CD4+ CD25+ regulatory T cell repertoire formation in response to varying expression of a neo-self-antigen

We have examined the development of self-peptide-specific CD4+ CD25+ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-E(d)-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4+ CD25+ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4+ CD25+ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4+ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4+ CD25+ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4+ CD25+ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4+ CD25+ regulatory T cells in all cases coexist with clonally related CD4+ CD25- T cells that lack regulatory function. These findings show that the formation of the CD4+ CD25+ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.     

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.     

CD25+ immunoregulatory CD4 T cells mediate acquired central transplantation tolerance

Transplantation tolerance is induced reliably in experimental animals following intrathymic inoculation with the relevant donor strain Ags; however, the immunological mechanisms responsible for the induction and maintenance of the tolerant state remain unknown. We investigated these mechanisms using TCR transgenic mice (TS1) that carry T cells specific for an immunodominant, MHC class II-restricted peptide (S1) of the influenza PR8 hemagglutinin (HA) molecule. We demonstrated that TS1 mice reject skin grafts that have transgene-encoded HA molecules (HA104) as their sole antigenic disparity and that intrathymic but not i.v. inoculation of TS1 mice with S1 peptide induces tolerance to HA-expressing skin grafts. Intrathymic peptide inoculation was associated with a dose-dependent reduction in T cells bearing high levels of TCR specific for HA. However, this reduction was both incomplete and transient, with a full recovery of S1-specific thymocytes by 4 wk. Peptide inoculation into the thymus also resulted in the generation of immunoregulatory T cells (CD4+CD25+) that migrated to the peripheral lymphoid organs. Adoptive transfer experiments using FACS sorted CD4+CD25- and CD4+CD25+ T cells from tolerant mice revealed that the former but not the latter maintain the capacity to induce rejection of HA bearing skin allografts in syngeneic hosts. Our results suggest that both clonal frequency reduction in the thymus and immunoregulatory T cells exported from the thymus are critical to transplantation tolerance induced by intrathymic Ag inoculation.     

Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection

CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

Survival and homeostatic proliferation of naive peripheral CD4+ T cells in the absence of self peptide:MHC complexes

TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.     

In a transgenic model of spontaneous autoimmune diabetes, expression of a protective class II MHC molecule results in thymic deletion of diabetogenic CD8+ T cells

H-2(d) mice expressing both the influenza virus hemagglutinin (HA) as a transgene-encoded protein on pancreatic islet beta cells (InsHA), as well as the Clone 4 TCR specific for the dominant H-2K(d)-restricted HA epitope, can be protected from the development of spontaneous autoimmune diabetes by expression of the H-2(b) haplotype. Protection occurs due to the deletion of K(d)HA-specific CD8+ T cells. This was unexpected as neither the presence of the InsHA transgene nor H-2(b), individually, resulted in thymic deletion. Further analyses revealed that thymic deletion required both a hybrid MHC class II molecule, Ebeta(b) Ealpha(d), and the K(d) molecule presenting the HA epitope, which together synergize to effect deletion of CD4+CD8+ thymocytes. This surprising example of protection from autoimmunity that maps to a class II MHC molecule, yet effects an alteration in the CD8+ T cell repertoire, suggests that selective events in the thymus represent the integrated strength of signal delivered to each cell through recognition of a variety of different MHC-peptide ligands.     

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").     

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.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

In vivo CD4+ T cell tolerance induction versus priming is independent of the rate and number of cell divisions

In vitro studies have suggested that tolerance induction (i.e., anergy) is associated with an inability of T cells to proliferate vigorously upon Ag recognition. In vivo, the relationship between T cell proliferation and tolerance induction is less clear. To clarify this issue, we have been studying a model system in which naive CD4+ T cells specific for the model Ag hemagluttinin (HA) are adoptively transferred into different transgenic founder lines of mice expressing HA as a peripheral self-Ag. When transferred into two lines whose HA expression differs by at least 1000-fold, HA-specific T cells undergo multiple rounds of cell division before reaching a nonresponsive (i.e., tolerant) state. While the proliferative response is more rapid in mice expressing higher levels of HA, the T cells become tolerant regardless of the level of peripheral HA expression. When the T cells encounter HA expressed as a viral Ag, they proliferate at a similar rate and undergo the same number of divisions as with self-HA, but they do not become tolerant. These results indicate that a tolerizing stimulus can induce similar T cell mitotic rates as a priming stimulus. Therefore, CD4+ T cell tolerance induction in vivo is not the result of an insufficient proliferative response elicited upon TCR engagement.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

Peptide-specific intercellular transfer of MHC class II to CD4+ T cells directly from the immunological synapse upon cellular dissociation

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-E(k) molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-E(k)-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56(lck), phosphotyrosine, and polarization of the actin cytoskeleton.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

Cutting edge: size and diversity of CD4+CD25high Foxp3+ regulatory T cell repertoire in humans: evidence for similarities and partial overlapping with CD4+CD25- T cells

Both differentiation and function of CD4+CD25(high) naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the alphabetaTCR repertoire sizes of human peripheral blood Treg and CD4+CD25- T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted alphabeta TCR repertoire, the size and diversity of which are closely similar to those of CD4+CD25- T cells, with a mean estimated size of 3.5 x 10(6) distinct alphabeta TCR vs 4.7 x 10(6) distinct alphabetaTCR for CD4+CD25- T cells. In addition, a 24% overlap between the repertoires of these two CD4+ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.     

Altered selection of CD4+ T cells by class II MHC bound with dominant and low abundance self-peptides

We have investigated the development of CD4(+) T cells in mice expressing low levels of transgenic class II MHC molecules (A(b)) preoccupied with covalent peptide (Ep), which in the presence of invariant chain (Ii) is extensively cleaved and replaced with self-derived peptides. In these mice, the transgenic A(b) molecules, bound with predominant peptide (Ep) and with multiple self-peptides, selected more CD4(+) T cells than A(b)/self-peptide complexes expressed in wild-type mice. The enhanced outcome of thymic selection was a result of impaired negative selection, rather than more efficient positive selection by an overall lowered abundance of self-derived A(b)/peptide complexes. Peripheral CD4(+) T cells in the A(b)EpIi(+) mice had memory phenotype, often followed by polyclonal activation of B cells. The A(b)EpIi(+) mice preserved their good health and had a normal life span despite the profound number of activated CD4(+) T cells and B cells in peripheral lymphoid organs, moderate hypergammaglobulinemia, and deposited complexes in the kidneys. We propose that CD4(+) T cells positively selected due to low avidity for high abundant A(b)Ep complex avoid negative selection on A(b) molecules loaded with low abundant peptides and become self-reactive in the peripheral lymphoid organs.     

CD4+ T cells recognizing a single self-peptide expressed by APCs induce spontaneous autoimmune arthritis

We have examined processes leading to the spontaneous development of autoimmune inflammatory arthritis in transgenic mice containing CD4+ T cells targeted to a nominal Ag (hemagglutinin (HA)) and coexpressing HA driven by a MHC class II promoter. Despite being subjected to multiple tolerance mechanisms, autoreactive CD4+ T cells accumulate in the periphery of these mice and promote systemic proinflammatory cytokine production. The majority of mice spontaneously develop inflammatory arthritis, which is accompanied by an enhanced regional immune response in lymph nodes draining major joints. Arthritis development is accompanied by systemic B cell activation; however, neither B cells nor Ab is required for arthritis development, since disease develops in a B cell-deficient background. Moreover, arthritis also develops in a recombinase activating gene-deficient background, indicating that the disease process is driven by CD4+ T cells recognizing the neo-self HA Ag. These findings show that autoreactive CD4+ T cells recognizing a single self-Ag, expressed by systemically distributed APCs, can induce arthritis via a mechanism that is independent of their ability to provide help for autoantibody production.     

Regulatory CD4+ CD25+ Foxp3+ T cells expand during experimental Plasmodium infection but do not prevent cerebral malaria

Pathogenic CD8+ T cells are implicated in the physiopathological mechanisms leading to experimental cerebral malaria (CM) in Plasmodium berghei ANKA (PbA) infected mice. Therefore, we hypothesised that in CM susceptible mice the neuropathology could be, at least in part, the result of an inefficient control of pathogenic effector T cells by CD4+ CD25+ Treg cells. Remarkably, the number of CD4+ CD25high T cells expressing Foxp3 increased in the spleen during the course of infection. These cells displayed an activated phenotype and consistent with that, CD4+ CD25high Treg cells isolated from PbA-infected mice showed an enhanced regulatory activity in vitro. Surprisingly, these cells do not migrate to the brain at the time of neurological symptoms as the conventional CD4+ T cells do. CM was not exacerbated in anti-CD25 treated mice when infected with PbA one month after treatment, even if splenic CD8+ T cells expressing CD69 increased in these mice. Taken together, these results show that P. berghei infection leads to an increase of the number of splenic CD4+ CD25high Treg cells exhibiting in vitro suppressive function, but they do not seem to be involved in vivo in the protection against CM.