Ir Gene Control Research Articles

Identification of distinct predominant epitopes recognized by myoglobin-specific T cells under the control of different Ir genes and characterization of representative T cell clones.

We characterized the fine specificity of antigen recognition of myoglobin-immune T cells from B10.D2, B10.GD, and B10.A(5R) mice. Polyclonal H-2d T cells show a predominant response to an epitope centering on Glu 109 and His 116. These residues localize a dominant epitope to one segment of the myoglobin surface, but probably are not the only amino acid residues involved. This response pattern maps genetically to I-Ad (or Kd) based on the B10.GD recombinant strain. A different epitope specificity was detected in B10.A(5R) T cells mapping to I-E beta b E alpha k or I-Cd, but no difference was detected between strains differing at the Igh locus. Thus, epitope specificity varied with Ia haplotype but not Igh allotype. Myoglobin-specific B10.D2 T cell lines were established, and five clones specific for the Glu 109, His 116 epitope were isolated; these were all restricted to I-Ad antigen-presenting cells. These clones represent the dominant specificity in the polyclonal T cell response to myoglobin and will be useful in characterizing the structure and function of T cell receptors for antigen and Ia. The differences in number and nature of T cell epitopes compared to B cell epitopes of myoglobin are discussed.

Formula omitted] hierarchy in the [formula omitted]-gene controlled responses to hapten-modified mouse serum albumin 1

Five hapten-modified autogenous mouse serum albumins (MSA), TNP 11MSA, FITC 8MSA, DNP 64MSA, DNP 5MSA, and PC 2MSA, were tested for their ability to induce hapten-specific antibody responses in H-2 congenic mice of the H-2 d , H-2 a , and H-2 f haplotypes. Four of the five modified MSAs, TNP 11MSA, FITC 8MSA, DNP 64MSA, and PC 2MSA, stimulated responses found to be under H-2 -linked Ir gene control, whereas DNP 5MSA failed to induce responses in any strain tested. H-2 d , H-2 b , and H-2 a mice responded to FITC 8MSA; H-2 d and H-2 b responded to TNP 11MSA; and only H-2 d mice were high responders to DNP 64MSA and PC 2MSA. H-2 f mice failed to respond to any of the stimulating antigens. Thus, a hierarchy of H-2 haplotypes was observed in the responsiveness to modified MSA with H-2 d > H-2 b > H-2 a > H-2 T . Primed lymph node cells from PC 2MSA immunized H-2 d mice were challenged with antigen in vitro to assess the nature of the determinant recognized by T cells in the response to modified MSA. Lympn node cells proliferated only when challenged with PC 2MSA. Unmodified MSA or TNP 11MSA did not stimulate a proliferative response, suggesting that the immune T cells recognize a neo-determinant present on the modified PC 2MSA.

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses.

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.

Insulin-specific tolerance induction. I. Abrogation of helper T cell activity is controlled by H-2-linked Ir genes.

Murine antibody responses to heterologous insulins are under H-2-linked immune response (Ir) gene control. We have found that the immune response to insulin in adjuvant can be inhibited by prior i.v. injection of soluble insulin. The effect of i.v. injection of insulin is antigen-specific and dose-dependent and requires the same doses of insulin that are immunogenic if administered with adjuvant. In addition, the inhibitory effect of soluble insulin is dependent upon the route of injection; if soluble insulin is injected i.p., the subsequent response to insulin in adjuvant is augmented rather than inhibited. Unresponsiveness requires at least 4 days after i.v. injection to develop and once induced, it is maintained for 4 wk or more. Unresponsiveness is caused by T cell, but not B cell, tolerance, and we have been unable to demonstrate any role for suppressor T cells in this unresponsiveness. More importantly, analysis of the ability of numerous insulin variants to induce unresponsiveness in several H-2k and H-2b strains of mice has demonstrated that only the variants that were immunogenic in a given strain when administered with adjuvant were able to cause tolerance. This report is, to our knowledge, the first describing that induction of helper T cell tolerance, like the induction of immunity, is controlled by H-2-linked Ir genes.

Human helper T-cell clones that recognize different influenza hemagglutinin determinants are restricted by different HLA-D region epitopes.

Human T-lymphocyte clones ( TLCs ) were generated against the hemagglutinin (HA) of A/Texas/1/77 influenza virus by limiting dilution. TLCs were then screened for antigen specificity on chemically synthesized peptides representing the HA1 molecule. It has been hypothesized that different T cells that recognize the identical antigenic determinant are controlled by (restricted by) the same class II epitope. Two TLCs , HA1.4 and HA1.7, both recognized the same HA peptide and in proliferation studies exhibited identical restriction patterns. Two other clones, HA 1.9 and HA 2.43, recognized different HA determinants and also had distinct restriction patterns. Proliferation inhibition studies with monoclonal antibodies against human class II molecules demonstrated three unique patterns of blocking with the clones, suggesting that clones may be restricted to a unique class II epitope depending on the HA determinant recognized. These data can be interpreted as supporting the argument that human immune responses to influenza hemagglutinin are under Ir gene control exerted at the level of the viral antigenic determinant recognized in association with particular D-region restricting elements. The determinant selection and clonal deletion theories are compared for their capacity to best explain these findings.

B cell activation potential of insulin-reactive T cells in H-2b mice.

The murine T cell response to heterologous insulins provides a good model system for studying the mechanism of immune response (Ir)-gene function, since insulin is a small, chemically well-defined molecule. H-2b mice respond predominantly to A chain loop determinants of beef insulin, presented by the I-A epitope Ia. W39. However, using a library of insulin-specific T cell hybridomas (THy), we previously found that immunization of H-2b mice with beef insulin activates a much wider population of T cells than are detected in T cell proliferation assays. Using such cloned THy we were able to study Ir-gene control at the level of antigen presentation. We compared the ability of the various THy to induce differentiation in I-A-matched B cells in response to antigen. Although both A and B chain-reactive clones respond with interleukin 2 production, they differ markedly in their potential to activate B cells in that only the former are able to induce B cell differentiation in the presence of the intact beef insulin molecule. The latter, however, can serve as helper cells in the presence of isolated B chain, and can synergize with a suboptimal concentration of A chain-reactive THy to induce an optimal B cell response. These results suggest that the insulin molecule is presented by I-Ab antigen-presenting cells in a very specific configuration that allows more effective T cell recognition of the A chain loop than the B chain determinants. To explain the discrepancy between the interleukin 2 assay and the induction of polyclonal activation, it can be assumed that in the former assay antigen is presented by macrophages, while presentation by B cells is necessary for induction of polyclonal activation. Macrophages are able to process the intact beef insulin molecule and, therefore, present B chain determinants, while nonimmune B cells may be unable to process antigen and could present B chain determinants only when the isolated B chain is given as antigen.

Accessory cell function of liver granuloma macrophages of Schistosoma mansoni-infected mice.

In murine schistosomiasis mansoni, the inflammatory macrophage comprises 30% of the granuloma which forms around parasite eggs in the tissue. These granuloma macrophages (GR-Mphi) displayed dense Fc and C3 receptors, and about 50% expressed H-2I region-encoded determinants (Ia antigens). These GR-Mphi were able to effectively reconstitute the burro erythrocyte-specific immunoglobulin M and G antibody response of primed macrophage-depleted spleen cells. However, in contrast to splenic macrophages, GR-Mphi gave only minimal reconstitution of the primary immunoglobulin M response. The reconstitution of the T-cell proliferative response to L-glutamic60-L-alanine30-L-tyrosine10, an antigen under Ir gene control, was also observed when GR-Mphi were added to purified lymph node T-cells. The addition of a monoclonal antibody recognizing a determinant on the Ia complex effectively blocked L-glutamic60-L-alanine30-L-tyrosine10 presentation by GR-Mphi. These studies demonstrated that inflammatory GR-Mphi could function as antigen-presenting cells and that this accessory function was mediated by H-2I region gene products.

Genetic control and effector cells in host-versus-graft responses to H-Y antigen in mice.

T cell responses to the male-specific H-Y antigen in mice include skin graft rejection, delayed-type hypersensitivity and cytotoxic T cell responses; these are under complex H-2 and non-H-2 Ir gene control. The effector cells for these two in vivo responses are Ly 1+2-, and the cytotoxic T cell effectors generated in secondary mixed lymphocyte reactions in vitro are Ly1+2+, although their development requires the presence of Ly 1+2- T helper cells. We investigated the Ir gene control of another in vivo response to H-Y, the host-versus-graft response (HVGR), measured by popliteal lymph node enlargement. The strain distribution pattern (SDP) of primary and secondary HVG responses to H-Y indicates that there are both H-2 and non-H-2 Ir genes involved in controlling responsiveness. Cell transfer of the secondary response identifies an Ly 1+2- effector T cell for this response: this information together with the SDP of cytotoxic T cell responses suggests that the HVGR may represent activation of the T helper population involved in the generation of cytotoxic T cells.

Electrophoretic analysis of human HLA-DR antigens from HLA-DR4 homozygous cell lines: correlation between beta-chain diversity and HLA-D.

Two-dimensional gel electrophoresis of immunoprecipitated human HLA-DR antigens from cells expressing the HLA-DR4 haplotype shows distinct clustering of beta-chain patterns. Six unique electrophoretic variants were observed among 17 HLA-DR4 homozygous cell lines (HCL) analyzed. These patterns correlate precisely with the HLA-D phenotype of the HCL donor as determined by reactivity in mixed lymphocyte culture. All DR4 HCL that belong to one of the well-defined HLA-D antigen groups (Dw4, Dw10, LD "40", LD "DYT", LD "KT2", or LD "TAS") have identical DR beta-chain patterns; DR4 HCL belonging to different HLA-D antigen groups do not. The concordance of the functional expression in mixed lymphocyte culture of a specific D phenotype with a distinct DR beta-chain pattern on gel analysis provides a direct structural basis for understanding the genetic control of HLA-D polymorphisms; HLA-D specificities as revealed by T-cell recognition in mixed lymphocyte culture thus might be accounted for by DR beta-chain polymorphisms. The extent of this beta-chain diversity within a single DR haplotype may aid in understanding variations in Ia-regulated functions, such as Ir gene control and certain disease susceptibilities.

IR gene regulation of the response to trinitrophenyl-polysaccharides. Two independent genes are required for antibody production.

The primary in vitro antibody response to TNP-Ficoll was found to be under H-2-restricted Ir gene control. Strains B10(H-2b), B10.A(H-2a), and B10.S(9R) (H-2t4) were consistently low responders while strains D2.GD(H-2g2), B10.GD(H-2g2), and B10.S(H-2s) were high responders. The in vitro TNP-Ficoll response in congenic recombinant and F1 hybrid mice demonstrated the requirement for complementation of two independent Ir genes. One Ir gene mapped in or to the left of the I-A subregion with high responder alleles being s or d. The second Ir gene mapped to the right of the I-E subregion and required b or s alleles for complementation. These results were further supported by the ability to block the TNP-Ficoll response by appropriate anti-Ia serum pretreatment of the antigen-presenting macrophages. When a structurally different polysaccharide antigen TNP-dextran was used, an identical pattern of restriction was observed.

Ir gene control of immune responses to insulins. II. Phenotypic differences in T cell activity among nonresponder strains of mice.

Immune responses by mice to heterologous insulins are controlled by H-2 linked Ir genes. In studies to determine the mechanisms responsible for nonresponsiveness, we found that although pork insulin failed to stimulate antibody or proliferative responses in H-2b mice, it did prime T cells that can express helper activity in adoptive recipient mice. This helper activity was insulin-specific in both elicitation and expression. In studies presented in this paper, we have extended this analysis to the response patterns of helper T cells stimulated by sheep, horse, and rat insulins in mice bearing different H-2 haplotypes. The results demonstrate that nonresponder forms of insulin, including rat insulin, prime T cells in H-2b and H-2d, but not H-2k, mice. These results suggest that regulation of nonresponsiveness to insulin appears to be through different pathways in mice bearing different H-2 haplotypes.

Neonatal tolerance of major histocompatibility complex antigens alters Ir gene control of the cytotoxic T cell response to vaccinia virus.

The K region of H-2 controls the Tc cell response to vaccinia-Db. The Kb, Kd, and Kq alleles allow good Tc cell responses against vaccinia-Db. In contrast, the presence of Kk in H-2 recombinants 2R (Kk,Db) and 4R (Kk,Db) or in F1 hybrids greatly reduces the anti-vaccinia-Db response. The defect does not lie in antigen presentation, as infected 4R cells can stimulate anti-vaccinia-Db Tc cells in vitro. Furthermore, nonresponder animals possess Tc cell precursors for vaccinia-Db, as transfer of F1 nonresponder spleen cells into infected, lethally irradiated responder recipients allowed generation of anti-vaccinia-Db effector Tc cells. Secondary responses to vaccinia-Db can also be obtained in vitro from T cells of 4R animals. Feedback inhibition was excluded in experiments with mixed chimeras in which Kk and Db were expressed on separate cell populations. Neonatal tolerance of B10 animals to Kk suppressed the anti-vaccinia-Db response but did not affect anti-vaccinia-Kb, anti-lymphocytic choriomeningitis virus, or anti-H-2d responses. In cold target competition experiments, H-2k competitors inhibited vaccinia-Db-specific target cell lysis by Tc cells, which suggests that anti-vaccinia-Db and anti-H-2Kk Tc cells may cross-react. Therefore, we propose that the suppressive influence of Kk on anti-vaccinia-Db Tc cell responses is a consequence of self-tolerance and that suppression of anti-Kk Tc cells results in cross-reactive suppression of anti-vaccinia-Db Tc cells.

MHC-linked Ir gene control of T cell responses: GAT-specific proliferating T cells and helper T cells are elicited in nonresponder mice immunized with soluble GAT.

The immune response to the synthetic terpolymer GAT is controlled by MHC-linked Ir gene(s). We show in this paper that antigen-presenting cells and T cells from mice belonging to two nonresponder strains (SJL and DBA/1) can present and recognize GAT, respectively. This has been measured with a T cell proliferation assay of GAT-primed lymph node cells. In order to detect T cell proliferation among GAT-primed lymph node cells from DBA/1 mice, it is necessary to treat the cells with monoclonal anti-Lyt-2 antibodies and complement (C) before the assay. These conclusions were further verified with SJL mice, when a T cell line derived from LN cells was used. We have shown that after immunization with GAT, specific T helper cells can be generated in the lymph nodes of SJL mice but not in the lymph nodes of DBA/1 mice. Furthermore, GAT-specific T helper cells can be detected in the spleen of SJL mice after immunizations with GAT, provided these spleen cells are pretreated with monoclonal anti-Lyt-2 antibodies + C or mild irradiation. Together, these results support the general idea that nonresponsiveness can be explained by a regulatory imbalance rather than by discrete cellular "defects."

Elicitation and detection of in vitro H-2-linked Jr gene regulated antibody responses to poly ( LTyr, Glu)-Poly( dlALa)--poly( lLys)

A microculture system is described in which secreted antibody responses to the synthetic polypeptide (T,G)-A--L were obtained in vitro. Responses were highly reproducible, antigen-dependent, antigen-specific, and under H-2-linked Ir gene control. Critical elements in the system include the schedule of in vivo antigen-priming, removal of the stimulating antigen after 3 days of culture, and a sensitive detection system (double-antibody ELISA). This system should be useful in the analysis of the mechanism of action of Ir genes as well as the mechanisms by which anti-idiotype antibodies modulate immune responses.

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.

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.

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.

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.