HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.

BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.

Enhanced interferon-gamma by CD8+ CD28- lymphocytes from HIV+ patients.

Flow cytometric analysis of T cells from HIV+ and normal individuals activated for 15 hr showed that the percentage of cells producing interferon-gamma (INFgamma) was enhanced approximately threefold (39 compared to 14%) in the HIV+ CD8+ population. Activation modes, other than anti-CD3 with PMA, were ineffective, and in no case did the percentage of HIV+ CD4+ T cells show increased INFgamma production over controls. Enhanced INFgamma production was not induced by either anti-CD3 or PMA alone, or anti-CD3 or ConA with anti-CD28, or enhanced by N-acetylcysteine. In contrast to INFgamma production, the percentage of CD4+ T cells producing interleukin-2 (Il-2) greatly exceeded that of the CD8+ T cells. The results from flow cytometry analyses of HIV+ CD8+ T cells was supported by quantitative analysis of INFgamma mRNA (by PCR) and INFgamma secretion by ELISA. These methods showed a sixfold and three- to fivefold increase, respectively, on a per cell basis. As HIV infection progresses, as shown by loss of CD4+ T cells, the proportion of CD8+ CD28- T cells increases, and it is this T cell subset that is responsible for 80% or more of the enhanced INFgamma production. The enhanced INFgamma in HIV+ patients derives from two factors: the increase in CD8+ CD28- cells to 70% and the percentage producing INFgamma (60%, compared to 21% for CD8+ CD28+ cells). Our findings of a substantial increase in INFgamma production in HIV infection arising from the increased number of CD8+ CD28- T cells are compatible with clinical studies which show elevated INFgamma in HIV+ serum and INFgamma producing CD8+ T cells dominating HIV+ lymph nodes. We also found a significantly decreased proliferative response of the HIV+-derived CD8+ T cell fraction with coactivator anti-CD-28, in contrast to PMA (with anti-CD3), which is probably a reflection of the diminished population of CD8+ CD28+ T cells in HIV+ donors compared to normal donors (30.7 compared to 67.9%).

Cancer gene therapy mediated by CTS1, a p53 derivative: advantage over wild-type p53 in growth inhibition of human tumors overexpressing MDM2.

Recently, a new p53 derivative has been designed, namely chimeric tumor suppressor 1 (CTS1), in which the p53 domains that are known to mediate p53 inactivation have been replaced. In this study, the antitumoral activity of CTS1 mediated by adenovirus vector has been evaluated in comparison with a p53 adenovirus vector in various human tumor cell lines. In vitro, in terms of cell growth inhibition, the CTS1 vector was significantly (P < .01) more efficient (2- to 7-fold) than the p53 vector in tumor models overexpressing an inhibitor of p53, murine double minute-2. This result was confirmed in vivo in a pre-established tumor developed in nude mice. In an osteosarcoma model overexpressing murine double minute-2, we have shown a significantly (P < .05) higher tumor growth delay with the CTS1 vector compared with the p53 vector (25.6 days compared with 12.4 days). Furthermore, both in vitro and in vivo, we have shown that this higher inhibition of tumor growth with the CTS1 vector was correlated with a higher induction of apoptosis. Therefore, CTS1 is a potentially improved tumor suppressor gene for the treatment of human tumors resistant to wild-type p53 gene therapy.

The proliferative response of HIV+ T-cells (CD4+ and CD8+) are severely suppressed via CD28 coactivation.

Several studies have suggested that regulation of expression of the costimulatory molecule CD28 on the T-cell surface may play an important role in AIDS pathogenesis. In a study of T-cells from HIV+ donors, we find that activation with anti-CD3 plus anti-CD28 results in a mitogenic response which was approximately 86% suppressed for both CD4+ and CD8+ T-cells when compared to normal control cells. With PMA costimulation (instead of anti-CD28), the anti-CD3 response was suppressed much less, by 64 and 61%, respectively. With Con A as opposed to CD3 stimulation, the degree of suppression was less with either coactivator but still more severe with CD28 than with PMA coactivation. It has been reported that the CD28 subset of CD8+ T-cells is diminished in HIV+ individuals and could account for these results. It is possible as well that the CD28 costimulatory pathway in the CD4+ T-cells particularly is altered due to intervention by the HIV. While our data do not differentiate between these two possibilities, it show that the immune status is compromised in the HIV+ individual not only in terms of number of CD4+ T-cells, but in their activation response as well.

Mechanisms of action of intravenous immune globulin in immune-mediated diseases.

Intravenous immune globulin (IVIG) exhibits a number of immunomodulatory properties that are mediated by the Fe portion of IgG and by the spectrum of variable (V) regions contained in the immune globulin preparations. Five predominant and non-exclusive mechanisms of action have been proposed to account for the immunomodulatory effects of IVIG in immune-mediated diseases: (i) functional blockade of Fc receptors on splenic macrophages; (ii) inhibition of complement-mediated damage, an effect that is dependent on the ability of IgG to bind C3b and C4b and thus reduce the number of activated complement fragments that may deposit on target surfaces of complement activation; (iii) modulation of the production of cytokines and cytokine antagonists; (iv) neutralization of circulating autoantibodies by complementary (e.g. anti-idiotypic) antibodies in IVIG, a mechanism that accounts for the rapid decrease in titre of circulating autoantibodies that is often observed within hours following the infusion of IVIG; (v) selection of immune repertoires, a complex set of effects that may be observed in individuals receiving IVIG far beyond the half-life of the infused immunoglobulin and that is directly relevant to the ability of IVIG to, for example, suppress autoantibody-producing clones in patients with antibody-mediated autoimmune disease and modulate graft versus host disease (GVHD). IVIG has been shown to downregulate or activate B-cell clones expressing surface IgG that is complementary (anti-idiotypic) to V regions of antibodies present in IVIG. IVIG has been shown also to interact with surface molecules of T cells that are essential to immune regulation, such as the alpha beta TCR, CD5, CD4, non-polymorphic determinants of MHC class I molecules and adhesion molecules of T and B cells. The complex interactions of IVIG with functional molecules of cells of the immune system are relevant to its therapeutic effects in T cell- as well as B cell-mediated diseases and indeed, to our understanding of the physiological role of normal IgG and antibody networks in controlling autoreactivity in healthy individuals.

Antibodies to a conserved region of HLA class I molecules, capable of modulating CD8 T cell-mediated function, are present in pooled normal immunoglobulin for therapeutic use.

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune diseases and the prevention of infections and of graft versus host reactions in recipients of allogeneic bone marrow transplants. The immunomodulatory effects of IVIg are largely dependent on their ability to interact with membrane molecules of lymphocytes. We report here that IVIg recognizes the B07.75-84 peptide, corresponding to a conserved region of the alpha I helix of the first domain of HLA-B7 01, which represents a nonpolymorphic determinant of HLA class I molecules. Intact IVIg and its F(ab')2 fragments bound to the peptide as well as to purified soluble HLA and to HLA on a human T cell line. Binding of IVIg to HLA was assessed by ELISA, immunofluorescence, and real-time analysis of the interaction using the BIAlite system. The binding of antipeptide antibodies to HLA was inhibited by free peptide. Antipeptide antibodies isolated from IVIg by affinity chromatography inhibited CD8 cell-mediated cytotoxicity of an influenza virus-specific human T cell line. The presence in IVIg of antibodies to critical regions of HLA class 1 molecules suggests a possible role for IVIg in modulation of class-I-restricted cellular interactions in the immune response.