Ir Gene Research Articles

HLA and disease 1982--a survey.

Within the last 7 years, HLA and disease studies have made it clear that most of the diseases previously known to be HLA-A- or B-associated do in fact show stronger associations with HLA-D/DR antigens. This observation strengthens the assumption that Ir and/or Is determinants are responsible for these associations in agreement with the fact that many of these diseases are characterized by autoimmune phenomena. However, some diseases, ankylosing spondylitis in particular, still show stronger associations with HLA-ABC than with DR antigens. Among the conditions which have been shown to be HLA-associated more recently, four deserves special mention: (i) maternal immunization against the Zwa antigen because this is a good candidate for an antigen-specific Ir gene action; (ii) IgA deficiency in blood donors because this is a non-antigen-specific immunodeficiency; (iii) idiopathic hemochromatosis and (iv) congenital adrenal hyperplasia due to 21-OH deficiency because immune mechanisms are unlikely to be involved. HLA studies and new genetic methodology have significantly advanced our knowledge about the inheritance of some diseases. Thus, HLA-B27 or a B27-associated HLA factor confers a dominant susceptibility to ankylosing spondylitis. HLA plays a definite and strong role in the susceptibility to IDDM, but simple genetic models (dominant, recessive, and intermediate) have been made unlikely on the basis of HLA results; the hypothesis that there are two different susceptibility genes within the HLA system still remains viable, but the demonstration of clinical heterogeneity and/or (better) of different pathogenetic pathways for DR3- and DR4-associated IDDM is required to substantiate it.

HLA-DR antigens in Mexican patients with Guillain-Barré syndrome

The distribution of HLA-DR antigens was investigated in 38 Mexican Mestizo patients with Guillain-Barré syndrome (GBS) and in 100 healthy controls belonging to the same population. IgG, IgM, IgA, CH50, C3, C4 and the number of T and B lymphocytes were also evaluated in the patients. Only DR3 was significantly increased in the patients (Yates' χ 2 = 0.943, P c = 0.014) and the relative risk for developing the disease was 3.49. These findings support the hypothesis that DR# or a closely linked Ir gene may play some role in the susceptibility to GBS.

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B.

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.

Bone marrow-derived macrophage as accessory cells in antigen-induced T cell proliferation. H-2I region requirements for L-glutamic60-L-alanine30-L-tyrosine10 response.

Marrow stem cells cultured with L cell-conditioned medium were used to produce large numbers of macrophages free of contaminating lymphoid cells or granulocytes. Experiments were performed by using these bone marrow-derived macrophages (BMDM) as antigen-presenting cells (APC) for the terpolymer antigen L-glutamic60-L-alanine30-L-tyrosine10 (GAT). By using antigen-specific T cell proliferation, it was demonstrated that BMDM were equal to splenic macrophages in their capacity to present GAT. Furthermore, when BMDM were pretreated with alloantibodies specific for Ia antigens of the I-A subregion, the T cell proliferative response to GAT was inhibited. The I-A subregion is known to be the site of Ir gene regulation of the GAT response, and antibodies to other subregions had no inhibitory effect. Monoclonal antibodies recognizing a beta-chain product of the I-A subregion (Ia. 17) also inhibited the BMDM from effective antigen presentation. These results were similar to those obtained with purified splenic macrophages.

Virus-induced immune complex disease: genetic control of C1q binding complexes in the circulation of mice persistently infected with lymphocytic choriomeningitis virus.

Virus-antibody immune complex formation, deposition, and disease is a common manifestation in most mice persistently infected with lymphocytic choriomeningitis virus (LCMV). Although mice of several strains persistently infected with LCMV mount continuous anti-LCMV immune responses to the three virion structural polypeptides, the amount of antibody(s) made varies among strains. The formation of antibody to LCMV correlates with the detection of C1q binding materials (immune complexes) in the circulation; however, there is no correlation between the total amount of IgG and the C1q binding material. SWR/J mice (H2qq) are high responders (high levels of anti-LCMV antibodies, high C1q binding responses), whereas BALB/W mice (H-2dd) are low responders (low levels of anti-LCMV antibodies, low C1q binding responses). Sera from 37 SWR/J mice, 12 wk of age, bound 55.1% of offered 125I C1q compared to 8.4% for 37 age and sex-matched BALB/W mice. SWR/J mice made approximately 60-fold more antibody to LCMV than did BALB/W mice. To define the genetic factors involved, we used the C1q binding assay, high responder and low responder mice, their hybrid offspring, backcrosses of the hybrids to both high and low responder parents, and selected recombinant inbred mouse strains. From studies with BALB/W mice persistently infected with LCMV, we defined low C1q responder mice as those having binding activity of 18.4% or less (BALB/W mean C1q binding value + 2 SD). By using this criteria for 12-wk-old mice, 55/57 (96%) of SWR/J, 27/37 (73%) of F1(SxB or BxS), 13/21 (62%) of qq or 11/20 (55%) qd from F1 x SWR/J, and 6/13 (45%) qd from F1 x BALB/W were high C1q responders. In contrast, none of the 17 dd mice from the F1 x BALB/W cross were high responders. Thus, F1 hybrid mice are high responders (dominant) and data from backcrossing F1 hybrids to either high or low responder parents suggested the complexes associated with C1q binding was controlled by gene(s) in the H-2 complex. Support for the role of Ir gene(s) was provided by experiments showing LCMV persistently infected BALB/kae (H-2 KkIkDk) and recombinant inbred strains B10.A (KkIkDd) and A.TL (KsIkDd) were high C1q responders, whereas B10.A(5R) (KbIbDd) and BALB/cby (KdIdDd) were not. The wide scatter in C1q binding levels observed among C1q high responder mice and the production of C1q binding levels in F1 mice that are intermediate between high and low responder strains suggested that, in addition to H-2-linked genes, other non-H-2-linked genes also play a role in this response. The amount of C1q binding complexes in LCMV persistently infected mouse strains and their crosses was unrelated to the amounts of infectious virus carried in their sera. Despite the low C1q binding by sera of H-2dd mice that originated from mating F1 hybrid (qd) x BALB/WEHI (dd), these mice carried equivalent amounts of infectious virus as their qd littermates or qd and qq mice originated from F1 x SWR/J mice.

Epitope specificity and Ia restriction of T cell responses to insulin in a system of complementing Ir genes: analysis with primed lymph node T cells and a long-term cultured T cell line.

The antibody response of (H-2b X H-2k)F1 mice to pig insulin (PI) has previously been shown to be under the control of H-2-linked, complementing Ir genes. In addition, this response was reported to depend on the genetic background of the parental strains (Keck, K., Eur. J. Immunol. 1977. 7: 811). Here it is demonstrated that the secondary in vitro response of proliferating T cells shows the same dependence on H-2-linked Ir genes yet an influence of the background genes could not be detected. The complementing genes were mapped to the Kb, I-Ab and Kk, I-Ak regions. For restimulation of F1 T cells by PI, the Ir genes of both parental chromosomes have to be expressed in the same antigen-presenting cell, suggesting complementation at the molecular rather than at a cell interaction level. With a long-term cultured, PI-specific T cell line (ST2) of (B10 X B10.BR)F1 origin the complementation data could be confirmed by mapping the Ia restriction elements to Kb, I-Ab and I-Ak. The reactivity pattern of this line towards species variants of insulin and the isolated A and B polypeptide chains in the presence of syngeneic accessory cells suggests that the glutamic acid residue in position 4 of the A polypeptide chain (Asp in mouse insulin) is essential for recognition in conjunction with an (I-Ab X I-Ak)F1 hybrid Ia complex. I-Ab-encoded molecules carrying specificity Ia. W39 which, according to Rosenwasser, L. J. and Huber, B. T. are essential for the presentation of BI to (CBA/N X C57BL/6)F1 T cells, are not required as components of the F1-unique restriction element recognized by the F1 T cells of the ST2 line in conjunction with PI. This is indicated by the fact that accessory cells of (CBA/N X B10)F1 hybrids, regardless of their sex, could present PI as well as beef, sheep and horse insulin to the F1-restricted ST2 cells.

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. Ir gene regulation of the T cell-dependent activation of distinct B cell subpopulations.

Antibody responses to (T,G)-A--L and (H,G)-A--L or to the TNP conjugates of these polypeptides, are regulated in the mouse by Ir genes encoded in the I-A subregion of the mouse MHC. To study the mechanism by which Ir genes function, it is essential to identify the cell interactions that are regulated by these genes. In the present report, it was specifically determined whether Ir gene function is expressed differently in the activation of B cell subpopulations that participate in two different pathways of B cell activation for in vitro antibody responses to TNP-(T,G)-A--L and TNP- (H,G)-A--L. The results of these studies demonstrate that under conditions that activate the Lyb-5- b cell subpopulation via MHC-restricted TH cell-B cell and TH cell-APC interactions, Ir gene function regulates TH cell interactions with these B cells as well as with APC. In addition, under conditions that activate the Lyb-5+ B cell subpopulation via MHC-restricted TH cell-APC but not TH cell-B cell interaction, Ir gene function regulates only TH cell interactions with APC but not with B cells.

Genetic dependence of IgE antibody production in mice infected with the nematode Nippostronglyus brasiliensis. I. Modulation of the IgE antibody response in vivo by serum factors.

We investigated the IgE-antibody response in 36 inbred mouse strains during infection with Nippostrongylus brasiliensis. With regard to the N. brasiliensis-specific IgE-antibody activity responder and nonresponder mice were obtained. Mice with the H-2-f haplotype (A.CA, B10.M, A.TFR 5) are high specific responders. It is suggested that one Ir gene for the N. brasiliensis-induced IgE-antibody response is localized within the K to J region of the mouse major histocompatibility complex. To obtain a medium IgE-antibody response it appears that a complementation between two Ir genes is necessary. An additional genetic control beyond the H-2 complex cannot be excluded. As to the total nonspecific serum IgE levels the parasitic infection leads to a 20- to 40-fold increase in high and low specific responder strains as well. Pretreatment of high responder mice (A.TFR 5, B10.M) with sera that were obtained from normal or complete Freund's adjuvant treated low (B10.G) and nonresponder (A.TFR 1) mice led to significant inhibition or enhancement of the N. brasiliensis-specific IgE-antibody response.

Molecular events in the processing of avidin by antigen-presenting cells (APC). II. Identical processing by APC of H-2 high- and low-responder mouse strains.

The differences between the immune response (Ir) phenotypes of H-2 gene-controlled high- and low-responder mice have been attributed to events occurring at the interaction between antigen-presenting cells (APC) and lymphocytes. To investigate this interaction we undertook a study of molecular events in the processing of avidin, a molecule whose uniquely strong affinity for binding to biotin renders it traceable at very low concentrations, and a molecule to which the T-lymphocyte immune response is controlled by Ir genes. In this paper we describe the generation of processed avidin by APC and characterize it biochemically and immunologically. We found that APC of H-2 genetically high- and low-responder mice were indistinguishable in their capacity to generate immunogenic processed avidin (PA). Immunogenic PA differed from native avidin in size and carbohydrate moieties, but preserved its capacity to bind biotin, and was 1000-fold more efficient than NA as an immunogen for primed T lymphocytes. Primed T lymphocytes appeared to recognize PA that was conformationally intact. Highly immunogenic PA was not H-2 restricted. Thus, differences in the Ir phenotype of the response to avidin could not be attributed to determinant selection by APC.

RT1-linked Ir and Is genes control the immune response to bovine insulin in the rat.

The immune response to bovine or pork insulin (BI or PI, respectively) was studied in the rat using the in vitro insulin-induced lymphocyte-proliferation assay. Results indicated that 11 inbred rat strains were divided into categories of high and low responders. Two high responders, SDJ (RT1u) and BN(RT1n) inbred rat strains, appeared to recognize different antigenic determinant(s) on the insulin molecule. The results of linkage and segregation analyses in F1, F2, backcross, and partially congenic rats showed that the Ir gene (Ir-BI), which encodes the high responsiveness in the SDJ rats, is inherited associated with RT1u, whereas the immune suppression gene (Is-BI), which encodes the low responsiveness in the WKA(RT1k) rats, is inherited together with RT1k. The Is-BI is the first major histocompatibility complex (MHC)-linked Is gene reported in the rat. The LEJ(RT1-AuBb) inbred rat strain showed a low response to BI, indicating that Ir-BI is closer to RT1-B/RT1-D region than to RT1-A.

Ir gene control of T and B cell responses to determinants in poly(Glu, Ala, Phe).

Ir gene control of T and B cell responses to determinants in poly(Glu, Ala, Phe).

Splenic B cells function as immunogenic antigen-presenting cells for the induction of effector T cells.

Previous studies performed in our laboratory have revealed that an ordered, sequential, tricellular interaction is obligatory for the antigen-driven induction of a specific effector memory T cell. Thus, it was found that antigen-pulsed peritoneal macrophages signal, in spleen cells, the generation of antigen-specific initiator lymphocytes. These lymphocytes, following injection to syngeneic recipients, recruit, in the draining lymph nodes, "virgin" antigen-reactive T lymphocytes. Although the nature of the first and last cell in the interacting sequence was well characterized, the identity of the intermediary initiator splenic cell was obscure. Studies were carried out to characterize the nature of the splenic initiator cells. It was found that spleen cells from nu/nu, adult thymectomized and neonatal thymectomized, or spleen cells from normal donors which had been subjected to cytolysis using anti-Thy-1.2 antibodies in the presence of complement, did generate, following interaction with keyhole limpet hemocyanin (KLH)-fed macrophages, specific initiator cells. Carrageenan impairment of spleen macrophages did not affect the generation of initiator cells, nor did the depletion of dendritic cells from the spleen. On the other hand highly enriched B cell, but not highly enriched T cell populations, when seeded on KLH-pulsed macrophages, generated antigen-specific initiators, which, in vivo, recruited antigen-reactive T cells. It thus appeared that B lymphocytes can function as intermediary obligatory antigen-presenting cells and actively transfer immunogenic signals from peritoneal antigen-presenting cells to T lymphocytes. These findings may therefore suggest that antigen-specific B cells do not function solely as antibody-producing cells, but, once activated by macrophages, may control the induction and differentiation of some antigen-reactive T cell subsets. Thus, one can view the B cell as an important regulatory cell of both cellular and humoral immune functions. The significance of this observation with regard to Ir gene control at the level of B lymphocytes is discussed.

Molecular events in the processing of avidin by antigen-presenting cells (APC). I. The immune response of T lymphocytes to avidin is regulated by H-2-linked Ir genes.

The immune response of T lymphocytes to avidin was measured by proliferative assays, antibody production and delayed-type hypersensitivity. Mice of H-2k haplotypes were found to be low responders, whereas mice of other haplotypes, and particularly of H-2s, were high responders. Ir genes controlling this response were mapped to the I subregion of H-2. Helper T cells were found to be responsible for the Ir phenotype of antibody production. These results indicate the feasibility of using the avidin-biotin complex as a tool for studying molecular mechanisms by which antigens under Ir gene control are processed and presented to T lymphocytes.

Genetic control of immune response to myoglobin. Ir gene function in genetic restriction between T and B lymphocytes.

We studied the genetic restrictions on the interaction between T cells, B cells, and antigen-presenting cells (APC) involved in the H-2-linked Ir gene control of the in vitro secondary antibody response to sperm whale myoglobin (Mb) in mice. The B cells in this study were specific for Mb itself, rather than for a hapten unrelated to the Ir gene control, as in many previous studies. Low responder mice immunized in vivo with Mb bound to an immunogenic carrier, fowl gamma globulin (F gamma G), produced B cells competent to secrete anti-Mb antibodies in vitro if they received F gamma G-specific T cell help. However, (high-responder X low responder) F1 T cells from Mb-immune mice did not help these primed low responder (H-2k or H-2b) B cells in vitro, even in the presence of various numbers of F1 APC that were demonstrated to be component to reconstitute the response of spleen cells depleted by APC. Similar results were obtained with B6 leads to B6D2F1 radiation bone marrow chimeras. Genotypic low responder (H-2b) T cells from these mice helped Mb-primed B6D2F1B cells plus APC, but did not help syngeneic chimeric H-2b B cells, even in the presence of F1 APC. In contrast, we could not detect any Ir restriction on APC function during these in vitro secondary responses. Moreover, in the preceding paper, we found that low responder mice neonatally tolerized to higher responder H-2 had competent Mb-specific helper T cells capable of helping high responder but not low responder B cells and APC. Therefore, although function Mb-specific T cells and B cells both exist in low responder mice, the Ir gene defect is a manifestation of the failure of syngeneic collaboration between these two cell types. This genetic restriction on the interaction between T cells and B cells is consistent with the additional new finding that Lyb-5-negative B cells are a major participant in ths vitro secondary response because it is this Lyb-5-negative subpopulation of B cells that have recently been shown to require genetically restricted help. The Ir gene defect behaves operationally as a failure of low responder B cells to receive help from any source of Mb-specific T cells either high responder, low responder, or F1. The possible additional role of T cell-APC interactions, either during primary immunization in vivo or in the secondary culture is discussed.

Involvement of Mhc loci in immune responses that are not Ir-gene-controlled.

Twenty-nine randomly chosen, soluble antigens, many of them highly complex, were used to immunize mice of two strains, C3H and B10.RIII. Lymph node cells from the immunized mice were restimulated in vitro with the priming antigens and the proliferative responses of the cells was determined. Both strains were responders to 28 of 29 antigens. Eight antigens were then used to immunize 11 congenic strains carrying different H-2 haplotypes, and the T-cell proliferative responses of these strains were determined. Again, all the strains responded to seven of the eight antigens. These experiments were then repeated, but this time antibodies specific for the A (A alpha A beta) or E (E alpha E beta) molecules were added to the culture to block the in vitro responsiveness. In all but one of the responses, inhibition with both A-specific and E-specific antibodies was observed. The response to one antigen (Blastomyces) was exceptional in that some strains were nonresponders to this antigen. Furthermore, the response in the responder strains was blocked with A-specific, but not with E-specific, antibodies. The study demonstrates that responses to antigens not controlled by Ir genes nevertheless require participation of class II Mhc molecules. In contrast to Ir gene-controlled responses involving either the A- or the E-molecule controlling loci (but never both), the responses not Ir-controlled involve participation of both A- and E-controlling loci. The lack of IR-gene control is probably the result of complexity of the responses to multiple determinants. There is thus no principal difference between responses controlled and those not controlled by IR genes: both types involve the recognition of the antigen, in the context of Mhc molecules.

Chemically related antigens compete for presentation by accessory cells to T cells.

Immune responsiveness of guinea pigs to dinitrophenyl-poly-L-lysine (DNP-PLL) and to the lysine-rich random copolymer of L-glutamic acid and L-lysine (GL) are both controlled by the "poly-L-lysine gene." We demonstrate that accessory cells of responder strains can be made incapable of presenting DNP-PLL to response T cells in assays for proliferation by in vitro exposure of the cells to GL before and during their exposure to DNP-PLL. The inhibition was not rapidly reversible, because GL preexposed accessory cells that were cultured for 2 hr in GL-free medium were still refractory to pulsing with DNP-PLL. In contrast, DNP-PLL had only a moderate inhibitory effect on accessory cell presentation of GL. Unconjugated poly- and oligo-lysines also inhibited the ability of accessory cells to present DNP-PLL, but inhibitory activity was displayed only by homopolymers containing eight to 12 or more residues in the chain. The homopolymers of D-lysine, L-arginine, and L-glutamic acid, and lysine-free glutamic acid-rich copolymers had little or no inhibitory effect. The results are interpreted to mean that antigens to which responsiveness is regulated by the same Ir gene compete for presentation by accessory cells. This may reflect a competition for the Ir gene product of the antigen-presenting cell.

Immune response to the p-azobenzenearsonate (ABA)-GAT conjugate: role of I region genes in the selective activation of ABA-specific or GAT-specific T helper cells.

Immunization of GAT non-responders with ABA-GAT leads to the activation of ABA-specific T cells. These hapten specific T cells are Lyt-1+2- helper cells capable of inducing anti-ABA antibody responses in vivo or B cell activation in vitro. However, their activation does not modify the GAT non-responder phenotype. Immunization of GAT responder mice with ABA-GAT activates GAT-specific T cells, which can help anti-ABA and anti-GAT antibody responses. Since the responder and non-responder strains used in these experiments differ only in the alleles present in the I region, the results suggest that the selective activation of hapten- or carrier-specific T cells is controlled by I region genes. Yet sensitization of the two strains with ABA-KLH or ABA-Tyr induces KLH-specific or ABA-specific T cells, respectively. This provides further evidence that the use of an immunogenic carrier prevents the expression of the hapten-specific T cell clones present in the repertoire of both responder and non-responder animals. Macrophages from responder animals pulsed with ABA-GAT can present ABA and GAT determinants to T cells. Thus, the absence of ABA-specific T cells in responders primed with ABA-GAT and their presence in GAT non-responders reflects a competition between hapten- and carrier-specific T cells and not an epitope selection by macrophages. We discuss the significance of the results in terms of Ir genes determining the self-plus-antigen-specific T cell repertoire rather than controlling antigen presentation by macrophages.

Immune responses to complex protein antigens I. MHC control of immune responses to bovine albumin.

Murine T cell proliferative and antibody responses to the multi-determinant protein bovine serum albumin (BSA) are controlled by Ir genes mapping within the H-2 gene complex. Strains possessing the H-2k, H-2a, and H-2d haplotypes are classified as high responders to BSA. In contrast, H-2b strains are low responders to BSA. Genetic mapping experiments employing strains with recombinant H-2 haplotypes indicate that both T cell proliferative and antibody responses are at least in part regulated by genes within the I-A subregion. Studies on the inhibition of T cell proliferation by monoclonal anti-Ia antibodies are consistent with the assignment of an Ir gene for BSA to the I-A subregion and strongly suggest a role for genes within the I-E/C subregions as well. The MHC-mediated control of antibody responses did not affect the affinity or the isotype of the antibody produced. The relative quantities of antibody specific for each of the three domains of BSA appears to be regulated by H-2-linked BSA Ir genes, and domain III antigenic determinants were found to be dominant in the responses of low-responder mice and in the early response of high-responder mice. This domain III epitope dominance essentially disappears by the tertiary response of high-responder mice.

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. III. Ir gene control of lymphocyte transformation correlates with binding of the mitogen to specific Ia-bearing cells.

The supernatant from Mycoplasma arthritidis broth cultures (MAS) contains a T cell mitogen that is under Ir gene control. Responsiveness to this mitogen is dictated by the I-E/I-C subregion of the major histocompatibility complex and is dependent upon adherent radioresistant Ia+ accessory cells from responding haplotype animals. In this study, we established that MAS could be removed from culture supernatants by absorption with spleen cells from mice that themselves are responsive to the mitogen (k and d haplotypes), but activity is not removed by spleen cells from mouse strains that are nonresponsive to the mitogen (b, q, and s haplotypes). Absorption studies with lymphoid cells from congenic and recombinant strain mice established that absorption of the mitogen was itself linked to the I-E/I-C subregion of the major histocompatibility complex. Thymocytes from responding haplotype strains were incapable of removing MAS activity, and spleen cells devoid of Thy-1-positive cells retained their full absorbing capacity. The ability to effectively absorb MAS activity was abrogated by the pretreatment of spleen cells with anti-Ia antiserum and complement. Furthermore, the ability of spleen cells from responding haplotype strains to respond to MAS was blocked by the addition of anti-Ia serum to the cell cultures. Whereas the latter treatment resulted in an almost complete elimination of MAS responsiveness, the ability of similarly treated spleen cells to respond to the mitogens PHA and Con A was only minimally depressed. These results are consistent with our hypothesis that the mitogenic moiety of MAS actually binds to I-E/I-C-coded Ia antigens.

Genetic control of the immune response to myoglobins. Both low and high responder T cells tolerant to the other major histocompatibility complex help high but not low responder B cells.

We sought to examine the role of immune response (Ir) genes in helper T cells. To eliminate allogeneic effects, we used neonatally tolerized mice. The results bear not only on the mechanism of Ir genes, but also on the development of the T cell repertoire. B 10.BR (H-2(k)) or C57BL/10 (H-2(b)) mice, which were low responders to myoglobin (Mb), were neonatally tolerized to high responder H-2(d) alloantigens, and B10.D2 mice, which were high responders to Mb, were neonatally tolerized to low responder H-2(k) or H-2(b) alloantigens. Spleen cells from these tolerized mice did not show any reactivity in mixed-lymphocyte reaction or cell-mediated lympholysis against alloantigens used in tolerization. Mb-immune F(1) B cells were helped comparably by Mb-immune tolerized low or high responder T cells. Thus, low responder T cells functioned equivalently to high responder T cells. The failure of nonimmune T cells from tolerized low responder mice to help F(1) B cells and antigen-presenting cells (APC) indicated that collaboration between B10.BR or C57BL/10 T cells and F(1) B cells was not caused by a positive allogeneic effect. Spleen cells from tolerized mice were contaminated with 2-4 percent chimeric F(1) cells, as judged by fluorescence-activated cell sorter analysis, and no F(1) alloantigens were detectable in the thymus. However, removal of chimeric F(1) T cells from the tolerized cell population by treatment with anti-H-2 and complement did not change the helper activity of tolerized low responder T cells. These data indicated that helper activity in the T cell population from low responder mice was not due to F(1) cells. Also, the level of contamination was not sufficient to quantitatively account for the help. In examining the genetic restriction of these tolerized T cells, we found that T cells from tolerized low responder B10.BR or C57BL/10 mice helped F(1) or high responder B10.D2 B cells and APC but not syngeneic B10.BR or C57BL/10 B cells and APC, which were immunized with Mb-coupled fowl gamma globulin instead of Mb to prime low responder B cells with Mb. On the other hand, high responder B 10.D2 tolerized T cells helped syngeneic B 10.D2 B cells but not allogeneic low responder B10.BR B cells. These data indicated that clones of helper T cells specific for Mb exist in low responder mice, and these are not phenotypically different from those in high responder mice, in that both help high responder and F(1) but not low responder B cells and APC. These data are discussed in terms of the mechanism for Ir gene control, and the mechanism of T cell repertoire development- whether intra- or extrathymically-in neonatally tolerized mice.