T cells from patients with chronic liver diseases: abnormalities in PHA-induced expression of HLA class II antigens and in autologous mixed-lymphocyte reactions

The expression of HLA Class II antigens by resting and phytohemagglutinin (PHA)-activated T cells and their functional properties in autologous mixed-lymphocyte reactions (MLR) were investigated in patients with chronic active hepatitis, with alcoholic cirrhosis, and with primary biliary cirrhosis. In all groups of patients the percentage of resting T cells expressing HLA Class II antigens was significantly higher than that in controls. The percentage of T cells which acquired HLA Class II antigens following PHA stimulation was reduced in patients with chronic active hepatitis, serum hepatitis B surface antigen (HBsAg) positive, and in those with alcoholic cirrhosis, HBsAg negative, although the level of [3H] thymidine incorporation was within normal limits. The degree of proliferation in autologous MLR with PHA-T cells was significantly reduced in patients with chronic active hepatitis, HBsAb positive, and in those with alcoholic cirrhosis, HBsAg positive. A reduced proliferation was also detected in autologous MLR with non-T cells, in patients with chronic active hepatitis, HBsAg positive. The abnormalities of autologous MLR are selective, since the proliferative and stimulatory activities of cells from patients with chronic liver diseases in allogeneic MLR were within normal ranges. The immunoregulatory role of HLA Class II antigens and of autologous MLR suggests that the abnormalities we have identified may play a role in the immunological dysfunctions underlying chronic liver diseases.     

PHA-T cells in systemic lupus erythematosus and in rheumatoid arthritis: abnormalities in HLA class II antigen induction and in autologous mixed lymphocyte reactions

Analysis of T cells from patients with systemic lupus erythematosus (SLE) and with rheumatoid arthritis (RA) identified a deficit in the induction of HLA Class II antigens by PHA although the proliferative response was normal and in the [3H]thymidine incorporation in autologous mixed lymphocyte reactions (MLR) with PHA-T cells as stimulators. In RA these abnormalities were more marked in patients with active disease than in those in clinical remission. The deficit of autologous MLR with PHA-T cells was more marked than that of autologous MLR with non-T cells and of allogeneic MLR. Serum from patients with SLE and with RA did not display any detectable inhibitory activity on the induction of HLA Class II antigens by PHA, on the proliferative response of lymphocytes to PHA, on autologous MLR with PHA-T cells and with non-T cells as stimulators and on allogeneic MLR. These results suggest that the abnormalities we have identified reflect an intrinsic defect of T cells.     

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

Major histocompatibility antigens are not detectable on post-meiotic human testicular germ cells

Neither HLA Class I nor Class II transplantation antigens were detected on human testicular germ cells by immunohistologic or immunoprecipitation techniques. This unusual characteristic of human germ-line cells could help to explain their immunologically privileged status in potentially hostile autologous and allogeneic host environments.     

Interferon-gamma regulates the T cell response to precursor nevi and biologically early melanoma

To examine the potential regulatory role of interferon-gamma in the cellular immune response to melanoma and its precursor lesions, we have tested the capacity of this lymphokine to enhance HLA class II antigen-dependent T lymphocyte blastogenesis, its in vitro production by autologous T cells stimulated by melanoma, and its presence in melanocytic lesions in situ. Cell lines derived from a dysplastic nevus, a radial growth phase primary tumor, a vertical growth phase primary, and metastatic lesions were induced by recombinant interferon-gamma to express increased amounts of HLA class II antigens. Such cells were then examined in radioimmunoassay for expression of HLA-DR antigens and in co-culture for their ability to stimulate proliferation of autologous T cells. Interferon-gamma treatment of melanocytic cells increased their expression of HLA-DR antigens threefold to sixfold. In parallel with these findings, co-culture of T cells with interferon-treated cells of a dysplastic nevus and a radial phase melanoma led to augmented T cell incorporation of tritiated thymidine, and this stimulation was inhibited with a monoclonal antibody to HLA-DR antigens. Despite augmented expression of HLA class II antigens (HLA-DR, -DQ, and -DP), vertical growth phase and metastatic melanoma cells failed to stimulate autologous T cells. When T cells were co-cultured with stimulating melanoma cells, culture supernatants contained significantly increased amounts of interferon-gamma (12 U/ml) in comparison with supernatants of T cells alone (4 U/ml). No interferon was detectable in cultures of melanoma cells alone. To link these in vitro phenomena to in situ events, we used murine monoclonal antibodies to interferon-gamma, the interleukin 2 receptor, and HLA-DR antigens in an immunoperoxidase system to detect interferon production and lymphocyte activation in frozen sections of lesions representative of melanocytic tumor progression. In these studies, precursor dysplastic nevi and radial phase melanomas contained the highest numbers of activated lymphocytes and stained positively for interferon-gamma. These results suggest that interferon-gamma plays a central role in the regulation of the cellular immune response to melanoma. It is produced by T cells, likely activated by tumor antigens seen in the context of HLA class II antigens. In turn, interferon-gamma production enhances expression of HLA class II antigens by melanoma and precursor cells, and such enhancement is associated with additional T cell activation in a positive feed-back loop.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Monoclonal antibody OKT3-induced T cell proliferation: differential role of HLA class II determinants expressed by T cells and monocytes.

Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role of the corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CR10-343 and Q5/6 with high inhibitory effect and MAb CR11-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR10-343 and CR12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect of the combination of MAb CR10-343 and Q5/6 and of the MAb CR11-462 and CR12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3-induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli.     

Modulation of the Antigenic Phenotype of Human Melanoma Cells by Differentiation-inducing and Growth-suppressing Agents

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-β), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-β results in an induction and/or increased expression of ICAM-1. HLA Class I antigens and HLA Class II antigens. IFN-β and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-γ), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-β plus IFN-γ which synergistically but reversibly suppresses HO-1 growth. to induce melanin synthesis or terminal differentiation in HO-1 cells. The inhibitor of protein kinase C, H-7, only marginally alters 72 hr growth suppression induced by MEZ or the interferons, used alone or in combination. In several experiments, H-7 only partially and variably inhibited the enhanced expression of ICAM-1, HLA Class I antigens and HLA Class II antigens in HO-1 cells treated with MEZ. IFN-β or IFN-γ, used alone or in various combinations. This model system will be useful in defining the biochemical, genomic and antigenic changes associated with the chemical induction of terminal differentiation and the loss of proliferative capacity in human melanoma cells.     

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Expression of class II antigens by subsets of activated T cells

Gene products coded for within the HLA complex play an important role in the control of immune responses. Class I antigens, coded for by the HLA-A, B, and C loci, are expressed by virtually all mononuclear blood cells. Class II antigens, coded for by the DR, DQ, and DP loci, have a more limited tissue distribution. They are expressed by B cells, monocytes, and by activated, but not by resting, T cells. The class II molecules of B cells and antigen-presenting cells have long been of interest to immunologists, since they are involved in the presentation of antigen, in communication between T cells and B cells and between T cells and adherent cells, and in susceptibility to certain diseases. The class II antigens expressed by activated T cells, however, remain largely uncharacterized in terms of their specificity, functional significance, and molecular nature. We have studied the expression of DR and DQ antigens by activated T cells and then examined the expression of DR versus DQ antigens by Leu 2a and Leu 3a subsets of mitogen-activated populations. Our results demonstrated that, as for class II-positive macrophages, the intensity of staining with monoclonal antibodies directed against DR antigens was much greater than that obtained with those directed against DQ antigens. Interestingly, the percentages of Leu 2a- and Leu 3a-positive cells which expressed DR antigens were quite similar, as were the percentages of Leu 2a and Leu 3a cells which expressed DQ. Thus, there does not seem to be preferential expression of DR versus DQ antigens by mitogen-activated T-cell subsets. Finally, though both DR-positive-DQ-positive and DR-positive-DQ-negative populations were detected, few or no DR-negative-DQ-positive cells were observed in these populations.     

Human myelin basic protein-specific cytolytic T lymphocyte clones are functionally restricted by HLA class II gene products

Cellular immune reactions against the autoantigen myelin basic protein (MBP) are strongly implicated in the occurrence of postinfectious and postvaccination encephalomyelitis. Clinical autoimmune encephalomyelitis in experimental animals can be transferred with cloned MBP-specific cytolytic major histocompatibility complex Class II-restricted T lymphocytes. The HLA restriction pattern of specific proliferative and cytolytic functions of two human MBP-specific cytotoxic T lymphocyte clones, derived from two different multiple sclerosis patients, was analyzed in detail. Using monoclonal antibodies against various HLA gene products and allogeneic Epstein-Barr virus-transformed B cells as antigen-presenting cells and as targets for cytolysis, it was found that MBP-specific functions of the T cell clones was restricted by HLA class II antigens, and, more specifically, by molecules encoded for by DR locus genes.     

Antigenic profile and functional characterization of human peritoneal macrophages

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.     

Depletion of protein kinase C induced by an anti HLA class I monoclonal antibody in phytohemagglutinin activated human T cells

Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the proliferative response of T cells to lectins are discussed.     

Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.