Development of an antigen-specific CD8 suppressor effector clone in man

A long-term cultured IL-2-dependent keyhole limpet hemocyanin (KLH)-specific CD8 (T8) suppressor clone (5B9) was generated from a healthy donor hyperimmunized with KLH. The 5B9 clonal population suppressed in vitro anti-KLH antibody response but did not suppress anti-TT antibody response or PWM-driven IgG synthesis. Moreover, 5B9 cells could not suppress anti-TT antibody response even in the presence of free KLH. 5B9 cloned cells suppressed the anti-KLH antibody response of B cells cultured with CD4+4B4+ cells without requiring the presence of CD8+ cells. This KLH-specific CD8 suppressor clone is an effector type rather than an inducer type of suppressor T cell. The cloned cells expressed alpha- and beta-TCR proteins (defined by WT-31 antibody) on their cell surface. More importantly, the CD3:TCR complex was functionally important in the suppression induced by this clone, because after CD3 antigen modulation from its cell surface, the suppressor effector function was abolished.     

Antigen-specific human B-cell responses: modulation by immunoregulatory T-cell subsets

In vitro T-cell requirements for and modulation of human B-cell responses were studied in individuals immunized in vivo to the protein antigen keyhole limpet hemocyanin or tetanus toxoid. T cells were required for antibody synthesis in both antigen-driven and pokeweed mitogen (PWM)-driven cultures. T cells were separated into T4+ and T8+ subpopulations using monoclonal antibodies, and their modulation of antibody synthesis was studied. T4+ cells functioned as helper cells in both antigen-driven and PWM-driven cultures in a dose-dependent manner. Whereas T8+ cells suppress both total and specific immunoglobulin secretion in PWM-stimulated cultures, in antigen-stimulated cultures T8+ cells do not suppress unless activated by another cell population present in peripheral blood mononuclear cells (PBMNC). This cellular requirement was further investigated by prestimulation of cells prior to addition to optimally stimulated antigen-driven cultures of PBMNC or B cells, monocytes, and helper T cells. No suppression of these optimally stimulated cultures was seen when T8+ cells were precultured with antigen or PWM. However, after 3-5 days preculture of total T cells with PWM or antigen and then selection of T4+ cells, these cells were able to induce fresh autologous T8+ cells to suppress optimally stimulated antigen-driven cultures. Addition of a precultured mixture of T8+ cells with 20% T4+ cells also resulted in antigen-induced suppression. These data indicate that T8+ cells can suppress antigen-driven cultures but require the presence of preactivated T4+ cells for induction of this suppression of antigen-specific T-cell-dependent human B-cell responses.     

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.     

Cell-marker studies in CLL with monoclonal OKT antisera and lactic dehydrogenase isoenzyme analysis

In 16 patients with B-type CLL, the T and B cell compartment was analysed using the monoclonal anti-T-cell antisera OKT1, 3, 4 and 8 and the lactic dehydrogenase enzyme marker. Pertinent findings were: the presence on the CLL cells in 15 out of 16 patients of the antigen determined by the OKT1 antiserum, and, in all patients, a lower total LDH content on a per cell basis combined with a higher percentage-activity in the LDH-3 band as compared to the normal B-cell. Furthermore, the T-cell compartment was also disturbed in these patients, as the ratio of OKT4+ to OKT8+ cells was significantly depressed accompanied by a significant decrease of the LDH-1 percentage-activity in favour of LDH-3 and 4. These findings argue for the B-cell being immature and confirm the recent evidence that the T-cell compartment is changed in B-CLL.     

Efficient induction of peptide-specific cytotoxic T lymphocytes by LPS-activated spleen cells

Lipopolysaccharides of gram-negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages. We have investigated the use of LPS-activated spleen cells as antigen-presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides. Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS-activated spleen cells. Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells. The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B-cell blasts were largely responsible. This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T-cell repertoire contains reactive T cells.     

The helper and suppressor functions of primate T cells elicited by a 185K streptococcal antigen, as compared with the helper function elicited by a 4K streptococcal antigen

Helper and suppressor functions of human T lymphocytes that act on antibody-forming B cells were elicited by a large 185K streptococcal cell wall antigen. However, a small 4K streptococcal peptide elicited helper but no suppressor function. These differences in the functional activities of the large and small m.w. streptococcal antigens (SA) were confirmed by direct immunisation of rhesus monkeys with the 185K-SA and 4K-SA. Sequential studies have shown that whereas the 185K-SA elicits dose-dependent helper and suppressor activities, the 4K-SA elicits only helper function. Cell-depletion studies with human cells suggest that removal of T8+ cells by killing with OK.T8 and complement leads to a loss of suppressor and a broadening in the concentration of 185K-SA, which elicits helper activity. Because the 4K-SA does not elicit suppression, removal of T8+ cells does not affect this function. However, similar depletion of T4+ cells results in loss of the helper activities, both with the 185K-SA and 4K-SA, and again a broadening in the concentration of the 185K-SA, which elicits suppression. Direct comparison by autoradiography between 125I-labeled 185K-SA and 4K-SA suggests that both antigens can bind directly to monocytes or T8+ VV+ cells. Furthermore, both antigens can induce helper function if T4+ cells are reconstituted with either monocytes or T8+ VV+ cells. Attempts will now be made to sequence the amino acid determinants of the 185K-SA, so as to define the epitopes responsible for the two major regulating functions elicited by this antigen.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.     

Role of monocytes in pokeweed mitogen-induced differentiation of human peripheral blood lymphocytes

Separate stimulation (“pulsing”) method of different cell populations with pokeweed mitogen (PWM) was used to study the regulatory role of monocytes in the PWM-induced plaque-forming cell response of human peripheral blood lymphocytes. T cells, B cells, and monocytes were separated, pulse-stimulated with PWM, extensively washed, and cocultured with unstimulated cell populations without additional PWM. Pulse-stimulated T cells helped unstimulated B cells to differentiate into immunoglobulin-secreting cells. This generation of helper T cells by PWM-pulsing was enhanced by monocytes in the presence of free PWM, as well as by PWM-pulsed monocytes in the absence of free PWM. A coculture of pulse-stimulated B cells and unstimulated T cells produced more substantial B-cell differentiation than the coculture of stimulated T cells and unstimulated B cells. Further enhancement of the latter response was obtained when B cells were pulse-stimulated in the presence of monocytes. However, pulse-stimulated B cells did not differentiate in the absence of T cells, and monocytes were unable to replace this T-cell function. It appears that there are several pathways by which PWM induces B-cell differentiation and in each, monocytes play an enhancing role.     

Induction of suppressor cells to T- and B-cell proliferative responses and immunoglobulin production by monoclonal antibodies recognizing the CD3 T-cell differentiation antigen

Monoclonal antibodies (mAb's) recognizing the CD3 T-cell differentiation antigen induced the generation of suppressor cells. These cells inhibited (1) proliferative responses of human peripheral blood mononuclear cells (PBMC) to PHA and allogeneic cells in mixed leukocyte culture; (2) proliferative responses of purified E-rosette-negative cells to Staphylococcus aureus Cowans I; and (3) de novo immunoglobulin synthesis and secretion in the pokeweed mitogen (PWM)-induced differentiation system. Monoclonal antibodies recognizing other T-cell differentiation antigens (anti-Leu 2a, anti-Leu 3a, and anti-Leu 5) did not induce the generation of suppressor cells, even at very high antibody concentrations. Statistically significant differences were not observed in the ability of the OKT3 and anti-Leu 4 mAb's to induce suppressor cells. Monocytes were not required for the generation of anti-CD3-induced suppressor cells. F(ab')2 fragments of the OKT3 mAb's were equally effective when compared with intact antibody molecules in inducing suppressor cells, although they did not induce proliferative responses. Proliferation was not required for the induction of suppressor cells. Irradiation (2500 rad) of PBMC before incubation with the anti-CD3 mAb did not affect the generation of suppressor cells. Furthermore, anti-CD3-induced suppressor cells were radioresistant. Addition of recombinant IL-2 to the cultures of responding cells and suppressor cells did not reverse the suppression. In vitro treatment of anti-CD3-induced suppressor cells with either the OKT4 mAb plus complement or the OKT8 mAb plus complement partially decreased the suppression of proliferative responses of PBMC to PHA or allogeneic cells in mixed lymphocytes culture. However, treatment with both OKT4 and OKT8 mAb's plus complement or the OKT11 mAb plus complement completely abolished the suppression. These results suggest that the suppressor cells are of the T11+T4+T8- and T11+T4-T8+ phenotypes. In other experiments, T4+T8- and T8+T4- cells were isolated from PBMC treated for 48 hr with anti-CD3 mAbs. Both these two populations significantly inhibited proliferative responses of autologous PBMC to PHA and de novo immunoglobulin synthesis and secretion by mixtures of purified T4 and B cells from normal donors, in the PWM-induced differentiation system. These results demonstrate that anti-CD3-induced suppressor cells are of the T4 or T8 phenotype. Treatment of purified T4+T8- and T8+T4- cells with anti-CD3 mAb's resulted in the generation of suppressor cells, suggesting that the precursors of the anti-CD3-induced suppressor cells can belong to either of these two populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Effect of antigen priming on T-cell suppression. I. Activity of Ly 1+2+ feedback suppressor T-cell precursors after isolation from competing Ly 2-T cells

Previous experiments have demonstrated that feedback suppression of murine antibody responses occurs in vitro after exposure of unprimed T-cell subsets to suppression-inducing signals from primed cells, resulting in suppression of primary and secondary IgM as well as IgG anti-SRBC responses. However, following priming with antigen when cells appear which are capable of inducing feedback suppression, the ability of unfractionated splenic T-cell populations to mediate detectable feedback suppression in vitro rapidly disappears, suggesting that priming alters the expression of feedback suppression at the same time as providing for its induction. In the present study, we have succeeded in isolating active feedback suppressor T-cell precursors (preTs) in the Ly 1+2+ and L3T4- T-cell populations from SRBC-primed as well as from unprimed mice, demonstrating that preTs are not lost after priming. The preTs isolated from primed mice resemble those isolated from unprimed mice in Ly and L3T4 phenotype, cell dose requirements, kinetics, level of suppression, and requirement for in vitro activation by primed cells. These results imply that antigen priming neither significantly depresses nor enhances the ability of Ly 1+2+ preTs to participate in feedback suppression and that activated suppressor effector cells are not detectable in the Ly 1+2+ splenic T-cell subset. Priming does, however, induce an enhancing activity in Ly 2-, L3T4+ T cells which appears to compete with feedback suppression and thus may account for the absence of detectable feedback suppression when unfractionated T cells from primed mice are the only source of preTs.     

Cellular immune response to hog cholera virus (HCV): T cells of immune pigs proliferate in vitro upon stimulation with live HCV, but the E1 envelope glycoprotein is not a major T-cell antigen.

T-cell responses of pigs to hog cholera virus (HCV) have reportedly been absent or difficult to detect. Therefore, little is known about cellular immunity to HCV. In this study, we used an attenuated strain of pseudorabies virus expressing the envelope glycoprotein E1 of HCV and purified recombinant E1 to examine whether the E1 protein is a target antigen recognized by the T cells of HCV-immune pigs. We were unable to identify the E1 protein as a major target antigen recognized by the T cells of HCV-immune animals. However, such cells proliferated in vitro upon stimulation with viable HCV antigen. The lymphoproliferative response to HCV was strictly time and dose dependent and could be induced upon stimulation by live but not by UV light-inactivated HCV. Depletion studies demonstrated that lymphoproliferation depended on the presence of CD2+CD8bright+ lymphocytes, but CD2+CD4+ cells also contributed to the lymphoproliferative response. The primary lymphoproliferative response in animals inoculated with 10(7) 50% tissue culture infective doses of strain Brescia 2.1.1 was stronger than that observed in animals inoculated with 10(3) 50% tissue culture infective doses of the Cedipest strain. A remarkable finding was the increase in non-antigen-specific lymphoproliferation upon inoculation of the animals with HCV strains. This immunological phenomenon may mask a specific T-cell response to the virus.     

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Presence of suppressor HIV-specific CD8+ T cells is associated with increased PD-1 expression on effector CD8+ T cells

Mechanisms leading to the observed immune dysregulation in HIV-1 infection are not well understood. HIV-specific IL-10-positive CD8(+) T cells are increased in advanced HIV disease. We have previously reported that Gag-specific IL-10-positive CD8(+) T cells suppressed cytolysis. In this study we describe the suppressive effect of Nef-specific IL-10-positive CD8(+) T cells. Interestingly, simultaneous removal of both Gag- and Nef-specific IL-10-positive CD8(+) T cells led to higher HIV-specific cytolysis compared with the removal of Nef-specific IL-10-positive CD8(+) T cells alone. We also examined the level of programmed cell death-1 (PD-1) as a measure of immune dysfunction in association with IL-10-positive suppressor CD8(+) T cells. The level of PD-1 expression on CD107-positive effector CD8(+) T cells was significantly increased when IL-10-positive suppressor CD8(+) T cells were present (p < 0.05). Our results suggest that IL-10-positive suppressor CD8(+) T cells contribute to the immune dysfunction observed in advanced HIV infection and that the concomitant presence of multiple IL-10-positive CD8(+) T cell populations may have an additive suppressive effect.     

T cell regulation of polyclonally induced immunoglobulin secretion in humans

We measured the pokeweed mitogen (PWM)-induced secretion of IgG by the unfractionated mononuclear cells (MNC) of young adult donors, and correlated the results with the functional activity of cell suspensions enriched for T helper (T4+) and T suppressor/cytotoxic (T8+) cells. The distribution of IgG levels secreted by MNC differs from a Gaussian curve, implying that the group is composed of distinct heterogeneous populations. When donors were compared who were judged to be very low responders or very high responders on the basis of IgG secretion levels by MNC (less than 700 ng/ml or greater than 2500 ng/ml), no differences were found in the capacity of T4+-enriched cells to support PWM-driven IgG secretion by a common B cell pool. In contrast, the addition of 0.2 X 10(5) T8+ cells from these low responders to PWM-stimulated cultures of 0.5 X 10(5) T4+ cells plus 0.5 X 10(5) B cells resulted in significantly less IgG secretion (389 +/- 121 ng/ml) than did the addition of the same number of T8+ cells from the high responders (2241 +/- 548 ng/ml, p less than 0.01). Normalized percent suppression by T8+ cells was higher in low responders than in high responders (77.0 +/- 9.9% vs 33.0 +/- 8.5%, p less than 0.01). Both high and low responders markedly suppressed IgG secretion when 0.5 X 10(5) T8+ cells were added. No correlation was found either between proportion of T3+, T8+, T4+, or M1+ cells within the MNC population and levels of IgG secretion by MNC or between T8+ numbers and levels of suppression induced by a constant number of T8+-enriched cells. Our data indicate that differences in the functional activity of T8+ cells, rather than quantitative differences, account for the wide range of PWM-induced IgG secretion by MNC.     

Differential effects of leukotriene B4 on T4+ and T8+ lymphocyte phenotype and immunoregulatory functions

Leukotriene B4 (LTB4) can regulate several lymphocyte functions, including the augmentation of cytotoxic activity and the induction of suppressor cells. When T lymphocytes were preincubated with picomolar concentrations of LTB4, they would suppress the proliferative response of unfractionated peripheral blood mononuclear leukocytes to concanavalin A in a subsequent co-culture system. Such a suppression did not occur when the responding population was depleted of monocytes. Furthermore, the effect was reversed to an enhancement when the responding unfractionated population was treated with indomethacin, suggesting a role for monocytes and cyclooxygenase products in the effector phase of LTB4-induced suppressor activity. When sorted into T4+ and T8+ cells before preincubation with LTB4, both T cell subsets could be induced by LTB4 to exert suppression. T4+ cells, however, required the presence of monocytes in the responder population in order to manifest suppressor activity, whereas T8+ cells were active even in the absence of monocytes. When LTB4-preincubated T cells or T4+ cells were sorted into T4+ and T8+ subsets after preincubation, suppressor cell activity was found only in the T8+ subset. Furthermore, T8+ cell-depleted T lymphocyte cultures, incubated for 24 hr with LTB4, showed a significant increase in the proportion of T8+ cells. Together, these data suggest that LTB4 induces suppressor T cells which can derive from either T4+ or T8+ subpopulations but which are phenotypically T8+ when exerting their suppressive activity. Thus, by interacting with both T4+ and T8+ lymphocytes, LTB4 can modulate immune responses with the cooperation of functionally competent accessory monocytes.     

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.     

Monocyte function in mixed lymphocyte reactions

Subpopulations of human peripheral blood lymphocytes were isolated by sequential separation techniques. The stimulating and responding capacity of these cells together with the T-cell population remaining after the removal of other populations was studied in one-way allogeneic mixed lymphocyte culture. Incorporation of [³H]thymidine was used as a measure of response. Monocytes, present in the stimulating or responding cell population, were necessary for lymphocyte response. T cells stimulated responding T-cell populations containing monocytes but not B cells. Stimulation by T cells could be inhibited with DRW antisera. Response was also inhibited by sera detecting DRw antigens on the monocytes of the responding cell population. It is concluded that monocytes play an important functional role in mixed lymphocyte reactions. In addition, it appears that the combination of anti-DRw sera and monocytes influences mixed lymphocyte reactions by an active process in that inhibition of response cannot be explained entirely by blocking DRw determinants.     

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) anti-genpulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.