Human suppressor T lymphocyte growth and differentiation.
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Antibodies, Monoclonal↗
Biomedical subjects
R R Rich
Publications and source records attributed to R R Rich.
Induction of suppressor T cells by cytokines.
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Biological Factors↗
T suppressor cell growth factor and anti-CD3 antibodies stimulate reciprocal subsets of T lymphocytes.
Because of the central role of IL-2 in clonal expansion of T cells, we have postulated that lymphocyte subpopulations with opposing regulatory functions might be independently regulated by differential requirements for expression of cell-surface IL-2-R. Purified CD4+ and CD8+ cells proliferated in an IL-2-dependent manner to crosslinked anti-T cell receptor antibodies (anti-CD3-Seph). Similarly, both CD4+ and CD8+ cells became IL-2 responsive after incubation in T suppressor cell growth factor (TsGF), a newly described approximately 8,000 Mr product of activated CD4+ cells. In support of our hypothesis, however, we observed that subpopulations of CD4+ and CD8+ cells, possessing distinct cell-surface antigens, showed differential responses to these stimuli. Those cells of suppressor-inducer or suppressor-effector phenotype failed to proliferate when cultured in anti-CD3-Seph plus IL-2, but did proliferate in an IL-2-dependent manner to TsGF. Furthermore, the suppressor-effector population was unresponsive to TsGF plus IL-2 when cocultured in anti-CD3-Seph, suggesting that functionally induced Ts may be refractory to growth stimuli. Conversely, cells with helper-inducer or cytolytic phenotype proliferated when incubated in anti-CD3-Seph and IL-2, while remaining essentially unresponsive to TsGF and IL-2. The results could not be explained by differences in the level of CD3 expression by the T cell subsets. Thus, cells within the helper and suppressor lineages appear to have distinct and reciprocal patterns for the induction of IL-2 responsiveness.
Antigens, Differentiation, T-Lymphocyte↗
Sequential effects of prostaglandins and interferon-gamma on differentiation of CD8+ suppressor cells.
We have previously demonstrated that differentiation of CD8+ Tp44- suppressor cells in pokeweed mitogen (PWM)-stimulated cultures requires soluble factors elaborated by CD4+ cells and monocytes, and that the monocyte signal for such differentiation can be replaced by prostaglandin E2 (PGE2). In this study, we explored the ability of interleukin 2 (IL 2) and interferon-gamma (IFN-gamma) to replace the CD4+ signal. When IL 2 or IFN-gamma was used at concentrations equivalent to those present in supernatants of PWM-pulsed cultures of CD4+ cells, no effect on differentiation of CD8+ cells was observed. However, a potent suppressor inducing activity was detected when IFN-gamma, but not IL 2, was mixed with supernatants derived from cultures of PWM-pulsed purified monocytes (M phi sup) or with 10(-8) M PGE2. Differentiated CD8+ suppressor cells (Ts) inhibited both PWM-stimulated proliferative response of CD4+ cells and immunoglobulin production by B cells. The signals mediated by the M phi sup or PGE2 and IFN-gamma were shown to act sequentially. That is, M phi sup or PGE2 was required initially, followed by an IFN-gamma-dependent differentiative step. These studies thus suggest a cascade of cellular interactions involving monocytes, CD4+ cells, and CD8+ Ts precursors that are required for the differentiation of CD8+ suppressor effector cells.
Antibody Formation↗
Differential presentation of HLA-DR, DQ, and DP restriction elements by interferon-gamma-treated dermal fibroblasts.
IFN-gamma has been reported to induce expression of HLA class II (DR, DQ, DP) antigens on cultured human dermal fibroblasts (FB) by stimulating the de novo transcription of the alpha and beta chain genes of HLA-DR, -DQ, and -DP in these cells. We examined the relative nominal and alloantigen-presentation capacity of each HLA class II gene product on FB by using CD4-positive, TNP-specific T cell clones restricted by determinants on DR, DQ, or DP molecules, as well as allospecific, CD4-positive T cell clones recognizing DR-, DQ-, or DP-lymphocyte activating determinants. After IFN-gamma exposure, FB strains used for antigen presentation displayed a high percentage of DR-positive cells and a much smaller percentage of DP-positive cells, but no detectable DQ-positive cells by immunofluorescent techniques. FB stimulator cells supported proliferative responses of two DR-allospecific T cell clones and one TNP-specific, DR-restricted clone, but not another TNP-specific, DR-restricted clone. Despite only modest DP expression, FB stimulated both a TNP-specific, DP-restricted clone and a DP-allospecific T cell line. However, IFN-gamma treated FB failed to stimulate a TNP-specific, DQ-restricted clone and a DQ-allospecific clone. Our data indicate that IFN-gamma differentially regulates expression of functional class II lymphocyte activating determinants on FB antigen-presenting cells and that FB may fail to support DQ-directed T cell responses due to insufficient expression of DQ molecules on the FB cell surface. However, the quantity of DR or DP expressed on FB did not directly correlate with their ability to support T cell responses, indicating that additional factors, such as differences in T cell clone activation requirements, contribute to the capacity of FB to present class II allo- and antigen-restricting epitopes.
Antigen-Presenting Cells↗
Differential antigen-presenting cell requirements of hapten-specific T cell lines restricted to class II determinants.
We used a panel of class II-restricted T cell lines (TCL), generated against trinitrophenyl (TNP)-modified autologous peripheral blood mononuclear cells (PBMC), to examine the antigen-presenting functions of various PBMC-derived class II-positive cell types, including adherent cells, B + null cells, and activated T cells. However, activated T cells and transformed or activated B cells differed in their ability to present TNP to the TCL; TNP-modified activated lymphocytes stimulated only a subset of the class II-restricted TCL that responded to class II-positive resting cells. Moreover, certain antigen-specific TCL distinguished between antigen presented on activated T cells and transformed B cells. The differences in stimulatory capacity for particular TCL did not appear to reflect differences in the expression of class II molecules or in the ability of these cells to deliver hormonal signals or process antigen. Instead, the data suggest that differences in the ability of the cells to recognize antigen on the surface of different class II-positive cells may be a function of a secondary cell surface interaction.
Antigen-Presenting Cells↗
Maternally transmitted antigens are codominantly expressed by mouse cells containing two kinds of mitochondrial DNA.
Mtf, a cytoplasmic, probably mitochondrial factor, controls Mta polymorphism. We tested for dominance between two forms of Mtf to determine whether Mta is controlled by positive or negative genetic mechanisms. We fused Mtf-disparate cells containing distinct mtDNA markers and selected for hybrids containing both. Such mtDNA heteroplasmons codominantly and stably express alternative Mta antigens. Stable codominance excludes negative genetic mechanisms as well as a model of induced nuclear compensation, and implies Mtf controls Mta expression through a positive genetic mechanism.
Animals↗
Trinitrophenyl reactive T-cell clones in functional and molecular analysis of the HLA-D region.
We believe that the approach we have utilized contributes to more complete understanding of the role of HLA class II molecules in presentation of antigen to T lymphocytes and helps to resolve paradoxes presented by earlier studies in which class II-associated restrictions were not apparent. The approach has depended upon development of a library of cloned TCL reactive to a particular haptenic determinant in various class II MHC contexts. It employed initial screening for MHC restriction by analysis on allogeneic stimulator cell panels that could be confirmed or clarified by antibody blocking experiments employing mAb of defined specificities. This has led to clear identification of TCL restricted to recognition of TNP in the context of each of the major class II molecular species, DR, DQ and DP. It has also raised the intriguing and novel possibility that in some instances, at least for DP, T-cells may employ restricting elements, as revealed by mAb blocking, that are either nonpolymorphic or of very low polymorphism. Finally, where important functional polymorphisms have been identified at apparent variance from defined serological specificities, it has been possible to correlate antigen recognition with molecular polymorphisms defined at both the protein level, by amino acid sequencing, and the genomic level by restriction enzyme polymorphisms. As these approaches are combined with new molecular genetic techniques for generation of novel class II constructs, it seems likely that important, but as yet unresolved, questions relating to the nature of the ternary complex involving antigen, MHC molecule and T-cell receptor will be resolved.
Epitopes↗
Unglycosylated Mtaa expresses an Mtab-like determinant.
Antigenic polymorphism of the class I-like maternally transmitted antigen (Mta) is controlled by a maternally transmitted factor (Mtf) thought to reside in mitochondria. However, the mechanisms by which Mtf generates antigenic polymorphism are not known. To address this issue, we investigated a possible role of posttranslational oligosaccharide addition in the formation of Mta determinants. We examined the expression of Mta on cytotoxic T lymphocyte (CTL) target cells cultured in tunicamycin (TM), a known inhibitor of asparagine(N)-linked glycosylation. Of 18 Mtab-specific CTL lines, 8 lysed TM-treated Mtaa targets. Furthermore, a subclone of one of these eight lines, 17D5.G2, lysed TM-treated targets from all Mtaa strains tested, regardless of H-2K/D haplotype. On the other hand, this CTL clone did not lyse TM-treated target cells from the Mta null, but H-2 expressing strain B10.CAS2. Therefore expression of this Mtab-like determinant is concordant with the expression of Mtaa and seems unlikely to represent a cross-reactive H-2K/D epitope. Our data suggest that an Mtab-like determinant is expressed on unglycosylated Mtaa molecules. Thus, N-linked oligosaccharides probably prevent the expression of an Mtab-like determinant on the Mtaa molecule. We discuss how Mtf may contribute to Mta polymorphism through glycosylation.
Animals↗
Defective antigen presentation and novel structural properties of DR1 from an HLA haplotype associated with 21-hydroxylase deficiency.
We have segregated DR1+ individuals into two categories according to whether or not their class II+ cells stimulated T lymphocyte clones specific for or restricted to DR1. In a majority of cases (87%), failure to stimulate was a property of cells having the B14;DR1 haplotype and/or nonclassical 21-hydroxylase deficiency. Absence of clonal proliferation could not be explained by release of an intercellular suppressor factor or by stimulator cell absorption of interleukin 2. Homozygous cells inheriting both stimulatory (DR1n) and nonstimulatory (DR1x) haplotypes did not successfully mediate clonal expansion, implying that a trans acting factor operates intracellularly to modify both DR1 alleles or their products. Other DR alleles did not appear to be affected as evidence by normal proliferative responses of T lymphocyte clones restricted to DR2 or DR7 and stimulated by DR1x,2 and DR1x,7 cells, respectively. By two-dimensional gel analysis, we have further identified a 50-kD surface glycoprotein contained in anti-DR immunoprecipitates of DR1x, but not DR1n or non-DR1 cellular lysates. This 50-kD structure had antigenic and peptide identity to DR alpha and beta chains but was resistant to dissociation under conditions that normally separate DR alpha and beta (8 M urea plus 5% 2-mercaptoethanol); boiling in sodium dodecyl sulfate was required to segregate the component polypeptides of the 50-kD heterodimer. We postulate that a product of a novel combinatorial association between constitutive chains of DR may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and a restricting element. We further propose that gene abnormalities within the class III region of a haplotype associated with nonclassical 21-hydroxylase deficiency may extend into the DR subregion of the major histocompatibility complex with consequent aberrations in DR1 presentation.
Adrenal Hyperplasia, Congenital↗
Unusual functional and biochemical properties of DR molecules from the HLA-DR1, B14 haplotype.
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Cell Line↗
Opposing immunoregulatory functions of CD8+ lymphocytes: a requirement for monocytes in suppressor cell induction.
We have found that the induction of suppressor T cell (Ts) function in pokeweed mitogen (PWM)-stimulated cultures of peripheral blood mononuclear cels (PBMC) depends on the concentration of monocytes in inductive cultures. When cultured for 7 to 8 days in a physiologic concentration of monocytes (10 to 15%) suppressor cells differentiated with the surface phenotype CD8+ DR+ Leu-7- Leu-8+ Tp44-. Suppressor function, as assessed in secondary cultures of fresh PWM-stimulated CD4+ cells, was partially radiosensitive, and its induction could be partially inhibited by treatment with indomethacin. Culture supernatants of PWM-pulsed CD4+ cells could induce CD8+ suppressor cells only when monocytes were included in cultures for supernatant production. In marked contrast, if the monocyte concentration of PWM-stimulated T cells was reduced to less than 5%, CD8+ cells harvested from such cultures substantially augmented proliferation of PWM-stimulated CD4+ cells in assay cultures. Amplifier cells from monocyte-depleted cultures, were also DR+Tp44-. Suppressive activity of the CD8+ subset was restored simply by addition of monocytes to purified T cells, reconstituting the concentration present among unfractionated PBMC. Addition of IL 1 could not replace the requirement of monocytes for suppressor cell induction either in PWM-stimulated primary cultures or in culture supernatants. The data thus demonstrate that the regulatory activity of CD8+ lymphocytes is critically dependent on the conditions of cellular activation; at concentrations of monocytes normally present in peripheral blood, PWM stimulation induced potent suppressor activity, whereas under conditions of moderate monocyte depletion the regulatory activity of the same phenotypic subset of CD8+ cells was reversed. The experiments thus suggest that the functional consequences of an activating stimulus may depend on regulatory effects of monocytes in CD8+ suppressor cell induction.
Antigens, Surface↗
Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells.
Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.
Animals↗
Molecular characterization of a subtype of DQw1 recognized by hapten-specific T cells.
Previous studies of HLA-restricted antigen recognition by cloned T cells have frequently demonstrated reactivity that did not correlate precisely with the expression of serologically defined HLA specificities. To further explore such discrepancies, we utilized monoclonal antibody (MoAb) blocking, partial NH2-terminal amino acid sequencing, and Southern blot hybridization techniques to analyze the fine specificity of four autologous trinitrophenyl-specific T cell lines restricted to DR2-linked epitopes. MoAb blocking studies demonstrated that two of these lines recognized determinants on DR molecules while the other two recognized determinants on the same molecule that expresses the DQw1 determinant. However, these latter two lines appeared to recognize a DQw1-related determinant found primarily in association with DR2, but not the other DQw1-associated DR alleles, DR1 and DRw6. To ascertain whether these lines were defining a functional split of DQw1, we performed partial NH2-terminal amino acid sequencing of the molecules precipitated with a DQw1-specific MoAb (Genox 3.53) from different stimulator lines. The results showed that these T cell lines recognized a subtype of DQw1 that is in linkage disequilibrium with DR2. Moreover, we identified characteristic restriction fragment length polymorphisms with a DQ beta-specific cDNA that correlated with stimulatory capacity for the DQw1-restricted lines. These results demonstrate that: DQ molecules may provide restriction determinants that are incorrectly assigned to DR molecules on stimulator panel analyses; cloned antigen-specific T cell lines recognize polymorphic regions of class II molecules not distinguished by either conventional typing antisera or xenogeneic MoAb; and the DQw1 epitope(s) is located on a heterogeneous group of DQ molecules that differ from each other in the primary sequence of their beta chains.
Alleles↗
Human suppressor T cells: induction, differentiation, and regulatory functions.
T-cell-mediated suppression of human immune responses involves a complex interaction between distinct lymphocyte subsets with suppressor-inducer and suppressor-effector functions. Recent studies with subset-specific monoclonal antibodies have defined a characteristic phenotype of suppressor-inducer cells (CD4+ Leu8+ 2H4+ 4B4-) that can be distinguished from that of helper cells for antibody synthesis (CD4+ Leu8- 2H4- 4B4+). Similarly, suppressor-effector cells (CD8+CD11+Tp44-) can typically be defined as a subset separable from cytotoxic T cells (CD8+CD11-Tp44+). Both antigen-specific and nonspecific interactions are important in suppressor T-cell activation and function. Soluble signals required for differentiation of CD8+ suppressor cells include an indomethacin-sensitive monocyte product and interferon gamma. In contrast, proliferation of the CD8+ suppressor cell subset depends on stimulation first by a product of CD4+Leu8+ cells, T suppressor cell growth factor, and second by interleukin 2. Although the molecular basis of antigen-specific interactions between CD4+ and CD8+ cells in suppressor cell generation has not been defined, it may involve both conventional, presumably MHC-restricted, interactions between antigen and antigen receptors, as well as anti-idiotypic interactions of suppressor-effectors with determinants on suppressor-inducer receptors. Progress in elucidating requirements for activation, growth, and differentiation of suppressor cells should facilitate long-term culture of such cells and lead to clearer understanding of mechanisms of suppressor-cell mediated immunoregulation.
Antibodies, Monoclonal↗
Maternally transmitted antigen.
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Animals↗
Proliferative signals for suppressor T cells. Helper cells stimulated with pokeweed mitogen in vitro produce a suppressor cell growth factor.
To define molecular signals elaborated by inducer populations supporting growth or differentiation of T8+ cells, we collected supernatants of pokeweed mitogen (PWM)-stimulated cultures depleted of T8+ cells. When added to purified T8+ cells, these supernatants caused significant proliferation. PWM plus interleukin 2 (IL-2) in amounts equivalent to those in the supernatant could not reconstitute the response caused by the supernatant. T8+ cells activated by supernatants obtained from PWM-pulsed T4+ cells suppressed fresh PWM cultures. Although exhibiting little proliferation, T8+ cells cultured for 6 d in PWM plus IL-2 still suppressed a fresh PWM response. The supernatants therefore contain an additional T suppressor cell growth factor (TsGF). Elaboration of TsGF required radiosensitive T4+Leu8+ cells. Molecular weight determination by high performance liquid chromatography gave a single peak of TsGF activity at approximately 8,000. Finally, whereas TsGF in the absence of IL-2 could not support the proliferation of T suppressor cells, it did cause T8+ cells to become strongly IL-2 receptor-positive.
Antibodies, Monoclonal↗
Clonal analysis of the anti-Qa-1 cytotoxic T lymphocyte repertoire: definition of the Qa-1d and Qa-1c alloantigens and cross-reactivity with H-2.
To characterize the four common Qa-1 allelic products, we examined in detail the CTL-defined determinants encoded by Qa-1. In previous studies with anti-Qa-1 CTL and alloantisera, investigators have described antigenic determinants present on Qa-1a and Qa-1b antigens, but they have defined Qa-1c and Qa-1d exclusively by their cross-reactivity with Qa-1a and/or Qa-1b determinants. To delineate further the CTL-defined determinants encoded by Qa-1d, we generated CTL clones with Qa-1d specificity and demonstrated that the Qa-1d molecule expressed determinants that were not detected on Qa-1a, Qa-1b, or Qa-1c target cells. Other CTL clones derived from anti-Qa-1d MLC recognized new antigenic determinants on Qa-1c that cross-reacted with Qa-1d. Each of the four common Qa-1 phenotypes was shown to exhibit unique antigenic determinants. In addition, Qa-1d anti-Qa-1a and Qa-1d anti-Qa-1b CTL confirmed extensive cross-reactivity among these Qa-1 alloantigens. Analysis of CTL from these four immunizations also resulted in the isolation of Qa-1a-specific and Qa-1d-specific CTL clones that cross-reacted with H-2Df and H-2Ks, respectively.
Animals↗