T cell responses specific for subregions of allogeneic MHC molecules.

The repertoire of C3H (H-2k) CD4+ T cells for I-Ab allopolymorphisms was analyzed by studying the responses of unprimed populations of T cells and of I-Ab-specific T cell clones for recombinant MHC molecules containing combinations of polymorphic subregions of the alpha- and beta-chains from the I-Ab and I-Ak molecules. In this system, polymorphisms in the predicted MHC alpha-helices were more potent than polymorphisms in the beta-strands in stimulating unprimed alloreactive T cells. Similarly, 75% of I-Ab-specific T cell clones responded to recombinants containing b polymorphisms in both the alpha- and beta-chains helices and tolerated the substitution of k polymorphisms in the beta-pleated sheet. Furthermore, 20% of the clones responded to a molecule containing allogeneic b residues in just the beta-chain helix. The results demonstrate that the T cell response to allogeneic MHC molecules consists largely of sets of T cells with overlapping specificities for subregions of the MHC molecule. In addition, they highlight the importance of the alpha-helices in these responses and a diminished role for polymorphisms in the beta-strands when, as in the present case, MHC structure and conformation is tolerant of beta-sheet substitutions. These results sharply contrast with observations made in the analysis of Ag-specific T cells and lead to the suggestion that a subset of alloreactive T cells are not peptide specific and can directly recognize MHC polymorphisms.     

The diversity of T cell receptors specific for self MHC gene products

Cytolytic and helper T cells exhibit, in addition to their specificity for foreign antigen, a restriction specificity for self MHC gene products. The present study was designed to assess the degree of diversity within the repertoire of receptors that are involved in T cell recognition of self MHC gene products. For this purpose, we generated a series of murine cytolytic T lymphocyte (CTL) clones specific for a hapten antigen and restricted to the self MHC gene product H-2Kb. An analysis of the hapten fine specificity of these clones by using hapten analogues revealed the presence of substantial diversity within the repertoire of CTL receptors specific for the hapten. The degree of diversity within the repertoire of self H-2 recognition structures on these clones was assessed by testing clones on panels of syngeneic, congenic H-2K disparate, and H-2Kb mutant target cells bearing varying amounts of antigen. A striking degree of heterogeneity in H-2K recognition was found among these H-2Kb restricted CTL. We estimate that there are probably a minimum of 65 different patterns of H-2K recognition among these clones. Our results suggest a high degree of diversity exists within the repertoire of self MHC recognition structures on antigen-specific T cells restricted to a single self MHC gene product.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Missing HLA class I expression on Daudi cells unveils cytotoxic and proliferative responses of human gammadelta T lymphocytes

The major subset of human blood gammadelta T lymphocytes expresses the variable-region genes Vgamma9 and Vdelta2. These cells recognize non-peptidic phosphoantigens that are present in some microbial extracts, as well as the beta(2)-microglobulin-deficient Burkitt's lymphoma Daudi. Most cytotoxic human Vgamma9/Vdelta2 T cells express inhibitory natural killer cell receptors for HLA class I that downmodulate the responses of the gammadelta T lymphocytes against HLA class I expressing cells. In this study we show that transfection of the human beta(2)-microglobulin cDNA into Daudi cells markedly inhibits the cytotoxic and proliferative responses of human Vgamma9/Vdelta2 T cells. This provides direct evidence that the "innate" specificity of human Vgamma9/Vdelta2 T-lymphocytes for Daudi cells is uncovered by the loss of beta(2)m by Daudi. However, Daudi cells that express HLA class I in association with mouse beta(2)m at the cell surface are recognized by human Vgamma9/Vdelta2 T cells close to the same degree as the parental HLA class I deficient Daudi cell line. Thus, proper conformation of the HLA class I molecules is required for binding to natural killer cell receptors. Cloning of the HLA class I A, B, and C molecules of Daudi cells and transfer of the individual HLA class I molecules of Daudi cells into the HLA class I deficient recipient cell lines.221 and C1R demonstrate that for some human gammadelta T-cell clones cytolysis can be entirely inhibited by single HLA class I alleles while for other clones single HLA class I alleles only partially inhibit cytotoxicity. Thus, most human Vgamma9/Vdelta2 T cells represent a population of killer cells that evolved like NK cells to destroy target cells that have lost expression of individual HLA class I molecules but with a specificity that is determined by the Vgamma9/Vdelta2 TCR.     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

Cell-mediated destruction of human leukemic cells by MHC identical lymphocytes: requirement for a proliferative trigger in vitro.

These experiments have investigated cellular mechanisms involved in the generation of cellular immune responses to human acute leukemic blasts. Because normal human lymphocytes are not able to recognize immunologically, in vitro, lymphocytes from MHC identical siblings, the present studies have examined the in vitro proliferative and cytotoxic responses of normal lymphocytes to MHC identical AML and ALL blasts. In those cases where acute leukemic cells were unable to induce a proliferative response by MHC identical lymphocytes, the generation of effective anti-leukemic cytotoxicity required the addition of unrelated stimulating cells to the sensitization culture. In contrast, leukemic blasts that induced a proliferative response by MHC identical lymphocytes were also able to stimulate anti-leukemic cytotoxicity. This could be augmented by the addition of unrelated stimulating cells to the sensitization culture. The specificity of anti-leukemic cell cytotoxicity was demonstrated in all instances by simultaneous testing of putative killer cells on 51Cr leukemic blasts as well as 51Cr-labeled MHC identical phytohemagglutinin blasts or normal lymphocytes. Simultaneous sensitization to MHC identical leukemic blasts and unrelated stimulating lymphocytes did not invariably generate anti-leukemic cytotoxicity even when allogeneic cytotoxicity was observed; the absence of demonstrable suppressor activity in these nonreactive combinations suggested that some individuals may be specifically immunoincompetent, and thereby unable to generate effective anti-leukemic CML.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

Intrinsic defects explain altered proliferative responses of T lymphocytes and HVS-derived T-cell lines in gastric adenocarcinoma

We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.APV and MP-B should be considered as joint first authors     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

Defective MLR capacitation in the human: Low xenogeneic MLR capacity associated with HLA antigens AW25 B18, and DW2

In tests with 72 human donors, the outcome of the xenogeneic human-mouse MLR was influenced more by the general capacity of the human responder cells to react than by the MHS genotype of the mouse stimulating cells. While the response of the human lymphocytes of the same donor to the lymphocytes of six congenic mouse strains with differentH-2 haplotypes was generally rather similar, there were pronounced differences among individual human donors in the reactivity in these tests. These differences correlated with HLA, since the low reactivity was associated with the presence of the antigens HLA-AW25, HLA-B18, and HLA-DW2. The effect of the presence of antigens AW25, B18 was much more pronounced in males than in females. The low capacity to react in xenogeneic MLR in individuals with antigens HLA-AW25 and HLA-B18 may be the result of the general immune insufficiency associated with these antigens, which also includes C2 defficiency and association with immunopathological diorders.     

Phenotypic analysis and proliferative responses of human endometrial granulated lymphocytes during the menstrual cycle

The in vivo function of the unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in human endometrium is unknown; their increased numbers in the secretory phase of the menstrual cycle suggests that they may play a role in the immunobiology of nonpregnant endometrium. In the present study, the phenotype and proliferative responses of eGLs at various phases of the menstrual cycle were compared with those in early pregnancy. Endometrial GLs were highly purified (> 98% CD56+) using immunomagnetic separation, and the expression of cell surface antigens was examined in smears using a double immunohistochemical labeling technique. Proliferative responses to mitogens and interleukin 2 (IL-2) were assessed in hanging drops in 60-well Terasaki plates. There was low to no expression of CD3, CD8, CD16, HML-1, L-selectin, and CD25 (IL-2 receptor alpha) on CD56+ cells isolated from nonpregnant and pregnant endometrium. The expression of CD2, CD49a, and CD122 (IL-2 receptor beta, IL-2Rbeta), however, increased from the proliferative to the late secretory phase of the menstrual cycle. In contrast, CD11a, CD69, and CD49d expression was high and did not vary with menstrual cycle phase; CD49d levels were significantly reduced in early pregnancy. Unlike early-pregnancy eGLs, none of the CD56+ eGL cultures throughout the menstrual cycle displayed phytohaemagglutinin (PHA)-induced lymphoproliferation. In contrast, eGLs from nonpregnant endometrium in the presence of 5 or 100 U/ml IL-2 after 48- and 120-h incubation showed significant proliferative responses, as did eGL cultures from early pregnancy. A significantly reduced number of proliferative phase eGL cultures proliferated in response to IL-2 compared to secretory phase and early-pregnancy eGL cultures. The IL-2-induced proliferative responses of CD56+ eGLs were associated with increased IL-2Rbeta (CD122) expression. These findings demonstrate 1) differential eGL expression of CD2, CD49a, and CD122 during the menstrual cycle; 2) differential IL-2-induced eGL proliferative responses during the menstrual cycle; and 3) differences between eGLs from nonpregnant and pregnant endometrium in CD49d expression and their ability to respond to PHA.     

Mechanisms that limit the in vitro proliferative potential of human CD8+ T lymphocytes

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16(INK4a) is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16(INK4a) and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16(INK4a) expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16(INK4a) and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16(INK4a) limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.     

Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.