Intrathymic Negative Selection Research Articles

Low-Level MHC Class II Expression Leads to Suboptimal Th Cell Response, Increased Autoaggression, and Heightened Cytokine Inducibility.

The development and activation of MHC class II (MHC-II)-restricted CD4+ T cells are distinct immunological processes that are strictly MHC-II-dependent. To address their relative dependence on MHC-II, we established a novel ENU-induced mutant mouse on the C57BL/6 background, named I-A12%, with ∼8-fold reduced I-A expression on the surface of B cells, dendritic cells, cortical thymic epithelial cells, and medullary thymic epithelial cells. I-A100% and I-A12% mice are highly similar with respect to the numbers of double-positive thymocytes, CD4+CD8- T cells, regulatory T cells, CD4+ T cell marker expression, lifespan, and Th/regulatory T cell function. Despite the demonstration of functional intrathymic negative selection in I-A12% mice, transfer of I-A12% CD25-CD4+ T cells into RAG-knockout hosts revealed increased autoaggression activity against the liver. Compared to I-A100% mice, infection of I-A12% mice with graded doses of Listeria monotcytogenes or influenza virus revealed comparable and significantly reduced generation of Ag-specific CD4+ T cells at high and low infection doses, respectively. A significantly weakened Ag-specific recall cytokine production response was also found for I-A12% mice previously infected with a relative low dose of L. monocytogenes CD44hiCD4+ T cells from I-A100% and I-A12% mice previously infected with a relatively high L. monocytogenes dose displayed highly similar Ag-specific multicytokine production profiles. In contrast, polyclonal activation of endogenous memory-like I-A12% CD44hiCD4+ T cells revealed highly elevated production of multiple cytokines. Our results demonstrate that there exist distinct thresholds for different MHC-II-dependent immunological processes. The I-A12% mutant mouse model we describe in the present study is a valuable tool for investigations on the quantitative cause-effect relationship in MHC-II-dependent normal and autoimmune responses.

Central tolerance spares the private high-avidity CD4(+) T-cell repertoire specific for an islet antigen in NOD mice.

Although central tolerance induces the deletion of most autoreactive T cells, some autoreactive T cells escape thymic censorship. Whether potentially harmful autoreactive T cells present distinct TCRαβ features remains unclear. Here, we analyzed the TCRαβ repertoire of CD4(+) T cells specific for the S100β protein, an islet antigen associated with type 1 diabetes. We found that diabetes-resistant NOD mice deficient for thymus specific serine protease (TSSP), a protease that impairs class II antigen presentation by thymic stromal cells, were hyporesponsive to the immunodominant S100β1-15 epitope, as compared to wild-type NOD mice, due to intrathymic negative selection. In both TSSP-deficient and wild-type NOD mice, the TCRαβ repertoire of S100β-specific CD4(+) T cells though diverse showed a specific bias for dominant TCRα rearrangements with limited CDR3α diversity. These dominant TCRα chains were public since they were found in all mice. They were of intermediate- to low-avidity. In contrast, high-avidity T cells expressed unique TCRs specific to each individual (private TCRs) and were only found in wild-type NOD mice. Hence, in NOD mice, the autoreactive CD4(+) T-cell compartment has two major components, a dominant and public low-avidity TCRα repertoire and a private high-avidity CD4(+) T-cell repertoire; the latter is deleted by re-enforced negative selection.

Monocyte-derived dendritic cells can induce autoreactive CD4(+) T cells showing myeloid lineage directed reactivity in healthy individuals.

T cells against self-antigens can be detected in peripheral blood of healthy individuals, although intrathymic negative selection removes most high-avidity T cells specific for self-antigens from the peripheral repertoire. Moreover, spontaneous T-cell proliferation following stimulation with autologous monocyte-derived dendritic cells (autoDCs) has been observed in vitro. In this study, we characterized the nature and immunological basis of the autoDC reactivity in the T-cell repertoire of healthy donors. We show that a minority of naive and memory CD4(+) T cells within the healthy human T-cell repertoire mediates HLA-restricted reactivity against autoDCs, which behave like a normal antigen-specific immune response. This reactivity appeared to be primarily directed against myeloid lineage cells. Although cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against autoDCs. AutoDC reactivity was also observed in the CD8(+) T-cell compartment, but this appeared to be mainly cytokine-induced rather than antigen-driven. In conclusion, we show that the presence of autoreactive T cells harboring the potential to react against autologous and HLA-matched allogeneic myeloid cells is a common phenomenon in healthy individuals. These autoDC-reactive T cells may help the induction of primary T-cell responses at the DC priming site.

Novel Process of Intrathymic Tumor-Immune Tolerance through CCR2-Mediated Recruitment of Sirpα+ Dendritic Cells: A Murine Model

Immune surveillance system can detect more efficiently secretory tumor-specific antigens, which are superior as a target for cancer immunotherapy. On the contrary, immune tolerance can be induced in the thymus when a tumor antigen is massively secreted into circulation. Thus, the secretion of tumor-specific antigen may have contradictory roles in tumor immunity in a context-dependent manner. However, it remains elusive on the precise cellular mechanism of intrathymic immune tolerance against tumor antigens. We previously demonstrated that a minor thymic conventional dendritic cell (cDC) subset, CD8α−Sirpα+ cDCs, but not the major subset, CD8α+Sirpα− cDCs can selectively capture blood-borne antigens and crucially contribute to the self-tolerance. In the present study, we further demonstrated that Sirpα+ cDCs can capture a blood-borne antigen leaking inside the interlobular vascular-rich regions (IVRs). Blood-borne antigen selectively captured by Sirpα+ cDCs can induce antigen-specific Treg generation or negative selection, depending on the immunogenicity of the presented antigen. Furthermore, CCR2 expression by thymic Sirpα+ cDCs and abundant expression of its ligands, particularly, CCL2 by tumor-bearing mice prompted us to examine the function of thymic Sirpα+ cDCs in tumor-bearing mice. Interestingly, tumor-bearing mice deposited CCL2 inside IVRs in the thymus. Moreover, tumor formation induced the accumulation of Sirpα+ cDCs in IVRs under the control of CCR2-CCL2 axis and enhanced their capacity to take up antigens, resulting in the shift from Treg differentiation to negative selection. Finally, intrathymic negative selection similarly ensued in CCR2-competent mice once the tumor-specific antigen was secreted into bloodstream. Thus, we demonstrated that thymic Sirpα+ cDCs crucially contribute to this novel process of intrathymic tumor immune tolerance.

Crucial Contribution of Thymic Sirpα+ Conventional Dendritic Cells to Central Tolerance against Blood-Borne Antigens in a CCR2-Dependent Manner

Thymic dendritic cells (DCs) as well as thymic epithelial cells are presumed to be major sentinels in central tolerance by inducing the apoptosis of autoreactive T progenitor cells. The thymic DC population is composed of heterogeneous subsets including CD11c(+)B220(+) plasmacytoid DCs, CD11c(+)B220(-)CD8alpha(+) signal regulatory protein alpha (Sirpalpha)(-) and CD11c(+)B220(-)CD8alpha(-)Sirpalpha(+) conventional DCs (cDCs). However, the distinctive role of each DC subset remains undefined. We show herein that Sirpalpha(+) cDCs, a minor subpopulation, was disseminated in the thymic cortical area with some of them uniquely localized inside perivascular regions and nearby small vessels in the thymus. The Sirpalpha(+) but not Sirpalpha(-) cDC subset can selectively capture blood-circulating Ags. Moreover, in CCR2-deficient mice, the thymic Sirpalpha(+) cDC subset, but not other thymic cell components, was moderately decreased especially in the perivascular regions. Concomitantly, these mice exhibited a modest impairment in intrathymic negative selection against blood-borne Ags, with the reduced capacity to uptake blood-borne Ags. Given their intrathymic cortical localization, CD11c(+)B220(-)CD8alpha(-)Sirpalpha(+) cDCs can have a unique role in the development of central tolerance against circulating peripheral Ags, at least partially in a CCR2-dependent manner.

No requirement for TRAIL in intrathymic negative selection

The contribution of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway to intrathymic negative selection is a controversial subject with two studies suggesting a key role for TRAIL, while others demonstrated normal negative selection, in TRAIL- and TRAIL receptor-deficient mice. The basis of these discrepancies is unclear and may in part reflect differences in the negative selection models under investigation. Considering the importance of the negative selection process in the establishment of a competent immune system, it is essential that these discrepancies be fully resolved. In this study, we failed to identify a role for TRAIL in an acute model of peptide antigen-specific negative selection using a TCR transgenic system as well as antibody-mediated TCR/CD3 ligation in vitro and in vivo. Moreover, thymic dendritic cells, the main cellular mediators of negative selection in the thymus, did not constitutively express TRAIL, and TRAIL receptor (DR5) expression was negative or extremely low on thymocytes. Furthermore, in vitro thymocyte deletion was normal in C57BL/6 TRAIL(-/-) gld mice, suggesting that TRAIL and FasL do not function cooperatively to induce negative selection. These results, combined with the fact that aged C57BL/6 TRAIL(-/-) mice showed no signs of spontaneous autoimmunity, strongly indicate that intrathymic negative selection occurs normally in the absence of TRAIL signaling.

Normal thymocyte negative selection in TRAIL-deficient mice.

The molecular basis of thymocyte negative selection, which plays a critical role in establishing and maintaining immunological tolerance, is not yet resolved. In particular, the importance of the death receptor subgroup of the tumor necrosis factor (TNF)-family has been the subject of many investigations, with equivocal results. A recent report suggested that TRAIL was a critical factor in this process, a result that does not fit well with previous studies that excluded a role for the FADD-caspase 8 pathway, which is essential for TRAIL and Fas ligand (FasL) signaling, in negative selection. We have investigated intrathymic negative selection of TRAIL-deficient thymocytes, using four well-established models, including antibody-mediated TCR/CD3 ligation in vitro, stimulation with endogenous superantigen in vitro and in vivo, and treatment with exogenous superantigen in vitro. We were unable to demonstrate a role for TRAIL signaling in any of these models, suggesting that this pathway is not a critical factor for thymocyte negative selection.

Despite efficient intrathymic negative selection of host-reactive T cells, autoimmune disease may develop in porcine thymus-grafted athymic mice: evidence for failure of regulatory mechanisms suppressing autoimmunity.

CD4 T-cell reconstitution and xenogeneic tolerance is achieved in T cell-depleted, thymectomized C57BL/6 (B6) mice and nude mice by grafting of fetal pig thymus (FP THY). Sixty percent of grafted nude mice and 10% of grafted thymectomized B6 mice develop a clinical illness resembling chronic graft-versus-host disease. Negative selection of mouse T cells in FP THY grafts was studied in "AND" TCR transgenic mice with a negative selecting MHC. Pathologic and immunohistochemical examinations and adoptive transfer assays were performed to determine the role of mouse CD4+ cells in the occurrence of autoimmune disease in this model. Marked clonal deletion of mouse thymocytes bearing a transgenic TCR ("AND"), which recognizes H2s expressed by host hematopoietic cells, was observed in FP THY grafts. Pathologic and immunohistochemical examinations of the liver, skin, lungs, and kidneys of mice with wasting syndrome showed marked mouse CD4+ T-cell infiltration without detectable pig cells. After adoptive transfer of splenocytes, but not of CD4+ cell-depleted splenocytes, from sick mice along with B6 bone marrow cells to lethally irradiated syngeneic B6 mice, the secondary recipients developed a similar autoimmune syndrome as the donors. Cotransfer of naïve syngeneic splenocytes prevented the occurrence of autoimmune disease in secondary recipients of splenocytes from healthy FP THY-grafted BALB/c nude mice. These results demonstrate a key role for mouse CD4+ T cells in causing autoimmune disease in this model and suggest the importance of regulatory mechanisms in addition to intrathymic clonal deletion for the maintenance of tolerance to recipient antigens.

Vav1-deficient mice are resistant to MOG-induced experimental autoimmune encephalomyelitis due to impaired antigen priming

Mice that lack the guanine nucleotide exchange factor (GEF) Vav1 exhibit particular defects in antigen-triggered T cell activation but may have an autoreactive T cell repertoire due to impaired intra-thymic negative selection. MOG 35–55-induced experimental autoimmune encephalomyelitis (EAE) was used to test the susceptibility of Vav1 −/− mice to organ-specific autoimmunity. Vav1 −/− animals were found to be resistant to MOG 35–55–EAE since the priming and in vivo expansion of myelin oligodendrocyte glycoprotein (MOG)-specific T cells was inefficient despite fully functional antigen presentation. Protection from cell-mediated autoimmunity was not due to a Th2 bias, to the lack of IL-2 or a failure of Vav1 −/− T cells in terms of chemotactic mobility.

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection.

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.

A role for accessibility to self-peptide-self-MHC complexes in intrathymic negative selection.

Whether intrathymic-positive and -negative selection of conventional alpha beta T cells occur in anatomically distinct sites is a matter of debate. By using a system composed of two distinct immune receptors, the Y-Ae mAb and the 1H3.1 (V alpha 1/V beta 6) TCR, both directed against the 52--68 fragment of the I-E alpha-chain (E alpha 52--68) bound to I-A(b), we examined the occurrence of negative selection imposed in vivo by a self-peptide-self-MHC class II complex with differential tissue expression. 1H3.1 TCR-transgenic (Tg) mice were bred to mice having an I-E alpha transgene with expression directed to all MHC class II-positive cells, restricted to thymic epithelial cells, or restricted to B cells, dendritic cells, and medullary thymic epithelial cells. All 1H3.1 TCR/I-E alpha double-Tg mice revealed a severely diminished thymic cellularity. Their lymph node cells were depleted of V beta 6(+)CD4(+) cells and were unresponsive to E alpha 52--68 in vitro. The absolute number of CD4(+)CD8(+) thymocytes was drastically reduced in all combinations, indicating that negative selection caused by an endogenously expressed self-determinant can effectively occur in the thymic cortex in vivo. Moreover, both cortical epithelial cells and, interestingly, the few cortical dendritic cells were able to support negative selection of CD4(+)CD8(+) thymocytes, albeit with a distinct efficiency. Collectively, these observations support a model where, in addition to the avidity of the thymocyte/stromal cell interaction, in vivo negative selection of autoreactive TCR-Tg T cells is determined by accessibility to self-peptide-self-MHC complexes regardless of the anatomical site.

Abrogation of Negative Selection by GVHR Induced by Minor Histocompatibility Antigens or H-2D Antigen Alone

Allogeneic bone marrow chimeras were prepared by donor and recipient combinations that differed in minor histocompatibility loci or H-2D locus aloneWhen 1×10 5 splenic T cells were inoculated in addition to T cell-depleted bone marrow cells (l×10 7), clinically detectable GVHR was inducedIn these GVHR chimeras, substantial numbers of T cells reactive to either donor or recipient antigens were both phenotypically and functionally detectedThe mechanisms underlying the abrogation of intrathymic negative selection are discussed.

Functional and phenotypic evidence for presentation of E alpha 52-68 structurally related self-peptide(s) in I-E alpha-deficient mice.

The Y-Ae mAb and the 1H3.1 TCR-alpha beta (V alpha 1/V beta 6) are two immune receptors specific for I-Ab MHC class II molecules complexed to the 52-68 fragment of the alpha-chain of I-E class II molecules (the E alpha 52-68 peptide). A profound intrathymic negative selection occurs in 1H3.1 TCR transgenic mice in the presence of an I-E alpha transgene. The administration of mAbs to 1H3.1/I-E alpha double-transgenic newborn mice reveals that Y-Ae, but not the isotype-matched anti-I-E Y17 mAb, rescues a significant number of mature (V beta 6highCD4+CD8-) thymocytes and allows the detection of E alpha 52-68-reactive T cells in the periphery. These observations indicate that deletion of autoreactive T cells can be specifically inhibited in vivo by an mAb specific for the deleting self-peptide:self-MHC class II complex. Similar inhibition experiments indicate that C57BL/6 (I-Ab+/I-E alpha-) mice constitutively express an E alpha-independent, Y-Ae-recognizable epitope(s). This finding is confirmed by the phenotypic analysis of mature (MHC class II high) C57BL/6 bone marrow-derived dendritic cells. Collectively, these observations further illustrate the peptide specificity of negative selection and demonstrate that MHC class II-positive cells from unmanipulated C57BL/6 mice that lack a functional I-E alpha gene can assemble one or more self-peptide:I-Ab complexes recognizable by the E alpha 52-68:I-Ab complex-specific Y-Ae mAb.

Role of IL-12 in Intrathymic Negative Selection

AbstractCytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R β2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the αβTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40−/− mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high “self” Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.

Cyclosporin A-Induced Autologous Graft-Versus-Host Disease: A Prototypical Model of Autoimmunity and Active (Dominant) Tolerance Coordinately Induced by Recent Thymic Emigrants

Cyclosporin A (CSA)-induced autologous graft-vs-host disease (autoGVHD) is an autoimmune syndrome initiated by autoeffector T cells presumed to be exported from the thymus during CSA treatment. The appearance of noncytotoxic immunoregulatory T cell activity after cessation of CSA treatment is also thymus dependent. In the present study, we have tested the hypothesis that both autoeffector and immunoregulatory T cells in CSA-treated rats are recent thymic emigrants (RTEs). Local syngeneic graft-vs-host reaction (synGVHR) and timed thymectomy (Tx) assays revealed that autoeffector T cells appear initially in the thymus and are promptly exported to lymph nodes (LN) during the first week of CSA treatment. In contrast, immunoregulatory thymocytes are first detectable by local synGVHR inhibition assays during the second week of CSA treatment but are not exported to LN until approximately 4 days post-CSA. Both the autoeffector and immunoregulatory T cells in LN express Thy-1, a selective marker for RTEs in the rat. However, the autoeffector RTEs have a CD4+8+ phenotype, whereas the immunoregulatory RTEs have a CD4+8- phenotype. Thus, the coordinate formation in and release from the thymus cortex and medulla of autoeffector and immunoregulatory T cells in CSA-treated rats directly demonstrates that centrally induced, nondeletional tolerance can serve as a fail-safe mechanism by which clones of autoeffector T cells that have escaped intrathymic negative selection for self-MHC class II Ag can be suppressed postthymically.

Central T Cell Tolerance in Lupus-Prone Mice: Influence of Autoimmune Background and the lpr Mutation

Abstract Lupus-prone mice develop a systemic autoimmune disease that is dependent upon the B cell help provided by autoreactive αβ CD4+ T cells. Since autoreactive T cells with high affinity for self peptides are normally deleted in the thymus, their presence in these mice suggests the possibility that intrathymic negative selection may be defective. Here, we directly compared central T cell tolerance in response to a conventional peptide Ag in lupus-prone MRL/MpJ mice with a nonautoimmune strain using an MHC class II-restricted TCR transgene. Our results did not demonstrate any defects after Ag exposure in the induction of intrathymic deletion of immature CD4+CD8+ thymocytes, in TCR down-regulation, or in the number of apoptotic thymocytes in MRL/MpJ compared with nonautoimmune mice. Furthermore, we found that the lpr mutation had no influence upon the Ag-induced thymic deletion of immature thymocytes. These data support the notion that T cell autoreactivity in MRL/MpJ mice is caused by defects in peripheral control mechanisms.

NADPH diaphorase-positive dendritic profiles in rat thymus are discrete from autofluorescent cells, immunoreactive for inducible nitric oxide synthase, and show strain-specific abundance differences.

Predisposition to autoimmune disorder in Lewis rats has been associated with abnormal hypothalamic regulation of circulating steroids, leading to inadequate suppression of T helper 1 (Th1) cell-mediated inflammatory reactions. In addition, autoimmune syndromes can be triggered within formerly resistant animals, following damage to the negative selection process of the thymus. A contribution to the autoimmune-susceptible phenotype may therefore derive from the status of thymic tolerance. One mechanism of intrathymic negative selection may involve nitric oxide. Because inducible nitric oxide synthase (iNOS) is known to be inhibitable by steroids, its expression might be different within strains having neuroendocrine disturbance. We report on a study to compare intrathymic iNOS expression in autoimmune-prone Lewis rats with other resistant strains. Interdigitating cells (IDC), darkly stained for diaphorase, were confirmed as immunoreactive for iNOS. They were located towards the medullary side of an accumulation of unstained, but autofluorescent cells (presumed to be macrophages) that circumscribes the corticomedullary zone. The role of iNOS+ IDC in the apoptotic deletion of T cells has been suggested by other studies. Despite the blunted steroidal condition reported for Lewis, nitrergic cell abundance was shown, by quantitative analysis of histochemical stain, to be on average approximately twofold lower compared with resistant strains (Fischer and Sprague-Dawley). This trend was evident in males and females, and confirmed by independent observers. We hypothesize that an intrathymic, iNOS-dependent mechanism may be important for the suppression of potentially autoreactive T-cell clones.

No evidence for emergence of autoreactive V β6 + T cells in Mls-1 a mice following exposure to a thymotoxic dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin

Tolerance to minor lymphocyte stimulatory-1 a (Mls-1 a) antigens is associated with clonal deletion of cells carrying the T cell receptor variable region Vβ6. Thymic epithelial cells may contribute to the intrathymic negative selection of potentially autoreactive Vβ6 + cells. Because 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) acts on thymic epithelial cells, we hypothesized that it may interfere with intrathymic selection processes. In the present study, this was addressed by exposing Mls-1 a DBA/2 mice to a single thymotoxic dose of TCDD. The emergence of Vβ6 + cells in thymus, spleen, and mesenteric lymph nodes was investigated during the subsequent recovery of TCDD-induced thymic atrophy. In addition, the extrathymic differentiation of T lymphocytes in the liver was studied. TCDD exposure resulted in a severe thymic atrophy, and an increase in hepatic mononuclear cells. However, we were not able to demonstrate any emergence of potentially autoreactive mature Vβ6 + T cells, that differentiated either intrathymically or extrathymically, in TCDD-exposed DBA/2 mice.

Involvement of CD45 in Dexamethasone- and Heat-Shock-Induced Apoptosis of Rat Thymocytes

CD45 is a transmembrane tyrosine-specific phosphatase which participates in lymphoid cell signal transduction during T cell activation, as well as in intrathymic negative and positive selection. In mammals, this molecule exhibits a variety of isoforms of different molecular weight, whose roles have still to be fully elucidated. We report here that apoptosis of rat thymocytes after in vitro dexamethasone and heat shock treatment was accompanied by an early significative increase of cells expressing CD45RC, the high molecular weight isoform of CD45 molecule. The same phenomenon was observed in thymocytes derived from in vivo dexamethasone-treated rats. However, the increase of CD45RC + cells was not apparently characteristic of cells undergoing apoptosis, as the same phenomenon was also observed in rat thymocytes induced to proliferate by Concanavalin A. On the whole, these results suggest that CD45 modulation can be added to the list of early molecular events, such as the increased expression of genes (ornithine decarboxylase), proto-oncogenes ( c-fos, c-jun, c-myc) and activation of transcription factors (AP-1, NFκB), we previously demonstrated in the same experimental model to occur and to be shared by these two apparently opposite biological processes, i.e.,cell proliferation and apoptosis, both likely depending on a complex balance of protein phosphorylation and dephosphorylation.

Collagen-induced arthritis and TCRs in SWR and B10.Q mice expressing an Ek alpha transgene.

B10.Ek alpha transgenic mice were mated with H2-E B10.Q and SWR mice. F1 and F1 x parental strain backcross progeny were tested for arthritis and autoimmune reactivity to mouse type II collagen (MII) after immunization with bovine, chick, deer, or human type II collagen. The results were correlated with the H-2 haplotype (b/q vs q/q) and the TCR V beta profile of peripheral blood T cells in each mouse. Hybrid progeny expressed TCR profiles different from either parent because of the TCR V beta genomic deletions of SWR mice (V beta a), the wild-type TCR allele of C57Bl/10 (B10) mice (V beta b), and the intrathymic negative selection processes resulting from cell surface expression of Ek alpha-A q beta or Eb beta-Ek alpha, together with the integrated retroviral genes Mtv-9 originating in B10 mice and Mtv-7 (Mls-1a) from SWR mice. (B10.Ek alpha x SWR)F1 mice developed higher IgG anti-MII Ab titers, but much milder arthritis than (B10.E x B10.Q)F1 mice. Expression of Ek alpha did not change the level of IgG anti-MII Ab nor the degree of susceptibility to collagen-induced arthritis (CIA) in the H-2q/q and H-2b/q progeny of (B10.Ek alpha x B10.Q)F1 x B10.Q matings, indicating that the Mtv-9-reactive, TCR V beta 5+, and V beta 11+ T cells are not critical to CIA. Among bovine type II collagen-immunized (B10.Ek alpha x SWR)F1 x SWR backcross mice: 1) arthritis severity is associated with the presence of V beta b (p < or = 0.01) and expression of Ek alpha (p < or = 0.05), but not with the MHC haplotype (b/q vs q/q); 2) regression analysis showed a significant association (R = 0.99) between IgG anti-MII Ab titers and the level of Mtv-7-reactive V beta 6+ T cells that was detectable in the IgG1, but not the IgG2a subclass. The data prompt the speculation that Mtv-7-reactive V beta 6+ (or V beta 7+) T cells in (B10.EK alpha x SWR)F1 x SWR mice express Th2-type properties, and thus contribute to the combination of mild arthritis but high anti-MII Ab titers that characterize mice of SWR heritage.