Genetic analysis of complementing H-linked Ir genes.
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Biomedical subjects
B Benacerraf
Publications and source records attributed to B Benacerraf.
Specificity of rat xenogeneic cell-mediated cytolysis for the products of the K and D loci of the mouse H-2 complex.
Lewis rat lymph node (LN) cells, when cultured with irradiated mouse spleen cells, develop cytotoxic effectors that were shown to be specific for the sensitizing mouse strain. Alloantisera directed to the products of the K and D loci of the target inhibited cytolysis whereas an antiserum to the I region gene products did not inhibit cytolysis even though LPS-induced spleen blasts were used as targets. Thus, it appears that even when effector cells from another species are generated to mouse spleen cells, the gene products of the K and D loci continue to play a predominant role.
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Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT). I. Production, characterization, and lack of H-2 restriction for activity in recipient strain.
The random synthetic copolymer of L-glutamic acid50-L-tyrosine50 (GT) fails to elicit a GT-specific antibody response in all inbred strains of mice tested. Preimmunization with GT specifically inhibits a GT-MBSA response in certain H2d,k,s, but not other, H-2a,b,q, nonresponder mice. This unresponsiveness is mediated by GT-specific suppressor T cells. Extracts prepared from lymphoid cells of GT-primed suppressor haplotype mice inhibit the development of primary GT-specific antibody responses to GT-MBSA in normal syngeneic mice. Nonsuppressor haplotype mice do not produce GT-specific suppressor factor. The GT-suppressive extract has affinity for antigen and a m.w. of less than 50,000 daltons, thus, resembling antigen-specific immunosuppressive factors already described. However, the GT-suppressive extract does not appear to have H-2 restrictions since it works across allogeneic barriers. Evidence is presented that two genes are required for factor-mediated suppression.
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Development of a hemolytic plaque assay for glutamic acid, lysine-containing polypeptides: demonstration that nonresponder mice produce antibodies to these peptides when conjugated to an immunogenic carrier.
Fowl gamma-globulin, when chemically conjugated to GLO or GL, functions as a T-dependent immunogenic carrier and stimulates anti-GLO and anti-GL antibody production in nonresponder mice. The conjugation procedure utilizes the Schiff base reaction. The anti-GL and anti-GLO responses were detected by hemagglutination and hemolytic plaque assays by using GL-coated erythrocytes. The coupling of GL to erythrocytes utilizes a novel procedure in which a palmitoyl derivative of GL is adsorbed onto red blood cells. The optimal conditions for preparing the palmitoyl derivative and for coupling to SRBC are presented. With the hemolytic plaque assay, we have verified that GLO responder animals make both IgM and IgG responses, whereas nonresponder mice fail to make either IgM or IgG plaque-forming cells.
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Cross-reactive lysis of trinitrophenyl (TNP)-derivatized H-2 incompatible target cells by cytolytic T lymphocytes generated against syngeneic TNP spleen cells.
Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.
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Regulation by the H-2 gene complex of macrophage-lymphoid cell interactions in secondary antibody responses in vitro.
The ability of antigen-bearing syngeneic and allogeneic peptone-induced peritoneal exudate macrophages to support development of primary and secondary antibody responses by murine lymphoid or spleen cells in vitro has been investigated. The antigen used was the terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Syngeneic and allogeneic macrophages supported development of comparable primary antibody responses to GAT, indicating that genetic restrictions do not limit efficient macrophage-lymphocyte interactions in primary responses. By contrast, immunized spleen or lymphoid cells developed secondary antibody responses preferentially when stimulated in vitro with GAT on macrophages syngeneic to the macrophages used to present GAT during in vivo immunization. Thus, genetic restrictions regulate efficient macrophage-lymphocyte interactions in secondary antibody responses. These restrictions have been demonstrated from 2 to 8 wk after a single immunization with limiting quantities of GAT and are controlled by the H-2 gene complex. The implications that immune lymphocytes selectively recognize and respond to antigen presented in the context of the macrophage membrane-antigen complex which sensitized the lymphocytes initially are considered.
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Genetic control of specific immune suppression. III. Mapping of H-2 complex complementing genes controlling immune suppression by the random copolymer L-glutamic acid50-L-tyrosine50 (GT).
Earlier studies from our laboratory demonstrated that the terpolymer of L-glutamic acid, L-alanine, and L-tyrpsine (GAT) stimulated the development of T cells capable of specifically suppressing the antibody responses in vivo and in vitro of nonresponder strains (bearing the H-2(s), H-2(q), and H-2(p) haplotypes) to GAT complexed with an immunogenic carrier, methylated bovine serum albumin, MBSA (1,2). We then extended these findings to another antigen, the copolymer of L-glutamic acid and L-tyrosine (GT). None of 19 inbred or congenic resistant mouse strains developed antibody responses to GT after immunization with this synthetic polypeptide in adjuvants. All the strains investigated, however, developed IgG plaque-forming cells (PFC) primary responses to GT complexed with MBSA (3). This permitted us to determine that: (a) preimmunization with GT suppressed the response to GT-MBSA in certain but not in all strains; (b) the suppression could be transferred by thymocytes and spleen cells from GT-primed animals; (c) the development of GT-specific suppressor cells is under dominant control of H-2- linked gene(s) which have been designated specific immune suppressor genes (Is genes); (d) the Is genes are antigen specific since GAT-MBSA responses are suppressed by GAT in strains carrying the H-2(q) haplotype, while GT-MBSA responses are not suppressed by the related polymer GT in these same strains (3,4). The experiments reported in this study map the Is genes responsible for GT-specific suppression within the H-2 complex. The data indicate that the K and D loci are not concerned with GT-specific suppression, and that this phenomenon is controlled by complementing or interacting genes which map on either side of cross-over events between the IB and IC subregions.
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Genetic control of specific immune suppression. IV. Responsiveness to the random copolymer L-glutamic acid50-L-tyrosine50 induced in BALB/c mice by cyclophosphamide.
Previous reports from our laboratory have demonstrated the stimulation of specific suppressor T cells in genetic nonresponder mice after immunization with the terpolymer of L- glutamic acid, L-alanine, and L-tyrosine (GAT) (1,2) and with the copolymer of L-glutamic acid and L-tyrosine (GT) (3-5). These findings raise two important questions: (a) do the specific suppressor T cells inhibit an antibody response which would otherwise develop in nonresponder mice; and, (b) can specific helper T cells inhibit an antibody response which would otherwise develop in nonresponder mice; and, (b) can specific helper T-cell activity be detected in these animals. Responsiveness appears to be completely dominant over suppression in (responder x suppressor)F(1) hybrids, therefore, we have been unable to detect suppressor cells in these hybrids after conventional immunization with GAT (2). However , using special conditions of antigen administration, GAT helper activity could be demonstrated in nonresponder DBA/1 ("suppressor") mice. Thus, GAT-specific helper activity was not detected in these nonresponder animals after immunization with GAT irrespective of the adjuvant used, but could be stimulated by macrophage-bound GAT or by GAT complexed with methylated bovine serum albumin GAT-MBSA (6). In the current report we have taken advantage of the fact that suppressor T-cell activity is more sensitive to cyclophosphamide treatment than T-cell helper activity (7) to demonstrate the presence of GT-specific helper activity in "nonresponder" BALB/c mice. We describe: (a) the dose of cyclophosphamide and conditions of treatment which inhibits the well-documented stimulation of specific suppressor T cells in BALB/c mice injected with GT previous to immunization with GT-MBSA, and (b) the ability of cyclophosphamide to permit the development of primary PFC responses to GT in these "nonresponder" mice. These cyclophosphamide-induced responses are not characterized by the high levels of antibody detected in genetic responder animals.
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Inclusion group systems and cis-trans effects in responses controlled by the two complementing Ir-GLphi genes.
The immune responses to the random linear terpolymers of L-amino acids, poly-(glu57, lys38, tyr5), poly-(glu51, lys34, tyr15,), and poly-(glu53, lys36, phe11) are each controlled by two dominant H-linked Ir genes. The immune responses to these three related terpolymers demonstrate different H-2 distributions, however, the H-2 patterns are part of a single inclusion group system. The alpha- and beta-genes are dominant; however, most effective gene interactions occur when the two genes are in the cis configuration. The potential significance of this cis-trans effect is discussed.
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The requirement for two complementing Ir-GLphi immune response genes in the T-lymphocyte proliferative response to poly-(Glu53Lys36Phe11).
The antibody response to poly-(Glu53Lys36Phe11) (GLphi) has been shown to be under the control of two independent, major histocompatibility-linked immune response genes, designated alpha and beta. In the present work we demonstrate that the T-lymphocyte proliferative response is also under the control of these two immune response genes. Thus, mice of the H-2a, H-2b, H-2k, and H-2s haplotypes were all nonresponders to GLphi. In contrast F1 hybrids between these strains, such as (B10 X B10.A)F1 and (C3H X SJL)F1, as well as several recombinant mice derived from the nonresponder haplotypes, such as B10.1(5R), B10.HTT, and B10.S(9R), were all responders to GLphi. The complementation between nonresponder genomes appeared to be stronger in the cis position than in the trans position for some strain combinations. The failure of strains bearing only one of the two responder alleles to show a T-lymphocyte proliferative response to GLphi, argues strongly that neither gene can be expressed exclusively in B lymphocytes. This conclusion is discussed in relation to another two gene model which has recently been proposed.
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Control of t-lymphocyte and B-lymphocyte activation by two complementing Ir-GLphi immune response genes.
The possibility that the two complementing alpha- and beta-Ir-GLphi genes are independently responsible for controlling events in T lymphocytes and B lymphocytes, respectively, has been tested in double adoptive transfer experiments utilizing cells from appropriate inbred strains of mice. The results of these studies show that the functions of T lymphocytes and B lymphocytes and the cooperative interactions between T and B cells require the presence of both alpha- and beta-genes in each respective cell type. Moreover, evidence has been obtained in these studies that indicates a preference for the alpha- and beta-Ir-GLphi genes in the cis position to obtain the most effective T-B-cell interactions. The possible implications of these findings are discussed.
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Genetic control of the immune response to the radom linear terpolymer of L-glutamic acid, L-lysine and L-leucine (GLleu) by complementing Ir genes.
Three levels of humoral responsiveness (none, high or marginal) were noted following secondary immunization with random linear terpolymer of L-glutamic acid, L-lysine and L-leucine (GLleu). These immune responses were under dual H-2-linked Ir gene control. Gene complementation was observed in appropriate F1 offspring between two nonresponder strains and in selected intra-I region recombinants derived from crossing over between nonresponder parental haplotypes. One of the Ir-GLleu genes, beta, was localized within the I-A or I-B subregions. The other Ir-GLleu locus, termed alpha, was mapped in the I-C or S subregions. Differences in the strain distribution of immune responsiveness to GLleu and related terpolymer GLphe (which is also under complementing Ir gene control) are primarily attributable to differences among the Ir-GLleu and Ir-GLphe beta alleles. A correlation exists between the presence of the Ir-GLleu alpha(¿allele and the gene coding for specificity Ia.7.
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Determinants of antigenic molecules responsible for genetically controlled regulation of immune responses.
The ability of mice bearing the H-2S haplotype to develop helper responses to the random copolymer of Glu,Ala while they developed suppressor responses to the terpolymer of Glu,Ala,Tyr suggested the crucial role of tyrosine in these peptides. On the basis of various considerations, it was postulated that many of the tyrosine residues in Glu,Ala,Tyr would be localized at the NH2-terminal end of the molecule. To verify this hypothesis, a block terpolymer composed of a short sequence of homopolymer tyrosine covalently bound to the random copolymer of Glu,Ala was synthesized. The present studies, using this block terpolymer, demonstrated that the chemical determinants stimulating helper and suppressor responses are distinct and can be present simultaneously in the same molecule. Thus, addition of COOH-terminal tyrosine residues to the Glu,Ala polypeptide converted this immunogenic molecule to an immunosuppressive molecule in mice bearing the H-2S haplotype. The mechanism by which these short sequences of tyrosine influence H-2-linked immune responses remains to be determined.
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Analysis of the cross-reactive immune suppression induced by the random copolymers L-glutamic acid50-L-tyrosine50 (GT), L-glutamic acid60-L-alanine40 and L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).
Cross-reactivity in the induction of immune suppression by the structurally related polymers of L-glutamic acid, L-alanine, and/or L-tyrosine (GAT, GA, GT) was studied. SJL (H-2S) and DBA/1 (H-2q) mice are nonresponders to GAT and GT, whereas SJL, but not DBA/1 responds to GA. Both strains respond to these polymers when complexed to the immunogenic carrier methylated bovine serum albumin (MBSA). Preimmunization with GA suppresses GA-MBSA as well as GAT-MBSA and GT-MBSA plaque-forming cell (PFC) responses in DBA/1 mice. Likewise, GAT suppresses GAT-MBSA and GA (SJL) and GA-MBSA (DBA/1) PFC responses in both strains while being totally ineffective on GT-MBSA responses. GT preimmunization suppressed GA, GAT-MBSA, and GT-MBSA PFC responses in SJL but not DBA/1 responses. This study demonstrates that cross-reactive immune suppression does not follow predictable patterns based upon cross-reactivity of specific antibodies and that cross-suppression is not reciprocal.
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Immunosuppressive factor(s) extracted from lymphoid cells of nonresponder mice primed with L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT).
The synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) not only fails to elicit a GAT-specific antibody response in nonresponder mice, but also prior injection of GAT specifically decreases the ability of nonresponder mice to develop a GAT-specific antibody response to a subsequent challenge with GAT-MBSA. This inhibition is mediated by GAT-specific suppressor T cells. Further, a suppressive factor can be extracted from lymphoid cells of GAT-primed nonresponder mice that inhibits the development of primary GAT-specific antibody responses to GAT-MBSA and to GAT-PRBC- by normal syngeneic mice. The suppressive activity is dose-dependent and absorbed by GAT-Sepharose, but not by BSA-Sepharose. The suppressive activity elutes from a G-100 Sephadex column in the same fraction as ovalbumin, suggesting its m.w. is approximately 45,000 daltons.
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Specific and nonspecific antitumor immunity. III. Specific T lymphocyte-mediated cytolysis of P815 mastocytoma and SL2 lymphoma by draining lymph node cells from syngeneic tumor-bearing DBA/2J mice.
Tumor-specific cytolytic activity, as measured by the 51Cr release assay, has been demonstrated in the draining lymph node cells from DBA/2 mice bearing the syngeneic P815 mastocytoma or SL2 lymphoma. This lytic activity is mediated by cytolytic T lymphocytes (CTL), since cytotoxicity is eliminated by treatment of the effector cells with anti-Thy 1.2 (theta) serum plus complement but is enhanced or unaffected by anti-Thy 1.2 serum alone, antimouse immunoglobulin plus complement, normal or aggregated mouse immunoglobulin, or removal of adherent cells. The time course of the CTL response has been analyzed and is similar for both P815 and SL2, with a peak around Days 10 to 12 after tumor grafting. Detectable CTL activity then wanes despite continued antigenic stimulation from the growing tumor. The ability of the immunotherapeutic agent Corynebacterium parvum to augment such specific CTL responses is documented as one antitumor pathway by which this agent may act.
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Genetic control of specific immune suppression. I. Experimental conditions for the stimulation of suppressor cells by the copolymer L-glutamic acid50-L-tyrosine50 (GT) in nonresponder BALB/c mice.
In the present studies we have confirmed that the random copolymer of L-glutamic acid50-L-tyrosine50 (GT) fails to induce an antibody response in a large number of inbred strains of mice. Nevertheless, GT complexed to methylated bovine serum albumin (MBSA) elicits a GT-specific IgG PFC response in vivo. Furthermore, injection of BALB/c mice with 10 to 100 mug of GT specifically decreases their ability to develop anti-GT PFC responses to a subsequent challenge with GT-MBSA. GT-specific tolerance can be transferred to normal, syngeneic recipients by spleen cells or thymocytes of GT-primed animals. These results indicate that the stimulation of suppressor cells can be observed in nonresponder mice with another synthetic polypeptide besides GAT. Various parameters of GT-specific immunosuppression in BALB/c mice are described. The application of these techniques to the study of the genetic factors controlling the stimulation of specific immune suppression is discussed.
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Genetic control of specific immune suppression. II. H-2-linked dominant genetic control of immune suppression by the random copolymer L-glutamic acid50-L-tyrosine50 (GT).
Several inbred as well as congenic resistant strains of mice, which fail to respond to the random copolymer of L-glutamic acid50-L-tyrosine50 (GT), were shown to develop specific PFC responses when stimulated by GT complexed to an immunogenic carrier such as methylated bovine serum albumin (MBSA). In these studies we have found that GT preimmunization has a tolerogenic effect on the response to GT-MBSA in some mouse strains; whereas in other strains of mice, GT fails to inhibit the GT-MBSA response. We may, therefore, conclude that immune suppression cannot account for nonresponsiveness in all cases. The development of specific immune suppression in response to GT was shown to be inherited as a dominant trait in F1 hybrids resulting from the mating of suppressor with nonsuppressor strains. This trait is, therefore, under the control of a gene or genes that we have designated as specific immune suppression gene(s) Is genes. The strain distribution of GT induced suppression demonstrates that Is genes are coded for in the H-2 complex. Furthermore, immune suppression by the two related copolymers, GT and GAT, are distinct in different strains of mice. The significance of these data for our understanding of the regulation of the immune response is discussed.
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