Analysis of neonatally induced tolerance of H-2 alloantigens

Neonatal inoculation of mice with semi-allogeneic lymphohematopoietic cells produces a state of highly specific allograft tolerance. Phenotypically, by both in vivo and in vitro criteria, antigen-reactive cells specific for the tolerated antigens appear to be clonally deleted from intact, tolerant mice. However, a series of adoptive transfer experiments using mice rendered tolerant of variousH-2 alloantigens revealed that tolerance of Ia (class II) antigens is maintained by a different mechanism than tolerance of K/D (class I) antigens. Long-term acceptance of Ia-disparate grafts by recipients of Ia-tolerant lymphoid cells suggested that an active process (rather than passive clonal deletion) mediates and maintains this type of tolerance. No comparable success was achieved when tolerance of isolated class I or entireH-2 haplotype disparity was examined, suggesting that clonal deletion might be operative in these combinations. Modest prolongation of skin-graft survival was observed in adoptive transfer recipients of lymphoid cells from donors tolerant ofI-JECSD disparity. These data are compatible with the hypothesis that the centralI region (JE) promotes tolerance induction to associated strong IA- and D-region alloantigens by activating a suppression mechanism.With the technical assistance of Phoebe Strome.     

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.     

Tolerance to class II major histocompatibility complex molecules is maintained in the presence of endogenous, interleukin-2-producing, tolerogen-specific T lymphocytes

We have examined the requirement for clonal reductions of tolerogen-reactive lymphocytes in mice of the A strain background rendered neonatally tolerant of class II major histocompatibility complex molecules. Tolerogen-specific mixed lymphocyte reactivity of lymphocytes obtained from 130 adult, class II tolerant mice, bearing a healthy skin allograft, was examined. Lymphocytes obtained from 86 mice responded to the tolerogen, in vitro, with a positive mixed lymphocyte response (MLR) indicating that a large proportion (75%) of adult class II tolerant mice on the A strain background are not clonally deleted for tolerogen-reactive lymphocytes. In addition, lymphocytes from 29 mice were MLR-negative to the tolerogen, and lymphocytes from 15 mice demonstrated such high amounts of proliferation to syngeneic stimulators that their specific response to the tolerogen could not be determined. In view of the discordance between the in vivo and in vitro expressions of tolerance in the MLR-positive mice, lymphocytes from these mice were compared with normal lymphocytes by several assays. 1) Tolerogen-specific proliferative responses obtained from both normal and tolerant lymphocytes could be inhibited by the addition of monoclonal antibodies specific for the relevant class II antigens; 2) quantitative differences in the ability of normal, as compared with tolerant cells, to respond to the tolerogen in the MLR were not apparent; 3) no evidence of qualitative differences in the cell-surface phenotype of the proliferating cell was observed, (i.e., the cells were Thy-1+, L3T4+, Lyt-2-); and 4) lymphocytes from both normal and MLR-positive tolerant mice produced substantial amounts of interleukin-2 in response to the tolerogen. Thus, clonal deletion of helper cells is not required for tolerance to class II major histocompatibility complex antigens and we propose that tolerance may be maintained by either 1) in vivo suppression of the tolerogen-specific helper cells or 2) selective deletion or suppression of class II specific effector cells.     

The role of peripheral T-cell deletion in transplantation tolerance

The apoptotic deletion of thymocytes that express self-reactive antigen receptors is the basis of central (thymic) self-tolerance. However, it is clear that some autoreactive T cells escape deletion in the thymus and exist as mature lymphocytes in the periphery. Therefore, peripheral mechanisms of tolerance are also crucial, and failure of these peripheral mechanisms leads to autoimmunity. Clonal deletion, clonal anergy and immunoregulation and/or suppression have been suggested as mechanisms by which 'inappropriate' T-lymphocyte responses may be controlled in the periphery. Peripheral clonal deletion, which involves the apoptotic elimination of lymphocytes, is critical for T-cell homeostasis during normal immune responses, and is recognized as an important process by which self-tolerance is maintained. Transplantation of foreign tissue into an adult host represents a special case of 'inappropriate' T-cell reactivity that is subject to the same central and peripheral tolerance mechanisms that control reactivity against self. In this case, the unusually high frequency of naive T cells able to recognize and respond against non-self-allogeneic major histocompatibility complex (MHC) antigens leads to an exceptionally large pool of pathogenic effector lymphocytes that must be controlled if graft rejection is to be avoided. A great deal of effort has been directed toward understanding the role of clonal anergy and/or active immunoregulation in the induction of peripheral transplantation tolerance but, until recently, relatively little progress had been made towards defining the potential contribution of clonal deletion. Here, we outline recent data that define a clear requirement for deletion in the induction of peripheral transplantation tolerance across MHC barriers, and discuss the potential implications of these results in the context of current treatment modalities used in the clinical transplantation setting.     

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.     

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.     

Allo-I-J determinants participate in maintenance of neonatal H-2 tolerance

To evaluate the role of IJ antigens in maintenance of the tolerant state in adult H-2 tolerant mice, we have attempted to abolish tolerance by injecting monoclonal antibodies (mab) specific for host, donor, or third party IJ antigens into adult H-2 tolerant mice. Abolition of tolerance was evidenced by the rejection of fresh test skin grafts bearing the tolerated antigens. Whole H-2 tolerant mice treated with anti-IJ mab specific for donor (allo) IJ antigens rejected their test skin grafts, indicating that tolerance had been abolished. When two other types of tolerant mice were tested, we found that mice tolerant of class II antigens alone, but not mice tolerant of an IJ thru D disparity, were susceptible to the anti-donor IJ mab treatment. In addition, adult tolerant mice were unaffected by treatment with either anti-host or anti-third party IJ mab. When tested in vitro, lymphoid cells from tolerant mice, the tolerance of which was abolished by anti-IJ mab, remained unresponsive to the tolerogen, just as untreated (control) tolerant mice, in several in vitro assays (e.g., mixed lymphocyte reaction, cytotoxic T cell precursor frequency and bulk cell-mediated lysis without growth factor). Mice treated with antidonor IJ mab, however, unlike mice treated with anti-host or third party IJ mab, were capable of generating tolerogen-specific T cells in the absence of exogenous growth factor. Thus in the strain combinations we used, adult mice tolerant of either the entire H-2 region or of the class II major histocompatibility complex region alone are susceptible to abolition of the tolerant state by treatment with anti-donor IJ mab. Coincidentally, lymphoid cells from these mice generate sufficient endogenous T helper activity to activate the tolerogen-specific cytotoxic T cells. We suspect that these latter cells may be responsible for rejection of grafts bearing the tolerated antigens.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Sequential mechanisms of cyclophosphamide-induced skin allograft tolerance including the intrathymic clonal deletion followed by late breakdown of the clonal deletion

The cellular basis of the transplantation tolerance in a model system of BALB/c (Mls-1b) mice rendered cyclophosphamide (CP)-induced tolerant to DBA/2 (Mls-1a) skin allograft was investigated by assessing V beta 6+ T cells. From our results, three major mechanisms that are essential to the CP-induced skin allograft tolerance were sequentially elucidated. The first mechanism was destruction of donor-Ag-stimulated T cells in the periphery by CP treatment. The second mechanism was intrathymic clonal deletion of donor-reactive T cells, such as V beta 6+ T cells, correlating strongly with intrathymic mixed chimerism. The clonal deletion, however, was not always essential for the maintenance of the skin allografts, because DBA/2 skin survived even after the clonal deletion terminated and V beta 6+ T cells reappeared in the periphery of the recipient BALB/c mice. The third mechanism was generation of tolerogen-specific suppressor T cells, especially in the late stage of the tolerance. In contrast, the clonal anergy that is evidenced by the specific suppression of mixed lymphocyte reaction in the recipient BALB/c mice after injecting with DBA/2 spleen cells alone was not considered as a significant mechanism in prolonging skin allograft survival because such anergic mice showed accelerated rejection of the skin allografts. These results may suggest practical hierarchy of the mechanisms of CP-induced allograft tolerance.     

Neonatal tolerance of H-2 alloantigens. II. I region dependence of tolerance expressed to K and D antigens

Third-party skin allografts were employed to test the specificity of transplantation tolerance achieved by neonatal inoculation of cells bearing H-2 alloantigens. Tolerant animals rejected with normal vigour third-party grafts expressing strong Class I alloantigens foreign to the host and to the donor of the tolerance-conferring inoculum. However, these animals rejected with exceptional vigour third-party grafts expressing weak Class II alloantigens plus the tolerated Class I alloantigen; even third-party grafts comprised of the host's own Class II antigens in conjunction with the tolerated Class I alloantigen were acutely rejected. It is proposed, but there is no direct evidence to prove, that rejection of these third-party grafts is mediated by killer T cells directed at the tolerated Class I alloantigens and that these cells are activated by the presentation of the putative tolerogen in an inappropriate I region context. Inconsistency of these data with a clonal deletion mechanism is discussed.     

Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self tolerance is maintained in mature T cell populations. We have reported that murine bone marrow cells cultured in the presence of high-dose IL-2 (activated bone marrow cells) mediate strong veto suppressor function in vitro and in vivo, and that such veto activity is effected through clonal deletion of cytotoxic T cell precursors. In our studies, we have determined that bone marrow cell populations from athymic NCr-nu mice (H-2d) mediate strong veto cell activity without exposure to exogenous IL-2 in vitro. To examine mechanisms by which these naturally occurring veto cell populations in BM suppress precursor CTL (pCTL) responses, we used as a responding cell population in MLC, spleen cells of transgenic mice expressing at high frequency TCR specific for H-2 Ld encoded Ag with stimulation by H-2d-expressing cells in culture. Flow cytometric analysis was performed by staining the responding MLC cell population with the mAb 1B2 specific for the transgene-encoded TCR and determined changes of 1B2+ T cells. Such experiments demonstrated that the anti-H-2d cytotoxic response by these cell populations was specifically suppressed by NCr-nu (H-2d) bone marrow, and that 1B2+ pCTL were in fact specifically deleted from the responding cell population by incubation with such naturally occurring veto cell populations expressing the appropriate target Ag. In addition, to further understand the interactions of pCTL and veto cells and possible contributions by the latter to peripheral tolerance, we evaluated the effect of cyclosporine A (CsA) on veto cell-mediated suppression of pCTL of the transgenic mice. CsA inhibited veto cell-mediated suppression of cytotoxic T cell responses, and this inhibition correlated with a lack of clonal deletion of pCTL by veto cells in the presence of CsA. Furthermore, CsA exerted its effect through pCTL and not through veto cells, indicating that pCTL may play an active role in their own deletion by veto cells.     

Evidence for involvement of clonal anergy in MHC class I and class II disparate skin allograft tolerance after the termination of intrathymic clonal deletion

Mechanisms of cyclophosphamide (CP)-induced tolerance to class I (D) and class II (IE) alloantigens were studied. Transplantation tolerance across H-2D plus IE Ag-barriers has been achieved when B10.Thy-1.1 (Kb,IAb,IE-,Db; Thy-1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) mice (5R; kb,IAb,IEb,Dd; Thy-1.2) and treated i.p. with 200 mg/kg of CP 2 days later. The tolerant state in the early and the late stage was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor Ag. In the tolerant B10.Thy-1.1 mice treated with 5R cells 28 days earlier and followed by CP, intrathymic clonal deletion of V beta 11+ T cells reactive to IE-encoded antigens was observed in association with intrathymic mixed chimerism. 5R skin survived, however, even after the clonal deletion of V beta 11+ T cells terminated by 180 days after tolerance induction. V beta 11+ T cells, which reappeared in the periphery of the recipient B10.Thy-1.1 mice bearing 5R skin at this stage, were not capable of proliferating in response to receptor cross-linking with V beta 11-specific mAb. Furthermore, the CTL activity against class I (Dd) alloantigens of spleen cells from these tolerant mice was restored by the addition of IL-2 to MLC. Thus, our experiments provide direct evidence that tolerance to both class I (Dd) and class II (IEb) alloantigens by clonal allergy occurs during the termination of intrathymic clonal deletion. These results clearly show practical hierarchy of the mechanisms of transplantation tolerance.     

Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice

To address the mechanisms of tolerance to extrathymic proteins, we have generated transgenic mice expressing the lymphocytic choriomeningitis viral (LCMV) glycoprotein (GP) in the beta islet cells of the pancreas. The fate of LCMV GP-specific T cells was followed by breeding the GP transgenic mice with T cell receptor transgenic mice, specific for LCMV and H-2Db. These studies suggest that "peripheral tolerance" of self-reactive T cells does not involve clonal deletion, clonal anergy, or a decrease in the density of T cell receptors or accessory molecules. Instead, this model indicates that self-reactive cytotoxic T cells may remain functionally unresponsive, owing to a lack of appropriate T cell activation. Infection of transgenic mice with LCMV readily abolishes peripheral unresponsiveness to the self LCMV GP antigen, resulting in a CD8+ T cell-mediated diabetes. These data suggest that similar mechanisms may operate in several so-called "T cell-mediated autoimmune diseases."     

Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants

Transplantation tolerance can be induced in mice by grafting under the cover of nondepleting CD4 plus CD8 or CD154 mAbs. This tolerance is donor Ag specific and depends on a population of CD4(+) regulatory T cells that, as yet, remain poorly defined in terms of their specificity, origin, and phenotype. Blocking of the Ag-specific response in vitro with an anti-CD4 mAb allowed T cells from monospecific female TCR-transgenic mice against the male Ag Dby, presented by H-2E(k), to express high levels of foxP3 mRNA. foxP3 induction was dependent on TGF-beta. The nondepleting anti-CD4 mAb was also able to induce tolerance in vivo in such monospecific TCR-transgenic mice, and this too was dependent on TGF-beta. As in conventional mice, acquired tolerance was dominant, such that naive monospecific T cells were not able to override tolerance. Splenic T cells from tolerant mice proliferated normally in response to Ag, and secreted IFN-gamma and some IL-4, similar to control mice undergoing primary or secondary graft rejection. High levels of foxP3 mRNA, and glucocorticoid-induced TNFR superfamily member 18 (GITR)(+) CD25(+) T cells were found within the tolerated skin grafts of long-term tolerant recipients. These data suggest that regulatory T cells maintaining transplantation tolerance after CD4 Ab blockade can be induced de novo through a TGF-beta-dependent mechanism, and come to accumulate in tolerated grafts.     

Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity

The mechanism by which T lymphocytes are tolerized to self or foreign antigens is still controversial. Clonal deletion is the major mechanism of tolerance for immature thymocytes; for mature T cells, tolerance is considered to reflect anergy rather than deletion, and to be a consequence of defective presentation of antigen. This paper documents a novel form of tolerance resulting when mature T cells encounter antigen in immunogenic form. Evidence is presented that exposure of mature T cells to Mlsa antigens in vivo leads to specific tolerance and disappearance of Mlsa-reactive V beta 6+ T cells. Surprisingly, the clonal elimination of V beta 6+ cells is preceded by marked expansion of these cells. Thus, tolerance induction can be the end result of a powerful immune response. These data raise important questions concerning the relationship of tolerance and memory.     

Antigen presentation in acquired immunological tolerance

In acquired tolerance, previous exposure to antigen under certain conditions induces specific unresponsiveness instead of specific immunological memory. It has been studied as an approach to the mechanisms of self-tolerance that operate on immunocompetent T and B lymphocytes once they leave their sites of origin in the thymus and the bone marrow. Possible mechanisms involve induction of specific suppressor cells or inactivation of antigen-specific lymphocytes (clonal anergy) as a consequence of abortive antigen presentation, in which the antigen receptor is effectively engaged but certain poorly defined accessory signals the T lymphocytes require are lacking. We propose that small, resting B lymphocytes, which lack these accessory signals, are the inactivating antigen-presenting cells in acquired tolerance to proteins and to the class II transplantation antigens. B lymphocytes, which can use their antigen receptors to gather and process antigens that are present at very low concentrations, may play a role in self-tolerance. In addition, B lymphocytes and T lymphocytes rendered anergic by encounter with self antigens could persist as self-specific suppressor cells to block an autoimmune response of autoreactive clones that had escaped deletion or anergy.     

Clonal analysis of helper and effector T-Cell function in neonatal transplantation tolerance: Clonal deletion of helper cells determines lack of in vitro responsiveness

Mice rendered tolerant at birth of H-2 alloantigens display concordant in vivo and in vitro phenotypes: they fail to reject skin grafts bearing the tolerated antigens, and their lymphoid cells fail to participate in tolerogen-specific mixed lymphocyte reactions (MLRs) and cell-mediated lympholysis (CML). Tolerant animals normally reject third-party skin allografts and develop positive MLRs and CML to third-party antigens. It has been suggested that clonal deletion of antigen reactive cells is the basis for this spectrum of responses. To investigate further the basis for the lack of in vitro alloreactivity, we conducted limiting dilution studies with lymph node cells from adult mice tolerant of various H-2 disparities. When the frequencies of (a) cells responding to the tolerogen in MLR and (b) interleukin-2-producing cells against the tolerogen were determined, it appeared that both types of cells were functionally deleted, that is, the frequency of cells responding to tolerogen-bearing stimulator cells was identical with that of cells stimulated with syngeneic cells. On the assumption that cells from H-2 tolerant mice are deficient in helper cell activity toward the tolerogen, we performed CML cultures under conditions in which exogenous help was provided in the form of supernatants derived from concanavalin A stimulated rat spleen cell cultures. Lymphoid cells from H-2 tolerant mice generated significant cytotoxicity toward the tolerogen under these conditions, although the absolute level of killing was reduced compared with that of cells from normal mice. Limiting dilution assays confirmed that T_c precursors were present in tolerant mice, and that they were reduced to less than 10% of normal numbers; however, tolerogen-specific T_c precursors were present in frequencies significantly greater than self-reactive T_c precursors. These data indicate that a deletion mechanism operates in neonatal transplantation     

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Role of Mls-1 locus and clonal deletion of T cells in susceptibility to collagen-induced arthritis in mice

The role of T cell-mediated and humoral immunity to type II collagen has been well documented in collagen-induced arthritis (CIA). Previous work from our laboratory has indicated that genomic deletions of TCR V beta genes may play a role in CIA resistance in mice. This indicated a selectivity of TCR usage by autoreactive T cells in CIA in mice. Certain strains of mice, although having a normal genomic V beta TCR repertoire, can show clonal deletion of peripheral T cells that bear specific V beta gene products in their TCR. These clonally deleted T cells are reactive with self-Ag such as minor lymphocyte stimulation (Mls) Ag. An Mls-congenic strain, BALB.D2.Mlsa, which differs only at the Mls-1 a locus from BALB/c (Mls-1b), was used to examine the effect of clonal deletion of Mls-1a-reactive T cells in CIA. These two strains were crossed to three CIA-susceptible strains, B10.RIII (H-2r, Mls-1b), DBA/1 (H-2q, Mls-1a), and B10.Q (H-2q, Mls-1b), and the crosses were injected with type II collagen. A significantly decreased incidence of arthritis was observed in the (BALB.D2.Mlsa x B10.Q)F1 hybrids, compared with (BALB/c x B10.Q)F1 hybrids, upon immunization with chick type II collagen. The BALB.D2.Mlsa cross mice also had significantly lower levels of antimouse collagen antibodies. Flow cytometric analysis confirmed the clonal deletion of Mls-1a-reactive V beta 8.1, V beta 6, V beta 7, and V beta 9 subsets in the (BALB.D2.Mlsa x B10.Q)F1 hybrids. The study of H-2q/d mice in (BALB.D2.Mlsa x B10.Q) x B10.Q back-crosses demonstrated a significant correlation between CIA resistance and Mls-1a locus. On the other hand, B10.RIII crosses showed only a modest decrease in CIA incidence in the presence of Mls-1a. As expected, all the DBA/1 crosses had an equal incidence of CIA, which was somewhat less than that seen in DBA/1 mice themselves. These studies point out that the Mls-1a locus could play a role in decreasing CIA incidence by clonal deletion of T cells bearing specific V beta TCR, which may be involved in the pathogenesis of CIA. The influence of the clonal deletion of T cells on CIA, and hence the usage of specific V beta TCR by autoreactive anti-type II collagen T cells, however, depends not only on the source of the type II collagen and the MHC class II molecules involved but also on other background genes in mice.