Thymic expression of a gastritogenic epitope results in positive selection of self-reactive pathogenic T cells

Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase beta subunit(253-277) (H/Kbeta(253-277)), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kbeta(253-277)) expressed in mice under control of the Ii promoter. The Ii-H/Kbeta(253-277) fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kbeta(253-277) transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kbeta(253-277)-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kbeta(253-277) enhanced positive selection of 1E4-TCR cells in Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kbeta(253-277) mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.     

CD4+CD25+ regulatory T cells inhibit the antigen-dependent expansion of self-reactive T cells in vivo

A deficiency of CD4+CD25+ regulatory T cells (CD25+ Tregs) in lymphopenic mice can result in the onset of autoimmune gastritis. The gastric H/K ATPase alpha (H/Kalpha) and beta (H/Kbeta) subunits are the immunodominant autoantigens recognized by effector CD4+ T cells in autoimmune gastritis. The mechanism by which CD25+ Tregs suppress autoimmune gastritis in lymphopenic mice is poorly understood. To investigate the antigenic requirements for the genesis and survival of gastritis-protecting CD25+ Tregs, we analyzed mice deficient in H/Kbeta and H/Kalpha, as well as a transgenic mouse line (H/Kbeta-tsA58 Tg line 224) that lacks differentiated gastric epithelial cells. By adoptive transfer of purified T cell populations to athymic mice, we show that the CD25+ Treg population from mice deficient in either one or both of H/Kalpha and H/Kbeta, or from the H/Kbeta-tsA58 Tg line 224 mice, is equally effective in suppressing the ability of polyclonal populations of effector CD4+ T cells to induce autoimmune gastritis. Furthermore, CD25+ Tregs, from either wild-type or H/Kalpha-deficient mice, dramatically reduced the expansion of pathogenic H/Kalpha-specific TCR transgenic T cells and the induction of autoimmune gastritis in athymic recipient mice. Proliferation of H/Kalpha-specific T cells in lymphopenic hosts occurs predominantly in the paragastric lymph node and was dependent on the presence of the cognate H/Kalpha Ag. Collectively, these studies demonstrate that the gastritis-protecting CD25+ Tregs do not depend on the major gastric Ags for their thymic development or their survival in the periphery, and that CD25+ Tregs inhibit the Ag-specific expansion of pathogenic T cells in vivo.     

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.     

Identification of a new type of invariant V alpha 14+ T cells and responsiveness to a superantigen, Yersinia pseudotuberculosis-derived mitogen.

We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-alpha beta+ CD4+ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4int thymic T cells, which express a skewed V beta repertoire of V beta 2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44high, Ly6Chigh, NK1.1+, and TCR-alpha beta low. H4high thymic T cells also show a skewed V beta repertoire, V beta 2, -7, and -8, and predominantly express an invariant V alpha 14-J alpha 281+ alpha-chain in their TCRs but constitute a distinct population in that they are CD44int, Ly6C-, NK1.1-, and TCR-alpha beta high. Thus, invariant V alpha 14+ thymic T cells consist of ordinary NKT cells and a new type of T cell population. V beta 7+ and V beta 8.1+ invariant V alpha 14+ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, V beta 7+ invariant V alpha 14+ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced V beta 7+ invariant V alpha 14+ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V alpha 14+ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.     

Endogenous H/K ATPase beta-subunit promotes T cell tolerance to the immunodominant gastritogenic determinant

A CD4(+) T cell response to the gastric H/K ATPase beta-subunit (H/Kbeta) is required for the onset of experimental autoimmune gastritis in BALB/c mice. The extent to which endogenous H/Kbeta contributes toward the tolerance of the H/Kbeta-specific T cell repertoire in normal individuals is not known. By comparison of T cell responses in H/Kbeta-deficient (o/o) and H/Kbeta-expressing BALB/c mice, in this work we show that the endogenous H/Kbeta autoantigen plays a major role in the tolerance of pathogenic H/Kbeta-specific T cells. First, T cell-dependent Ab responses to the H/Kbeta Ag were enhanced in H/K ATPase-immunized H/Kbeta-deficient mice compared with wild-type mice. Second, peptide immunization experiments indicated that immune responses to the major gastritogenic epitope of the H/K ATPase, namely H/Kbeta(253-277), were significantly more vigorous in H/Kbeta-deficient mice compared with wild-type mice. Third, unfractionated splenocytes from H/Kbeta-deficient mice, but not H/Kbeta-expressing mice, induced autoimmune gastritis after adoptive transfer to BALB/c nude mice. The enhanced responses to H/Kbeta in H/Kbeta-deficient mice were shown to be intrinsic to CD4(+)CD25(-) T cells rather than a change in status of CD4(+)CD25(+) regulatory T cells. We conclude from these studies that the H/Kbeta-specific T cells in wild-type mice represent the residue of a T cell repertoire, directed toward a single determinant, that has been subjected to partial tolerance induction.     

Chagasic thymic atrophy does not affect negative selection but results in the export of activated CD4+CD8+ T cells in severe forms of human disease

Extrathymic CD4+CD8+ double-positive (DP) T cells are increased in some pathophysiological conditions, including infectious diseases. In the murine model of Chagas disease, it has been shown that the protozoan parasite Trypanosoma cruzi is able to target the thymus and induce alterations of the thymic microenvironment and the lymphoid compartment. In the acute phase, this results in a severe atrophy of the organ and early release of DP cells into the periphery. To date, the effect of the changes promoted by the parasite infection on thymic central tolerance has remained elusive. Herein we show that the intrathymic key elements that are necessary to promote the negative selection of thymocytes undergoing maturation during the thymopoiesis remains functional during the acute chagasic thymic atrophy. Intrathymic expression of the autoimmune regulator factor (Aire) and tissue-restricted antigen (TRA) genes is normal. In addition, the expression of the proapoptotic Bim protein in thymocytes was not changed, revealing that the parasite infection-induced thymus atrophy has no effect on these marker genes necessary to promote clonal deletion of T cells. In a chicken egg ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic system, the administration of OVA peptide into infected mice with thymic atrophy promoted OVA-specific thymocyte apoptosis, further indicating normal negative selection process during the infection. Yet, although the intrathymic checkpoints necessary for thymic negative selection are present in the acute phase of Chagas disease, we found that the DP cells released into the periphery acquire an activated phenotype similar to what is described for activated effector or memory single-positive T cells. Most interestingly, we also demonstrate that increased percentages of peripheral blood subset of DP cells exhibiting an activated HLA-DR+ phenotype are associated with severe cardiac forms of human chronic Chagas disease. These cells may contribute to the immunopathological events seen in the Chagas disease.     

Tolerance to proinsulin-2 is due to radioresistant thymic cells

Proinsulin is a key Ag in type 1 diabetes, but the mechanisms regulating proinsulin immune tolerance are unknown. We have shown that preproinsulin-2 gene-deficient mice (proins-2(-/-)) are intolerant to proinsulin-2. In this study, we analyzed the mechanisms underlying T cell-mediated tolerance to proinsulin-2 in 129/Sv nonautoimmune mice. The expression of one proinsulin-2 allele, whatever its parental origin, was sufficient to maintain tolerance. The site of proinsulin-2 expression relevant to tolerance was evaluated in thymus and bone marrow chimeras. CD4+ T cell reactivity to proinsulin-2 was independent of proinsulin-2 expression in radiation-sensitive bone marrow-derived cells. A wt thymus restored tolerance in proins-2(-/-) mice. Conversely, the absence of the preproinsulin-2 gene in radioresistant thymic cells was sufficient to break tolerance. Although chimeric animals had proinsulin-2-reactive CD4+ T cells in their peripheral repertoire, they displayed no insulitis or insulin Abs, suggesting additional protective mechanisms. In a model involving transfer to immunodeficient (CD3epsilon(-/-)) mice, naive and proinsulin-2-primed CD4+ T cells were not activated, but could be activated by immunization regardless of whether the recipient mice expressed proinsulin-2. Furthermore, we could not identify a role for putative specific T cells regulating proinsulin-2-reactive CD4+ T in transfer experiments. Thus, proinsulin-2 gene expression by radioresistant thymic epithelial cells is involved in the induction of self-tolerance, and additional factors are required to induce islet abnormalities.     

Autophagy in the thymic epithelium is dispensable for the development of self-tolerance in a novel mouse model

The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.     

Lymphotoxin beta receptor is required for the migration and selection of autoreactive T cells in thymic medulla

How organ-specific central tolerance is established and regulated has been an intriguing question. Lymphotoxin beta receptor (LTbetaR) deficiency is associated with autoimmune phenotypes characterized by humoral and cellular autoreactivity to peripheral organs. Whether this results from defective negative selection of T cells directed at tissue-restricted Ags has not been well understood. By tracing the development of OT-I thymocytes in rat insulin 2 promoter-mOVA transgenic mice on either Ltbr+/+ or Ltbr-/- background, we demonstrate that LTbetaR is necessary for thymic negative selection. LTbetaR deficiency resulted in a dramatic escape of "neo-self" specific OT-I cells that persist in circulation and lead to development of peri-insulitis. When the underlying mechanism was further explored, we found interestingly that LTbetaR deficiency did not result in reduced thymic expression of mOVA. Instead, LTbetaR was revealed to control the expression of thymic medullary chemokines (secondary lymphoid tissue chemokine (SLC) and EBV-induced molecule 1 ligand chemokine (ELC)) which are required for thymocytes migration and selection in medulla. Furthermore, RIP-mOVA transgenic mice on SLC/ELC deficient background (plt) demonstrated significant impaired negative selection of OT-I cells, suggesting that the dysregulation of SLC/ELC- expression alone in Ltbr-/- thymi can be sufficient to impair thymic negative selection. Thus, LTbetaR has been revealed to play an important role in thymic negative selection of organ-specific thymocytes through thymic medullary chemokines regulation.     

NF-kappaB2 is required for the control of autoimmunity by regulating the development of medullary thymic epithelial cells

Medullary thymic epithelial cells function as antigen-presenting cells in negative selection of self-reactive T cell clones, a process essential for the establishment of central self-tolerance. These cells mirror peripheral tissues through promiscuous expression of a diverse set of tissue-restricted self-antigens. The genes and signaling pathways that regulate the development of medullary thymic epithelial cells are not fully understood. Here we show that mice deficient in NF-kappaB2, a member of the NF-kappaB family, display a marked reduction in the number of mature medullary thymic epithelial cells that express CD80 and bind the lectin Ulex europaeus agglutinin-1, leading to a significant decrease in the extent of promiscuous gene expression in the thymus of NF-kappaB2(-/-) mice. Moreover, NF-kappaB2(-/-) mice manifest autoimmunity characterized by multiorgan infiltration of activated T cells and high levels of autoantibodies to multiple organs. A subpopulation of the mice also develops immune complex glomerulonephritis. These findings identify a physiological function of NF-kappaB2 in the development of medullary thymic epithelial cells and, thus, the control of self-tolerance induction.     

Split tolerance in nude mice transplanted with 2'-deoxyguanosine-treated allogeneic thymus lobes

To elucidate the acquisition of self tolerance in the thymus, full-allogeneic thymic chimeras were constructed. Athymic C3H and BALB/c nude mice were reconstituted with the thymic lobes of BALB/c and B10.BR fetuses, respectively, that were organ cultured for 5 days in the presence of 2'-deoxyguanosine. T cells in these chimeras were tolerized to the host MHC in both MLR and CTL assays. In contrast, T cells in the chimeras exhibited split tolerance for the thymic MHC haplotype. CTL specific for class I MHC of the thymic haplotype were generated not only from the peripheral T cells of the chimeras but also from thymocytes re-populated in the engrafted thymic lobes. However, T cells in these chimeras responded poorly to the class II MHC of the thymic haplotype in a standard MLR assay. In a syngeneic MLR culture upon stimulation with enriched APC of the thymic haplotype, only 22 to 48% of the responses were mediated by CD4+ cells, and proliferations of CD4- cells were prominent. There were no haplotype-specific suppressor cells detected which would cause the unresponsiveness to the thymic class II MHC. These results indicated that the thymic lobes treated with 2'-deoxyguanosine were defective in the ability to induce the transplantation tolerance for the class I MHC expressed on the thymus, although the same thymic lobes were able to induce the transplantation tolerance for the thymic class II MHC.     

B cells participate in thymic negative selection of murine auto-reactive CD4+ T cells

It is well documented that thymic epithelial cells participate in the process of negative selection in the thymus. In recent years it was reported that also dendritic cells enter the thymus and contribute to this process, thus allowing for the depletion of thymocytes that are specific to peripherally expressed self-antigens. Here we report that also B cells may take part in the elimination of auto-reactive thymocytes. Using a unique mouse model we show that B cells induce negative selection of self-reactive thymocytes in a process that leads to the deletion of these cells whereas regulatory T cells are spared. These findings have direct implication in autoimmunity, as expression of a myelin antigen by B cells in the thymus renders the mice resistant to autoimmune inflammation of the CNS.     

Through regulation of TCR expression levels, an Idd7 region gene(s) interactively contributes to the impaired thymic deletion of autoreactive diabetogenic CD8+ T cells in nonobese diabetic mice

When expressed in NOD, but not C57BL/6 (B6) genetic background mice, the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose the ability to mediate the thymic deletion of autoreactive CD8+ T cells contributing to type 1 diabetes (T1D). This indicated some subset of the T1D susceptibility (Idd) genes located outside the MHC of NOD mice interactively impair the negative selection of diabetogenic CD8+ T cells. In this study, using both linkage and congenic strain analyses, we demonstrate contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chromosome 7 predominantly, but not exclusively, determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells as illustrated using the AI4 TCR transgenic system. The polymorphic Idd7 region gene(s) appears to control events that respectively result in high vs low expression of the AI4 clonotypic TCR alpha-chain on developing thymocytes in B6.H2g7 and NOD background mice. This expression difference likely lowers levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. These findings provide further insight to how susceptibility genes, both within and outside the MHC, may interact to elicit autoreactive T cell responses mediating T1D development in both NOD mice and human patients.     

CD83 expression influences CD4+ T cell development in the thymus

T lymphocyte selection and lineage commitment in the thymus requires multiple signals. Herein, CD4+ T cell generation required engagement of CD83, a surface molecule expressed by thymic epithelial and dendritic cells. CD83-deficient (CD83-/-) mice had a specific block in CD4+ single-positive thymocyte development without increased CD4+CD8+ double- or CD8+ single-positive thymocytes. This resulted in a selective 75%-90% reduction in peripheral CD4+ T cells, predominantly within the naive subset. Wild-type thymocytes and bone marrow stem cells failed to differentiate into mature CD4+ T cells when transferred into CD83-/- mice, while CD83-/- thymocytes and stem cells developed normally in wild-type mice. Thereby, CD83 expression represents an additional regulatory component for CD4+ T cell development in the thymus.     

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

Clonal deletion of simian virus 40 large T antigen-specific T cells in the transgenic adenocarcinoma of mouse prostate mice: an important role for clonal deletion in shaping the repertoire of T cells specific for antigens overexpressed in solid tumors

In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2K(k). Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus.