CD24 IN T LYMPHOCYTE HOMEOSTATIC PROLIFERATION AND
AUTOIMMUNE DISEASE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Ou Li, Bachelor of Medicine
The Ohio State University 2005
Dissertation Committee:
Yang Liu, Ph.D., Advisor Approved By
Pan Zheng, M.D., Ph.D.
W. James Waldman, Ph.D. ______
Xuefeng Bai, M.D., Ph. D. Advisor
Jianxin Gao, M.D., Ph.D. Department of Pathology
ABSTRACT
In this thesis, we show that CD24 can regulate naïve T cell homeostatic proliferation and autoimmune disease. The activation of T lymphocyte requires two signals from antigen presenting cells, peptide and self-MHC complex to TCR and co stimulatory signals. CD24, a glycosyl-phosphatidylinositol (GPI) arched glycoprotein, is an important co-stimulatory molecule. Although it is not essential for induction of T cell response, CD24 is required to express on T cells and for the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS).
Here, we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or non-hematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE. At the same time, it is well established that T lymphocytes undergo homeostatic proliferation in lymphopenic environment. The homeostatic proliferation requires recognition of major histocompatibility complex on the host. Recent studies have demonstrated that co-stimulation, mediated by CD28, 4-1BB, and CD40, is not required for T cell homeostatic proliferation. Here, we report that T cells from mice with a targeted mutation of CD24 have a remarkably reduced rate of proliferation when adoptively transferred into syngeneic lymphopenic hosts. The reduced proliferation cannot be attributed to abnormal survival and homing properties of the CD24-deficient T cells. T
ii cell proliferation in allogeneic hosts is less affected by this mutation. Thus, although distinct co-stimulatory molecules are involved in antigen-driven proliferation and homeostatic proliferation, both processes can be modulated by co-stimulatory molecules.
Surprisingly, in the lymphopenic CD24-deficient mice, T cells launch an uncontrollable proliferation that results in rapid death of the recipient mice. The dividing T cells have the phenotypes similar to those activated by cognate antigens. In addition, the CD24- deficient DC have superior capacity to drive homeostatic proliferation of the syngeneic cells. Thus, our data demonstrate that CD24 expressed on the host antigen-presenting cells limits T cell response to homeostatic cue and prevents fatal damage associated with the uncontrolled homeostatic proliferation. Taken together, in this thesis, we demonstrate that although CD24 is not essential for the induction of T cell response, it can regulate T cell homeostatic proliferation and its function in autoimmune disease.
iii Dedicated to Ming
iv ACKNOWLEDGMENT
Foremost, I would like to thank my advisor, Dr. Yang Liu, for his intellectual support, encouragement and patience over the years. His dedication to research and his working ethic inspired me beyond scientific field.
Also, I would like to express my appreciation to all present and past members of
Drs. Liu’s and Zheng’s lab for their scientific support and friendship. I truly appreciate their generous assistance that make this dissertation possible: Huiming Zhang, Jian-xin
Gao, Ken May, Qunmin Zhou, Yin Wang, Yan Liu, Ergun Kocak, Xing Chang, Xingluo
(Tom) Liu, Jing Wen, John Richard, Ping Lu, Penghui Zhou, Lizhong Wang, Runhua
Liu, Cindy Wu, Jinqing Liu, Tao Zuo, Yi Xiao, Beth McNally, Ken Lute and Pramod
Joshi, Tianyu Yang, Peishuang Du, Lisa Yin and Jennifer Kiel. I am particularly grateful for Lynde Shaw’s friendship and enthusiastic support. I also want to especially thank Dr.
Xuefeng Bai for helping me start on the right track in the lab. The data presented in
Chapter 2 were obtained by collaboration with Dr. Bai.
At the Department of Pathology, I would especially like to thank Dr. James
Waldman and Mrs. Gretchen Staschiak, both for organizing the graduate curriculum and for keeping in touch with me all the time to help my Ph.D. works progress very well. I would like to give special thanks to Dr. Peter J. Nielsen for providing CD24 knockout
v mice. Moreover, my research works are supported by grants from the National Institute of Health (CA82355, CA69091, CA58033 and AI51342).
Finally, I couldn’t be more thankful to my devoted parents, who provide me unconditional love and support. Mostly, I am very grateful to my husband, Ming Jiang, whose love and patient help me through out the years.
vi VITA
June 7, 1976………………………………………………Born-Changchun, P.R. China
1995-2000 ……………………...... Bachelor of Medicine, Beijing
Medical University
2000-presemt…………………………...... Graduate Research Associate, Department of
Pathology, The Ohio State University
PUBLICATIONS
Research Publications:
1. Ou Li, Jin-Qing Liu, Pan Zheng, Yang Liu, and Xue-Feng Bai. L-selectin is required for the induction but not effector function of T cells in the experimental autoimmune encephalomyelitis. Submitted.
2. Ou Li, Pan Zheng, and Yang Liu. CD24 expression on T cells is required for optimal T cell proliferation in lymphopenic host. Journal of Experimental Medicine. 200(8):1083- 1089, 2004.
3. Ou Li, Xue-Feng Bai, Qunmin Zhou, Huiming Zhang, Pramod S. Josh, Xincheng Zheng, Yan Liu, Yin Wang, Pan Zheng, and Yang Liu. CD24 controls expansion and persistence of autoreactive T cells in the central nervous system during experimental autoimmune encephalomyelitis. Journal of Experimental Medicine. 200(4):447-458, 2004.
4. Qunmin Zhou, Kottil Rammohan, Shili Lin, Nikki Robinson, Ou Li, Xingluo Liu, Xue- Feng Bai, Lijie Yin, Bruce Scarberry, Peishuang Du, Ming You, Kunliang Guan, Pan Zheng, and Yang Liu. CD24 is a genetic modifier for risk and progression of multiple sclerosis. Proceedings of the National Academy of Sciences. 100(25):15041-15046, 2003. vii 5. Xingluo Liu, Jian Xin Gao, Jing Wen, Lijie Yin, Ou Li, Tao Zuo, Thomas F. Gajewski, Yang-Xin Fu, Pan Zheng, and Yang Liu. B7DC/PDL2 promotes tumor immunity by a PD-1-independent mechanism. Journal of Experimental Medicine. 197(12):1721-1730, 2003.
6. Xue-Feng Bai, Jin-Qing Liu, Ou Li, Pan Zheng, and Yang Liu. Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes. Journal of Clinical Investigation. 111(10):1487-1496, 2003.
7. Xue-Feng Bai, Jonathan Bender, Jinqing Liu, Huiming Zhang, Yin Wang, Ou Li, Peishuang Du, Pan Zheng, and Yang Liu. Local costimulation reinvigorates tumor- specific cytolytic T lymphocytes for experimental therapy in mice with large tumor burdens. Journal of Immunology. 167(7):3936-3943, 2001.
FIELD OF STUDY
Major Field: Pathology
viii TABLE OF CONTENTS
Page
Abstract……………………………………………………………………………….….ii Dedication…………………………………………………………………………….….iv Acknowledgements……………………………………………………………………....v Vita…………………………………………………………………………………...... vii List of Tables………………………………………………………………………...... xi List of Figures…………………………………………………………………………..xii Statement…………………………………………………………………………….....xiv
Chapters:
1. Introduction……………………………………………..…………………...... 1
1.1 T lymphocyte and antigen presentation…………….....1
1.2 CD24……………………………………………………..2
1.3 MS, EAE and CD24………………………………….....4
1.4 T cell homeostatic proliferation……………………...... 8
2. CD24 controls expansion and persistence of autoreactive T cells in the central
nerve system during experimental autoimmune
encephalomyelitis…………………………………………………………..12
2.1 Abstract………………………………………………...12
2.2 Introduction…………………………..………………..13
2.3 Material and Methods………………..……………….15
2.4 Results…………………………………..……………...21
ix 2.5 Discussion………………………………..…………….30
3. CD24 expression on T cells is required for optimal T cell proliferation in
lymphopenic host………………..…………………………………………59
3.1 Abstract………...………………………………………59
3.2 Introduction……...…………………………………….60
3.3 Material and Methods…...……………………………61
3.4 Results and Discussion……...…………………………63
4. Massive and destructive T-cell response to homeostatic cue in the CD24-
deficient lymphopenic hosts……………………...………………………..81
4.1 Abstract………………………...………………………81
4.2 Introduction……………………...…………………….82
4.3 Material and Methods……………..………………….83
4.4 Results………………………………...………………..86
4.5 Discussion……………………………..……………….92
5. Conclusion………………………………………………………………………118
Bibliography………………………………………………………………………123
x LIST of TABLE
Table Page
Table1. Fetal destruction of T cell proliferation in CD24-/- host……………………....113
xi LIST OF FIGURES
Figure Page
2.1 CD24 is required for persistence but not recruitment of autoreactive T cells in the
EAE model…………………………………...... 36
2.2 Quantitation of T cells in the brain of WT and CD24-/- mice………………...…….38
2.3 CNS infiltration of the MOG-active T cells from Thy1.1 congenic mice……..……41
2.4 Requirement for CD24 for optimal T cell proliferation in the CNS……………..….43
2.5 Antigen-specific T cells undergo apoptosis at similar rates in WT and CD24-/-
CNS………………………………………………………………………………..…45
2.6 Cytokine gene expression in brains of WT and CD24-/- mice on day 12 and 29 after
adoptive transfer of T cells……………………………………………………….….47
2.7 CD24-/-CNS astrocytes had a reduced capacity to stimulate MOG-specific T cell
proliferation…………………………………………………………..………………52
2.8 CD24-/-CNS microglia had a reduced capacity to stimulate MOG-specific T cell
proliferation…………………………………………………………...……………...54
2.9 Expression of CD24 on either hematopoietic or nonhematopoietic cells is sufficient
to confer susceptibility to adoptively transferred MOG-specific T cells……...……..56
3.1 Expression of CD24 on T cells does not affect the distribution and survival of T cells
upon adoptive transfer to lymphopenic host……………………………………...….69
xii 3.2 A critical role for CD24 on T cells in homeostatic proliferation in a lymphopenic
host………………………………………………………………………………...…72
3.3 Alloantigen-driven T cell proliferation is less dependent on CD24 expression on T
cells…………………………………………………………………………………..75
3.4 Expression of CD24 on T cells promotes acquisition of memory cell markers…….77
3.5 CD24 dificiency promotes neither apoptosis of dividing T cells nor their migration
into the liver………………………………………………………………………….79
4.1 Naïve T cells underwent uncontrolled homeostatic proliferation in lymphopenic
CD24-deficient host………………………………….………………………………95
4.2 In the CD24-deficient mice, cells undergoing homestatic proliferation displayed
markers of T cells induced by cognate antigen………..……………………………..99
4.3 The rapid dividing T cells underwent apoptosis in CD24-/- recipients ….………..101
4.4 Expression of CD24 on T cells does not restore regulation of homeostatic
proliferation in the CD24-/- host…………………………………………..………..104
4.5 The identification of the cells that are responsible for T cell uncontrolled
proliferation in CD24-/- host……………………………………………….………106
4.6 Super stimulatory activity of the CD24-/- dendritic cells………………….………109
4.7 Fatal destruction of CD24-/- host associated with mass T cell proliferation…..….114
xiii STATEMENT
The data presented in Chapter 2 were obtained by collaboration with Dr.
Xuefeng Bai.
xiv CHAPTER 1
INTRODUCTION
1.1 T lymphocytes and antigen presentation:
T lymphocytes play a central role in adaptive immunity. T cell receptor (TCR) is the defining marker for a T cell. Most TCR is composed by alpha chain and beta chain, which can form about 1016 potential repertoires.
T cells are derived from bone marrow. The progenitor cells migrate from bone marrow to thymus, where they rearrange their TCR and become mature T cells. All the T cells have to go through thymic maturation, which includes positive selection and negative selection. Positive selection provides surviving signals to T cells with TCRs that bind with low avidity to peptide-self-MHC (Major Histocompatibility Complex). It ensures the self-MHC restriction of T cells. Positive selection also ensures matching of
TCR restriction and expression of co-receptors. As a result of positive selection, only the
T cells with class I restricted TCR generally express CD8 and those with class II restricted TCR express CD4. Furthermore, there is a functional dichotomy between CD4
1 and CD8 T cells. CD4 T cells are helper cells and CD8 T cells are cytolytic effectors.
Negative selection will induce the T cells with TCRs that have high affinity to self- antigen-MHC complexes to undergo apoptosis. It can deplete the T cells that have the tendency to develop into auto-reactive T cells and cause autoimmune diseases. The two selection processes eliminate more than 90% of T cells produced in the thymus.
The mature T cells will reside in the peripheral lymph organs, spleen and lymph nodes. Upon activation, both subsets of T cells can perform their unique function in a highly co-operative fashion [1].
Mature T cells can not optimally respond to antigen in response to
MHC:peptide complex alone. Antigen-presenting cells (APCs) provide two signals necessary for efficient T cell activation. Dendritic cells, B cells and macrophages are all known as APC [2, 3]. Dendritic cell attracts the most attention due to its strong antigen presenting ability [2, 4-6]. The first signal from APC is the recognition of peptide-MHC complex by TCR. The major function for antigen presenting cells is to process pathogens and present them to T cells as their recognizable form. The other signal is termed costimulatory signal. The antigen presenting cells up regulate costimulatory molecules, like B7-1 and B7-2, upon pathogen stimulation [7]. Without proper costimulation, T cells may either become anergenic to the antigens or undergo apoptosis. When T cells in the peripheral lymphoid organs receive both signals, they become activated. The activated helper T cells will secret cytokines to activate macrophages to destruct phagocytosed antigens or B cells to secret antibodies. The activated cytotoxic T cells will secret perforin and lyse the target cells [1]. Due to the importance of the two signals for T cell
2 activation, researchers have been focused on manipulating either signal to regulate T cell response [8, 9]. It has been postulated that the need for two signals provides a theoretical framework to explain tolerance to peripheral organs [10, 11]. In this context, numerous studies have demonstrated that blockage of costimulation can be tolerant to allogeneic grafts and prevent induction of autoimmune diseases [12].
1.2 CD24:
CD24, also known as Heat-Stable Antigen (HSA), consists of a short peptide with extensive glycosylation [13]. The protein core has about 30 amino acids and is anchored to membrane with a glycosyl-phosphatidylinositol (GPI) tail [14]. It has been identified by various antibodies, M1/69, 20C9 and J11d [13, 15, 16]. CD24 was first cloned in 1990 [13]. Although 3 similar genes called CD24a, b and c have been identified in mouse, CD24b and CD24c are actually inactive, intronless pseudo genes. CD24a, as the functional gene, locates in chromosome 10 in mouse and 6q21 in human [17]. CD24 is expressed on various cell types, including T and B lymphocytes, dendritic cells and myeloid precursors [18, 19]. In addition, non-hematopoietic cells, such as epithelial cells, endothelial cells and various cell types in the central nervous system also express high level of CD24 [20-22]. CD24 is down-regulated as cell mature. This down-regulation is most striking in T cells [23]. CD4 /CD8 double-positive thymocytes express highest level of CD24, whereas mature positive thymocytes and peripheral T cells express diminished level of CD24 [23]. Therefore, CD24 is often used as marker for T cell
3 maturation in the thymus. Due to its widespread expression on hematopoetic cells, CD24 has commonly been used as differentiation marker.
CD24 also provides a CD28-independent costimulation for both CD4 and CD8
T cells [7, 23]. It has been reported that tumors transfected with CD24 induce better priming of anti-tumor CTL responses [24]. Constitutive expression of CD24 on B and T cells can augment a secondary antibody response [25, 26]. However, the costimulatory ligand for CD24 has not been identified [27]. Another function of CD24 is adhesion molecule. It has been reported that CD24 interacted with P-selectin [28], which has been indentified as its ligand. It also appears to modify very late antigen-4 (VLA-4) interaction with its ligands, vascular cell adhesion molecule-1 (VCAM-1) and fibronectin [29].
Since CD24 has high expression during the maturation of most bone marrow- derived cells [30], an interesting issue is whether CD24 is essential for the development of hematopoetic cells. However, although only 1/3 of pre-B cells are detected in CD24 knockout mice, the numbers of mature T and B cell are normal [31]. Other bone marrow derived cell types also appear unaffected by targeted mutation of CD24. Moreover, both cellular immune response and humor immune responses are normal in CD24-/- mice [31].
Thus, CD24 is not essential for immune response in the lymphoid organs.
1.3 MS, EAE and CD24
Multiple Sclerosis (MS) is a chronic disease in central nerve system (CNS) with multiple foci of inflammation, demyelination, and glial scarring (sclerosis), which usually involves CNS white matter. Inflammatory cells enter CNS, attack the nerve
4 myelin sheath and cause demyelination and damage to neurons, which lead to the
blockade of neuron signal transmission [32]. T lymphocytes and macrophages are the
major cells in perivascular inflammatory cuffs and parenchyma infiltrated cells in the
CNS sections from MS patients [33]. T cells that respond to myelin antigens are found in the cerebrospinal fluid (CSF) and in the lesions [34]. However, the etiology of MS is unknown. Genetic susceptibility [35], autoimmune mechanism [36], and viral infections
[37] may all contribute to pathogenesis. Although drugs that partially ameliorate MS symptoms are available [38-40], there is still no cure. The research using animal model is still needed to find potential therapeutic targets [41].
Experimental autoimmune encephalomyelitis (EAE) is the best available animal model for MS. EAE is a Th1 cell mediated autoimmune demyelinating disease
[42]. The main auto-antigens for EAE are myelin proteins. The major peptides for EAE induction are those derived from myelin oligodendrocyte glycoprotein (MOG), prolipid protein (PLP), and myelin basic protein (MBP) [32]. Various animal models have been used, including C57B6, SJL and Lewis rat.
There are several check points for EAE development. The first is the clonal expansion of MOG-immunized T cells. The second is the migration of activated T cells cross the blood brain barrier (BBB) to enter CNS [43]. The third is local antigen presentation to re-stimulate T cells so that T cells can establish inflammatory sites and recruit inflammatory cells [44, 45].
The induction of Th1 response is critical for EAE development. In addition to peptide and proper major histocompatibility complex (MHC) class II, co-stimulatory
5 molecules are required to active T cells [46]. The epitopes of the peptides that can induce
EAE are varied from strain to strain. In our experiment, we are using MOG 35-55 and B6
mice, whose MHC II is H-2b [32]. The absence or reduced severity of EAE has been
observed in the mice deficient for various co-stimulatory molecule. However, not all of
the mice showed defect in the priming of antigen specific T cells [47-49].
Activated T cells start rolling along the blood vessel at a reduced speed,
primarily through interaction between selectins and their ligands. The interaction of
chemokines with their receptor on T cells activates integrins and thus promotes firm
adhesion and migration of T cells [43, 44]. The cytokines produced by activated T cells,
such as interferon (IFN)-Ȗ and tumor necrosis factor (TNF), can upregulate lymphocyte function associated molecule (LFA)-1 and very late antigen (VLA)-4 ligand on the endothelium, namely intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 [13, [50]. Antibody treatment against adhesion molecules can reduce the severity of EAE [51]. Usually, all the activated T cells can migrate across
BBB. However, only the T cells who can find the antigens that they are respond to will stay in CNS [43].
A local antigen presentation in the CNS is critical for activated T cell to survive and develop EAE. Astrocytes, microglia and cerebral vascular endothelial cells can present antigens in the CNS [50]. Upon stimulation by cytokines, such as IFN- Ȗ,
these local APCs increase the expression of MHC class II and co-stimulatory molecules,
such as B7-1, B7-2 [52]. Perivascular and meningeal macrophages [53], which can also
express MHC II and co-stimulatory molecules, are believed to act as APC in the CNS
6 [54]. Co-stimulation in the CNS is critical for EAE development. Without co-stimulatory signal, antigen specific T cells will not be fully activated and will stay in the perivascular area, where they appear to undergo apoptosis [48, 49].
When B6 mice are immunized with MOG, EAE develops around day 8, reaches peak rapidly, lasts for about 20 days and is usually remitted with few relapses. CD24 (-/-) mice are resistant to either active or passive EAE. Both the recipients and the T cells need to express CD24 to induce EAE [55]. There are several possibilities for the absence of
EAE from the knockout mice. First, CD24, as a co-stimulatory molecule, may affect the production of antigen specific T cells. Second, CD24 may act as an adhesion molecule
[28, 29]. The deficiency of CD24 may affect T cell attachment to the endothelium, which is critical for T cells to enter CNS. Third, the absence of CD24 in the host may affect later events at the effector phase of EAE. Local antigen presentating cells may have defect from the absence of CD24.
Although CD24 was known as a co-stimulatory molecule, it was found to be redundant in the induction of T cell response. Specifically, the induction MOG-specific response in CD24 knockout mice was completely normal [55]. The fact that CD24 (-/-) mice are resistant to EAE despite abundance of autoreactive T cells makes this model ideal to study the function of CD24 and the pathogenesis of EAE. Unlike most experimental treatments for EAE, which have to start before the onset or even before the immunization, blocking CD24 may have therapeutic effects even after self-reactive T cells are produced, which make it more relevant to clinical settings. Thus, CD24 may emerge as a possible target for effective treatment of MS.
7 1.4 T cell homeostatic proliferation
Immune system has the tendency to maintain a constant number of lymphocytes. When pathogens attack, immune cells are activated to eliminate them. Both
T and B cell numbers are increased dramatically. After the number of lymphocytes increase drastically, the majority of the activated cells will undergo apoptosis and only a small part of the activated cells become memory cells and survive in peripheral lymphoid organs. The lymphocyte number will return to normal over time [1].
In animal models, lymphopenia can be seen in new-born mice [56], irradiated mice and mice with certain gene mutation [57]. Normal individuals have a number of fresh T lymphocytes coming from thymus to peripheral. However, at an older age, the thymus function decreases, so does the T cell output. Stress and lost of blood can also reduce peripheral lymphocyte number. Under disease conditions, virus infection, corticosteroid administration, chemotherapy, or radiation are some of the reasons for lymphocyte decreases. When the lymphocyte number decreases, the naïve lymphocytes will undergo proliferation, which is called homeostatic proliferation. Therefore, homeostatic proliferation is the host’s attempt to maintain immune cell number and its ability for immune response [57].
T cells and B cells undergo homeostatic proliferation independently. The studies from knockout models showed that the absence of B cells does not lead to T cell expansion. At the same time, the absence of T cells does not affect B cell number in the peripheral. Even among T cells, a reduction in the number of naïve T cells does not
8 trigger proliferation of memory T cells and vice versa. However, CD4 and CD8 T cells do appear to partially compensate each other [57].
In lymphopenic environment, the antigens that are essential for homeostatic proliferation are self antigens [58]. Many studies showed they are the same antigens that have been presented to T cells during thymic selection [59]. However, not all the naïve T cells have the same ability to proliferate in a lymphopenic host. In lymphopenic mice, only about 30% of the transferred naïve T cells will proliferate [58]. It is decided by the affinity of TCR to self-peptide and MHC complexes [58]. It has been predicted that the affinity between TCR and peptide and self-MHC in homeostatic proliferation is higher than during thymic selection [58].
Early studies appear to suggest that, in contrast to T cell response to pathogens, which requires stimulation by self MHC: foreign antigens and costimulatory molecules, homeostatic proliferation may initiate when only TCR is engaged by MHC. The absence of B7-CD28, CD40-CD40L and 4-1BB-4-1BBL does not affect homeostatic proliferation
[58]. However, one group did report that CTLA4-Ig treatment can slow TCR transgenic
T cell homeostatic proliferation [60]. Besides TCR and MHC contact, cytokines like IL-7,
IL-15, and IL-21 can also regulate T cell homeostatic proliferation. As reported, IL-7 is required for naïve T cell proliferation in lymphopenic hosts [61]. IL-21 and IL-15 can accelerate the division of T cells in vitro, but rather dispensable in vivo [62, 63].
“The space” that has been discussed about in the immune system is also referred as niche, an environment for lymphocytes to obtain signals for survival and proliferation
[64]. Since T cells and B cells have different niches, their survival and homeostatic
9 proliferation are independent of each other. T cell niche is consist of MHC, costimulatory molecules, adhesion molecules, cytokines, chemokines and other growth factors [64]. At the same time, limitation of the resources in the niche can also be used to control the proliferation. T cell proliferation depends on the availability of the resources in the niche.
In lymphopenic host, CD8 T cells divide faster than CD4 T cells, which can be explained by the abundant expression of MHC I in the peripheral [65]. Moreover, the cells that share niche compete for the resources. Although requiring different MHC, CD4 and CD8
T cells still share other resources. Thus, the number of one subset may affect that of another [64].
Since the affinity of TCR may decide the ability for T cell to undergo homeostatic proliferation and T cells have to compete for the limited resources, the self-reactive T cells that are not depleted during thymic selection may have the advantage to proliferate and suppress other subsets, which will increase the frequency of self-reactive T cells [65].
Moreover, T cells that have underwent homeostatic proliferation become memory cell and can respond to the stimulation more rapidly and have large amounts of cytokines produced in a short period of time. These will increase the possibility of developing autoimmune diseases. Studies have shown connections between lymphocytes homeostatic proliferation and rheumatoid arthritis[66], lupus [66]and MS [65, 67].
Recent publication about the study on NOD (Nonobese diabetic) mice showed that the
NOD mice suffered lymphopenia and the homeostatic proliferation of the self-reactive T cell may contribute to the spontaneous development of diabetes [68]. Thus, understanding the checkpoints to lymphopenia-induced lymphocyte proliferation may
10 provide targets for the intervention of autoimmune diseases. In chapter 3, we will show that CD24, as a costimulatory molecule, is required to express on T cells for T cell proliferation in lymphopenic hosts. In chapter 4, we found that CD24 expression on lymphopenic host can limit naïve T cell proliferation. This is a novel function for CD24, which may provide a possible mechanism to control naïve T cell homeostastic proliferation and further new therapeutic approach for auto immune diseases.
11 CHAPTER 2
CD24 CONTROLS EXPANSION AND PERSISTENCE OF AUTOREACTIVE T
CELLS IN THE CENTRAL NERVE SYSTEM DURING EXPERIMENTAL
AUTOIMMUNE ENCEPHALOMYELITIS
2.1 Abstract:
In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen
(CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on
either hematopoietic cells or non-hematopoietic antigen-presenting cells in the recipient is
sufficient to confer susceptibility to EAE. 12 2.2 Introduction:
MS (multiple sclerosis) is a chronic disease in CNS with multiple foci of
inflammation, demyelination, and glial scarring (sclerosis), which usually involves CNS
white matter. EAE (experimental autoimmune encephalomyelitis) is the best available
animal model for MS [41]. EAE is a Th1 cell mediated autoimmune demyelinating
disease. There are several check points for EAE development. The first is the clone
expansion of MOG-immunized T cells. The second is the migration of activated T cells
cross the blood brain barrier (BBB) to enter CNS. The third is local antigen presentation
to re-stimulate T cells so that T cells can establish inflammatory sites and recruit inflammatory cells [32]. More recently, it has been elegantly demonstrated that the
autoreactive T cells express new activation markers after their entry into the CNS,
presumably by interacting with local APCs [41, 69]. It is unclear, however, whether the
cells in the CNS undergo further clonal expansion. This issue has an important bearing on
how the autoimmune disease is sustained in the CNS, as T cells in the CNS appear to be
prone to programmed cell death. [70, 71]
CD24 is a GPI-anchored cell surface glycoprotein and is expressed in a broad
range of cell types, including developing hematopoietic cells and mature B cells [31]. It is
also known that CD24 is abundantly expressed in the CNS [21, 22], although its
expression on the cells in the CNS that have immune function has yet to be carefully
defined. We have reported that mice with a targeted mutation of CD24 are resistant to the
induction of both actively induced and adoptively transferred autoreactive T cells and that
the development of EAE requires a functional CD24 gene in both T cells and non–T host
13 cells [55]. More recently, our genetic analysis revealed that CD24 polymorphism is a
genetic modifier for risk and progression of MS [72]. Because CD24 is a costimulatory
molecule that functions independently of CD28 [7, 16], it is possible that CD24 in the
CNS mediates local costimulation of T cells [69]. Alternatively, CD24 also modulates
very late antigen (VLA) 4–vascular cell adhesion molecule (VCAM)-1 interaction that
regulates T cell recruitment to the CNS [28, 29]. Therefore, CD24 may regulate the
recruitment of autoreactive T cells to the CNS.
To test these two hypotheses, we adoptively transferred myelin oligodendrocyte
glycoprotein (MOG)-reactive T cells into WT and CD24–/– mice, and analyzed the
recruitment of T cells. We found that although the pathogenic T cells can be recruited at a
comparable efficiency to the CNS of the WT and CD24–/– mice, the T cells persist and
expand only in WT CNS. In consistency with this, we found that susceptibility to
adoptively transferred T cells can be conferred by cells of either hematopoietic or non-
hematopoietic origin. Interestingly, CD24 deficiency significantly reduced the in vitro
costimulatory activity of microglia and astrocytes, two major APCs in the CNS, and that
the CD24 deficiency in the hosts leads to a significantly reduced local proliferation in the
CNS. Finally, we found that expression of CD24 does not affect apoptosis of T cells in
the CNS. These results demonstrate that CD24 controls a novel checkpoint for EAE, most likely by controlling local proliferation of autoreactive T cells in the CNS.
14 2.3 Material and Method:
Experimental animals:
CD24-deficient mice were produced using embryonic stem cells from C57BL6/j
mice as described previously. C57BL6/j mice were purchased from the National Cancer
Institute. All mice were maintained in animal facilities at the Ohio State University
Medical Center under specific pathogen-free conditions. Thy1.1 congenic mice were
purchased from The Jackson Laboratory.
Bone Marrow Chimera Mice:
Bone marrow cells were isolated by flushing femur and tibia bones with PBS.
Recipient mice were lethally irradiated (1,000 rads) and constituted with 10–20 x 106 bone marrow cells by intravenous injection. Engraftment took place over a 6–8-wk period.
Culture Medium and Reagents:
Click's EHAA medium supplemented with 10% FCS, 2 mM L-glutamine, 100
µg/ml penicillin/streptomycin, and 1 mM 2-ME were used for all lymph node T cell
cultures. For culture of glial cells, DMEM supplemented with 10% FCS, 2 mM L-
glutamine, and 100 µg/ml penicillin/streptomycin was used. Recombinant murine IFN- ,
IL-12, and recombinant human IL-2 were purchased from PeproTech. The immunogen
MOG peptide 35–55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from Genemed
Synthesis, Inc. The purity of the peptide was >90%.
Adoptive Transfer EAE:
8–12-wk-old C57BL/6j or B6.PL/J (Thy1.1) mice were immunized subcutaneously with 100 µg MOG peptide in CFA in a total volume of 100 µl. 10 d after
15 immunization, draining lymph nodes were harvested and stimulated at a density of 2 x
106/ml in Click's EHAA medium supplemented with 15% FCS, 20 ng/ml recombinant IL-
12, and 50 µg/ml MOG peptide for 4 d. Two different protocols were used in this study.
Protocol A was used for most of the studies, except for those described in Fig. 7, which
used protocol B. Protocol A: 10 x 106 MOG peptide–activated lymph node cells were
injected i.v. into each recipient mouse in a total volume of 200 µl PBS. Each mouse
received 160 ng of pertussis toxin (Sigma-Aldrich) in 200 µl PBS in the tail vein
immediately after the cell transfer and again 48 h later. Protocol B: 20–50 x 106 MOG- activated T cells were injected i.p. into each recipient mouse that had been -irradiated
(350 rads) 1 h earlier. The mice were observed every day and scored on a scale of 0–5
with gradations of 0.5 for intermediate scores: 0, no clinical signs; 1, loss of tail tone; 2,
wobbly gait; 3, hind limb paralysis; 4, moribund; and 5, death.
Immunohistochemistry:
Data shown here were representative of two serial sections from the same brain,
stained by H&E and immunohistochemical staining.
Brain was collected from each group and embedded in Optical Cutting
Temperature (OCT) compound. The tissues were frozen at-70C and cut into 7mm
sections. All the sections were blown dried overnight, fixed by cold Aceton and stored at
-20C.
For immunohistochemical staining, the tissue sections were put into 1% H2O2
for 15 minutes and 10% goat serum for 30 minutes. Rat anti-mouse anti-CD3 antibody
was incubated with tissue sections for 60 minute at RT. After washed with PBS, tissue
16 sections were incubated with second antibody (Goat anti-Rat antibody) for 30 minutes.
Avidin and Biotin Complex was incubated with the sections for 30 minutes. After three
washes with PBS, DAB kit was used to develop color. Hematoxilyn was used to stain
nuclear as counter staining.
H&E staining: The serial sections from the CD3 staining were put into
hemotoxilyn for 4 minutes, washed with water; and put into acid alcohol for 2 minutes,
washed with water; and put into NH4.H2O for 1 minute, washed with water. Then the
sections were put into eosin for 1 minute and washed with water. The sections went
through 70%, 90%, 100% alcohol and sealed with cytoseal XYL.
RNase Protection Assay (RPA):
Total RNA from each individual brain was isolated with Trizol reagent (Life
Technologies). The concentration of RNA in each sample was assessed by
spectrophotometry. The multiprobe RPA kit (RiboQuant; BD Biosciences) was used with
the assay performed according to the manufacturer's protocol. In brief, a set of 32P-labeled
RNA probes synthesized from DNA templates using T7 polymerase was hybridized with
20 µg total RNA, after which free probes and other single strand RNA were digested with
RNase. The remaining RNase-protected probes were purified, and then resolved on denaturing polyacrylamide gels. The template set mCD1 for mouse cell surface antigen was used in this study, which detects mRNA of TCR- , TCR- , CD3 , CD4, CD8 ,
CD8ß, CD19, F4/80, CD45, L32, and GAPDH. For the detection of cytokines, we used template set mCK3b, which detects mRNA of TNF-ß, LTß, TNF- , IL-6, IFN- , IFN-ß,
TGF-ß1, TGF-ß2, TGF-ß3, MIF, L32, and GAPDH; mCK-1b, which detects mRNA of
17 IL-4, IL-5, IL-10 IL-15, IFN-Ȗ, L32 and GAPDH; mCK-5c, which detects mRNA of Ltn,
RANTES and IP-10;mCR-5, which detects mRNA of CCR1, CCR3, CCR5 and CCR2 .
Isolation of Mononuclear Cells from the CNS:
Fresh brain or spinal cord tissues were removed from mice and cut into 2-mm pieces and incubated in 10 mM Hepes/NaOH buffer containing 1mg/ml of collagenase
(Sigma-Aldrich) for 1 h at 37°C. The tissues were dispersed with a syringe, filtered through a 100-mm wire mesh, and centrifuged at 2,000 rpm for 5 min at 4°C. After centrifugation, cell pellets were resuspended in 15 ml of 30% Percoll (Amersham
Biosciences), and centrifuged against 70% Percoll in a 50-ml tube for 15 min. The cell monolayer at the 30–70% Percoll interface was collected and washed once for further staining.
Culture of Microglia and Astrocytes from Brains of Newborn Mice:
Primary glial cell cultures were prepared from brains of newborn CD24+/– or
CD24–/– mice as described previously. In brief, after removal of the meninges, the brains
were mechanically dissociated by nylon sieves. The cells were seeded in culture medium
(DMEM) containing 20% FCS in 75 cm2 tissue culture flasks. On day 4 the medium was replaced with DMEM containing 10% FCS and medium was changed every 3 d thereafter.
For isolation of microglial cells, confluent cultures between days 12 and 14 were vigorously shaken and the floating cells were collected and incubated at 37°C in flasks for another 2 h. The cultures were then shaken at 90 rpm and the nonadherent cells were washed out. The adherent cells were identified as microglial cells by positive staining with isolectin IB4 conjugated with Alexa Fluor 488 (Molecule Probes) and anti-CD45
18 antibodies (BD Biosciences). The firmly adherent cells were stained with anti–glial
fibrillary acidic protein (GFAP) antibody to confirm their identity as astrocytes.
MOG-specific T Cell Line:
Draining lymph node cells were isolated 10 d after immunization of C57BL6 mice
with MOG peptide. 2 x 106 cells/ml were stimulated with 50 µg/ml MOG peptide in the presence of 20 ng/ml IL-12 for 4 d. The viable cells were isolated and cultured in Click's
EHAA medium in the presence of 5 ng/ml recombinant human IL-2 for another 2 wk before they were used for the proliferation assay.
Antibodies and Flow Cytometry:
FITC- or PE-conjugated antibodies against CD3 (2C11), CD80 (16-10A), CD86
(GL1), CD24 (M1/69), Db (KH95) I-Ab (AF6-120), IFN- (XMG1.2), IL-4 (11B11), and
rat IgG1 isotype control were all purchased from BD Biosciences. APC-Thy1.1 (HIS51)
was purchased from eBioscience. Alexa Fluor 488–labeled isolectin IB4 was purchased
from Molecular Probes. Purified mouse anti-GFAP cocktail was purchased from BD
Biosciences. The antibody against CD24 (20C9) has been described. For cell surface
staining, cells were incubated with antibodies in staining buffer for 30 min on ice. Cells
were then washed three times and fixed. For intracellular staining of GFAP, astrocytes
were first fixed with Cytofix/Cytoperm buffer (BD Biosciences) followed by washing
with Perm/Wash buffer (BD Biosciences), and incubated with anti-GFAP in Perm/Wash
buffer for 30 min on ice. After washing with Perm/Wash, cells were incubated with FITC-
conjugated rabbit anti–mouse IgG antibody (Accurate Chemical & Scientific Corp.). For
the staining of intracellular cytokines, lymphocytes were incubated with GolgiStop (BD
19 Biosciences) in the last 5 h of culture. Viable cells were first fixed in the
Cytofix/Cytoperm buffer followed by staining with PE-labeled anti–mouse cytokine antibodies and isotype control in Perm/Wash buffer. Cells were analyzed on a
FACSCaliburTM cytometer (Becton Dickinson).
To analyze T cell apoptosis, mononuclear cells isolated from the CNS of WT or
CD24–/– mice were stained with anti-Thy1.1 antibody in conjunction with 7-AAD and
FITC-labeled annexin V. The annexin V+ 7AAD+ cells are at late stages of apoptosis, whereas the annexin V+ 7AAD– cells are at early stages of apoptosis.
Bromodeoxyuridine (BrdU) Incorporation:
MOG-specific T cells were injected into mice i.v. At various times after adoptive transfer, mice were given an i.p. injection of 1 mg BrdU (BD Biosciences). 12 h later, mice were killed and perfused with PBS through the left ventricle. The brain and spinal cord were removed from each mouse. Mononuclear cells were prepared from the brain and spinal cord, and were stained with anti-BrdU antibody using a BD Biosciences kit.
T Cell Proliferation Assay:
To assess the antigen-presenting functions of cultured astrocytes and microglia, the WT MOG-specific T cell line was used as a responder. In brief, irradiated (3,000 rads) astrocytes or microglia were cultured in round-bottomed microtiter plates in 200 µl
DMEM medium containing 100 U/ml IFN- . 3 d later, the medium was removed and 2–5 x 104 T cells in Click's EHAA medium and the indicated concentrations of MOG peptide were added into each well. After 48 h, the cultures were pulsed with 1 µCi/well
20 [3H]thymidine (ICN Pharmaceuticals) for another 12 h, and incorporation of
[3H]thymidine was measured in a liquid scintillation ß plate counter.
2.4 Results:
Persistence and Expansion of MOG-reactive T Cells in the CNS of CD24+/+ But Not
CD24–/– Mice.
Because CD24 is known to modulate the VLA4–VCAM-1 interaction [29], an
attractive hypothesis is that CD24 may determine the pathogenicity of autoreactive T
cells by controlling their recruitment to the CNS. To test this hypothesis, we injected
MOG peptide–immune CD24+/+ T cells into WT or CD24-deficient mice and followed the
clinical scores in the recipient mice. In addition, the T cells in the brain were analyzed by
flow cytometry and quantitative RPA.
The T cells used were of the Th1 cell type as they produced IFN- , but not IL-4, in response to MOG peptide stimulation in vitro. As shown in Fig. 2.1A, when we adoptively transferred MOG peptide–activated draining lymph node cells into CD24+/+ syngeneic recipient mice, all mice developed EAE at 12–14 d after cell transfer. The clinical scores peaked at 18 d after T cell transfer and declined gradually over the next 3 weeks. In contrast, none of the CD24–/– recipient mice developed EAE, which is in agreement with our previous report [55].
Because CD24 reportedly modulates the interaction between VLA4 and VCAM-
1 [29], we tested whether expression of CD24 in the host is required for T cell migration into the CNS. We generated MOG-specific T cells from Thy1.1 congenic mice and
21 adoptively transferred them into WT and CD24–/– hosts. Based on published observations
by others, most of the adoptively transferred T cells migrate into the CNS by the third day
after adoptive transfer [44]. Therefore, we harvested recipient brains after perfusion at 3 d
after adoptive transfer and analyzed the number of T cells by flow cytometry. As shown
in Fig. 2.1B, despite their low percentages, a well-defined population of donor cells was
found in the brains of WT recipients. The same subset was found in the CD24–/– brains at a comparable frequency. Thus, CD24 expression in the host is not required for T cell migration into the brain.
We used RPA to measure the amount of T cells in the brain. At various time points after adoptive transfer of T cells, groups of two mice were perfused with PBS through the left heart ventricle immediately after death, and the brains were harvested for
RNA isolation. Expression of several T cell–specific genes was determined by RPA. As directly shown in Fig. 2.2, CD3 and CD4 mRNA were undetectable in the CNS of normal
CD24+/+ and CD24–/– mice that received no transfer, which was expected. By day 8 after
the adoptive transfer, weak but significant signals for the T cell–specific genes were
detected in both groups of recipients. High and comparable amounts of CD3 and CD4
mRNA transcripts were detected on days 10–12 after T cell transfer, again in both groups.
In the CD24+/+ brain, the amount of T cells continued to rise on day 20 and remained
detectable on day 40 after the adoptive transfer. In contrast, the T cells were greatly
reduced on day 20 and became undetectable by day 40 in the CD24–/– brain. The majority
of the T cells in the brain belonged to the CD4 subset, as the CD4 mRNA was more
abundant than that of CD8. Thus, the autoreactive T cells persist and expand in CD24+/+
22 but not in the CD24–/– CNS. These data demonstrate a critical role of CD24 in sustaining
T cells in the CNS. The mRNA for macrophage marker F4/80 also increased with that of
CD3 and CD8, consistent with an ongoing T cell–initiated inflammation in the host.
It has been established that in the adoptive transfer model, the host T cells are also
recruited into the CNS after initiation of inflammation by the donor T cells[43, 73]. To
distinguish the adoptively transferred T cells versus T cells that were nonspecifically
recruited into the CNS secondary to the antigen-specific T cells, we immunized Thy1.1
congenic mice and immune lymph node cells were cultured in the presence of MOG
peptide and IL-12 for 4 d. The T cells were mostly of Th1 cell type, as the majority of
them produced IFN- in response to PMA and ionomycin, although a small portion of T cells were capable of producing IL-4. After adoptive transfer of these cells into either
CD24+/+ or CD24–/– recipient mice, we could detect Thy1.1+ T cells in the CNS of both
CD24+/+ and CD24–/– mice on day 10 after transfer (Fig. 2.3). The immunohistochemical
analysis of Thy1.1+ T cells revealed several interesting points. First, despite comparable
levels of CD3 mRNA in the brains of WT and CD24–/– mice, a significant difference was
observed in the number of Thy1.1+ T cells at early time points. Second, in addition to the
difference in the number of Thy1.1+ T cells, there was also a significant difference in their distribution. In the WT host, a substantial number of T cells were found in the brain parenchyma. In contrast, essentially all of the donor T cells were localized in the meninges and perivascular regions in the CD24–/– brain and spinal cord. Third, as shown
in Fig. 2.3B, the donor T cells appeared in clusters in the WT CNS. In the CD24–/– hosts,
the donor cells rarely formed clusters.
23 Reduced Local Proliferation in the CNS of the CD24–/– Mice.
The failure of CD3+ T cells to persist in the CNS of CD24–/– mice could be due to
reduced proliferation of CD24+/+ T cells, or due to accelerated apoptosis of T cells. To
address this issue, we adoptively transferred MOG-specific Thy1.1 congenic T cells into
CD24+/+ and CD24–/– mice, and determined the proliferation of the donor T cells by a
pulsing of BrdU on days 3–20 after T cell transfer. 12 h after BrdU injection, the
recipients were killed and perfused through the left ventricle to avoid blood
contamination in the CNS. Mononuclear cells were isolated from the brain, spinal cord,
and spleen, and stained for BrdU incorporation. As shown in Fig. 2.4 A, on day 3 after T
cell transfer, comparable numbers of Thy1.1+ T cells were detected in the spleens of both
CD24+/+ and CD24–/– mice (0.45 vs. 0.57% of total lymphocytes). These cells were
undergoing vigorous proliferation, as 19.1 versus 23% of Thy1.1+ T cells were
incorporating BrdU within 12 h. At this point, 0.39 and 0.33% of Thy1.1+ T cells were
detected among the CNS mononuclear cells of CD24+/+ and CD24–/– mice, respectively.
Interestingly, in WT mice, 43.2% of Thy1.1+ T cells were incorporating BrdU in the CNS
of WT mice. This is more than twofold higher than those Thy1.1+ T cells in the spleen,
presumably reflecting the effect of local antigen stimulation in the CNS. However, only
27.1% of Thy1.1+ T cells in the CD24–/– mouse brains were BrdU+. Because the rate of
BrdU incorporation in the CNS is comparable to that of the spleen, it is less clear to what extent their proliferation was in response to stimulation in the periphery or in the CNS, as
T cells are known to be able to continue their proliferation if a threshold proliferation has been observed[74].
24 By day 12 after T cell transfer, fewer donor T cells were detected in the CD24–/– spleen than in the CD24+/+ spleen (0.8% in CD24+/+ mice vs. 0.018% in CD24–/– mice).
About 2% of Thy1.1+ T cells in the CD24+/+ spleen were BrdU+. However, none of the
Thy1+ T cells in the CD24–/– spleen were incorporating BrdU in the spleen. We observed a significant increase in the number of Thy1.1+ T cells in both CD24+/+ and CD24–/– CNS on day 12 compared with day 3. More than 4% of the donor T cells in the CD24+/+ mouse brain were actively incorporating BrdU, whereas only 1.5% of donor T cells underwent division in the CD24–/– mouse brain. Similar differences were observed in the T cells
isolated from the spinal cord. The significance of CD24-mediated T cell clonal expansion
is further substantiated by a >10-fold difference in the number of T cells accumulated in
CD24+/+ (26%) versus CD24–/– (2%) mouse brains on day 12.
A comparison between the total number of T cells in the CNS detected by RPA
(Fig. 2.2) and those detected by Thy1.1-specific antibody in both immunohistochemistry
(Fig. 2.3) and flow cytometry (Fig. 2.4 A) suggests that the overwhelming majority of T
cells detected by RPA in the CD24–/– mouse brain are of host origin. This is confirmed by
flow cytometry, which revealed that more than 95% of T cells in the CD24–/– mouse brain are of host origin, whereas about an equal number of host and donor cells were found in the CD24+/+ brain (Fig. 2.4 B).
Local Activation and Apoptosis of T Cells in the CNS.
In addition to proliferation, a major factor that determines the number of T cells is
apoptosis. To test whether the lack of T cell persistence in the CNS of CD24–/– mice is
due to accelerated cell death, we stained CNS-infiltrating cells for apoptosis markers 7-
25 AAD and annexin V, as shown in Fig. 2.5. On day 10 after T cell transfer, 30% of
Thy1.1+ brain-infiltrating T cells were undergoing apoptosis in both CD24+/+ and CD24–/–
mice. More than 50% of Thy1.1+ T cells were undergoing apoptosis in the spinal cords in
both CD24+/+ and CD24–/– mice. However, no difference was observed between WT and
CD24–/– hosts. Thus, the failure of T cell persistence in the CNS of CD24–/– mice is not due to accelerated T cell apoptosis.
Because expression of cytokines in the CNS is widely used to measure T cell activation in situ [45], we used the RPA assay to measure inflammatory cytokine mRNA expression in the CNS. As shown in Fig. 2.6 A, the cytokines that play important roles in inhibiting EAE development as IL-4 and IL-10, don’t showed any difference between the two groups on the initiation stage of the disease, day 12 and on the peak of the disease, day 29. At the same time, we also tried to detect mRNA level by using RPA templates
(figure 2.6 B, C) that contain chemokines that have been known affect T cell inflammation and recruitment, RANTE and IP-10 [75] and their receptors, CCR-1, 3, 5 and 2.There were little difference between the two groups. At last, we used RPA template mCK-5 to detect IFN- , TNF- , TNF-ß, and TGF-ß expression [75]. Although little IFN-
, TNF- , TNF-ß, and TGF-ß were detected in both CD24+/+ and CD24–/– CNS on day 12
after T cell transfer, significant up-regulation of all of these cytokine genes was detected
in WT mouse brains on days 29 (figure 2.6 D). With a notable exception of TNF-ß,
significant induction of IFN- , TNF- , and TGF-ß was also observed in the CD24–/– brains, although the overall levels were somewhat lower than in the WT mice. The reduction in TNF-ß mRNA that can be attributable to CD24 deficiency was not as drastic
26 as that in the number of donor T cells, perhaps because some of the host T cells also
synthesize cytokine mRNA.
CD24-deficient CNS APCs Have Reduced Capacity to Stimulate Proliferation of
Autoreactive T Cells.
Because T cells proliferated less in the CNS of CD24–/– recipient mice, we
reasoned that CD24 may contribute to EAE development by regulating the functions of
the CNS APCs. To test this hypothesis, we compared astrocytes and microglia from
brains of newborn CD24+/– or CD24–/– mice for their ability to stimulate WT T cells. As
shown in Fig. 2.7 A, essentially all of the cells in the astrocyte culture expressed GFAP.
CD24+/– astrocytes constitutively expressed CD24, and after stimulation with IFN- , the level of CD24 increased about threefold. The levels of CD80 and CD86 were low even after IFN- stimulation. As previously reported by others, IFN- induced significant
expression of MHC class I and II in both groups [76].
Although both groups of astrocytes stimulated proliferation of these T cells, WT
astrocytes were approximately fourfold more efficient in inducing the proliferation than
the CD24–/– astrocytes on a cell to cell basis (Fig. 2.7 B). When the MOG peptide was
titrated, the WT astrocytes were again about fourfold more potent. The enhanced
proliferation was blocked by the addition of an anti-CD24 antibody, 20C9 (Fig. 2.7 C),
which is known to block the costimulatory activity of the CD24 molecule [77].
We also isolated microglia cells from newborn CD24+/– or CD24–/– mice. As shown in Fig. 2.8 A, comparable levels of the CD24+/– and CD24–/– microglia were
strongly positive for isolectin IB4 staining and CD45 (not depicted; references [78] and
27 [79]). CD24+/– microglia constitutively expressed high levels of CD24 and after
stimulation with IFN- , they expressed even higher levels of CD24. IFN- induced
significant expression of MHC class I and II and low levels of CD80 and CD86 on
microglia. To compare the costimulatory activity of the CD24+/– and CD24–/– microglia cells, we stimulated MOG-specific T cell lines with these cells in the presence of different concentrations of MOG peptide. Based on the titration of antigen, WT microglia were approximately fivefold more efficient in inducing T cell proliferation (Fig. 2.8 B). These results suggest that CD24 expressed on microglia can promote proliferation of MOG- reactive T cells.
Expression of CD24 on Radiosensitive and Radioresistant Host Cells Confers
Susceptibility to Pathogenic T Cells in the CNS.
The resistance of the CD24–/– mice to EAE induction by WT T cells suggests that at least some host cells are required to express CD24. Because local APCs can be of either hematopoietic or non-hematopoietic origin, we created radiation bone marrow chimeras to determine whether expression of CD24 on hematopoietic cells is sufficient to convey EAE susceptibility. As shown in Fig. 2.9 A, a single round of bone marrow transplantation resulted in an essentially complete replacement of CD24-expressing cells in the spleen, which demonstrates the efficacy of irradiation. Reciprocal chimeras were used to determine the nature of the host cells required for EAE susceptibility, whereas syngeneic chimeras were used as controls.
As shown in Fig. 2.9 B, all CD24+/+ > CD24+/+ bone marrow chimeras developed
EAE after CD24+/+ T cell transfer. In contrast, none of the CD24–/– > CD24–/– recipient
28 chimeras were susceptible to pathogenic T cells. All CD24+/+ > CD24–/– chimeras developed EAE with similar kinetics to that of the CD24+/+ > CD24+/+ mice. These results
demonstrate that CD24+/+ bone marrow–derived cells corrected the local antigen
presentation defects in the CD24–/– mice. Interestingly, all CD24–/– > CD24+/+ chimeras also developed EAE. Although the onset was delayed by 4 d in this experiment, the peak
EAE scores were equal to those observed in the CD24+/+ > CD24+/+ and CD24+/+ > CD24–
/– chimera mice.
It is possible that the susceptibility of the CD24–/– > CD24+/+ mice to EAE was
due to residual bone marrow–derived CD24+/+ cells. Here, we first detect CD24
expression in the CNS of all the bone marrow chimera groups. As shown in Fig. 2.9 C,
we can detect CD24 expression on the cells in CD24+/+ > CD24+/+ and CD24+/+ > CD24–/–
CNS. Those cells have been identified by the H&E staining on the consecutive sections
as blood vessel endothelial cells. At the same time, there were no CD24 positive cells in
CD24–/– > CD24–/– CNS. However, in CD24–/– > CD24+/+ CNS, a considerable amount of
CD24 positive cells were found, which can also be co located with blood vessel
endothelial cells as showed in figure 2.9 D. It is therefore likely that although WT bone
marrow can convey EAE susceptibility to CD24–/– mice, replacement with CD24–/– bone marrow in WT mice does not abolish the EAE susceptibility of the CD24+/+ mice. Thus,
either hematopoietic or non-hematopoietic CD24-expressing cells can confer
susceptibility to pathogenic T cells in the CNS. However, it is likely that the two cell
types can act synergistically, as revealed by the partial efficacy of a single cell type in
recipients after two rounds of bone marrow replacement.
29 2.5 Discussion:
We have previously demonstrated that CD24-deficient hosts resist the pathogenic
T cells adoptively transferred from WT mice. Because resistance to the adoptively transferred T cells was observed when the hosts were immune suppressed by irradiation before adoptive transfer [55], it is likely that the resistance is not due to host immunity to adoptively transferred T cells. To avoid complications associated with homeostatic proliferation in an irradiated host, we reproduced the above observation in nonirradiated recipients and followed the recruitment and local activation of autoreactive T cells in the
CNS during EAE. The data presented here make three points.
CD24 Expression in the Host Is Required for T Cell Persistence in, But Not
Recruitment to, the CNS.
Our previous studies have demonstrated that expression of CD24 on host cells is critically important for the development of EAE [55]. Because CD24 was not required for the priming of autoreactive T cells, and because CD24 was reported to modulate VLA4 interaction with VCAM-1 [29], which is known to be required for T cell recruitment , we tested whether CD24 is critical for the recruitment of T cells to the CNS by immunohistochemistry, RPA, and flow cytometry.
At 3 d after adoptive transfer, a similar number of Thy1.1+ donor cells were detected in the CNS of both CD24+/+ and CD24–/– recipients. On day 12, the number of donor T cells in the CNS was substantially less in CD24–/– mice in comparison to WT mice. However, the number of CD3+ T cells recruited to the CNS was comparable in the
30 two groups, as revealed by quantitative RPA, largely due to recruitment of host T cells.
These results demonstrate that expression of CD24 in the CNS is not required for T cell
migration into the CNS, regardless of their origin.
Despite the lack of a significant difference in T cell recruitment, we observed major
differences in the persistence of autoreactive T cells in the CNS. By day 12, the number
of donor T cells was reduced by 10-fold in the CD24–/– brain in comparison to WT
recipients. The reduction of donor autoreactive T cells was followed by a disappearance
of bystander host T cells, such that no T cells were detected in the CD24–/– mouse brain at
a time when the WT brain had seen a significant increase in total T cells. Thus, CD24
expression is required for the persistence of both antigen-specific donor cells and host T
cells with unknown specificity.
A potential caveat associated with the adoptive transfer experiment is that the
CD24 may serve as a rejection antigen when the WT T cells are injected into the CD24–/– host. We performed two types of experiments to rule out this possibility. First, we obtained serum from CD24-deficient hosts at 3 wk after they received WT T cells, and then tested for the presence of anti-CD24 antibodies. No such antibodies could be detected in the serum. Second, at 3 wk after adoptive transfer, we reinjected carboxyfluorescein succinimidyl ester–labeled, MOG-reactive WT T cells into the WT and CD24–/– recipient that had been "primed" by the previous round of adoptive transfer
and determined the amounts of donor cells in the two types of recipients. The analysis
indicated that CD24–/– recipients did not preferentially eliminate the donor T cells. Both
31 approaches suggest that it is highly unlikely that the failure of WT T cells to persist in the
CD24–/– CNS is due to host immunity specific for CD24.
The Rate of Local Expansion of Autoreactive T Cells in the CNS Is Controlled by
CD24.
Theoretically, the differential persistence of T cells in the WT and CD24–/– hosts can be attributed to two different factors. The first is the local expansion of T cells and the second is their programmed cell death. It is well established that the T cells in the
CNS are prone to programmed cell death [71, 80]. However, it is unknown whether T cells clonally expand in the CNS and if so, whether CD24 plays a role in this process.
Using a short-term BrdU labeling in vivo, we were able to clearly demonstrate substantial
T cell division in the CNS of WT mice. Because the antigen-specific T cells migrated into the CNS en masse shortly after adoptive transfer, and more importantly, because the rate of T cell division was substantially higher in the CNS than in the spleen, it is likely that the majority of BrdU incorporation in the CD24+/+ CNS took place locally. In contrast,
even though the donor T cells divided at a slightly higher rate in the spleen of CD24–/– recipients than the WT counterpart on day 3, the rate of division in the CD24–/– CNS was
less than that in the WT CNS. The reversion of the relative BrdU incorporation rates
between the spleen and the CNS of the two strains of mice reveals two interesting points.
First, the BrdU incorporation in the CNS reflects local T cell proliferation, and second,
CD24 plays a critical role in the process. Consequently, the number of donor T cells was
10-fold higher in the WT CNS on day 12 when the total numbers of T cells in CNS were
comparable between WT and CD24–/– mice.
32 Our study extends the elegant work by Flugel et al., who reported de novo
expression of activation markers on autoreactive T cells after migration into the CNS. It is
of interest to note that these authors also showed, in an adoptive transfer model of EAE,
that T cell migration into the CNS is complete within 3 d of adoptive transfer. Given the
rapid T cell death seen in WT CNS [71], it is highly likely that a more rapid T cell clonal
expansion is required for a sustained immune response. Our study indicated that by
controlling the efficacy of this process, CD24 expression on host cells constitutes a major
checkpoint of EAE pathogenesis.
Two Lineages of CD24-expressing APCs in CNS.
Recognition of CNS autoantigen is considered a prerequisite for the initiation of T
cell–mediated CNS inflammation. Recent evidence suggests that local antigen processing
and presentation in the CNS is essential for EAE development because mice deficient for
Ii and H-2M failed to develop EAE [81].. Because our work established the importance of
local T cell clonal expansion, it also raised the possibility that T cell costimulation might
be required for the process.
Our comparison of T cell proliferation in CD24+/+ and CD24–/– recipients demonstrates that CD24 is required for optimal local T cell clonal expansion in the CNS.
In support of this hypothesis, we also demonstrated that activation of T cells by astrocytes and microglia in vitro depends on CD24, as CD24+/– APCs are substantially more
efficient in inducing T cell proliferation, and that proliferation of CD24+/+ astrocytes are
blocked by an anti-CD24 antibody.
33 Our bone marrow chimera experiments revealed that expression of CD24 on one of
the two lineages of APCs is sufficient for EAE development. One of the two is bone
marrow derived. Although no genetic model is available to define other cell lineages as
the potential local APC, our in vitro analysis demonstrated that the ability of microglia to
present antigen to T cells is diminished if the CD24 gene is defective. Because
perivascular microglia, although not parenchyma microglia, are mainly bone marrow
derived [82], it is possible that this lineage of the microglia is the radiosensitive bone
marrow–derived APC.
Interestingly, we also found that CD24 expression on radioresistant cells in the
host was sufficient to convey EAE susceptibility. The identity of the radioresistant cells is
unknown. However, at least two candidates can be proposed from our in vitro data. The
first is the parenchyma microglia that has been shown to be radioresistant [83], and the
other is astrocytes. Both cell types constitutively express CD24, which can be further
elevated by inflammatory cytokines such as IFN- . Moreover, both cell types require the
CD24 gene for optimal induction of T cell proliferation in vitro.
In summary, we have shown that CD24 is a novel checkpoint for local clonal expansion of T cells in the CNS. Because there is no difference in apoptosis of donor T cells, the defective proliferation in the CD24–/– CNS is likely the primary cause for the
drastic reduction of donor T cells at the time when clinical signs of EAE were observed.
The lack of effect on T cell apoptosis indicated that the function of CD24 differs from B7-
1 and B7-2 [47]. However, it should be pointed out that this study has not definitively
proven that defective proliferation alone prevented the induction of EAE in the adoptive
34 transfer model. CD24 may mediate other functions essential for EAE development. For
instance, the transferred autoreactive T cells reside primarily in the meninges in CD24–/– mice, whereas many of them migrate into the brain parenchyma in WT mice. The significance of this observation is unknown at this point, nor is it clear if this is a direct consequence of defective T cell proliferation. Nevertheless, given the high death rate of T cells in the CNS, it is highly likely that a gene that controls the rate of proliferation of autoreactive T cells in the CNS may play an essential role for pathogenesis. This checkpoint might be particularly relevant for immunotherapy of MS for two reasons. First, this checkpoint controls the function of T cells after they are primed and have migrated to the CNS. This resembles a clinical setting, as patients who seek care already have autoreactive T cells in the lymphoid organ and/or in the CNS. Second, the significance of
CD24 in MS is demonstrated by our recent observation that CD24 polymorphism in the human is a genetic modifier for the risk and progression of MS [72].
35 Figure 2.1. CD24 is required for persistence but not recruitment of autoreactive T cells in the EAE model. (A) CD24–/– recipient mice are resistant to EAE induced by adoptively transferred T cells. WT and CD24–/– mice (five per group) received 10 x 106 MOG peptide–activated lymph node cells i.v. The clinical signs were scored daily. The data shown are means and SD of EAE scores at different times after adoptive transfer and are representative of three independent experiments. (B) Migration of adoptively transferred T cells into the brain of WT and CD24–/– mice. A MOG-immune T cell line prepared from Thy1.1 congenic mice was adoptively transferred into WT or CD24–/– hosts. At 3 d after adoptive transfer, the brains were perfused with PBS and single cell suspensions were analyzed by FACS® using anti-Thy1.1 and anti-CD4 antibodies. Data shown have been repeated at least three times.
36 Figure 2.1
37 Figure 2.2. Quantitation of T cells in the brains of WT and CD24–/– mice. (A) RPA. Mice that had received MOG-specific T cell transfers were killed at the indicated time points. Immediately after death, the mice were perfused with PBS through the left heart ventricle to wash blood out of the brain. The brain was removed and total RNA was isolated by the Trizol method. Each lane represents RNA samples mixed from two individual mice at a 1:1 ratio. A total of 20 µg RNA was used per lane. Data are representative of four independent experiments. (B) Quantitation of the relative amount of T cells based on intensity of the CD3 bands shown in A. Intensity in the negative control samples was artificially set as 1.0. (C) RPA. As described before, each lane represents RNA sample from individual mouse that had received MOG-specific T cell transfers at different time points.
38 Figure 2.2 continued
39 Figure 2.2 continued
C Day29 Day 12 WT -/- WT CD24 (-/-) WT CD24 (-/-) ctrl + - 1 2 3 4 1 2 3 4 1 2 3 1 2 3
CD3 CD4 CD8a
F4/80
CD45
Figure 2.2
40 Figure 2.3. CNS infiltration of the MOG-activated T cells from Thy1.1 congenic mice. MOG peptide–activated Thy1.1+ T cells were injected into either CD24+/+ or CD24–/– recipient mice, and 160 ng of pertussis toxin was injected i.v. at the time of T cell transfer at 48 h. On day 8, 10, 12, 20 and 40 after T cell transfer, the mice were killed and frozen brain tissue sections were prepared and stained with anti-Thy1.1 antibody. A and B represent either WT or CD24-/- mice that received MOG peptide–activated Thy1.1+ T cells. A, 50. B, 200.
41 A WT CD24(-/-)
Day 8
Day 10
Day 12
Day 20
Day 40
B
Figure 2.3
42 Figure 2.4. Requirement for CD24 for optimal T cell proliferation in the CNS. WT and CD24–/– mice adoptively transferred with congenic T cells received an i.p. injection of BrdU (1 mg/mouse) 12 h before they were killed. Immediately after death, the mice were perfused with PBS through the left heart ventricle. The brain, spinal cord, and spleen were removed, and mononuclear cells were prepared and stained for Thy1.1 marker and BrdU incorporation. (A) BrdU incorporation in antigen-specific donor T cells. Data shown are profiles of gated Thy1.1+ T cells. The percentage of donor T cells among mononuclear cells in the spleen or brain is presented in the parentheses on top of each panel, and the proportion of BrdU+ T cells are indicated underneath the gates. (B) T cell accumulation in the CNS at day 12 after T cell transfer. Note that both donor-derived and host-derived T cells accumulated in the CNS. The substantially reduced incorporation of BrdU and the accumulation of donor cells in the brain of CD24–/– mice have been reproduced in three independent experiments.
43 Figure 2.4
44 Figure 2.5. Antigen-specific T cells undergo apoptosis at similar rates in WT and CD24–/– CNS. MOG peptide–activated Thy1.1+ T cells were injected into either CD24+/+ or CD24–/– recipient mice, and 160 ng of pertussis toxin was injected i.v. at the time of T cell transfer and at 48 h. On day 10 after T cell transfer, mice were killed and CNS- infiltrating cells were prepared as described in Fig. 2.3. The CNS mononuclear cells were triple labeled for Thy1.1, 7-AAD, and annexin V markers, as described in Materials and Methods. Data presented here are gated on Thy1.1+ T cells. Two independent experiments were performed with similar results.
45 Figure 2.5
46 Figure 2.6. Cytokine gene expression in brains of WT and CD24–/– mice on days 12 and 29 after adoptive transfer of T cells. (A) An autoradiograph of a representative RPA for cytokines. (B) An autoradiograph of a representative RPA for chemokines. (C) An autoradiograph of a representative RPA for chemokine receptors. (D) An autoradiograph of a representative RPA for inflammatory cytokines.
47 A Day 29 Day 12 WT -/- WT CD24-/- WT CD24-/- + - ctrl 1 2 3 4 1 2 3 4 1 2 3 1 2 3
IL-4 IL-5 IL-10 IL-13 IL-15 IL-9
IL-2 IL-6 IFN-r
Figure 2.6 continued
48 Figure2.6 continued
Day12 Day29 B WT -/- WT CD24-/- WT CD24-/- Ctrl + - 1 2 3 1 2 3 1 2 3 4 1 2 3 4
RANTES
IP-10
Figure2.6 continued
49 Figure 2.6 continued
Day 29 Day 12 C WT -/- WT CD24-/- WT CD24-/- ctrl 1 2 3 4 1 2 3 4 3 2 1 3 2 1
CCR1
CCR3
CCR5
CCR2
Figure 2.6 continued
50 Figure 2.6 continued
D Day 29 Day 12 WT -/- WT CD24-/- WT CD24-/- + - ctrl ctrl 1 2 3 4 1 2 3 4 1 2 3 1 2 3
LTb TNFĮ
IFN-r
TGFb
MIF
Figure 2.6
51 Figure 2.7. CD24–/– CNS astrocytes had a reduced capacity to stimulate MOG- specific T cell proliferation. (A) Characterization of astrocytes prepared from newborn CD24+/– and CD24–/– littermates. GFAP was stained after permeabilization and the intact cells were used for analysis of other markers. Red lines and black lines represent profiles of fluorescence intensity of CD24+/– astrocytes when stained with either specific antibodies (red) or controls (black), and blue lines (specific staining) and green lines (control staining) depict the fluorescence intensity of CD24–/– astrocytes, as indicated by arrows. CD24 staining of IFN- –treated and –untreated astrocytes is shown, whereas only IFN- –stimulated astrocytes were analyzed for CD80, CD86, Db, and I-Ab expression. (B) The MOG-reactive T cell line (5 x 104/well) was stimulated by serial titrated astrocytes in the presence of 50 µg/ml MOG peptide. Data are representative of two independent experiments with similar results. (C) Blocking of T cell proliferation by CD24-specific antibody. CD24+/– or CD24–/– astrocytes were irradiated (3,000 rads) and seeded into U- bottomed 96-well plates in DMEM culture medium containing 100 U/ml IFN- . 3 d later, medium was discarded and a T cell line specific for MOG peptide was added into the indicated wells at a concentration of 2.5 x 104/well in the presence of given concentrations of MOG peptide. Anti-CD24 mAb 20C9 and a control hamster IgG were used at 1 µg/ml. Data are means and SD of cpm and are representative of three experiments.
52 Figure 2.7
53 Figure 2.8. CD24–/– CNS microglia had a reduced capacity to stimulate MOG- specific T cell proliferation. (A) Characterization of microglia prepared from newborn CD24+/– and CD24–/– mice. Microglia preparation was either left untreated or stimulated with 100 U/ml IFN- for 48 h. Cell surface IB4, CD80, CD86, Db, and I-Ab were analyzed by flow cytometry. Red lines and black lines represent profiles of fluorescence intensity of CD24+/– astrocytes when stained with either specific antibodies (red) or controls (black), and blue lines (specific staining) and green lines (control staining) depict the fluorescence intensity of CD24–/– astrocytes, as indicated by arrows. (B) CD24–/– brain microglia had a reduced capacity to stimulate MOG-specific T cell proliferation. Irradiated microglia were stimulated with 100 U/ml IFN- for 3 d and were used to present MOG peptide to a MOG-specific T cell line (5 x 104/well). Data are means and SD of cpm and are representative of five individual experiments.
54 Figure 2.8
55 Figure 2.9. Expression of CD24 on either hematopoietic or nonhematopoietic cells is sufficient to confer susceptibility to adoptively transferred MOG-specific T cells. (A) FACS® profiles of bone marrow chimeras at 8 wk after reconstitution. Splenocytes were stained with FITC-labeled anti-CD3, B220, CD11b and PE-conjugated anti-CD24 antibodies. (B) Induction of EAE in bone marrow chimeras. 40 x 106 MOG peptide– activated lymph node cells were injected into mice that were irradiated (350 rads) 1 h before receiving pathogenic T cells. Data are means and SEM of EAE score in groups of four mice each and are representative of three independent experiments with similar results. (C) CD24 expression on CNS endothelial cells. Frozen sections of the CNS from all the groups were stained with boitinylated anti-CD24 antibody. The consecutive sections were stained with hemotoxilyn and eosin. (D) Blood vessel endothelial cells were double stained with both biotinylated anti-CD24 antibody and FITC-conjugated anti-CD31 antibody.
56 A
WT
CD24 (-/-)
WT>WT
WT>CD24 (-/-)
CD24 CD24 (-/-)>CD24 (-/-)
CD24 (-/-)>WT
CD3 B220 Mac-1 B
4
WT n=2 WT>WT n=4 3 WT>-/- n=4 -/->WT n=4 -/->-/- n=4
2 EAE Score
1
0 0 5 10 15 20 Days after T cell transfer Figure2.9 continued
57 Figure2.9 continued
Figure 2.9
58 CHAPTER 3
CD24 EXPRESSION ON T CELLS IS REQUIRED FOR THE OPTIMAL T CELL
PROLIFERATION IN LYMPHOPENIC HOST
3.1 Abstract:
It is well established that T lymphocytes undergo homeostatic proliferation in lymphopenic environment. The homeostatic proliferation requires recognition of the major histocompatibility complex on the host. Recent studies have demonstrated that costimulation, mediated CD28, 4-1BB, and CD40, is not required for T cell homeostatic proliferation. It has been suggested that homeostatic proliferation is costimulation independent. Here, we report that T cells from mice with a targeted mutation of CD24 have a remarkably reduced rate of proliferation when adoptively transferred into syngeneic lymphopenic hosts. The reduced proliferation cannot be attributed to abnormal survival and homing properties of the CD24-deficient T cells. T cell proliferation in allogeneic hosts is less affected by this mutation. These results demonstrate a novel function of CD24 expressed on T cells. Thus, although distinct costimulatory molecules
59 are involved in antigen-driven proliferation and homeostatic proliferation, both processes can be modulated by costimulatory molecules.
3.2 Introduction:
The immune system maintains a relatively constant number of lymphocytes by two distinct mechanisms. Activation-induced cell death results [84, 85] in the removal of lymphocytes after massive antigen-induced clonal expansions. When the lymphocytes are depleted, naive T cells undergo rapid expansion to replenish the T lymphocyte pools [56,
86]. This phenomenon is termed homeostatic proliferation. Homeostatic proliferation appears to be driven by self antigens [87]. In addition, activated T cells acquire features of memory T cells [88, 89]. However, homeostatic T cell proliferation is distinct from antigen-driven proliferation in its requirements for costimulatory molecules. Although
CD28–B7 interaction is required for optimal antigen-driven proliferation [90], several groups have reported that the lack of CD28 on T cells did not have an appreciable impact in homeostatic T cell proliferation [58]. Moreover, interaction between 4-1BB and 4-
1BBL, which is required for allogeneic T cell response in vivo , is unnecessary for homeostatic proliferation [58]. Likewise, CD40–CD40L interaction, which is known to be indirectly involved in antigen-specific T cell response, is also not required for homeostatic proliferation [58]. These observations lead to the hypothesis that homeostatic proliferation is driven by TCR signal only and does not require costimulation.[41]
CD24 (heat-stable antigen) is a cell surface glycosyl-phosphatidylinositol– anchored protein [14] with a broad expression on a variety of cell types, including
60 developing T and most B lymphocytes and a variety of APCs [31]. Although CD24 is down-regulated when T cells reach maturity [15], it is rapidly induced after the engagement of the TCR–CD3 complex [23]. Although we and others have demonstrated that CD24 on APCs mediates a CD28-independent costimulatory pathway for both CD4 and CD8 T cell responses, the role of CD24 on T cells is largely unknown.[7] Here, we report that CD24 expression on T cells is required for optimal homeostatic proliferation of both CD4 and CD8 T cells. Our results reveal an important function of CD24 expressed on T cells and suggest that homeostatic proliferation of T cells is regulated by
T cell costimulation.
3.2 Material and methods:
Mice and Antibodies:
C57BL6/J and BALB/c mice were purchased from Charles River Laboratories through a contract with the National Cancer Institute. B6.Thy1.1 mice were purchased from The Jackson Laboratory. Mice with the targeted mutation of CD24 were produced using embryonic stem cells from C57BL/6 mice as described previously and were maintained under specific pathogen-free conditions in the animal facility at the Ohio
State University. All studies involving animals have been approved by the Ohio State
University Institutional Laboratory Animal Care and Use Committee.
61 All conjugated antibodies used here were purchased from either eBioscience
(Thy1.2, CD44, and CD62L) or BD Biosciences (CD4, CD8, H-2Kb, CD25 and IFN- , and isotype control).
Analysis of T Cell Division In Vivo:
T lymphocytes were purified from pooled spleen and lymph node cells by negative selection. In brief, pools of spleen and lymph node cells were incubated with a cocktail of antibodies specific for CD11b (Mac-1), Fc receptor (2.4G2), B220, and
CD11c. The Dynal beads coated with goat anti–rat IgG were used to negatively select T cells. The purity of the cells was checked by flow cytometry to be >95%. The purified T cells (Thy1.2+, H-2b) were labeled with CFSE and injected intravenously into irradiated
(600 R) recipient mice that were either congenic in Thy1 locus (B6. Thy1.1+) or allogeneic BALB/c (H-2d) mice, at a dose of 5 x 106/mouse. At given times after adoptive transfer, spleen cells were harvested and analyzed for the intensity of CFSE dye and other cell surface markers. In some experiments, mononuclear cells were harvested from the liver after perfusion with PBS as described previously.
Flow Cytometry:
The cell surface markers, including CD4, CD8, CD24, CD44, CD62L, Thy1.2, and H-2Kb, were analyzed by three- or four-color flow cytometry, using fluorochrome- conjugated monoclonal antibodies purchased from BD Biosciences. Apoptosis of T cells was analyzed by staining with PE–annexin V.
62 To assess intracellular cytokine production, spleen or lymph node cells were cultured for 4–6 h with 50 ng/ml PMA, 500 ng/ml ionomycin, and 2 µM GolgiStop (BD
Biosciences). Cells were stained for cell surface markers CD4 and CD8 followed by intracellular staining for IFN- and/or isotype control using the CytoFix/CytoPerm kit
(BD Biosciences).
3.3 Results and discussion:
Expression of CD24 on T Cells Is Not Required for Its Survival upon Adoptive
Transfer.
We analyzed CD24 expression among WT T cells before and after adoptive transfer. CD24–/– T cells were used as a control. As shown in Fig. 3.1 A, in comparison with CD24–/– T cells, WT T cells expressed significant levels of CD24 before adoptive transfer. The specificity of the antibody binding was confirmed both by the uses of isotype control and mice with targeted mutation of CD24. Upon adoptive transfer into lymphopenic hosts, the levels of CD24 were maintained from days 1 to 4 (Fig. 3.1 B).
To determine if CD24 was required for T cell survival and homing to the spleen, we compared the number of WT and CD24–/– T cells at 6 and 18 h after adoptive transfer. As shown in Fig. 3.1 C, the percentage of donor T cells in the spleen was essentially identical in the two groups at 6 h after adoptive transfer. Although the number of CD24–/– T cells was slightly lower at 18 h, the difference was not statistically
63 significant (P = 0.085). We also analyzed the rate of cell death by staining the spleen cells with Annexin V. As shown in Fig. 3.1 D, low and comparable apoptotic cells were observed in both groups. Thus, the data presented in this section demonstrated that expression of CD24 does not affect distribution and survival of T cells upon adoptive transfer.
CD24 Expression on T Cells Is Necessary for Homeostatic Proliferation, but Less So for Response to Allogeneic Antigens.
To test whether CD24 expression on T cells plays a role in homeostatic proliferation, we isolated T cells from WT and CD24–/– C57BL/6 mice and injected them into irradiated Thy1.1 congenic mice. At 4 d after adoptive transfer, we isolated the spleen cells and compared them for the rate of division based on CFSE intensity. As shown in Fig. 3.2 A, a substantial proportion of WT CD4 T cells had undergone one or more divisions, whereas few CD24–/– CD4 T cells had divided. In comparison, CD8 T cells divided faster than CD4 T cells. The majority of WT CD8 T cells accumulated was the product of two to four divisions. In contrast, the majority of CD24–/– CD8 cells had not undergone any divisions. Correspondingly, the percentage of WT donor cells was fourfold greater than that of the CD24–/– donors (Fig. 3.2, B and C), although it is unclear whether the difference can be solely attributed to the rates of proliferation. Over a
6-d period, the difference in division further increased (Fig. 3.2 D). The differential division rate resulted in an almost 20-fold difference in the number of donor T cells in the recipients at 3 wk after adoptive transfer (Fig. 3.2 E). Consistent with previous papers [58,
64 86], targeted mutation of CD28 had no effect on T cell homeostatic proliferation in our
model.
An interesting issue is whether CD24 is required for antigen-driven proliferation.
To analyze this issue in a polyclonal T cell pool, we transferred CFSE-labeled WT and
CD24–/– T cells into irradiated BALB/c mice. Because of a strong allogeneic-reactive T
cell response, a robust proliferation of donor T cells was observed, regardless of whether
WT or CD24–/– T cells were used as donors. As shown in Fig. 3.3 A, >30% of the spleen cells in the host spleen are of donor origin. In the allogeneic hosts, the donor cells were larger in size in comparison with the host cells. Although the amount of WT T cells was consistently higher than that of the CD24–/– T cells, the difference was usually not >40%
(Fig. 3 B). When the number of T cell divisions was analyzed by CFSE intensity of the donor cells, it was clear that most of the WT and CD24–/– donor T cells had undergone
more than seven divisions. Among CD4 T cells, the distribution of CFSE intensity was
grossly similar between WT and CD24–/– donors (Fig. 3 C, left). However, among the
CD8 compartment, more WT than CD24–/– T cells reached the maximal number of
divisions that can be traced by CFSE (Fig. 3 C, right). Thus, CD24 is less crucial for the
division of T cells in response to allogeneic antigens in vivo, especially for CD4 T cells.
The difference of 40%, mostly among CD8 T cells, can be attributed to the role of CD24
in homeostatic proliferation and/or antigen-specific proliferation. Because the host is both
allogeneic and lymphopenic, the contribution of each component cannot be easily
determined at this point. The lesser contribution of CD24 to antigen-driven proliferation
65 is consistent with our previous reports that the targeted mutation of CD24 alone does not
have a significant impact on CD4 and CD8 T cell responses [77, 91].
It has been reported that homeostatic proliferation of T cells is accompanied by the
acquisition of memory markers. To test whether CD24 plays a role, we analyzed the
memory markers on WT and CD24–/– T cells, including CD44 and CD62L. The data are
presented in Fig. 3.4 A. Consistent with previous reports, the majority of WT CD8 T cells
expressed high levels of CD44 upon adoptive transfer. A significant proportion of donor
cells also down-regulated CD62L. Compared with CD8 T cells, there was less up-
regulation of CD44, but more down-regulation of CD62L among WT CD4 T cells. As
expected, the CD24–/– cells were less activated. This was demonstrated by substantially
fewer CD62LlowCD44high cells and more CD44lowCD62Lhigh cells.
Memory T cells differ from naive T cells in their ability to produce cytokine within
hours of stimulation. We stimulated spleen cells from mice that received WT or CD24–/–
T cells with PMA and ionomycin and analyzed the intracellular IFN- production. As
shown in Fig. 3.4 B, 12% of the WT CD4 cells synthesized IFN- within 6 h of
stimulation. In the same experiments, the percentage of IFN- –producing cells among the
CD24–/– CD4 T cells was 2.5–3-fold less than their WT counterpart. Although more
CD8 T cells produced IFN- , there was also a two- to threefold reduction among CD24–/– donor cells in the percentage of cytokine-producing cells.
66 –/– Reduction of Dividing CD24 T Cells Was Not Due to Accelerated Apoptosis.
A potential explanation for the reduced number of divided CD24–/– T cells in the
lymphopenic host is that CD24 may be required for the survival of dividing T cells. To
test this possibility, we analyzed the apoptosis of the dividing T cells based on their
binding to annexin V and intensity of CFSE. As shown in Fig. 3.5 A, very few, if any,
annexin V+ donor T cells were found in the spleen, and the proportion of annexin V+ cells was similar in the two groups. Because the liver is the site for apoptosis of activated T cells, one may expect an increase in CD24–/– T cells in the liver if CD24 deficiency
promoted T cell death. Therefore, we analyzed migration of donor T cells in the liver. As
shown in Fig. 3.5 B, approximately three- to fivefold fewer CD24–/– than WT T cells
were detected in the liver. Moreover, the majority of WT T cells in the liver were
products of multiple rounds of divisions, whereas CD24–/– T cells barely divided. Thus,
the reduction of the dividing CD24–/– T cells was neither due to accelerated apoptosis nor trafficking to liver. Nevertheless, it is unclear at this stage whether the difference in the accorded numbers of accumulated T cells in the spleen is solely due to differential proliferation of T cells.
Together, the data reported here revealed a crucial role for CD24 in homeostatic
proliferation of T cells in a lymphopenic environment. Although CD24 is widely used as
a marker for T cell differentiation, its function has remained elusive. Hubbe et al. reported
that anti-CD24 can synergize with anti-CD28 in inducing T cell proliferation in vitro [23],
which suggests that the CD24 expressed on T cells is capable of transducing a
costimulatory signal. In addition, we showed that although CD24 was not essential in the
67 priming of autoreactive T cells in the mouse model of experimental autoimmune
encephalomyelitis, CD24–/– T cells failed to induce experimental autoimmune
encephalomyelitis when transferred in WT hosts [55]. It would be of interest to determine
whether its contribution to homeostatic proliferation explains its critical role in the
pathogenesis of CD4 T cells in the experimental autoimmune encephalomyelitis model.
Much like antigen-driven proliferation, homeostatic proliferation requires the
expression of MHC molecules and intact antigen-processing machinery [87]. Thus, both
processes require engagement of the TCR. However, unlike antigen-specific T cell
responses, homeostatic proliferation was not affected by blocking costimulation mediated by CD28–B7, CD40–CD40L, and 4-1BB–41BB-L interactions [87]. The latter observation leads to the hypothesis that homeostatic proliferation is independent of costimulation [58]. Our results demonstrated that CD24 on T cells plays a major role in the homeostatic proliferation of T cells in the lymphopenic environment. To our knowledge, this is the first demonstration of any costimulatory molecules playing a significant role in homeostatic proliferation.
68 Figure 3.1. Expression of CD24 on T cells does not affect the distribution and survival of T cells upon adoptive transfer to lymphopenic host. (A) WT (top) and CD24–/– (bottom) spleen cells were stained with anti-CD3 in conjunction with either anti- CD24 or isotype control. (B) Levels of CD24 expression on T cells on days 1 (left) and 4 (right) after adoptive transfer into a lymphopenic environment. Purified T cells (Thy1.2+) were adoptively transferred into Thy1.1+ congenic mice that received 600 R of - irradiation before adoptive transfer. At different times after adoptive transfer, the spleen cells were analyzed. Data shown are FACS profiles of a representative mouse in each group and have been reproduced at least three times. (C) Percentage of donor T cells in the recipient at 6 and 18 h after adoptive transfer. Data shown are a summary of three to five independent experiments involving at least eight mice per group. The apparent reduction observed at 18 h is not statistically significant (P = 0.085). (D) Apoptosis of newly transferred T cells was not affected by CD24. Spleen cells were harvested at 18 h after adoptive transfer and stained with Thy1.2 (to marker donors), CD4, and annexin V. Data shown are profiles of gated Thy1.2 donor cells and have been reproduced three times.
69 A Total WT
10000 10000
34.1 0.71 1000 23.2 21.1 1000
CD3 100 100
10 1.13 54.6 10 1.39
1 1 10 100 1000 10000 1 1 10 100 1000 10000 CD24 Total CD24-/- Isotype 10000 10000 31.5 0.78 1000 31 1.08 1000
100 100
10 10 4.12 2.02 1 1 1 10 100 1000 10000 1 10 100 1000 10000 CD24 Isotype B Day 1 Gated WT donor T Day 4
10000 10000 46.2 15.5 41.8 8.35 1000 1000
100 100 CD4
10 10 11.7 17.4 1 1 1 10 100 1000 10000 1 10 100 1000 10000 CD24 CD24
Figure 3.1 continued
70 Figure 3.1 continued
D
C 10000
1000 1.55 1.69 7 6 CD24+/+ 100 Annexin V CD24-/- WT 5 10 4 44.5 1 3 1 10 100 1000 10000
2 10000 1.84 1.45 % of Total Splenocytes 1 1000 0
6 h 18 h 100 Time after Adoptive transfer
10 CD24-/- 37.5 1 1 10 100 1000 10000 CD4
Figure 3.1
71 Figure 3.2. A critical role for CD24 on T cells in homeostatic proliferation in a lymphopenic host. Purified T cells (Thy1.2+) were adoptively transferred into Thy1.1+ congenic mice that received 600 R of -irradiation before adoptive transfer. At different times after adoptive transfer, the spleen cells were analyzed for the rate of division (A and D) and accumulation of donor cells (B, C, and E). Donor cells used in A–D were labeled with CFSE before adoptive transfer, whereas those in e were not labeled. Data in A are representative profiles of groups of two mice and have been repeated four times. Data in B are representative profiles of gated Thy1.2+ T cells from five independent experiments, each involving three mice per group. Data in d are representative profiles of an experiment with three mice per group. Data in c are summary of three experiments in which the initial "take" of T cells was confirmed identical (Fig. 3.1). Similar data were confirmed with four other experiments in which the initial takes of T cells were not monitored. Data in e are a summary of an experiment involving four mice per group.
72 A 100 100
80 80 WT 60 60
40 40 CD24-/-
Cell Number 20 20
0 0 1 10 100 1000 10000 1 10 100 1000 10000 CFSE intensity
B C 10000
1000 1 9.58 100 1 Thy1.2 10 1 tes
1 y 0 200 400 600 800 1000 8 lenoc
10000 p 6 P=0.0002 1000 % of S 1.42 4 100 2 10
1 0 0 200 400 600 800 1000 CD24+/+ CD24-/- Forward Figure 3.2 continued
73 Figure 3.2 continued
D 100
80 CD24-/- Day 4
60
40 WT
Cell numbers 20
0 100 1 10 100 1000
80 Day 6
60
40
20
0 1 10 100 1000 10000 E CFSE intensity
16 800
14 700 )
12 -4 600
10 500
8 400 Splenocytes % Splenocytes %
+ 6 300
4 P=0.019 200 Thy1.2 Total Cell Number(X10 P=0.048 2 100
0 WT CD24-/- 0 WT CD24-/- Figure 3.2
74 Figure 3.3. Alloantigen-driven T cell proliferation is less dependent on CD24 expression on T cells. T cells from WT or CD24–/– C57BL/6 mice were labeled with CFSE and adoptively transferred into irradiated BALB/c mice. At 3 d after adoptive transfer, the spleen cells were analyzed for the accumulation (A and B) and proliferation of donor T cells. (A) Percentage of donor T cells in the host spleen cells at 3 d after adoptive transfer. (B) Summary of experiments involving three mice per group. (C) Division of CD4 (left) and CD8 (right) donor T cells in the host spleen at 3 d after adoptive transfer. Data shown are representative of three independent experiments, each involving three mice per group.
75 A B
10000 7 1000 56.2 b 100 6 H-2 P=0.013 10 5
1 0 200 400 600 800 1000 4
10000 3 1000 33.4 2 100 Percentage of Splenocytes
10 1
1 0 200 400 600 800 1000 0 WT CD24-/- Forward scatter C CD4 CD8 100 100 WT CD24- 80 80
60 60
40 40 Cell numbers 20 20
0 0 1 10 100 1000 10000 1 10 100 1000 10000 CFSE
Figure 3.3
76 Figure 3.4. (A) Expression of CD24 on T cells promotes acquisition of memory cell markers. WT or CD24–/– T cells were adoptively transferred into the irradiated congeneic mice. At 4 d after adoptive transfer, the spleen cells were harvested and analyzed for acquisition of memory markers. The differential expression of activation markers has been reproduced in three independent experiments, each involving two to three mice per group. (B) Production of IFN- after short-term stimulation with PMA and ionomycin. Spleen cells from 4-d reconstituted mice were harvested and stimulated for 6 h. The intracellular IFN- was analyzed by flow cytometry. Pools of three spleens were used in each group. Data shown are representative of two independent experiments.
77 CD4 T cells CD8 T cells A 10000 10000 23.9 47 13. 56.8 1000 1000 FL 1- H: 100 C 100 D 44
CD4 FI 10 10 WT 6.3 22.7 6.3 22.9 1 1 1 10 100 1000 10000 1 10 100 1000 10000 FL2-H: CD62L PE FL2-H: CD62L PE
10000 10000
8.9 52.2 6.31 28.3 1000 1000
100 100
10 10
4.3 34.5 7.51 57.8 CD24-/- 1 1 1 10 100 1000 10000 1 10 100 1000 10000
B CD62 10000 10000 10000
1000 11.6 1000 46.3 1000 1.38
100 J 100 100 WT Isotype Isotype IFN 10 10 10
1 1 1 1 10 100 1000 10000 1 10 100 1000 10000 1 10 100 1000 10000
10000 10000 10000 0.73 1000 4.71 1000 22.4 1000
100 100 100
10 10 10 CD24-/-
1 1 1 1 10 100 1000 10000 1 10 100 1000 10000 1 10 100 1000 10000 CFSE Figure 3.4
78 Figure 3.5. CD24 deficiency promotes neither apoptosis of dividing T cells nor their migration into the liver. WT or CD24–/– T cells were adoptively transferred into the irradiated congeneic mice. At 4 d after adoptive transfer, the spleen cells (A) or liver mononuclear cells (B) were harvested and analyzed for apoptosis (A), accumulation (B, top), or rate of division in the liver (B, bottom). Data shown in A and B (bottom) are gated donor cells, whereas those in B (top) are gated liver mononuclear cells. Data shown are representative of two to three independent experiments, each with two to three mice per group.
79 10000 A 1000 4.48
Annexin V 100 WT
10
1 1 10 100 1000 10000 10000
1000 3.54 100
10 CD24-/-
1 1 10 100 1000 10000 CFSE intensity WT CD24-/- B. 10000 10000 2.47 0.7 1000 1000
100 100 Thy1.2
10 10
1 1 0 200 400 600 800 1000 0 200 400 600 800 1000 Forward scatter
100
80 WT CD24-/- 60
40 Cell numbers 20
0 1 10 100 1000 10000 CFSE intensity
Figure 3.5
80 CHAPTER 4
MASSIVE AND DESTRUCTIVE T-CELL RESPONSE TO HOMEOSTATIC CUE
IN THE CD24-DEFICIENT LYMPHOPENIC HOSTS
4.1 Abstract:
In response to lymphopenic cue, T lymphocytes undergo a slow-paced homeostatic proliferation in an attempt to restore the T cell cellularity. The molecular interaction that maintains the pace of homeostatic proliferation is unknown. Here, we report that in the lymphopenic CD24-deficient mice, T cells launch an uncontrollable proliferation that results in rapid death of the recipient mice. The dividing T cells have the phenotypes similar to those activated by cognate antigens. In addition, the CD24- deficient DC have superior capacity to drive homeostatic proliferation of the syngeneic cells. Taken together, our data demonstrate that CD24 expressed on the host antigen- presenting cells limits T cell response to homeostatic cue and prevents fatal damage associated with the uncontrolled homeostatic proliferation.
81 4.2 Introduction:
The immune system employs multiple mechanisms to maintain a relatively
constant number of lymphocytes [64]. Expansion of antigen-specific lymphocytes during
the immune response to infection results in a large increase in the cellularity of the
secondary lymphoid organ, which is normally followed by activation-induced cell death
[84, 85]. On the other hand, T lymphocytes spontaneously divide when the hosts are
lymphopenic. Lymphopenia is found in new borne animals [56, 92, 93], baring certain
gene mutation, and in those exposed to chemotherapy or irradiation [58]. Because the
latter event was viewed as host attempts to restore the lymphocyte cellularity, it is often
referred as homeostatic proliferation [1].
Homeostatic proliferation is similar to antigen-driven proliferation in their requirement for the MHC-TCR interaction [58]. Additional evidence demonstrates that the peptide presented by MHC matters as a diversity of peptide is apparently involved
[64]. However, these two types of T cell-proliferation differ in several important ways.
First, the homeostatic proliferation appears to be polyclonal and results in preservation of the TCR repertoire for the future combat of infection, while antigen-driven proliferation resulted in expansion of T cells specific for the antigens involved. Second, homeostatic proliferation is significantly slower than antigen-driven proliferation [64]. This, together with acute activation markers, such as CD44 and CD62L on the dividing T cells suggests that the signals received by the T cell undergoing homeostatic proliferation is lower than those delivered by a typical antigenic engagement [88]. Thirdly, homeostatic proliferation uses distinct costimulatory pathways than antigen-driven proliferation [58].
82 For instance, while B7-CD28 interaction has a major impact in antigen-driven proliferation [87], it is dispensable for antigen-driven proliferation. Like wise, CD40-
CD40L [58], 4-1BB-4-1BBL [94] interactions are also not required for homeostatic proliferation. On the other hand, we have recently reported that CD24-expression on T cells appears essential for homeostatic proliferation [95], but not obligatory for antigen- driven proliferation [55].
Since polyclonal activation of T cells by TCR agonists are usually associated with deleterious consequences [94], homeostatic proliferation, while essential, must be under strict control. The molecular machinery that controls the rate of homeostatic proliferation is unknown at this stage. A systematic approach to identify such mechanism is hampered by the fact that the nature of the molecular cues that leads to homeostatic control remains poorly understood [64]. We have recently reported that CD24 expression on the T cells is essential for homeostatic T cell proliferation [95]. It was unclear whether CD24- expressed on host cells is involved for T cell homeostasis. Here we report a serendipitous observation that in the lymphopenic CD24-deficient host, the T cells undergo uncontrolled homeostatic proliferation that leads to rapid death of the recipients. The uncontrolled proliferation is due to superior stimulatory activity of dendritic cells generated from the CD24-deficient mice. Our results identify CD24 as a key regulator for T cell response to homeostatic cue in the lymphopenic host.
83 4.3 Materials and methods:
Mice and antibodies:
C57BL6/J and BALB/c mice were purchased from Charles River Laboratories
through a contract with the National Cancer Institute. B6.Thy1.1 mice were purchased
from The Jackson Laboratory. Mice with the targeted mutation of CD24 were produced
using embryonic stem cells from C57BL/6 mice as described previously [25]. Mice with
CD24 expression on T cells were produced as described previously and were breeding
into CD24 -/- background. All the mice were maintained under specific pathogen-free
conditions in the animal facility at the Ohio State University. All studies involving
animals have been approved by the Ohio State University Institutional Laboratory
Animal Care and Use Committee.
All conjugated antibodies used here were purchased from either eBioscience
(Thy1.2, CD44, and CD62L) or BD Biosciences (CD4, CD8, H-2Kb, CD25 and IFN- , and isotype controls).
Analysis of T cell division in vivo:
T lymphocytes were purified from pooled spleen and lymph node cells by negative selection. In brief, pools of spleen and lymph node cells were incubated with a cocktail of antibodies specific for CD11b (Mac-1), Fc receptor (2.4G2), B220, and
CD11c. The Dynal beads coated with goat anti–rat IgG were used to negatively select T cells. The purity of the cells was checked by flow cytometry to be >95%. The purified T cells congenic in Thy1 locus (B6. Thy1.1+) were labeled with CFSE and injected intravenously into irradiated (600 R) recipient mice that were WT or CD24-/-(B6,
84 Thy1.2+) mice, at a dose of 5 x 106/mouse. At given times after adoptive transfer, spleen
cells were harvested and analyzed for the intensity of CFSE dye and other cell surface
markers.
Bone marrow reconstitution:
Bone marrow cells from WT B6 mice and CD24-/- mice were transferred back to
1,000 rad irradiated CD24-/- mice. 4 weeks later, the CD24 expression was detected in
both groups to determine the reconstitution efficiency.
Dendritic cells culture from bone marrow:
Bone marrow cells from WT B6 mice or CD24-/- mice were cultured with 10%
RPMI and recombine murine GM-CSF as described before [96]. LPS was added into
culture after day 10. The non-adherent cells harvested from the culture are mature
dendritic cells.
Dendritic cells from spleen:
Spleens were harvested from sublethally irradiated WT B6 mice and CD24-/-
mice. The spleens were dissected into small fragments and digested with 10 Pg/ml of
Type IV collagenase for 30 minutes at 37oC. The cells were harvested and analyzed by
flow cytometry.
T Cell-DC Coculture:
For suspension cultures, CFSE-labeled Thy1.1 T cells (usually 400,000/well)
were mixed with various numbers of purified DC in 24-well plates in RPMI
1640 medium. Cells were cultured at 37° in a humidified 5% CO2 incubator for 5 days
before analysis.
85 Flow Cytometry:
The cell surface markers, including CD4, CD8, CD24, CD44, CD62L, and Thy1.1, were analyzed by three- or four-color flow cytometry, using fluorochrome-conjugated monoclonal antibodies purchased from BD Biosciences. Apoptosis of T cells was analyzed by staining with PE–annexin V.
To assess intracellular cytokine production, spleen or lymph node cells were cultured for 4–6 h with 50 ng/ml PMA, 500 ng/ml ionomycin, and 2 µM GolgiStop (BD
Biosciences). Cells were stained for cell surface markers CD4 and CD8 followed by intracellular staining for IFN- and/or isotype control using the CytoFix/CytoPerm kit
(BD Biosciences).
4.4 Results
Uncontrolled homeostatic proliferation of syngeneic T cells in lymphopenic CD24- defieicnt host
We transferred CFSE labeled, purified naive T cells from Thy1.1 congenic mice into sublethally irradiated WT B6 mice and CD24-/- B6 mice. The spleens were harvested from the recipients 4 days after adoptive transfer. Cell division was measured by CFSE dilution by flow cytometry. As shown in Fig. 4.1 A, the transferred CD4 T cells from WT B6 recipients underwent two divisions. At the same time, CD8 T cells underwent 4 divisions. On the other hand, both populations of the transferred T cells in
CD24-/- mice underwent much accelerated division, more than 7 generations. We then
86 carried out a kinetic study to substantiate this surprising observation. On day one, the T
cells in both recipients maintained the same single CFSE fluorescence density (Fig. 4.1
B). However, on day 2, when little change happened to the transferred T cells in WT B6
recipients, the T cells in CD24-/- recipients already started dividing. By day 3, in WT B6
mice, when CD8 T cells divided 2 times and CD4 T cells just started to divide, both CD4
and CD8 T cells in CD24-/- recipients already divided 5 times. Thus, although the same
amount of naïve T cells were transferred into both groups, T cells started to divide much
earlier and faster in CD24-/- recipients. Consistent with previous papers, targeted
mutation of both B7 molecules had no effect on T cell homeostatic proliferation in our
model (Figure 4.1 C).
In the CD24-deficient mice, cells undergoing homeostatic proliferation displayed
markers of T cells induced by cognate antigen.
Previous studies have demonstrated that T cells undergoing homeostatic
proliferation have the markers of central memory cells, namely, they express high levels
of CD44, but generally fail to down regulate CD62L [88]. On the other hand, most of the
antigen-induced T cells down-regulates CD62L [93]. As shown in Fig. 2, regardless of
the CD24-status in the host, T cells acquired memory cell phenotype with high level
CD44 expression (Fig. 4.2 A). However, the majority of T cells from CD24-/- recipients
but not those from the WT mice have down-regulated 62L (Fig. 4.2 B). Nevertheless,
comparable T cells from both groups can produce IFN- Ȗupon a short-term stimulation with PMA and ionomycin (Fig. 4.2 C). Thus, in the CD24-/- lymphopenic host, T cell response to homeostatic cue resembles those to antigenic stimuli.
87 The highly divided T cells underwent apoptosis in CD24-/- recipients.
With T cells high division rate, we expected to recover more transferred cells from CD24-/- recipients. However, there is no difference for the total spleen cells recovery. The percentages of CD4+ and CD8+ T cells among total splenocytes were comparable between the two groups. As shown in figure. 4.3 A, we observed totally 41
WT B6 recipients and 67 CD24-/- recipients. The difference in all the subpopulations between both groups was not statistically significant. The P values were P= 0.36 in CD4
T cells, P= 0.25 in CD8 T cells and P=0.92 in total T cells.
To determine whether the low yield of T cells from CD24-/- recipients is due to
T cell apoptosis [85], we used Annexin V staining to detect apoptotic cells. As shown in figure. 4.3 B, on day one, about 5% of T cells in both recipients are Annexin V positive.
However, on day four, although the percentage of apoptotic cell was still 5% in WT recipients, there were more than 30% of transferred T cells that underwent apoptosis in
CD24-/- recipients. Thus, a large portion of the highly activated T cells in CD24-/- recipients underwent apoptosis. The data indicates that, at least at early stage, the absence of CD24 on lymphopenic host caused T cell to divide rapidly and become vulnerable for apoptosis.
Expression of CD24 on T cells does not restore regulation of homeostatic proliferation in the CD24-/- host
We have reported that CD24 expressed on T cells is required for T cell homeostatic proliferation. An interesting possibility is that the lack of CD24 expression on T cells might have increased the levels of its ligand in the host cells. To test the
88 consequence of CD24 overexpression on T cells alone, we used a transgenic mouse developed in our lab to address this question. The transgenic mouse has CD24 gene under the proximal lck promoter which resulted in higher expression of CD24 in T cells [27].
The transgenic allele was then bred into the CD24-/- background to generate mice that express CD24 in T cell lineage. We transferred CFSE-labeled purified Thy1.1 congenic
T cells into this transgenic CD24TGCD24-/- model. As shown in figure 4.4, both CD4 and
CD8 T cells in CD24-/- recipients divided more than 7 generations, which was comparable to CD24-/- recipients divided at the same rate. Thus, CD24 expression on T cells doesn’t dampen the host’s ability to drive homeostatic proliferation.
Superior stimulatory activity of the CD24-/- dendritic cells.
To determine the cell type that is required to express CD24 to regulate T cell proliferation in lymphopenic environment, we transferred WT and CD24-/- bone marrow into lethally irradiated CD24-/- mice. After 4 weeks, we transferred CFSE labeled, purified naïve T cells from Thy1.1 congenic mice into both recipients, which have been sublethally irradiated before T cell transfer. We harvested spleens from both groups and analyzed CFSE dilution in the adoptive transferred T cells. As shown in Fig 4.5 A, in
CD24-/- bone marrow reconstituted recipients, both CD4 and CD8 T cells had undergone massive proliferation over the four day period, as expected. In contrast, much less proliferation was observed in the chimera mice reconstituted with WT bone marrow cells.
Expression of CD24 on bone marrow-derived cells was both necessary and sufficient to control the pace of homeostatic proliferation.
89 Among the bone-marrow-derived antigen presenting cells, B cells and dendritic cells constitutively express CD24, while macrophage expressed CD24 after their phagocytosis. To determine whether CD24 expression on B cells is required to modulate
T cell homeostatic proliferation, we transferred CFSE-labeled naïve T cells into sub- lethally irradiated RAG-1-/- and WT mice. After 4 days, the spleens were harvested from both groups and analyzed by flow cytometry. As shown in Fig. 4.5 B, T cell proliferation was essentially indistinguishable in WT and RAG-1-/- host. Thus, the pace of homeostatic proliferation is maintained in the absence of CD24-expressing B cells.
Two laboratories have established that both mouse and human DC can induce T cell homeostatic proliferation in vitro [97, 98]. To determine whether lack of CD24 on the dendritic cells promote their ability to induce homeostatic proliferation, we generated dendritic cells from both WT and CD24-/- bone marrow. We then cultured CFSE-labeled purified naïve Thy1.1 congenic T cells with the DC at 1:2 ratio. After 5 days, we analyzed CFSE dilution to determine T cell proliferation. As shown in figure 4.6 A, T cells cultured with WT B6 bone marrow derived dendritic cells only had undergone two divisions, while those co-cultured with CD24-/- dendritic cells divided more than 5 times.
Thus, CD24-deficient DC has superior stimulatory activity for homeostatic T cell proliferation.
To test the possibility that CD24-/- DC may express higher level of other molecules that are important for T cell homeostatic proliferation, we compared the expression level of co-stimulatory molecules and MHCs on both WT and CD24-/- bone marrow derived DC. As showed in Figure 4.6 B, both groups express similar level of
90 CD40, B7-1, B7-2, I-Ab and H-2Db. The similar result was also found from the dendritc
cells harvested from sub lethal irradiated WT and CD24-/- spleens (Figure 4.6 C).
Therefore, our data indicated that it is the CD24 on dendritic cells that regulated T cell
homeostatic proliferation.
Thus, our data demonstrated lack of CD24 on DC, but not those on B cells and
macrophages explain the massive homeostatic proliferation of T cells in the CD24-
deficient host.
Fatal destruction of CD24-deficient host associated with mass T cell proliferation
Surprisingly, around 60% of the CD24-/- recipients died within 2 weeks after T
cell transfer, while all the WT recipients remained healthy (Fig. 4.7 A). The death is
caused by adoptively transferred T cells as the control mice that received no T cells are
healthy. We have systematically examined the host for histological sign of acute graft vs
host diseases [85, 99, 100]. As shown in Fig. 4.7 B, no inflammation is observed in the
gut of the CD24-deficient host. Similarly, no inflammation was observed in any other
organs examined. Thus, the destruction of the recipient is pathologically distinct from
graft vs. host diseases.
Our analysis of the moribound mice revealed a massive increase of activated T
cells in the spleen (Fig. 4.7 D). Thus, it is likely that the lethality is associated with
massive T cell activation. It has been reported that TNF-a, which is produced by activated
T cells, can cause cytokine shock when presented at large amount in vivo [101]. We first
used intracellular staining to detect TNF-a and IFN-Ȗ production in the transferred T cells from the surviving mice in the long term experiment. As shown in figure. 4.7 C, large
91 amounts of TNF-Į expression was detected in both groups upon a short-term stimulation with PMA and ionomycin. However, further experiments are needed to identify the particular cytokine that is responsible for the fatal effect in the CD24-/- mice.
4.5 Discussion:
Although CD24 has been widely used as hematopoietic cell differentiation marker
[13], it can also provide CD28-independent costimulation for both CD4 and CD8 T cells.
However [7, 16], CD24 is not essential for foreign antigen-driven T cell priming. The data published by our group showed that CD24 provides co-stimulation to the antigen specific T cells for secondary activation in the target organs [55]. Here, our data showed the expression of CD24 on hosts as a novel check point for T cell homeostatic proliferation in lymphopenic environment.
T cells proliferation in lymphopenic environment does not depend on costimulatory signals through B7-CD28, CD40-CD40L or 4-1BB-4-1BBL [58, 94, 97].
CD24, when expressing on T cells, is the first co-stimulatory molecule to be reported to play a important role on T cell proliferation induced by lymphopenia [95]. However, the expression of CD24 on lymphopenic hosts has totally different function. It has been reported that the expression level of MHC and some cytokines like IL-21 can regulate naïve T cell homeostatic proliferation [62, 92]. CD24 here showed more effective function, for T cells have undergone more than 7 divisions in CD24-/- mice in 4 days, which is comparable to the proliferation induced by foreign antigen. As showed by other groups, the divided T cells acquire activated/memory T cell phenotype and can produce
92 large amount of cytokines with short-term stimulation. The T cells from CD24-/- recipients showed even stronger cytokine production. Moreover, the large amounts of inflammatory cytokine production may lead to fatal shock in the recipient. However, further experiments are still needed to identify the putative cytokine that is responsible.
As dendritic cells showing dominant ability to stimulate antigen specific T cell response, other groups also showed that dendritic cells can also regulate naïve T cell homeostatic proliferation[97] . Although there is no difference on the expression level of MHC and other costimulation molecules on DC between WT and CD24-/- mice, CD24 expression on dendritic cells is required to control naïve T cell homeostatic proliferation in the lymphopenic environment.
We presented here a novel function of CD24 and provide a unique regulation mechanism for T cell homeostatic proliferation. However, CD24 is expressed in a broad range of cell types, including mature B cells [30]. As reported, B cell and T cell homeostatic proliferations are independent of each other [64]. As another major effecter in adaptive immunity, the further exploration of CD24 effect on B cell homeostatic proliferation may provide us better understanding on how immune system maintains its ability to response to attacks.
Homeostatic proliferation has been a major concern in organ transplantation
[94]. Since the patients are usually lymphopenic, induced for acceptance of the transplant
[94], the expression level of CD24 on the donor T cells and recipients may play an important role in GVHD and be equally important. Recent studies have been focusing on the connection between homeostatic proliferation and autoimmune diseases. In human,
93 lymphopenia is observed from patients suffering from IDDM [68], rheumatoid arthritis
[66] or lupus [66, 102]. HIV infected patients often show signs of autoimmunity [65]. We presented here a novel function of CD24 and provide a unique regulation mechanism for
T cell homeostatic proliferation. We hope the unique function of CD24 presented in this paper can provide some new information for future studies.
94 Figure 4.1. Naïve T cells underwent uncontrolled homeostatic proliferation in lymphopenic CD24-deficient host. Purified T cells (Thy1.1+) were labeled with CFSE and adoptively transferred into Thy1.2+ either WT or CD24-/- mice that received 600 R of -irradiation before adoptive transfer. (A)When gated on Thy1.1 positive T cells, in WT recipients, CD4 T cell divided two times and CD8 T cells divided 4 times. However, both CD4 and CD8 T cells in CD24-/- recipients divided more than 7 times. (B) At different times after adoptive transfer, the spleen cells were analyzed for the rate of division. The transferred T cells in CD24-/- recipients started proliferation on day 2 and divided much faster that the T cells in WT recipients.
95 A 100
80 CD24-/- WT 60
40 CD4
20
0 100 101 102 103 104
100
Relative Cell Number 80
60 CD8
40
20
0 100 101 102 103 104
CFSE Figure 4.1 continued
96 Figure 4.1 continued
B Thy1.1 T > WT Thy1.1 T >CD24-/-
1000 1000
800 800 600 600 Day 1 400 400
200 200
0 0 100 101 102 103 104 100 101 102 103 104
1000 1000
800 800
600 600
400 400 Day 2
200 200
0 0 100 101 102 103 104 100 101 102 103 104
FSC 1000 1000
800 800 600 600 Day 3 400 400
200 200
0 0 100 101 102 103 104 100 101 102 103 104 CFSE
Figure 4.1 continued
97 Figure 4.1 continued
C 100
80 WT
60
40 CD4
20 Relative Cell Number 0 100 101 102 103 104
100
80
60 CD8
40
20
0 100 101 102 103 104 CFSE
Figure 4.1
98 Figure 4.2. In the CD24-deficient mice, cells undergoing homestatic proliferation displayed markers of T cells induced by cognate antigen. CFSE-labeled Thy1.1+ T cells were adoptively transferred into the irradiated WT and CD24-/- mice. At 4 d after adoptive transfer, the spleen cells were harvested and analyzed for acquisition of memory markers. (A, B) The divided T cells in CD24-/- group express high level of CD44 and down-regulated 62L. The differential expression of activation markers has been reproduced in three independent experiments, each involving two to three mice per group. (C) Production of IFN- after short-term stimulation with PMA and ionomycin. Spleen cells from 4-d reconstituted mice were harvested and stimulated for 6 h. The intracellular IFN- was analyzed by flow cytometry. Pools of three spleens were used in each group. Data shown are representative of two independent experiments.
99 A Thy1.1 T > WT Thy1.1 T >CD24-/- 104 104
103 103 CD44
102 102
101 101
100 100 100 101 102 103 104 100 101 102 103 104 B CFSE 4 10 104 16 54.8 54.8 28.1
103 103 CD44
102 102
101 101
1.08 21.82 4.42 12.7 100 100 100 101 102 103 104 100 101 102 103 104 CD62L
C CD4 CD8
21.88 10.62 37.35 Thy1.1 T > WT
0.49 Ȗ IFN- 27.42 22.94 39.97
Thy1.1 T >CD24-/-
CFSE Figure 4.2
100 Figure 4.3. The rapid dividing T cells underwent apoptosis in CD24-/- recipients. T cells from Thy1.1 mice were labeled with CFSE and adoptively transferred into irradiated WT and CD24-/- mice. At 4 d after adoptive transfer, the spleen cells were analyzed for the accumulation and proliferation of donor T cells. (A) Percentage of donor T cells in the host spleen cells at 4 d after adoptive transfer. (B) The percentage of Annexin V positive donor cells. On day 1, the apoptotic cells were comparable between WT and CD24-/- groups. On day 4, more that 30% of donor cells in CD24-/- recipients underwent apoptosis, comparing to 5.4% in WT recipients. Data shown are representative of two independent experiments, each involving three mice per group.
101 A 60
50
40
30
20 % of total splenocytes
10
0 CD24-/- WT CD24-/- WT CD24-/- WT CD4 CD8 Total T cells Figure 4.3 continued
102 Figure 4.3 continued
Day 1 Day 4 B
80
50 60 7.16 7.81 40
Thy1.1 T >WT 40 30 20 20 10
Relative Cell Number 0 0 1 10 100 1000 10000 1 10 100 1000 10000
15
30 5.58 46.2 10 Thy1.1 T >CD24-/- 20
5 10
0 0 1 10 100 1000 10000 1 10 100 1000 10000 Annexin V
Figure 4.3
103 Figure 4.4. Expression of CD24 on T cells does not restore regulation of homeostatic proliferation in the CD24-/- host. T cells from Thy1.1 mice were labeled with CFSE and adoptively transferred into irradiated CD24-/- and CD24TG-/- mice. At 4 d after adoptive transfer, the spleen cells were analyzed for the proliferation of donor T cells. The top panel showed that CD24-/- mice as recipients. The bottom penal showed that CD24TG-/- mice as recipients. Data shown are representative of two independent experiments, each involving three mice per group.
104 50
40
30 Thy1.1 T >CD24-/- 20 Relative Cell Number 10
0 1 10 100 1000 10000
80
Thy1.1 T >CD24TG-/- 60
40
20
0 1 10 100 1000 10000 CFSE
Figure 4.4
105 Figure 4.5. The identification of the cells that are responsible for T cell uncontrolled proliferation in CD24-/- host. (A) WT and CD24-/- bone marrows were transferred into lethally irradiated CD24-/- recipients. 4 weeks later, CFSE-labeled purified Thy1.1 T cells were transferred into both groups, which has been sublethally irradiated before transfer. 4 days after the adoptive transfer, the spleen cells were analyzed for the proliferation of donor T cells. (B) T cells from Thy1.1 mice were labeled with CFSE and adoptively transferred into irradiated WT and RAG-1(-/-) mice. At 4 d after adoptive transfer, the spleen cells were analyzed for the proliferation of donor T cells. Data shown are representative of two independent experiments, each involving three mice per group.
106 A 100
80 CD24-/- BM
60
CD4 40
20
0 100 101 102 103 104 Relative Cell Number 100
80
60 CD8 40
20
0 100 101 102 103 104 CFSE Figure 4.5 continued
107 Figure 4.5 continued B Thy1.1 T >WT Thy1.1 T >RAG-/-
25 300 20
200 15 CD4 10 100 5 Relative Cell Number
0 0 1 10 100 1000 10000 1 10 100 1000 10000
20 80
15 60 CD8 40 10
20 5
0 0 1 10 100 1000 10000 1 10 100 1000 10000
CFSE Figure 4.5
108 Figure 4.6. Super stimulatory activity of the CD24-/- dendritic cells. (A) Bone marrow cells from both WT and CD24-/- mice were cultured in 10% DMEM with 20ng/ml GM-CSF for 10 days. 10ug/ml LPS was added to mature the dendritic cells. CFSE-labeled Thy1.1 T cells were put into culture with bone marrow derived dendritic cells for 5 days at 1:2 ratio. (B) The expressions of CD24, CD40, CD80, CD86, I-Ab and H-2Db on dendritic cells were analyzed by flow cytometry. (C) Spleens from sub lethal irradiated WT and CD24-/- mice were treated with collagenase IV for 30 minutes. All the cells were harvested and analyzed by flow cytometry. (Blue, WT. Red, CD24-/-.)
109 A 100
80
60
40
20 Relative Cell Number 0 10 0 10 1 10 2 10 3 10 4 CFSE Figure 4.6 continued
110 Figure 4.6 continued
B 100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 CD24 CD40
100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 CD86 CD80
100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 I-Ab H-2Db
Figure 4.6 continued
111 Figure 4.6 continued
C 100 100
80 80 WT 60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 CD24 CD40
100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 CD86 CD80
100 100
80 80
60 60
40 40
20 20
0 0 100 101 102 103 104 100 101 102 103 104 I-Ab H-2Db Figure 4.6
112 Recipient Dead Alive Total
Exp 1 WT 0 5 5 CD24-/- 4 1 5
Exp 2 WT 1 1 CD24-/- 2 2 4
Exp 3 WT 0 3 3 CD24-/- 1 2 3
Exp 4 WT 0 5 5 CD24-/- 3 2 5
Radiation only WT 6 6 CD24-/- 6 6
Table 1 Fatal destruction of T cell proliferation in CD24-/- host.
113 Figure 4.7. Fatal destruction of CD24-/- host associated with mass T cell proliferation. (A) The statistics of survival distribution between WT and CD24-/- groups is significant. P=0.0007. (B) The gut tissues from both groups were harvested and fixed by 10% formalin. There were no signs of T cell infiltration in both groups. (C) Production of TNF-Į and IFN- after short-term stimulation with PMA and ionomycin. Spleen cells from 4-d reconstituted mice were harvested and stimulated for 3 h. The intracellular TNF-Į and IFN- were analyzed by flow cytometry. Three mice were used in each group. Data shown are representative of two independent experiments. (D) The spleen cells were harvested and analyzed for acquisition of memory markers. The differential expression of activation markers has been reproduced in three independent experiments, each involving two to three mice per group.
114 A
Test Statistics for Equality of Survival Distributions for TYPE
Statistic df Significance
Log Rank 11.47 1 .0007
Survival Functions 1.0
.9
.8
.7
.6
.5 TYPE .4 Cum Survival .3 w
.2 w -censored
.1 k 0.0 k-censored 0 10 20 30 40 50 60 70
TIME Figure 4.7 continued
115 Figure 4.7 continued B Thy1.1 T >WT
intestine kidney liver lung
Thy1.1 T >CD24-/-
intestine kidney liver lung
Figure 4.7 continued
116 Figure 4.7 continued C Thy1.1 T >WT Thy1.1 T>CD24-/-
0.36 73.23 51.67
TNFĮ
0.22 52.69 64.52 IFN
FSC
D Thy1.1 T >WT Thy1.1 T >CD24-/-
10000 31 56.3 10000 86.4 6.63
1000 1000
100 100 CD44
10 10
1.06 11.7 0.58 6.42 1 1 1 10 100 1000 10000 1 10 100 1000 10000 CD62L Figure 4.7
117 CHAPTER 5
CONCLUDING REMARKS
The finding presented here further the understanding of CD24 and its effect on T cell homeostatic proliferation and autoimmune disease. CD24 has been known as a differentiation marker for hematopoitic cells [30], adhesion molecule that can affect
VLA-4 and VCAM-1 interaction [29] and costimulatory molecule that can provide
CD28-independent co-stimulation for both CD4 and CD8 T cells [7, 23]. Although CD24 is not required for normal T cell response and antibody response in peripheral lymphoid organs [55], we showed that CD24 affects T cell response in the target organ in our study on EAE [103]. We also made a serendipitous discovery on CD24 roles in T cell homeostatic proliferation.
EAE, as the best available animal model for the human disease, multiple sclerosis, has been widely used to test Th1 response and cell migration [41]. Our lab showed that CD24-/- mice are resistant for EAE development, induced by either active immunization or adoptive transfer [55]. There are several steps that are critical for EAE
118 development: T cell priming, T cell attachment to BBB endothelial cells, T cell migration to CNS, re-stimulation of T cells in situ and recruitment of inflammatory cells [43, 44].
The published data showed that T cell priming in CD24-/- mice was comparable to WT.
We hypotheses that the absence of CD24 may affect T cell migration into CNS or CD24 expression is essential for T cell secondary activation in target organs [41]. Our data showed adoptively transferred WT T cells can enter the CNS of CD24 (-/-) mice. The cell numbers in the CNS of knockout and WT mice were comparable at early stage of the disease. We also analyzed the ability of the local antigen presenting cells, microglia and astrocyte [69, 104] to prime T cells. Upon IFN-J induction, there were no difference on the up-regulation of MHC and both B7 molecules between the two groups [49].
Therefore, it is the CD24 expression on local antigen presenting cells as costimulatory molecule that affects EAE development [50]. According to the data from the human study by Dr. Zhou, CD24 is also a genetic modifier for risk and progression of multiple sclerosis [72]. Taken together, our study reveals a possible target to develop effective treatment for MS. The drugs available for MS can partially ameliorate symptoms, yet there is still no cure [41]. Most experimental treatments have to start before or at the point of EAE immunization [105]. According to our data, although the absence of CD24 may not eliminate autoreactive T cells, blocking CD24 has therapeutic effect even after self-reactive T cells are produced, which makes it more relevant to clinical settings.
Unlike the medication available now [39, 106-109], the absence of CD24 in the peripheral doesn’t affect normal immune response or normal cellular development [31].
119 The discovery of a role for CD24 in T cell homeostatic proliferation came rather unexpected. When CD24-/- T cells were transferred into lymphopenic environment, they divided slower, comparing with WT T cell [95]. However, the absence of CD24 on the recipients led to rapid proliferation of transferred WT T cells. These data provide us great insight on the regulation of naïve T cell proliferation in lymphopenic host.
A great deal of research on homeostatic proliferation reveals that this process involves TCR and self-peptide MHC contact [58]. The involvement of major costimulation molecules has been ruled out [58, 94]. The possible ways to regulate lymphocyte homeostatic proliferation are quite limited [57, 61]. However, our data indicated that CD24 expression on T cells showed an intriguing impact on T cell homeostatic proliferation. CD24 expression on T cells is required for lymphopenia induced T cell proliferation. At the same time, the absence of CD24 on lymphopenic host led to T cell massive proliferation, which has lethal effect on the recipients. Our observation indicates that CD24 is the regulator that can control T cell homeostatic proliferation.
Lymphopenia induced T cell proliferation can is a mixed blessing. In current practice, before organ transplantation, lymphopenia is induced prior to the surgery [94].
Massive T cell proliferation in these patients may lead to rapid organ rejection [110]. The expression level of CD24 on both the donor and the recipient may well predict the acceptance of an organ. Blocking CD24 on T cells can reduce GVHD [111]. However, for cancer patients, radiation therapy and chemotherapy induce lymphopenia [112]. The damage on the immune system usually leaves the patients vulnerable for pathogen attacks.
120 Moreover, the defect in T cell homeostatic proliferation may result the failure of anti- tumor immunity [113]. On the other hand, blocking CD24 expression in cancer patients with radiation therapy or chemotherapy, can replenish the T cell pool in short time.
Moreover, the T cells acquired stronger ability to produce inflammatory cytokines, which may improve T cell anti-tumor immunity in those patients [60].
At the same time, recent studies have been focusing on the connection between homeostatic proliferation and auto immune diseases [65]. In human, lymphopenia is observed from patients suffering IDDM (Insulin Dependent Diabetes Mellitus) [68], rheumatoid arthritis [66] or lupus [102]. HIV-infected patients often show signs of autoimmune diseases [110, 114, 115]. The study of NOD mice by Dr. Sarvetnick suggest that lymphopenia may facilitate destructive autoimmunity [68]. Most interestingly, one of the causes for MS is believed to be virus infection [116], some of which may induce lymphopenia in patients [117]. CD24 effects on lymphocyte homeostatic proliferation may provide an explaination for the resistance of CD24-/- mice to EAE. However, further experiments are needed to determine whether CD24 can act as a regulatory factor to alter the development of autoimmunity.
The study about CD24 and its effect on T cell homeostatic proliferation and autoimmune disease in the thesis has provided us great insight information. Here, we presented a novel function for CD24 as costimulatory molecule for T cell secondary stimulation in target organs and as regulator for naïve T cell homeostatic proliferation.
Our findings may lead to new therapeutic approach for transplantation, autoimmune diseases and more.
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