RE-ENGINEERING THE MICROENVIRONMENT OF MICE THYMUS TO

PROMOTE TOLERANCE TO A VCA TRANSPLANT

by Jialu Wang

A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering

Baltimore, Maryland May 2019 Abstract

Nowadays, patients with a solid organ transplant or a vascularized composite allotransplant (VCA) transplant rely on life-long immunosuppression or a highly controversial bone marrow transplant to induce immune tolerance and to maintain their grafts. With only a few novel methods that attempt to induce donor-specific tolerance such as the splenocyte induced donor-specific tolerance, such methods target the peripheral tolerance mechanism instead of central tolerance.1 This study searches for a novel tolerance induction method that does not compromise the immune system with long-term immunosuppression, focuses on the practicality of inducing donor-specific central tolerance and aims to reduce graft-versus-host-disease seen in VCA. We hypothesize that through intrathymic injection of donor thymic epithelial cells after a hindlimb transplant, a possible donor-recipient hybrid thymus may achieve immunotolerance without the risk of graft-versus-host-disease induced by a bone marrow transplant. Some possible mechanisms that are impacted by the hybrid thymus are investigated. Although with many obstacles before this hybrid thymus idea could be translated into clinical treatment, the concept remains a potential option for the induction of VCA transplant tolerance.

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Acknowledgment

I would like to thank the entire Vascularized Composite Allotransplantation Lab. The PIs,

Dr. Giorgio Raimondi and Dr. Gerald Brandacher, are tremendously resourceful, guided me patiently through all the trouble-shooting and pointed me in the right direction when encountered obstacles. The postdoctoral fellow, Dr. Marcos Iglesias, has accommodated my requests no matter how busy he is or how late at night. I am grateful for all the hours

Dr. Yinan Guo put into microsurgeries so I could complete this project. The lab has a dynamic and supportive environment, without which I would not accomplish what I have accomplished. I would like to thank every member of the VCA lab for making my stay in the lab so enjoyable.

I would like to thank the Biomedical Engineering department faculty and staff, all of whom are extremely helpful and encouraging. I hold great gratitude to Sam Bourne, the

BME Master’s Program Manager, who has given me invaluable advice in the past two years and supported all of my decisions along the journey.

I would like to thank my fiancé, Alexander Samuel Kaplitz, and his family, who have provided me a home away from home here in the US. Alex knows me better than myself and offered me the emotional support that I needed to complete my study.

Last but not least, I would like to thank my parents, who have financially and emotionally supported me all these years. They had never doubted my potential even in situations when I had no faith in myself.

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Table of Content

Title i

Abstract ii

Acknowledgment iii

List of Figures vi

1. Introduction 1-14

1.1 Current Treatment Challenges in Transplantation 1-4

1.2 T cell development 4-9

1.2.1 Early Stage T Cell Development 5-6

1.2.2 Late Stage T Cell Development 6-7

1.2.3 T Cell Activation 7-8

1.2.4 Regulatory T Cells 8-9

1.3 Thymic Epithelial Cells 9-12

1.3.1 cTEC 10-11

1.3.2 mTEC 11-12

1.4 Antigen Presenting Cells 12-13

1.5 Purpose of the Study 13-14

2. Optimal Donor TEC Processing 15-25

2.1 Animals Used 15

2.2 Method 15-17

2.2.1 Digestion 15-16

2.2.2 Negative Selection 16-17

2.2.3 Density Gradient Selection 17

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2.2.4 Positive Selection 18

2.2.5 FACS Sorting 18

2.3 Material and Reagents 19

2.4 Results 20-25

3. Donor TEC Engraftment 26-31

3.1 Animals Used 26

3.2 Method 26-27

3.3 Material and Reagents 27

3.4 Results 27-31

4. Donor TEC’s Interaction with the Recipient 32-35

4.1 Animals Used 32

4.2 Method 32

4.3 Material and Reagents 32

4.4 Results 33-35

5. Allogeneic TEC Survival in a Hindlimb Recipient 36-37

5.1 Animals Used 36

5.2 Method 36

5.3 Material and Reagents 36

5.4 Results 36-37

6. Discussion 38-42

7. Bibliography 43-47

8. Curriculum Vitae 48

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List of Figures

Figure 1. A model for cTEC contributions to T cell development 5

Figure 2. A flow cytometry analysis of the effect of different digestion buffer

compositions 20

Figure 3. A flow cytometry analysis of the effect of different medium 20

Figure 4. Cell aggregates formed due to thermic shock 21

Figure 5. Comparison between different panning strategies 22

Figure 6. TEC yield after using different density solutions 23

Figure 7. The positive enrichment consistently yields around 10% TEC in the

population 24

Figure 8. The two-step positive enrichment yields satisfactory percentage of TEC 24

Figure 9. Conditions for optimal engraftment 28

Figure 10. TECs from donors older than 20 days old do not survive in the recipients

28

Figure 11. Allogeneic TEC survival in BALB/c animals 29

Figure 12. Donor TEC percentage in congenic animals 31

Figure 13. Long-term effect of donor TECs on the recipient TEC population 33

Figure 14. Short-term effect of donor TECs on the recipient TEC population 34

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Introduction

Current Treatment Challenges in Transplantation

Transplantation, such as solid organ transplantation, has long been an accepted standard therapeutic method to patients with end-stage organ failures and has saved countless lives. Although not life-saving, the vascular composite allotransplantation (VCA) has also increasingly become a valid option for patients who seek to restore normality after devastating injuries. VCA refers to a form of transplant with multiple tissue types and fills the gap where conventional reconstruction would be unable to regain the form and function.2 Until the end of 2017, 61 VCA programs were approved in the United States with 50 patients completing the surgery before 2014, over 100 upper extremity transplants and 30 face transplants have been performed worldwide.3,4

With the ever-increasing rates of both solid organ and VCA transplant, how to suppress the allograft rejection remains the central problem. The most common treatment after kidney transplantation, for example, is to use a life-long immunosuppressive regimen.

Immunosuppression is a crucial component in the prevention of immune-related allograft rejection, and it dramatically decreases acute rejection after a transplant.5 Common immunosuppression can be achieved in three ways: lymphocytes depletion, diverting lymphocyte traffic and blocking the lymphocyte response pathways.6 The introduction of immunosuppressants in the 1990s such as cyclosporine Neoral, tacrolimus, and mycophenolate mofetil has reduced the incidence of acute rejection episodes during the first year after solid organ transplantation. Unfortunately, these agents not only fail to demonstrate efficacy on long-term graft survival or the prevalence of chronic rejection,

1 but also fail to prevent acute rejections. Acute rejection is common in solid organ transplants, with 50-70% patients experience at least one episode, and due to the complex tissue types in a VCA transplant, as high as 85% of all VCA patients experience one or more episodes of acute rejection in the first year.7,8,9,10 The commonly used immunosuppressants are all non-specific, and each has its agent-specific side effects, for instance, studies have identified the usage of immunosuppressants as a risk factor for high cardiovascular morbidity, diabetes onset, nephrotoxicity, hypertension, increased infectious, and cancer risks following a liver transplant.11,12

An alternative treatment to increase graft survival is through bone marrow transplantation. The original purpose for bone marrow transplantation was to provide the recipient with a functional stem cell population to aid a defected immune system.13

However, a broader application of donor bone marrow was adopted in order to accompany a solid organ or VCA transplant; the donor bone marrow is transplanted into the recipient with the expectation to derive donor-specific immune cells and thus induce tolerance to the graft. Mixed chimerism, a phenomenon observed in bone marrow recipients, refers to the co-existence of cells from two different subjects in one body is called chimerism.14 Induction of mixed chimerism includes but not limited to the use of cyclophosphamide, anti-CD2 mAb, TBI, and hATG.15 In a kidney transplant model, transient mixed chimerism could promote solid organ allograft tolerance, although durable mixed chimerism can be observed in almost half of HLA-matched bone marrow transplants, where the grafts are tolerated without any immunosuppressants.16,15

Nonetheless, bone marrow transplantation has its problem: the graft-versus-host disease

(GVHD). GVHD can be observed in 30-70 percent of bone marrow recipients when the

2 donor lymphoid cells actively conduct an immunologic assault against host target organs, and the consequences of GVHD can result in infection and affect graft function.17

In an effort to induce peripheral tolerance, several mechanisms were extensively studied.

Fas ligand (FasL)’s interaction with Fas receptor on T cells is the main mechanism for cell apoptosis. Since naïve T cells do not express Fas receptors, activated T cells with upregulated Fas receptors are extremely susceptible to FasL-induced apoptosis, therefore administering FasL together with alloantigen in nanoparticles can induce systematic cell death in the alloreactive T cell population.18 This peripheral tolerance induction was proven effective with a single protein antigen and induced total depletion of reactive

CD8+ T cells. However, it has not been tested in the transplantation model, and the limitation is obvious: there are simply too many donor-specific antigens to be encapsulated in nanoparticles. Other models such as pancreatic islet transplant have used hydrogel to mediate immune responses. Veiseh et al. reported the use of a hydrogel capsule could abrogate the foreign-body response for up to 180 days in pancreatic islet transplant. This technique seems promising when done on a micro-scale and optimized for insulin and nutrient exchange, but can encounter difficulties when scaling up for a hand transplant where vessels and nerves need to be connected, thus unable to encapsulate the whole graft within.19

T cell costimulation blockade treatment has been trending in transplantation since the

1900s. T cells are activated by the engagement of both T cell receptor and costimulatory molecules CD28; the lack of either signal is sufficient to prevent T cell activation.

Cytotoxic T-lymphocyte-associated-antigen 4 (CTLA4)-Ig binds CTLA4, a molecule similar to CD28 but only present on activated T cells. By binding CTLA4, the CD28

3 pathway is blocked, and an inhibitory signal is transmitted to terminate the immune response. CD40 ligands are considered early T cell activation markers, by targeting

CD40L on T cells, the antigen-specific response can be weakened and thus delay allograft rejections.20 CTLA4-Ig together with anti-CD40L monoclonal antibody is recognized as one form of costimulation blockade that is proven to be useful in the prevention of acute rejection after transplantation.21,22,23,24,25,26 However, CTLA4-Ig is incapable of long-term tolerance induction by itself in primates, and like other immunosuppressants, the long-term administration of anti-CD40L could show severe side-effects and lead to thromboembolic complications both in monkeys and human.27,28

Short-term administration of such form of costimulation blockade has reversible side- effects but is not sufficient to provide long-term protection to the graft alone.

Although immunosuppressant, bone marrow transplantation and peripheral tolerance induction are the most popular transplant treatments, new options need to be investigated to overcome the limitations and undesired side-effects of commonly considered approaches.

T Cell Development

The rejection of a VCA graft, similar to solid organ rejection, can be attributed to antigen recognition and alloantigen-specific T-cell activation, a function of both innate and adaptive immune responses.9 T cells are lymphocytes that are essential for cell-mediated adaptive immunity and have the ability to positively or negatively control other cells involved in immune responses.29 In a T-cell mediated transplant rejection, effector T cells infiltrate the graft, recognize either the donor MHC (direct recognition) or donor peptide presented on recipient MHC (indirect recognition), activate other immune cells through

4 increasing chemokine and cytokine production, and ultimately facilitate the deterioration of graft function.30,6 Therefore, T cells have commonly been the target of immunosuppressants to protect the graft and the importance of T cells in allograft rejection cannot be overstated.

Early Stage T Cell Development

Hematopoietic stem cell in the bone marrow give rise common myeloid progenitors and common lymphoid progenitors, which then produce T cells, B cells and NK cells depending on the commitment to different cell lineages under different environments within the thymus. T cells are educated to no react against “self” via thymic selection; such tolerance is achieved through deletion of autoreactive T cells or conversion of

autoreactive cells into regulatory T cells

(Treg).31 More specifically, the lymphocyte

progenitors migrate through the blood to

the thymus, where the heterodimeric T cell

receptors (TCR) undergo somatic gene

rearrangement, and the cells go through so-

called positive selection and negative

selection. When the lymphocyte Figure 1 A model for cTEC contributions to T cell development.44 progenitors first arrive in the thymus, they lack most of the surface cell markers compared to a mature T cell, and are given the name

“thymocytes.” At this stage, the progenitors still possess the ability to differentiate into B cells or NK cells if they exit from the thymus. The CD4-CD8- double negative thymocytes migrate across the cortex, enter the thymic corticomedullary junctions where

5 they encounter the stroma and initiate the T-cell lineage differentiation and given the first

T-cell lineage marker in mouse model: Thy1. A minor population of T cell that express

γ:δ T-cell receptor (TCR) may exit into the periphery while the majority of the T cell population with α:β TCR goes through additional four steps of differentiation called

DN1, DN2, DN3, and DN4 based on the expression of adhesion molecule CD44, CD25, and Kit, shown in Figure 1. During the development of double negative thymocytes, the stromal cells produce IL7 as a survival signal to thymocytes with IL7 receptors. After T cell progenitors have successfully rearranged the TCR genes, they are differentiated into

CD4+CD8+ double positive in the thymic cortex and interact with MHC molecules presented on the cortical thymic epithelial cells (cTEC) for the selection of CD4+CD8- or

CD4-CD8+ single positive cells based on the recognition of self MHC-peptide-restricted

TCR.32 The single positive thymocytes gain the expression of chemokine receptor CCR7 and migrate inversely across the cortex to the medulla region, where the medullary thymic epithelial cells (mTECs) produce CCR7 ligand to attract the cells for negative selection.

Late Stage T Cell Development

During negative selection, thymocytes get selected via interaction with mTECs which present tissue-specific antigen to the cells. T cell precursors that have high affinity to these self-antigens are deleted while the rest of the population survive, and mature, into self-tolerant T cells. mTECs do not perform negative selection alone. Antigen-presenting cells (APCs) that are present in the periphery are also extensively spread throughout the thymus. APCs such as dendritic cells and macrophages present a target of possible autoimmune responses to single positive T cells that need to be eliminated. Mature CD4+

6 or CD8+ single positive T cells that have gone through multiple layers of positive and negative selections then exit the thymus and migrate to secondary lymphoid organs such as lymph nodes, where they can be activated and proliferate to eliminate infection.33

Mature T cells that exit the thymus can be either CD4+ or CD8+ single positive, the two types of cell have separate functions. CD4+ T cells recognize antigens presented by class

II MHC molecule and function mainly as helper cells, while CD8+ T cells identify class I

MHC and have cytolytic effector functions.34 Subcategories of CD4+ cells include helper

T (Th) 1, Th2, and Th17, whereas CD8+ T cells lineage is categorized into cytotoxic effector cell and memory cells. Almost every cell in the body presents class I molecule, which gives CD8+ cytotoxic cells power to recognize allospecific cells and destroy cells of a foreign origin in the transplant. Despite different functions, CD4+ and CD8+ T cells still communicate with each other after their departure from the thymus.

T Cell Activation

T cells recognize antigen through T cell receptors on the surface of the cells, commonly termed Signal 1. Once an antigen binds to TCR, the TCR and CD3 chain together can trigger the activation cascade.34 One of the earliest activation-induced surface molecules on T cells is CD40L. CD40L interacts with CD40 presented on antigen-presenting cells, such as dendritic cells and macrophages, to trigger upregulation of CD80 and CD86 on the surface that binds to CD28 or CTLA4 on T cells. Such interaction promotes the appearance of what is commonly termed Signal 2.35 Blocking CD40/CD40L pathway results in the inhibition of CD80 and CD86 expression, promoting a condition when an antigen triggers Signal 1 without Signal 2, resulting in an unresponsive state of T cells— anergy. The usage of anti-CD40L monoclonal antibodies or CTLA4-Ig to block either

7

CD40L or CTLA4 on T cell surface results in a temporary unresponsive state in T cells and have been successfully used in animal transplant models to prevent graft rejection.

Regulatory T Cells

After negative selection in the thymus, the majority of self-reactive T cells are removed due to autoreactivity. However, due to the stochastic nature of interactions, this process is not complete. Moreover, the remaining T cells with low avidity to self-antigen may still be activated by self-antigen and proliferate in the periphery. Regulatory T cells (Tregs), a special T cell population that constitute 5-15% of all T cells in the periphery, regulate and suppress these autoreactive cells.36

Tregs are autoreactive CD4+T cells that express the CD25 surface marker and suppress the activation of Ths and the release of cytokines through multiple different mechanisms.

Thymic Tregs (tTregs) in particular, are cells that recognize self-antigens and are induced in the thymus in either autoimmune regulator (AIRE)-dependent or independent fashion.30 Tregs can also be induced in the periphery (iTregs) after autoreactive T cells have escaped the central tolerance. Evidence showed that early infusion of Tregs helped reduce GVHD in bone marrow transplant patients, and effort has been made to induce tolerance by inducing Tregs in the periphery with immature allogeneic APCs.37,38,39

Compared to Ths that can be activated and expanded, Treg’s default state is anergic and non-proliferative.40

Transplant rejection is dependent on the expansion of high avidity allospecific T cells,30 and the maturation of high avidity allospecific T cells is associated with the skewed allospecific TCR sequence that favors binding to allospecific peptide-MHCs. Under the

8 appropriate environment, the high avidity CD4+ T cells may become anergic, upregulate

FoxP3 transcription factor, become Tregs, and gain the ability to suppress the activation of naïve T cells.41 Therefore, if the thymic microenvironment can be engineered in a way to either reduce the alloreactive TCR frequency or induce regulatory T cells to prevent the expansion of these high avidity T cell population after a solid organ or VCA transplant, lasting transplant tolerance could be achieved.

Thymic Epithelial Cells

The thymus is a primary lymphoid organ with a key function to enforce tolerance to self by discrimination between self-antigens and non-self-antigens. The thymus has spatially distinct lobules, including the outer thymocyte-dense cortical regions, inner thymocyte- sparse medullary regions and corticomedullary junctions (CMJ), each has its unique microenvironment. The thymus is organized in a way so that a subset of cells such as thymic epithelial cells (TECs) can participate in and regulate maturation of thymocytes.32

Within the network of epithelia known as the stromal population of the thymus, TECs play an essential role in the thymic selection of T cells, contributing to what is referred to as central tolerance. TECs, together with the thymic microenvironment, support both negative and positive selection through the interaction of MHC molecules on the epithelial cells with the TCR of the developing T cell.42 Some important molecules expressed in TECs include IL-7, DLL-4, β5t, autoimmune regulator, CCR7 ligand chemokines, and self-peptide-presenting MHC molecules, all of which play a role in T cell selection and development.43 With the demonstrated plasticity of thymocytes, TECs are able to fine-tune the microenvironment in the thymus and thus shift the TCR repertoire.29

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cTEC

The thymus induces T cell generation from the lymphoid progenitors in the cortical region of the thymus, and the most important cell population within the thymic cortex is cortical thymic epithelial cells (cTECs).44 cTECs are a network of cells originated from endodermal epithelial progenitors, localized in the cortical region of the thymus, they selectively induce survival in only the low-affinity TCR arrangement during positive selection.44,43 There are two populations of cTECs: MHC II high cTECs and MHC II low cTECs, both of which are important for the development of MHC-restricted T cells and the positive selection.45

T cell lineage commitment depends on Notch 1 receptor-mediated signaling.46 Although expressed by both cTECs and mTECs, Delta-like 4 ligands (DLL4) expressed on cTEC is indispensable for Notch1-dependent thymopoiesis, such expression is one of the ways cTEC influences T cell development.47 Aside from the expression of DLL4, the thymoproteasome subunit β5t that facilitates the development of CD8+ T cells is known to be exclusively expressed by cTEC. The function of thymoproteason subunit

β5t is to catalyze the generation of self-peptide that feeds into class I MHC, therefore the lack of β5t would block a major path of T cell development.48

Interleukin 7 (IL7) is critical for early T cell development. Although both cTECs and mTECs display traits of IL7, cTECs are shown to express a higher level IL7 than mTEC and the IL7-high subpopulation are localized in the CMJ.49 Comparable to IL7,

Kit ligand, a stem cell factor receptor, is a cytokine expressed by both the cTECs and mTECs, but highly expressed in cTECs to promote the survival and proliferation of immature thymocytes.50 Some key chemokines expressed by TECs but less relevant to

10 this topic are CXCR4 ligands that help to localize lymphoid progenitors to the thymic cortex and CCR7 ligand that is responsible for the migration of positively selected thymocyte from cortex to the medulla.51,52 cTECs also produce some other cytokines such as CCR9 ligands that instruct the colonization of lymphoid progenitors prenatally, which are not relevant to the scope of this study and thus will not be discussed.

Interestingly, autophagy is prevalent among TECs even though the conventional autophagy is caused by deficient nutrient. Nevertheless, this process helps cTECs in class II MHC peptide presentation and is an unusual phenomenon that can rarely be observed anywhere outside of the thymus.43

mTEC

Autoimmune regulator (AIRE) is a vital transcription regulator within the thymic medulla. AIRE regulates medullary thymic epithelial cells (mTECs) on the ectopic expression of tissue-specific-antigen, induces controlled apoptosis in autoreactive T cells and promotes the production of regulatory T cells to maintain immune tolerance.53,54

TECs have been the target of cell therapies to treat immunity related disorders due to the unique property of mTEC to present tissue-specific self-antigens. mTECs can be characterized into two subpopulations: one carries AIRE and functions as self-antigen producing cells, the other bares CCR7 ligand and its function is to attract thymocytes.

While the thymic cortex provides the proper microenvironment for immature T cell development and positive selection, mTECs are critical in developing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire.44,55

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Besides the developing lymphocytes, two other major types of cells reside within the thymic medulla: dendritic cells (DCs) and macrophages of bone marrow origin. Since there is a differential distribution of DCs and macrophages in the medulla compared to the thymic cortex, we have a reason to believe that these antigen presenting cells cooperate with the mTECs in antigen presentation and negative selection. The presence of DCs and macrophages are necessary for late-stage T cell development in the thymic medulla.

Antigen Presenting Cells

Out of the two classic types of antigen presenting cell (APCs), macrophages and dendritic cells (DCs), DCs are the more potent cell type in our bodies. The DC network is involved in immune surveillance as well as antigen capture, processing, and presentation to T cells.56 As mentioned in the T cell development section, APCs assist

TECs in the thymus during positive and negative selections. Peripheral DCs are specialized in activation of both CD4+ and CD8+ T cells despite their different functions. To activate CD8+ T cells, APCs process antigen by degrading cytosolic proteins with the help of proteasomes before presenting in class I MHC molecules. In the thymus, cTEC-specific β5t subunit catalyzes thymoproteosome, a type of proteasome, in antigen processing and therefore helping the development of CD8+ T cells, as previously mentioned. APCs can also process extracellular pathogens, and presented antigens on class II MHCs to activate CD4+ T helpers cell, initiate the cytokine secretion and start an immune cell activation cascade. During thymic selection, the influx of peripheral DCs present blood-born antigen or organ-specific antigens to the thymic medulla, helping to complete the thymic selection.57

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To summarize, the involvement of APCs in T cell development and immunoregulation cannot be underestimated. Therefore, having donor bone-marrow-derived APCs to work in concert with donor TECs would result in ideal immunotolerance in a VCA transplant model.

Purpose of the Study

TECs are undeniably one of the most important cells in central tolerance. Realizing the importance of TECs, we hypothesized that the careful manipulation of this cell population by engineering a hybrid thymus that consists of TECs from both the donor and recipient origin would work synergetically with donor bone marrow-derived antigen presenting cells, result in transplant tolerance with little to no side-effects.

Studies have shown that in an MHC-mismatched donor-recipient pair, alloreactive T cells require engraftment of donor-type DCs to expand donor-type Tregs, which encourages tolerogenic properties of host-type DCs that leads to expansion of host-type Tregs, resulting in peripheral tolerance.58 DCs are essential for mTEC to perform negative selection and controls the late stage of T cell development. Evidence supports the idea of a hybrid thymus treatment in a VCA recipient, due to the viable bone marrow from a donor limb can derive donor-type DCs that aids the expansion of both donor and host- type Tregs.59

In this study, the feasibility of this donor-specific central tolerance induction in a VCA transplant model is evaluated in several aspects: 1, what is the necessary treatment for donor TEC to engraft and how long does the protective effect last, 2, is there any unexpected adverse effect of donor TEC on the recipient, 3, can this mouse model be

13 easily translated into another animal, or even human, models. Through re-engineering the recipient thymic microenvironment with donor TEC engraftment, and the help of donor dendritic cells derived from the VCA bone marrow, we expect to create a hybrid thymus model that is easily translatable and have prolonged protective effect compared to other immuno-modulatory treatment used in VCA transplantation.

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Optimal Donor TEC Processing

Animals Used

A C57BL/6 transgenic mouse model with constitutive expression of firefly luciferase

(B6luc;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J, stock number: 025854), were purchased from the Jackson Laboratory and setup into breeding pairs in the Johns

Hopkins Miller Research Building animal facility. B6luc animals produced by B6luc breeding pairs were euthanized between 8 to 12 days after birth and their thymi were carefully dissected out for TEC processing with IACUC approval.

Method

Digestion

Papain enzymes were activated in activation buffer of 0.24% β-Mercaptoethanol, 0.44%

EDTA, and 2% L-Cysteine in cell-culture-grade water for 30 minutes, and frozen in single-use aliquots prior to use. Donor thymi were carefully harvested after euthanizing a litter of B6luc animals between 8 and 12 days after birth. Each thymic lobe was cut into two pieces and kept in 5 ml plain RPMI-1640 or DMEM/F12 medium in a 15 ml Falcon tube at room temperature or on ice. The thymic pieces were gently rocked in the medium under the solution was cloudy, aspirate the cloudy supernatant after letting the pieces rest and sink to the bottom. Such process was repeated 2-4 times until the supernatant was clear, then the thymic pieces were transferred to a well with 2 ml plain RPMI-1640 or

DMEM/F12 medium in a 12-well plate. In a separate well, 1 ml digestion buffer and 1 ml extra digestion buffer was prepared for every three additional thymi. Different compositions of digestion buffer were tested. Composition 1: 1mg/ml activated papain,

15

0.1% BSA, 0.1mg/ml DNase I, 0.25 mg/ml Collagenase IV in either RPMI-1640 or

DMEM/F12 medium. Composition 2: 0.5mg/ml activated papain, 1mg/ml DNase I, 0.25 mg/ml Collagenase IV. The outcome of these two buffer compositions can be found in the result section. The thymic pieces were transferred into the well with digestion buffer and incubated in the 37℃ incubator for 30 minutes. During the 30-minute-incubation, the

12-well plate was taken out of the incubator every 10 minutes so the thymic pieces could be pipetted and well mixed with the digestion buffer; p1000 pipettes with tips cut off were used for pipetting the thymic pieces for best results. 2 ml MACS buffer was added to the well by the end of the 30-minute-incubation to stop the digestion, an additional 3 ml MACS buffer was used to wash the well. After the 3 ml mixture was strained through

70 um cell strainer into either a 15 ml or 50 ml Falcon tube, the wash buffer was also strained through the strainer into the same tube. The centrifuge was kept open and allowed to reach room temperature before using. The collected cells were centrifuged at

300 g at room temperature for 5 minutes while the centrifuge temperature was set to 4℃, allowing the temperature to gradually decrease as the cells are being spun down.

Negative Selection

Panning negative selection can be used immediately following the thymic digestion.

However, the panning plates need to be prepared 12 hours before using. Sterile 100 mm diameter Petri dishes were coated evenly with 6 ml of NaHCO3 solution with either 1:100 dilution of anti-rat IgG or 1:250 dilution of goat anti-mouse IgG antibody and stored at

4℃ overnight. The plates were washed with 5 ml 1x PBS twice before cells were plated.

The cells were resuspended in plain RPMI-1640 medium at a concentration of 2*108 cells/ml, either 1:300 rat anti-mouse CD90 and 1:400 rat anti-mouse or 1:200 mouse anti-

16 mouse CD90.1 antibodies were added to the cell suspension. After incubating at 4℃for

20 minutes, the cells were washed with up to 10 ml of RPMI-1640 and resuspended in 6 ml RPMI-1640 for every 1*108 cells prior to plating on the pre-coated plates. The plates were left incubating on a level surface at 4℃for 30 minutes before gently washed twice with 5 ml MACS buffer and transferred to a 50 ml Falcon tube.

Density Gradient Selection

Right after thymic digestion, washing buffer with 1mg/ml BSA and 2 mM EDTA in PBS was prepared. 3 different density solutions were made by mixing the washing buffer and

1.32 g/ml OptiPrep: 9.6 ml washing buffer + 2.4 ml OptiPrep to make 1.064 g/ml solution, 9.375 ml washing buffer + 2.625 ml OptiPrep to make 1.07 g/ml solution, and 9 ml washing buffer + 3 ml OptiPrep to make 1.08 g/ml solution. The recovered thymic cells were resuspended homogenously in 20 ml plain RPMI-1640 medium with 2.5 ml washing buffer in a 50 ml Falcon tube. Each condition received the appropriate10 ml density solution by pipetting the solution directly to the bottom of the tube to avoid mixing between solutions; then the tubes are gently transferred into the centrifuge to be spun at 600g, at room temperature with deceleration set to 0, for 20 minutes. After centrifugation, the tubes were taken out of the centrifuge with minimal disturbance, and the top layer of liquid together with the interface was removed from the tube and transferred to a new 50 ml Falcon tube. The cells were washed with up to 30 ml of washing buffer then centrifuged at 400 g for 6 minutes, with deceleration set back to maximum.

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Positive Selection

Once the thymic pieces were fully digested and strained into single cells suspension, the cells were resuspended at a concentration of 5*108 cells/ml in PBS with 2% FBS.

EpCAM-PE antibodies were added at a 1:200 dilution then incubated on the fridge door for 15 minutes to reduce trauma to the cell due to sudden temperature drop. After incubation, the cells were washed with up to 10 ml of PBS then centrifuged at 300g, at

4℃ for 5 minutes. Resuspend again in a concentration of 5*108 cells/ml in PBS with 2%

FBS, and add anti-PE microbeads at 1:40 dilution, incubate at 4℃ for 15 minutes. Wash and centrifuge the cells as described previously, then cells were resuspended in 1 ml PBS with 2% FBS and sent to AutoMACS machine, where they were positively selected under sensitive mode.

FACS Sorting

Upon recovering the cells from enrichment, the single cell suspension was then stained with EpCAM antibody and CD45 antibody in the color PE and PerCp, respectively. The cells were strained through 70 μm strainer again and resuspended in sorting buffer after antibody staining to make a final concentration of 3*107 cells/ml. Sorting buffer was made with 25 mM HEPES, 1.25 mM EDTA, 0.1% FBS and 0.1 mg/ml DNase I in PBS.

Cells were gated on CD45-EpCAM+ population and sorted using 100 μm nozzle at 3000

± 1000 events/second. The sorted TECs were collected in 5 ml collection buffer with

30% FBS in plain RPMI-1640 or plain DMEM/F12.

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Material and Reagents

L-Cysteine-HCl (catalog: C7477-25G, Sigma), EDTA (catalog: 46-034-CL, Cellgro),

55mM β-Mercaptoethanol (catalog: 21985-023), DMEM/F12 (catalog: 11330-032, Life technologies), RPMI-1640 (catalog: 112-301-101, Quality Biological). Lyophilized papain (catalog: LS003119), Deoxyribonuclease I (catalog: LS002058), and Collagenase

IV (catalog: LS004188) from Worthington Biochemical Corporation. OptiPrep (catalog:

07820, Stemcell Technologies), Sodium bicarbonate (catalog: S5761-500G, Sigma),

HEPES (catatlog: 15630-080, Life Technologies), FBS (Atlanta),10% PBS (catalog: 46-

013-CM, Corning), BSA (catalog: 9048-46-8, US Biological) Anti-PE Microbeads

(catalog: 130-048-801, Miltenyi Biotec)

Sterile straight forceps, curved forceps, and scissors. Pipettes and pipette tips, 15 ml

Falcon tubes, sterile 100 mm petri dishes (catalog: FB0875712, Fisher Scientific), Sterile non-treated 12-well cell culture plate (Nest Biotechnology), 70 μm cell strainer.

Purified anti-CD16/32 (clone: 2.4G2), purified mouse anti-mouse CD45 (clone: 30-F11) purified rat anti-mouse CD45.1 (clone:), purified rat anti-mouse CD90 (clone: G7) from

BD Biosciences. Anti-Mo/Rat CD90.1 (clone: HIS51) and eF450-conjugated Streptavidin from eBiosciences. Phycoerythrin (PE)-conjugated anti-EpCAM (clone: G8.8), PerCP-

Cy5.5-conjugated anti-CD45 (clone: 30-F11) and unconjugated goat anti-mouse IgG

Highly Cross-Adsorbed Secondary Antibody (catalog: 16068) from Invitrogen,

Biotinylated Ulex Europaeus Agglutinin 1 (catalog: B-1065, Vector) and purified anti-rat

IgG.

19

Results

The thymic digestion protocol has been revised many times since its adaptation from

Serwold’s Lab, Joslin Diabetes Center.60 Upon optimization, this protocol does not need to be changed between different strains of mice and therefore produces consistent reliable results. This digestion protocol has the potential to be modified to accommodate other animal models or be used for a different application.

Some of the factors that may affect A B the release of TEC include: 1, the

usage of RPMI-1640 or DMEM/F12

medium, 2, digestion buffer

composition, and 3, the speed of

Figure 2 A flow cytometry analysis of the effect of different temperature transition. With digestion buffer compositions. Cells were on CD45-EpCAM+ TEC cell. A, thymus digested in composition 1 digestion buffer with 1mg/ml activated papain, 0.1% BSA, 0.1mg/ml DNase I concerns regarding the viability of and 0.25 mg/ml Collagenase IV. The yield of TEC is 0.18%. B, thymus digested in composition 2 digestion buffer with TECs in different medium, 0.5mg/ml activated papain, 1mg/ml DNase I and 0.25 mg/ml Collagenase IV. The yield of TEC is 0.35%. DMEM/F12 medium replaced

RPMI-1640 during thymic digestion based on the data shown in Figure 2.

We desire a high yield of TEC since

a higher yield in the initial digestion

step can affect the yield of TEC after Figure 3 A flow cytometry analysis of the effect of different medium. Digested thymic cells with gating on CD45-EpCAM+ cell population to identify the percentage of TEC. A, thymus enrichment. Digestion buffer digested in DMEM/F12 medium using composition 1 digestion buffer. The yield is 0.59% TEC. B, thymus digestion in RPMI- 1640 medium. The yield is 0.34% TEC. compositions were also compared as shown in Figure 3, where composition 2 has a noticeable advantage over composition 1

20 and releases more TEC in total. The third important factor that affects the recovery of A B C

Figure 4 Cell aggregates formed due to thermic shock. A, an example of a sponge-textured cell aggregate after sudden temperature drop when the cells transit from 37℃ to 4℃ in 5 minutes. B, a normal thymic digestion without cell aggregate yields 0.48% TEC. C, a thymic digestion where cell aggregate, such as shown in Figure 4A, appears, yields 0.096% TEC.

TEC is the pace of temperature transition. We have observed that TECs are sensitive to thermic shock, a decrease of temperature from 37oC to 4 oC in 5 minutes is directly correlated to over 50% cell death, especially in thymi harvested from animals younger than 15 days old. In an environment with many dead cells, centrifugation creates a spongy cell aggregate in the cell pellet and reaggregates even after disaggregation and straining through a 70 μm cell strainer. Demonstrated in Figure 4 is evidence of cell aggregate decreasing the yield of TEC to 1/5 of average yield. Hence, the optimized digestion protocol prioritizes a smoother and much slower temperature transition from

37℃ to 4℃ to obtain alive and healthy TECs.

The idea of a negative selection is to remove cell population with surface markers that are not presented on the target cell, TECs. The commonly displayed markers on cTEC are

CD205, CD249 (Ly 51) and CD326 (EpCAM), and mTEC markers include EpCAM, keratin 5, keratin 14, MTS-10, ER-TR5, Aire and UEA. It is known that TECs do not express CD90 or the strain-specific CD90.1 in C57BL/6; therefore, capturing cells that are stained with CD90 and restraining them on the plate should theoretically remove only non-TEC cells.61 In reality, TECs are a type of epithelium and consequently retains the

21

A B sticky property that’s

common for epithelial cells,

resulting in either a massive

loss in TECs if the panning C D E plates were washed gently,

or poor enrichment result if

the panning plates were

Figure 5 Comparison between different panning strategies. A, 9.3*107 washed vigorously. Figure digested thymic cells with 0.59% TEC yield. B, 1.5*107 thymic cells recovered from a one-step panning of cells from Figure 5A, washed gently, 5 reveals that, if the plates yields 1.87% TEC. C, 1.2*108 digested thymic cells with 0.51% TEC yield. D, 6.4*107 thymic cells recovered from a one-step panning of cells from Figure 5C, washed vigorously, yields 0.73% TEC. E, 3.3*107 thymic cells are washed gently, a single- recovered from a one-step panning of cells from Figure 5D (a two-step panning of cells from Figure 5C), washed vigorously, yields 1.13% TEC. step negative selection Thymic could enrich the cells three folds, but half of the TECs are lost. On the other hand, if the plates were washed vigorously, some of the non-TECs detaches from the plate together with TECs that results in less TEC loss but also in poor enrichment even with two-step panning. The negative panning enrichment is deemed as a non-practical way to enrich

TECs due to its technique-dependent nature, an example being one person’s gentle wash might be harsh for another, in addition to inadequate enrichment result.

Density gradient enrichment is not dependent on a cell’s immunological property, but on its physical density, which means, in T cell population for example, unhealthy, dying cells with higher density will be selected differently from their healthy counterpart. In theory, cells with higher density will collect at the bottom of the tube whereas lower density cells collect on the interface between washing buffer and density solution. This method would be more reliable if applied to isolate a less heterogeneous group of cells;

22

A B however, due to the diversity observed within the TEC

population, we have a reason to

believe certain TECs are being

selected for, while others are being C D selected against. Flow cytometry

analysis in Figure 6 shows that

although 1.068 g/ml density

solution is satisfactory in enriching

Figure 6 TEC yield after using different density. A, 8.5*107 TECs approximately ten folds, digested thymic cells for each condition, the starting percentage of TEC is 0.42%. B, 6.5*105 cells with 6.32% TEC recovered from almost half of the TECs was lost. the enrichment with 1.064 g/ml solution. C, 3.4*106 cells with 1.24% TEC recovered from the enrichment with 1.07 /ml solution. D, 5.8*107 cells with 0.21% TEC recovered from Interestingly, we detected a drop in enrichment with 1.08 g/ml solution. TEC percentage in the 1.08 g/ml

density enrichment group, leaving us to wonder if some TECs are denser than 1.08 g/ml but other TECs are less dense than 1.064 g/ml. If our assumption is true, this could create a bias against either cTEC or mTEC, or even a subpopulation of cTEC or mTEC that we have previously not noticed. With our goal to recover the whole TEC population in mind, the density gradient enrichment did not satisfy the requirement of our goal to isolate the entire TEC population. We then quickly ruled out the density gradient as an enrichment option for TEC preparation due to the significant loss in TEC after enrichment.

The final optimized thymic processing protocol consisted digestion, positive enrichment, and FACS sorting. Positive enrichment outperforms both negative enrichment and density enrichment in the percentage of TEC yield and maximum recovery of TECs. We

23

have been able to maintain a A steady level of TECs after

microbeads positive

enrichment such as shown in

Figure 7. Even though the

C specific production batch of

anti-PE microbeads is a

deciding factor for enrichment

result, as we have experienced

lately, a bad batch can enrich

Figure 7 The positive enrichment consistently yields around 10% TEC TECs well by running through in the population. A, 1.75*108 digested thymic cells with 0.31% TEC go through the positive enrichment process. B, 5*106 cells are recovered from the enrichment with 8.46% TEC. C, positive enrichment can AutoMACS machine twice produce up to 13.5% TEC with a single enrichment step. without losing any TECs.

A B C Figure 8 compares a single-

step enrichment and a two-step

enrichment with a less

effective batch of anti-PE Figure 8 The two-step positive enrichment could yield satisfactory percentage of TEC. A, 1.68*108 digested thymic cells with 0.11% TEC go through the positive enrichment process. B, 9.5*106 cells are microbeads. The high recovery recovered from the first-step enrichment with 3.39% TEC. C, 1.5*106 cells are recovered from the second-step enrichment with 21.5% TEC. of TEC after the second enrichment is valuable to the next step: FACS sorting.

FACS sorting is the final step of the thymic processing but is undeniably an important step that can impact the viability of the recovered TECs. As mentioned before, TECs are sensitive to thermic changes, and we have discovered that TECs are also sensitive to the

24 change in pressure during fast-speed sorting. Evidence of diminished signal from in vivo injected TECs suggests that the viability of recovered TECs decrease once the speed of sorting is over 4000 events per second. Cells that were sorted between 2000 and 3500 events per second recovers much better in vivo than those sorted at higher than 4000 events per second. Because the cells need to be sorted at a low speed, longer sorting time is needed to collect the desired amount of cells, and therefore a good enrichment is crucial before sorting to shorten the amount of time that cells spend in the unfriendly environment of the sorting buffer.

25

Donor TEC Engraftment

Animals Used

B6luc (B6;FVB-Ptprca Tg(CAG-luc,-GFP)L2G85Chco Thy1a/J, stock number: 025854) as donors were purchased from the Jackson Laboratory and breeding pairs were set up in the

Johns Hopkins Miller Research Building animal facility. 8 weeks old NCI CB6F1/Cre mice (strain code: 566), an F1 generation produced by C57BL/6 and BALB/c breeding pairs, were purchased from Charles River. 6 weeks old BALB/c mice and C57BL/6 mice were purchased from the Jackson Laboratory and grew in the Johns Hopkins Miller

Research Building animal facility until they reach 8-12 weeks for optimal intrathymic injections.

Method

TECs recovered from FACS sorting were centrifuged at 300 g, at 4 ℃ for 5 minutes, and resuspended in PBS at a concentration of 6.6*106 cells/ml. The recipient animals, F1, or

C57BL/6, or BALB/c, were intraperitoneally injected with 500 μl Avertin (2,2,2-

Tribromoethanol). An experienced microsurgeon in the lab performed intrathymic injection on the recipients by anesthesize the animals with avertin, opening the thoracic cavity and using a fine needle to push the cell suspension into each lobe in a timely fashion to ensure the survival of the animals. F1 animals received a various amount of

TECs ranging from 75,000 to 250,000 in either 15 μl or 25 μl PBS in each thymic lobe,

BALB/c and C57BL/6 animals received 100,000 TECs in 15 μl PBS in each thymic lobe.

BALB/cs were pretreated with 2mg anti-Thy1.2 with post-injection 500μg anti-CD40L and 500μg CTLA4-Ig on post-operative day (POD) 0, 500μg anti-CD40L and 250μg

26

CTLA4-Ig on POD 2, 4, and 6, or post-injection anti-CD40L and CTLA4-Ig alone, or received no treatment to observe rejection trend.

In vivo imaging system (IVIS) was used to examine the dynamics of donor TEC in the recipient. 200 μl of D-luciferin solution at 15mg/ml was injected in the recipient 10 minutes before anesthetizing the animal, and IVIS luminescence exposure time was set to

60 seconds. The total signal count from each animal was recorded longitudinally after gating on the thymus.

F1 animals were euthanized at determined time points, 6 to 10 weeks, following IVIS imaging; the thymi were carefully dissected, digested, and stained with indicated antibodies before analyzing via flow cytometry.

Material and Reagents

Anti-mouse Thy-1.2 (clone: 30H12, BioCell), anti-mouse CD40L (clone: MR1, BioCell),

Abatacept (CTLA4-Ig, Bristol-Myers Squibb), 2,2,2-Tribromoethanol (catalog: T48402-

25G, Aldrich Chemistry), XenoLight D-Luciferin Potassium Salt (catalog: 122799,

PerkinElmer)

Results

Using the F1 animals where no rejection response against B6luc is observed, we studied the optimal engraftment condition for TECs and concluded that 100,000 TECs injected in

15 μl PBS in each thymic lobe is the ideal condition for TECs to survive. If the thymic lobes receive less than 100,000 cells, the luminescent signal is not as strong, but the signal strength does not get any higher if each thymic lobe receives more than 100,000 cells, as shown in Figure 9. This could be explained by the spatial limitation in the

27 thymus that only permits the engraftment and growth of 100,000 cells. The age of the

POD 2 POD 5 POD 7 POW 2 POW 3 POW 4 POW 5 donor is also important A to the engraftment of the

B cells. Donor TECs older

C than 20 days could not

survive in the recipient D microenvironment, and E by post-operative day Figure 9 Conditions for optimal engraftment. Less than 15 days old donor B6luc cells were sorted at different rates, and different amount of cells were injected (POD) 2 we observed in the recipient F1 animals. A, 250,000 TECs sorted at 6000 cells/second were injected in each thymic lobe in 25 μl PBS. B, 200,000 TECs sorted at 3000 cells/second were injected in each thymic lobe in 15 μl PBS. C, 75,000 TECs complete disappearance sorted at 3000 cells/second were injected in each thymic lobe in 15 μl PBS. D,E, 100,000 TECs sorted at 3000 cells/second were injected in each thymic of donor cells shown in lobe in 15 μl PBS.

POD 2 POD 5 Figure 10. Since the

A natural rejection of TEC takes over two weeks, this indicates that the cells are not rejected but simply

B unable to proliferate in the new environment.

Figure 10 TECs from donors older than 20 days old do not survive in the recipients. The purpose of the treatment regimen with anti- Conditions are otherwise identical to Figure 9D,E. A, B, 21 days old donor B6luc CD40L and CTLA4-Ig (costimulation blockade) cells were sorted at 3000 cells/second and 100,000 cells injected in each thymic lobe in 15 μl PBS. with or without anti-Thy1.2 T cell depletion is to protect the injected TECs for a short period before the newly exported lymphocytes are educated by the donor TECs. Although costimulation blockade can protect certain transplants for up to 16 weeks in some mouse strains, studies have reported that costimulation blockade treatment is effective in the periphery but not to the intrathymic allospecific T cells.62 Alternatively, the researchers suggested thymic irradiation as an

28 option to eliminate all allospecific T cells in the thymus to induce even longer tolerance.16 Not only is thymic irradiation traumatic for the recipient, the use of long-term A

B

C

Figure 11 Allogeneic TEC survival in BALB/c animals. BALB/c animals received 100,000 sorted B6luc TEC per thymic lobe and were left untreated or received the manipulation indicated below. Numbers under each image indicates the bioluminescent signal captured by IVIS. The signals were monitored on POD2, 5, and 7, then monitored weekly after post-operative week (POW)2, until no signal was detected in two consecutive weeks. A, 10-week-old BALB/c mice received 200,000 pure B6luc TEC, without any treatment. B, 10-week-old BALB/c mouse received 200,000 pure B6luc TEC and were also treated with Anti-Thy1.2 mAb on POD-1, CTLA4-Ig and anti-CD40L on POD0,2,4,6, all injections done intraperitoneally. C, 10-week-old BALB/c mouse received 200,000 pure B6luc TEC and were treated with CTLA4-Ig and anti-CD40L on POD0,2,4,6, all injections done intraperitoneally

29 anti-CD40L and CTLA4-Ig also display disappointing therapeutic effects in a human trial, and are associated with thromboembolic events.63 We have designed a more effective treatment with hybrid thymus in combination with costimulation blockade that brings less trauma to the recipient as well as eliminating the need for long-term immunosuppression. The addition of anti-Thy1.2 helps to prolong the effect of costimulation blockade by creating a transient depletion of T cells. Since anti-CD40L and

CTLA4-Ig work better in rodent models compared to in human, if the hybrid thymus treatment were to be used in a clinical setting, another short-course costimulation blockade treatment with better results in human might substitute the treatment used in the mouse model.

The thymus is an immune-privileged organ where cell-mediated rejection mechanism is different from the rest of the body and not well understood. Studies have reported that a hindlimb rejection first displays symptoms between POD 10 and POD 12, much shorter compared to the 3 to 6 weeks rejection time noted in the allogenic TEC intrathymic injection.64 Large variation in rejection time is observed among the six rejection control animals; we speculated that this is due to the exact location of the injection, some cells may have attached to a less-immunogenic region within the thymus, in contrast to others that have landed in unfavorable locations. The same reason could also explain the big variation between signal strength in the treated animals. Figure 11 compares the 6 subjects in the rejection control group with the 4 subjects from the costimulation blockade treatment group and 5 subjects from the ATG with costimulation blockade treatment group. The trendlines show that the ATG with costimulation blockade treatment group has promising graft protection and the engrafted donor TECs continue to

30 thrive in the thymic microenvironment even after the protective effect of the treatment wears off. With more subjects ongoing, we anticipate reaching a statistically significant

TEC survival time compared to the control animals and analyze the functions of donor

TEC in the recipient. A B C

The fundamental

question for a TEC

engraftment is how

Figure 12 Donor TEC percentage in congenic animals. TECs are gated based much of the donor on the CD45-EpCAM+ population. Donor TECs are identified as MHC-I Kd- Kb+ in the recipients. A, F1 recipient of 250,000 donor TECs per thymic lobe euthanized 11 weeks after the intrathymic injection. B, F1 recipient of 200,000 TECs survived in the donor TECs per thymic lobe euthanized 9 weeks after the intrathymic injection. C, F1 recipient of 100,000 donor TECs per thymi lobe euthanized 5 recipient thymus. The weeks after the intrathymic injection.

dynamics of donor

TECs can be analyzed with IVIS, however, IVIS signal does not entail the number of cells alive. The flow cytometry analysis in Figure 12 shows that the percentage of donor

TEC is low in the total TEC population, and only 2000 donor TECs are approximated to be alive in the recipient, a calculation based on 0.5% donor TEC in 0.17% total TEC from 200 million digested recipient cells. The low number of the surviving donor TECs is not necessarily discouraging if the small population have a physiological or functional impact on the recipient thymus, which is discussed in detail in the next section.

Nevertheless, to translate this model into a clinically relevant model, substrates promoting the proliferation of TEC should be co-administrated with TECs during the intrathymic injection to ensure the maximum effect of donor cells on the recipient microenvironment.

31

Donor TEC’s Interaction with the Recipient

Animals Used

NCI CB6F1/Cre mice (strain code: 566), BALB/c mice and C57BL/6 mice wild type or those received B6luc TEC intrathymic injections were used.

Method

F1 animals receiving either donor TECs or sham were euthanized. Their thymi were carefully harvested, digested, and the single thymic cell suspension was stained with

CD45, EpCAM, UEA, Streptavidin, Kd, and Kb in appropriate fluorophores.

Wild type C57BL/6 receiving B6luc TECs or nothing were euthanized. Their thymi were carefully harvested, digested, and the single thymic cell suspension was stained with

CD45, EpCAM, UEA. Flow cytometry analysis was done on the population gated on

CD45-EpCAM+ TECs.

Material and Reagents

Purified anti-CD16/32 (clone: 2.4G2, BD Biosciences). eF450-conjugated Streptavidin

(eBiosciences). Phycoerythrin (PE)-conjugated anti-EpCAM (clone: G8.8), PerCP-

Cy5.5-conjugated anti-CD45 (clone: 30-F11) from Invitrogen. Biotinylated Ulex

Europaeus Agglutinin 1 (catalog: B-1065, Vector). Pacific Blue anti-mouse H-2Kb

(clone: AF6-88.5, Biolegend), APC anti-mouse H-2Kd (clone: SF1-1.1, Biolegend)

32

Results

Aiming to find the impact of donor TECs on the recipient microenvironment, we have carefully harvested thymi from recipients of B6luc TECs and identified the cTEC and mTEC subpopulation with UEA staining. Figure 12 in the previous section stated that the

donor TECs only make up less than 1% of the

recipient TEC population. Therefore the

contribution of donor TEC to any change in

the recipient is not due to the changing

number of total TEC but due to its

manipulation in the recipient thymic

microenvironment. In the F1 recipients, we

have observed that young TECs helps

maintain the thymic shape compared to age- Figure 13 Long-term effect of donor TECs on the recipient TEC population. cTEC is characterized as EpCAM+UEA- cells, mTEC is characterized matched control animals, in agreement with as EpCAM+UEA+ cells. A. The thymus from a 20 weeks old F1 animal was harvested, digested and stained with surface antibodies. B. Two animals Kim et al., its molecular composition was then who previous received B6 luc TEC cells through intrathymic injection were sacrificed on week 10 examined by differentiating the UEA- cTECs and week 6 respectively after the injection, the thymi were digested and stained with surface 60 antibodies. Gated on recipient TEC population. C. from the UEA+ mTECs via flow cytometry. Donor B6 luc TEC cells were identified from the respective recipient in figure 3B. Gated on donor In Figure 13 we can observe a dramatic population. increase in the percentage of cTEC. Although some microscopic assessment is needed to confirm the finding, the increasing cTEC implies the donor TECs causes an architectural change in the recipient and instructs the expansion of thymic cortex. This effect is obvious shortly after the intrathymic injection. On day 7 after the intrathymic injection, a

33

A syngeneic C57BL/6 recipient is euthanized,

and we found evidence of trauma in the

thymus judged by the reduction in size. A

higher than normal percentage of TEC is B recovered in Figure 14B suggesting that the

reduction in the thymic size is caused by a loss

in a population other than TECs while the

total number of TEC experiences little impact C from such trauma. By POD14, the recipient

thymus returns to a similar size compared to

the age-matched control, but the shifted

D cTEC:mTEC ratio was still apparent. The

functional effect of an increased cTEC

percentage is unknown since most self-

tolerant education is performed in the thymic Figure 14 Short-term effect of donor TECs on the recipient TEC population. The cells are gated on CD45-EpCAM+ population as TECs. cTEC is medulla. characterized as UEA- cells, mTEC is characterized as UEA+ cells. A, A wild-type, age- matched C57BL/6 was euthanized. The thymus It is clear that at least 2000 donor TECs are was digested and stained, yielding 28.6% cTEC and 71.4% mTEC. B, On POD7 after the intrathymic injection, the C57BL/6 animal was firmly attached and integrated into the euthanized. The thymus was digested and stained, yielding 46.4% cTEC and 53.6% mTEC. C,D, recipient thymic architecture, or the non- POD14 after the intrathymic injection, two C57BL/6 animals were euthanized. The thymi were digested and stained, yielding 47.5% cTEC, integrated cells would be lost during the 52.5% mTEC, or 58% cTEC, 42% mTEC, respectively. preparatory steps before thymic digestion.

More visual confirmation is required to see if the donor TECs are evenly distributed or

remain in a cell aggregate in the injection site. In summary, we have confirmed that the

34 donor and recipient TECs communicate and the recipient thymic microenvironment could be altered by the donor TECs soon after the intrathymic injection. With the aid of modern technologies to reduce the surgical trauma from the intrathymic injection, this procedure is feasible, and even if the hybrid thymus cannot provide enough graft protection alone, it can be in combination with other long-term treatments to reduce the incidence of acute or chronic rejection.

35

Allogeneic TEC Survival in a Hindlimb Recipient Animals

B6luc were used and donor TECs, C57BL/6 wild type mice were used as the hindlimb donor. BALB/c animals were used as the hindlimb recipient.

Method

A total of four BALB/c animals between the age of 8 to 10 weeks received an orthotopic

C57BL/6 hindlimb transplant.65 On post-operative day 0, 2, 4, and 6, each hindlimb recipient received anti-CD40L and CTLA4-Ig treatment via intraperitoneal injection. On

POD7 or POD8 of the hindlimb surgery, each animal received an intrathymic injection of

200,000 sorted B6luc TECs. The hindlimb recipients were imaged under IVIS weekly for the first two weeks, then imaged bimonthly to avoid the frequent use of anesthesia.

Material and Reagent

Anti-mouse Thy-1.2 (clone: 30H12, BioCell), anti-mouse CD40L (clone: MR1, BioCell),

Abatacept (CTLA4-Ig, Bristol-Myers Squibb)

Results

One animal was lost shortly after the intrathymic injection, bringing the overall survival rate for combined hindlimb and intrathymic injection procedure to 75%. The cause of death was unknown but reasoned that the amount of trauma from the combined procedure might lead to the death of this animal, since no apparent bleeding was observed. We then moved the procedure to POD8 to separate the intrathymic injection from the hindlimb transplantation even more, with the expectation to lower the mortality rate in this

36 hindlimb transplant group. Out of the three animals that survived both procedures, only one graft was functional due to unexpected self-mutilation. Thus we were not able to tell the starting point of graft rejection. One remaining intact hindlimb was rejected at week two post-transplant, shown in Figure 15, the animal displays symptoms of severe erythema, swelling and formation of dry skin.66,67 With only one subject remaining, more subjects need to be tested in order to reach a conclusion on the rejection progression in a

C57BL/6 into BALB/c transplant model. If the short-course of CTLA4-Ig and anti-

CD40L proves to be non-effective in such a transplant model, we should consider the use of another short-course immunosuppression to protect the graft until donor TECs start exporting tolerogenic T cells.

37

Discussion

The procedure for donor TEC processing is still in its preliminary stage and is not ready to be translated into clinical use in the near future while the transition into another animal model is possible. However, the hybrid thymus concept could be promising. Studies in rodent models have shown intrathymic alloantigen grants long-term protection to an allotransplant graft.59 Donor TECs have more dynamic and more representative antigens compared to splenocytes, while Goss et al. has successfully induced donor tolerance with the injection of donor splenocytes along, the future of the hybrid thymus is not to be underestimated.

Major adjustments need to be made in our protocol for clinical relevance, such as the inclusion of older donors. In the United States, approximately 57% of deceased donors for organs or VCA grafts are over the age of 35.68 Since we have to exclude donors that have past the initial thymic development stage, more than half of the organ or VCA recipient will not be able to benefit from the hybrid thymus treatment. Some adjustment needs to be implemented due to anatomical differences between the mouse model and human, and the prolonged ischemic time bearing in mind that the thymus would come from a deceased donor. However, we have identified the key factor that predicts the yield of donor TEC in this procedure, which is the gradual thermic changes between each step.

With the appropriate modifications to the protocol, we can consistently recover healthy, alive donor cells that are suitable for injection.

A parallel approach to the same problem can be considered. Stem cell-derived TECs or harvested TEC in 3D culture can overcome the limitations of older deceased donors, but time has to be invested in developing an operational protocol for suitable cell culture

38 conditions. Boukamp et al. suggests a simplified 3D culture of TEC with human skin fibroblasts with thrombin and fibrinogen is sufficient enough to maintain TEC survival, proliferation and differentiation.69

Application of the hybrid thymus is not limited to one type of transplantation. In addition to being an immunotolerance treatment following a VCA transplant, it is also practical for the hybrid thymus to be used in a bone marrow transplant or a solid organ transplant.

TECs are susceptible to bone marrow transplant-conditioning-induced damage, and therefore the thymus is unable to produce T cells for an extended period after bone marrow transplant.70 Intrathymic injection of donor TEC into the bone marrow recipient could both help reduce GVHD and help regenerate normal TEC functions quickly after transplant to regain a functional immune system.

The short-course costimulation blockade treatment is a practical and reasonable component of the hybrid thymus treatment. The thymocytes can bear the distinctive T- cell lineage marker, Thy1, but remain in the thymus for weeks before they receive CD3,

CD4 or CD8 markers. The gradual expansion and development of T cells suggest the need for a temporary immunosuppressive treatment in the initial period after the intrathymic injection before TECs could have a systemic effect on the peripheral T cells.

This fact justifies the four administrations of costimulation blockade to inhibit peripheral

T cell function before donor TECs can educate and export tolerogenic T cells. The costimulation blockade with T cell depletion regimen protects donor TECs for up to 22 weeks, long-enough for the donor cells to recover from the shock and start modifying the recipient thymic microenvironment.

39

The thymic development lasts approximate 3-4 weeks in murine models, then once the murine animal reaches adulthood, the thymus stops expanding and the process known as thymic involution occurs. With the decreased production of T cells following thymic involution, the inverted ratio of cTEC versus mTEC that we observed following an intrathymic injection should help with the proliferation and development of immature thymocyte in the thymic cortex, exporting more tolerogenic T cells into the periphery and educating peripheral alloreactive T cells. According to Rode et al., involution is marked by the decline of cTEC, as cTEC supports early-stage T cell development and governs the overall lymphopoiesis, therefore we hypothesize an increased percentage of cTEC in injected animals states the treatment has regenerative properties and stimulates T cell production in the thymus.71

Another variability added by the change of TEC composition is the changing level of IL7 production. As mentioned in the introduction, IL7 is a crucial cytokine that determines the fate of early T cell development. Studies have described the phenomenon of decreased IL7 expression due to aging but have also suggested IL7 levels are dynamically regulated under different physiological states.72 This leads us to believe that a change in TEC composition due to physical damages dealt to the thymus following intrathymic injection may alter the level of IL7 production; the T cell populations are subsequently affected accordingly. Adding on top of the change in IL7,

DLL4 level may also be forced to change by the changing TEC composition. DLL4 level, similar to IL7 level, is a chief player that supports TECs on thymic selection but declines as mice age. Injecting foreign TECs into a system at equilibrium may break the existing balance and trigger a change in DLL4 level, influencing the whole T cell development

40 cascade. Although it is not yet defined if IL7 and DLL4 are upregulated or down- regulated post intrathymic injection, and if the shift in cTEC:mTEC ratio contributes to the changing level of IL7 and DLL4, this is indeed a subject that needs investigation.

The initial shock to the recipient thymus is an adverse effect that needs attention. Our finding of the initial decrease in thymic size and TEC signal matches the published literature. Rode et al. reported that the cTEC compartment of TEC does not recover from total ablation on POD8 but can mostly recover by POD14. Although not given total ablation of the cells, our intrathymic injection introduces trauma to the thymus, and it is possible that a large thymic population dies; in our experience, the thymus recovers completely by POD14, which is comparable to the result described by Rode et al.

In addition to the thymic trauma caused by the intrathymic injection, we have to consider that donor TECs may have an opposite effect than what we expected. Through unknown mechanisms, the TECs might educate the recipient T cells to recognize the graft instead of inducing tolerance. However, we have not collected enough data to either support or reject this idea.

It is known that growth factors such as fibroblast growth factor (FGF)-7, FGF-10, and insulin-like growth factor (IGF)-1 promote the proliferation of both cTECs and mTECs.

Regarding the current findings that a significant portion of donor TECs was lost in the initial 48 hours, and the fact that mTECs have a half-life of 2 to 3 weeks, maintaining a healthy and proliferative donor TEC population is our top priority. The next stage of the project should incorporate the use of these growth factors to support the engraftment and expansion of donor-specific TECs.

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In conclusion, the idea of a hybrid thymus has many obstacles to overcome before its adaptation into clinical use. The short-course costimulation blockade treatment has reversible side-effects and can be substituted with other short-course immunosuppressants. Most importantly, we have observed many impacts of donor TECs on the recipient thymic microenvironment but have not yet discovered any harmful effects. By adding more subjects to this study, we will be able to reach a conclusion on whether or not donor TECs have a positive impact on hindlimb graft survival.

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Curriculum Vitae

Jialu Wang, born in Beijing, China on April 19, 1994, came to the United States of

America in 2010 during her sophomore year of high school to pursue an education that fits her best interests. She attended Lehigh University in Bethlehem, PA from 2013 to

2017, where she majored in Integrated Degree of Engineering, Arts, and Sciences

(IDEAS) with concentrations in Bioengineering and Chemistry. She graduated from

Lehigh with a Bachelor of Science degree. She enrolled in the Biomedical Engineering master’s program at the Johns Hopkins University Whiting School of Engineering in

2017 and is determined to earn a thesis-track MSE degree. Jialu joined the Vascularized

Composite Allotransplantation lab in January 2018 and has been doing immunological research under Dr. Giorgio Raimondi since then.

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