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R R Rich

Publications and source records attributed to R R Rich.

At least 91 records · Page 5Linked to original sources
PubMed1985

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-1-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1a. Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1b- and Qa-1c-encoded determinants. Qa-1d target cells are unique in that they express determinants recognized by anti-Qa-1a CTL and by anti-Qa-1b CTL. Killing of Qa-1d targets by anti-Qa-1a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1d cells by one anti-Qa-1b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1a CTL and Qa-1.1-specific antibodies did not have identical specificities.

Animals↗
PubMed1985

Class II determinants recognized by TNP-specific cloned human T cell lines.

Cloned human T lymphocyte lines were generated against trinitrophenyl (TNP)-modified autologous cells from four different individuals. By examining the reactivity patterns of 58 of these T cell clones (TLC) on panels of HLA-typed antigen presenting cells and employing monoclonal antibodies (MoAb) for inhibition studies, we have demonstrated that TNP may be recognized in the context of different DR and DP associated determinants. We did not identify any TLC restricted by determinants associated uniquely with DQ. Determinants associated with the expression of DR1,2,4,5, and 7 as well as the supertypic specificities DRw52 (MT2) and DRw53 (MT3) functioned as restriction elements. In addition, at least two antigenically distinct regions of the DP4 molecule appeared to function in the presentation of TNP. One TNP-specific TLC recognized a determinant associated with the expression of DP4 while another recognized TNP in the context of a highly nonpolymorphic determinant on DP which was expressed on all stimulators tested. Monoclonal antibody blocking studies suggest that these two determinants lie on different portions of the DP molecule. These studies demonstrated that both polymorphic and relatively nonpolymorphic restriction determinants on DR and DP may function in the presentation of conventional antigen to autologous lymphocytes.

Antibodies, Monoclonal↗
PubMed1985

Class II positive human dermal fibroblasts restimulate cloned allospecific T cells but fail to stimulate primary allogeneic lymphoproliferation.

Recently, a number of laboratories have shown that gamma interferon (IFN-gamma) is a potent modulator of HLA class II antigen expression in a variety of cell types ranging from classical antigen presenting cells to those not expected to participate in physiological antigen presentation such as fibroblasts. In order to examine the role of HLA class II expressing fibroblasts in antigen presentation, we established dermal fibroblast (FIB) strains from five HLA typed donors. After optimal preculture with IFN-gamma, class II positive FIB were fully competent to restimulate proliferative responses of two DR specific T cell clones and one DP specific T cell line. However, they failed to elicit strong primary allogeneic proliferation from fully DR mismatched fresh PBMC. This failure was not due to a direct suppressive effect of FIB and could not be corrected by exogenous IL1 or by factors contained in conventional mixed leukocyte culture supernatants.

Antigen-Presenting Cells↗
PubMed1985

Mitochondrial modulation of maternally transmitted antigen: analysis of cell hybrids.

Maternally transmitted antigen (Mta) is a murine cell surface class I-like antigen that is defined by specific cytotoxic lymphocyte reactivity. Mta is unique in that its expression requires cooperation between genetic elements both in the Qa/Tla region of chromosome 17 and in the cytoplasm. In view of the known cytoplasmic, and thus maternal, inheritance of mitochondria, we have directly assessed their potential involvement in Mta expression. The mitochondria-specific lethal dye rhodamine 6G (R6G) was used to control the input of mitochondria into cell hybrids. The parental lines, one of BALB/c and one of NZB origin, were known to differ in Mta and mtDNA phenotype. Our data show that most control BALB/c-NZB hybrids expressed the BALB/c Mta phenotype and likewise contained only BALB/c-type mtDNA. The NZB Mta phenotype was not coexpressed in the control hybrids. However, when the mitochondrial contribution from BALB/c was prevented by R6G treatment, the majority of the resultant hybrids expressed only the NZB Mta type and likewise contained only NZB mtDNA. The exceptional R6G-treated hybrids that continued to express the BALB/c Mta phenotype likewise contained only BALB/c mtDNA. Thus, in every case the mtDNA phenotype correlated with the Mta phenotype of the cells. Together, the data support the remarkable conclusion that mitochondria modulate the phenotypic expression of a cell surface molecule.

Animals↗
PubMed1985

Multiparameter analysis of human lymphocyte subpopulations using flow cytometry.

This review has emphasized several recent advances in our understanding of human lymphocyte biology made possible by mAbs that recognize specific functional subpopulations of lymphocytes, coupled with multiparameter flow-cytometric analysis. In the last 3 years, both the T4+ and T8+ subsets of T cells have been further divided according to function and appearance of specific cell surface antigens. In addition, subpopulations of NK cells with different maturation states and B cells with distinct functional abilities have been discovered. Multiparameter flow-cytometric analysis of lymphocyte subpopulations can provide clues to the pathogenetic mechanisms of immune disorders, as has been demonstrated in AIDS patients and children with JRA. The use of multiparameter flow-cytometric analysis to study human immune responses in vitro has enabled a clearer understanding of the complex lymphoid interactions and, as we believe, may play an important role in the analysis of immune-associated disease processes.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Polymorphism and tissue distribution of maternally transmitted antigen defined by cytotoxic T lymphocyte lines.

We have developed cytotoxic T lymphocyte (CTL) lines specific for two determinants of the maternally transmitted antigen (Mta) and have used these CTL lines to study the tissue distribution of Mta. In previous reports, we characterized CTL lines specific for the Mta.1 determinant. Here, we describe CTL lines specific for the newly defined Mta.2 determinant. Mta.2-specific CTL lines lysed target cells from F1 mice of an NZB (Mtf beta) mother but did not lyse target cells from reciprocal F1 mice of any Mtf alpha mother. Backcross mice were used as the source of target cells to demonstrate that the Mta.2-specific CTL were H-2 nonrestricted in their recognition. Mta.-1- and Mta.2-specific CTL lines were used to demonstrate Mta expression on lymphoid cells taken from spleen, thymus, lymph nodes, and bone marrow. In addition, Mta was expressed on cultured cell lines of myeloid, epithelial, and mesenchymal origin. Our results suggest that Mta is not a differentiation marker restricted to lymphocyte subpopulations.

Animals↗
PubMed1985

Oligosaccharide-dependent and independent Qa-1 determinants.

A contribution of N-linked oligosaccharides to determinants recognized by alloreactive cytotoxic T lymphocytes has not been demonstrated. Employing cloned CTL and tunicamycin, an inhibitor of protein glycosylation, we found that carbohydrate addition was required for the formation of two of six Qa-1 determinants. The other determinants were detectable on nonglycosylated Qa-1 molecules, similar to observations in most reports that allodeterminants on class I molecules are not dependent on glycosylation for serologic detection. Examination of TM-treated, Con A-activated lymphoblasts revealed a direct correlation between the determinants defined by the reactivity of CTL clones with target cells from four Qa-1 genotypes and their dependence on carbohydrate side chains for expression. Most anti-Qa-1b CTL clones recognized either a glycosylation-dependent determinant found only on Qa-1b cells or glycosylation-independent determinants on both Qa-1b and Qa-1c cells. Similarly, clones that killed only Qa-1a cells recognized a glycosylation-independent determinant. However, clones reactive with both Qa-1a and Qa-1d cells recognized a glycosylation-dependent determinant on Qa-1a molecules and a glycosylation-independent determinant on Qa-1d molecules. This result indicates that such clones recognize cross-reactive conformational determinants, not carbohydrate itself. Thus, N-linked oligosaccharides serve to stabilize the conformation of some Qa-1 determinants, but others remain intact on nonglycosylated molecules. The absence of similar data for H-2K/D/L molecules suggest that a reexamination of other class I antigens with cloned CTL is in order to determine whether Qa-1 molecules are unique.

Animals↗
PubMed1985

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens.

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.

Animals↗
PubMed1985

Differential glycosylation requirements for the cell surface expression of class I molecules.

The importance of asparagine-linked glycosylation in the cell surface expression of several class I molecules was examined. C57BL/6 (B6) T cell blasts were treated with tunicamycin (TM), an antibiotic that inhibits N-linked glycosylation. The levels of various class I molecules on these cells were examined by flow cytometry and were compared to the levels of the same molecules on untreated cells. A 12-hr TM treatment did not significantly alter the levels of H-2Kb, Db, or Qa-2; however, such treatment decreased the surface expression of the Qa-1b allelic product to undetectable levels. A time-course study indicated that a decrease in the level of Qa-1.2 expression was apparent after only 4 hr of TM treatment. An examination of T cell blasts prepared from mouse strains possessing the Qa-1a, Qa-1c, and Qa-1d alleles indicated that all allelic products of this locus demonstrated a marked decrease in cell surface expression on TM treatment, whereas other class I molecules (H-2Ks, TL) exhibited slight or no decrease. Two-dimensional polyacrylamide gel electrophoresis analysis of immunoprecipitates from detergent lysates of surface-iodinated TM-treated B6 blasts revealed the presence of the unglycosylated form of the H-2Kb molecule on the cell surface. No such form of the Qa-1.2 molecule could be detected by similar analysis. To establish that the above observations were not simply a result of the inability of the Qa-1-specific alloantisera to react with the unglycosylated Qa-1 molecule, lysates of surface-iodinated B6 blasts were digested with endoglycosidase F, which cleaves N-linked carbohydrate moieties. Immunoprecipitation analysis indicated that the antisera could react with the unglycosylated form of the Qa-1 molecule. These results indicate that N-linked glycosylation has differential importance in the cell surface expression of class I molecules.

Animals↗
PubMed1985

The Qa-1 alloantigens. III. Biochemical analysis of the structure and extent of polymorphism of the Qa-1 allelic products.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to examine and compare the products of the Qa-1 locus. Analysis of Qa-1 isolated from detergent lysates of surface labeled cells indicated this molecule was a slightly acidic 48,000 to 50,000 dalton glycoprotein that displayed little charge heterogeneity on resting lymphocytes. The level of expression and degree of charge heterogeneity were both increased on activated lymphocytes. Direct comparison of the Qa-1b, Qa-1c, and Qa-1d allelic products by 2-D PAGE revealed that these three molecules could be distinguished from one another on the basis of isoelectric point, indicating that they were distinct at the molecular level. Comparison of Qa-1 isolated from several Qa-1b strains did not detect additional polymorphism. Removal of asparagine-linked oligosaccharides by treatment with endoglycosidase F indicated that carbohydrate contributed 10,000 to 12,000 to the m.w. of these allelic products. Comparative 2-D PAGE analysis could not distinguish between the deglycosylated Qa-1b, Qa-1c, and Qa-1d allelic products, implying that these molecules have similar primary structures. Peptide mapping supported this conclusion. Proteolytic digestion of the deglycosylated Qa-1b and Qa-1c allelic products resulted in identical peptide map patterns; such treatment of the deglycosylated Qa-1d allelic product produced a slightly different pattern. Peptide mapping analysis also demonstrated that the Tlaa and Qa-1a allelic products were distinct from one another, as well as being very different from the other three Qa-1 allelic products.

Alleles↗
PubMed1984

Cloned human T cells synthesize Ia molecules and can function as antigen presenting cells.

TNP-specific proliferative cloned human T cell lines were investigated for their synthesis and cell surface expression of HLA-DR molecules and for their capacity to function as antigen presenting cells. Utilizing radioactive amino acid precursors for metabolic labeling, these studies demonstrated endogenous synthesis of HLA-DR molecules by cloned T cells, which by two-dimensional gel electrophoresis were similar to HLA-DR molecules expressed by B cells and monocytes. Moreover, when TNP-modified, the irradiated cloned T cells functioned very effectively to stimulate TNP-specific proliferation by cloned responders; when unmodified they were potent stimulators of allogeneic mixed leukocyte responses. Thus, for haptens covalently attached to cell membrane proteins and for allogeneic HLA antigens, Ia+ cloned T cells can function as effectively for antigen presentation and T cell activation as other Ia+ populations to which such properties have been ascribed.

Antigen-Presenting Cells↗
PubMed1984

Cellular requirements for induction of human primary proliferative responses to trinitrophenyl-modified cells.

Cellular requirements for induction of primary proliferative responses by human T cells to trinitrophenylated autologous stimulators have been characterized. Substantial proliferative responses were observed with each of the Ia+ stimulator populations tested. Nevertheless, major differences in the hapten specificity of such responses were observed. Thus purified macrophages/monocytes (M phi) when TNP-modified induced responses that were relatively modest in absolute magnitude, but were highly hapten specific. This reflected the very limited capacity of purified M phi to induce proliferation when unmodified, i.e., an autologous mixed leukocyte response (AMLR). In contrast, unmodified M phi-depleted B plus null cells were potent stimulators of AMLR, but hapten modification did not significantly enhance the responses induced by these cells. Moreover, when M phi were added to B plus null cell stimulators AMLR responses were reduced and, with TNP-modified stimulators, hapten-specific responses were restored. The data thus suggest that M phi may have important roles in induction of primary T cell responses to conventional antigens but function largely as regulators rather than stimulators of AMLR. Finally, we have introduced a novel antigen-presenting cell population, the irradiated Ia+ TNP-specific cloned T cell. The possibility that such cells may utilize autostimulatory positive feedback circuits for activation of naive T cells and in interactions between subpopulations of hapten-reactive T cells is discussed.

Clone Cells↗
PubMed1984

Regulatory interactions governing the proliferation of T cell subsets stimulated with pokeweed mitogen.

Serial phenotyping of human peripheral blood mononuclear cells (PBMC) cultured with pokeweed mitogen (PWM) demonstrated an excess of T8+ cells after stimulation. Preferential expansion of the T8+ cell compartment was a result of T8+ cell blast transformation while T4+ cells generated fewer blasts and tended to remain as small resting cells. When the proliferative behavior of T cell subsets in PWM-stimulated PBMC with physiologic proportions of T4+ and T8+ cells was compared with that of cultures depleted of T4+ or T8+ cells, two levels of regulation of proliferation were found: without T4+ cell help, T8+ cells were unable to divide; however, in the presence of T4+ cells, PWM-stimulated T8+ cells became potent feedback inhibitors of T4+ cell proliferation. The mechanism of suppression by PWM-activated T8+ cells of T4+ cell proliferation, not only to PWM, but also to tetanus toxoid, was pursued by measuring decreased interleukin 2 (IL2) recovery from cultures containing suppressors. Although passive absorption of IL2 by PWM-activated cells could contribute to the suppression of fresh proliferative responses, as shown directly with isolated T4+ cells induced by PWM to express IL2 receptors, a much more profound suppression was mediated by PWM-activated T8+ cells. The regulation of proliferative responses of helper and suppressor T cell subsets may determine the magnitude of their subsequent interactions and thus control the ultimate outcome of in vivo physiologic and pathologic immune responses.

Antigens, Differentiation, T-Lymphocyte↗