A cell-free extract of Salmonella typhimurium inhibits mitogen-induced proliferation of murine splenic T lymphocytes

Abstract In this study, a cell-free extract of Salmonella inhibited T cell mitogen-induced proliferation of spleen cells from non-immunized mice. The proliferation of murine spleen cells stimulated with a T cell mitogen, such as phytohaemagglutinin (PHA) or concanavalin A (ConA) was suppressed significantly when the cells were treated with a sonicate of S. typhimurium , but not of E. coli . The agent(s) responsible for the suppressive effect existed mainly in the soluble fraction of S. typhimurium , whereas the membrane fraction possessed minimal activity. The T cell proliferation suppression paralleled the level of interleukin-2 (IL-2) secretion. Addition of phorbol 12-myristate-13 acetate (PMA) to the cultures restored IL-2 secretion to normal levels, although proliferation remained suppressed and was not reversed by treatment with recombinant IL-2. These results suggest that the suppression of T cell proliferation induced by a soluble Salmonella fraction is associated with inhibition of IL-2 secretion and the response of T cells to IL-2 and the former effect is dependent upon the inhibition of the stimulatory activity of protein kinase C on IL-2 secretion. This type of suppression may explain a mechanism of immunosuppression induced by murine typhoid fever.     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

Effects of purified measles virus components on proliferating human lymphocytes.

Preparations of purified measles virus and both core and membrane-rich fractions suppressed mitogen-induced proliferation of human lymphocytes without reducing viability or the number of T lymphocytes in culture. The suppressive activity of the measles virus was heat labile. The virus preparations by themselves did not induce lymphocyte proliferation.     

Measles virus-induced suppression of lymphocyte proliferation

The mechanism by which measles virus induces immunosuppression was investigated using an in vitro system employing phytohemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a multiplicity of infection of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the kinetics of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and irradiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologous PBMC caused significant inhibition of lymphoproliferation over a wide range of infected:fresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Virus recovered from the irradiated, infected cells was 100-fold lower than the virus titer needed to cause inhibition by direct addition of measles virus. However, antibody to measles virus reversed the inhibition. Virus-free supernatant fluids from the infected irradiated cells caused immunosuppression of the PHA response. This immunosuppressive material induced by the measles virus was maximally produced after 72 hr and did not appear to require viral replication. This factor was not prostaglandin E or interferon-alpha or -gamma. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be detected.     

Suppression of human lymphocyte mitogen response by disrupted primate retroviruses of type C (baboon endogenous virus) and type D (PMFV)

Disrupted primate retroviruses of type C (baboon endogenous virus, BaEV) and type D (human cell line-derived isolate PMFV) considerably suppressed Concanavalin A - induced blastogenic response of human lymphocytes. Rauscher mouse leukemia virus (RLV) displayed a suppressive activity on murine splenic lymphocytes when tested under analogous conditions. The immunosuppressive activities were shown not to result from cytotoxicity or from virus-mitogen binding.     

Cell lines derived from human autorosette-forming cells with suppressive activity

Concanavalin-A-stimulated human T lymphocytes from healthy donors and from patients suffering from diverse immune disorders were fractionated into rosette-forming (R) and nonrosette-forming (NR) cells. The separation method is based upon the ability of the lymphocytes to bind autologous erythrocytes and form autorosettes. Long-term cultures of the R and NR subpopulations were established. The activity of the culture supernatants on the T cell proliferation of normal human phytohemagglutinin (PHA)-induced lymphocytes and of a murine, interleukin-2 (IL-2)-dependent cytotoxic T cell line (CTLL) was investigated. Only the R cell line-derived supernatants from almost all patients tested evinced potent suppressor activity, those from healthy donors less so. The suppressive function was demonstrated not to be due to a cytotoxic effect since preincubation of the PHA-induced lymphocytes and CTLL cells with the factor did not diminish their proliferative capacity. Our study indicates the existence of a competitive relationship between the suppressor factor and IL-2. We found that inhibition of the proliferation decreased with the addition of increasing quantities of exogenous IL-2. We also observed that preincubating the CTLL cells with IL-2 prior to exposing them to the suppressive factor precludes inhibition of their proliferation. Phenotypic analysis of the suppressor cell line revealed that they were comprised of a T cell population which included OKT4+ and OKT8+ cells and that 99% of the cells formed autorosettes. Preliminary purification of the suppressive factor was performed by ultrafiltration and maximal suppression was exhibited by the fraction of less than 10,000 daltons. The development of suppressor cell lines from the unique population of autologous rosette-forming cells may be very helpful in studying the immunoregulatory properties of these cells and their suppressor activity.     

Antiproliferative effect of IFN-gamma on proliferation of mouse connective tissue-type mast cells

Mouse rIFN-gamma suppressed clonal growth of tissue-type mast cells (CTMC) separated from mouse peritoneal cells (greater than or equal to 98% purity) in methylcellulose culture containing IL-3 + IL-4 in a dose-dependent fashion. The IFN-gamma-mediated suppression was also observed both in serum-free culture and in single-cell culture. In addition, mAb to mouse IFN-gamma neutralized the suppressive effect of IFN-gamma on CTMC proliferation supported by the combination of IL-3 + IL-4. These observations suggest a direct antiproliferative effect of IFN-gamma on CTMC. No suppression of CTMC proliferation was observed when CTMC were washed with alpha-medium after 24 h of preincubation with IL-3 + IFN-gamma and then cultured in methylcellulose medium containing IL-3 + IL-4. IFN-gamma had suppressive effects on CTMC after preincubation with IL-3 + IL-4. These preincubation studies suggest that IFN-gamma exerts a reversible inhibitory effect on CTMC which have entered into a proliferative state by IL-3 + IL-4, and has neither cytotoxic nor irreversible suppressive effects on resting CTMC in cultures containing IL-3 alone. CTMC proliferation after stimulation with the cross-linking of cell-bound IgE by specific Ag in the presence of IL-3 was also suppressed by IFN-gamma. By contrast, proliferation of CTMC induced by the combination of 12-O-tetradecanoyl phorbol-13-acetate + IL-3 was not affected by the addition of IFN-gamma to the culture. Based on these observations, we suggest that IFN-gamma antagonizes the colony inducing effect of IL-4 and IgE-Ag stimulation which act synergistically with IL-3 on CTMC.     

Characteristics of the specific cell-mediated immune response in human immunodeficiency virus infection.

The human immunodeficiency virus (HIV)-specific lymphocyte proliferation response was determined for 40 persons at different stages of HIV infection. The specific response to purified HIV virion antigens from strain HTLV-IIIB was poor, occurred in only 9 of the 40 subjects, was not improved with the addition of interleukin-2, and was more frequent in symptom-free individuals (46%) than in patients with lymphadenopathy syndrome (10%). Reactivity to subcomponent p24 was better than that to whole HIV; reactivity was present in five of six infected persons and increased with the addition of exogenous interleukin-2. Reactivities to subcomponents (g)p41 and gp120 were also measured. This is the first evidence of a specific cell-mediated immune response to HIV antigen in HIV-infected persons. Monkeys immunized with purified HIV or with purified p24 displayed cellular immunoreactivity both to whole HIV and to subcomponents. In contrast to the poor reactivity to HIV antigen, the lymphocytes of the patients had good specific cell proliferation responses to cytomegalovirus and herpes simplex virus challenge and a normal response to the addition of phytohemagglutinin. The results suggest a functional defect in peripheral lymphocytes of some HIV-infected individuals on the basis of their response to whole HIV antigen and a better response to gag protein.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

运动员剧烈运动后血中应激免疫抑制蛋白的产生

我们曾经报道,大鼠或小鼠在束缚应激后血中产生了一种能抑制免疫功能的应激免疫抑制蛋白,（又称Ｎｅｕ－ｒｏｉｍｍｕｎｅｐｒｏｔｅｉｎ,ＮＩＰ,神经免疫蛋白）。本工作证明,运动员在大运动量的训练后血清中也产生一种能抑制淋巴细胞转化的物质,它的生化特性及分子量与前述大鼠和小鼠中的应激免疫抑制蛋白相同。在体外实验中,应激大鼠的血清培养人淋巴结细胞,获得了与大鼠实验相同的结果,即人淋巴结细胞也能产生应激免疫抑制蛋白。同时小鼠束缚应激的血清和大运动量的人类血清可以分别抑制人正常淋巴细胞和正常小鼠由ＣｏｎＡ诱导的淋巴细胞转化,以上结果表明,这种应激免疫抑制蛋白的种属特异性不强。     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Phytohemagglutin-stimulated human T cell: prothymosin alpha as an accessory signal

Thymic peptide factors are known to modulate proliferation of normal human lymphocytes. In this work, we studied the effect of Prothymosin alpha (Pro alpha) on PHA-stimulated PBMC and PBLC. The observed effects of Pro alpha and thymosin alpha 1 (alpha 1) on PBMC were found to depend on the degree of cell stimulation, dose, and preincubation-time. Thymosin beta 4 (beta 4) had no effect on either cell type, regardless of the degree of stimulation, which shows that beta 4 may be used as a control peptide to work in this area. Induction of lymphoproliferation also depended on the presence of macrophages. Addition of monocytes or a cell-free monocyte culture supernatant (not containing IL-2) to the PHA-stimulated PBLC cultures resulted in T cell proliferation. Although IL-1 could not restore the PHA-induced proliferative response of isolated T cells by itself, it would enhance the helper effect of Pro alpha. Moreover, a polyclonal goat anti-human IL-2R (Tac Ag) did block the proliferative response induced by combined rIL-1 and Pro alpha, suggesting that an IL-2-dependent pathway of T cell proliferation was involved.     

Immunologic dysfunction during viral oncogenesis. I. Nonspecific immunosuppression caused by malignant rabbit fibroma virus

Malignant rabbit fibroma virus (MV) is a potent oncogenic poxvirus that produces a rapidly progressive syndrome of disseminated myxosarcoma, immunosuppression, and fatal gram-negative infection. MV is probably a recombinant between Shope fibroma virus (SFV) and rabbit myxoma virus, and is capable of preventing or aborting the in vitro proliferative responses of rabbit lymphocytes to B and T lymphocyte mitogens. Proliferative responses to sheep erythrocytes (SRBC) are similarly affected, although MV does not alter ongoing antibody responses to SRBC. Splenic lymphocytes from MV tumor-bearing rabbits suppress antibody and proliferative responses to SRBC when added to lymphocytes from SRBC-primed rabbits. Finally, lysates of cultured splenic lymphocytes from rabbits given MV suppress both proliferative and antibody-forming responses to SRBC. When MV is removed from these lysates by UV inactivation or by centrifugation, the suppressive activity remains. We therefore conclude that MV induces immunologic unresponsiveness in rabbits by at least two mechanisms. First, a direct suppressive effect of added virus on in vitro lymphocyte proliferation is seen. There is no effect in this situation if an antibody response is already in progress. Second, spleen cells exposed to MV in vivo produce one or more soluble factors capable of suppressing both proliferative and antibody responses of normal lymphocytes.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.