Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide

Despite accumulating evidence that regulatory T cells play a crucial role in preventing autoimmunity, the processes underlying their generation during immune repertoire formation are unknown. We show here that interactions with a single self-peptide can induce thymocytes that bear an autoreactive T cell receptor (TCR) to undergo selection to become CD4+CD25+ regulatory T cells. Selection of CD4+CD25+ thymocytes appears to require a TCR with high affinity for a self peptide because thymocytes that bear TCRs with low affinity do not undergo selection into this pathway. Our findings indicate that specificity for self-peptides directs the selection of CD4+CD25+ regulatory thymocytes by a process that is distinct from positive selection and deletion.     

Impact of effector cell differentiation on CD4+ T cells that evade negative selection by a self-peptide

We have used a transgenic mouse system to examine how differing reactivities of TCRs expressed by naive versus effector cells can shape the functional potential of autoreactive CD4+ T cells. Transgenic mice expressing TCRs that exhibit either high (TS1) or low [TS1(SW)] reactivity toward the I-Ed-restricted determinant S1 from the influenza virus PR8 hemagglutinin (HA) were mated with transgenic mice expressing HA under the control of different promoters. HACII mice express HA driven by an MHC class II promoter, and both the TS1 and TS1(SW) TCRs underwent substantial deletion in this background. HA104 mice express HA driven by an SV40 promoter, and the highly reactive TS1 TCR was substantially deleted. By contrast, the less reactive TS1(SW) TCR underwent little or no deletion in TS1(SW) x HA104 mice, although CD5 up-regulation indicated that they had interacted with the S1 self-peptide. In adoptive transfer studies, naive CD4+ T cells expressing the TS1(SW) TCR failed to proliferate in response to the S1 peptide in HA104 mice, and were inefficient at providing help for HA-specific antibody responses. However, effector CD4+ T cells generated from TS1(SW) x HA104 mice acquired the ability to proliferate in response to the S1 peptide in HA104 mice, and were as efficient as CD4+ T cells expressing the high reactivity TS1 TCR in helping HA-specific antibody responses. Collectively, these studies demonstrate a basis by which CD4+ T cells expressing TCRs with low reactivity toward self-peptides can evade negative selection and acquire enhanced autoreactivity following activation by a cross-reactive antigen.     

An increased frequency of autoantibody-inducing CD4+ T cells in pre-diseased lupus-prone mice

Pathogenic autoantibody production in murine models of lupus is dependent on autoreactive CD4+ helper T cells. However, the mechanisms which permit the selection and maintenance of this autoantibody-inducing CD4+ T-cell repertoire are currently unknown. We hypothesized that the peripheral CD4+ T-cell repertoire of lupus-prone mice was enriched with autoantibody-inducing specificities. To test this, we utilized the splenic focus assay to determine if pre-diseased lupus-prone (NZB x NZW)F(1) mice have an elevated frequency of autoreactive CD4+ T lymphocytes capable of supporting autoantibody production. The splenic focus limiting dilution assay permits anti-nuclear antibodies to be generated from contact-dependent T-B interactions in vitro. We show that young, pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T cells. Interestingly, these autoantibody-inducing CD4+ T-cell responses are also present in the thymus. Therefore, an elevated frequency of autoantibody-inducing CD4+ T cells predisposes lupus-prone mice to the development of autoantibodies.     

T细胞在多发性硬化发病机制中作用的研究进展

多发性硬化（MS）是临床常见的炎性、脱髓鞘性中枢神经系统（CNS）退行性变,通常认为是T细胞介导的自身免疫性疾病,但其具体病因和发病机制尚不清楚.近年来研究表明,多个亚型的T细胞均参与了MS的发病及其病程的调控.其中包括CD4^＋Th1细胞、CD8^＋T细胞、Th17细胞、CD16＋γδ T细胞、Th2细胞、调节性CD4＋T细胞、NK T细胞、CD8＋调节性T细胞等,因而研究MS了发病机制具有重要意义.     

CD8+ suppressor-mediated regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65

Although potentially autoreactive T cells are present even in healthy subjects, most individuals do not develop autoimmune disease. It has been well demonstrated that CD4+ CD25+ regulatory T cells play a significant role in controlling the expansion of autoreactive T cells in the periphery. However, some healthy individuals exhibit measurable responses to self peptide even in the presence of CD4+ CD25+ regulatory cells. This article describes the regulation of human CD4+ T cell responses to glutamic acid decarboxylase 65 (GAD65), an autoantigen implicated in type-1 diabetes, by autologous CD8+ suppressor T cells. In cells cultured from healthy individuals, the inclusion of autologous CD8+ T cells at physiological levels resulted in a dramatic decrease in the magnitude of in vitro CD4+ T cell responses to GAD65 peptide. Based on transwell experiments, the observed suppression was cell contact-dependent. However, antibody blocking studies indicated that suppression was mediated by IL-10. Cell fractionation studies suggested that CD8+ suppressor T cells originate from the CD45RA+ CD27- population. The suppression of CD4+ T cell responses to GAD65 in healthy individuals raises the possibility that CD8+ suppressor T cells play an important role in controlling potentially autoreactive T cells in the general population.     

Human leukocyte antigen class I-restricted immunosuppression by human CD8+ regulatory T cells requires CTLA-4-mediated interaction with dendritic cells

We previously reported autoreactive CD8(+) regulatory T cells (Tregs) that were expanded and cloned from human peripheral blood by coculture with autologous dendritic cells (DC). Here we demonstrate that these CD8(+) Tregs require human leukocyte antigen (HLA)-class I restricted activation and then mediate cell-contact-dependent suppression of CD4(+) T cells. CD8(+) Tregs interacted with DC to suppress T-cell responses but DC were not irreversibly altered by this interaction because they could subsequently stimulate CD4(+) T cells normally. The ability of DC to form conjugates with CD4(+) T cells was reduced in the presence of CD8(+) Tregs. Suppression was blocked by Abs to CD80 and CTLA-4, implicating CTLA-4:CD80 interactions in the function of CD8(+) Tregs. CD8(+) Tregs rapidly express very high levels of surface CTLA-4 following activation compared with conventional T cells. Related to this, the expression of TRAT1 mRNA (T-cell receptor interacting molecule, or TRIM) was highly upregulated in microarray analysis of CD8(+) Tregs compared with conventional cytotoxic or nonregulatory CD8(+) T cells. TRIM acts to chaperone CTLA-4 transport to the cell surface; this function would be required to account for the phenotypic and functional properties of CD8(+) Tregs.     

Soluble, dimeric HLA DR4-peptide chimeras: an approach for detection and immunoregulation of human type-1 diabetes

Still there are no effective methods to predict or cure type 1 diabetes (T1D) in humans. Soluble, dimeric MHC class II-peptide (DEF) chimeras have potential for both early diagnosis and immunospecific therapy. DEF chimeras prevent and reverse diabetes in mice by stimulating antigen-specific type 1 T regulatory cell (Tr1)-like cells. We also showed that diabetes could be predicted by changes in the phenotype of autoreactive CD4 T cells in peripheral blood. Herein, we demonstrated that human DEF (HLA-DR*0401/Fcgamma1) chimeras expressing peptides of beta-cell antigens stimulate Tr1-like cells in blood of patients with T1D, non-diabetic relatives, and controls. Furthermore, the specific and stable binding of DEF chimeras to cognate TCR and CD4 coreceptor allowed quantification and phenotyping of autoreactive CD4 T cells in non-stimulated blood by FACS. Our results indicate that (1) autoreactive CD4 T cells to GAD65 autoantigen are commonly present in humans expressing diabetes-susceptible HLA-DR*0401 molecules; (2) these autoreactive T cells undergo avidity maturation upon encountering the self antigen early in life; (3) the disease is associated with an imbalance between autoreactive CD4+CD25+ and CD4+CD69+ T cells specific for GAD65. Based on this, we propose a model to explain the kinetics of autoreactive CD4 T cells in blood during the natural history of T1D.     

Autoreactive human peripheral blood CD8+ T cells with a regulatory phenotype and function

Despite substantial advances in our understanding of CD4+ CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCR alphabeta+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I-restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGFbeta1, but not IFN-gamma. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+ CD25+ regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN-gamma production and proliferation by CD4+ T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+ CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.     

Recognition of the peripheral self by naturally arising CD25+ CD4+ T cell receptors

Naturally arising CD25+ CD4+ regulatory T cells (TR) play an important role in the prevention of autoimmunity. TCR specificity is thought to play a critical role in TR development and function, but the repertoire and specificity of TR TCRs remain largely unknown. We find by sequencing of TRAV14 (Valpha2) TCRalpha chains associated with a transgenic TCRbeta chain that the TRand CD25- CD4+ TCR repertoires are similarly diverse, yet only partially overlapping. Retroviral expression of TCRalpha genes in TCR transgenic RAG-deficient T cells revealed that a high frequency of TCRs derived from CD25+ but not CD25- CD4+ T cells confers the ability to rapidly expand upon transfer into a lymphopenic host. Thus, these data show that a large proportion of naturally arising TR have substantially more efficient interactions with MHC class II bound peptides from the peripheral self than CD25- T cells.     

Structural basis for the killing of human beta cells by CD8(+) T cells in type 1 diabetes

The structural characteristics of the engagement of major histocompatibility complex (MHC) class II-restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8(+) T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A*0201-restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHC class I-restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8(+) T cell-mediated autoreactivity.     

TCRalphabeta repertoire diversity of human naturally occurring CD4+CD25+ regulatory T cells

We examined the alphabeta T cell receptor (TCR) repertoire of naturally occurring CD4+CD25+ regulatory T (Treg) cells isolated from healthy human blood. Three-color FACS analysis demonstrated that the usage of variable region segments of TCRbeta chains by CD4+CD25+ cells did not differ from those of CD4+CD25- cells. Complementarity-determining region 3 (CDR3) size distribution analyses demonstrated that the repertoire diversity of CDR3beta was almost identical between CD4+CD25+ and CD4+CD25- T cell subsets, and that there was no skewing of the CDR3beta repertoire of CD4+CD25+ T cells. In contrast, in vitro activated CD4+CD25+ T cells by cytomegalovirus-derived antigens showed a skewed CDR3 size distribution pattern. These findings support the hypothesis that naturally occurring CD4+CD25+ T cell subset in humans is 1argely composed of a T cell lineage positively selected in the thymus as a consequence of the interaction between self-peptides and TCRs and not derived from recent activation by a limited array of antigens.     

Role of TCR specificity in CD4+CD25+ regulatory T-cell selection

Summary: CD4⁺CD25⁺ regulatory T cells play a crucial role in preventing autoimmune disease and can also modulate immune responses in settings such as transplantation and infection. We have developed a transgenic mouse system in which the role that T‐cell receptor (TCR) specificity for self‐peptides plays in the formation of CD4⁺CD25⁺ regulatory T cells can be examined. We have shown that interactions with a single self‐peptide can induce thymocytes bearing an autoreactive TCR to undergo selection to become CD4⁺CD25⁺ regulatory T cells and that thymocytes bearing TCRs with low affinity for the selecting peptide do not appear to undergo selection into this pathway. In addition, thymocytes with identical specificity for the selecting self‐peptide can undergo overt deletion versus abundant selection to become CD4⁺CD25⁺ regulatory T cells in response to variations in expression of the selecting peptide in different lineages of transgenic mice. Finally, we have shown that CD4⁺CD25⁺ T cells proliferate in response to their selecting self‐peptide in the periphery, but these cells do not proliferate in response to lymphopenia in the absence of the selecting self‐peptide. These studies are determining how the specificity of the TCR for self‐peptides directs the thymic selection and peripheral expansion of CD4⁺CD25⁺ regulatory T cells.     

Molecular profile of the T cell receptors of regulatory and effector CD4+ T cells recognizing overlapping determinants on glutamic acid decarboxylase (524-543)

Glutamic acid decarboxylase (GAD65) is one of the autoantigens that initiates pathogenic T cell responses against insulin-secreting pancreatic beta cells in Type 1 diabetes (T1D). Previously it was shown that spontaneously arising pathogenic T cell responses in the NOD mouse model are confined to GAD530-543 (p530). However, regulatory T cell subpopulations, which can prevent diabetes, can also be generated, for example, by immunization with GAD524-538 (p524) or GAD524-543. Interestingly, two functionally distinct subpopulations of T cells which recognize overlapping determinants of GAD524-543, p524 and p530, utilize distinct TCR Vbeta families, Vbeta4 for pathogenic, and Vbeta12 for regulatory T cells. We characterized T cell receptors (TCRs) from each subpopulation of T cells and visualized p524-specific TCR/p524/I-A(g7) and p530-specific TCR/p530/I-A(g7) complexes via molecular modeling to help us understand, at a molecular level, the in vivo expansion of p524- or p530-specific T cells in the NOD model of T1D. The absolute restriction in Vbeta usage but not Valpha usage and conserved CDR3beta lengths for both T cell subpopulations demonstrates that the beta chains are main contributors in shaping both p524/I-A(g7) and p530/I-A(g7) restricted TCRs. However, only Vbeta4+ T cells but not Vbeta12+ T cells contain a common motif (DWG) in CDR3beta and may involve all of CDR1beta, CDR2beta, and CDR3beta in the recognition of the C-terminus of p530. These observations imply that the spontaneously arising p530-restricted TCRs may be selected under stringent structural frameworks to bind p530/I-A(g7) with high affinity. Thus, the pathogenic p530-specific T cells may arise from a small pool of autoreactive T cells upon breaking tolerance.     

Bmi-1 Regulates Autoreactive CD4+ T Cell Survival in Immune Thrombocytopenia Patients

Autoreactive T cells in immune thrombocytopenia(ITP) patients undergo a rapid clonal expansion and are resistant to apoptosis to maintain continuous effect in thrombocytopenia. As Bmi-1 is involved in memory CD4+ T cell survival and Th2 proliferation, we hypothesized that Bmi-1 may have a role in autoreactive CD4+ T cell clonal expansion and Th1/Th2 development in ITP patients. We found that CD4+ T cells from active ITP patients had a higher Bmi-1 expression in comparison with remission and healthy controls, and autoreactive CD4+ T cells had more capability to proliferate and resistance to apoptosis than that of healthy controls. We evaluated the part that Bmi-1 played in proliferation and Th1 bias condition of autoreactive CD4+ T cells in ITP. We used lentiviral transfer vectors containing Bmi-1 and shBmi-1 to infect CD4+ T cells from ITP patients and healthy controls during autologous platelets stimulation. Flow cytometry and ELISA were applied to detect various parameters. The results showed that suppression of Bmi-1 using short hairpin RNA inhibited the platelet-mediated proliferation and increased apoptosis of autoreactive CD4+ T cells from ITP patients.Increased Bmi-1 expression in CD4+ T cells from healthy controls promoted the proliferation and inhibited apoptosis of CD4+ T cells. Bmi-1 significantly promoted interleukin-4 secretion by CD4+ T cells. These findings suggest that Bmi-1 plays a part in autoreactive CD4+ T cell proliferative capability and apoptotic resistance in ITP patients.     

Characterization of CD8+ T cell repertoire in identical twins discordant and concordant for multiple sclerosis

Autoreactive CD4+ and CD8+ T cells directed against CNS autoantigens may play a role in the development of multiple sclerosis (MS). Identical twins share the same genetic background but not the TCR repertoire that is shaped by the encounter with self or foreign antigens. To gain insights into the interplay between MS and T cell repertoire, peripheral blood CD4+ and CD8+ T lymphocytes and their CCR7+/CCR7- subsets from five pairs of identical twins (four discordant and one concordant for MS; none of which had taken disease-modifying therapy) were compared by TCR beta-chain (TCRB) complementary-determining region 3 (CDR3) spectratyping. CD4+ T cells generally showed a Gaussian distribution, whereas CD8+ T cells exhibited subject-specific, widely skewed TCR spectratypes. There was no correlation between CD8+ T cell oligoclonality and disease. Sequencing of predominant spectratype expansions revealed shared TCRB-CDR3 motifs when comparing inter- and/or intrapair twin members. In many cases, these sequences were homologous to published TCRs, specific for viruses implicated in MS pathogenesis, CNS autoantigens, or copaxone [glatiramer acetate (GA)], implying the occurrence of naturally GA-responding CD8+ T cells. It is notable that these expanded T cell clones with putative pathogenic or regulatory properties were present in the affected as well as in the healthy subject, thus suggesting the existence of a "MS predisposing trait" shared by co-twins discordant for MS.     

Mechanisms,manifestations, and failures of self-tolerance

We have developed transgenic (Tg) mice that express the influenza virus A/PR/8/34 hemagglutinin (PR8 HA) as a well-characterized model antigen with which to analyze factors governing tolerance and autoractivity among CD4+ T and B cells. To analyze how the expression of self-antigens in varying amounts and in different cell types and tissues affects these processes, we have used a variety of promoters and enhancers to drive HA expression. By mating these HA Tg mice with Tg mice-expressing HA-specific major histocompatibility complex (MHC) class II-restricted T cell receptors (TCRs), we have shown that variations in the expression of the HA in different HA Tg line ages can cause CD4+ T cells with identical specificity for a self-peptide either to be deleted (to varying degrees) or to undergo selection to become CD4+ CD25+ regulatory T cells. In addition, a high intrinsic affinity of the TCR for a self-peptide appears to be required for thymocytes to undergo selection to become CD4+ CD25+ regulatory T cells. We have also shown that separate populations of HA-specific B cells that participate at distinct phases of the immune response to the HA in BALB/c mice differ significantly in their sensitivity to negative selection by the neo-self HA. Together, these studies demonstrate that both the diversity of the CD4+ T cell and B cell responses to the HA and variations in the expression of the HA and HA Tg mice can significantly affect the mechanisms and extent of CD4+ T and B cell tolerance induction.     

Autoreactive CD4+ T cells protect from autoimmune diabetes via bystander suppression using the IL-4/Stat6 pathway.

Targeted immune regulation can be achieved by use of tissue-specific T cells and offers the potential for organ-specific suppression of destructive autoimmune processes. Here, we report the generation and characterization of insulin B chain-specific "autoreactive" CD4+ regulatory T cells that locally suppress diabetogenic T cell responses against an unrelated self-antigen (viral transgene) in a virus-induced model for type 1 diabetes. Interleukin 4 (IL-4) is essential for prevention of diabetes since regulatory T cells cannot be induced in the absence of IL-4 or stat6 (IL-4 signaling pathway). Our observations demonstrate that autoreactive regulatory T cells can suppress autoreactive destructive T cell activity of differential antigenic specificity locally in the pancreatic draining lymph node, probably via cytokine-mediated modulation of antigen-presenting cells.