphocytes that are affected by WASp. B and T cells doubly deficient in

WASp and N-WASp results in lymphopenia and a block in development prior to the

21 CD4+ CD8+ double positive stage (Cotta-de-Almeida et al., 2007). Thymocytes from mice doubly deficient in WASp and N-WASp showed reduced proliferation and chemotactic responses to CCL12 (Cotta-de-Almeida et al., 2007). Colitis in mice doubly deficient for WASp and N-WASp was more severe than in WASp deficient mice (Cotta- de-Almeida et al., 2007). Since WASp deficient mice and WAS patients have lymphopenia and peripheral T cells proliferate in response to self-antigens in lymphopenic hosts, but such T cell proliferation does not occur when T cell numbers are normal, this severe colitis may have been due to lymphopenia and reduced numbers of T cells in the doubly deficient mice.

One possible mechanism that may contribute to the observed autoimmunity in

WASp deficient mice and WAS patients is defective homeostasis and function of regulatory T cells (Tregs). The numbers of FoxP3(+) Tregs are normal in the periphery and thymus of WASp deficient mice (Marangoni et al., 2007a). However, in bone marrow chimera experiments in mice, normal Tregs outcompete WASp deficient Tregs

(Humblet-Baron et al., 2007). This suggests a cell-intrinsic role for WASp in Treg survival. WASp deficient Tregs proliferate poorly and fail to produce TGF-when stimulated in vitro. IL-2 does not rescue this defect (Humblet-Baron et al., 2007)

(Marangoni et al., 2007a). WASp deficient Tregs have a reduced ability to suppress the proliferation of normal T cells (Marangoni et al., 2007a) and have a reduced ability to prevent colitis induced by naïve T cells transferred into immunodeficient hosts. Similar to other T cells, Tregs from WAS patients have activation and functional defects

(Marangoni et al., 2007a) (Adriani et al., 2007a). It is not clear how WASp deficiency affects Treg function, but defective Treg homeostasis may be due to the IL-2 dependence

22 of Tregs for survival. Additionally, functional defects in Tregs may be due to known T cell activation defects or due to problems with the secretion of cytotoxic granules.

Additionally, since programmed cell death of mature T cells requires TCR restimulation,

WASp deficiency may contribute to autoimmunity in WAS independent of the defects in

Tregs. TCR restimulation of activated T cells results in programmed cell death. This programmed cell death primarily occurs through the autocrine stimulation of the Fas receptor by Fas Ligand (FasL) (Siegel et al., 2000). WASp deficiency may contribute to the development of autoimmunity by affecting either TCR restimulation or FasL function. In order to help narrow down the mechanisms by which autoimmunity develops in WAS, one of the goals of this thesis is to resolve whether WASp deficiency reduces the function of FasL in T cells.

Overall, the cell intrinsic defects in platelets, macrophages, dendritic cells, neutrophils, NK cells, B cells and T cells may contribute to the complex immune dysfunction observed in WAS and WASp deficient mice (Figure 3) (Bosticardo et al.,

2009) (Orange et al., 2002). We intend to examine how the defects in WASp contribute to defects in programmed cell death. We hypothesize that the defects in programmed cell death contribute to the high percentage of autoimmunity in WAS patients.

Fas-FasL Deficiency Association with Autoimmunity

Autoimmune lymphoproliferative syndrome (ALPS) is a rare autosomal dominant disorder characterized by a defect in lymphocyte apoptosis that manifests in the accumulation of double negative (DN) T cells (Canale and Smith, 1967). This accumulation of up to 40% DN T cells in the blood results in lymphadenopathy (90% of

23 patients), splenomegaly (80% of patients), and the infiltration of potentially autoreactive lymphocytes in other organs throughout the body (Rieux-Laucat et al., 1995) (Bleesing et al., 2001). The diagnostic criteria for ALPS include chronic nonmalignant lymphadenopathy and/or splenomegaly, peripheral expansion of DN T cells and deficient lymphocyte apoptosis (Sneller et al., 2003). Severe autoimmune complications in ALPS patients tend to include the targeting and damaging of blood cells and manifest as autoimmune hemolytic anemia, thrombocytopenia and neutropenia (Rieux-Laucat et al.,

1995).

In 1995, patients with lymphocyte apoptosis defects were shown to have Fas mutations which lead to the understanding of Fas being the first molecular mechanism contributing to the development of ALPS (Rieux-Laucat et al., 1995) (Fisher et al., 1995).

Other mutations in other apoptotic pathway proteins have been identified in contributing to ALPS, i.e. FasL, caspase 10, caspase 8 (Wu et al., 1996) (Del-Rey et al., 2006) (Rieux-

Laucat et al., 2003a) (Rieux-Laucat et al., 2003b) (Chun et al., 2002) (Puck and Zhu,

2003) (Wang et al., 1999) and each protein is associated with specific characteristics of

ALPS. Humans rarely have a defect in FasL (Del-Rey et al., 2006) (Rieux-Laucat et al.,

2003b) and the majority of ALPS patients have a defect in Fas as opposed to other apoptotic proteins.

Lpr Fas deficient mice and Gld FasL deficient mice develop severe autoimmune complications due to the accumulation of potentially autoreactive double negative (DN)

T cells (Takahashi et al., 1994a) (Watanabe-Fukunaga et al., 1992). FasL knockout mice have a much more severe autoimmune phenotype and die early after birth (Karray et al.,

2004).

24 Intrinsic vs Extrinsic Cell Death Mechanisms

Activated T cells die through two distinct pathways, i.e, through the withdrawal of growth promoting cytokines (intrinsic) or by antigen or receptor induced apoptosis

(extrinsic). Intrinsic cell death occurs after effector T cells respond to an invading pathogen and levels of growth and survival promoting cytokines such as IL-2, IL-7 and

IL15 decrease at the conclusion of a successful T cell response to the pathogen. The decrease of IL-2 and other cytokines leads to the intrinsic death of the majority of effector T cells. This intrinsic cell death depends on the BH3 protein Bim promoting

Bcl-2-asociated X protein (BAX) and Bcl-2 antagonist or killer (Bak) oligomerization into pore forming complexes for inducing mitochondrial outer membrane permeabilization (MOMP). Bax and Bak form pores in the mitochondrial membrane leading to the release of cytochrome c. Cytochrome C binds to apoptotic protease- activating factor 1(APAF 1) leading to its oligmerization and subsequent formation of the apoptosome that recruits and activates caspase 9. Caspase 9 cleaves and activates caspase 3 and caspase 7 resulting in apoptosis. The mitochondrial release of second mitochondria-derived activator of caspase (SMAC) and OMI neutralizes the X-linked inhibitor of apoptosis protein (XIAP), which inhibits caspases. Anti-apoptotic B cell lymphoma 2 (Bcl-2) family members block BAX and Bak from inducing MOMP. Only a few memory T cells persist from the massive cytokine withdrawl induced death.

T cell specific death occurs in a process known as propriocidal regulation after simultaneous restimulation through the T cell receptor (TCR) and ligation between the

Fas receptor and Fas ligand (Hornung et al., 1997). Upon TCR ligation through a specific peptide:MHC Class II complex of a cycling CD4+ T cell, the TCR redistributes

25 into polarized focal aggregates on the membrane resulting in the polymerization of actin,

FasL and TNF gene activation, Fas redistribution into lipid rafts, and subsequent cell death (Combadiere et al., 1998) (Muppidi and Siegel, 2004). TCR ligation to specific antigens presented by MHC Class II sensitizes the CD4+ T cell to die and this sensitization is often referred to as the competency to die signal. Previous studies indicate that PKC-theta and Rac1 may contribute to the competency to die signal

(Manicassamy and Sun, 2007) (Ramaswamy et al., 2007). PKC-theta and WASp have opposing functions for the pSMAC of the immune synapse suggesting their role in possibly regulating the movement of Fas into lipid rafts of the immune synapse (Sims et al., 2007). While Rac1 is downstream of the Cdc42 activator for WASp, Rac1 has been shown to be involved in the translocation of Fas into lipid rafts suggesting that the upstream WASp function affects the competency for a T cell to die (Nobes and Hall,

1995). Fas oligomerization within lipid rafts leads to the Fas being receptive to FasL binding. The ligation of FasL to the oligomerized Fas receptor leads to the rapid recruitment of the adaptor protein FADD to Fas. Caspase-8 then associates with FADD and Fas, all of which form the death inducing signal complex (DISC) (Wang et al., 2010).

Depending on the level of caspase-8 activation upon DISC formation, extrinsic apoptosis can occur by direct cleavage of effector caspases in the cytosol (type I) or by proceeding through the intrinsic (mitochondrial) pathway to amplify the death signal

(type II) for apoptosis to occur (Scaffidi et al., 1998) (Lee et al., 2006). Type II cells have a slower DISC assembly and a subsequent low production of caspase 8 (Scaffidi et al., 1998). This caspase 8 cleaves BID on the surface of the mitochondria (Luo et al.,

1998). This cleaved BID (tBID) interacts with Bak and/or Bcl-XL on a separate complex

26 on the mitochondrial membrane to induce pore formation and MOMP. Activation of caspase 3 and caspase 7 directly by caspase 8 (type I) or through the apoptosome (type II) leads to nuclear and cytoplasmic condensation followed by cellular fragmentation into apoptotic bodies (Nagata, 1997). Figure 4 illustrates the intrinsic vs the extrinsic pathways for apoptosis.

27 Figure 4. The Intrinsic vs the Extrinsic pathways for apoptosis.

28 Figure 4. The Intrinsic vs the Extrinsic pathways for apoptosis. Activated T cells die through two distinct pathways, i.e, through the withdrawal of growth promoting cytokines (intrinsic) or by antigen or receptor induced apoptosis (extrinsic). Intrinsic cell death occurs after effector T cells respond to an invading pathogen and levels of growth and survival promoting cytokines such as IL-2, IL-7 and IL15 decrease at the conclusion of a successful T cell response to the pathogen. Intrinsic cell death depends on the BH3 protein Bim promoting Bcl-2-asociated X protein (BAX) and Bcl-2 antagonist or killer

(Bak) oligomerization into pore forming complexes for inducing mitochondrial outer membrane permeabilization (MOMP). Bax and Bak form pores in the mitochondrial membrane leading to the release of cytochrome c. Cytochrome C binds to apoptotic protease-activating factor 1(APAF 1) leading to its oligmerization and subsequent formation of the apoptosome that recruits and activates caspase 9. Caspase 9 cleaves and activates caspase 3 and caspase 7 resulting in apoptosis.

Extrinsic cell death occurs after simultaneous restimulation through the T cell receptor (TCR) and ligation between the Fas receptor and Fas ligand (Hornung et al.,

1997). Upon TCR ligation through a specific peptide:MHC Class II complex of a cycling CD4+ T cell, the TCR redistributes into polarized focal aggregates on the membrane resulting in the polymerization of actin, FasL and TNF gene activation, Fas redistribution into lipid rafts, and subsequent cell death (Combadiere et al., 1998)

(Muppidi and Siegel, 2004). TCR ligation to specific antigens presented by MHC Class

II sensitizes the CD4+ T cell to die and this sensitization is often referred to as the competency to die signal. Fas oligomerization within lipid rafts leads to the Fas being receptive to FasL binding. The ligation of FasL to the oligomerized Fas receptor leads to

29 the rapid recruitment of the adaptor protein FADD to Fas. Caspase-8 then associates with FADD and Fas, all of which form the death inducing signal complex (DISC) (Wang et al., 2010).

DISC assembly and a subsequent low production of caspase 8 (Scaffidi et al., 1998).

This caspase 8 cleaves BID on the surface of the mitochondria (Luo et al., 1998). This cleaved BID (tBID) interacts with Bak and/or Bcl-XL on a separate complex on the mitochondrial membrane to induce pore formation and MOMP. Activation of caspase 3 and caspase 7 directly by caspase 8 (type I) or through the apoptosome (type II) leads to nuclear and cytoplasmic condensation followed by cellular fragmentation into apoptotic bodies (Nagata, 1997).

30 Fas and FasL Function in Regulating Peripheral Tolerance

Restimulation Induced Cell Death (RICD) is a mechanism that regulates the population of clonal activated T cells. After TCR activation through a cognate antigen peptide presented by either MHC Class I or II, the specific T cells activated to respond to a specific antigen clonally expand for directing an appropriate immune response against the antigen. These clonally activated T cells upregulate cell surface Fas expression

(Wong et al., 1997). Fas is a member of the tumor necrosis factor receptor (TNF-R) family and contains an intracellular death domain (Suda et al., 1993). During the clearance of the antigen, the clonally expanded population of specifically activated T cells undergo TCR restimulation, which switches the T cells from an apoptosis resistant state to an apoptosis sensitive state. This apoptosis sensitive state upon TCR restimulation is due to the upregulation of FasL (Vignaux et al., 1995; Zheng et al.,

1998). A small fraction of the specifically activated T cells that were exposed to antigen survive to develop into memory T cells. The memory T cells rapidly respond with specific clonal expansion again when exposed to the same antigen to which the T cell originally encountered (Janssen et al., 2005).

Deficient Fas or FasL expression results in a defect in RICD and the accumulation of potentially autoreactive T cells and subsequent association of severe autoimmunity, including the rapid development of lymphadenopathy, splenomegaly in mice and humans. The mechanisms for FasL function during the expansion and contraction of clonal T cells responding to a specific antigen vary depending upon the different domains of FasL that interact with other molecules.

31 FasL functions in several roles during the response of a specific T cell to a specific antigen depending upon how FasL is regulated. FasL may function as a costimulatory molecule since FasL ligation with Fas inhibits the proliferation of CD4+ T cells in mice (Desbarats et al., 1998) while FasL ligation with Fas promotes proliferation of CD8+ T cells in mice (Suzuki and Fink, 1998) (Suzuki and Fink, 2000). Upon TCR restimulation, FasL accumulates on the plasma membrane (Zhang et al., 2000).

Membrane bound FasL aggregates into homo-trimeric or hexameric complexes (Holler et al., 2003) and is cytotoxic. Additionally, FasL may be shed from a T cell in either a membrane bound form on vesicles or a cleaved form known as soluble FasL (Jodo, 2001)

(Tanaka et al., 1995). ADAM-10 is the MMP that cleaves FasL into the soluble form

(Schulte et al., 2007). The membrane bound form of FasL shed from T cells is cytotoxic while the soluble form of FasL is not cytotoxic (O’ Reilly et al., 2009). The membrane bound form of FasL may be shed in vesicles that are generated from multivesicular bodies during exocytosis or the membrane bound form of FasL may be shed directly from vesicles that are generated from the plasma membrane during ectocytosis. These different mechanisms for generating vesicles may give insights into the observed differences in the functions of cytotoxicity between CD4+ T cells and CD8+ T cells

(Figure 5).

32 Figure 5. Different trafficking mechanisms that may produce the different post- translational forms of FasL.

33 Figure 5. Different trafficking mechanisms that may produce the different post- translational forms of FasL. After receptor stimulation, the Golgi either secretes FasL directly to be expressed on the plasma membrane or secretes FasL on vesicles through the exocytosis of multivesicular bodies. Alternatively, previously expressed FasL on the cell surface may be endocytosed and processed through multivesicular bodies in order to be secrete vesicular FasL. Depending upon the various receptors stimulated, FasL may be cleaved by metalloproteases on the cell surface or even on the surfaces of vesicular FasL.

FasL may also be secreted on vesicles through ectocytosis, which is direct budding from the plasma membrane.

34 Vesicle Trafficking

Cells may produce vesicles from either the process of exocytosis or ectocytosis.

Exocytosis is the process in which vesicles, which are generated from the cell, fuse with the plasma membrane of a cell for various extracellular functions. Previous studies have shown that there are three modes for exocytosis in secretory cells, i.e. full-collapse fusion, kiss-and-run and compound exocytosis. These different modes of exocytosis control the rate and amount of vesicular content release, which impact the strength of exocytosis. Ectocytosis, alternatively, is the process in which vesicles bud from the plasma membrane. Below, I will briefly explain the molecular mechanisms of exocytosis and ectocytosis. Additionally, I will explain how mutations in exocytosis and ectocytosis proteins associate with the development of primary immunodeficiency diseases.

The exocytosis of vesicles begins when the endoplasmic reticulum transports newly synthesized molecules specifically tagged for exocytosis to the Golgi apparatus.

The trans-Golgi network (TGN) constitutively packages the secretory vesicles (Gorr et al., 2005) (Jolly and Sattentau, 2007) (Mayorga et al., 2007) (Williams, 2010) (Wu et al.,

2006). Microtubules then primarily traffic secretory vesicles from the TGN to the plasma membrane (Chiang et al., 2012) (Nakata and Hirokawa, 2003). Once vesicles approach the plasma membrane, the actin cytoskeleton directs exocytic vesicles to the site of fusion

(Hume et al., 2011) (Schroeder et al., 2010) (Snider et al., 2004). The actin cytoskeleton regulates the fusion pore and provides the force to complete fusion (Sokac and Bement,

2006) (Masedunskas et al., 2012) (Nightingale et al., 2012). The actin cytoskeleton likely provides a scaffold for anchoring secretory vesicles in close proximity to the plasma membrane (Sankaranarayanan et al., 2003) and likely is a barrier that blocks exocytosis

35 (Brown et al., 2011). Actin associated secretory vesicles likely remain in the actin cytoskeleton until a stimulus provokes the detachment from the cytoskeleton (Abu-

Hamdah et al., 2006) (Bittins et al., 2009) (Bond et al., 2011) (Ceccaldi et al., 1995)

(Cesca et al., 2010) (Gaffield et al., 2006) (Giovedi et al., 2004) (Gotow et al., 1991)

(Landis et al., 1988) (Miyamoto, 1995) (Mizuno et al., 2011) (Sankaranarayanan et al.,

2003) (Sellers and Knight, 2007) (Wang et al., 2004).

TCR activation and T cell function involve the trafficking of vesicles. In order to understand how vesicular trafficking is involved in TCR activation and function, a brief description of T cell signaling is necessary. TCR activation occurs when the T cell receptor engages with a cognate peptide antigen that is presented by either MHC I or II.

The TCR ligation leads to the phosphorylation of ITAMs on the TCR/CD3 complex through either the CD4 or CD8 associated protein tyrosine kinase (PTK) p56lck. The phosphorylated TCR/CD3 complex recruits PTK ZAP-70. p56lck phosphorylates the

TCR/CD3/PTK ZAP-70 complex and/or autophosphorylation of the complex occurs.

ZAP-70 phosphorylates LAT (linker of activation) and SLP-76 (SH2 domain-containing leukocyte protein of 76kDA). GADS (Grb-2 related adapter downstream of Shc) assembles the complex at the plasma membrane to form a signalsome that recruits multiple downstream components of T cell activation.

TCR activation induces structural changes in LFA-1 (lymphocyte function- associated antigen 1) on T cells. LFA-1 on T cells can then bind to ICAM-1

(intercellular adhesion molecule 1) on APCs. This interaction between LFA-1 and

ICAM-1 between the T cell and APC stabilizes the interaction between the cells. This stabilized interaction initiates the formation of the immunological synapse. As few as 10

36 productive interactions between TCR and cognate peptide antigens presented by either

MHC Class I or II lead to the formation of the immunological synapse (Irvine et al.,

2002) (Purbhoo et al., 2004).

The immune synapse is differentiated into two main regions, i.e. the pSMAC and the cSMAC. The TCR is at the cSMAC and the integrins are at the pSMAC. A TCR and its signaling components assemble into microclusters throughout the region and then these microclusters migrate centrally to form the cSMAC over the course of minutes by cytoskeletal mechanisms (Yokosuka et al., 2005) (Campi et al., 2005) (Hashimoto-Tane et al., 2011). Newly formed clusters in the periphery then continue to move centrally.

These microclusters progressively decrease in their activity as they are centralized toward the cSMAC and then internalized (Varma et al., 2006). This process of centralization to the cSMAC may depend on WASp since CD28 endocytosis depends on WASp (Badour et al., 2007). Microclusters may compartmentalize distinct components involved in T cell activation (Lillemeier et al., 2010) (Sherman et al., 2011) as microclusters have been found to be dynamic and heterogeneous, e.g. VLA-4 (very late antigen 4) costimulation leads to enhanced TCR activation while costimulation by PD-1 (programmed cell death-

1) leads to suppressed downstream TCR signaling (Yokosuka et al., 2012).

Vesicles localize toward the immune synapse. TCR activation leads to the reorientation of the microtubule organizing center (MTOC) to a position below the immune synapse (Kupfer and Dennert, 1984). The MTOC distally localizes at the uropod when a T cell is migrating (Verschueren et al., 1991) (Ratner et al., 1997). Since vesicles move along microtubules in the cytoplasm, the reorientation of the MTOC toward the immune synapse creates a focal point for vesicular traffic toward the immune

37 synapse. Since the MTOC is linked to the Golgi apparatus, this reorientation of the

MTOC at the immune synapse in turn orients the Golgi apparatus at the immune synapse.

Since the Golgi apparatus generates vesicles for transport to the plasma membrane and since the Golgi apparatus recycles endosomes, the reorientation of the MTOC to the immune synapse allows for the efficient trafficking of vesicles to the immune synapse.

TCR activation induces actin polymerization at the immune synapse. MTOC reorientation leads to the clearance of filamentous actin (F-actin) from the central region of the synapse (cSMAC). The pSMAC has a characteristic ring of F-actin outside of periphery of the immune synapse (Bunnell et al., 2001) (Freiberg et al., 2002) (Sims et al., 2007). TCR activation leads to the recruitment of the adhesion-and-degranulation- promoting adapter protein (ADAP) to the actin rich periphery of the immune synapse.

ADAP links to the actin cytoskeleton and associates with microtubule motor Dynein at the periphery of the immune synapse (Combs et al., 2006). Microtubules from the

MTOC project toward the periphery of the immune synapse and localize with ADAP at the peripheral F-actin ring.

The maintenance of the polymerization of the F-actin ring at the immune synapse is a result of the Rho-family GTPase Cdc42 being recruited to the immune synapse. The

Cdc42 effector protein IQGAP (IQ motif containing GTPase activating protein) associates with the region of actin polymerization (Stinchcombe et al., 2006). IQGAP binds to actin and the plus end of microtubules and likely links MTOC reorientation to actin-reorganization at the immune synapse (Stinchcombe et al., 2006). While TCR activation and immune synapse formation depend on Cdc42 and IQGAP, MTOC reorientation does not depend on Cdc42 and IQGAP, indicating that redundancy is likely

38 involved in MTOC reorientation. WASp deficiency, therefore, may be confined to defects in TCR activation rather than on MTOC reorientation.

Evidence for the immune synapse being a location of increased vesicular activity include observations that receptors and membrane bound signaling complexes being internalized at the cSMAC once the receptors and membrane bound signaling complexes have been exhausted of their use (Varma et al., 2006). These receptors and membrane bound signaling complexes become part of early endosomes. Proteins in early endosomes either become part of late endosomes in which lysosomes degrade the components of late endosomes. The proteins in early endosomes can also become part of recycling endosomes in which the proteins are recycled back to the plasma membrane.

Endocytic recycling machinery polarizes toward the immune synapse during TCR activation (Das et al., 2004). Electron micrographs show that the Golgi apparatus can touch the plasma membrane (Stinchcombe et al., 2006) which may indicate that newly formed proteins in vesicles may be delivered directly to the immune synapse. Because T cells direct exocytic vesicles toward the APC at the immune synapse, this observation also shows how the immune synapse is a location of increased vesicular traffic. Defects in WASp may affect how exocytic vesicles are directed toward the APC at the immune synapse.

Distinct proteins are associated with vesicles. The most common marker for vesicles include the Rab (rat brain) proteins, which are monomeric GTPases involved in regulating many steps of membrane traffic. Early endosomes have Rab5 (Gorvel et al.,

1991) (Bucci et al., 1992). Rab7 replaces Rab5 as the early endosome matures into a late endosome. Recycling endosomes have Rab4 and/or Rab11 (Van Der Sluijs et al., 1991)

39 (Trischler et al., 1999). Golgi to membrane transport vesicles have Rab8 (Huber et al.,

1993). Specialized vesicles can include specific Rab proteins such as the secretory lysosomes of cytotoxic lymphocytes (CTLs) and natural killer cells (NK cells) having

Rab27 (Haddad et al., 2001). There exists only a partial understanding about how different vesicular components are targeted to the immune synapse. Much of this understanding has come from the studies on CTLs and NK cells.

The exocytic vesicles of CTLs and NK cells primarily associated with the lytic function of these cells are known as lytic granules or secretory lysosomes. Lytic granules contain perforin and granzymes, which induce cell death in the target cells. CTL and NK cells release lytic granules through the immune synapse toward the APC (Yannelli et al.,

1986) (Stinchcombe et al., 2001). Rab27 on the lytic granule interacts with MUNC 13-4, which then interacts with PI(4,5)P2 on the plasma membrane (Menager et al., 2007)

(Feldmann et al., 2003) (Haddad et al., 2001) (Stinchcombe et al., 2001). Rab27 and

MUNC 13-4 are involved in docking and priming of the lytic granule before exocytosis at the immune synapse.

Before TCR activation, different vesicular compartments keep the constituents of lytic granules separate, such that lysosome derived cytotoxic granules containing cytotoxic effector proteins (perforin, granzymes) are separate from endosome-derived exocytic vesicles containing mediators of exocytosis (MUNC 13-4). After TCR activation, both cytotoxic granules and exocytic vesicles move to the MTOC at the immune synapse. The different vesicular compartments likely fuse to form lytic granules

(Menager et al., 2007). This fusion process involves late endosomes having Rab7 and

Rab27 fusing with recycling endosomes having Rab11 and MUNC 13-4 (Menager et al.,

40 2007). This Rab7, Rab27, Rab11 and MUNC 13-4 containing exocytic endosome in turn fuses with an immature lytic granule containing cytotoxic effector protein(s) leading to the lytic granule being docked to the plasma membrane through the interaction between

MUNC 13-4 and PI(4,5)P2 (Shin et al., 2010).

Both microtubule and actin cytoskeleton rearrangement are involved in the delivery of the lytic granule to the cell surface of a CTL. Lytic granule release to the immune synapse requires the plus-end motor Kinesin-1, directs the growth of microtubules (Kurowska et al., 2012). NK cells, however, show that the final step of targeting a lytic granule to the cell surface involves the actin motor Myosin IIA (Andzelm et al., 2007) (Sanborn et al., 2009). Because vesicle movement depends on N-

WASp/WASp (Benesch et al., 2002), WASp may contribute to the movement of lytic granules to the plasma membrane. NK cell studies also show that the inner regions of the immune synapse has a fine mesh of F-actin and NK cell activation leads to granule sized holes at specific domains to which the MTOC locates and at which lytic granules dock

(Brown et al., 2011) (Rak et al., 2011). It is not known if such an actin mesh is present in the central regions of the T cell immune synapse.

Lytic granule delivery is altered when T cells are stimulated with sub-optimal antigenic signals. The MTOC and granules uncouple when there is a sub-optimal antigenic signal through the TCR (Jenkins et al., 2009). The cytotoxic granules move to the periphery of the immune synapse and then move to the secretory domain at the center of the immune synapse (Beal et al., 2009). This uncoupling likely reduces the ability for mature lytic granules to assemble from cytotoxic granules and exocytic vesicles because

41 the movement of the vesicle to the cSMAC after moving to the periphery leads to reduced cytotoxicity.

The thick meshwork of cortical actin near the plasma membrane sometimes allows some openings for secretory vesicles to access the plasma membrane (Giner et al.,

2005) (Nakata and Hirokawa, 1992). Disruptions in cortical actin occur when stimulation of exocytosis occurs leading to actin free zones that allow secretory vesicles to dock at the plasma membrane (Giner et al., 2007). NK cells have shown this phenomenon

(Brown et al., 2011). It is possible that CD4+ T cells and CD8+ T cells use a similar mechanism and that this mechanism may be WASp dependent.

Ectocytosis is the process in which vesicles bud from the plasma membrane.

Previous studies show that ectocytosis occurs in T cells (Booth et al., 2006). ESCRT proteins play a role in the membrane sorting, budding and fission involved in ectocytosis

(Bissig and Gruenberg, 2014) (Fang et al., 2007) (Van Engelenburg et al., 2014).

TSG101, a subunit of ESCRT1, localizes to the plasma membrane and interacts with Alix and ARRDC1 on ectosomes (Nabhan et al., 2012). ESCRT-III assists in pinching off the ectosome and releasing it into the extracellular space (Nabhan et al., 2012). Proteins bind to ectosomes by membrane anchors such as myristoylation, palmitoylation and high order polymerization (Shen et al., 2011) (Yang and Gould, 2013). Proteins that accumulate in the lumen of the ectosome include cytoskeletal proteins, heat-shock proteins, enzymes, RNAs (mRNAs, siRNAs, long non-coding RNAs) (Cocucci et al.,

2009) (Crescitelli et al., 2013) (Shen et al., 2011) (Yang and Gould, 2013). Proteins involved in the pinching off and release of vesicles include ceramide, Ca2+-dependent scramblase, annexin-2, chromosome segregation 1-like protein, and hyaluronan synthase

42 3 (Bianco et al., 2009) (Liao et al., 2012) (Rilla et al., 2013) (Trajkovic et al., 2008)

(Zhang et al., 2013). The cortical actin cytoskeleton rearranges during the pinching off and release of vesicles (Antonyak et al., 2012; de Curtis and Meldolesi, 2012)

(Muralidharan-Chari et al., 2010; Shilagardi et al., 2013; Wiesner et al., 2013) and depends on small G proteins of the Ras family such as Arf6, Cdc42 and various Rabs.

Ectosome release occurs within seconds after stimulation by ATP in dendritic cells and macrophages (Baroni et al., 2007; Bianco et al., 2005; Cocucci et al., 2009;

Shifrin et al., 2013) (Turola et al., 2012) (Yang and Gould, 2013). The increase in free

Ca2+ concentration sustains the release of ectosomes (Baroni et al., 2007) (Bianco et al.,

2005) (Cocucci et al., 2007) (Cocucci et al., 2009) (Yang and Gould, 2013). The activation of protein kinase C by phorbol esters induces strong ectosome release (Bianco et al., 2009; Cocucci et al., 2009). HIV likely co-opts the CD4+ T cell process of ectocytosis-- it is also possible that T cells process membrane bound vesicular FasL through ectocytosis.

Unlike exocytosis and ectocytosis, apoptotic blebs in T cells and other cells are formed from the increase in intracellular hydrostatic pressure followed by cellular contraction from the aid of actomyosin (Charras et al., 2005) (Charras et al., 2008).

Apoptotic blebs tend to be larger than ectosomes and exosomes indicating separate mechanisms from ecotocytosis and exocytosis for bleb formation.

Previous studies suggest that WASp mediates vesicular trafficking. It is not clear if WASp mediates processes in either exocytosis or ectocytosis. It is also not clear if

WASp mediates the specific processes involved in CD8+ T cell lytic granule secretion or if WASp mediates a different process for vesicle secretion in CD4+ T cells. We

43 specifically aim in these studies to determine how WASp contributes to the secretion of

FasL containing vesicles and whether WASp mediates the secretion of vesicles differentially between CD4+ and CD8+ T cells.

44 Chapter 2. Specific Aims

This research plan aims to investigate how monogenic primary immunodeficiency disease mouse models associated with the frequent development of autoimmunity can resolve cellular death mechanisms that may contribute to the development of autoimmunity. Wiskott-Aldrich Syndrome (WAS) is a monogenic primary immunodeficiency with 40-72% autoimmunity arising from defects in the Wiskott-

Aldrich Syndrome protein (WASp). WASp is only expressed in hematopoietic cells and is involved in the polymerization of branched F-actin. Defects in WASp lead to defects in various immune cell functions, including defects in T cell receptor (TCR) activation and immune synapse formation. IL-2 supplementation can overcome the TCR activation defects caused by WASp deficiency. Since T cells require TCR restimulation in order to induce restimulation induced cell death (RICD), we seek to understand how WASp affects the cellular death mechanisms of CD4+ and CD8+ T cells after TCR activation.

We aim to use known primary immunodeficiency disease mouse models for Rab27a deficiency (having defects in CTL cytotoxicity through perforin and granzyme exocytosis) and FasL deficiency (having defects in T cell apoptosis through the Fas-FasL pathway), both which have high prevalence of autoimmunity, to help resolve how WASp affects the functions of CD4+ and CD8+ T cells in undergoing RICD and in killing target cells.

Aim 1: To understand how WASp affects FasL function and secretion during RICD

via:

A) Quantitation of soluble and vesicular FasL secreted from TCR restimulated T

cells deficient in WASp function.

45 B) Quantitation of soluble and vesicular FasL secreted from TCR restimulated T

cells deficient in Rab27a function.

C) Quantitation of soluble and vesicular FasL secreted from TCR restimulated T

cells deficient in FasL function.

D) Assessment of WASp dependent CD4+ T cell instrinsic role in RICD.

E) Assessment for FasL associated microvesicular function in the CD4+ T cell

instrinsic role of RICD.

Aim 2: To assess CD4+ T cell and CD8+ T cell mechanisms for cytotoxic killing of target cells via

A) Assessment of requirements for WASp, Rab27a and FasL in CD4+ T cell

cytotoxic killing of target cells.

B) Assessment of requirements for WASP, Rab27a and FasL in CD8+ T cell

cytotoxic killing of target cells.

46 Chapter 3. Materials and Methods

Mice

WASP-deficient mice on the 129 background (129S6/SvEvTac-Wastm1Sbs/J) were obtained from The Jackson Laboratory. Some mice were backcrossed for 3 generations to

C3H/HeJ and for 8 generations to C57Bl/6J, and sera were sampled as described.

129S6/SvEvTac (129) controls were obtained from Taconic. FasL-deficient mice (Gld) on the B6 background (B6Smn.C3-Faslgld/J) and their C57BL/6J (B6) controls were from the Jackson Laboratory. Rab27a-deficient mice on the C3H background were a gift from

John Hammer of NHLBI. C3H/HeSnJ (C3H) controls were obtained from Jackson

Laboratories. Mice were maintained in SPF conditions, and experiments were carried out according to the Animal Care guidelines of the National Institutes of Health (NIH)

Intramural Research Program (IRP) and the Memphis VA Medical Center. The NIH IRP approved all mouse experiments.

Measurement of autoantibodies and circulating immune complexes

The presence of antinuclear antibodies (ANA) was determined by immunofluorescence staining of fixed HEp-2 cells (Antibodies). Cells were incubated with 1:40 diluted sera and then with Alexa 488–conjugated goat anti–mouse immunoglobulin G (IgG) antiserum (Invitrogen). Fluorescence was evaluated using fluorescent microscopy by 3 blinded observers (N.P.N., D.B., and R.M.S.) with very good inter-observer reproducibility. Positive staining was defined as a distinct staining within the nucleus brighter than the staining evident in the cytoplasm. Positive sera were

47 further serially diluted until they became negative for nuclear immunofluorescence.

ANA-positive sera were tested for anti-double–stranded DNA (anti-dsDNA) using mouse enzyme-linked immunosorbent assay (ELISA; Alpha Diagnostic International), following the manufacturer's instructions. The cutoff for a positive anti-dsDNA was 2 times the absorbance of the negative control corrected for blank values. ELISA measured serum circulating immune complexes according to the manufacturer's protocol (Alpha

Diagnostic International).

Kidney immunofluorescence and measurement of proteinuria

Sections of one of the kidneys were fixed in buffered formalin. Five-micron sections were stained with periodic acid–Schiff (PAS) and were processed for light microscopic evaluation. Immunofluorescence was performed on 4-μm cryostat sections of the other cryopreserved kidney with the use of goat anti–mouse fluorescein isothiocyanate (FITC)– conjugated polyclonal antibody to IgG, IgA, IgM (K&P Laboratories), and rat anti-mouse

FITC-conjugated monoclonal antibody to C3 (Cedarlane Laboratories, Ltd). At least 30 glomeruli from each sample were examined, and a semiquantitative grading from 0 to

4+was given as previously described. Examples are shown in Figure 6. Ig deposits were scored independently by 3 observers blinded to the origin of the samples and averaged.

Urine albumin was measured by using a mouse Albumin ELISA Quantitation kit (Alpha

Diagnostic International) according to the manufacturer's protocol. Urine creatinine was determined by using a urine Creatinine ELISA Quantitation kit (R&D Systems,

Minneapolis, MN) according to the manufacturer's protocol, and albumin/creatinine ratio was calculated. ELISA Quantitation kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol, and albumin/creatinine ratio was calculated.

48 Figure 6. Examples of fluorescence images used for scoring glomerular Ig subclass and C3 deposition.

49 Figure 6. Examples of fluorescence images used for scoring glomerular Ig subclass and C3 deposition. Sections of one of the kidneys were fixed in buffered formalin, stained with periodic acid–Schiff (PAS) and were processed for light microscopic evaluation. Immunofluorescence was performed on 4-μm cryostat sections of the other cryopreserved kidney with the use of goat anti–mouse fluorescein isothiocyanate (FITC)– conjugated polyclonal antibody to IgG, IgA, IgM (K&P Laboratories), and rat anti-mouse

FITC-conjugated monoclonal antibody to C3 (Cedarlane Laboratories, Ltd). At least 30 glomeruli from each sample were examined, and a semiquantitative grading from 0 to

4+was given as previously described. Ig deposits were scored independently by 3 observers blinded to the origin of the samples and averaged.

50 T-cell activation, proliferation, and apoptosis assays

Single cell suspensions were prepared from spleen and lymph nodes from 6- to 8- week-old 129Sv WASp-deficient mice and age- and sex-matched 129/SvEv controls

(Taconic), FasL deficient mice and age- and sex-matched B6 controls and/or Rab27a deficient mice and age and sex matched C3H controls. CD4+ T lymphocytes were prepared using CD4+ T-cell enrichment columns (R&D Systems) and were routinely >

90% pure. Alternatively, we obtained CD4+ T cells by negative selection of the T cell preparation from the T-cell enrichment columns using magnetic beads (cat no. 130-095-

248; Miltenyi Biotec). We collected the CD8+ T cells from the positive fraction separated by Automacs of the T cell preparation from the T-cell enrichment columns.

These cell preparations were routinely > 90% pure. CD4+ and CD8+ T cells were primed with plate-bound anti-CD3 antibody (2C11; BD Pharmingen) at 5 μg/mL and anti-CD28 antibody (BD Pharmingen) at 5 μg/mL at 37°C in complete RPMI medium

(RPMI 1640, supplemented with 10% fetal calf serum [FCS], pen/strep, 2.5mM L- glutamine, 10mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [HEPES], 1mM sodium pyruvate, and nonessential amino acids) in the presence of 50 U/mL recombinant human interleukin-2 (IL-2; National Cancer Institute [NCI]–Frederick). After 3 days, cells were washed twice and re-plated at 1 × 106/mL in complete medium with 50 U/mL

IL-2. After another 3 days, activation status was assessed by flow cytometry for CD4,

CD25, and Fas on a FACSCalibur flow cytometer (Becton Dickinson). We cultured cells 37°C in a 5% CO2-buffered incubator.

Proliferation was assayed by pulsing activated T cells for 24 hours with 1 uCi/well of 3H-thymidine and measuring incorporation of 3H-thymidine into DNA by

51 standard methods. For pre- and postactivation monitoring of proliferation, CD4+ T cells were labeled with 5μM carboxyfluorescein diacetate, succinimidyl ester (CFSE), and examined for CFSE dilution every 24 hours. The division index (average number of cell divisions in the population) was calculated with FlowJo Version 8.8.6 software

(TreeStar).

To induce apoptosis, activated CD4+ and CD8+ T cells were incubated for 6 hours with plate-bound anti-CD3 Ab (2C11; BD Pharmingen) or soluble Mega FasL (a generous gift from Apotech, Lausanne, Switzerland). We induced restimulation induced cell death by incubating CD4+ T cells on various concentrations of plate-bound anti- mouse CD3 (2C11; eBioscience) for 6 hours. Cells were then analyzed for viability and apoptosis by staining for annexin V and propidium iodide (PI) uptake on a FACSCalibur or alternatively we analyzed the cells for viability and apoptosis by staining for

MitoProbe DiIC1(5) and propidium iodide (PI) uptake on a FACS-canto. Anti-FasL

(MFL3; BD Pharmingen) was used to determine whether apoptosis was specifically caused by FasL.

For inducing cell death due to cytokine withdrawal, after initial activation for 48 hours with anti-CD3/CD28 and culture with IL-2 for 3 days, T cells were washed 3 times and placed in complete media with 50 U/mL IL-2 or without exogenous cytokines.

Viable cell number was determined by trypan blue exclusion, and cell viability was determined by flow cytometry using PI and the mitochondrial dye DiOC (Invitrogen).

Specific cell death was determined as follows: % specific death = 1 − (%viable treated/%viable untreated) × 100%.

52 Measurement of FasL mRNA, protein, and function

RNA was prepared using Trizol (Invitrogen) and RNeasy mini kit (QIAGEN) and amplified using one-step Superscript II RT-PCR (Invitrogen). Applied Biosystems primer/probe sets for FasL and β-2 microglobulin as standard were used. RNA induction

was quantified using the ΔCT method.

To evaluate FasL secretion, supernatants from the cell death assays were collected at 6 hours, and the amount of FasL was quantified using mouse ELISA (Fas

Ligand/TNFSF6 Duoset; R&D Systems) according to the manufacturer's recommendations. FasL surface levels were tested by FACS using anti-CD178-PE (BD

Pharmingen). FasL was collected from activated T cells cultured at 4 × 106cells/mL with anti-CD3 for 6 hours in the presence of IL-2. Supernatants were prespun at 10,000g for

30 minutes to remove cellular debris, and cell-free supernatant was then fractionated by centrifugation through Microcon YM-100 membranes after prewetting with a 1% bovine serum albumin (BSA) solution in phosphate-buffered saline (PBS). The concentrated fraction (> 100 kDa) was rediluted to the original volume.

FasL activity was assayed on WEHI-279 B cell lymphoma cells (ATCC) at 2.5 ×

105 cells/mL mixed at a 1:3 ratio with T-cell supernatants for 23 hours. Apoptosis was measured with the Cell Titer Glo cell viability assay (Promega). Anti-FasL (10 μg/mL,

MFL3; BD Pharmingen) in the presence of Fc receptor blockade with 2.4G2 was added to some samples to determine whether apoptosis was caused by FasL. Purified soluble

FasL was obtained from R&D Systems, and vesicular FasL was obtained from

Upstate/Millipore.

53 β-Hexosaminidase release assay

Degranulation induced via TCR restimulation was determined by β- hexosaminidase release. CD4+ or CD8+ T cells were incubated at 5.0 × 105 cells/mL in

Tyrode buffer (135mM NaCl, 5mM KCl, 1mM MgCl2, 1.8mM CaCl2, 5.6mM glucose,

20mM HEPES, pH 7.4). Cells were then restimulated with plate-bound murine anti-CD3e in the presence of 50 U/mL IL-2 for 4 hours at 37°C. The reaction was terminated by centrifugation at 4°C. The supernatant was collected, and the total hexosaminidase concentration was obtained by cell lysis in 1% Triton X-100. Aliquots of the supernatants and total cell lysates were incubated with 50μL 1mM p-nitrophenyl-N-acetyl-D- glucopytanoside (p-NAG; Sigma-Aldrich) substrate for 1 hour at 37°C in 0.1 M sodium citrate buffer (pH 4.5) at 37°C. The reaction was terminated by the addition of 100 μL 0.1

M carbonate/bicarbonate buffer. The release of the product 4-p-nitrophenol was read by optical absorbance at 405 nm. Percentage of β-hexosaminidase release was calculated as follows: (stimulated sup − unstimulated sup)/unstimulated total) × 100.

Assays for Cell Autonomy

We stained a pool of WASp deficient CD4+ T cells with CFSE and mixed CFSE stained WASp deficient CD4+ T cells with 129 wild type control CD4+ T cells in 1:1,

1:10 and 10:1 ratios. We induced restimulation induced cell death by incubating CD4+ T cells on various concentrations of plate-bound anti-mouse CD3 (2C11; ebioscience) for 6 hours. We analyzed the cells for viability and apoptosis by staining for MitoProbe

DiIC1(5) and propidium iodide (PI) uptake on a FACS-canto. Specific cell death was

54 determined as follows: % specific death = 1 – (%viable treated/%viable untreated) x

100%. We did similar experiments with FasL deficient CD4+ T cells stained with CFSE.

We isolated the vesicles from the culture supernatant of 129 control and WASp deficient restimulated CD4+ T cells and added the vesicles in a 10:1 ratio to either cells of the same genotype or the cells of the other genotype, which were plated on 1 µg/ml anti-mouse CD3 (2C11; eBioscience). We restimulated the CD4+ T cells with the vesicles for 6 hours and analyzed the cells for viability and apoptosis by staining for

MitoProbe DiIC1(5) and propidium iodide (PI) uptake on a FACS-canto. Specific cell death was determined as follows: % specific death = 1 – (%viable treated/%viable untreated) x 100%. We did similar experiments for FasL deficient CD4+ T cells.

Redirected Kill Assays

We stained the mouse lymphoblast-like mastocytoma line, P815, with CFSE and then incubated the cells at 2 x 105 cells/ml with either 100 ng/ml of anti-mouse CD3

(2C11: eBioscience) or Armenian Hamster IgG isotype control (eBioscience) for 20 minutes at 37°C in 5% CO2. We added CD4+ T cells or CD8+ T cells at 1:1, 3:1 or 10:1 ratios such that the T cells were equal to or exceeded the P815, L1210 B lymphocytes and L1210 Fas B lymphocytes in all the ratios. We incubated the cells for 3-4 hours at

37°C in 5% CO2. We analyzed the cells for viability and apoptosis by staining for

MitoProbe DiIC1(5) and propidium iodide (PI) uptake on a FACS-canto. Specific cell death was determined as follows: % specific death = 1 – (%viable treated/%viable untreated) x 100%.

55 Statistical analysis

Quantitative statistics were computed by Student t test. Categorical variables were assessed using Fisher exact and χ2 tests. Two-sided P values were used for all analyses, and the level of statistical significance was set a priori at .05. Statistical analyses were performed using SAS E-Guide, Version 3 for Windows (SAS Institute) and Prism

Version 4 and 5b for Macintosh (GraphPad Software).

56 Chapter 4. Systemic autoimmunity and defective Fas ligand secretion in the

absence of the Wiskott-Aldrich syndrome protein

Introduction

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency that affects the development and function of multiple hematopoietic cell lineages, including T cells, B cells, natural killer (NK) cells, dendritic cells, and platelets (Bosticardo et al.,

2009; Sullivan et al., 1994). WAS patients present with thrombocytopenia, eczema and susceptibility to infection. WAS patients have a high prevalence of autoimmunity between 40-72% (Dupuis-Girod et al., 2003; Sullivan et al., 1994). WAS patients often present with autoimmune hemolytic anemia, thrombocytopenia, nephritis, vasculitis and inflammatory bowel disease (Dupuis-Girod et al., 2003; Schurman and Candotti, 2003;

Sullivan et al., 1994). These autoimmune complications raise questions about what how

WAS affects the functions of hematopoietic cells to cause such complications.

The loss of function of the WAS protein (WASp) has been associated with most cases of WAS (Derry et al., 1994). WASp is a multidomain 502 amino acid cytoplasmic protein specifically expressed in hematopoietic cells (Derry et al., 1994; Parolini et al.,

1997; Stewart et al., 1996) and induces branched-actin polymerization through interactions with the ARP2/3 complex. In T cells, upon T-cell antigen receptor (TCR) interaction with MHC Class II presenting cognate peptide, WASp gets activated through the small G protein Cdc42. WASp deficiency leads to defective formation of the immune synapse in WASp deficient T cells and NK cells (Badour et al., 2003; Cannon et al.,

2001; Derry et al., 1994; Orange et al., 2002; Snapper et al., 1998). WASp deficient T

57 cells proliferate poorly after initial TCR activation, but addition of exogenous interleukin-

2 rescues the defect (Zhang et al., 1999f).

A defect in regulatory T cell (Treg) homeostasis and function occurs in WASp deficiency suggesting a possible mechanism that could predispose WASp patients to develop autoimmunity (Adriani et al., 2007b; Humblet-Baron et al., 2007; Maillard et al.,

2007; Marangoni et al., 2007e). Another mechanism of peripheral immune tolerance includes death of activated T cells after TCR restimulation, termed restimulation induced cell death (RICD), and whether WASp deficiency results in RICD defects is unknown

(Lenardo, 1991; Ramaswamy and Siegel, 2007; Siegel et al., 2000). This apoptotic pathway can eliminate T cells responding to chronically expressed antigens, such as autoantigens and pathogens in persistent infections (Critchfield et al., 1994; Ettinger et al., 1995; Weant et al., 2008). RICD in CD4+ T cells mostly depends on autocrine interactions of the tumor necrosis family member Fas ligand (FasL) and the Fas receptor

(Green et al., 2003; Siegel et al., 2000). Fas or FasL deficiency results in systemic autoimmunity in humans and mice (Cohen and Eisenberg, 1992a; Straus et al., 1999).

Fas deficiency in T cells, B cells or dendritic cell lineages can independently lead to autoantibody production in animal models (Ramaswamy and Siegel, 2007; Stranges et al., 2007b). Since WASp deficient T cells have T cell signaling defects, we hypothesized that in addition to affecting aspects of T cell activation, WASp deficiency impairs the

RICD pathway, contributes to the breakdown of self tolerance and leads to the development of autoimmunity. We show here that T cells from Was knockout (KO) mice have defection production of biologically active FasL after restimulation through the TCR. These defects may contribute to the development of age-dependent production

58 of autoantibodies and immune-complex nephritis that we have seen in these mice and likely play a role in the onset of autoimmunity disease in WAS patients.

Results

We screened for manifestations of autoimmunity in a genetically defined model of WAS by first evaluating autoantibody production in WASp deficient mice. We screened for ANA from the sera of WASp deficient mice on a 129 SvEv/Tac background and WT mice on the same 129 SvEv/Tac background. A total of 266 serum samples were tested and 47% of WASp deficient mice older than 6 months has an ANA titer of greater than 1:640 (as high as 1:20,480) vs 25% in 129 age-matched controls (Figure 7A).

The ANA titers were significantly higher in older WASp deficient mice. (Figure 7A).

The prevalence of ANA seropositivity remained significantly higher in WASP deficient mice compared with WT controls at all titers higher than 1:640 (P<0.002, Wicoxon 2- sample test). We noticed that the pattern of ANA was typically homogeneous and was similar to the pattern seen in Fas deficient lpr/lpr control sera (Figure 7B). ANA production increased with age in WASp deficient mice (Figure 7C) and logistic regression analysis showed significant and independent contributions of WASP genotype

(P=0.0138) and age (P=0.046).

59 Figure 7. Autoantibody Production in WASp deficient mice.

60 Figure 7. Autoantibody Production in WASp deficient mice. (A) Fluorescent ANA titers in WASp-deficient and age-matched control mice on 129SvEv genetic background divided into 2 groups based on age (> 6 months and <6 months). Statistical comparison was performed using the Fisher exact test. A total of 266 serum samples were tested. For the group less than or equal to 6 months old (n = 48), the mean age +/- SD in days was

WASp-deficient, 119 +/- 35; WT controls, 104 +/- 33 (P=.18). For the group more than 6 months old (n = 218), the mean age +/- SD in days was WASp-deficient, 325 +/- 98; WT controls, 303 +/- 83 (P = .08).

(B) Representative images of 129 background WASp ANA (serum dilution 1:640) with positive (B6.lpr/lpr 1:640) and negative 129 SvEv WT controls (serum dilution 1:40).

Note the staining of mitotic figures by serum from the WASp-deficient mouse, suggesting the presence of anti-chromatin antibodies.

(C) Kaplan-Meier analysis of the proportion of mice becoming ANA-positive at 1:640 cutoff titers over time (solid line, WT controls; dashed line, WASp-deficient mice).

(D) Sera from ANA-positive mice tested for anti-dsDNA specificity by ELISA and expressed as a percent of the total tested samples per genotype. Mean age in days +/- SD

(not statistically significantly different) and number of mice per group are indicated (anti- dsDNA–negative, white box; anti-dsDNA–positive, black box).

61 In order to determine if the significant ANA production was due to the genetic background of the mice, we backcrossed WASp deficient mice for 8 generations to

C56Bl/6 and 3 generation to C3H, respectively. We found significantly higher ANA prevalence (P=0.005, Fisher exact test, n=16) and titers (P=0.31, Wilcoxon 2 sample test, n=16) in WASp deficient mice compared with WT controls older than 6 months. Similar trends were seen in sera from WASp deficient mice backcrossed for 3 generations to C3H

(50% ANA positive WASp-KO sera vs 0% WT controls, n=13, P=0.07) in mice older than 6 months.

In order to determine the specificity of the autoantibodies produced in WASp deficient mice, we measured anti-dsDNA antibody titers in ANA positive mice. ANA positive sera from WASp deficient mice were positive for anti-dsDNA in 64% vs 31% n

WT controls (Figure 7D, P,0.001, 2 tailed Fisher exact test, n=116). Female WASp deficient mice were more likely to have positive anti-DNA titers compared with controls

(85% vs 33%, P=0.003, Fisher exact test, n=62), but males did not have a significant difference (48% vs 28%, P=0.16, Fisher exact test, n=54).

In order to determine whether the autoantibodies are pathogenic, we examined the kidneys from 129 SvEv WASp deficient mice for evidence of immune complex nephritis.

We saw mild mesangial cell proliferation and glomerular basement membrane thickening was observed in PAS-stained sections from WASp deficient mice older than 6 months and increased in severity with age (Figure 7A). Indirect immunofluorescence showed significantly more intense glomerular IgG, IgA and C3 complement deposition in the kidneys of WASp deficient mice compared with WT controls (Figure 8A). Although

IgM deposition could also be seen, this was nonspecific as IgM deposition could also be

62 seen in kidneys from normal mice (Figure 8A). We quantitated the immunofluorescence results from 20 WASp deficient mice and an equal number of matched controls to confirm the observations (Figure 8B). In order to assess the kidney functions of WASp deficient mice, we measured the circulating immune complexes (CIC) capable of binding

C1q in the serum of WASp deficient mice compared to WT controls and found that only the WASp deficient mice older than 6 months had higher C1q binding CIC (Figure 8C).

For testing whether kidney disease has developed, we measured the urinary albumin/creatine ratio in WASp deficient mice that were older than 6 months. We found that WASp deficient mice older than 6 months had a greater than 5 fold elevation of this measure of proteinuria compared to WT controls (Figure 8D). These results suggest that autoantibody production affects kidney function.

63 Figure 8. Immune Complex Deposition and Mesangial Cell Proliferation in WASp deficient mice .

64 Figure 8. Immune Complex Deposition and Mesangial Cell Proliferation in WASp deficient mice .

(A) Representative PAS stained glomeruli from WASp-deficient and 129 SvEv control mice and immunofluorescence images of IgA, IgG, IgM, and C3 deposition in glomeruli from the same animals.

(B) Quantitation of immunofluorescence results from mice of the indicated genotype more than 6 months old measured as in panel B.

(D) Urinary albumin/creatinine ratios in a cohort ofWASp-deficient and 129 age-matched control mice.

65 Defects in T cell apoptosis can contribute to the loss of self tolerance and to the formation of autoantibodies. In order to determine whether WASP contributes to RICD, we examined the ability of activated CD4+ T cells from WASp deficient mice to undergo apoptosis in response to TCR restimulation (Figure 9A). Purified CD4+ T cells were activated for 2 days and expanded in the presence of IL-2 for 3 days. RICD was then induced by restimulation with plate-bound anti-CD3. RICD was significantly reduced in

WASp deficient T cells over a wide range of concentrations of anti-CD3 (Figure 9A). In order to assess whether the cell death under these conditions depended on FasL-Fas interactions, we blocked with anti-FasL to find that such blocking completely abrogated cell death (data not shown). We also sought to assess whether WASp deficiency affected

Fas expression and found that the surface expression of Fas on activated T cells from WT and WASp deficient mice were similar (Figure 9B).

Because TCR induced apoptosis requires cell cycle progression through G1/S phase, we sought to understand whether WASP deficiency lead to reduced cycling of the

T cells by measuring thymidine incorporation in WT and WASp deficient T cells. We found that WT and WASp deficient T cells incorporate thymidine similarly after 3 days of activation (Figure 9C). We also assessed how WASp deficiency affects cell division in T cells by examining CFSE dilution over time. We found that WASp deficient T cells initially had delayed proliferation at 48 hours after initial activation, but was similar to

WT during the expansion phase of culture (Figure 9D).

Because TCR induced cell death can occur through the Fas-FasL pathway, we sought to understand if Fas signaling was affected by WASp deficiency. In order to assess whether WASp deficiency affects Fas signaling, we added the biologically active

66 form of oligomerized FasL to TCR restimulated cultures of WT and WASp deficient

CD4+ T cells (Figure 9F). We found that TCR induced cell death was not affected differently from WT in WASp deficient CD4+ T cells. This indicates that Fas signaling was intact.

Because previous studies showed that WASp deficient lymphocytes from WAS patient had accelerated spontaneous apoptosis, we sought to determine if the absence of survival promoting cytokines affected this acceleration in apoptosis. We cultured activated CD4+ T cells in the absence of cytokines and monitored viable cell number and viability (Figure 9E-F). Cell numbers in WASp deficient CD4+ T cells were slightly lower but viability was similar to WT CD4+ T cells. Significant cell death occurred in both WT and WASp deficient CD4+ T cells after IL-2 was withdrawn from the culture.

These results indicate that the intrinsic cell death pathway does not compensate for defective TCR induced cell death of WASp deficient T cells.

67 Figure 9. Impaired TCR-mediated apoptosis of activated WASp-deficient CD4+

T lymphocytes

68 Figure 9. Impaired TCR-mediated apoptosis of activated WASp-deficient CD4+

T lymphocytes

(A) Specific cell death of activated CD4+ T cells from WT and WASp deficient mice restimulated with the indicated concentrations of plate-bound anti-CD3 mAb for 6 hours and measured by annexin V and PI staining. The data are average and SEM of 5 independent experiments with age- and sex-matched mice on the 129 background.

Apoptosis measurements were performed in triplicate for each sample. Results of unpaired t test comparisons of cell death at each dose of anti-CD3 are shown as *P <.05,

**P < .005, ***P <.001.

(B) Surface TCR, Fas on activated CD4+ T cells from WASp-deficient and control mice activated as in panel A.

(C) Proliferation of WT and WASp-deficient T cells activated in the presence of exogenous IL-2 as in panel A measured by 3H-thymidine incorporation at 72 hours.

(D) Cell death of WASp-deficient and control T-cell blasts after addition of the indicated concentrations of Mega-FasL (an oligomerized biologically active form of soluble FasL).

(E) Viable cell number during IL-2 cytokine deprivation (cRPMI) or with IL-2 measured by trypan blue exclusion.

(F) Cell viability measured by PI and DiOC of WASp-deficient and control CD4+ T cells during IL-2 cytokine withdrawal.

(E-F) Data are representative of 2 independent experiments.

69 Since Fas signaling was normal in WASp deficient CD4+ T cells, we hypothesized that WASp affects FasL production or secretion after TCR restimulation.

We measured FasL mRNA in response to TCR stimulation of activated CD4+ T cells from WT and WASp deficient mice. We found that there was no difference in FasL mRNA up regulation between WT and WASp deficient CD4+ T cells that have been

TCR restimulated (Figure 10A). These results indicate that the components of TCR signaling responsible for the transcriptional induction of FasL. In examining whether

WASp deficieny affected FasL expression on the cell surface of TCR restimulated CD4+

T cells, we saw that there was not a difference in the cell surface FasL expression among

WT and WASp deficient CD4+ T cells that have been TCR restimulated. These results indicate that WASp deficiency does not affect the TCR signaling that leads to the transcriptional induction of FasL and the transport of FasL to the plasma membrane.

70 Figure 10. Normal up-regulation of FasL mRNA and surface expression in WASp- deficient T cells

71 Figure 10. Normal up-regulation of FasL mRNA and surface expression in WASp- deficient T cells

(A) Induction of FasL mRNA measured by real-time quantitative polymerase chain reaction (RT-qPCR) after exposure of activated CD4+ T lymphocytes to 1 μg/mL plate- bound anti-CD3 antibody for 6 hours. Induction of mRNA is shown relative to stimulation with isotype control antibodies. Average and SD of mRNA induction is shown. Similar results were observed in 2 independent experiments.

(B) WT and WASp-deficient T cells were restimulated with anti-CD3 for the indicated number of hours, and surface FasL was quantitated by FACS. The mean change in geometric mean fluorescence is shown for WT and WASp-KO T cells restimulated for the indicated periods of time with anti-CD3. The data are the average ± SEM of 3 independent experiments.

72 Since FasL can be regulated into several post-translational forms, i.e. non- cytotoxic, soluble form and either the cytotoxic cell membrane form or the cytotoxic form on extracellular vesicles, we sought to understand if WASp deficiency affected the post-translational expression of soluble, plasma membrane or extracellular vesicle FasL.

Metalloproteinases on the cell surface cleave FasL to produce soluble FasL, which does not induce cell death (O' Reilly et al., 2009; Schulte et al., 2007). FasL may be on the surfaces of vesicles that are either secreted from exocytosis with secretory lysosomes containing granzymes and -hexosaminidase or from ectocytosis (Blott et al., 2001).

Vesicular FasL is potently cytotoxic to Fas-expressing target cells (Jodo et al., 2001). In order to determine if WASp deficiency affects the post-translational regulation of FasL, we measured FasL present in the supernatant of TCR restimulated CD4+ T cells from

WT and WASp deficient mice (Figure 11A). We found reduced amount of FasL in the supernatant of TCR restimulated CD4+ T cells from WASp deficient mice. In order to determine if WASp deficiency affects the regulation of vesicular FasL, we filtered the

TCR restimulated supernatants of WT and WASp deficient CD4+ T cells through 100- kDa cutoff membranes to concentrate FasL in high molecular weight vesicles (HMW

FasL) while soluble FasL flowed through the membrane (Figure 11B). WASp deficient

TCR restimulated CD4+ T cells secreted less FasL in both the soluble flow through and the concentrated HMW FasL fractions (Figure 11C).

In order to determine whether the reduced FasL secretion was functionally significant, we added the TCR restimulated CD4+ T cell supernatants from WT and

WASp deficient mice to the Fas-sensitive WEHI-279 lymphoma cell line. As previously described, purified vesicular, but not soluble, FasL induced cell death in these cells and

73 FasL blocking antibody inhibits the cell death induced by vesicular FasL (Figure 11D)

(Jodo et al., 2001). We applied the TCR restimulated CD4+ T cell supernatants from WT and WASp deficient mice to the WEHI-279 cells to measure the cytotoxicity of these fractions. We found that the HMW fractions of the WT TCR restimulated CD4+ T cells induced significant cell death and that this cell death could be blocked by anti-FasL indicating that FasL was the principal cytotoxic molecule present in the fraction. The

HMW fraction from the WASp deficient TCR restimulated CD4+ T cells induced significantly less cell death compared with WT supernatants. The residual cytotoxicity in the supernatant of the WASp deficient TCR restimulated CD4+ T cells was partially inhibited by anti-FasL blockade, but not as strongly as in WT supernatants. The low molecular weight fractions of both WT and WASp deficient TCR restimulated CD4+ T cells induced less than 10% cytotoxicity (data not shown).

In order to determine whether WASp deficiency more generally affects the release of secretory lysosomes, we measured the specific release of -hexosaminidase by

WT and WASp deficient CD4+ and CD8+ T cells after TCR restimulation. Secretion of

-hexosaminidase was significantly reduced in both WASp deficient CD4+ and CD8+ T cells after TCR restimulation (Figure 11E). This result suggests that the defect in FasL secretion is likely due to the reduced secretion of exosomes by WASp deficient TCR restimulated T cells. Overall, our data suggests that decreased cell death in WASp deficient TCR restimulated CD4+ T cells results at least in part from deficiency in the production of biologically active FasL.

74 Figure 11. Reduced bioactive FasL and granule secretion by WASp deficient T cells

75 Figure 11. Reduced bioactive FasL and granule secretion by WASp deficient T cell

(A) Secreted FasL measured by ELISA in supernatants after 6 hours of stimulation of activated CD4+ T lymphocytes with the indicated concentrations anti-CD3 antibody.

Values are the average ± SEM of data from 2 mice per group, and similar results were obtained in 3 independent experiments.

(B) Supernatants from CD4+ T cells restimulated for 6 hour with anti-CD3 were fractionated by centrifugation through 100-kDa cutoff membranes, and FasL was quantitated in each fraction by ELISA. No FasL was detected in the < 100-kDa fraction when purified vesicular FasL was filtered through identical membranes.

WEHI-279 cells, and cytotoxicity was quantitated by a luminescent cell viability assay.

Anti-FasL antibody was added to demonstrate specificity of this assay for bioactive FasL.

Specific cell death was quantitated as described in the methods.

(D) FasL-dependent apoptosis-inducing activity of the indicated fractions of supernatants collected from cells WT and WASp-KO T cells. Supernatants from cells restimulated in

C were assayed on WEHI-279 cells for apoptosis-inducing activity. Anti-FasL was added to the indicated samples to neutralize FasL activity. Asterisks mark the results of comparisons of WASp-KO with the identical WT cell supernatant fractions, and anti-

FasL–treated samples compared with the same samples without anti-FasL. Results of P values from comparisons using Student unpaired t test are denoted as *P < .05,

**P < .005, ***P < .001.

(E) β-Hexosaminadase release from activated WASp-deficient and control T cells restimulated with the indicated concentrations of anti-CD3 mAb. The curve of percent

76 specific release was significantly different in WASp-deficient mice for both CD4 and

CD8 than controls (P < .001, 2-way analysis of variance).

77 Discussion

Our study shows a high incidence of autoantibodies and immune-complex nephritis in WASp deficient mice. The pathological features or renal impairment in

WASp deficient mice observed in this study are not as severe as observed in complete

Fas deficiency on a Murphy Roths Large (MRL) background or the NZB/NZW mouse model, but are similar to human IgA nephropathy in WAS patients (DeSanto et al., 1988).

Anti-dsDNA antibodies are also increased in the serum of ANA-positive WASp deficient mice indicating autoimmune manifestations. Since we observed apoptosis defects in mice less than 3 months of age, before the development of autoantibodies, and autoantibodies are not seen in every animal, environmental factors may play a role in the production of autoantibodies from WASp deficiency. Although our results show an apoptosis defect in T cells, other cell lineages may have reduced apoptosis in response to the FasL defect we show here and also a play a role in the pathogenesis of autoimmunity in WASp deficiency. Other mechanisms, such as defects in regulatory T cells in WASp deficiency may also contribute to the development of autoimmunity (Maillard et al.,

2007) (Humblet-Baron et al., 2007) (Adriani et al., 2007b) (Marangoni et al., 2007e).

Our results suggest that WASp deficient mice represent a useful model for the autoimmune and renal manifestations in WAS.

These results show for the first time that WASP affects not only antigen driven primary T cell activation, but also affects TCR mediated restimulation and apoptotic cell death of previously activated CD4+ T cells. Unlike the situation in lpr mice or the autoimmune lymphoproliferative syndrome, Fas induced apoptosis is intact in WASp deficient T cells. The defect in FasL function in WASp deficient mice is not as severe as

78 the defect seen in FasL mutant gld mice (Cohen and Eisenberg, 1992b). Because WASp deficiency does not have as severe defects in Fas or FasL function, it is not surprising that

WASp deficient mice do not show all the features of Fas or FasL deficiency such as generalized lymphadenopathy and accumulation of peripheral CD4-CD8- “double negative” T cells. Although we examined for anti-red blood cell (RBC) antibodies seen in WAS patients, we did not see anti-RBCs. We also did not see the colitis associated with WASp deficiency observed by others in WASp deficient mice. Because colitis associated with WASp deficiency has recently been shown to be transferable by T cells

(Nguyen et al., 2007), we seek to re-express WASP in T cells of WASp deficient mice to determine whether the autoantibody production seen in WASp deficient mice stems from a T cell intrinsic defect.

Our data suggests that reduced secretion of biologically active FasL contributes to inefficient TCE induced apoptosis in the absence of WASp. Soluble FasL was reduced in the supernatants of WASp deficient T cells suggesting that metalloprotease mediated cleavage of FasL may also be reduced in WASp deficiency, although this did not result in measurable increases in surface FasL. Reduced apoptosis of WASp deficient T cells was more likely due to the deficiency in either membrane bound FasL from the CD4+ T cell surface or from the surface of extracellular vesicles since only this form of FasL induced significant cytotoxicity and protects against autoimmunity (O' Reilly et al., 2009). The fact that surface expression of FasL was normal despite reduced RICD suggests that the vesicular form of FasL may be important in autocrine T cell death.

WASp may influence trafficking of FasL into ectosomes or into secretory lysosomes. WASp may more generally affect trafficking of secretory lysosomes into

79 multivesicular bodies and the fusion of these structures with the plasma membrane (Blott et al., 2001; Blott and Griffiths, 2002). Because WASp deficiency lead to a reduced release of secretory lysosome component -hexosaminidase from T cells, WASp deficiency may affect a more generalized deficiency in granule secretion. WASp deficient T cells have defects in secretion of other cytokines such as IL-2 and interferon-

(IFN-) (Morales-Tirado et al., 2004). It is not known if these defects in secretion are part of the same mechanism that impairs vesicular FasL secretion. Other cell types such as mast cells and NK cells have defects likely related to abnormal granule secretion from

WASp deficiency (Orange et al., 2002; Pivniouk et al., 2003).

Although B cell specific deletion of Fas can result in autoantibody production, defective vesicular FasL production may not likely affect B cell cells since B cells likely do not produce FasL in sufficient quantity to induce autocrine apoptosis within the B cell compartment. Other immunodeficiencies with reduced granule exocytosis such as

Griscelli syndrome associated with Rab27a deficiency and Hermansky-Pudlak syndrome due to AP3 deficiency, predispose patients to immunopathologic complications such as hemphagocytic syndrome (Enders et al., 2006; Menasche et al., 2000). It is not known if

FasL is defective in these diseases.

Other mechanisms may also contribute to the pathogenesis of autoimmunity in

WASp deficiency. The delayed phagocytic uptake of apoptotic cells by WASp deficient macrophages may be another potential mechanism of loss of peripheral tolerance

(Leverrier et al., 2001). However, we have not found significant cell uptake defects in macrophages from WASp deficient mice (Figure 4-6A). The lymphopenia report in

WAS patients and WASp deficient mice may also play an independent role in

80 predisposing toward autoimmunity, although the degree of lymphopenia in WAS is not as severe as other human syndromes of mouse models associated with autoimmunity (King et al., 2004). The function and homeostasis of Treg cells are also affected by WASp deficiency (Adriani et al., 2007b; Humblet-Baron et al., 2007; Maillard et al., 2007;

Marangoni et al., 2007e). Since Tregs may exert some of their suppressive effects through secretory lysosome components, WASp may regulate Treg function similar mechanisms by which it controls TCR induced cell death (Cao et al., 2007; Grossman et al., 2004a). Better understanding of these mechanisms may aid in the design of specialized therapies for autoimmune complications in WAS that avoid generalized immunosuppression, which is especially important in patients with concomitant immunodeficiency.

81 Chapter 5. Distinct secretory pathways govern CD4+ and CD8+ T cell cytotoxicity

and restimulation-induced cell death

Introduction

As observed in the previous chapter, WASp deficient T cells have a defect in the secretion of FasL. This observation raises a number of questions: 1) Is the FasL secretion defect a result of generalized problem in lytic granule exocytosis; and 2) Is the FasL secretion defect the only problem that impairs RICD in WASp deficient cells? We set out to answer these questions by: 1) examining whether a known mutation in the lytic granule secretion pathway phenocopies WASp deficiency; and examining how WASp deficiency compares to FasL deficiency in regard to cell death mechanisms such as RICD and the killing of target cells.

Our previous experiments in the previous chapter showed that WASp deficiency leads to reduced Restimulated Induced Cell Death (RICD) in CD4+ T cells. Since RICD may remove T cells responsive to persistently expressed antigens, such as autoantigens,

WASp deficiency may be a contributing factor toward the development of autoimmunity

(Carneiro-Sampaio and Coutinho, 2007; Notarangelo et al., 2009). We showed in our experiments discussed in the previous chapter, that WASp deficiency leads to not only a reduction in RICD, but reduced secretion of granules and FasL. We also showed that

WASp deficiency results in the decreased secretion of the cytotoxic, HMW form of FasL, likely in vesicles. In addition, the reduction of cytotoxic activity by CD4+ T cells likely contributes to the development of autoreactive CD4+ T cells and the subsequent development of autoimmunity.

82 Because CD4+ T cells also regulate the development of B cells for antibody production, WASp deficiency may affect the ability of CD4+ T cells to help in eliminating autoreactive B cells. Since WASp deficient mice have increased levels of autoantibodies (Nikolov et al., 2010; Shimizu et al., 2012), WASp deficiency in CD4+ T cell function may contribute to the development of autoimmunity. Since WAS patients have an increased risk of B-cell lymphoma, the inability of T cells, particularly CTLs, to kill B-cell lymphoma targets may also contribute to the development of autoimmunity.

In order to understand the various mechanisms by which autoimmunity can develop from WASp deficiency in various immune cells, we compared how proteins associated with vesicle trafficking and cell death affected RICD, FasL secretion and target cell killing. Rab27a is a protein involved in late exocytosis by docking vesicles to the plasma membrane. Rab27a mutations cause Griscelli Syndrome 2, which is an autosomal recessive disorder resulting in immune deficiency and hypopigmented skin and hair (Menashe et al., 2000). Griscelli Syndrome type 2 patients start developing light silvery-gray hair in infancy, are prone to recurrent infections and develop an immune condition called hemophagocytic lymphistiocytosis (HLH). HLH patients produce excessive activated T cells and macrophages, which can damage various organs and tissues throughout the body if they are not cleared by apoptotic mechanisms. Previous studies by other labs have shown that Rab27a deficiency results in a decrease in CTL cytotoxicity (Haddad et al., 2001). Because WASp deficient mice and humans have been shown to have autoimmunity and WASp-deficient CD4+ T cells have in reduced RICD,

HMW FasL, and potentially vesicular FasL, which may contribute to the breach in tolerance and rise in autoreactive T cells, we sought to understand if WASp deficiency

83 phenocopied Rab27a deficiency in CTL. The presence or absence of RICD in Rab27a deficient CD4+ T cells had not been previously determined, and we sought to resolve whether Rab27a deficient CD4+ T cells undergo reduced RICD. We expected that

WASp and Rab27a would have the same defects in RICD, HMW FasL secretion and defects in CTL target cell killing.

In this chapter, to determine whether WASp deficiency involves more than just a

FasL secretion problem, we also compared WASp deficient cells directly to FasL deficient (Gld) T cells in terms of their ability to kill target cells and undergo TCR induced apoptosis. Mutations in FasL result in defects in Fas-FasL mediated death and autoimmunity. The Fas-FasL pathway regulates the homeostasis of mature lymphocytes by limiting lymphocyte accumulation and maintaining self-antigens reactions to low levels (Cohen and Eisenberg, 1991) through cell mediated death mechanisms such as

RICD. Patients and mice with mutations in Fas or FasL develop Autoimmune

Lymphoproliferative Syndrome (ALPS) (Sneller et al., 1992). In 1967, Canale and Smith described a syndrome with nonmalignant lymphadenopathies associated with autoimmune features in children (Canale and Smith, 1967), which was later named ALPS after mutations in Fas and FasL were identified (Sneller et al., 1992). ALPS usually develops in patients by 5 years of age. Patients with ALPS have lymphoproliferative defects, such as lymphadenopathies and hepatosplenomegaly, with a specific disorder of hypergammaglobulinemia G and an expanded population of TCR alphabeta+CD4-CD8- double negative T cells (Sneller et al., 1992). Most ALPS patients have autoimmunity

(Straus et al., 1999). Fas deficient mice, aka Lpr mice, have lymphadenopathy, splenomegaly and autoimmunity (Watanabe-Fukunaga et al., 1992). FasL deficient mice,

84 aka, Gld mice, have lymphadenopathy, splenomegaly and autoimmunity (Takahashi et al., 1994a). While all cells contain Fas, only T cells have FasL (Takahashi et al., 1994c).

Therefore, using FasL deficient T cells from Gld mice serves as a control for comparing cellular mechanisms for death between WASp and Rab27a, which are two different proteins associated with reduced granule secretion. Because defects in the Fas-FasL pathway result in reduced RICD, we used FasL deficient T cells from Gld mice as a control to examine RICD, HMW FasL secretion, HMW FasL cytotoxicity and CTL killing. By comparing WASp deficient, Rab27a deficient and FasL deficient CD4+ and

CD8+ T cell functions, and comparing whether each defective protein results in a phenocopy of the other proteins, this helps resolve the mechanisms by which CD4+ and

CD8+ T cells act to regulate the elimination of self reactive lymphocytes.

Finally, we specifically sought to understand how CD4+ and CD8+ T cells differ in their ability to kill other CD4+ T cells and other CD8+ T cells, respectively. As a result, we were able to differentiate between mechanisms by which CD4+ T cells undergo RICD compared to CD8+ T cells. This observation also lead us to differentiate between mechanisms by which CD4+ T cells kill target cells compared to CD8+ T cells.

Therefore, this study helps resolve different mechanisms by which CD4+ T cells and

CD8+ T cells act in order to regulate the proliferation of self-reactive lymphocytes.

Results

The Predominant Form of FasL in RICD is in High-Molecular Weight Complexes

Previous studies have shown that there are three post-translational forms of FasL with varying degrees of cytotoxicity: 1) oligomerized, full-length FasL on the plasma

85 surface of cells; 2) oligomerized, full-length FasL on microvesicles (HMW FasL) secreted from cells; and 3) FasL cleaved by metalloproteases (LMW FasL). Both the plasma membrane and vesicular forms of FasL are cytotoxic, but metalloproteinase cleaved soluble FasL is not cytotoxic and in some circumstances has been found to block apoptosis induced by oligomerized FasL. We also showed in previous studies that apoptosis of T cells restimulated through the TCR (Restimulation Induced Cell Death,

RICD) and Fas Ligand secretion depends on WASp. In order to confirm the dependence of RICD on FasL function, we restimulated the TCR of activated gld/gld CD4+ T cells, which have a point mutation in the extracellular domain that interacts with the Fas receptor. We found that gld/gld CD4+ T cells have significantly reduced apoptosis compared to isogenic controls (Figure 12A, left two bars in the graph). This result confirms that that RICD of CD4+ T cells depends to some extent on intact FasL.

Because the FasL point mutation did not completely obliterate cell death upon TCR restimulation, this result suggested that either the FasL point mutation has a partial defect on the function of FasL or that mechanisms beside FasL mediate cell death during TCR restimulation. In order to determine whether the cell death in gld/gld CD4+ T cells is due to residual function of the gld mutant, FasL we blocked the remaining FasL function

(using the anti-mouse FasL antibody, MFL3) during TCR restimulation. We found that anti-FasL treatment reduced the cell death among WT CD4+ T cells to the same level of cell death seen among TCR restimulated gld/gld CD4+ T cells (Figure 12A, third bar from the left) while the level of cell death among TCR restimulated gld/gld CD4+ T cells treated with anti-FasL was not reduced from untreated TCR restimulated gld/gld CD4+ T cells (Figure 12A, fourth bar from the left). While these results showed that RICD

86 partially depends on FasL, other death factors beside FasL contribute or compensate when Fas-FasL interactions are blocked genetically or with antibodies.

We also measured FasL secreted by TCR restimulated gld/gld CD4+ T cells

(Figure 12B). TCR stimulation lead to increased Fas, which is mostly secreted in the high molecular weight (HMW) form (Figure 1B labeled as CS>100kD). Surprisingly, we found that the TCR restimulated gld/gld CD4+ T cells secrete less total, HMW and LMW

FasL compared to isogenic WT CD4+ T cells (Figure 12B black bars compared to dark grey bars, respectively). We were not able to resolve whether there is either a detection problem by anti-FasL for gld FasL, whether gld FasL secretion may be affected by the gld mutation, or whether reduced FasL secretion may be affected by reduced death occurring in FasL mutant T cells.

87 Figure 12. RICD and the secretion of FasL in gld FasL mutant T cells.

88 Figure 12. RICD and the secretion of FasL in gld FasL mutant T cells.

A) FasL deficiency results in significantly reduced RICD. WT and gld/gld CD4+ T cells were TCR restimulated with plate-bound anti-CD3 for 6 hours. Some WT and gld/gld

CD4+ T cells were additionally treated with anti-FasL in order to confirm that FasL specific cell death is only part of the total specific cell death. Less cell death occurs with the FasL deficiency and this cell death cannot be further eliminated upon anti-FasL treatment. N=10 mice. These experiments are averages of 2 separate experiments that were done in triplicate on each day.

B) The FasL secreted from 6 hour TCR restimulated WT and gld/gld CD4+ T cells was measured by ELISA. N=10 mice. This experiments is one representative of 2 separate experiments done in duplicates.

89 Because our results suggested that FasL secretion may be affected by reduced cell death occurring in mutant FasL CD4+ T cells, we tested whether FasL secretion occurs when caspases are inhibited during restimulation induced cell death. We treated plate- bound anti-CD3 TCR restimulated CD4+ T cells with the pan caspase inhibitor, z-VAD and then measured FasL secretion in the supernatants. As expected, we found that less cell death occurs in z-VAD treated, TCR restimulated CD4+ T cells than z-VAD untreated TCR restimulated WT CD4+ T cells, indicating that cell death in this system is caspase dependent (Figure 13A). We found that less total (CS) and HMW FasL

(CS>100kD) was secreted by z-VAD treated, TCR restimulated CD4+ T cells compared to z-VAD untreated TCR restimulated WT CD4+ T cells (Figure 13B, black bars compared to dark grey bars). However, because the HMW FasL (CS>100kD) was not affected by caspase inhibition, the effect of the gld mutation on FasL secretion during

RICD in Figure 13B is likely due to altered recognition of the mutant FasL by the antibodies.

90 Figure 13. FasL secretion does not require caspase-dependent apoptosis.

91 Figure 13. FasL secretion does not require caspase-dependent apoptosis.

A) CD4+ T cells were TCR restimulated with plate-bound anti-CD3 for 6 hours and treated with z-VAD in order to test for caspase dependence by RICD. Less cell death occurs during RICD with z-VAD treatment. N=10 mice. These experiments are averages of 2 separate experiments that were done in triplicate on each day.

B) The FasL secreted from 6 hour TCR restimulated WT CD4+ T cells was measured by

ELISA. Less FasL was found to be secreted by TCR restimulated necroptotic CD4+ T cells. N=10 mice. This experiments is one representative of 2 separate experiments done in duplicates.

92 Distinct roles of WASp vs Rab27a in CD4+ T Cell RICD

Since our previous studies have found that RICD, FasL and granule secretion of

TCR restimulated CD4+ T cells depends on WASp, this brought up the question of whether FasL is secreted through the general process of exocytosis of secretory lysosomes. Previous studies have shown that Rab27a, a component of endosomes, mediates exocytosis of secretory granules by localizing the granules to the immune synapse through interactions with Rab 11 and Munc l3-4 (Elstak et al., 2011). In order to determine whether RICD and FasL secretion is Rab27 dependent, we examined RICD and FasL secretion in TCR restimulated Rab27a deficient CD4+ T cells from Ashen mice, which have a naturally occurring mutation in Rab27a which has been shown to disrupt CTL granule secretion (Haddad et al., 2001). We found that Rab27a deficient

CD4+ T cells underwent comparable RICD compared to isogenic WT CD4+ T cells

(Figure 14A, bold black circle and bold red square). Cell death of Rab27a deficient T cells was still partially dependent on Fas-FasL interactions, as a similar, but not complete reduction in RICD occurred in restimulated Ashen and wild-type T cells after addition of the anti-FasL mAb MFL3 (Figure 14A, bold black triangle and dotted red square). In order to test whether microvesicular FasL depends on Rab27a for secretion, we measured

FasL in the supernatants of TCR restimulated Rab27a deficient CD4+ T cells. We found

Rab27a deficient T cells were able to secrete normal amounts total (CS), HMW

(CS>100kD) and soluble FasL (CS<100kD) after TCR restimulation (Figure 13B, compare dark gray bars to dark black bars). We found that FasL does not depend on

Rab27a for secretion during RICD and that RICD depends on WASp, but not Rab27a.

Because we showed in Chapter 4 that WASp deficiency reduces RICD and HMW FasL

93 secretion, these results suggested that WASp and Rab27a act independently in RICD and in FasL secretion of CD4+ T cells.

94 Figure 14. RICD and FasL secretion by CD4+ T cells does not require Rab27a.

95 Figure 14. RICD and FasL secretion by CD4+ T cells does not require Rab27a.

A) Restimulation Induced Cell Death (RICD) of Rab27a deficient CD4+ T cells is not different from WT CD4+ T cells. The addition of anti-FasL during RICD reduces RICD, but not completely. N=10 mice. These experiments are averages of 2 separate experiments that were done in triplicate on each day.

B) FasL secreted by anti-CD3 restimulated Rab27a deficient CD4+ T cells does not differ from WT CD4+ T cells. N=10 mice. This experiments is one representative of 2 separate experiments done in duplicates.

CD8+ T cell Cytotoxicity is Rab27a Dependent whereas CD4+ T cell Cytotoxicity

Depends on WASP

Rab27a is known to be important in lytic granule secretion and CD8+ T cell cytotoxicity. In order to test whether Rab27a and WASp affect the same pathway of lytic granule exocytosis in relation to CD8+ cytotoxicity, we assayed the ability of Rab27a and

WASp deficient CD8+ T cells to kill anti-CD3-coated P815 mastocytoma cells.

Consistent with previous studies, we found, that Rab27a deficient CD8+ T cells kill target cells significantly less than isogenic WT CD8+ T cells (Figure 15A, left panel).

This result suggests that the lysis of target cells, likely by the exocytosis of lytic granules depends on Rab27a. Although Rab27a deficiency significantly reduces the lysis of target cells, complete obliteration of target cell death does not occur as a result of Rab27a deficiency (Figure 15A, left panel), suggesting that other cytotoxic death factors beside

Rab27a also contribute to the lysis of target cells.

96 Because TCR restimulated CD8+ T cells undergo cell death (Russell, 1991,

PNAS 88:2151), we hypothesized that Rab27a deficient CD8+ T cells restimulated through their TCR in target cell assays will exhibit less cell death compared to isogenic

WT CD8+ T cells. In order to test whether CD8+ T cell RICD depends on Rab27a mediated exocytosis of lytic granules among CD8+ T cells, we examined the CD8+ T cell death occurring in the target cell lysis assay described above. We found that there is not a significant difference in the RICD of Rab27a deficient CD8+ T cells and their isogenic WT controls (Figure 15A, right panel). Although CD8+ T cells induced high levels of cell death in the P815 target cells, cell death of the CD8+ T cells was remarkably low, and there was no difference between n Rab27a deficient and WT cells.

These results suggest that in this system, where CD8+ T cells are restimulated in the presence of target cells RICD of CD8+ T cells does not depend on Rab27a mediated exocytosis of lytic granules for cell death.

To ask whether WASp participates in CTL target cell lysis in this system, we assayed target cell death and T cell death in cultures in which wild-type or WASp deficient T cells were mixed with P815 target cells as we did for Rab27a deficient T cells. We found that WASp deficient CD8+ T cells lysed anti-CD3 conjugated P815 target cells with the same efficiency as isogenic WT CD8+ T cells (Figure 15B, left panel). This result suggests that WASp is not necessary for target cell lysis by CD8+ T cells, at least in the CD3 stimulated killing of P815 cells. Additionally, this result suggests that WASp and Rab27a do not mediate the lysis of target cell death via the same mechanism. We also examined RICD among WASp deficient CD8+ T cells in culture with target cells to determine whether CD8+ T cell RICD is dependent on WASp.

97 Similar to the experiments with Ashen mice, we found very little cell death in the effector cells mixed with P815 targets. We found that there was only a very minimal defect in

RICD by WASp deficient CD8+ T cells compared to isogenic WT CD8+ T cells, with statistical significance only at the 3:1 E:T ratio (Figure 15B, right panel). This result suggests that RICD among CD8+ T cells is only very minimally dependent on WASp.

Because CD8+ T cells have been shown to mediate the lysis of target cells by the

Fas-FasL pathway and FasL microvesicles depend on WASp for secretion during TCR restimulation, we also sought to determine whether CD8+ T cell mediated lysis of target cells depends on FasL. In order to test whether FasL affects CD8+ T cell cytotoxicity, we assayed target cell death and FasL point mutated gld/gld CD8+ T cell death in cultures with CD3-coated P815 mastocytoma cells, which are Fas positive. We did see a small reduction in P815 target cell lysis, with the lowest E:T ratio being significant (Figure

15C, left panel). This result may differ from other studies in which a role for Fas-FasL interactions was found in CD8+ cytotoxicity due to P815 mast cells expressing a much lower level of Fas compared to other cell lines where Fas is transfected into them resulting in much higher surface levels (Simon et al., 2000).

We also examined RICD among Gld mutated CD8+ T cells in culture with target cells to determine whether CD8+ T cell RICD is dependent on FasL. Similar to the experiments with Ashen and WASp KO mice, we found very little cell death in the effector cells mixed with P815 targets. We found that there was only a very minimal defect in RICD by Gld mutated CD8+ T cells compared to isogenic WT CD8+ T cells and there was not any statistical significance between the isogenic WT CD8+ T cells and

98 the Gld mutated CD8+ T cells (Figure 15C, right panel). This result suggests that RICD among CD8+ T cells is not dependent on FasL.

99 Figure 15. CD8+ T cell cytotoxicity is dependent on Rab27 but not WASp; partially dependent on FasL.

100 Figure 15. CD8+ T cell cytotoxicity is dependent on Rab27 but not WASp; partially dependent on FasL.

A) Rab27a deficient CD8+ T cells (Ashen) kill target cells less than WT CD8+ T cells

(left panel) and undergo minimal RICD in the same culture (right panel).

B) WASp deficient CD8+ T cells kill P815 target cells similar to WT CD8+ T cells (left panel) and undergo reduced RICD compared to WT CD8+ T cells in the same culture

(right panel).

C) FasL deficient (Gld) CD8+ T cells kill P815 target cells less than WT CD8+ T cells

(left panel) and undergo reduced RICD compared to WT CD8+ T cells in the same culture (right panel). N=10 for each genotype. These experiments are averages of 2 separate experiments done on separate days.

101 We observed that cell death among CD8+ T cells was relatively low compared to the target cell death in the target cell killing cultures, and lower than CD4+ T cell death in

TCR restimulated T cells. Therefore we sought to understand how CD8+ T cells respond to TCR restimulation in the absence of target cells. We restimulated Rab27a deficient,

WASp deficient and FasL deficient CD8+ T cells along with their isogenic WT control cells with plate-bound anti-CD3 (Figure 16). We saw minimal RICD among the isogenic

WT CD8+ T cells and the Ashen, WASp KO and Gld mutated CD8+ T cells even without the presence of target cells. Under these conditions, none of the mutant cells underwent reduced apoptosis compared to their wild-type counterparts. While Ashen underwent more apoptosis, this increased apoptosis observed may be due to either systematic error or possibly to spontaneous death in culture.

102 Figure 16. CD8+ T cell RICD in the absence of target cells.

103 Figure 16. CD8+ T cell RICD in the absence of target cells.

N=10 for each genotype . These graphs represent averages of 2 experiments done on separate days in triplicates.

104 Although not as well-studied as CD8+ T cell cytotoxicity, CD4+ T cells have been found to kill target cells via a FasL dependent mechanism and via perforin and granzyme based exocytosis. To determine whether the distinction between WASp and

Rab27 dependent mechanisms we found in CD8+ T cells also applies to CD4+ cells, we performed cytotoxicity assays on P815 target cells with CD4+ T cells from WASp deficient, Rab27 deficient and Gld FasL mutant strains. Unlike CD8+ T cells Rab27a deficient CD4+ T cells were able to lyse P815 target cells at similar levels to wild-type isogenic CD4+ T cells (Figure 17A, left panel).

We also examined RICD among Ashen mutated CD4+ T cells in culture with target cells to determine whether CD4+ T cell RICD is dependent on Rab27a. We found very little cell death in the effector cells mixed with P815 targets (Figure 17A, right panel) compared to the high amount of RICD observed in Figure 14A when CD4+ T cells are not mixed with P815 target cells. We found that there was a slight reduction of RICD of Rab27a deficient CD4+ T cells mixed with P815 target cells. We saw no significant defect in RICD by Rab27a deficient CD4+ T cells compared to isogenic WT CD4+ T cells in the higher CD4+ T cell:P815 Target Cell Ratios, but there was a significant defect in RICD among the 1:1 CD4+ T cell:P815 Target Cell Ratio (Figure 17A, right panel).

Since the level of RICD was so low in these assays, the biological significance is unclear.

To determine if CD4+, like CD8+ T cells diverge in their use of WASp and

Rab27a in cytotoxicity, we sought to determine if the target cell killing of CD4+ T cells depends on WASp. We found that WASp deficient CD4+ T cells kill significantly fewer target cells at CD4+ T cell: P815 target cell ratios of 3:1 and 10:1 (Figure 17B, left panel). Similar to the RICD in WASp deficient CD4 T cells cultured with anti-CD3, we

105 observed less CD4+ T cell death at E:T cell ratios of 1:1 and 3:1 (Figure 17B, right panel). As in the other cultures, the presence of P815 target cells reduced the amount of

CD4+ T cell death compared to cultures of T cells alone. This data shows that unlike in

CD8+ T cells, WASp is important for the cytotoxicity of CD4+ T cells.

In order to determine if CD4+ T cell target cell killing depends on FasL, we assayed the ability of gld/gld FasL point mutant CD4+ T cells to lyse anti-CD3 coated

P815 target cells. We found that gld/gld CD4+ T cells have significantly reduced ability to lyse target cells only at the 1:1 CD4+ T cell:P815 Target Cell Ratio but not at higher

E:T ratios (Figure 17C, left panel). In order to test whether CD4+ T cell RICD depends on FasL in target cell assays, we examined the CD4+ T cell death occurring in the target cell lysis assay described above. Although the levels were much lower than when T cells were cultured alone, (Figure 17C, right panel compared to Figure 12A). We found that

FasL mutant CD4+ T cells have significantly less RICD at the CD4+ T cell: P815 target cell ratios of 1:1 and 10:1 (Figure 17C, right panel). These results suggest that CD4+ T cells partially depend on FasL for the lysis of target cells and for RICD in target cell cytotoxicity assays. This suggests that at higher E:T ratios, mechanisms other than

FasL:Fas interactions compensate for the FasL deficiency in the Gld CD4+ T cells. This is distinct from the situation with CD8+ T cells where gld/gld T cells were defective in killing at all E:T ratios.

106 Figure 17. CD4+ T cells depend on WASp but not Rab27 and partially on FasL for target cells lysis.

107 Figure 17. CD4+ T cells depend on WASp but not Rab27 and partially on FasL for target cells lysis.

A) Rab27a deficient CD4+ T cells kill target cells similar to WT CD4+ T cells (right panel) and undergo minimal RICD in the same culture (right panel).

B) WASp deficient CD4+ T cells kill target cells less than WT CD4+ T cells (right panel) and undergo less RICD than WT CD4+ T cells in the same culture (right panel)

C) FasL deficient (Gld) CD4+ T cells kill target cells less than WT CD4+ T cells (right panel) and undergo less RICD than WT CD4+ T cells in the same culture (right panel).

N=10 for each genotype. These graphs represent averages of 2 experiments done on separate days in triplicates.

108 In order to test whether the FasL dependency CD4+ T cell cytotoxicity also applies to TCR-induced autocrine cell death, we performed RICD experiments at varied

T cell densities in the absence of target cells. We restimulated the TCR of CD4+ T cells at three different cell densities: 1) 5x106 cells/mL (Figure 18, White Bars); 2)

1x106cells/mL (Figure 17, Gray Bars) and 3) 1x104 cells/mL (Figure 18, Black Bars).

Cell death at all densities varied between 20-40% with no clear density dependence.

However, blockade by anti-FasL was highly concentration dependent with almost complete inhibition at the highest cell concentration (Figure 18, right White bar), and no inhibition at the lowest cell concentration (Figure 18, right black bar). These results suggest that unlike target cell killing, Fas-FasL interactions are the predominant mechanism of TCR-induced cell death at high cell concentrations, but at low cell concentrations other Fas-independent mechanisms of apoptosis can compensate for blockade of Fas-FasL interactions. This may imply that cell-cell contact makes RICD more efficient because this may happen more at high cell density.

109 Figure 18. High CD4+ T cell density during restimulation results in less cell death.

110 Figure 18. High CD4+ T cell density during restimulation results in less cell death.

129 CD4+ T cells restimulated at 5x106 cells/ml +/ anti-FasL (white bars). 129 CD4+ T cells restimulated at 1x106 cells/ml +/- anti-FasL (grey bars). 129 CD4+ T cells restimulated at 1x104 cells/ml +/- anti-FasL (black bars). N=10 for each genotype. These graphs represent averages of 2 experiments done on separate days in triplicates.

111 Because Fas-bearing P815 cells are a mast cell line and CD4+ T cells usually recognize and regulate dendritic cells as APC’s, we cultured wild-type and WASp deficient T cells crossed to the OT-II TCR transgenic background. The OT-II TCR recognizes a peptide derived from ovalbumin complexed to MHC class II. We monitored cell death in the T cells and dendritic cells in these cultures. We found that OT-II WT

CD4+ T cells killed dendritic cell targets with increasing OVA concentration while

WASp deficient OT-II CD4+ T cells were significantly defective in killing dendritic cells

(Figure 19, left panel). These results suggest that the secretion of cytotoxic microvesicles to kill antigen-presenting dendritic cells depends on WASp When we examined the ability of the OT-2 CD4+ T cells to undergo RICD in these cultures, unlike the situation with plate bound anti-CD3, we found that apoptosis of OT-II CD4+ T cells reached a plateau and then declined at OVA concentrations above 0.1 M (Figure 19, right panel).

This suggests that higher peptide concentrations may shift the behavior of CD4+ T cells from dying by TCR restimulation to killing target cells upon TCR restimulation. Like the

RICD cultures with polyclonally stimulated T cells, we found that WASp deficient CD4+

T cells do have reduced apoptosis compared to WT CD4+ T cells upon TCR restimulation at all OVA peptide concentrations (Figure 19, right panel). Thus, WASp is genetically required for CD4+ T cells to kill target cells and undergo RICD when peptide antigen is presented more physiologically in the context of class II MHC.

112 Figure 19. Role for WASp in CD4+ T cell cytotoxicity and RICD in presence of

APC presenting cognate antigen.

% Dendritic Cell Death

113 Figure 19. Role for WASp in CD4+ T cell cytotoxicity and RICD in presence of

APC presenting cognate antigen.

A) In order to determine whether WASp deficient CD4+ T cells can kill target cells differentially from WT CD4+ T cells, we used WASp (+/-) dendritic cells that present ovalbumin to ovalbumin specific CD4+ T cells. WASp deficient CD4+ T cells significantly kill ovalbumin targets with less efficiency than WT CD4+ T cells at ovalbumin concentrations of 0.01, 0.1 and 1 microM. This data suggests that CD4+ T cells can kill physiologic target cells similarly to the trend seen in CD4+ T cells killing

P815 cell line target cells.

N=10 for each genotype. These graphs represent averages of 2 experiments done on separate days in triplicates.

114 Cell Intrinsic Role for WASp in CD4+ T cell Restimulation-Induced Cell Death

In our previous experiments with WASp deficient T cells, we found that FasL secretion and RICD was reduced. However, these experiments did not address the question of whether FasL secretion was the only defect in WASp deficient T cells, or whether some other aspect of TCR function, such as the ability of the TCR to sensitize cells to FasL dependent killing, was affected by WASp deficiency. The ability of WASp deficient T cells to undergo cell death in response to highly cross-linked FasL showed that there was no cell intrinsic defect in Fas signaling in WASp deficient T cells, but left open the question of whether defective FasL secretion was the only feature of WASp deficient T cells that explained their resistance to TCR-induced cell death, or whether there were other, cell autonomous mechanisms governing TCR-induced cell death. To determine whether FasL produced by WT T cells could rescue the apoptosis defect in

WASp deficient T cells we mixed WT and WASp deficient CD4+ T cells in the same culture, labeling one population with the tracking dye CFSE, and stimulated both populations of cells with plate-bound anti-CD3 at three different ratios of wild-type to

WASp deficient cells. We found that TCR restimulated WT CD4+ T cells could not rescue the RICD defect in WASp deficient CD4+ T cells, even at a 10:1 WT:WASp deficient cell ratio (Figure 20). These results suggested that WASp deficient CD4+ T cells have a cell intrinsic defect in their ability to die that cannot be bypassed in the absence of TCR signaling, and that WASp deficient CD4+ T cells are incapable of being killed in trans by either plasma membrane bound cytotoxic factors that mediate cell death by cell contact or cytotoxic secretory vesicles, including FasL microvesicles.

115 Figure 20. WASp deficiency results in a cell autonomous defect in RICD.

116 Figure 20. WASp deficiency results in a cell autonomous defect in RICD.

1:1 mixture of WT CD4+ T cells with WASp deficient CD4+ T cells restimulated with anti-CD3 for 6 hours (left panel). 10:1 mixture of WT CD4+ T cells with WASp deficient CD4+ T cells restimulated with anti-CD3 for 6 hours (center panel). 1:10 mixture of WT CD4+ T cells with WASp deficient CD4+ T cells restimulated with anti-

CD3 for 6 hours (right panel).

N=10 for each genotype. These graphs represent averages of 2-3 experiments done on separate days in triplicates.

117 FasL is Able to Induce Cell Death in Trans during RICD

The inability of WASp deficient T cells to be killed by FasL secreted from co- cultured T cells could also be due to inefficiency of FasL cytotoxicity delivered in trans from other cells. To determine if this was the case, we designed mixing experiments in which WT and gld T cells harboring a disabling point mutation in Fas Ligand were mixed at various ratios and stimulated with plate-bound anti-CD3 under similar conditions as the WASp deficient T cell mixing experiments, with one population labeled with CFSE.

We expected the WT CD4+ T cells to rescue the death defect observed in gld/gld CD4+

T cells during TCR restimulation. Only if cell death is “intracrine,” e.g. intracellular

FasL triggering cell death, would we expect that FasL derived from wild-type T would not be able to rescue the cell death defect in TCR restimulated gld T cells. In these mixing experiments, we found that WT CD4+ T cells could rescue the death defect in gld/gld CD4+ T cells during TCR restimulation at all WT:gld/gld ratios (Figure 21).

Even with the WT:gld/gld ratio of 1:10, WT CD4+ T cells significantly rescued the death defect observed in gld/gld CD4+ T cells (Figure 21, right panel). These results suggest that death factors from one CD4+ T cell can impact the cell death of another CD4+ T cell. Since the only genetic difference between the gld T cells and isogenic B6 controls is the point mutation in Fas Ligand, these data suggest that FasL is the cytokine that rescues the gld T cells from cell death in these experiments.

118 Figure 21. WT FasL can rescue gld/gld RICD defect in trans.

119 Figure 21. WT FasL can rescue gld/gld RICD defect in trans.

10:1 WT CD4+ T cells mixed with FasL deficient (Gld) CD4+ T cells while restimulated through the TCR for 6 hours (left panel). 1:1 WT CD4+ T cells mixed with FasL deficient (Gld) CD4+ T cells while restimulated through the TCR for 6 hours (center panel). 1:10 WT CD4+ T cells mixed with FasL deficient (Gld) CD4+ T cells while restimulated through the TCR for 6 hours.

N=10 for each genotype. These graphs represent averages of 2 experiments done on separate days in triplicates.

120 While the previous experiments suggests that death factors from one CD4+ T cell can impact the cell death of another CD4+ T cell, it is not known if the cytotoxic secretory vesicles of one CD4+ T cell can mediate the cell death of another CD4+ T cell.

In order to determine whether the cytotoxic secretory vesicles of one CD4+ T cell can mediate the cell death of another CD4+ T cell, we TCR restimulated either WT or gld/gld

CD4+ T cells with plate bound anti-CD3 while making available CD4+ T cells of the same genotype available in a transwell during TCR restimulation. We expected the TCR restimulated WT CD4+ T cells to induce cell death of the WT CD4+ T cells in the transwell while the TCR restimulated gld/gld CD4+ T cells should not induce as much cell death in the gld/gld CD4+ T cells within the transwell. We found that TCR restimulated WT CD4+ T cells in the presence of a transwell do not induce RICD among the WT CD4+ T cells as efficiently as when TCR restimulation occurs without the presence of a transwell (Figure 22A, left panel compared to Figure 12A). Despite this observation, TCR restimulated WT CD4+ T cells induce cell death of the WT CD4+ T cells in the transwell while the TCR restimulated gld/gld CD4+ T cells induce significantly less cell death in the gld/gld CD4+ T cells within the transwell (Figure 22A, left panel). These results suggest that TCR restimulated CD4+ T cells can induce cell death in other CD4+ T cells without cell contact during TCR restimulation.

Since our previous results suggest that TCR restimulated CD4+ T cells can induce cell death in other CD4+ T cells without cell contact, we sought to understand if the cytotoxic secretory vesicles of the WT CD4+ T cells could rescue the cell death defect observed in gld/gld CD4+ T cells during TCR restimulation. We predicted that WT

CD4+ T cells could rescue the cell death defect observed in gld/gld CD4+ T cells during

121 TCR restimulation based on our previous results where death factors from one CD4+ T cell can impact the cell death of another CD4+ T cell when WT and gld/gld CD4+ T cells are mixed during TCR restimulation. We found that TCR restimulated WT CD4+ T cells induce significantly more death in gld/gld CD4+ T cells within a transwell than TCR restimulated gld/gld CD4+ T cells can induce death in WT CD4+ T cells within a transwell (Figure 22A, right panel). Additionally, we found that TCR restimulated WT

CD4+ T cells induce significantly more cell death in gld/gld CD4+ T cells within a transwell than WT CD4+ T cells induced amongst each other underneath a transwell

(Figure 22A, outer left bar compared to the outer right bar). These results suggest that cell contact is not necessary for the death factors of one TCR restimulated CD4+ T cell to induce death in another CD4+ T cell that has not been TCR restimulated.

Since we encountered the limitation of the transwell experiments such that TCR restimulated WT CD4+ T cells in the presence of a transwell do not induce RICD among the WT CD4+ T cells as efficiently as when TCR restimulation occurs without the presence of a transwell, we decided to devise another experimental method for showing that cell contact is not necessary for inducing CD4+ T cell death during TCR restimulation. In our second method, we first TCR restimulated WT and gld/gld CD4+ T cells separately, and prepared a cell-free supernatant from these cells. We then applied that applied that TCR restimulated supernatant to either wild-type or gld/gld CD4+ T cells, which are unable to synthesize their own functional FasL. We found that the supernatant of TCR restimulated WT CD4+ T cells induced more cell death in TCR restimulated WT CD4+ T cells than the supernatant of gld/gld CD4+ T cells induced in

TCR restimulated gld/gld CD4+ T cells (Figure 22B, outer left and outer right bars on the

122 graph). Although these results do not eliminate the possibility that FasL delivered through cell-cell contact can contribute to RICD, these results do show that cell-contact is not necessary for the FasL secreted by a restimulated CD4+ T cell to induce death in another CD4+ T cell in trans, and highlight the importance of FasL in secreted microvesicles which are likely to be the apoptosis inducing factor in the transferred supernatants.

123 Figure 22. Secreted WT FasL can rescue gld/gld RICD defect in trans.

124 Figure 22. Secreted WT FasL can rescue gld/gld RICD defect in trans.

A) Specific cell death of WT (white bars) and FasL deficient (black bars) CD4+T cells

TCR restimulated with anti-CD3 for 6 hours at the bottom of the transwell (RICD).

Either WT or FasL deficient CD4+ T cells were subjected to the TCR restimulated supernatant in control or mixed culture through a transwell. Specific cell death of WT

CD4+ T cells in a transwell above TCR restimulated WT CD4+ T cells (white bar of

Transwell on left panel). Specific cell death of FasL deficient CD4+ T cells in a transwell above TCR restimulated FasL deficient CD4+ T cells (black bar of Transwell on left panel). Specific cell death of WT CD4+ T cells above TCR restimulated FasL

CD4+ T cells (light gray bar of Transwell on right panel). Specific cell death of FasL deficient CD4+ T cells above TCR restimulated WT CD4+ T cells (dark gray bar of

Transwell on right panel).

B) Supernatants from TCR restimulated WT or FasL deficient CD4+ T cells were concentrated and added to the cultures of TCR restimulated WT or FasL deficient CD4+

T cells in control or mixed situations.

N=10 for each genotype. These graphs represent averages of 2 experiments done on separate days in triplicates.

125 Discussion

Overall our results show that CD4+ and CD8+ T cells use different mechanisms to not only execute RICD, but also use different mechanisms for facilitating the killing of target cells. CD8+ T cells do not rely on WASp for RICD or the killing of target cells.

Instead CD8+ T cells depend on Rab27a for the killing of target cells and CD8+ T cells do not undergo RICD to the extent that CD4+ T cells. We were not able to identify the mechanism that CD8+ T cells depend on for RICD. Our results also show that CD4+ T cells depend on WASp for RICD and the killing of target cells, but do not depend on

Rab27a for RICD or the killing of target cells. We also found that CD4+ T cells depend on the density of other CD4+ T cells for their function in mediating RICD and target cell death and that WASp mediates a cell intrinsic defect in CD4+ T cells to undergo RICD.

Our results together help resolve the distinct cell death mechanisms that T cells use to maintain immune cell homeostasis. Future implications of our studies will help in distinguishing what appear to be highly specialized mechanisms that each T cell lineage and subset develop for the complex role each immune cell has in the regulation of immune cell homeostasis.

CD8+ T cells Rely on Rab27a Instead of WASp for Mediating Cell Death

We found that CD8+ T cells do not depend on WASp for the killing of target mast cells and instead depend on Rab27a for the killing of target mast cells. This result implies that CD8+ T cells specifically have a distinct mechanism from CD4+ T cells in mediating the killing of target mast cells. Beyond target mast cells, it is possible that

CD8+ T cells may also kill any peptide:MHC I presenting target cell through the same

Rab27a mediated mechanism. While CD8+ T cells have not been widely examined for

126 WASp dependency, our results are consistent with previous studies showing that WASp

CTLs express normal levels of lytic molecules, perforin and Granzyme B, and “efficient exocytosis” to target B cells while the “lytic granules appeared to not fully polarize toward the center of the CTL/tumor target cell contact area” (De Meester et al., 2010).

Our results are likely not consistent with previous results that used higher CD8+ T cell:target cell ratios (Lang et al., 2013) because we did not examine higher CD8+ T cell:target cell ratios. The discrepancy between our study and the study that examined higher CD8+ T cell:ratios may be resolved by our examination of higher CD8+ T cell:target cell ratios or an examination with lower total cell concentrations per well. This discrepancy also suggests that the WASp dependent function of CD8+ T cells additionally depends on the density of CD8+ T cells as CD8+ T cells become less dependent on WASp as the CD8+ T cell:target cell ratio decreases to 1:1 (Lang et al.,

2013). While our examination of TCR restimulated WASp deficient CD8+ T cells did not result in any significant defect in RICD from WT TCR restimulated CD8+ T cells, we also observed that RICD did not occur above a level of ~25% suggesting that CD8+ T cells do not undergo RICD readily at the cell concentration of 1 million cells/mL. Since the Lang et al, 2013 study used total cell concentrations/well between 900,000-8000 cells/culture (Lang et al, 2013 do not report the well size nor the total volume of the culture), it is possible that my cultures may have not been dense enough to see the effect observed in Lang et al., 2013. Further exploration of RICD in CD8+ T cells at total cell concentrations above 250,000 cells/culture may show that CD8+ T cells RICD function depends on cell density as much as WASp. Because CD8+ T cells proliferate faster than

CD4+ T cells (Schlub et al., 2011), it is likely that CD8+ T cells function more efficiently

127 in the killing of target cells by WASp mediated mechanisms at higher CD8+ T cells densities. Further imaging studies on WASp deficient CD8+ T cells encountering various target cells to determine whether WASp differentially associates with perforin and granzymes or vesicular FasL will help in resolving how CD8+ T cells likely kill through a Rab27a dependent mechanism of lytic granule exocytosis to target cells while

CD4+ T cells likely kill through a WASp dependent mechanism of vesicular FasL exocytosis to target cells.

We also found that CD8+ T cells do not depend on functional FasL to kill target mast cells except at the lowest E:T ratio of 1:1. This suggests that CD8+ T cells kill target cells primarily through the exocytosis of lytic granules, such as perforin and granzymes, which is a result that is consistent with the majority of previous studies

(Peters et al., 1991). This also suggests that cell density affects the function of FasL mediated cytotoxicity while Rab27a mediated cytotoxicity is not as cell density dependent.

CD4+ T cells Depend on WASp for Mediating Cell Death

Previous studies have examined how cytotoxic CD4+ T cells kill target cells.

However, these previous studies often only examined perforin/granzyme expression or only examined FasL expression (Appay et al., 2002; Brien et al., 2008; Chtanova et al.,

2001; Eshima et al., 2012; Gagnon et al., 1999; Grossman et al., 2004b; Haigh et al.,

2008; Jellison et al., 2005; Kaneko, 2000; Niiya et al., 2005; Norris et al., 2001; Stalder et al., 1994; Sun et al., 2002; van Bergen et al., 2009; Wahid et al., 2005; Yawalkar et al.,

2001; Zajac et al., 1996). Our study examined CD4+ T cells for the mechanism known to

128 facilitate the exocytosis of lytic granules such as perforin and granzymes during the killing of target cells. We found that the CD4+ T cells do not use Rab27a for the killing of target cells while we confirmed that CD8+ T cells depend on Rab27a for target cell killing. Previous studies have not associated Rab27a with the killing of target cells by

CD4+ T cells and we also confirmed this (Haddad et al., 2001).

We found in our results in this chapter that WASp deficiency resulted in defective target cell killing at higher E:T ratios while Gld FasL dependent killing of targets only happened at the lowest 1:1 E:T ratio. Because Gld FasL deficiency in mice leads to a more rapid and debilitating disease than WASp deficiency in mice, this result, such that

WASp deficiency leads to more target cell killing only at the higher E:T ratios and FasL deficiency leads to more target cell killing only at the lowest E:T ratio, suggested that the ratio of cytotoxic CD4+ T cells to their target cells affects the function of CD4+ T cells.

Perhaps because Gld FasL deficiency results in significantly less killing of target cells at lower E:T ratios, the function of FasL in the killing of target cells depends more on the cellular environment having more target cells present than effector cells. Because WASp deficiency affects the secretion of FasL, at least from cultures of restimulated CD4+ T cells, we expected a similar pattern in the killing of target cells by WASp deficient CD4+

T cells and Gld FasL deficient CD4+ T cells. However, our results show that FasL deficiency and WASp deficiency in CD4+ T cells do not phenocopy each other in the case of the killing of target cells. Perhaps because WASp deficiency affects more cellular functions through the branched actin cytoskeletal rearrangements in the killing of target cells than just FasL, we see this discrepancy in the expected phenocopying.

Further studies examining how WASp deficient and Gld FasL CD4+ T cells kill target

129 cells at broader ranges of E:T ratios ranging from, 1:3 to1:100 may provide some clues about how the function of FasL depends on the population density of target cells. Also, because FasL functions in several forms with target cells, i.e. soluble, cellular membrane bound and vesicular membrane bound, vesicular cytotoxic forms of FasL may more efficiently function in the killing of target cells when target cells outnumber effector

CD4+ T cells. Because cultures of TCR restimulated CD4+ T cells are close to 100% effector CD4+ T cells, we would have expected more target cell killing among higher

E:T ratios from Gld FasL deficient CD4+ T cells. It is likely that non-CD4+ T cell targets, especially these P815 mast cells and other antigen:MHC II presenting cells, promote different responses in CD4+ T cells that result in CD4+ T cells being more dependent on WASp for cytotoxicity than simply FasL. Previous studies have been done to image the immune synapse between CD4+ T cells and mast cells, but FasL was not examined (Gaudenzio et al., 2009). Further imaging studies examining conjugates of

CD4+ T cells with these P815 target cells can help resolve whether WASp is affecting the trafficking of vesicular or cellular membrane bound FasL for the efficient killing of target cells or whether perhaps FasL on the cellular membrane is mediating the target cell killing which may be independent of WASp localization.

Since other studies have found that CD4+ T cells increase both their FasL and

CD63 expression on the cell surface after ionomycin stimulation (Bossi and Griffiths,

1999), it is possible that CD4+ T cells use a mechanism to degranulate FasL in vesicles to peptide:MHC II presenting target cells or to be released into the environment for the killing of Fas expressing bystander cells, including clonal CD4+ T cells in RICD. Our results showing that the supernatants of TCR restimulated WT and Gld FasL deficient

130 CD4+ T cells can kill activated CD4+T cells through a transwell and through application of the supernatant directly onto TCR restimulated CD4+ T cells indicate that TCR restimulated CD4+ T cells depend on FasL for killing activated CD4+ T cells at a distance. This mechanism for TCR restimulated CD4+ T cells to kill activated CD4+ T cells at a distance helps resolve how CD4+ T cells mediate immune cell homeostasis.

Fas+ immune cells such as clonal CD4+ T cells, dendritic cells, B cells and mast cells may be abrogated through TCR restimulation. If there is a break in the ability for TCR restimulation to reduce the population of immune cells responding to a particular antigen, especially a self-antigen, then the development of autoimmunity will likely occur.

Considering that the development of autoimmune symptoms develops more rapidly in

Gld FasL and Lpr Fas deficient mice compared to WASp deficient mice, it is clear that the mechanism for cell death mediated through FasL is critical to ensure immune cell homeostasis. Because WASp deficiency affects more than apoptotic CD4+ T cellular functions, including the reduction in activation of T cells and therefore the reduction in peripheral T cells in WASp patients (Molina et al., 1992) (Molina et al., 1993), this may be the reason for the less rapid development of autoimmune symptoms in WASp mice and patients compared to ALPS patients and Gld FasL deficient and Lpr Fas deficient mice. Further imaging studies, such as intravital microscopy which has been optimized for tracking secretory vesicles in mice, on the trafficking of FasL after TCR restimulation in CD4+ T cells is needed to resolve how CD4+ T cells mobilize FasL for the killing of other immune cells.

While we showed that WASp deficiency affects the killing of dendritic cell targets just as its affects the killing of P815 mast cell targets, we did not examine how

131 WASp deficiency affects the killing of target B cells. Other studies in other labs have shown that WASp deficiency reduces the killing of B cell targets and reduces the secretion of granzyme B by regulatory T cells (Adriani et al., 2011). Because regulatory

T cells are a subset of CD4+ T cells, it is likely that cytotoxic CD4+ T cells depend on

WASp for killing B cell targets. Additionally, because CD4+ T cells kill B cell lymphoma cells through a WASp dependent manner (De Meester et al., 2010), CD4+ T cells may kill non-lymphoma B cells in a WASp dependent manner. Further studies examining how the killing of B cell targets by cytotoxic CD4+ T cells is WASp dependent is worth examining as well as a comparison of FasL transfer to B cell targets in imaging studies. The killing of B cell targets by CD4+ T cells has implications in preventing the production of autoantibodies contributing to the development of autoimmunity. Since WASp deficient CD4+ T cells do not kill likely kill mast cell, dendritic cell, B cell or CD4+ T cell targets efficiently, this deficiency in WASp mediated target cell killing likely contributes to the development of autoimmunity since dendritic cells presenting self antigens may not be eliminated and B cells producing autoantibodies may not be eliminated. The defects in target cell killing in Fas and FasL deficiency likewise contribute to the development of autoimmunity from the presentation of self- antigens by dendritic cells and the subsequent lack of elimination of autoantibody producing B cells. Because Fas deficient dendritic cells and B cells lead to the development of autoimmunity (Chen et al., 2006; Stranges et al., 2007a), the inability of

WASp deficient CD4+ T cells to kill dendritic ell and B cell targets through what is likely a FasL mediated mechanism likely contributes to the development of autoimmunity. Additionally, since WASp deficient mast cell, dendritic cell and B cell

132 targets may not respond as efficiently to being targets of CD4+ T cell killing, this may be one of the reasons for the observation that WASp deficient mice do not develop symptoms of autoimmune disease as severely as Gld or Lpr mice.

WASp Mediates a Cell Intrinsic Defect in CD4+ T cell RICD

In testing for whether WASp deficient CD4+ T cells phenocopied the functions of

Gld FasL deficient CD4+ T cells, we also found that the presence of WT CD4+ T cells and their supernatants with and without a transwell rescued FasL Gld deficient CD4+ T cells from the defect in restimulation induced cell death (RICD) while the presence of

WT CD4+ T cells did not rescue WASp deficient CD4+ T cells from the defect in RICD.

This suggests that there is a cell intrinsic defect in WASp deficient CD4+ T cells to undergo RICD. This cell intrinsic defect is likely due to defects in actin polymerization affecting the competency to die signal upstream from the function of FasL after TCR restimulation. Since we observed this cell intrinsic defect in WASp deficient CD4+ T cells, we did not test for the presence of TCR restimulated WT CD4+ T cell supernatants without or without a transwell on WASp deficient CD4+ T cells. Further tests may likely confirm the cell intrinsic defect we observed from mixing CD4+ T cells with WASp deficient CD4+ T cells in a well at different ratios. Because these WASp deficient CD4+

T cells do not die by RICD at the same frequency of TCR restimulated WT CD4+ T cells, this cell intrinsic phenotype is similar to the phenotype of TCR restimulated Rac1 deficient CD4+ T cells which could not be rescued by TCR restimulated WT CD4+ T cells (Ramaswamy et al., 2007). Because Rac1 is downstream of WASp, it is possible that the cell autonomous defect in WASp deficient CD4+ T cells is similar to the cell

133 autonomous defect in Rac1 deficient CD4+ T cells during restimulation because the same pathway is being affected. Further studies such as microarray and ChIP-Seq analysis for

TCR restimulated CD4+ T cells compared to activated CD4+ T cells examining how the different proteins involved in the signaling cascade involved for TCR restimulation induced death need to be examined for cell autonomous defects in order to resolve how defects in CD4+ T cells specifically contribute to autoimmunity.

134 Chapter 6. Conclusions

Overall, we found that WASp deficient mice develop autoantibodies in an age- dependent manner similar to the autoantibodies in Wiskott-Aldrich Syndrome (WAS) patients (Schurman and Candotti, 2003) (Dupuis-Girod et al., 2003). These autoantibodies in WASp deficient mice were associated with immune complex deposition and mesangial cell proliferation in the kidneys (Nikolov et al., 2010). Since defects in T cell apoptosis may contribute to loss of self-tolerance and autoantibody formation, we examined restimulation induced cell death (RICD). Our analyses showed that WASp deficiency results in significantly reduced RICD (Nikolov et al., 2010). In seeking to understand whether WASp affected the Fas-FasL pathway that contributes to RICD, we found that blockage of FasL by antibody during RICD reduced specific cell death in

WASp deficient CD4+ T cells compared to WT CD4+ T cells. This RICD defect from

WASp deficiency was not due to Fas since surface Fas was not affected while the secretion of cytotoxic granules from TCR restimulated CD4+ and CD8+ T cells were reduced in WASp deficient mice (Nikolov et al., 2010). We found that WASp deficiency reduced the secretion of high molecular weight (HMW) FasL from TCR restimulated

CD4+ T cells (Nikolov et al., 2010).

From this observation that WASp deficient TCR restimulated CD4+ T cells do not secrete HMW FasL, we sought to find how TCR restimulated CD4+ and CD8+ T cells function in RICD and in the killing of target cells through the secretion of FasL vesicles. We used other primary immunodeficiency mouse models with highly associated autoimmunity as controls to understand how WASp deficiency affects vesicular FasL function. We found that WASp deficiency affects RICD in CD4+ T cells

135 and the killing of target cells by CD4+ T cells, but it does not affect the killing of target cells by CD8+ T cells. We found that WASp deficiency has a further cell intrinsic effect on CD4+ T cell RICD since WT CD4+ T cells could not rescue WASp deficient CD4+ T cells from the defect in RICD (Figure 23). We also found that Rab27a deficiency only affects the killing of target cells by CD8+ T cells suggesting that CD8+ T cells and CD4+

T cells have different primary mechanisms for the killing of target cells and for undergoing RICD. By using FasL deficient CD4+ and CD8+ T cells as a control to compare cell death mechanisms among CD4+ and CD8+ T cells, we found that FasL deficiency, like WASp deficiency, affects RICD, but does not affect the killing of target cells by CD4+ T cells to the extent that WASp deficiency does nor does FasL deficiency affect the killing of target cells by CD8+ T cells. This observation leads to several questions about the differences between the severity of autoimmunity among WASp deficient mice and FasL deficient (Gld) mice. Since Gld mice develop severe lymphadenopathy and splenomegaly that WASp deficient mice do not develop to the same extent at the same age, it is possible that the actin polymerization defects in WASp deficiency affect not only cell death mechanisms involved in T cell homeostasis in the periphery, but also affect potentially autoreactive or even normal T cells from ever getting activated.

Overall, the insight into how WASp, Rab27a and FasL deficiency affects the cell death mechanisms of TCR restimulated CD4+ and CD8+ T cells allows for more targeted discovery into how T cell death mechanisms contribute to the development of autoimmunity. Because WASp and FasL are necessary for Th2 CD4+ T cell effector function (Fuss et al., 1997; Morales-Tirado et al., 2010), the characteristics of Th2

136 cellular function likely provide insight regarding how autoimmunity develops from a primary immunodeficiency. Th2 CD4+ T cells undergo more plasticity than Th1 cells in regulating the functions of T cell subsets (Geginat et al., 2014) such that Th2 cells differentiate into T follicular helper cells and/or Th9 cells for helping B cells or regulating an inflammatory response to allergens, respectively. Th2 cells do not undergo

RICD as readily as Th1 cells and Th2 cells regulate Granzyme B and FasL expression through vasoactive intestinal peptide (VIP) while Th1 cells do not (Sharma et al., 2006).

Fas deficient Th2 cells also undergo TCR restimulated cell death mediated by Granzyme

B (Sharma et al., 2006) and Granzyme B inhibition of TCR restimulated Th2 cells suggests that Th2 cells have independent mechanisms for Granzyme B mediated and

FasL mediated death upon TCR restimulation. Because Th2 cells mediate Granzyme B and FasL through independent mechanisms upon TCR restimulation and this mechanism is likely due to differential intracellular expression and storage of Granzyme B in Th2 memory cells compared to CD8+ T cells (Medina et al., 2012), the regulation of

Granzyme B by WASp and Rab27a likely differs between CD4+ and CD8+ T cells.

Because Granzyme B is regulated through the secretion of vesicles, we expect vesicular

FasL to also be differentially regulated between CD4+ and CD8+ T cells as other studies indicate that FasL is in distinct vesicles from Granzymes in human T cells (Schmidt et al., 2011). Our studies confirmed that WASp mediates FasL secretion in CD4+ T cell

RICD and the killing of target cells by Cd4+ T cells, but does not mediate the killing of target cells by CD8+ T cells. Conversely, Rab27a deficiency does not affect RICD in

CD4+ or CD8+ T cells, does not affect the killing of target cells by CD4+ T cells, but reduces the ability for CD8+ T cells to kill target cells. Our results are consistent with

137 other studies regarding how different vesicles are mediated by distinct cellular mechanisms. Further intravital microscopy studies of how these vesicles are trafficked in

WASp, Rab27a and FasL deficient animal models will likely resolve how WASp and

Rab27a affect the distinct trafficking of the FasL containing and Granzyme B containing vesicles, respectively, through the cell.

Our results in resolving the cell intrinsic, WASp dependent mechanism, which

CD4+ T cells use to undergo RICD, may lead to a better understanding of how immune cell homeostasis operates. The FasL in TCR restimulated CD4+ T cells that is necessary to effect Fas mediated death in clonal, self-reactive CD4+ T cells likely is not efficiently trafficked in vesicles to the cell surface for release. Our finding that WASp deficiency results in less killing of target mast cells also helps to resolve the specific apoptotic mechanisms that CD4+ T cells use for maintaining immune cell homeostasis. Because previous studies on either CD4+ T cells or CD4+ T cell subsets showed that WASp deficiency lead to less killing of antigen-presenting dendritic cell and B cell targets, our results consistently support the concept that CD4+ T cells operate in a WASp dependent manner to kill antigen presenting cells including self-presenting dendritic cells, B cells and mast cells and therefore eliminate a self-reacting cascade after TCR restimulation.

We have resolved that CD8+ T cells do not depend on WASp for RICD or cytotoxicity toward target mast cells. These results support previous studies that show CD8+ T cells do not depend on WASp for killing target cells, except when CD8+ T cells are highly dense. Moreover, our work showing that CD8+ T cells do not depend on WASp for

RICD and that TCR restimulated CD8+ T cells do not undergo RICD efficiently (at levels above 40% of the total CD8+ T cell population undergoing restimulation), reveals

138 that CD4+ T cells and CD8+ T cells likely operate in distinct manners in terms of eliminating potential self-reactive cells. Deficiency in Rab27a, a molecule shown to affect the trafficking of lytic granules such as perforin and Granzymes, does not result in reduced CD4+ T cell or CD8+ T cell RICD, but results in less efficient killing of target mast cells by CD8+ T cells. Deficiency in Rab27a does not affect how CD4+ T cells kill target mast cells. These findings from our studies in our last chapter show an apoptotic mechanism affecting the function of CD8+ T cells that CD4+ T cells do not use. Our findings reveal that CD4+ and CD8+ T cells do not use the same apoptotic mechanisms for eliminating self-reactive cells. This is significant because our results support the previous research showing that CD8+ T cells specialized to respond to MHC I presenting cells differ in their functions from CD4+ T cells specialized to respond to MHC II presenting cells. Our work shows that the same apoptotic mechanisms do not apply to all

T cell lineages and subsets. This is a significant finding that must be resolved even further with research on how the different T cell lineages and subsets are specialized in their apoptotic mechanisms. While CD4+ and CD8+ T cells use FasL and Granzyme B as apoptotic mechanisms, each T cell subset uses different mechanisms for achieving apoptosis. These different mechanisms are likely distinct kinetically in order to regulate

T cell homeostasis. We show here that different T cell subsets use very different mechanisms to effect cell death. Continued studies into these different mechanisms such as microarray, ChIP Seq screens and further resolution for how the presence or absence of identified proteins from the screens affect RICD and/or target cell killing in monogenic primary immunodeficiency animal models will help resolve why some immune deficiencies lead to more rapid development of autoimmunity.

139

Overall, in the grand scheme for how WASp deficiency may lead to the development of autoimmunity, my contribution to the field has shown that WASp affects

FasL function in RICD and that WASp affects the killing of dendritic cells by CD4+ T cells (Figure 23).

Because other studies in other labs, including Fabio Candotti’s lab, have shown that WASp deficiency can contribute to autoimmunity in failing to kill target cells by NK cells (Figure 23), failing to regulate the proliferation of autoreactive native T cells by T regs, failing to kill B cells by Tregs (Fabio’s lab), and failing to phagocytose apoptotic blebs from RICD or B cell death by macrophages, my thesis project has shown that

WASp deficiency in CD4+ T cells leads to significant reductions in the ability for CD4+

T cells to not only die, but kill target cells. These defective cellular death mechanisms may contribute to the frequent development of autoimmunity among WAS patients just as the frequent autoimmunity among ALPS patients have similar defects in cellular death mechanisms.

140 Figure 23. Different cells that WASp deficiency could impact for the development of autoimmunity.

Macrophage

T Cell Apoptotic Blebs F

A B Cell

Apoptotic Target Restimulated T Cell Blebs

B Cell C Ag/MHC Dendritic Activated T CellCell

NK Cell E

Treg

Naïve T Cell

141 Figure 23. Different cells that WASp deficiency could impact for the development of autoimmunity.

The red arrows indicate that WASp deficiency affects the cellular function of the particular hematopoietic cell.

A) My work in this thesis shows that WASp affects FasL function in RICD.

B) My work in this thesis shows WASp affects the killing of dendritic cells by activated

CD4+ T cells.

C) WASp deficiency can contribute to autoimmunity in failing to kill target cells by NK cells.

D) WASp deficiency can contribute to autoimmunity by failing to regulate the proliferation of autoreactive naive T cells by T regs.

E) WASp deficiency can contribute to autoimmunity by failing to kill B cells by Tregs.

F) WASp deficiency can contribute to autoimmunity by failing to phagocytose apoptotic blebs from RICD by macrophages.

G) WASp deficiency can contribute to autoimmunity by failing to phagocytose apoptotic blebs from B cell death by macrophages.

My thesis project has shown that WASp deficiency in CD4+ T cells leads to significant reductions in the ability for CD4+ T cells to not only die, but kill target cells. These defective cellular death mechanisms may contribute to the frequent development of autoimmunity among WAS patients.

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