Failure to remove autoreactive Vbeta6+ T cells in Mls-1 newborn mice attributed to the delayed development of B cells in the thymus.

Clonal deletion of autoreactive T cells in the thymus is one of the major mechanisms for establishing tolerance to self-antigens, and self-reactive T cells bearing Vbeta6 T-cell receptors are usually deleted before their maturation in Mls-1a mice. However, these T cells develop transiently in the neonatal thymus, and migrate to the periphery. In order to understand the mechanisms which permit these potentially auto-toxic T cells to generate, we investigated in vivo the physiological or functional properties of the elements involved, such as neonatal T cells, antigens and antigen-presenting cells (APC). Confirming the previous findings that each of these elements per se is already completed in function in neonates, we investigated the possibility of the absence or immaturity of particular APC with Mls antigens of their own products in the neonatal thymus. In the search for the cellular and histological changes occurring in the newborn thymus, we found that the elimination of Vbeta6+ T cells progressed in parallel with the development of thymic B cells. Involvement of B cells in purging the autoreactive T cells from the newborn thymus was shown by prevention of the deletion of Vbeta6+ T cells after the removal of B cells by the treatment of neonates with anti-immunoglobulin M antibodies. The restricted and stable expression of CD5 on the thymic B cells, but not on the splenic cells, suggests that these B cells are not postnatal immigrants from the periphery. Finally, it is concluded that the deficiency in the deletion of self-reactive T cells in the thymus of Mls-1a neonates is due to the delayed development of B cells.

Production of minor lymphocyte stimulatory-1a antigens from T cell subsets.

T cell subsets that produce minor lymphocyte stimulatory (Mls) antigens were analyzed using mixed lymphocyte reaction (MLR) in vitro or clonal elimination assay in vivo. When lymph node T cells from B10.BR(Mls-1b) mice were stimulated with various T cell subsets from AKR (Mls-1a) mice in the presence of B10.BR antigen presenting cells (APC), proportions of Mls-1a reactive T cell blasts (V beta 6+, V beta 8.1+) increased. The stimulatory potency of CD8+ T cells was higher than that of CD4+ T cells. Furthermore, among either CD8+ or CD4+ T cell subset, CD44+ T cells appeared to produce larger amounts of Mls-1a antigens than CD44- T cells. More marked difference was demonstrated, when stimulator AKR T cells were being activated by immobilized anti-T cell antigen receptor (TCR) antibody during MLR. Thus, AKR T cells appeared to produce large amounts of Mls-1a antigens on appropriate stimulations. These findings were confirmed by the semiquantitative analysis of mRNA levels of MTV-7 in the AKR T cell subsets. When CD8+CD44+ T cells from (AKR x B10.BR)F1 mice were injected intravenously into [B10.BR-->B10.BR] syngeneic bone marrow (BM) chimeras 1 week after BM reconstitution and proportions of V beta 6+ T cells were quantitated 7 weeks later, significant clonal elimination of V beta 6+ T cells was induced among both thymocyte population and lymph node T cell population in a dose-dependent manner of the inoculated F1 T cells. Inoculation of CD8+CD44-F1 T cells eliminated V beta 6+ T cells less efficiently from lymph node T cells and inoculation of CD4+F1 T cells induced no significant clonal elimination of the V beta 6+ T cells. The present findings demonstrate clearly that CD8+CD44+ T cells represent the cells producing large amounts of Mls-1a antigens and inducing clonal elimination of V beta 6+ T cells in vivo.

Mls-1 and Mls-2 superantigens do not control susceptibility to collagen-induced arthritis in HI and HII mice.

The HI mouse line is sensitive to collagen-induced arthritis (CIA), whereas HII is refractory, although both express the H-2q permissive haplotype. The two lines also share the same T-cell receptor (TcR) gene haplotypes for alpha and beta chains. The distribution of mouse mammary tumour viruses (MMTV), which encode endogenous superantigens (SAg) such as minor leucocyte-stimulating antigens (Mls) known to modulate the available TcR-V beta repertoire, was investigated in the two lines. Mls-1 is present in HI-susceptible mice, while Mls-2 and Mls-2-like SAg are absent in both lines. This suggests that Mls antigens play no significant role in the resistance to CIA. Moreover, HI and HII exhibit close V beta gene usage as assessed by fluorescence staining with 11 V beta-specific monoclonal antibodies (mAb). These results indicate that mechanisms other than clonal deletion based on V beta expression and induced by SAg are involved in the resistance of H-2q-positive mice to experimental arthritis. Yet, a slightly reduced level of V beta 5+ T cells is observed in HII animals which might correlate with the presence of Mtv-6 and Mtv-9 proviruses.

A kinetic study on the deletion of thymic, peripheral, and gut-associated V beta 6+ T cells in an Mls-1b BALB/c colony infected with an exogenous mouse mammary tumor virus.

Mls-1a expression results in the deletion of T cells bearing V beta 6 chains of the TCR. However, V beta 6+ T cells are also deleted in Mls-1b BALB/c mice that have been infected with an exogenous mouse mammary tumor virus (swiss mice) via maternal milk intake, and whose open reading frame region is markedly similar to that of the provirus Mtv-7. In this report we describe the kinetics of V beta 6+ T cell deletion in the thymus, spleen, lymph nodes, and gut-associated lymphoid populations of these BALB/c mice from the early weeks of life to 6 mo of age. Deletion of V beta 6+ T cells within the CD4+ T cell population was more obvious in the thymus than in the spleen at 8 wk of age. However, the earliest incidence of deletion was observed in the gut intraepithelial lymphocyte population at 5 wk of age. Furthermore Mtv-7 (SW) transcripts were only found in the gut in the first wk of life, whereafter they could be detected in the thymus, spleen, and lymph nodes. This report suggests that after entering the intestinal tract of host mice, mouse mammary tumor virus (swiss mice) is subsequently transferred to the thymus and peripheral lymphoid organs resulting in the deletion of CD4+V beta 6+ T cells in that order.

Population movement and fate of autoreactive V beta 6+ T cells in Mls-1a mice.

In order to demonstrate the precise fail-safe mechanisms involved in the prevention of autoreactive T-cell functions, we analysed the movement of the population of self-reactive V beta 6+ cells in Mls-1a mice. T cells bearing V beta 6 T-cell receptor (TcR) could be detected in the thymus at birth. They increased in number during the next few days, then decreased and disappeared by 1 week after birth. These cells are autoreactive and capable of eliciting a syngeneic graft-versus-host reaction (GVHR). The autoreactive V beta 6+ cells in the thymus on day 3 were abolished by a previous injection of Mls-expressing syngeneic adult spleen cells, showing that the tolerance-inducing antigens had probably not yet developed in newborn mice. These autoreactive V beta 6+ cells escaping clonal deletion may leave the thymus and become appreciable as their percentages rise in the periphery in mice thymectomized 3 days after birth (d3-ThX). However, the 'autoreactive' T cells seemed to be neither cell cycling nor proliferating even after exogenous antigenic stimulation. The proportion of these peripheralized V beta 6+ cells in an 'anergy' state decreased gradually to a half-life of about 50 days in adults, in contrast to the complete deletion in a few days of V beta 6hi cells in the developing thymus. On the other hand, in weanlings the percentage of V beta 6+ T cells was reduced to a half-life of less than 20 days, probably because of the diluting out of these cells by the physiological expansion of the irrelevant T-cell population and probably by an increase of body fluid by a factor of 10. In contrast, V beta 8+ T cells, Mls-1a-unrelated, maintained a constant proportion, as in non-thymectomized mice. Thus, T-cell repertoire shaping may not always be achieved in the thymus, but may be completed after the cells leave the thymus a few days after birth in a developmentally programmed process.

Influence of the T cell receptor alpha-chain on T cell reactivity and tolerance to Mls-1 in T cell receptor beta-chain transgenic mice.

Previous analyses of a TCR V beta 8.1 transgenic mouse revealed multiple mechanisms of tolerance to the retroviral superantigen, Mls-1. Whereas some T cells were clonally deleted in the thymus, others became anergic in the periphery, or remained unaffected by the expression of Mls-1. In addition, a strong correlation between TCR alpha-chain usage and Mls-1 reactivity of individual transgenic V beta 8.1+ T cell hybridomas was established. Based on these observations, we speculated that the different mechanisms of tolerance were a consequence of the alpha-chain-mediated differences in Mls-1 reactivity. In the current studies, we make use of a V alpha 2-specific mAb to directly examine the role of the alpha-chain on tolerance in this transgenic model. We show, first, that V alpha 2+ CD4+ T cells, as a group, are relatively less Mls-1-reactive, and are elevated twofold in the periphery of Mls-1+ compared with Mls-1-V beta 8.1 transgenic mice. This elevated expression is also seen in the V alpha 2+ CD4+ population of mature thymocytes, but not in immature thymocytes. Second, Mls-1-induced neonatal tolerance in Mls-1-negative mice caused an increase of V alpha 2+ CD4+ T cells, comparable with the frequency of expression in transgenic mice that endogenously expressed Mls-1. Third, we have demonstrated a general correlation between the age-dependent increase in Mls-1-expression and the levels of V alpha 2+ CD4+ T cells during the first 4 wk of life. Taken together, these data suggest that the over-expression of V alpha 2+ CD4+ T cells in Mls-1+ mice is a consequence of mechanisms of tolerance, predominantly mediated by preferential lack of clonal deletion in the thymus. These data support the idea that clonal deletion is a competitive process and the influence of the TCR alpha-chain on the strength of Mls-1 reactivity of individual V beta 8.1+ transgenic T cells controls their susceptibility to clonal deletion.

Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features.

Mouse mammary tumor virus (MMTV)-encoded superantigens (SAGs) influence the murine T cell repertoire and stimulate a strong mixed lymphocyte response in vitro. These SAGs are encoded by the open reading frame of the 3' long terminal repeat of MMTV, termed MMTV SAGs. The T cell response to MMTV SAGs is V beta restricted and requires expression of the class II molecules of the major histocompatibility complex (MHC) on the presenting cells. While human T cells respond to bacterial SAGs, it is not known if human T cells or human MHC class II molecules can interact with MMTV SAGs. A fibroblastic cell line expressing the human MHC class II molecule HLA-DR1 and the Mtv-7 sag gene encoding Mls-1 was used to stimulate human T cells. We show here that human T cells efficiently proliferate in response to Mls-1 presented by HLA-DR1. This T cell response was inhibited by mAbs directed against CD4 or MHC class II molecules but not by mAbs specific for CD8 or MHC class I molecules. Moreover, the response to Mls-1 was limited to human T cells expressing a restricted set of T cell receptor V beta chains. Human T cells expressing V beta 12, 13, 14, 15, and 23 were selectively amplified after Mtv-7 sag stimulation. Interestingly, these human V beta s share the highest degree of homology with the mouse V beta s interacting with Mls-1. These results show a strong evolutionary conservation of the structures required for the presentation and the response to retrovirally encoded endogenous SAGs, raising the possibility that similar elements operate in humans to shape the T cell repertoire.

Deletion of donor-reactive T lymphocytes in adult mice after intrathymic inoculation with lymphoid cells.

Clonal deletion of self antigen-reactive T lymphocytes is known to be a dominant mechanism of tolerance induction in the normal immune system. This report considers whether deletion of antigen-reactive T cells is also the immunologic basis for the recently described model of transplantation tolerance that follows intrathymic inoculation with allogeneic lymphoid cells. We found that the outcome of injecting Mlsa- hosts with lymphocytes from Mlsa+ donors was depletion of V beta 6+ T cells (which are known to be reactive with the Mlsa superantigen). The process was found to be specific in that a similar reduction was not seen in an irrelevant T cell population (V beta 8+) in IT injected hosts. Deletion was observed in this model only if immunosuppression with ALS or anti-CD4 accompanied intrathymic injection. When the inoculum of allogeneic lymphocytes was administered intravenously instead of intrathymically only minimal deletion was observed. The induction of transplantation tolerance by intrathymic injection of donor lymphoid cells may prove especially efficacious since it relies on deletion of only those T cells specifically reactive to donor antigens, a process analogous to tolerance induction to self antigens.

Minor lymphocyte stimulatory antigen-bearing stimulator cells require lipopolysaccharide activation to induce programmed cell death in T cell hybridomas.

T cell hybridomas respond to conventional peptide Ag associated with self major histocompatibility restriction elements, as well as to alloantigens, activating lectins, and stimulatory forms of mAb by producing lymphokines and undergoing programmed cell death (PCD). We show here that the level of PCD and IL-2 production correlate well in responses to CD3 or allostimulation. The response to minor lymphocyte-stimulatory (Mls) Ag, members of the family of endogenous superantigens, however, are marked by divergence in the levels of the PCD and lymphokine responses. Specifically, PCD in response to Mls activation is achieved poorly despite vigorous IL-2 production. B lymphoma cell stimulators induced PCD in alloreactive T cell hybridomas but not in Mls-reactive T cell hybridomas. This suggests that the absence of PCD in the Mls response is a function of superantigen recognition rather than the stimulator cell type. LPS-preactivated Mls+ stimulators, either splenic B or B lymphoma cells, are shown to trigger PCD in the T cell hybridomas. These results imply that T cell interaction with Mls presented by untreated stimulator cells is not sufficient for induction of PCD and thus is distinct from interactions with conventional Ag.

Expression of Mls determinants in mice exhibiting the severe combined immunodeficiency (scid) mutation or X-linked immunodeficiency (xid) defect.

While Ig+ B cells appear to be the principal cell type expressing immunogenic minor lymphocyte stimulatory (Mls) determinants, both T cells and B cells are capable of mediating deletion of developing Mls-reactive thymocytes. In addition, levels of mouse mammary tumor proviral transcripts are increased after B or T cell stimulation, and expression of functional Mls determinants is augmented by activation of B cells. These findings suggest Mls determinants are present on B and T lymphocytes, and that activation of B and T cells augments Mls expression. In the present study, we wished to determine whether B and T cells were required for expression of Mls determinants by examining mice with severe combined immunodeficiency (SCID) containing no detectable Ig+ B cells or TCR+ T cells, as well as animals that expressed the X-linked immunodeficiency (xid) defect and lacked a subset of mature B cells. We found Mlsa-reactive V beta 6hi T cells were deleted from thymi of male (CBA/NxAKR/J)F1 xid mice, and that spleen cells from these animals stimulated anti-Mlsa mixed lymphocyte responses by unprimed B10.BR spleen T cells. In addition, Mlsc-reactive V beta 3hi AKR/J thymocytes and spleen T cells were deleted in AKR/J----SCID bone marrow chimeras, and spleen cells from SCID mice stimulated proliferation by an Mlsc-specific T cell clone. These results demonstrate that both xid mice and SCID animals express Mls determinants that mediate deletion of developing, Mls-responsive thymocytes and stimulate proliferation of mature, Mls-reactive T cells. Hence, mature B cells and T cells are not essential for Mls expression.

Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7.

Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.

An exogenous mouse mammary tumor virus with properties of Mls-1a (Mtv-7).

The classical minor lymphocyte stimulating (Mls) antigens, which induce a strong primary T cell response in vitro, are closely linked to endogenous copies of mouse mammary tumor viruses (MMTV). Expression of Mls genes leads to clonal deletion of T cell subsets expressing specific T cell receptor (TCR) V beta chains. We describe the isolation and characterization of a new exogenous (infectious) MMTV with biological properties similar to the Mls antigen Mls-1a. In vivo administration of either Mls-1a-expressing B cells or the infectious MMTV (SW) led to an increase of T cells expressing V beta 6 followed by their deletion. Surprisingly, different kinetics of deletion were observed with the exogenous virus depending upon the route of infection. Infection through the mucosa led to a slow deletion of V beta 6+ T cells, whereas deletion was rapid after subcutaneous infection. Sequence analysis of the open reading frames in the 3' long terminal repeat of both this exogenous MMTV (SW) and of Mtv-7 (which is closely linked to Mls-1a) revealed striking similarities, particularly in the COOH terminus, which has been implicated in TCR V beta recognition. The identification of an infectious MMTV with the properties of a strong Mls antigen provides a new, powerful tool to study immunity and tolerance in vivo.

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.

Physiologic expression of two superantigens in the BDF1 mouse.

The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.