Reduced production of both Th1 and Tc1 lymphocyte subsets in atopic dermatitis (AD).

An imbalance of interferon-gamma (IFN-gamma)-bearing CD4+ T (Th1) cells in the pathogenesis of AD is well recognized; however, a possible role in AD for CD8+ T cells secreting Th1-like cytokines (Tc1) has not been properly addressed. In this study, two- and three-colour FACS analysis allowed us to discriminate the Th1 from the Tc1 subset. AD patients had half the number of IFN-gamma-producing circulating T cells (P < 0.005; 13.6 +/- 1.9% (mean +/- s.d.)) compared with normal donors (25.0 +/- 2.4%). Specifically, both Th1 (4.8 +/- 0.7%) and Tc1 (8.1 +/- 1.1%) cells in AD were decreased compared with Th1 (8.8 +/- 0.8%) and Tc1 (15.0 +/- 1.5%) cells in controls. Moreover, at the mRNA level, the ratios of IFN-gamma/IL-4 and IFN-gamma/IL-10 were lower in cells from AD patients compared with controls. In conclusion, the decrease of IFN-gamma-producing T lymphocytes in AD is due to a reduction in both Th1 and Tc1 IFN-gamma-secreting cells; this may not only contribute to the over-production of IgE, but also explain the high incidence of cutaneous infections observed in AD patients.

HIV p17 enhances lymphocyte proliferation and HIV-1 replication after binding to a human serum factor.

OBJECTIVE: To analyse the role of recombinant HIV-1 protein p17 in the modulation of cell activity. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence or absence of p17 with mitogens such as phytohaemagglutinin or interleukin-2 and their response assayed by cell proliferation. Cross-linking experiments were employed to investigate the presence of a binding between p17 and factor(s) present in human serum. An immunoenzymatic assay for p24 antigen detection was used to analyse the effect of the addition of exogenous p17 to cultures of PBMC infected with HIV-1 in vitro. RESULTS: Purified recombinant p17 protein at a concentration of 0.25 microg/ml significantly increased the proliferation of preactivated PBMC obtained from healthy donors. This effect was obtained by binding p17 to factor(s) present in human serum and observed on both CD4+ and CD8+ T cells. Recombinant p17 also induced an increased rate of HIV-1 replication, probably due to enhanced T-cell proliferation. The activity of p17 protein was inhibited by anti-p17 antibodies generated by injecting recombinant p17 in rabbits, but not by human antibodies generated during the natural course of HIV infection. CONCLUSION: Characterization of the human factor(s) and identification of the interacting p17 epitope(s) will improve our understanding of the mechanisms used by HIV to efficiently replicate in our organisms.

Lack of polarized type 1 or type 2 cytokine profile in asymptomatic HIV-1-infected patients during a two-year bimonthly follow-up.

The production of type 1 (interferon or IFN-gamma) and type 2 (interleukin or IL-4 and IL-10) cytokines by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic human immunodeficiency virus-seropositive (HIV+) patients untreated with any antiviral, antibacterial or antimycotic drugs, and from healthy individuals, was evaluated by quantitative ELISA. Patients who were HIV+ were characterized by the absence of abnormal cytokine production. The level of each cytokine differed among individuals in the same group with intersubject variations greater for HIV+ patients than for healthy individuals. The longitudinal evaluation of IFN-gamma, IL-4 and IL-10 production showed intrasubject variations which were particularly marked in HIV+ patients. Accordingly, HIV+ patients and, to a lesser extent, healthy individuals were characterized by a wide spectrum of possible profiles, which were confined to type 0 phenotype. In HIV+ patients no correlation was found between each cytokine level and the number of CD4+ T cells, not even in those with a falling CD4+ T-cell count and clinical symptoms.

Contribution of CD4+, CD8+CD28+, and CD8+CD28- T cells to CD3+ lymphocyte homeostasis during the natural course of HIV-1 infection.

The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.

T cells from individuals in advanced stages of HIV-1 infection do not proliferate but express activation antigens in response to HIV-1-specific antigens.

Like T cells from healthy subjects, those of HIV-1-infected patients are capable of expressing activation antigens on their surface after antigenic or mitogenic stimulation, but their proliferative activity is strongly reduced or even absent, especially in patients with advanced stages of the disease. The characteristic of expressing activation antigens in response to different stimuli in the absence of cell proliferation is shared by CD4+ and CD8+ T-cell subsets from HIV-1-infected patients. The number of T cells capable of expressing CD25 and CD71 in response to HIV-1-related antigens but not of proliferating increased significantly with the progression of the disease, but the number of T cells capable of expressing the two activation antigens in response to the classic tetanus toxoid recall antigen decreased. The higher numbers of T cells capable of responding to HIV-1-related antigens in conjunction with a reduction in the number of T cells responding to recall antigens may explain the occurrence of different infections, including opportunistic microorganisms, during the more advanced stages of HIV-1 infection. Because the increase in the number of HIV-1 antigen-responding T cells (defined by CD25 and CD71 activation antigen expression) is a characteristic of symptomatic HIV-1-infected patients, expression (by flow cytometry) of these activation antigens on T cells in response to HIV-1 antigens could be used as a new marker of disease progression.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.

The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.

Heat-killed Bacillus subtilis inhibits T-cell proliferative response to mitogens and recall antigens.

Heat-killed vegetative forms of Bacillus subtilis were found to impair considerably the capacity of human T-lymphocytes to secrete interleukin-2 (IL-2) and to proliferate (in terms of [3H]thymidine incorporation) after phytohaemagglutinin (PHA) stimulation. B. subtilis was also found to interfere with T-cell proliferation induced by concanavalin A (Con A) and the recall antigen tetanus toxoid (TT). The suppressive activity was dependent on bacterial concentration, and was not ascribed to mitogen, medium-nutrient absorption or cell killing. Moreover, B. subtilis did not interfere with mitogen-induced IL-2 receptor expression on the T-cell surface. On the other hand, B. subtilis did not interfere with T-cell proliferation induced by phorbol myristate acetate (PMA) and ionomycin stimulation. All data obtained suggest the binding of B. subtilis subcomponents to- or very close to-the T-cell receptor (TCR). Identification and purification of the basic structure(s) or component(s) of B. subtilis with TCR antagonist activity in vitro will help to exploit different aspects of T-cell activity and development, and possibly, will provide a means of specific control or modification of the immune response.

Characterization of T cell subsets involved in the production of IFN-gamma in asymptomatic HIV-infected patients.

Cells capable of interferon (IFN)-gamma synthesis following mitogenic stimulation can be detected and quantified by a recently developed immunofluorescence assay and flow cytometric analysis. The production of IFN-gamma was investigated in a cohort of 20 asymptomatic human immunodeficiency virus (HIV)-seropositive patients with normal numbers of CD4+ lymphocytes, and in 10 healthy subjects. About 60% of asymptomatic stage A1 patients had increased percentages of blood lymphocytes capable of IFN-gamma synthesis, as compared to healthy subjects. The difference reflected the relatively higher numbers of CD8+ cells, in particular the CD8+ T cell subset lacking CD28 antigen expression. The strong correlation between the CD4+/CD8+ ratio and the CD8+CD28+/CD8+CD28- ratio suggests either a role for CD4+ cells in controlling the CD28+ phenotype or a role for CD8+CD28- cells in the decline of CD4+ lymphocytes. The peculiar ability of CD8+CD28- cells to produce high amounts of IFN-gamma, as compared to CD8+CD28+ cells, supports the hypothesis that the CD8+CD28- lymphocytes constitute a population that is functionally distinct from their double-positive counterparts.

IFN-gamma restores HIV- and non-HIV-specific cell mediated immune response in vitro and its activity is neutralized by antibodies from patients with AIDS.

The addition of IFN-gamma to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV-infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), and allogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV-seropositive patients specifically interfered with IFN-gamma-induced cell proliferation in response to recall antigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN-gamma antibodies. Data obtained demonstrate that IFN-gamma can restore the cell-mediated immunity of a number of asymptomatic HIV+ individuals in vitro, while IFN-gamma antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.

CD4+ and CD8+ lymphocytes of patients with AIDS synthesize increased amounts of interferon-gamma.

Individual cells capable of interferon-gamma (IFN-gamma) synthesis are easily detected by immunofluorescence and flow cytometric analysis using an anti-IFN-gamma monoclonal antibody as specific reagent. By IFN-gamma flow cytometry assay, we demonstrated that HIV-seropositive patients, starting at the early stage of viral infection, generally have an increased percentage of lymphocytes potentially able to produce IFN-gamma, compared with healthy blood donors. IFN-gamma expression in patient lymphocytes was observed to increase with the progressive stages of HIV infection, with the highest figures occurring in stage C patients. Such increased IFN-gamma expression involved both CD4+ and CD8+ T cell subsets. Most interestingly, we found patients at the same stage of HIV infection who had similar numbers of total and CD4+ lymphocytes but highly different percentages of lymphocytes potentially capable of producing IFN-gamma.

Monoclonal antibodies against antigens exposed on the surface of vegetative forms and spores of Myxococcus virescens.

Twelve monoclonal antibodies (mAbs) directed against cell-surface antigens of Myxococcus virescens cells were developed and partially characterized. All of them recognized multiple, diffuse proteic bands in Western blot and four were also reactive to living bacteria, as assessed by flow cytometry. The four latter mAbs recognized antigens common to a number of vegetative forms and spores. The selective expression of proteins recognized by mAbs on the microorganisms and the possible applications of mAbs to the study of myxobacterial cell interaction are discussed.

Expression of CD28 on CD8+ and CD4+ lymphocytes during HIV infection.

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, co-stimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4+ and CD8+ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4+ and CD8+ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8+CD28- cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.