Orsola Pugliese

HIV-1 gp120 Accelerates Fas-Mediated Activation-Induced Human Lamina Propria T Cell Apoptosis

Intestinal mucosa represents an important portal of entry of HIV and a site of virus reservoir and active replication. Recently, in HIV patients, an early depletion of intestinal lamina propria T lymphocytes (LPT) has been described. HIV-1 gp120 has been demonstrated to promote apoptosis in noninfected isolated peripheral blood T cells, therefore we investigated whether gp120 modulates apoptosis of normal human intestinal lamina propria T cells. Purified T cells were obtained by immunomagnetic negative selection from human lamina propria mononuclear cells isolated from surgical specimens by enzymatic procedure. Cells were incubated with or without recombinant gp120 (10 μg/ml) and cultured either in the absence of any stimulus or in the presence of plate-bound anti-CD3 Ab (OKT3) or soluble anti-CD2 Ab (T112 + T113). Apoptosis was assessed by flow cytometric analysis after propidium iodide staining. We demonstrated that preincubation of normal LPT cells with HIV-1 gp120 accelerates the apoptosis observed during CD2-pathway stimulation of LPT cells. This process is mediated by Fas/Fas ligand interaction and related to an increased induction of Fas ligand mRNA by gp120. Therefore HIV-1 gp120 could contribute to the depletion of noninfected LPT cells inducing a premature cell death.

Cholera toxin B subunit promotes the induction of regulatory T cells by preventing human dendritic cell maturation

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. There is also good evidence that CTB acts as an immunosuppressant, as it is able to down‐modulate human monocyte/macrophage cell line activation and to suppress Th1‐type responses. In the present study, we examined the possibility that recombinant CTB (rCTB) may affect human dendritic cell (DC) functions in response to LPS stimulation and may induce the generation of DC with the capacity to generate CD4+ regulatory T cells (Tregs). Our findings show that rCTB partially prevents the LPS‐induced maturation process of monocyte‐derived DC (MDDC) and decreases their IL‐12 production with no relevant effect on IL‐10 production. LPS‐stimulated MDDC pretreated with rCTB are able to promote the induction of low proliferating T cells, which show an enhanced IL‐10 production associated with a reduced IFN‐γ production and the same high levels of TGF‐β as the control. These T cells suppress proliferation of activated autologous T cells. Transwell experiments and blockade of IL‐10R and TGF‐β showed that the immunomodulatory effect is mediated by soluble factors. Thus, T cells induced by rCTB‐conditioned MDDC acquire a regulatory phenotype and activity similar to those described for type 1 Tregs.

A gp120 HIV peptide with high similarity to HLA class II β chains enhances PPD-specific and autoreactive T cell activation

The recent report that anli‐gp120 antibodies can be induced by allogeneic stimuli in experimental animals in the absence of HIV, has focused attention on the structural similarities between gp120 and MHC. Here we report that some HIV+ individuals develop antibodies which similarly react with the gp120 HIV sequence (aa 254–263) and with the HLA‐DR β chains (aa 142–151). As these two peptides share a high level of similarity, we have investigated the role of this gp120 region on HLA class II mediated T cell recognition. The synthetic peptide corresponding to the gp120 HIV sequence aa 254–263 has been tested on T cell line (TCL) activation. Both the PPD‐specific and the self‐HLA reactive TCL proliferation increased in the presence of this peptide. Prepulsing experiments indicate that this enhancing effect carried out by HIV peptide is exerted at the level of antigen presentation. Moreover, the specificity of this interaction is supported by the fact that a MoAb specific for this HIV peptide blocked the autoreactive TCL proliferation, similarly to the inhibition carried out by anti‐class II antibody. These data support the hypothesis that the functional homology between the HIV peptide and the HLA β chain described may be involved in the pathogenesis of AIDS.

Mechanism of action of an antigen nonspecific inhibitory factor produced by human T cells stimulated by MPPS and PPD

Human T lymphocytes cultured in vitro for 5 days with C. albicans purified polysaccharide (MPPS) and with purified protein derivative (PPD) from M. tuberculosis produce an antigen nonspecific inhibitory factor(s) (nsINH). nsINH blocks antigen-driven cell proliferation and the development of natural killer cells (NK) when added at the beginning of peripheral blood mononuclear cell culture. Analysis of the mechanism of action shows that nsINH inhibits the production of interleukin 2 (IL-2), the expression of IL-2 receptor (Tac antigen), and the synthesis of immune interferon (IFN). The biochemical characterization of nsINH shows that the suppressive activity is acid (pH 2.5) and temperature (56 degrees C) resistant. Gel filtration analysis indicates a molecular weight of 30-35K and 60-65K. These results suggest a role for nsINH in the down regulation of the lymphokine cascade.

A Limiting-Dilution Analysis of Activated Circulating B Cells in Crohn's Disease

In the present study the spontaneousin vitro production of immunoglobulins G, A, and M by peripheral mononuclear cells was evaluated, in patients with Crohns disease, in relation to the state of B-cell activation and further characterized by limiting-dilution analysis. A total of 25 patients with Crohns disease and 10 healthy controls was studied. The proportion of the transferrin receptor-bearing cells in the B7+ subset was higher in active Crohns disease patients than in either those with quiescent disease or controls. There was a significant rise in thein vitro IgG, IgM, and IgA production in patients with untreated active Crohns disease compared to either those with untreated quiescent disease or controls. When patients were followed up from the active phase to clinical remission, a significant decrease in the production of IgG and IgM was observed. IgA levels also showed a decrease, although not reaching statistical significance. When the Ig production was analyzed by limiting dilution, no difference was observed between patients and controls in terms of either precursor frequency of Ig-producing cells or patterns of frequency distribution. In both patients and controls a biphasic limiting-dilution profile was observed. This study shows that a significant B-cell activation is present in Crohns disease patients, which is accompanied by an increase in immunoglobulin production. This study also indicates that in Crohns disease the increased immunoglobulin production is related to an augmented B-cell clone size rather than to an increased precursor frequency.

Increased autoreactive T cell frequency in tuberculous patients.

The development of putative self-MHC-reactive T cells and their precursor frequency was estimated in peripheral blood lymphocyte cultures stimulated in vitro with PPD. The role of foreign antigen in the generation of self-MHC-reactive T cells in vivo was analyzed by comparing the frequency of autoreactive T cells in the peripheral blood of tuberculous patients with that observed in healthy individuals. It was found that PPD in vitro and Mycobacterium tuberculosis infection in vivo increased substantially the generation of autoreactive T cells. Autoreactive T cell clones were shown (1) to recognize self MHC class II products; (2) to release gamma interferon in the absence of exogenous antigen, and (3) to express autocytotoxic activity. All these findings suggest that self-MHC-reactive T cells may be involved in the inflammatory response to M. tuberculosis.

The antigen-specific induction of normal human lymphocytes in vitro is down-regulated by a conserved HIV p24 epitope

Synthetic peptides containing amino acid sequence 218-238 of the core protein p24 of human immunodeficiency virus type 1 (HIV-1) and progressively shorter sequences at its C-terminus, were tested for their effect on antigen dependent in vitro responses of peripheral blood lymphocytes (PBL) from normal human donors. A peptide as short as 7 amino acids, corresponding to a highly conserved sequence, was able to inhibit in a dose-dependent manner the induction of a specific primary antibody response to the sheep red cell (SRC) antigen, as well as the proliferative response to recall microbial antigens. The results of this study constitute additional evidence of the immunoinhibitory effects of HIV components and may help to unravel some of the pathogenic mechanisms of AIDS. Moreover, they are of potential relevance for the development of immunoprophylactic and therapeutic strategies.

A purified polysaccharide isolated from Candida albicans induces antibody response in vitro by human peripheral blood lymphocytes and discriminates between sera from normal and Candida albicans-infected individuals.

A purified polysaccharide extracted from Candida albicans (MPPS), stimulates in vitro synthesis of specific antibodies by human peripheral blood lymphocytes. These antibodies can be detected by a sensitive enzyme-linked immunoassay. The same assay can be applied to the quantitation of anti-candida antibodies in serum. Statistically significant differences were found between sera of normal and candida-infected individuals.

Further evaluation of autoreactive T cells in hydatid patients

We evaluated the peripheral autoreactive response in patients with Echinococcus granulosus who showed a negative humoral response as compared to seropositive patients and healthy controls. For this purpose, a limiting dilution analysis (LDA) of autologous mixed lymphocyte cultures was established to both estimate frequency of autoreactive T cells and, by analysing the shape of the curves, to clarify the mechanisms that underlie the autoreactive response. Different LDA curves were observed between healthy controls and patients, suggesting that different cell interactions are involved in the two populations. More interestingly, all hydatid patients, independent of their humoral response, showed a higher number of autoreactive T cells than controls. Precisely, subjects with a negative humoral response showed a range of values for autoreactive T cells exactly between the value ranges observed in seropositive and normal subjects. The present data also show that the increase of autoreactive T cells in hydatid patients correlates with the production of specific antibodies.

Regulation of self-major histocompatibility complex reactive human T-cell clones

The proliferative response of human T-lymphocyte clones, (TLC) specific for self-major histocompatibility complex (MHC) products either alone or associated with PPD epitopes are inhibited in vitro by dexamethasone (DEX) and by a non-specific inhibitory factor(s) (nsINH) produced by PPD-activated T-cells. The inhibiting effect has been investigated by preincubating autoreactive and PPD-specific TLC with nsINH or DEX. Results obtained indicate that T-lymphocytes are the target of these two immunoregulatory molecules. Moreover, the addition of exogenous recombinant interleukin 2 (rIL-2) substantially reverses the inhibition observed in both nsINH- or DEX-treated cultures.