Neonatal tolerance induction to Mlsa. II. T cells induce tolerance to Mlsa

We have investigated the ability of murine T cell lines to induce neonatal tolerance to Mlsa (minor lymphocyte stimulating). Mlsb mice were injected within 24 hr of birth with MHC (major histocompatibility complex) identical T cell lines generated by culturing responders from Mlsa strains with stimulators from Mlsb strains. Injected mice were tested at 6 to 8 weeks of age for responses in either primary mixed leukocyte reaction or IL-2 limiting dilution analysis. Mlsa specific responses by injected tolerant mice relative to noninjected controls were reduced by 92-98% in MLR and by 2- to 10-fold in IL-2 LDA. In contrast, responses against third-party MHC antigens by either the injected or the noninjected mice were identical. Fifty percent of all mice injected with the T cell lines were tolerant to Mlsa. These results strongly suggest that murine T cells express the Mlsa gene product.     

T cell recognition of Mls. T cell clones demonstrate polymorphism between Mlsa, Mlsc, and Mlsd

The determinants encoded by the minor lymphocyte stimulating locus (Mls) are defined as determinants that induce strong T cell proliferative responses in primary mixed lymphocyte reactions. Although the Mls locus was originally described as having four alleles, a, b, c, and d, a number of recent observations have led several investigators to challenge the idea that Mls is truly a polymorphic system. To better define this system of determinants recognized at high frequency by T cells, the present studies were undertaken to evaluate the polymorphism of Mls products. In the present study, the in vitro proliferative responses of Mlsa- and Mlsc-specific T cell clones were employed to analyze Mls products. The identification of determinants recognized by Mlsa- and Mlsc-reactive clones was established by the pattern of responses to stimulators derived from congenic strains, recombinant inbred strains, and backcross mice. T cell clones and unprimed T cells gave concordant responses that confirmed the Mlsa or Mlsc specificity of the cloned populations. With the use of these two sets of Mls-specific T cell clones, the existence or absence of polymorphism of Mls-encoded gene products was examined. It was found that Mlsa-specific cloned T cells responded to Mlsa but not Mlsc stimulators, whereas Mlsc-specific clones responded to Mlsc but not Mlsa. This reciprocal pattern of specificity indicates that the Mls system as currently defined is therefore truly polymorphic. In addition, it was observed that both Mlsa- and Mlsc-specific clones were stimulated by Mlsd stimulators. In particular, the possibility that Mlsa and Mlsc are not alleles but products of different loci and that Mlsd strains are those that express both Mlsa and Mlsc is considered.     

Macrophages and T cells do not express Mlsa determinants

In order to test the tissue distribution of Mlsa determinants, we have generated highly purified stimulator cell populations. First, Mlsa expression in bone marrow derived macrophages (M phi) of Mlsa genotype was tested in primary MLR and on Mlsa-specific T cell hybridomas (THy). Second, a similar experimental approach was used to analyze thioglycolate, peptone or Con A elicited peritoneal M phi. In all cases, these M phi cell populations were able to generate an excellent alloresponse, whereas no functional Mlsa determinants could be detected. Third, to further investigate whether the expression of Mlsa is lymphocyte specific, but dependent on expression of class II molecules, we have transfected I-Ek alpha and beta cDNA into a panel of thymomas of Mlsa genotype. Although we achieved a high level of surface I-Ek expression in all of these T cell tumors, none of them was able to trigger the Mlsa-specific THy. These results strongly suggest that Mlsa expression is limited to B cells. It is likely that Mlsa is a tissue-specific self-peptide that associates with class II molecules.     

Activation requirements of cloned inducer T cells. III. Need for two stimulator cells in the response of a cloned line to Mls determinants

To gain insight into the nature of Mls determinants, we examined the stimulator cells responsible for the activation of inducer T cell clones by Mls determinants. Two types of clones responding to Mls determinants were identified. One type responded to purified B cells, but not to splenic adherent cells (SAC), from mice bearing Mls stimulatory determinants. The other type of Mls-reactive T cell clone, including the representative clone Ly1-N5, demonstrated a vigorous response to unfractionated spleen cells, but showed little or no response to B cells alone or to SAC alone from mice bearing the Mlsa or Mlsd stimulatory determinant. The response of these clones to Mls determinants required stimulation by two cell types. The failure of clone Ly1-N5 to respond to Mlsa-bearing B cells was reversed by the addition of SAC taken from mice bearing the Mlsa allele. In addition, SAC from mice bearing the nonstimulatory Mlsb allele could synergize with B cells from Mlsa-bearing animals. B cells were required to provide the Mlsa determinant, because the combination of Mlsa-bearing SAC and Mlsb-bearing B cells did not activate the clone. The response of clone Ly1-N5 to Mls is restricted by Ia determinants (shared by H-2b, H-2d, and H-2k haplotypes but not by the H-2q haplotype). The permissive H-2 alleles can be present either on the stimulator B cell or on the SAC. The optimal response of the clone was obtained by using B cells bearing Mlsa and the permissive Ia epitopes. However, a significant response of the clone to B cells bearing Mlsa but an inappropriate Ia (Iaq) was also seen in the presence of SAC bearing the nonstimulatory Mlsb allele but the permissive Ia epitopes.     

T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice.

To study T cell tolerance, transgenic mice were generated that expressed the Mlsa-reactive T cell receptor (TCR) beta chain V beta 8.1 (cDNA) under the control of the H-2Kb promoter/immunoglobulin heavy chain enhancer on approximately 90% of peripheral T cells. In transgenic mice bearing Mlsa, thymocytes expressing the TCR at a high density were deleted and the percentage of Thy 1.2+ lymph node cells was reduced. The CD4/CD8 ratio of mature T cells was reversed in Mlsa and Mlsb transgenic mice independent of the H-2. RNA analysis and immunofluorescence with TCR V beta-specific antibodies revealed that expression of endogenous TCR beta genes was suppressed. Both Mlsa and Mlsb TCR beta chain transgenic mice mounted a T-cell-dependent IgG response against viral antigens, whereas the capacity to generate alloreactive and virus-specific cytotoxic T cells was impaired in TCR beta chain transgenic Mlsa, but not in transgenic Mlsb mice.     

The Mlsd-defined primary mixed lymphocyte reaction: a composite response to Mlsa and Mlsc determinants

Considerable disagreement exists among immunologists regarding the polymorphic nature of the murine Mls system. An estimate of the capacity of a given putative Mls allelic gene product expressed on a stimulator population to elicit proliferation of H-2-compatible Mls-disparate unprimed T cells may vary widely among different groups of investigators. This laboratory has shown previously that preactivation of B lymphocytes in a splenocyte stimulator population by exposure to goat anti-mouse IgD (GaMD) before irradiation dramatically enhanced the in vitro presentation not only of the strongly stimulatory (and highly cross-reactive) Mlsa and Mlsd, but also the more poorly stimulatory Mlsc specificity. Therefore, by the use of GaMD-treated splenocytes that optimally present the various Mls non-H-2 stimulatory epitopes, we attempted in this study to obtain a clearer understanding of Mls polymorphism by re-examining the conflicting claims associated with the mixed lymphocyte reaction (MLR) stimulatory capacity of different Mls specificities. Among H-2k responder cells of the Mls null, Mlsa, Mlsb, or Mlsd genotypes, only T cells from Mlsd-bearing CBA/J mice did not respond to Mlsc determinants present on GaMD-treated C3H/HeJ stimulator cells. Crossing CBA/J with an Mlsc-responsive mouse strain yielded an F1 animal in which nonresponsiveness to Mlsc was dominant. Although Mlsa (AKR/J) and Mlsc (C3H/HeJ) parental T cells both proliferated vigorously to Mlsd (CBA/J) stimulator cells, the Mlsa/c (AKR X C3H)F1 T cells responded poorly to GaMD-treated Mlsd stimulator cells. In addition, Mlsd (CBA/J) T cells were nonresponsive to Mlsa (AKR/J), Mlsc (C3H/HeJ), and Mlsa/c (AKR X C3H)F1 GaMD-treated stimulator cells. Because Mlsa (AKR/J) and Mlsc (C3H/HeJ) specificities are mutually stimulatory, at least limited polymorphism must exist in the Mls system. However, because Mlsa/c (AKR X C3H) and Mlsd (CBA/J) specificities are mutually nonstimulatory, T cell proliferation in an Mlsd-defined primary MLR is most likely due to a composite response to Mlsa and Mlsc epitopes present on CBA/J stimulator cells.     

Nonresponsiveness to Mlsd in F1 hybrid mice carrying Mlsa and Mlsc genes

Neither the biological function nor a basic understanding of the enigmatic chromosome 1-encoded Mls locus of the mouse has yet been uncovered despite extensive investigations. The present report is a continuation of our genetic analyses of the Mls locus in an attempt to better define the system. Data presented here indicate that in contrast to cells of mice expressing either the Mlsa or Mlsc allele which respond in mixed leukocyte reactions to cells expressing the Mlsd allelic products, cells from (Mlsa X Mlsc)F1-hybrid mice do not. In addition, the nonresponder phenotype appears to segregate as a single autosomal genetic system in backcross animals. These findings fail to support two recently advanced hypotheses: first, that the Mls locus is nonpolymorphic, or second, that the Mls locus controls differential expression of Ia antigenic determinants. Although the mechanism by which a (responder X responder) converts to a nonresponder remains unknown, three models involving gene complementation are discussed.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing cells.     

Analysis of T hybridomas prepared from a T cell clone with three specificities. Recognition of self + X and allo-H-2 determinants segregates from recognition of Mlsa determinants

To see k information on T cell recognition of Mlsa determinants, hybridomas were prepared from a well-characterized F23.2+ (V beta 8.2+) T cell clone specific for three different ligands, i.e., 1) Mlsa determinants, 2) fowl gamma-globulin (F gamma G) plus self-H-2 (H-2d), and 3) allo-H-2, e.g., H-2p, determinants. Fusion of the clone to the BW5147 thymoma line produced a triple-reactive T hybridoma which generated two types of spontaneous variants. One type of variant (type I) lost Mlsa reactivity but retained reactivity to both F gamma G/H-2d and allo-H-2p. These variants, which were generated at high frequency, stained strongly with a mAb, A1.57, with idiotypic specificity for the TCR molecules of the parental clone. The second type of variant (type II) reacted to Mlsa determinants but showed no reactivity to F gamma G/H-2d or to allo-H-2p. These variants failed to express the A1.57 idiotypic determinants of the parent clone, but were F23.2+ (V beta 8.2+); nonequilibrium pH gradient electrophoresis analysis suggested that these hybrids expressed a mixed TCR heterodimer composed of the parental clone beta-chain and the BW5147 alpha-chain. Three aspects of the data are very difficult to accommodate with the view that Mlsa, F gamma G, and allo-H-2 determinants are all recognized via a common TCR molecule: 1) the independent (and frequent) segregation of Mlsa reactivity from F gamma G/H-2d and allo-H-2p reactivity, 2) the retention of Mlsa reactivity by the type II variants despite loss of the parental clone alpha-chain, and 3) the loss of Mlsa reactivity by the type I variants despite high expression of the A1.57+ TcR molecules derived from the parental clone. The data support a model in which Mlsa determinants are recognized by a separate T cell structure, which we envisage as a monomorphic accessory molecule unrelated to the TCR. Since the type II hybridoma variants invariably retained quantitatively normal TcR expression, the triggering phase of anti-Mlsa responses appears to be TCR dependent. The model we favor is that anti-Mlsa/Mlsa interaction increases TCR binding with Ia epitopes to above the threshold required for cell triggering. A key feature of this model is that Mlsa and Ia determinants are recognized as separate structures rather than as a complex.     

The immunobiology of the T cell response to Mls-locus-disparate stimulator cells. I. Unidirectionality, new strain combinations, and the role of Ia antigens

The primary mixed lymphocyte reaction of T cells to Mls-locus-disparate stimulator cells differs from that to non-self Ia antigens in several respects. In the present experiments, the unidirectional nature of this response is shown in several strain combinations, including the newly detected Mlsa and Mlsa-like alleles expressed by strains PL/J, RF/J, and SM/J. All of these strains stimulate MHC-identical T cells strongly. In addition, they stimulate a variety of cloned T cell lines specific for Mlsa,d, which can thus be shown to respond to Mlsa,d stimulators of the H-2b,d,k,u, and v haplotypes. Although these results suggest that primary T cell responses to Mlsa,d are unlikely to be MHC restricted, these primary responses are readily inhibited by monoclonal antibodies specific for the I-A and especially the I-E products borne by the stimulator cells, as well as by monoclonal antibodies specific for L3T4a on the responding T cells. This effect of anti-Ia antibodies is not overcome by exogenous interleukin 1. Thus, I-A and especially I-E molecules are centrally involved in the unidirectional primary T cell response to the potently stimulating Mlsa and Mlsd alleles expressed by cells of several different MHC haplotypes.     

T cell responses to Mls determinants are restricted by cross-reactive MHC determinants

The studies presented here investigated the relationship between T cell recognition of MHC-encoded products and non-MHC-linked Mls determinants. The first aspect addressed whether Mls-reactive T cells recognize Mls-encoded products alone or in association with MHC-encoded determinants. Initial studies used Mlsa-specific T cell clones that were generated by repeated stimulation of C57BL/6 or B10.A(5R) spleen cells with DBA/2 lymphoid cells. These clones recognized Mlsa on cells expressing MHC products of the H-2b, H-2d, and H-2k haplotypes, but not the H-2q haplotype. Thus, these cloned T cells were found to recognize Mlsa products in association with public but demonstrably polymorphic H-2 determinants. The question of whether T cell clones that were specific for self-H-2 determinants (autoreactive) or soluble antigen plus syngeneic H-2 (antigen-specific) could also be stimulated by Mlsa determinants was also addressed. A substantial proportion of the antigen-specific or autoreactive T cell clones tested were stimulated by Mlsa determinants. Furthermore, stimulation of these clones by Mlsa was H-2 restricted. The pattern of H-2-restricted recognition of Mlsa by these clones was not distinguishable from that observed in the Mlsa-specific T cell clones, nor was it influenced by the primary specificity or H-2 restriction pattern of a given clone. Although these findings provide a means of explaining the observation that Mls-reactive T cells exist at extremely high precursor frequencies, they also raise questions regarding the nature of the receptor structures which are used by a single T cell in the recognition of two or more apparently distinct stimuli.     

Bidirectionality of mixed lymphocyte stimulation (Mls) response. Effects of Mlsb stimulator cells on Mlsa helper cells

The activation of BALB/c lymphocytes in the mixed lymphocyte reaction to Mls-disparate APC has been shown to encompass up to 20% of the mature resting helper T lymphocyte population. In addition to these overtly Mls-responsive cells, our studies have revealed a second population that respond to the Mls difference of DBA/2 spleen cells in conjunction with the mitogen Con A. This part of the Mls response is therefore latent. As mitogen and Mls-stimulating effect act in synergy, it is likely that both stimuli act on the same cell, and hence the Mls effect can be regarded as a regulatory interaction between APC and Th cell. By use of congenic BALB.Mlsa mice, the regulatory effect has been mapped to the Mls locus. The regulatory influence has also been demonstrated in DBA/2 Th cells (Mlsa) stimulated simultaneously with mitogen and Mls-disparate (Mlsb) APC, consistently causing inhibition of mitogen-induced proliferation in this reverse Mls direction. This antagonistic effect has also been linked to the Mls locus. We conclude that the Mls reaction governed by the a and b alleles is bidirectional, producing synergy with class II-dependent activation signals in the direction of Mlsa----Mlsb, and antagonism in the direction Mlsb----Mlsa. Both the classical Mls and the reverse Mls effects have been demonstrated at the clonal level. These results are in accord with the previously proposed hypothesis that the Mls molecule serves as a down-regulatory stimulus in the activation of Th cells. Mls responses of Mlsb T cells are explained as the consequence of a diminished down-regulation by Mlsa APC. Conversely, the reverse Mls response described here can be considered a consequence of inordinately high down-regulation of the Mlsa T cell responses by Mlsb APC.     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

Transient T and B cell activation after neonatal induction of tolerance to MHC class II or Mls alloantigens.

The neonatal injection of semiallogeneic F1 spleen cells into newborn parental mice results in the induction of tolerance to the corresponding alloantigen (alloAg) and chimerism. In these F1 cell-injected mice, we have previously observed that this state of specific tolerance is associated with the development of a transient lupus-like autoimmune syndrome. In this study, we show that neonatal injection of mice with spleen cells differing from the host at major histocompatibility complex (MHC) class I, class II, class (I + II), or minor lymphocyte stimulating (Mls) alloAg induced a state of specific tolerance characterized by the absence of alloreactive CTL and/or Th cell responses in the spleen and the thymus of 6- to 12-week-old injected mice. However, in mice rendered tolerant to MHC class II or class (I + II) alloAg, the presence of high levels of IgG1 antibodies, of circulating immune complexes, of anti-ssDNA autoantibodies, and of tissue lesions were transiently observed. In these mice, an increased Ia Ag expression on lymphoid spleen cells was also detected at 1 wk. The elevated production of IgG1 and the overexpression of Ia Ag were almost completely prevented by treatment with an anti-IL-4 mAb. Such manifestations of B cell activation and autoimmunity were not observed in mice neonatally injected with F1 cells differing from the host only at MHC class I Ag. In mice neonatally tolerized to Mls Ag, a transient increase in IgG2a production and an overexpression of Ia Ag were detected without features of autoimmunity, and were prevented by anti-INF-gamma mAb treatment. In mice rendered tolerant to MHC class II, class (I + II), or Mls alloAg at birth, the manifestations of B cell activation were associated with the presence of in vivo-activated alloreactive CD4+ T cells in the spleen--but not the thymus--of 1-wk-old injected mice. Together, these results suggest that in mice neonatally injected with semiallogeneic F1 cells, the process of tolerance induction is not efficient during the early postnatal period, and could allow the maturation and peripheralization of some alloreactive CD4+ T cells, leading to transient B cell activation and, depending on the alloAg, to autoimmunity.     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

Effect of Mlsa on antigen presentation to class II-restricted T cells

The nature of the gene products encoded or regulated by the minor lymphocyte-stimulating (Mls) loci remains enigmatic despite extensive experimental evaluation. This work tested the hypothesis that the Mlsa genotype, when compared to the Mlsb genotype, facilitates Ag presentation to class II-restricted T cells. Titrated numbers of H-2-identical, Mls-disparate APC were used to stimulate proliferation of autoreactive, alloreactive, or Ag-specific class II-restricted T cell clones or lines. Apparent preferential presentation by Mlsa vs Mlsb APC obtained from H-2-identical strains was seen infrequently, and when observed, analysis with the use of APC from recombinant inbred lines revealed that preferential presentation did not correlate with the Mls genotype of the APC. These studies show that the Mlsa genotype does not influence overall Ag presentation to class II-restricted T cells.     

Specific neonatal induction of functional tolerance to allogeneic Mls determinants occurs intrathymically and the tolerant state is Mls haplotype-specific

The studies described show that functional Mls specific tolerance, which we previously reported in peripheral spleen cells of mice injected within 24 h of birth with Mls incompatible spleen cells, is observable in the thymus on day 6. At this time a significant positive response is not detectable in spleen cells of normal mice. In the limiting dilution assay, we are able to detect a more profound depletion than others have found with anti-TCR antibodies. The tolerance in the thymus is not due to active suppression or simple dilution of responders by nonresponsive cells of the neonatal inoculum. By tolerizing BALB/c (Mls(b,c] mice with spleen cells from Mls(a) congenic mice, we show that Mls(a) incompatibility alone is sufficient for tolerance induction. Data from these experiments also show that the T cells seen responding at high frequency to stimulators from mice expressing Mls(a) determinants, as well as many other non-H-2 encoded incompatibilities, are indeed responding to Mls(a) determinants. In addition, experiments involving neonatal injection of Mls(b) mice with Mls(a) and Mls(c) spleen cells show no cross-reactivity of tolerance between Mls(a) and Mls(c) haplotypes. Our findings also show coexpression of determinants common to both Mls(a) and Mls(c) haplotypes by the Mls(d) haplotype. In all, the described experiments elucidate a pattern of Mls determinant specific hyporesponsiveness, in mice neonatally injected with appropriate allogeneic spleen cells, which bears all the hallmarks of functional, alloantigen specific, clonal deletion type tolerance.     

T cell lines with dual specificity for strong Mls and H-2 determinants

To examine the relationship of T cell specificity for Mls vs H-2 determinants, BALB/c (H-2d,Mlsb)(d,b) T cells were stimulated repeatedly in vitro with H-2-compatible, Mls-incompatible DBA/2(d,a) stimulators. This line of T cells gave strong mixed-lymphocyte reactions to the priming Mlsa determinants but, in addition, gave appreciable responses to various foreign H-2 determinants. When this T cell line was subsequently stimulated over a period of 2 mo with Mlsa-negative cells of a particular foreign H-2 haplotype, e.g., H-2k, the cells gave high responses to H-2k determinants but only very low responses to third-party H-2 determinants. Significantly, the cells retained high reactivity for Mlsa determinants. In other experiments, BALB/c T cells positively selected to Mlsa,d-negative H-2-incompatible stimulator cells retained high reactivity for Mlsa determinants. The implications of these findings are discussed.     

Cell-mediated cytotoxicity against allogeneic mutant H-2 antigens: restricted and nonrestricted responses

Two "gain and loss" type mutations of the H-2D region, the H- 2bm13 and H- 2bm14 , resulted in the expression of noncross-reactive CML determinants that are unique to each mutation, the Dbm13 gains and Dbm14 gains, respectively. According to the results of direct cytolytic and competitive inhibition assays of in vitro induced primary cytotoxic T lymphocytes, allogeneic responses specific for Dbm13 gains are generated by responders bearing the H-2b ( KbIbDb ) haplotype, but not by responders bearing the H- 2bm14 ( KbIbDbm14 ), KbIbDd , KbIbDk , or KbIb / qDq haplotype. Responses by the non-H-2b responders against Dbm13 are limited to those determinants shared by the Dbm13 and Db molecules. Because congenic mice differing only at the H-2D region are either responsive or nonresponsive to Dbm13 gains, the responsiveness is controlled by gene(s) in the H-2D region. F1 hybrid offspring of responsive (H-2b) and nonresponsive (non-H-2b) parents are invariably responsive, indicating genetic dominance of the responsiveness. In contrast to the response against Dbm13 gains, cytotoxicity specific for Dbm14 gains is generated by responders bearing the H-2b, H- 2bm13 , KbIbDd , KbIbDk , or KbIb / qDq haplotype. These data indicate the existence of two types of allogeneic MHC determinants; one, represented by Dbm14 gains, is the classic type capable of eliciting CML responses in mice of a wide range of H-2 haplotypes, whereas the other, exemplified by Dbm13 gains, elicits CTL responses only in mice of a few related haplotypes. It is proposed that recognition of Dbm13 gains is restricted by structures shared by Db and Dbm13 but missing from other D (or L, R, etc.) molecules, such as Dbm14 , Dd, Dk, and Dq. Availability of various restricting structures in self MHC molecules may thus influence the alloreactive CTL repertoire.