Cellular and genetic control of antibody responses in vitro. II. Ir gene control of primary IgM responses to trinitrophenyl conjugates of poly-L-(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys and poly-L-(His,Glu)-poly-D,L- Ala--poly-L-Lys

The in vitro primary IgM anti-hapten responses to trinitrophenyl (TNP) conjugates of poly-L-(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys (T,G)-A--L and poly-L(His,Glu)-poly-D,L-Ala--poly-L-Lys (H,G)-A--L were shown to be T- cell dependent and under autosomal dominant H-2-linked Ir gene control which mapped within the K or I-A regions of the H-2 complex. The in vitro response to TNP-keyhole limpet hemocyanin, while T-dependent, was not under demonstrable genetic control. The genes governing the in vitro primary IgM anti-hapten responses to TNP-(T,G)-A--L and TNP-(H,G)- A--L resemble the Ir genes controlling the in vivo secondary IgG responses to (T,G)-A--L and (H,G)-A--L in that they are autosomal dominant, map identically within the H-2 complex, and have identical responder and nonresponder haplotypes. It is concluded that Ir genes can govern the ability to generate an IgM response upon initial exposure to antigen.     

The role of H-2-linked genes in helper T-cell function. III. Expression of immune response genes for trinitrophenyl conjugates of poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys in B cells and macrophages

Using lymph node T cells from poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys[(TG)-A--L]-primed animals and B cells from animals primed with trinitrophenylated (TNP) protein or lipopolysaccharide, we have obtained anti-TNP-(TG)-A--L direct plaque-forming responses in vitro. Response to this antigen was shown to be controlled by the H-2 haplotype of the animal studied. The strain distribution of in vitro response was very similar to that previously reported by others for in vivo secondary IgG responses to (TG)-A--L. We investigated the cell types expressing the Ir gene(s) for (TG)-A--L in our cultures. F1, high responder x low responder mice were primed with (TG)-A--L. Their T cells were active in stimulating anti-TNP-(TG)-A--L responses of high responder but not low responder B cells and macrophages (MPHI), even though both preparations of B cells and Mphi were obtained from mice congenic at H-2 with one of the parents of the F1. For three low responder strains tested, of the H-2h2, H-2k, and H-2f haplotypes, the anti-TNP-(TG)-A--L response of low responder B cells and Mphis in the presence of high responder, F1 T cells could not be improved by the addition of high responder, antigen-bearing Mphis to the cultures. In one strain of the H-2a haplotype, it was shown that neither the B cells nor Mphis could be functional in anti-TNP-(TG)-A--L responses. Our results therefore suggested the Ir genes for anti-TNP-(TG)-A--L responses were expressed at least in B cells in all the low responder strains we studied, and, in mice of the H-2a haplotype, in Mphis too.     

Antibody response of C3H in equilibrium (CKB X CWB)F1 tetraparental mice to poly-L(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys immunization

To test whether the antigen-specific stimulation of low responder-genotype B cells in tetraparental mice is due to a histoincompatibility reaction (allogeneic effect) against these B cells, tetraparental mice were constructed (a) between an Ir-1A low responder to the antigen poly-L(Tyr,Glu)-poly-D,L-Ala--poly-L-Lys. [(T,G)-A--L] and an Ir-1A F1 high responder and (b) between two histoincompatible Ir-lA low responders. In the first case the F1 high responder embryo shares the whole of the H-2 complex, including Ir, with the low responder embryo.     

Antigen-reactive T clones. III. Low responder antigen-presenting cells function effectively to present antigen to selected T cell clones derived from (High Responder x Low Responder)F1 mice

Long-term-cultured poly(Tyr, Glu)-poly-D,L,-Ala-poly-Lys [(T,G)-A--L]- reactive T cells and clones derived from (high responder x low responder)F1 [(C57BL/6 x A/J)F1] mice were shown to recognize (T,G)-A-- L presented by cells from low responder strain A/J mice. The antigen- presenting determinant(s) that allowed recognition of (T,G)-A--L by such T cell clones was controlled by the I-A subregion of the major histocompatibility complex. These results suggest that there is no functional defect in the ability of low responder Ir gene products (I-A antigens) to associate with (T,G)-A--L for effective recognition by T cells. Although these results might tentatively be interpreted to suggest that Ir gene-controlled low responsiveness is due to the inability of the T cell to recognize the association between (T,G)-A--L and low responder I-A gene products, it is similarly possible that there might be a defect in the functional capabilities of low responder antigen-presenting cells to effectively process (T,G)-A--L into immunodominant epitopes.     

The role of H-2-linked genes in helper T cell function. VII. Expression of I region and immune response genes by B cells in bystander help assays

The mode of action by bystander helper T cells was investigated by priming (responder X nonresponder) (B6A)F1 T cells with poly-L-(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(TG)-A--L] and titrating the ability of these cells to stimulate an anti-sheep red blood cell (SRBC) response of parental B cells and macrophages in the presence of (TG)-A--L. Under limiting T cell conditions, and in the presence of (TG)-A--L, (TG)-A--L-responsive T cells were able to drive anti-SRBC responses of high-responder C57BL/10.SgSn (B10) B cells and macrophages (M0), but not of low-responder (B10.A) B cells and M0. Surprisingly, the (TG)-A--L-driven anti-SRBC response of B10.A B cells was not restored by addition of high-responder acessory cells, in the form of (B6A)F1 peritoneal or irradiated T cell-depleted spleen cells, or in the form of B10 nonirradiated T cell-depleted spleen cells. These results suggested that (TG)-A--L-specific Ir genes expressed by B cells controlled the ability of these cells to be induced to respond to SRBC by (TG)-A--L-responding T cells, implying that direct contact between the SRBC-binding B cell precursor and the (TG)-A--L-responsive helper T cells was required. Analogous results were obtained for keyhold limpet hemocyanin (KLH)-driven bystander help using KLH-primed F1 T cells restricted to interact with cells on only one of the parental haplotypes by maturing them in parental bone marrow chimeras. It was hypothesized that bystander help was mediated by nonspecific uptake of antigen [(TG)-A--L or KLH] by SRBC-specific b cells and subsequent display of the antigen on the B cell surface in association with Ir of I-region gene products, in a fashion similar to the M0, where it was then recognized by helper T cells. Such an explanation was supported by the observation that high concentrations of antigen were required to elicit bystander help. This hypothesis raises the possibility of B cell processing of antigen bound to its immunoglobulin receptor and subsequent presentation of antigen to helper T cells.     

Gene complementation in the T-lymphocyte proliferative response to poly (Glu55Lys36Phe9)n. A demonstration that both immune response gene products must be expressed in the same antigen-presenting cell

The immune response (Ir) to the random copolymer GLphi depends upon the function of two Ir genes, Ir-GLphi-beta[beta] and Ir-GLphi-alpha[alpha], mapped to the I-A and I-E/C subregions of the major histocompatibility complex, respectively. In this paper, the site(s) of expression of the products of these two Ir genes was examined by evaluating T-lymphocyte proliferative responses of bone marrow radiation chimeras. Chimeras were created in [alpha+beta- X alpha-beta+]F1 responder mice by lethal irradiation and reconstitution with a mixture of bone marrow cells from both parental strains. These chimeras failed to respond to GLphi, although they were capable or responding to the much weaker antigens, (T,G)-A--L, TEPC-15, pigeon cytochrome c, and (H,G)-A--L. This failure to respond to GLphi was shown not to be the result of a cryptic mixed lymphocyte reaction, as similar chimeras created in (alpha+beta+ X alpha-beta+)F1 mice responded well to GLphi, although they possessed almost the same potential histoincompatibility. Furthermore, the lack of response to GLphi could not be attributed to a general failure of the two parental cell types in the chimeras to collaboratc with each other, as each chimeric parental cell type could respond to dinitrophenyl conjugated ovalbumin presented on nonimmune spleen cells from the other parent. Thus, the failure of low responder parental into F1 high responder chimeras to generate an immune response to GLphi suggests that immune competence for this antigen requires at least one cell type in the immune system to express gene products of both the Ir-glphi-alpha and -beta genes, i.e. one cell must be of high responder genotype. The the antigen-presenting cell is one such cell type was shown by experiments in which GLphi-primed T lymphocytes from responder F1 mice were stimulated with antigen bound to nonimmune spleen cells. Only spleen cells from responder F1 and recombinant mice could present GLphi. Neither of the two complementing nonresponder parental spleen cell populations, either alone or mixed together, could present GLphi, although both could present purified protein derivative of tuberculin. This was shown to be the case for T cells positively selected in vitro as well as freshly explanted T cells. Thus, both Ir-GLphi-alpha and Ir-GLphi-beta gene products must be expressed in the same antigen-presenting cell to generate a T-lymphocyte proliferative response to GLphi. The implications of these findings for models of two gene complementation are discussed.     

T-lymphocyte-enriched murine peritoneal exudate cells. IV. Genetic control of cross-stimulation at the T-cell level

Antibodies raised against many structurally related antigens have been shown to cross-react extensively. Manifestations of T-cell immunity, on the other hand, appear to be more restricted in their ability to be elicited by cross-reacting antigens, although examples have been reported. This paper explores the nature of the cross-reactions at the T-cell level among the branched-chain copolymers (T,G)-A--L, (phi,G)-A--L, (H,G)-A--L, and G-A--L, as well as a related linear terpolymer, GAT, in a variety of mouse strains using the peritoneal exudate T-lymphocyte-enriched cells (PETLES) proliferation assay. (T,G)-A--L, (phi,G)-A--L, and GAT could cross-stimulate cells immune to the other two antigens, whereas (H,G)-A--L, (T,G)-Pro--L, and G-A--L showed no cross-stimulations. The extent of the cross-reactions varied with the mouse strain and was shown to be under the control of immune response genes. It was necessary for the strain to be able to respond to both the immunogen and the cross-reacting antigen, when used as an immunogen, in order for cross-stimulation to occur; however, this was not always sufficient. Several examples of unequal or one-way cross-reactions were found. In addition, the immune responses to (H,G)-A--L and (phi,G)-A--L showed no cross-reactions with the other antigen even though their Ir genes were both mapped to the K region or I-A subregion. The problem of accounting for such fine specificity of T-cell recognition in lieu of the genetic evidence demonstrating only Ir gene control of the response is discussed.     

Self recognition in allogeneic radiation bone marrow chimeras. A radiation- resistant host element dictates the self specificity and immune response gene phenotype of T-helper cells

The specificity of the self-recognition repertoire in fully allogeneic (A {arrow} B), semiallogeneic (A {arrow} A x B and A x B {arrow} A), and double donor (A + B {arrow} A) radiation bone marrow chimeras was assessed by the ability of their spleen cells to generate in vitro primary plaque-forming cell (PFC) responses to trinitrophenyl- keyhole limpet hemocyanin. In contrast to spleen cells from semiallogeneic and double donor chimeras, intact spleen cells from fully allogeneic BI0 {arrow} B10.A and B10.A {arrow} B10 chimeras were not capable of generating responses to trinitrophenyl (TNP)-keyhole limpet hemocyanin. However, cultures containing a mixture of both B10 {arrow} B10.A and B10.A {arrow} B10 spleen cells did respond, demonstrating that all the cell populations required for the in vitro generation of T-dependent PFC responses were able to differentiate into functional competence in a fully allogeneic major histocompatibility complex (MHC) environment. The self recognition repertoire of T-helper cells from fully allogeneic A {arrow} B chimeras was determined to be specific for the recognition of host, not donor, MHC determinants in that they were able to collaborate with cells expressing only host MHC determinants but not with cells expressing only donor MHC determinants, even though the functional lymphocytes in these chimeras were shown to be of donor origin. Experiments utilizing double donor A + B {arrow} A chimeras further demonstrated that the ability of chimeric T cells to recognize allogeneic MHC determinants as self structures was a function of a radiation-resistant host element and not simply a consequence of the tolerization of T cell precursors to allogeneic MHC determinants, because strain A lymphocytes isolated from A + B {arrow} A chimeras were tolerant to both A and B MHC determinants but were restricted to the self recognition of syngeneic host type A MHC determinants. Finally, the Ir gene phenotype expressed by B10 {arrow} B10.A and B10.A {arrow} B10 chimeric lymphocytes was determined by their ability to function in the Ir gene controlled response to TNP-poly-L-(Tyr,Glu)-poly-D,L-Ala-poly- L-Lys [(T,G)-A--L]. The ability of lymphocytes to function in TNP-(T,G)-A--L responses was not determined by their genotype but rather paralleled the specificity of their self recognition repertoire for high responder (H-2 (b)) determinants. The possible degeneracy of the MHC-specific self recognition repertoire is discussed, and a model is proposed for Ir gene regulation in which expression of Ir gene function by lymphocytes is an antigen-nonspecific consequence of the specificity and cross-reactivity of their self recognition repertoire.     

Genetic regulation of delayed-type hypersensitivity responses to poly(LTyr,LGu)-poly(DLAla)--poly(LLys). I. Expression of the genetic defect at two phases of the immune process

Delayed-type hypersensitivity (DTH) responses served in this study as an experimental model for the analysis of genetic regulations of T-cell responses. Educated irradiated cells from H-2b mice mediated responses in syngeneic recipients, whereas mice of the a, d, f, k, and s haplotypes were nonresponders to poly(LTyr,LGlu)-poly(DLAla)-- poly(LLys)[(T,G)-A--L]. These results suggest that cell-mediated immune responsiveness to (T,G)-A--L is linked to the H-2 complex, as was shown for humoral responses. Educated irradiated T cells of F1 hybrids between high and low responders mediated DTH responses, which indicates that the gene(s) controlling the DTH responses is dominant. To analyze the genetic defect in DTH responses to (T,G)-A--L, we separated the T- cell activation phase from the effector phase that was determined in recipient mice. Two types of nonresponders were observed: (a) When lymphocytes of the a or k haplotypes were educated in a syngeneic environment and then transferred into hybrids between the parental (nonresponder x responder) F1 recipients, DTH responses could have been manifested. (b) On the other hand, no DTH responses could be mediated by transferring educated cells of the H-2s or H-2f origin into the appropriate F1 recipients. In addition, irradiated F1 cells that had been activated to (T,G)-A--L could not mediate DTH responses in both types of nonresponder recipients. These results suggest that T cells of H-2k or H-2a mice can be activated to generate DTH responses to (T,G)-A- -L and that the defect in these mouse strains is expressed in another cell population needed for the manifestation of the DTH reaction in the recipient mice. In contrast, T cells of H-2s and H-2f origin cannot be activated to (T,G)-A--L and, thus, fail to manifest DTH responses.     

Antigen-specific T-cell factors in the genetic control of the immune response to poly(Tyr,Glu)-polyDLAla--polyLys. Evidence for T- and B- cell defects in SJL mice

The cellular basis of the genetic control of the immune response to poly(LTyr, LGlu)-polyDLAla--polyLLys [(T,G)-A--L] in SJL (H-2s, low responder) mice has been investigated using T-cell factors. Thymocytes of SJL origin were educated to (T,G)-A--L and tested for their ability to produce an antigen-specific factor capable of cooperating in vivo with bone marrow cells of either SJL or C3H.SW (high responder) origin. SJL T cells were found to be incapable of producing such a cooperative factor, in contrast with results previously obtained with C3H/HeJ (low responders) and C3H.SW strains. Moreover, SJL bone marrow cells did not produce an antibody response to (T,G)-A--L, even when combined with factor produced by high responder (C3H.SW) mice. Thus, both T and B cells appear to be defective in the SJL strain in the response to (T,G)-A--L.     

The role of H-2-linked genes in helper T-cell function. VI. Expression of Ir genes by helper T cells

We examined the expression of (TG)-A--L specific Ir genes in helper T cells using T cells from low responder leads to (B10, high responder x low responder) F1 chimeric mice. In this paper, the low responder strain studied was B10.M, H-2f. B10.M T cells from these chimeric animals do not help anti-TNP-(TG)-A--L responses, even though they have matured in a high responder thymus and been primed and challenged with antigen on high responder Mphi and B cells. These findings indicate that in the H-2f haplotype an Ir-gene controlling anti-(TG)-A--L activity is expressed in helper T cells. The findings are in contrast to those we have obtained and previously reported with T cells of another low responder haplotype, H-2a. Taken together with our previous findings that (TG)-A--L specific Ir genes are expressed by B cells and Mphi of both the H-2a and H-2f haplotypes, the results indicate two sites of action for Ir genes, and suggest two different gene products acting at different stages of the response, both of which are defective in H-2f cells, and only one of which is defective in H-2a cells.     

GENETIC CONTROL OF THE IMMUNE RESPONSE : In Vitro Stimulation of Lymphocytes by (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4°C followed by washing and incubation at 37°C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti-H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition.     

GENETIC CONTROL OF IMMUNE RESPONSES IN VITRO : II. CELLULAR REQUIREMENTS FOR THE DEVELOPMENT OF PRIMARY PLAQUE-FORMING CELL RESPONSES TO THE RANDOM TERPOLYMER L-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 (GAT) BY MOUSE SPLEEN CELLS IN VITRO

The cellular requirements for the development of primary IgG GAT-specific PFC responses in cultures of spleen cells from responder, C57Bl/6, mice stimulated with GAT and GAT-MBSA and in cultures of spleen cells from nonresponder, SJL and B10.S, mice stimulated with GAT-MBSA were investigated. Macrophages were required for development of responses to GAT and GAT-MBSA in cultures of spleen cells from responder mice and for responses to GAT-MBSA in cultures of spleen cells from nonresponder mice. Macrophages from nonresponder mice supported the development of responses to GAT by nonadherent responder spleen cells, indicating that the failure of nonresponder mice to respond to GAT is not due to a macrophage defect. Furthermore, responder macrophages supported the responses of nonadherent, nonresponder spleen cells to SRBC and GAT-MBSA, but not to GAT. This indicates that the capacity to respond to GAT is a function of the nonadherent population which is composed of thymus-derived (T) helper cells and precursors of antibody-producing cells. Treatment of spleen cells with anti-theta serum and complement before culture initiation abolished PFC responses to GAT and GAT-MBSA thus establishing the requirement for T cells in the development of PFC responses to these antigens. Since precursors of antibody-producing cells in nonresponder mice are capable of synthesizing antibody specific for GAT after stimulation with GAT-MBSA and since the response to GAT is thymus-dependent, it appears that nonresponder mice lack GAT-specific helper T cell function.     

Genetic control of the immune response. The effect of graft-versus-host reaction on the antibody response to poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys in nonresponder mice

The transfer of parental (H-2^k/k) nonresponder lymphoid cells into heterozygous (H-2^k/q) nonresponder recipients at the time of primary challenge with aqueous poly-L(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys [(T,G)-A--L] elicited the production of both IgM and IgG anti-(T,G)-A--L antibody. Normally, the production of IgG anti-(T,G)-A--L antibody is restricted to strains possessing the responder Ir-1 allele. The timing and intensity of the graft-versus-host (GVH) reaction required for this effect were found to be critical. Injection of H-2^k/k cells into H-2^k/q recipients 1 wk before antigen challenge did not elicit IgG anti-(T,G)-A--L antibody production, and markedly suppressed IgM anti-(T,G)-A--L antibody production. The transfer of alloimmune (H-2^q-primed) H-2^k/k cells at the time of antigen challenge was also associated with no IgG and little IgM anti-(T,G)-A--L antibody production. These data are consistent with the model that nonresponder thymus-derived lymphocytes (T cells) activated in a GVH reaction can substitute for (T,G)-A--L-reactive T cells to induce a shift from IgM to IgG anti-(T,G)-A--L antibody production.     

Cellular requirements for lipopolysaccharide adjuvanticity. A role for both T lymphocytes and macrophages for in vitro responses to particulate antigens

By employing primary cultures of purified spleen cells from lipopolysaccharide (LPS) responder (C3H/HeN or C57BL/10Sn) or nonresponder (C3H/HeJ or C57BL/10ScN) mice incubated with particulate antigen and LPS prepared by phenol-water extraction (Ph), we have presented evidence that both T cells and macrophages (MO) are required for LPS-induced adjuvanticity. First, MO derived from C3H/HeN spleen cells, when mixed with responder, C3H/HeN lymphocytes and Ph-LPS, elicited enhanced antibody responses to sheep erythrocytes (SRC) antigen, whereas lymphocytes from the nonresponder, C3H/HeJ mouse strain did not evoke this response. Similarly, purified T cells from C3H/HeN spleens, when cultured with responder, nu/nu spleen cells, and Ph-LPS yielded enhanced anti-TNP PFC responses; whereas, C3H/HeJ T cells did not potentiate immune responses when mixed with optimal concentrations of Ph-LPS. LPS prepared by butanol-water extraction elicited significant adjuvant effects with all cell combinations. Finally, purified responder T cells and MO enabled either responder or nonresponder B cells to elicit LPS potentiation. These data indicate that T cells and MO are controlling LPS-induced augmentation of B-cell responses.     

Secondary antibody responses in vitro to L-glutamic acid60-L-alanine30- L-tyrosine10 (GAT) by (responder X nonresponder)F1 spleen cells stimulated by parental GAT-macrophages

The development of IgG L-glutamic Acid60-L-alanine30-L-tyrosine10 (GAT)-specific plaque-forming cell responses in vitro by virgin and immune (responder X nonresponder)F1 spleen cells after stimulation with responder and nonresponder parental GAT-macrophages (Mphi) was investigated. Virgin F1 spleen cells developed comparable primary responses to both parental GAT-Mphi. By contrast, F1 spleen cells from mice immunized with GAT or responder parental GAT-Mphi developed secondary responses after stimulation with only responder parental GAT-Mphi. Spleen cells from F1 mice immunized with nonresponder parental GAT-Mphi developed secondary responses to these GAT-Mphi, but failed to respond to responder parental GAT-Mphi. These results are discussed in the context of genetic restrictions regulating Mphi-T-cell interactions in secondary antibody responses and the possible expression of Ir-gene function in Mphi.     

Dendritic cells induce T cell proliferation to synthetic antigens under Ir gene control

Dendritic cells prepared by a modification of the method of Steinman and Cohn are I-A+ and FcR-. They are extremely potent at activating not only allogeneic T cell proliferation but also antigen-specific syngeneic T cell proliferation. Dendritic cells from nonresponder strains are unable to present antigens to responder X nonresponder T cells, suggesting that they may be a site of Ir gene product expression.     

Antigen-specific thymus cell factors in the genetic control of the immune response to poly-(tyrosyl, glutamyl)-poly-D, L-alanyl--poly-lysyl

The genetic control of the antibody response to a synthetic polypeptide antigen designated poly-L(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(T, G)-A--L] has been studied in congenic high responder C3H.SW (H-2^b) and low responder C3H/HeJ (H-2^k) strains of mice. This response is controlled by the Ir-1 gene and is H-2 linked. The method employed was to study the ability of specifically primed or "educated" T cells of each strain to produce cooperative factors for (T, G)-A--L in vitro. Such factors have been shown to be capable of replacing the requirement for T cells in the thymus-dependent antibody response to (T, G)-A--L in vivo. The T-cell factors produced were tested for their ability to cooperate with B cells of either high or low responder origin by transfer together with bone marrow cells and (T, G)-A--L into heavily irradiated, syngeneic (for bone marrow donor) recipients. Direct anti-(T, G)-A--L plaque-forming cells were measured later in the spleens of the recipients. The results showed that (a) educated T cells of both high and low responder origin produced active cooperative factors to (T, G)-A--L, and no differences between the strains in respect to production of T-cell factors could be demonstrated; and (b) such factors, whether of high or low responder origin, cooperated efficiently with B cells of high responder origin only, and hardly at all with B cells of low responder origin. The conclusion was drawn that the cellular difference between the two strains lies in the responsiveness of their B cells to specific signals or stimuli received from T cells. As far as could be discerned by the methods used, no T-cell defect existed in low responder mice and the expression of the controlling Ir-1 gene was solely at the level of the B cells in this case.     

Ir-genes in H-2 regulate generation of anti-viral cytotoxic T cells. Mapping to K or D and dominance of unresponsiveness

H-2 dependent and virus-specific Ir genes regulate the generation of primary virus-specific K or D restricted cytotoxic T-cell responses in vivo. The following examples have been analyzed in some detail: first, Dk restricted responses to vaccinia in Sendai viruses are at least 30 times lower than the corresponding K-restricted responses irrespective of the H-2 haplotypes (k, b, d, dxs, dxq) of K and I regions; in contrast, LCMV infection generates high responses to Dk. These findings are consistent with but do not prove that this Ir gene maps to D. Second, Db restricted responses to vaccinia and Sendai viruses are high in strains possessing the Kq or KbIb, KbaIb haplotype, are very low in strains with Kk, and relatively low in mouse strains of the KdI-Ad haplotype; LCMV generates high Db restricted response in the presence of Kk. This Ir gene for the response to vaccinia and Sendai viruses maps to K since B10.BYR (KqIkdDb) is a responder and B10.A (2R) is a nonresponder (KkIkdDb). Third, virus and K or D allele specific nonresponsiveness is dominant with variable penetrance; in heterozygous mice the nonresponder Kk allele over-rides responsiveness normally found in KbDb or KqDb combinations. Fourth, when (responder X nonresponder)F1 lymphocytes are stimulated in an environment expressing vaccinia virus plus only a high responder Kb or Kq allelle and Db, response to vaccinia Db is high; in contrast when the same F1 cells are stimulated in an environment expressing the low responder allele Kk, response to vaccinia Db is low. Thus absence of Kk during immunization allows generation of high responsive Db restricted vaccinia specific cytotoxic T cells. The Dk dependent low response to vaccinia Dk can be explained by a preclusion rule or by failure of vaccinia to complex with Db; however the analysis of Kk dependent low response to vaccinia Db does not support these explanations or that self-tolerance is responsible for this Ir effect but is compatible with the interpretation that Kk vaccinia is immunodominant over Db vaccinia. These results are discussed with respect to (a) possible mechanisms of regulation by Ir genes and (b) H-2 polymorphism and HLA-disease association.     

Histocompatibility linked immune responsiveness and restrictions imposed on sensitized lymphocytes

Delayed-type hypersensitivity (DTH) transfer to GAT was restricted by the I-A region of the major histocompatibility complex (MHC). Sensitized cells from F1 hybrid mice between responder and nonresponder strains transferred DTH to syngeneic F1 mice and to naive parental strain recipients of the responder but not of the nonresponder haplotypes. These results are interpreted to favor the postulate that the MHC-linked Ir genes exert their effects by coding for components which allow interactions between particular I region gene products and the region to form stable structures immunogenic for DTH T cells.