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.     

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.     

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.     

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.     

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.     

Altered expression of self-reactive T cell receptor V beta regions in autoimmune mice.

Intrathymic tolerance results in elimination of T cells bearing self-reactive TCR V beta regions in mice expressing certain combinations of I-E and minor lymphocyte stimulatory (Mls) phenotypes. To determine if autoimmune strains of mice have a defect in intrathymic deletion of self-reactive TCR V beta regions, expression of V beta 3, V beta 6, V beta 8.1, and V beta 11 were examined in lpr/lpr and +/+ strains of mice; MRL/MpJ(H-2K, I-E+, Mlsb,), C57BL/6J(H-2b, I-E-, Mlsb,), C3H/HeJ(H-2k, I-E+, Mlsc), AKR/J(H-2k, I-E+, Mlsa); and in autoimmune NZB/N(H-2d, I-E+, Mlsa) and BXSB(H-2b, I-E-, Mlsb) mice. The results suggest that, during intrathymic development, self-reactive T cells are deleted in autoimmune strains of mice as found in normal control strains of mice. However, the TCR V beta repertoire is skewed in autoimmune strains compared to normal strains of mice. For example, MRL-lpr/lpr mice, but not other lpr/lpr strains, had increased expression of V beta 6 relative to expression in control MRL(-)+/+ mice, which is associated with collagen-induced arthritis. These data are consistent with a model of normal affinity for negative selection of self-reactive T cells in the thymus of autoimmune strains of mice followed by expansion of autoreactive T cell clones in the peripheral lymphoid organs. The peripheral lymphoid organs of lpr/lpr mice contain an expanded population of abnormal CD4-, CD8-, 6B2+ T cells. Elimination of self-reactive peripheral T cells suggests that these abnormal cells are derived from a CD4+ subpopulation in the thymus. Flow cytometry analysis of peripheral lymph node T cells from MRL-lpr/lpr mice reveal three populations of CD4+ T cells expressing low, intermediate and high intensity of B220 (6B2). This supports the hypothesis that in lpr/lpr mice, self-reactive CD4+ T cells are eliminated in the thymus, and that these cells lose expression of CD4 and acquire expression of 6B2 in the periphery.     

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.     

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.     

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.     

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.     

Genetic analysis of the presentation of minor lymphocyte-stimulating determinants. II. Differing non-MHC control of super-stimulatory and more poorly stimulatory Mls phenotypes

Analysis of the capacity of splenocytes from non-prototypic Mlsa or Mlsc mouse strains to stimulate allogeneic H-2k-compatible T cells in a primary Mls-defined MLR provided interesting examples of exceptions to the usually stated characterization of Mlsa and Mlsc determinants as highly stimulatory of weakly stimulatory, respectively. Across the Mlsa barrier, MA/My stimulator cells had a significantly reduced capacity to elicit responder proliferation in comparison with prototypic AKR/J or less well studied C58/J, CE/J, or RF/J splenocytes. Across the Mlsc barrier, a gradient of stimulatory ability was observed with RF/J splenocytes being virtually nonstimulatory, prototypic C3H/HeJ splenocytes having an intermediate capacity, and CE/J and C58/J being highly stimulatory presenters of this non-MHC specificity. The differing capacity of each of these H-2k stimulator cells to elicit unprimed responder cell proliferation across an Mlsa or Mlsc difference correlated with the T cell growth factor activity that was secreted into the MLR supernatants. The super stimulatory form of Mlsc was expressed in an autosomal dominant fashion by (Mlsc poorly stimulatory x Mlsc super-stimulatory)F1 animals, (BALB.K x C58/J)F1 or (RF/J x CE/J)F1. The segregation of Mlsc stimulatory ability among first backcross and F2 animals derived from the former F1 was compatible with a single non-MHC gene controlling the expression and presentation of the super-stimulatory form of Mlsc. The regulatory nature of this gene was indicated by the observation that F1 animals generated from the Mlsc nonprototypic and poorly stimulatory BALB/c parental strain were self-tolerant to the super-stimulatory form of Mlsc. The existence of an Mls specificity other than a and c was suggested by positive non-MHC MLR responses in certain responder/stimulator cell combinations of Mls prototypic and nonprototypic mouse strains.     

Neonatal tolerance induction to Mlsa. I. Tolerance to Mlsa is restricted by shared MHC determinants

In this paper we have examined the influence of MHC (major histocompatibility complex) on neonatal tolerance to Mlsa (minor lymphocyte stimulating). By employing a novel approach we have shown that tolerance to Mlsa is restricted by shared MHC determinants. Thus, neonatal Mlsb mice, injected at birth with spleen cells from Mlsa mice, were tested as adults for Mlsa specific responses by interleukin-2 limiting dilution analysis, a technique which allows us to discriminate between responses to MHC + Mlsa and to MHC alone. Tolerance to Mlsa was in the context of any MHC type examined--donor, host, and third-party MHC products. These results show that tolerance to Mlsa is restricted by shared MHC determinants and extend previous studies indicating that activation of Mlsa responses is similarly restricted.     

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.     

Induction of transplantation tolerance in mice across major histocompatibility barrier by using allogeneic thymus transplantation and total lymphoid irradiation

The use of allogeneic thymus transplantation as a means of inducing tolerance across MHC barriers was investigated in thymectomized, total lymphoid irradiated BALB/c mice. In 90% of the animals long term outgrowth of histologically normal C57BL thymus grafts was observed. None of the latter animals was chimeric. All thymus graft-bearing mice showed specific nonresponsiveness for C57BL MHC Ag in mixed lymphocyte reaction and cell-mediated lympholysis. Spleen cells of the C57BL thymus-bearing mice were unable to induce lethal graft-vs-host disease in neonatal (BALB/c X C57BL) F1 mice but provoked a vigorous graft-vs-host disease reaction in (BALB/c x C3H) F1 neonates. Tolerant mice permanently accepted C57BL heart and pancreas grafts, but all rejected C3H grafts. Induction of tolerance of BALB/c pre-T cells through allogeneic thymus graft and/or specific suppressor cells seems to be involved. The present model offers new opportunities to study thymocyte maturation in a fully allogeneic environment and may yield applications for clinical organ transplantation.     

Immunogenetic studies of spontaneous abortion in mice. Preimmunization of females with allogeneic cells

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. IV. Significance of intrathymic chimerism of blood-born Ia+ cells in Mls tolerance.

The significance of thymus cell chimerism in the induction and maintenance of tolerance was investigated. Mls-1b BALB/c mice were neonatally tolerized by the intravenous administration of either bone marrow (BM) cells or peritoneal cavity (PerC) cells from Mls-1b/a (BALB/c x AKR) F1 mice. Tolerance was long-lasting in the BM cell group, but transient in the PerC cell group, probably because PerC cells lack hemopoietic stem cells required for a continuous supply of tolerance-inducing cells. The degree of anti-Mls-1a responsiveness of these BALB/c thymus cells was correlated with the degree of intrathymic distribution of the inoculated F1 cells. The effect of BM cell inoculation, resulting in a year-long deletion of Mls-1a-reactive V beta 6-bearing T cells is in marked contrast to that of PerC cell inoculation which causes only a transient loss of V beta 6+ mature thymocytes (for about 1 week after birth). This functional profile of the tolerant state correlates well with the degree and persistence of the intrathymic presence of F1 type Ia+ cells. The long-lasting presence of donor-derived cells throughout the thymus tissue in the BM cell group is also in marked contrast to the early disappearance of Ia+ cells (within 2-3 weeks) from the cortex and then from the medulla in the PerC cell group, although these Ia+ cells were once spread throughout the thymus tissue 4 days after the tolerance-inducing cell inoculation. Taken together with a failure to induce consistent unresponsiveness to Mls-1a determinants in Mls-1b thymocytes regenerating in Mls-1a-thymic epithelial environments, all the above data indicate that intrathymic chimerism caused by hemopoietic stem cell-derived MHC-class II-bearing cells is a requisite for the induction and maintenance of unresponsiveness by means of clonal deletion in experimentally as well as naturally induced tolerance to Mls determinants.     

Primary in vitro cytotoxic response of F1 T lymphocytes against parental antigens.

Spleen cells from adult female (AKR/J x BALB/(c)F1 mice can respond to mitomycin C-treated spleen from AKR/J mice and can generate effector CTL in a 5-day primary in vitro culture. The response is comparable in magnitude to the response to allogeneic H-2K or H-2D antigens. The response is T cell mediated and is directed to antigen(s) present only on the parental cells. The target cell must be homozygous at H-2Kk to be lysed and H-2Dk antigens do not serve as a target in this response. Spleen cells from (B10.BR x B10.D2) hybrids that have been stimulated with AKR/J lyse B10.Br as well as AKR/J target cells. Similar H-2k/d hybrid F1 anti-H-2k parent responses are seen in certain other strain combinations. A number of possible interpretations of these responses are discussed.     

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.     

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.     

Differential responsiveness to MIs locus antigens in graft-versus-host and host-versus-graft reactions

After (semi)allogeneic transplantation of lymphoid cells into lethally irradiated mice, the development of anti-host directed T effector cells can be demonstrated by means of a simple delayed-type hypersensitivity (DTH) assay. Using this assay we have shown that in H-2 compatible combinations Mls locus antigens can induce the generation of such T effector cells during a graft-versus-host (GvH) reaction. Other non-H-2 alloantigens are probably of minor importance. The capacity of Mls locus antigens to induce distinct anti-host DTH reactivity correlated with the capacity to induce a one-way mixed lymphocyte culture (MLC) response. Mls^a and Mls^c locus antigens initiated a positive MLC response as well as distinct GvH-related DTH reactivity. On the other hand, in the combination DBA/2 versus (BALB/c × DBA/2) F₁, the Mls^b locus antigen was not able to initiate in vitro proliferation, a lack of response which coincided with a marginal and short-lasting GvH-related DTH reactivity. In contrast, the host-versus-graft (HvG) DTH reaction of BALB/c and DBA/2 mice to subcutaneously injected (BALB/c × DBA/2) F₁ spleen cells was equally strong. Here antigens other than those coded for by the Mls locus were mainly responsible for the antigraft DTH response. These results suggest that T effector cells generated in GvH and HvG reactions are specific for largely different sets of minor histocompatibility antigens, with a selective stimulation by Mls locus antigens under GvH conditions.