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)     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.     

Human T lymphocyte activation in the presence of acute myelogenous leukaemia blasts; studies of normal polyclonal T cells and T lymphocyte clones derived early after allogeneic bone marrow transplantation

 T cell clones (CD4⁺CD8^–TCRαβ⁺γδ^–) derived from bone marrow transplant recipients were stimulated with phytohaemagglutinin (PHA) +interleukin-2 (IL-2) in the presence of irradiated (50 Gy) peripheral blood mononuclear cells (PBMC) derived from acute leukaemia patients(leukaemic PBMC containing more than 95% blast cells). Leukaemic PBMC could function as accessory cells during mitogenic T cell activation resulting in both T cell proliferation and a broad T cell cytokine response [IL-3, IL-4, IL-10, granulocyte/macrophage-colony-stimulating factor (GM-CSF) tumour necrosis factor α (TNFα) and interferon γ (IFNγ) secretion]. Blockade of IL-1 effects by adding IL-1 receptor antagonist together with PHA+IL-2+leukaemia blasts increased T cell proliferation, whereas IL-6-neutralizing antibodies did not alter T cell proliferation. A qualitatively similar T cell cytokine response and a similar cytokine profile (highest levels detected for GM-CSF and IFNγ) were detected when normal polyclonal T cell lines were stimulated with PHA in the presence of non-irradiated leukaemic PBMC. When leukaemic PBMC derived from 18 acute myelogenous leukaemia patients were cultured with PHA and cells from a polyclonal T cell line, increased concentrations of the T cell cytokines IFNγ and IL-4 were detected for all patients. We conclude that T cell activation resulting in proliferation and a broad cytokine response can take place in the presence of excess acute myelogenous leukaemia blasts. Received: 30 November 1995 / Accepted: 9 January 1996     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

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.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

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.     

Blockade of physiologically secreted IFN-gamma inhibits human T lymphocyte and natural killer cell activation

The role of physiologically secreted human IFN-gamma in T lymphocyte and NK cell activation has been probed with a panel of mouse mAb directed against various epitopes of the human IFN-gamma molecule, or human IFN-gamma R. Addition to the culture medium of those mAb that neutralize the antiviral activity of IFN-gamma or interact with its receptor inhibited proliferative and cytotoxic responses elicited in PBL by HLA alloantigens, anti-CD3 mAb, and IL-2, but not the proliferative response to PHA. The IFN-gamma blockade also inhibited IFN-gamma, IL-2, and TNF-alpha release during MLC. Kinetic experiments showed that reduction of proliferative and cytotoxic responses to HLA alloantigens is maximal when IFN-gamma is blocked within the first 48 h. Exogenous rIFN-gamma restored the proliferative response only when added at the beginning. Moreover, when IFN-gamma was blocked, T lymphocytes recovered from 6-day MLC displayed a profound decrease in their expression of p55 and p75 chains of the IL-2R, as well as in the number of high-affinity IL-2 binding sites. These findings strongly suggest that IFN-gamma is required in the early phases of induction of the oligo- and polyclonal proliferative and cytotoxic responses of lymphocytes.     

Role of CD2 in activation and cytotoxic function of CD8/Leu-7-positive T cells

CD3/CD8-positive, Leu-7-positive cells comprise about 3 to 5% of PBL in normal individuals, but the proportion of these cells is increased in patients with a variety of diseases including chronic viral infection, Crohn's disease, and AIDS. To study further the function of these cells, the proliferative and cytotoxic responses of highly purified CD8/Leu-7-positive cells were studied in vitro. These cells had low proliferative responses when exposed to PHA or mitogenic anti-CD3 mAb compared to CD8/Leu-7-negative cells, and their proliferative responses were significantly lower after addition of IL-2 or autologous adherent cells. However, the proliferative responses of both Leu-7-positive and Leu-7-negative CD8 cells were similar when stimulated with PHA, Ionomycin, or anti-CD3 in combination with phorbol ester. In addition, CD8/Leu-7-positive cells demonstrated high proliferative responses when exposed to a combination of both PHA and SRBC, and these responses could be inhibited by prior addition of non-stimulating anti-CD2.1 mAb. CD8/Leu-7-positive cells, but not CD8/Leu-7-negative cells, mediated lectin- and anti-CD3-induced cytotoxicity against K562 target cells. Cytotoxicity was in part dependent on the CD2 Ag because it was inhibited by anti-CD2.1 mAb. Finally, when small CD8-positive T cells having low cytotoxic potential were activated with PHA plus SRBC, but not PHA alone, there was significant enhancement of their cytotoxic function. Thus, the CD2 receptor may be an important activation pathway for cytotoxic cells.     

Transmission intensity determines lymphocyte responsiveness and cytokine bias in human lymphatic filariasis

Humans living in areas where filariasis is endemic vary greatly in their exposure to mosquito-borne infective third-stage larvae (L3) of these parasitic helminths. Because the intensity of exposure to Ags affects T cell differentiation and susceptibility to parasitic infections in murine models, we compared T cell and cytokine responses in 97 residents of two villages in Papua New Guinea, where transmission intensity of Wuchereria bancrofti differed by 63-fold (37 vs 2355 L3 per person per year). Residents of the high transmission village had 4- to 11-fold lower proliferation and IFN-gamma responses to filarial Ags, nonparasite Ag, and PHA by PBMC compared with the low transmission village (p < 0.01) even when subjects were matched for intensity of infection. In contrast, filarial Ag-driven IL-5 production was 5.5-fold greater (p < 0.001), and plasma IL-4 and TGF-beta levels were 4-fold and 34% higher, respectively, in residents of the high transmission village. IL-4 and IL-10 responses by PBMC differed little according to village, and increased production of the counterregulatory cytokines IL-10 or TGF-beta by PBMC did not correlate with weak proliferation and IFN-gamma responses. Plasma IL-5, IFN-gamma, and IL-10 levels were similar in the two villages. These data demonstrate that the intensity of exposure to L3 affects lymphocyte responsiveness and cytokine bias possibly by a mechanism that alters APC function.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

Antigen recognition and immunomodulation by gamma delta T cells in bovine tuberculosis

This report describes the in vitro proliferative responses of peripheral blood gammadelta T cells to defined mycobacterial protein Ags and the immunomodulatory effect of gammadelta T cells in cattle infected with Mycobacterium bovis. gammadelta T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating gammadelta T cell cultures produced enhanced levels of IFN-gamma and TGF-beta, but not IL-2 in response to the more immunodominant mycobacterial AGS: Depletion of gammadelta T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of gammadelta T cells upon other (alphabeta) T cell responses. Because gammadelta T cells constitute a major T cell population in the peripheral blood of cattle, the activities of gammadelta T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.     

Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity

Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.     

CD40 ligand trimer and IL-12 enhance peripheral blood mononuclear cells and CD4+ T cell proliferation and production of IFN-gamma in response to p24 antigen in HIV-infected individuals: potential contribution of anergy to HIV-specific unresponsiveness

It has been suggested that CD4+ T cell proliferative responses to HIV p24 Ag may be important in the control of HIV infection. However, these responses are minimal or absent in many HIV-infected individuals. Furthermore, while in vitro and in vivo responses to non-HIV recall Ags improve upon administration of highly active antiretroviral therapy, there does not appear to be a commensurate enhancement of HIV-specific immune responses. It is possible that CD4+ p24-specific T cells are deleted early in the course of infection. However, it is also possible that a discrete unresponsiveness, or anergy, contributes to the lack of proliferation to p24. To evaluate the possible contribution of unresponsiveness to the lack of CD4+ T cell proliferation to p24 in HIV-infected individuals, we attempted to overcome unresponsiveness. CD40 ligand trimer (CD40LT) and IL-12 significantly increased PBMC and CD4+ T cell proliferative responses to p24 Ag in HIV-infected, but not uninfected, individuals. No increase in proliferative response to CMV Ag was observed. CD40LT exerted its effect through B7-CD28-dependent and IL-12- and IL-15-independent mechanisms. Finally, the increase in proliferation with CD40LT and IL-12 was associated with an augmented production of IFN-gamma in most, but not all, individuals. These data suggest the possible contribution of HIV-specific unresponsiveness to the lack of CD4+ T cell proliferation to p24 Ag in HIV-infected individuals and that clonal deletion alone does not explain this phenomenon. They also indicate the potential for CD40LT and IL-12 as immune-based therapies for HIV infection.     

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.     

Complex effects of interferon-alpha on the cytokine network in HIV infection--possible contribution to immunosuppression

Since interleukin (IL-)2, IL-10 and IL-12 may contribute to the pathogenesis of human immune deficiency virus (HIV) infection we examined the effect of interferon (IFN)-alpha on these cytokines in cultures of various subsets of peripheral blood mononuclear cells (PBMC) in ten HIV-infected patients and ten healthy controls. Our main findings were: (1) IFN-alpha markedly enhanced IL-10 levels in a dose-dependent manner in both lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-stimulated PBMC, as well as in anti-CD3- and anti-CD3/anti-CD28-stimulated T cells in both HIV-infected patients and controls. (2) In contrast, IFN-alpha had a downregulatory effect on IL-10 levels in Candida -stimulated PBMC,with particularly strong suppressive effect in HIV-infected patients. (3) Furthermore, IFN-alpha had a significant but modest stimulatory effect on IL-2 levels in PHA- and Candida -stimulated PBMC and anti-CD3-stimulated T cells. (4) IFN-alpha enhanced IL-12 levels in a dose-dependent manner in LPS-stimulated PBMC in both patients and controls. Our findings that IFN-alpha markedly enhanced IL-10 and modestly enhanced IL-2 and IL-12, suggest a net immunosuppressive effect of IFN-alpha in HIV-infected patients, possibly contributing to progression of immunodeficiency in these patients.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

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.     

Regulation of pulmonary T cell responses to inhaled antigen: role in Th1- and Th2-mediated inflammation.

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323-339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-gamma. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.