Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.     

Peptide specificity of thymic selection of CD4+CD25+ T cells.

The CD4(+)CD25(+) regulatory T cells can be found in the thymus, but their need to undergo positive and negative selection has been questioned. Instead, it has been hypothesized that CD4(+)CD25(+) cells mature following TCR binding to MHC backbone, to low abundant MHC/peptide complexes, or to class II MHC loaded with peripheral autoantigens. In all these circumstances, processes that are distinct from positive and negative selection would govern the provenance of CD4(+)CD25(+) cells in the thymus. By comparing the development of CD4(+)CD25(-) and CD4(+)CD25(+) cells in mice expressing class II MHC molecules bound with one or many peptide(s), we show that the CD4(+)CD25(+) cells appear during natural selection of CD4(+) T cells. The proportion of CD4(+)CD25(+) cells in the population of CD4(+) thymocytes remains constant, and their total number reflects the complexity of selecting class II MHC/peptide complexes. Hence, thymic development of CD4(+)CD25(+) cells does not exclusively depend on the low-density, high-affinity MHC/peptide complexes or thymic presentation of peripheral self-Ags, but, rather, these cells are selected as a portion of the natural repertoire of CD4(+) T cells. Furthermore, while resistant to deletion mediated by endogenous superantigen(s), these cells were negatively selected on class II MHC/peptide complexes. We postulate that while the CD4(+)CD25(+) thymocytes are first detectable in the thymic medulla, their functional commitment occurs in the thymic cortex.     

Preferential recognition of self antigens despite normal thymic deletion of CD4(+)CD25(+) regulatory T cells

T cell tolerance to self Ags is in part established in the thymus by induction of apoptosis or anergy of potentially autoreactive thymocytes. Some autospecific T cells nevertheless migrate to peripheral lymphoid organs but are kept under control by the recently identified CD4(+)CD25(+) regulatory T cell subset. Because these cells inhibit autoimmunity more efficiently than useful non-self Ag-specific immune responses, they are probably autospecific, posing important questions as to how they develop in the thymus. In this study we show that significantly more peripheral CD4(+)CD25(+) regulatory T cells recognize self than non-self Ags. However, we also show for a large panel of endogenous superantigens as well as for self peptide/MHC complexes that autospecific CD4(+)CD25(+) thymocyte precursors are normally deleted during ontogeny. Combined, our data firmly establish that the repertoire of regulatory T cells is specifically enriched in autospecific cells despite the fact that their precursors are normally susceptible to thymic deletion.     

Peripheral tolerance to a nuclear autoantigen: dendritic cells expressing a nuclear autoantigen lead to persistent anergic state of CD4+ autoreactive T cells after proliferation.

It remains unknown why the T cell tolerance to nuclear autoantigens is impaired in systemic autoimmune diseases. To clarify this, we generated transgenic mice expressing OVA mainly in the nuclei (Ld-nOVA mice). When CD4+ T cells from DO11.10 mice expressing a TCR specific for OVA(323-339) were transferred into Ld-nOVA mice, they were rendered anergic, but persisted in vivo for at least 3 mo. These cells expressed CD44(high), CD45RB(low), and were generated after multiple cell divisions, suggesting that anergy is not the result of insufficient proliferative stimuli. Whereas dendritic cells (DCs) from Ld-nOVA (DCs derived from transgenic mice (TgDCs)), which present rather low amount of the self-peptide, efficiently induced proliferation of DO11.10 T cells, divided T cells stimulated in vivo by TgDCs exhibited a lower memory response than T cells stimulated in vitro by peptide-pulsed DCs. Furthermore, we found that repeated transfer of either TgDCs or DCs derived from wild-type mice pulsed with a lower concentration of OVA(323-339) induced a lower response of DO11.10 T cells in Ag-free wild-type recipients than DCs derived from wild-type mice. These results suggest that peripheral tolerance to a nuclear autoantigen is achieved by continuous presentation of the self-peptide by DCs, and that the low expression level of the peptide might also be involved in the induction of hyporesponsiveness.     

Development of memory-like autoregulatory CD8+ T cells is CD4+ T cell dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8(+) cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8(+) T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8(+) T cell memory is CD4(+) T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti-4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4(+) T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4(+) T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2(-/-) mice, but inhibited it in low-affinity TCR-TG NOD.Rag2(-/-) mice. Thus, in chronic autoimmune responses, CD4(+) Th cells contribute to both promoting and suppressing pathogenic autoimmunity.     

Negative regulation of CD4 lineage development and responses by CD5

CD5 deficiency results in a hyper-responsive phenotype to Ag receptor stimulation. Here we show that the development and responses of CD4 lineage T cells are regulated by the function of CD5. Thymocytes expressing the I-Ad-restricted DO11.10 TCR undergo abnormal selection without CD5. In H-2d mice, the absence of CD5 causes deletion of double-positive thymocytes, but allows for efficient selection of cells expressing high levels of the DO11.10 clonotype. By contrast, there is enhanced negative selection against the DO11.10 clonotype in the presence of I-Ab. T cell hybridomas and DO11.10 T cells are more responsive to TCR stimulation in the absence of CD5. Such hypersensitivity can be eliminated by expression of wild-type CD5, but not by a form of CD5 that lacks the cytoplasmic tail. Finally, CD5 deficiency partially suppresses the block of CD4 lineage development in CD4-deficient mice. Taken together, the data support a general role for CD5 as a negative regulator of Ag receptor signaling in the development and immune responses of CD4 lineage T cells.     

B7-CD28 interaction promotes proliferation and survival but suppresses differentiation of CD4-CD8- T cells in the thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25(+)CD4(+) regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4(-)CD8(-) (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25(+)CD44(-) (DN3) to CD25(-)CD44(-) (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

Spontaneous autoreactive memory B cell formation driven by a high frequency of autoreactive CD4+ T cells

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.     

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4⁺ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRβ chains, and a Foxp3-GFP reporter. We purified CD4⁺Foxp3^- and CD4⁺Foxp3⁺ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRβ chains. We identified >7,000 different TCRβ sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4⁺ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.     

Graded deletion and virus-induced activation of autoreactive CD4+ T cells

We have examined factors governing the negative selection of autoreactive CD4(+) T cells in transgenic mice expressing low (HA12 mice) vs. high (HA104 mice) amounts of the influenza virus hemagglutinin (HA). When mated with TS1 mice that express a transgenic TCR specific for the I-Ed-restricted determinant site 1 (S1) of HA, thymocytes expressing high levels of the clonotypic TCR were deleted in both HA-transgenic lineages. However, through allelic inclusion, thymocytes with lower levels of the clonotypic TCR evaded deletion in TS1 x HA12 and TS1 x HA104 mice to graded degrees. Moreover, in both lineages, peripheral CD4(+) T cells could be activated by the S1 peptide in vitro, and by influenza virus in vivo. These findings indicate that allelic inclusion can allow autoreactive CD4(+) thymocytes to evade thymic deletion to varying extents reflecting variation in the expression of the self peptide, and can provide a basis for the activation of autoreactive peripheral T cells by viruses bearing homologues of self peptides ("molecular mimicry").     

LFA-1 on CD4+ T cells is required for optimal antigen-dependent activation in vivo

The leukocyte-specific integrin, LFA-1, plays a critical role in trafficking of T cells to both lymphoid and nonlymphoid tissues. However, the role of LFA-1 in T cell activation in vivo has been less well understood. Although there have been reports describing LFA-1-deficient T cell response defects in vivo, due to impaired migration to lymphoid structures and to sites of effector function in the absence of LFA-1, it has been difficult to assess whether T cells also have a specific activation defect in vivo. We examined the role of LFA-1 in CD4(+) T cell activation in vivo by using a system that allows for segregation of the migration and activation defects through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 Ag-specific TCR transgenic mice into wild-type BALB/c mice. We find that in addition to its role in trafficking to peripheral lymph nodes, LFA-1 is required for optimal CD4(+) T cell priming in vivo upon s.c. immunization. CD18(-/-) DO11.10 CD4(+) T cells primed in the lymph nodes demonstrate defects in IL-2 and IFN-gamma production. In addition, recipient mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate a defect in OVA-specific IgG2a production after s.c. immunization. The defect in priming of CD18(-/-) CD4(+) T cells persists even in the presence of proliferating CD18(+/-) CD4(+) T cells and in lymphoid structures to which there is no migration defect. Taken together, these results demonstrate that LFA-1 is required for optimal CD4(+) T cell priming in vivo.     

Early events in peripheral regulatory T cell induction via the nasal mucosa

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.     

Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice

RasGRP1 and Sos are two Ras-guanyl-nucleotide exchange factors that link TCR signal transduction to Ras and MAPK activation. Recent studies demonstrate positive selection of developing thymocytes is crucially dependent on RasGRP1, whereas negative selection of autoreactive thymocytes appears to be RasGRP1 independent. However, the role of RasGRP1 in T regulatory (Treg) cell development and function is unknown. In this study, we characterized the development and function of CD4(+)CD25(+)Foxp3(+) and CD8(+)CD44(high)CD122(+) Treg lineages in RasGRP1(-/-) mice. Despite impaired CD4 Treg cell development in the thymus, the periphery of RasGRP1(-/-) mice contained significantly increased frequencies of CD4(+)Foxp3(+) Treg cells that possessed a more activated cell surface phenotype. Furthermore, on a per cell basis, CD4(+)Foxp3(+) Treg cells from mutant mice are more suppressive than their wild-type counterparts. Our data also suggest that the lymphopenic environment in the mutant mice plays a dominant role of favored peripheral development of CD4 Treg cells. These studies suggest that whereas RasGRP1 is crucial for the intrathymic development of CD4 Treg cells, it is not required for their peripheral expansion and function. By contrast to CD4(+)CD25(+)Foxp3(+) T cells, intrathymic development of CD8(+)CD44(high)CD122(+) Treg cells is unaffected by the RasGRP1(-/-) mutation. Moreover, RasGRP1(-/-) mice contained greater numbers of CD8(+)CD44(high)CD122(+) T cells in the spleen, relative to wild-type mice. Activated CD8 Treg cells from RasGRP1(-/-) mice retained their ability to synthesize IL-10 and suppress the proliferation of wild-type CD8(+)CD122(-) T cells, albeit at a much lower efficiency than wild-type CD8 Treg cells.     

Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.     

Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T cells

CD4(+)CD25(+) regulatory T cell selection is initiated by high-specificity interactions with self-peptides in the thymus, although how these cells respond to cytokine-derived signals and to re-exposure to self-peptide:MHC complexes in the periphery is not well understood. We have used a transgenic mouse system, in which the peptide that induces thymic selection of a clonal population of CD4(+)CD25(+) regulatory T cells is known, to show that CD4(+)CD25(+) T cells proliferate in response to their selecting self-peptide in vivo. Moreover, they do not proliferate in response to lymphopenia in the absence of the selecting self-peptide, reflecting a low level of expression of the high affinity receptor for IL-7 (CD127) relative to conventional CD4(+) T cells. That their selecting self-peptide is both required for and promotes the peripheral expansion of CD4(+)CD25(+) regulatory T cells may direct their accumulation in sites where the self-peptide is expressed.     

Inhibition of human CD4(+)CD25(+high) regulatory T cell function

CD4(+)CD25(+high) T cells are potent regulators of autoreactive T cells. However, it is unclear how regulatory CD4(+)CD25(+high) cells discriminate between desirable inflammatory immune responses to microbial Ags and potentially pathologic responses by autoreactive T cells. In this study, an in vitro model was created that allowed differential activation of regulatory CD4(+)CD25(+high) and responder CD4(+) T cells. If CD4(+)CD25(+high) regulatory cells were strongly activated, they maintained suppressive effector function for only 15 h, while stimulation with weaker TCR stimuli produced regulatory cells that were suppressive until 60 h after activation. In contrast, strongly activated CD4(+) responder T cells were resistant to regulation at all time points, while weakly stimulated CD4(+) cells were sensitive to suppression until 38 or 60 h after activation depending upon the strength of the stimulus. The extent of suppression mediated by CD4(+)CD25(+high) cells also depended on the strength of stimulation in an Ag-specific system. Thus, the stronger the TCR signal, the more rapidly and more completely the responder cells become refractory to suppression.     

Receptor revision in peripheral T cells creates a diverse V beta repertoire

In Vbeta5 transgenic mice, the age-dependent accumulation of Vbeta5(-)CD4(+) T cells expressing endogenous Vss elements represents an exception to the rule of strict allelic exclusion at the TCRbeta locus. The appearance of these cells is limited to the lymphoid periphery and is driven by a peripherally expressed tolerogen. Expression of the lymphoid-specific components of the recombinase machinery and the presence of recombination intermediates strongly suggest that TCR revision rescues tolerogen-reactive peripheral T cells from deletion. Here, we report that the appearance of Vbeta5(-)CD4(+) T cells is CD28-dependent. In addition, we find that the TCR repertoire of this unusual population of T cells in individual Vbeta5 transgenic mice is surprisingly diverse, both at the level of surface protein and at the nucleotide level within a given family of V(D)Jbeta rearrangements. This faithful recreation of the nontransgenic repertoire suggests that endogenous Vbeta-expressing populations do not arise from expansion of an initially rare subset. Furthermore, the undersized N regions in revised TCR genes distinguish these sequences from those generated in the adult thymus. The diversity of the revised TCRs, the minimal mouse-to-mouse variation in the expressed endogenous Vbeta repertoire, the atypical length of junctional sequences, and the CD28 dependence of the accumulation of Vbeta5(-)CD4(+) T cells all point to their extrathymic origin. Thus, tolerogen-driven receptor revision in peripheral T cells can expand the TCR repertoire extrathymically, thereby contributing to the flexibility of the immune repertoire.     

Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.     

CD4+CD25+ regulatory T cell repertoire formation shaped by differential presentation of peptides from a self-antigen

We have used TCR transgenic mice directed to different MHC class II-restricted determinants from the influenza virus hemagglutinin (HA) to analyze how specificity for self-peptides can shape CD4+CD25+ regulatory T (Treg) cell formation. We show that substantial increases in the number of CD4+CD25+ Treg cells can occur when an autoreactive TCR directed to a major I-E(d)-restricted determinant from HA develops in mice expressing HA as a self-Ag, and that the efficiency of this process is largely unaffected by the ability to coexpress additional TCR alpha-chains. This increased formation of CD4+CD25+ Treg cells in the presence of the self-peptide argues against models that postulate selective survival rather than induced formation as mechanisms of CD4+CD25+ Treg cell formation. In contrast, T cells bearing a TCR directed to a major I-A(d)-restricted determinant from HA underwent little or no selection to become CD4+CD25+ Treg cells in mice expressing HA as a self-Ag, correlating with inefficient processing and presentation of the peptide from the neo-self-HA polypeptide. These findings show that interactions with a self-peptide can induce thymocytes to differentiate along a pathway to become CD4+CD25+ Treg cells, and that peptide editing by DM molecules may help bias the CD4+CD25+ Treg cell repertoire away from self-peptides that associate weakly with MHC class II molecules.