Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.     

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.     

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.     

The potentiation of B lymphocyte responses through CD2/LFA-3 interactions involving erythrocytes is IL2 independent

The response of human B cells to pokeweed mitogen (PWM) stimulation is potentiated when autologous erythrocytes (E) are added to peripheral blood mononuclear cell (PBMC) cultures. This potentiation has been previously shown to be dependent on interactions between the CD2 molecule on T cells and the lymphocyte function-associated antigen 3 (LFA-3) expressed by autologous erythrocytes. Since in other experimental systems the activation of T cells by CD2/LFA-3 interactions has resulted in increased secretion of interleukin 2 (IL2), we were interested in studying the role of IL2 in PBMC cultures stimulated with PWM and autologous E. The addition of autologous E significantly depressed IL2 levels in PWM-stimulated PBMC cultures. This effect was not secondary to increased expression of IL2 receptors by activated cells, since the addition of anti-TAC antibodies did not result in a significant increase in measurable levels of IL2. The addition of anti-IL2 to PBMC failed to abrogate the potentiating effect of E and it actually further enhanced the production of IgM and IgG from cultures stimulated with PWM + E. These results suggest that the potentiation of B cell function induced by autologous E is not mediated by IL2, either directly or indirectly. It is possible that the effect of autologous E either is mediated by other interleukins or is dependent on cell-to-cell contact with directed release of IL2 and/or other lymphokines without detectable secretion to the extracellular compartment.     

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)     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

In vitro regulation of IgA subclass synthesis. II. The source of IgA2 plasma cells

In vitro regulation of IgA subclass synthesis was investigated in pokeweed mitogen (PWM)-stimulated cultures of peripheral blood lymphocytes. In past experiments we have demonstrated that 50% of the IgA plasma cells derived from PWM-stimulated cultures are positive for IgA1 and 50% are positive for IgA2. This observation is surprising because approximately 80% of the IgA B cells in the peripheral circulation bear IgA1 and 20% bear IgA2. To determine if the shift toward IgA2 predominance in PWM-stimulated cultures might be due to an enriched source for IgA2 plasma cells from a precursor pool of immature B cells, we used panning techniques to separate immature precursors that express surface IgM (sIgM+) from mature precursors that no longer express IgM (sIgM-). These separated B cells were cultured with equal numbers of T cells and PWM for 7 days. In all 10 experiments there was an enrichment for IgA2 in the sIgM+ cultures; 55 +/- 9.6% of the total IgA plasma cells were positive for IgA2 in the sIgM+ cultures vs 38 +/- 6.3% in the sIgM- cultures (p less than 0.001). These results indicate that both sIgM+ and sIgM- cells can give rise to IgA plasma cells in PWM-stimulated cultures and that there is an enrichment for IgA2 precursors in the sIgM+ population. Other possible regulatory mechanisms were also investigated. To determine if there was isotype switching from IgA1 to IgA2, monoclonal anti-IgA1 antibodies were added to PWM cultures. These antibodies resulted in a mean suppression of IgA1 plasma cell production of 82% with a concomitant 45% suppression of total IgA but only 4.6% suppression of IgA2. These results make it unlikely that IgA2 plasma cells in PWM-stimulated cultures are derived from cells that initially produced IgA1. To investigate the possibility that one IgA subclass might be more T cell dependent than the other, T and B cells were separated and B cells were reconstituted with T cells in ratios that varied from 1:10 to 10:1 or with irradiated T cells. These procedures did not alter the proportion of IgA plasma cells positive for IgA1 or IgA2, indicating that the two subclasses do not differ in their response to T cell signals in PWM-stimulated cultures.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

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.     

Effect of glucocorticoids on the development of suppressive activity in human lymphocyte response to a polysaccharide purified from Candida albicans

The action of glucocorticoids on the proliferative response of human lymphocytes stimulated in vitro by MPPS has been investigated. The effect of Dex was dependent on the time of steroid addition to the cultures. Dex added at the beginning of the culture period inhibited, cell proliferation and IL 1/IL 2 synthesis, although not completely. However, a delayed addition of 24 to 48 hr resulted in an enhancing effect on cell proliferative responses that was maximal at day 4. The effect of Dex on T suppressor cell activity was then investigated. Dex added 1 day before the appearance of suppressor cells resulted in a marked decrease or disappearance of the suppressive activity. Moreover, primed T lymphocytes treated with Dex in the presence of exogenous IL 2 enhanced the proliferative responses of fresh autologous PBMC stimulated by MPPS. Taken together, our data suggest that glucocorticoids inhibit the differentiation of T suppressor cells and that IL 2 is unable to reverse this inhibitory effect.     

Interleukin-2 reverses T cell unresponsiveness to Plasmodium falciparum-antigen in malaria immune subjects

Peripheral blood mononuclear cells (PBMC) from a large proportion of 34 healthy adult native residents in a malaria endemic area showed null or marginal proliferative response (low-responders) to schizont-enriched Plasmodium falciparum malaria antigen (M.Ag) but good response to pokeweed mitogen. In contrast, substantial proliferative response to M.Ag was observed in 8/8 adult temporary residents with a history of one to three acute malaria episodes. Purified CD4+ T cells preferentially responded to M.Ag, however in low-responders CD4+ T cell proliferation was poor. Moreover, no inhibition of CD4+ T cell proliferation was observed when graded numbers of CD8+ T cells were added in culture. The addition of recombinant interleukin 2 (rIL-2) to M.Ag restored the proliferative response of low-responders' PBMC. This response was M.Ag-specific when CD4+ T cells grown in M.Ag plus rIL-2, but not in rIL-2 alone, were tested in secondary cultures.     

Suppressor T cell activation by human leukocyte interferon

Murine fibroblast interferon (IFN beta) activates murine suppressor T lymphocytes in vitro, which suppress plaque-forming cell responses by spleen cells. Suppression of human in vitro immune responses by IFN was investigated to determine whether human IFN also activates suppressor T cells. Human leukocyte IFN (IFN alpha) suppressed pokeweed mitogen-induced polyclonal immunoglobulin production by human peripheral blood mononuclear cells (PBMC) by 80 to 90% at doses of 200 to 350 U/ml. Responses by IFN alpha-treated PBMC were suppressed in a dose-dependent manner; control cultures had maximal responses on day 7. PBMC incubated with 10,000 U/ml of IFN alpha contained activated suppressor cells that decreased pokeweed mitogen-stimulated, polyclonal immunoglobulin production by autologous cells by 70 to 80%. Suppression mediated by these cells was prevented by catalase, ascorbic acid, and 2-mercaptoethanol (2-ME). In murine systems, these reagents interfere with expression of suppressor T cell activity by preventing activation of soluble immune response suppressor. Selection procedures with monoclonal antibodies identified the suppressor cell as an OKT8+ (suppressor/cytotoxic) T lymphocyte. Selected OKT8+ cells required less IFN alpha (1000 U/ml) for activation and were effective in smaller numbers than unfractionated activated PBMC. IFN alpha-activated suppressor cells also inhibited proliferation in mixed lymphocyte and mitogen-stimulated PBMC cultures; again, catalase and 2-ME blocked suppression. These results indicate that IFN alpha activates suppressor T cells in human PBMC cultures; the ability of catalase, 2-ME, and ascorbic acid to block suppression suggests that these suppressor T cells have certain similarities to IFN beta or to concanavalin A-activated murine suppressor T cells.     

Human immunodeficiency virus type 1 gp120 induces anergy in human peripheral blood lymphocytes by inducing interleukin-10 production.

The effects of recombinant gp120 on the proliferative responses and cytokine production by normal peripheral blood mononuclear cells (PBMC) were investigated. gp120 inhibited in a dose-dependent fashion the anti-CD3 monoclonal antibody (MAb)- and concanavalin A-induced proliferative responses. The production of interleukin-2 (IL-2) and IL-4 was diminished by gp120 in the anti-CD3- and concanavalin A-stimulated cultures. In unstimulated PBMC, gp120 induced the production of considerable amounts of IL-10, gamma interferon, and tumor necrosis factor alpha. The gp120-induced reduction in the proliferative responses of PBMC was at least partially reversed by the addition of IL-2, anti-CD28 MAb, or transfectants expressing CD80, CD86, or CD40 but not with exogenous IL-4. Also, a neutralizing anti-IL-10 MAb reversed the inhibitory effect of gp120 on the proliferative responses whereas exogenous IL-10 further enhanced this inhibitory effect. These findings indicate that IL-10 plays an important role in the inhibitory effect of gp120 on PBMC proliferation. The ratio of CD3+CD4+ to CD3+CD8+ T cells was the same in gp120-treated and untreated cell cultures. No apoptosis in these two T-cell populations was observed. However, the number of activated CD3+CD4+ T cells and CD3+CD8+ T cells, as judged by CD25, CD69, and HLA-DR expression, was consistently reduced. gp120 induced the expression of IL-10 in the monocyte/macrophage population, and therefore gp120 also reduced the proliferative responses of CD4+ T-cell-depleted PBMC. Taken together, our observations point to the importance of the cytokine pattern changes and, in particular, the role of IL-10 (produced by the monocytes) in the inhibitory effect of gp120. This mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed immune responses associated with human immunodeficiency virus infection and thus have important implications for immunotherapeutic strategies to slow down disease progression in AIDS.     

T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.     

T cell surface antigen expression on lymphocytes of patients with AIDS during in vitro mitogen stimulation

Summary Surface marker expression on peripheral blood mononuclear cells (PBMC) was evaluated daily in PHA- and PWM-stimulated cultures of eight AIDS patients and eight normals. Before culture, the patients' cells showed the characteristic decrease in OKT 4+ cells (normals 40.4%, patients 22.3%; P<0.001), increase in OKT 8+ cells (normals 27.6%, AIDS 38.4%; P=0.002), increase in OKT 10+ cells (normals 15.5%, AIDS 42.8%; P=0.002), and increase in HLA-DR+ cells (normals 11.4%, AIDS 28.7%; P=0.01). The percentage of OKT 11+ cells remained unchanged, while the percentage of OKT 3+ cells dropped over the first 2 days in PHA but not in PWM cultures of both groups (PHA: normals 69.8% to 35.1%; P=0.001, AIDS 56.5 to 38.5%; P=0.001, PWM: normals 62.8%–65.9%, AIDS 66.8% to 63.9%), and recovered in both groups by day 5. In PWM cultures OKT 3+ cells increased significantly in normals but not in AIDS (normals 62.6%–77.7%; P=0.04, AIDS 61.8 to 48.7%). OKT 4 expression decreased in normal PHA cultures after 1 day (38.9% to 29.6%; P=0.05) and then recovered by day 5. Its expression increased in AIDS PHA cultures by day 5 (18.0%–41.1%; P<0.001). The final percentage of OKT 4+ cells in AIDS cultures was within the normal range (35.0%–49.0%). OKT 8 expression increased in both study groups after PHA stimulation (normals 29.5%–50.4%; P=0.002, AIDS 37.4%–50.7%; P=0.02) and in normals but not AIDS after PWM stimulation (normals 28.9%–35.5%; P=0.004, AIDS 38.5%–35.6%). Because of the relative changes in expression of OKT 4 and OKT 8, the 4/8 ratio declined in the normal PHA cultures (1.89 to 1.03; P=0.1) and increased in the AIDS cultures (0.68–1.18; P=0.09). Also, the sum of OKT 4+ and OKT 8+ cells in PHA cultures increased from 68% to 94% whist expression of OKT 11 remained unchanged, indicating co-expression of these antigens on individual cells. Both PHA- and PWM-stimulated normal cells showed an increase in OKT 10 (PHA 16.0%–53.4%; P=0.01, PWM 16.1%–33.9%; P=0.03) and HLA-DR (PHA 8.6%–27.3%; P=0.03, PWM 12.5%–26.6%; P=0.07). In AIDS PHA cultures this did not change, and in their PWM cultures OKT 10 expression declined (44.8 to 23.0%; P=0.05). The PHA- and PWM-stimulated cultures of AIDS patients showed a marked deficit in generation of Tac (PHA increased from 5.4% to 77.1% in normals and from 3.2% to 48.0% in AIDS; P=0.001; PWM increased from 6.1% to 35.3% in normals, and from 5.0% to 15.5% in AIDS; P=0.04). Analysis showed that this deficit was limited to a reduced expression on small lymphocytes and that those cells that did become lymphoblasts expressed Tac normally. These results indicate that the poor blastogenic responses in AIDS are related to failure of OKT 10, HLA-DR, and Tac to increase after stimulation.Abbreviations AIDS acquired immunodeficiency syndrome - PBMC peripheral blood mononuclear cells - PHA phytohemagglutinin - PWM pokeweed mitogen - Tac T cell activation antigen - ARC AIDS-related complex of symptoms - IL-2 interleukin 2 - GVHD graft-versus-host disease - HBSS Hank's balanced salt solution - RPMI 1640 Roswell Park Memorial Institute tissue culture medium 1640 - FITC fluorescein isothiocyanate     

Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.     

M. leprae and PPD-triggered T cell lines in tuberculoid and lepromatous leprosy

Proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and bacillus Calmette Guerin-derived purified protein derivative (PPD) were studied in the presence or absence of interleukin 2 (IL 2) in high M. leprae responders (tuberculoid leprosy patients and healthy subjects) and low M. leprae responders (lepromatous leprosy patients). High responders in most cases developed a strong proliferative response to both antigens in the absence of IL 2. Additional IL 2 and restimulation with antigen plus autologous antigen-presenting cells (APC) allowed the derivation of antigen-specific T cell lines. The lines were assayed for proliferative responses to several mycobacterial antigens. Both PPD and M. leprae-triggered T cell lines exhibited a good proliferative response to either antigen and showed in addition a broad cross-reactivity with other mycobacteria, suggesting a preferential T cell response to epitopes shared by several mycobacterial species. Within the lepromatous group, 50% of the patients studied could mount a proliferative response to PPD antigen in the absence of IL 2, but none of them was able to do so with M. leprae antigen. The addition of IL 2 increased the number of positive responders to PPD in this group, and in some patients IL 2 was able to restore M. leprae reactivity as well, suggesting that IL 2 had overcome a suppressor mechanism. PPD and M. leprae-triggered T cell lines were obtained from these subjects (with IL 2 added from the beginning of the culture when required). M. leprae lines exhibited variable and unstable pattern of specificity, most lines exhibiting, at least transiently, a cross-reactive response to other mycobacteria, but some displaying only M. leprae-specific response. In contrast, PPD lines from these subjects consistently exhibited a good response to PPD, a lesser response to various other mycobacteria and no response to M. leprae, a pattern differing from that obtained with PPD lines of high M. leprae responders. Co-cultures of irradiated lepromatous PPD triggered T cell lines with fresh autologous PBMC non-specifically reduced the proliferative response of the latter to PPD, as well as to unrelated antigens. A similar suppression was also observed when PPD lines from one of the tuberculoid patients were assayed. PPD and M. leprae T cell lines from both high and low responders initially exhibited the same CD4+ CD8- phenotype. In all cases, antigenic specificity declined and could not be maintained after 5 to 8 wk of continuous culture, a change associated with the progressive appearance of CD8+ and Leu8+ cells.     

HIV-induced immunodeficiency. Relatively preserved phytohemagglutinin as opposed to decreased pokeweed mitogen responses may be due to possibly preserved responses via CD2/phytohemagglutinin pathway

We studied the proliferative response of PBL to the mitogens PHA and PWM and Candida albicans Ag in 301 HIV seropositive homosexual men, of whom 55 had AIDS. The responses to PHA were reduced only in the clinically ill HIV seropositive subjects. In contrast, the responses to PWM were profoundly reduced in most HIV seropositive subjects including the asymptomatic group. Further analysis of 16 HIV seropositive subjects showed that the proliferative responses were reduced in both CD4 and CD8 T cell subsets. A total of 15 HIV seropositive individuals with low responses to PWM, of whom seven had AIDS and eight controls were chosen for the following studies. Expression of T3, Ti, delta receptors, and CD2 was investigated and showed an increased percentage of CD2 receptors positive cells in HIV seropositive subjects without AIDS. The proliferative responses of PBL to stimulation with PHA, PWM, antibodies to CD3, or antibodies to CD2 were investigated and showed significant correlation in controls, whereas in contrast, only the responses to PHA and CD2ab correlated in patients with AIDS. The proliferative responses to CD2ab and CD3ab in controls were larger than the responses to both PHA and PWM. In patients, these responses were less suppressed than the responses to PWM indicating that stimulation with mitogens is more complex than a simple stimulation of Ti/T3 and CD2 receptors. Further investigations were done on resting T cells, i.e., lymphocytes depleted of macrophages and pre-activated cells. Addition of PHA to these cells resulted in preactivation with expression of IL-2R (CD25) but not in proliferation. In contrast, addition of PHA plus SRBC, which bind to the CD2 receptors caused IL-2R expression, IL-2 production, and proliferation. Addition of PWM + SRBC did not result in proliferation. A comparison of the responses to PHA + SRBC of resting T cells from 26 HIV seropositive individuals, of whom seven had AIDS and 12 seronegative controls, showed that these responses were normal or only slightly decreased in the 19 seropositive men without AIDS whereas it was decreased in AIDS patients. Nevertheless, all AIDS patients showed clear-cut responses in this assay. Thus, the discrepancy between responses to PHA and PWM may be explained by an at least partially preserved function of the PHA/CD2-dependent pathway. We suggest that the defect induced by the HIV infection primarily concerns T3/Ti-induced responses.