Generation of antigen receptor-specific suppressor T cell clones in man.

We have shown previously that CD8+ T cells proliferate upon exposure to autologous, antigen primed CD4+ T cells, and suppress the response of fresh T cells to the priming antigen but not irrelevant antigens. The stimulus and target of suppression in this system appears to be the antigen receptor on the surface of CD4+ cells, rather than the nominal antigen. In the current study, alloantigen primed CD4+ inducer cells and IL-2-containing medium were used to generate clones of suppressor cells from several individuals. The clones inhibited the response of fresh autologous T cells only to the original allogeneic stimulator cell and to stimulator cells that shared HLA-DR antigens with the priming cell. The clones were also genetically restricted, since they inhibited the response of HLA-A,B-compatible but not HLA-A,B-incompatible individuals. The availability of a method for reproducibly generating antigen receptor-specific suppressor T cell clones in vitro should make it possible to clarify the mechanism, whereby such cells are activated and exert their suppressive effect.

Activation of antigen-specific suppressor T cells in the presence of cyclosporin requires interactions between T cells of inducer and suppressor lineage.

To probe the mechanism of suppressor T cell generation in man, we have carried out mixed leukocyte reactions (MLR) in the presence of cyclosporin (CsA), a fungal metabolite which prevents the generation of cytotoxic lymphocytes while permitting activation of suppressor cells. After a 12-d MLR in the presence of 1 microgram/ml CsA, T cells were fractionated into subsets with monoclonal antibodies, and each subset was tested for the ability to inhibit a second fresh MLR that is devoid of CsA. The results show that Leu 2+ T cells derived from the first culture suppress the second MLR in an HLA-DR antigen-specific manner and in the absence of detectable lysis of stimulator cells. However, Leu 2+ cells do not develop into suppressor cells unless acted upon by alloantigen-primed Leu 3+ inducer cells. Furthermore, only those Leu 3+ cells that also express the Leu 8 marker (Leu 3+, 8+) are capable of inducing suppressor cells. Thus, antigen-specific feedback inhibition of an immune response in man results from an ordered series of interactions between T cells of distinct phenotype.

Inhibition of antigen-presenting cell function by alendronate in vitro.

Bisphosphonates are potent inhibitors of bone resorption in vivo and are emerging as important and widely used drugs for the treatment of a variety of abnormal bone resorptive processes. In the current study we investigated the in vitro effects of 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), a recently developed, extremely potent bisphosphonate, on the immune functions of human peripheral blood mononuclear cells (PBMCs). PBMC proliferation induced by lectins, alloantigens, and a nominal antigen (tetanus toxoid) was inhibited in a dose-dependent manner by alendronate. Pretreatment of monocytes, but not T cells, with the compound at concentrations ranging from 10(-4) to 10(-8) M was inhibitory, indicating that alendronate acts selectively on antigen-presenting cells (APCs). Alendronate did not affect the viability of monocytes or T cells or the expression of cell surface molecules known to play critical roles in antigen presentation. Alendronate exhibited dose-dependent inhibition of the production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) by activated monocytes. The inhibitory effect of 10(-6) M alendronate on PBMC proliferation was reversed by 10 U/ml recombinant rIL-1 beta, whereas other cytokines such as IL-6, TNF-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) had no effect. Thus, alendronate acts on monocytes to inhibit their antigen-presenting/accessory cell functions through a mechanism that can be overcome by exogenous IL-1. The inhibitory effect of this agent on cytokine production may contribute to its inhibitory effect on bone resorption.

Immunoactivation enhances the concentration of luteinizing hormone-releasing hormone peptide and its gene expression in human peripheral T-lymphocytes.

An immunomodulatory role for LHRH was suggested when we reported the presence of immunoactive and bioactive LHRH and its mRNA in rat splenic and thymic lymphocytes. In this paper we report that human peripheral T-cells as well as its subsets CD4+ and CD8+ contained immunoactive and bioactive LHRH. Furthermore, analysis of phytohemagglutinin (PHA)-activated T-cell lysates for LHRH by RIA demonstrated that the mean concentration of LHRH in PHA-activated T-cells increased from 45 +/- 4.5 to 64 +/- 7 pg/10(6) cells after 24 h of culture and from 47 +/- 3.6 to 117 +/- 11.8 pg/10(6) cells (P < 0.01) after 48 h. While the LHRH concentration in PHA-activated cells increased over the last 24 h of culture h from 64 +/- 7 to 117 +/- 11.8 pg/10(6) cells (P < 0.001), there was no change in mean concentration of LHRH in T-cells kept in medium alone. In a preliminary study we found that fresh T-cells contain 20 +/- 1.4 pg pro-LHRH/10(6) cells, and PHA stimulation increased the pro-LHRH content similar to the increase in LHRH. As with unfractionated T-cells, a significant PHA-induced time-dependent enhancement of intracellular LHRH was noted in CD4+ and CD8+ T-cells. RNA extracted from lymphocytes was subjected to reverse transcription-polymerase chain reaction analysis using LHRH and histone-3.3, primers, the latter as an internal control. The polymerase chain reaction-generated data demonstrated that the relative amount of LHRH mRNA in cultured, but non-PHA-stimulated (resting), cells diminished dramatically between 5-24 h, but recovered by 48 h of culture. The relative amount of LHRH mRNA in PHA-stimulated cells revealed a markedly different pattern. LHRH message expression in PHA-activated cells increased slightly at 5 h of culture and was maximally stimulated by 24 h, but declined by 48 h of culture. The PHA activation-induced time-dependent enhancement of intracellular accumulation of LHRH peptide at 5 and 24 h was accompanied by increased LHRH message. However, the increased concentration of LHRH peptide at 48 h coincided with decreased LHRH message expression. The data from total protein synthesis in PHA-activated cells showed a progressive increase in protein synthesis, a pattern entirely similar to the changes in the cell content of LHRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Oral treatment with trimethoprim-sulfamethoxazole and zidovudine suppresses murine accessory cell-dependent immune responses.

Trimethoprim-sulfamethoxazole (TMP-SMX), commonly used for prophylaxis of Pneumocystis carinii pneumonia (PCP) in AIDS patients, often produces a high incidence of treatment-limiting reactions. We investigated the effect of oral administration of TMP-SMX alone or in combination with the antiretroviral drug zidovudine (ZDV) on hematopoiesis and cellular immunity in BALB/c mice. Daily treatment for 28 days with TMP-SMX (160:800 mg/kg) had no effect on hematopoiesis or the ex vivo proliferative response of splenic T lymphocytes to allogeneic tumor cells (EL-4) or to concanavalin A (ConA), or that of splenic B cells to lipopolysaccharide (LPS). ZDV at 240 mg/kg/day was not immunosuppressive but caused a mild macrocytic anemia. Combined treatment produced severe pancytopenia, a significant drop in splenic cellularity, and a 61% decrease in the percentage of splenic macrophages. The percentage of splenic CD3+ lymphocytes increased 150% in the TMP-SMX + ZDV group, but the ratios of T-cell subsets and the frequency of B cells remained unchanged. Combined drug treatment did not impair the proliferative response of B cells to LPS or that of T cells to EL-4 cells. In concert with the reduction in the percentage of macrophages, the proliferative response of T lymphocytes to ConA decreased significantly. Optimal ConA-induced T-cell proliferation requires the participation of accessory cells (AC) (e.g., macrophages); EL-4 cells are able to function as AC. These data indicate that ZDV synergizes with TMP-SMX, causing severe hematotoxicity and suppressing AC-dependent immune function, and suggest that this therapeutic regimen may contribute to the immune deterioration in AIDS patients.

Cellular immunity in malnourished mice.

The effects of malnutrition on the cellular immune response were studied in mice. Cellular immunity, measured by contact sensitivity to 2,4-dinitrochlorobenzene, was found to be impaired in protein energy deficient animals.

Effect of malnutrition on several parameters of the immune system of children.

In order to study the relationship between nutritional status and immunity, certain biochemical and immunological parameters were examined in 53 children with mild or severe malnutrition and 35 normal controls. The levels of hemoglobin, total serum protein and complement (C'3) were not affected by malnutrition. There was a significant depression of serum albumin, transferrin and ceruloplasmin in the severely malnourished children. Serum IgM, IgG and IgD were normal in both malnourished groups. The level of serum IgA was elevated only in the severely malnourished children. The proportion of B cells was the same in all groups; howver, the frequency of T cells was reduced in the severely malnourished cases.

Immunohematotoxicity studies with combinations of dapsone and zidovudine.

We investigated the immunohematoxicities of the antiparasitic drug dapsone (DDS) and the antiretroviral drug zidovudine (ZDV, AZT) given alone or in combination in BALB/c mice. DDS is used for prophylaxis and treatment of Pneumocystis carinii infection in AIDS patients. We examined the impact of concurrent administration of these drugs on the immune and hematopoietic systems because DDS causes hematotoxicity and ZDV therapy results in bone marrow toxicity. Daily oral administration of DDS at 25 and 50 mg/kg for 28 days caused a slight anemia, marked methemoglobinemia, reticulocytosis, and a moderate leukopenia (P < 0.01 for all parameters) but had no discernible effect on platelet count. In DDS-treated mice, the proliferative response of splenic T cells to concanavalin A was > or = 35% higher than that manifested by splenocytes from vehicle-treated control mice. ZDV at 240 and 480 mg/kg was not immunosuppressive but caused low-grade macrocytic anemia, thrombocytosis, and neutropenia; these effects were drug dose-dependent and statistically significant (P < 0.01). Concurrent administration of DDS and ZDV augmented the severity of ZDV-mediated macrocytic anemia, and 7 of 12 (58%) mice did not survive treatment with the high doses of DDS and ZDV (50 and 480 mg/kg, respectively). On the other hand, co-administration of ZDV mitigated DDS-induced methemoglobinemia and the DDS-associated elevation in lymphoproliferative response. These data suggest interaction between DDS and ZDV in mice and indicate a need for caution in using DDS as long-term therapy in AIDS patients receiving ZDV.

Effect of protein deficiency on suppressor cells.

The effects of moderate protein deficiency on the in vitro response of spleen cells to phytohemagglutinin in A/Jax mice were studied. The response of spleen cells from protein-deficient mice to phytohemagglutinin was found to be enhanced as compared with that of cells from control animals. Since inadequate development or function of suppressor cells in the protein-deficient mice offered a possible explanation for the enhanced lymphoproliferative activity, cocultures of spleen cells from protein-deficient and control animals were tested for their responses to phytohemagglutinin. Suppression of [3H]thymidine incorporation was detected in coculture of 25% mitomycin-treated spleen cells from control animals and 75% spleen cells from protein-deficient mice. The suppressor (regulator) elements in control spleens were found to reside in the adherent cell population.

HLA antigens in two Iranian populations: the Armenians and the Jews.

The distribution of HLA phenotype, genotype and haplotype frequencies was studied in two Iranian populations, the Armenians and the Jews. Although the frequencies of most antigens in the Armenians have approximately the average Jewish values, haplotype frequencies are quite different between the two populations. The frequencies of antigens in Iranian Jews are more similar to those in the Mazandaranies, a group of non-Jewish Iranians, than to the Jewish populations from Yemen and Cochin, India.

Distribution of HLA antigens in Zoroastrians.

The frequencies of HLA locus A, B and C antigens were determined in a previously unstudied population, the Zoroastrians. The phenotype and gene frequencies for this group are presented and contrasted with the distribution of HLA antigens among the Iranian Moslems. Comparison of the HLA profiles of these two Iranian populations suggests that the present Zoroastrians are a distinct group.

T cell defect in lepromatous leprosy is reversible in vitro in the absence of exogenous growth factors.

T lymphocytes from patients with lepromatous leprosy (LL) characteristically fail to respond to Mycobacterium leprae. This specific immunologic defect is thought to contribute to the aggressive clinical course that typifies patients with LL. We report that although fresh CD4+ (helper) T cells from most LL patients are specifically unresponsive to M. leprae, after culture in medium alone for 48 hr the same cells respond to M. leprae antigens. The recovery of T cell function is specific for M. leprae, occurs at the level of responder CD4+ T cells, and is not affected by monocytes or CD8+ (suppressor) T cells. Recovery of T cell reactivity is blocked by the presence of M. leprae bacilli in the preculture medium. These findings indicate that despite the apparent specific anergy seen in patients with LL, the T cells of most LL patients can respond to M. leprae. Their failure to do so, in vivo, may be due to the persistence of antigen, which renders antigen-reactive T cells nonresponsive either directly or via activation of CD4+ suppressor cells.

Soluble antigen-primed inducer T cells activate antigen-specific suppressor T cells in the absence of antigen-pulsed accessory cells: phenotypic definition of suppressor-inducer and suppressor-effector cells.

This study was undertaken to characterize interactions among human T cell subpopulations involved in the generation of suppressor T cells specific for a soluble antigen. Purified PPD-primed Leu-3+ cells, when co-cultured for 7 days with fresh autologous Leu-2+ cells, induced differentiation of Leu-2+ but not Leu-3+ cells into specific suppressor T cells, which subsequently inhibited the proliferative response of fresh Leu-3+ cells to PPD but not to tetanus toxoid or allogeneic non-T cells. The PPD-specific suppressor effect of activated Leu-2+ cells was not due to altered kinetics of the PPD response and also extended to the secondary response of PPD-primed Leu-3+ cells. Furthermore, only those Leu-2+ cells that lacked the 9.3 marker, an antigen present on the majority of T cells including the precursors of cytotoxic T cells, differentiated into suppressor T cells. To analyze the inducer population, fresh Leu-3+ cells were separated into Leu-3+,8- and Leu-3+,8+ subpopulations with anti-Leu-8 monoclonal antibody, activated with PPD, and then were examined for inducer function. Although both Leu-3+,8- and Leu-3+,8+ cells proliferated in response to PPD and upon activation expressed comparable amounts of HLA-DR (Ia) antigens, the Leu-3+,8+ subpopulation alone induced Leu-2+ cells to become suppressor-effectors in the absence of PPD-pulsed autologous non-T cells. Once activated, however, Leu-2+ suppressor cells inhibited the PPD response of both Leu-3+,8- and Leu-3+,8+ cells. These results indicate that antigen-primed Leu-3+,8+ inducer cells can directly activate Leu-2+, 9.3- precursors of antigen-specific suppressor T cells in the absence of antigen-pulsed autologous non-T cells.

Activation of antigen-specific suppressor T lymphocytes in man involves dual recognition of self class I MHC molecules and Leu-4/T3-associated structures on the surface of inducer T lymphocytes.

We showed previously that fresh Leu-2+ T cells respond to autologous antigen-primed Leu-3+ T cells by proliferation and differentiation into suppressor T cells (Ts) that specifically inhibit the response of fresh Leu-3+ cells to the original priming antigen. This study was undertaken to characterize the role of various cell surface molecules expressed by antigen-primed Leu-3+ cells in their activation of Leu-2+ Ts cells. Alloactivated Leu-3+ blasts were treated in the absence of complement with a variety of monoclonal antibodies recognizing distinct antigens on human lymphoid cells, and then were examined for their functional effects on fresh autologous T cells. Prior treatment of Leu-3+ blasts with anti-Leu-4 or anti-HLA-A,B,C framework antibodies, but not with anti-Leu-1, anti-Leu-3, anti-Leu-5, or anti-HLA-DR framework-specific antibodies, not only blocked proliferation of fresh Leu-2+ cells, it also prevented their differentiation into Ts cells. Furthermore, after their activation by Leu-3+ blasts, Leu-2+ Ts cells inhibited the response of fresh Leu-3+ cells from only those individuals who shared HLA-A,B phenotypes with suppressor-effector cells. These results suggest that both the inductive and effector phases of suppression involve dual recognition of autologous class I MHC molecules and structures associated with the Leu-4 (T3) molecule on the surface of antigen-reactive Leu-3+ cells.

The VLA-4/VCAM-1 molecules participate in gamma delta cell interaction with endothelial cells.

The accumulation of T lymphocytes at the site of chronic inflammation depends on a number of factors including adherence of T cells to vascular endothelial cells (EC) and endothelial permeability. We examined the effects of human gamma delta + T lymphocytes on the permeability of EC to macromolecules and characterized the cell surface molecules that are involved in these interactions. In this model, the flux of [125I]albumin was measured across the EC monolayer after a short-term culture with cloned gamma delta cells. Our results show that coculture of activated, but not resting, gamma delta cells with EC enhances endothelial permeability by a cytolytic process. Pretreating gamma delta cells with monoclonal antibodies directed at either LFA-1 or VLA-4 molecules or pretreating EC with monoclonal antibodies directed against either ICAM-1 or VCAM-1 molecules significantly inhibited gamma delta cell-mediated enhancement in endothelial permeability. This indicated that VLA-4/VCAM-1 and LFA-1/ICAM-1 adhesion pathways participate in gamma delta cell-EC interaction.

Activation of human CD8-positive T cells via the CD8/HLA class I complex.

Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMIg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and -DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM.1, Q1/28, and HC10 mAb were found inactive. Footprinting analysis of HLA class I molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CD8-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells.

Evidence that T cell activation is required for HIV-1 entry in CD4+ lymphocytes.

The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.