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Summary The bacterial superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes are potent stimulators of polyclonal T-cell proliferation. They are the causes of toxic shock syndrome but also induce CD25+ FOXP3+ regulatory cells in the CD4 compartment. Several studies have recently described different forms of antigen-induced regulatory CD8+ T cells in the context of inflammatory diseases and chronic viral infections. In this paper we show that bacterial superantigens are potent inducers of human regulatory CD8+ T cells. We used four prototypic superantigens of S. aureus (toxic shock syndrome toxin-1 and staphylococcal enterotoxin A) and Str. pyogenes (streptococcal pyrogenic exotoxins A and K/L). At concentrations below 1 ng/ml each toxin triggers concentration-dependent T-cell receptor Vβ-specific expression of CD25 and FOXP3 on CD8+ T cells. This effect is independent of CD4+ T-cell help but requires antigen-presenting cells for maximum effect. The cells also express the activation/regulatory markers cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumour necrosis factor receptor-related protein and skin homing adhesins CD103 and cutaneous lymphocyte-associated antigen. Superantigen-induced CD25+ FOXP3+ CD8+ T cells were as potent as freshly prepared naturally occurring CD4+ regulatory T cells in suppressing proliferation of CD4+ CD25− T cells in response to anti-CD3 stimulation. Although superantigen-induced CD8+ CD25+ FOXP3+ express interleukin-10 and interferon-γ their suppressive function is cell contact dependent. Our findings indicate that regulatory CD8+ T cells may be a feature of acute bacterial infections contributing to immune evasion by the microbe and disease pathogenesis. The presence and magnitude of regulatory CD8+ T-cell responses may represent a novel biomarker in such infections. Superantigen-induced regulatory CD8+ T cells also have therapeutic potential. [ABSTRACT FROM AUTHOR]

Peripheral blood CD4 CD45RO T cells activated in vitro are able to induce expression of tumour necrosis factor-α (TNF-α) in monocytes via a contact-dependent mechanism. Activation is achieved either with interleukin-2 (IL-2)/IL-6/TNF-α over an 8-day period or cross-linking CD3 using anti-CD3 antibody for 48 hr. In this paper, we show that the p38 mitogen-activated protein kinase (MAPK) signalling pathway played different roles in the generation of effector function in these two types of activated T cells. In anti-CD3 activated T cells, p38 MAPK is a negative regulator for anti-CD3 induced cell proliferation and has no significant effect on the acquisition of either the effector function (induction of monocyte-derived TNF-α) or production of T-cell cytokines. In contrast, the p38 MAPK signalling pathway is required for the acquisition of cytokine-induced effector function and promotes cell proliferation and cytokine production. [ABSTRACT FROM AUTHOR]

This paper describes the immunoregulatory effects of interleukin-10 (IL-10) on synovial cells in vitro. Synovial cells were cultured with IL-10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of IL-10 on the function of synovial cells as antigen-presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon-γ (IFN-γ), IL-lβ, or 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up-regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL-10. Autologous T-cell proliferation was stimulated by purified protein derivative (PPD) in IFN-γ-treated synovial cells and treatment of these synovial cells with IL-10 also suppressed T-cell, proliferation. Our results suggest that IL-10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

As described in an accompanying paper, trinitrophenyl (TNP) modification of pork insulin (PI) at the A1 glycine position allows this molecule to stimulate a proliferative response in H-2b (B10) mice. We now show that this antigen stimulates low IgG responses in the same strain of mice. Our results show that T-cell help and proliferation may therefore be regulated independently. [ABSTRACT FROM AUTHOR]

MRL-+, MRL-lpr and B6-lpr have been shown to be useful models in studying systemic lupus erythematosus. MRL-lpr and B6-lpr differ from their congenic counterparts by the presence and expression of the homozygous recessive lymphoproliferation (Ipr) gene. One manifestation of this gene is a massive Tall proliferation that results in a generalized lymphadenopathy in older animals. A paradox that has developed out of the work utilizing the congenic mice is that the gene responsible for lymphoproliferation also appears to be responsible for the inability of T cells to respond to proliferative signals in vitro. In this paper we investigate the basis for this hypo responsiveness in antigen-induced activation of proliferation and antibody synthesis. We have demonstrated that spleen cells from both MRL-+ and MRL-lpr mice gave minimal stimulation in a one-way mixed lymphocyte reaction against allogeneic T cells. These findings were extended to include antigen-specific proliferation involving antigen that must be processed and presented to responder lymphocytes in a H-2 restricted manner. Thus, MRL- + and MRL-lpr spleen cells pulsed with ferredoxin also failed to stimulate ferredoxin-primed T cells from B10.Br animals in vitro. We then investigated whether any T-cell defect(s) was also contributing to this proliferative hyporesponsiveness. T lymphocytes from the spleen of MRL- +, 2-month-old MRL-lpr, and 6-month-old MRL-lpr were tested in a one-way mixed lymphocyte reaction. It was found that only the MRL- + T cells gave responses approaching normal, suggesting lpr gene involvement in T-cell non-responsiveness. This was confirmed by the demonstration of an age-onset T-cell proliferative hyporesponsiveness in B6. lpr mice. This lpr gene-linked non-responsiveness was also shown to extend to T-cell helper function in a positive allogeneic effect assay. We can conclude from these studies that antigenic non-responsiveness in MRL congenic mice can be explained by two defects: (i) the failure of antigen-presenting cells in MRL- + and MRL-lpr mice to provide the necessary signal(s) to immunocompetent T cells, this defect not being associated with the lpr gene, and (ii) the Ip gene controlled outgrowth of a unique T-cell population that cannot respond in our assay systems. [ABSTRACT FROM AUTHOR]

In the autologous Theileria mixed leucocyte reaction (MLR), irradiated Theileriaparva-infected cells induce proliferative responses in autologous peripheral blood mononuclear leucocytes (PBM)II irrespective of the immune status of the donor animal. In this paper we have analysed the cellular basis of this response in naive and immune cattle to determine the Thelleria specificity of the response. The magnitude of proliferation is dependent on two parameters, namely the presence or absence of monocytes in the responder population, and the phenotype of the parasitized stimulator cells, both of which appeared to be independent of the immune status of the donor animal. Monocyte-depleted responders invariably gave stronger proliferative responses but generated cytotoxicity from immune cattle that tended to be less genetically restricted. Marked differences were observed in the stimulatory capacity of cloned parasitized T-cell and non-T cell lines. At least part of this variation was associated with differences in the capacity of the parasitized cells to secrete soluble suppressive factors and possibly also stimulatory factors. Two observations indicated that, in immune cattle, part of the proliferative response in the autologous Theileria MLR is parasite-specific. First, stimulator cells fixed with glutaraldehyde stimulated proliferative responses in monocyte-depleted PBM from immune animals but not naive animals. Second, in autologous Theileria MLRs with intact PBM, genetically restricted cytotoxic cells were generated from immune but not naive animals. While inonocytes seem not to be required for induction of the parasite-specific component of the response, their absence from the assay when viable stimulator cells are utilized appears to enhance the non- specific component of the proliferative and cytotoxic responses. [ABSTRACT FROM AUTHOR]

MRL-lpr mice and their congenic counterparts MRL- + spontaneously develop an autoimmune disease that resembles systemic lupus erythematosus in humans. The two strains, although congenic, differ by a considerable number of disease parameters, reflecting the expression of the lpr autosomal recessive gene. One paradox that has developed out of the work utilizing the congenic mice is that the gene responsible for lymphoproliferation also appears to be responsible for the inability of T cells to respond to proliferative signals in vitro. In this paper we investigated a possible lpr gene-encoded macrophage defect in these mice. It was found, however, that both the MRL- + and MRL-lpr mice failed to divide in response to Con A, the lack of division correlating with an inability to secrete the growth promoter interleukin-2. In MRL- + mice and young MRL-lpr mice this non-responsiveness was corrected by the addition of normal CRA PEC. The defect could not be explained by a failure of M RL- + or M RL-lpr peritoneal exudate cells to quantitatively or qualitatively provide a source of interleukin-1 to Con A-activated T cells or by the possibility that the peritoneal exudate cells were blocked in their function by the presence of sera-derived autoantibodies and/or immune complexes on their membranes. We postulate that the inability of T cells to proliferate in MRL congenic mice can be explained by two defects: (i) the failure of antigen-presenting cells in MRL- + and MRL-lpr to provide the necessary signals to immunocompetent T cells, this defect not being associated with the lpr gene, and (ii) the lpr gene controlled outgrowth of a unique T-cell population that cannot respond in our assay system. [ABSTRACT FROM AUTHOR]

In this paper we describe the first in vitro response to the liver alloantigen F. The anti-F response serves as a valuable model for autoimmune phenomena since priming appropriate strains of mice (responders) with allogeneic but not syngeneic type F leads to autoantibody production. The in vitro system is based on the proliferation of T cells, from mice primed in vivo with F, when coincubated with splenic adherent cells (SAC) prepulsed with F in vivo. The system displays two important correlates of the in vivo antibody response to F:1.T cells from mice primed with syngeneic F do not proliferate when incubated with SAC prepulsed with syngeneic F and 2. Mice that do not make antibody responses to allo F in vivo (DBA/2) do not show in vitro proliferative responses. These findings indicate that the proliferative assay is a good in vitro model for the F response. [ABSTRACT FROM AUTHOR]

Plaque-forming cell responses and antibody titres to sheep red cells (SRC) were elevated in normally-fed irradiated adult mice reconstituted with spleen cells from young mice which had received a 4% albumin diet, compared with those given cells from normally-fed littermates. This was independent of the dose of cells transferred (5 × 106-4 × 107), the day of assay (3–10) and the duration of protein-deficiency (1 or 3 weeks). It suggests that the depressed responses produced by protein-deficient (donor) mice reported in earlier papers resulted from an impairment of reticuloendothelial function or a shortage of protein reserves, not directly associated with the spleen. As protein-deficiency (of the donor) accelerated the initiation of the adoptive response and increased the cell yields of the recipients it is considered likely that an enhancement of cell proliferation during the first 3-5 days after transfer was responsible for the elevated responses. [ABSTRACT FROM AUTHOR]

This paper describes experiments under-taken to determine more uniform conditions for the generation of cytotoxic lymphokine -- 'lymphotoxin' (LT) in differently stimulated rat lymphocyte cultures, and to compare the sensitivity of different test systems for detecting rat LT activity in vitro. Wistar rat lymphoid cells were activated by culture with phytohaemagglutinin (PHA) and mixed lymphocyte cultures (MLC) with semi-allogeneic (Wistar × August) F1 lymphoid cells. Lymphocyte supernatants harvested between 1 and 8 days were tested for theft effect on the metabolism and viability of cultured mouse fibroblasts (L-929 cells) by four methods: (i) inhibition of [14C]leucine incorporation; (ii) total and viable cell counts in test tube cultures ('macro test'); (iii) viable cell counts in microtitre plates ('micro test'); and (iv) chromium (51Cr) release from chromated L-cell monolayers. Cytotoxic effects on target cells of lymphocyte supernatants were evident after 3 days of PHA stimulation and 6 days of mixed lymphocyte culture, and the most sensitive indication of cytotoxic activity provided by inhibition of amino-acid incorporation and by loss of viable L cells from monolayers in tube cultures. In dilutions greater than 1:16-1:32 both cytotoxic supernatants exhibited a stimulating effect on target-cell proliferation. Stimulation of L cells growth was also observed when monolayers were exposed for 24 h to 'early' (24 h) PHA undiluted supernatants, At a later time of exposure to these supernatants a considerable loss of total and viable cells in the monolayers was evident. The results indicated that both cytotoxic and growth-stimulating lymphokines could be generated during activation of rat lymphocytes. A hypothesis is suggested whereby `lymphotoxin' activity in vitro arises from the sequential effects of stimulating and cytotoxic lymphokine, and whereby the balance of these effects in vivo might determine the response of fibroblasts involved in reactions of chronic allergic inflammation. [ABSTRACT FROM AUTHOR]