Giovanna Lombardi

HIV-1 gp120-dependent induction of apoptosis in antigen-specific human T cell clones is characterized by ‘tissue’ transglutaminase expression and prevented by cyclosporin A

We investigated the effect of cyclosporin (CsA) on HIV‐gpl20‐dependent induction of cell death by apoptosis of human T cell clones specific for influenza virus haemagglutinin and restricted by HLA‐DR1. Preincubation of the clones with gp120 induced a large inhibition of their proliferation which was paralleled by the induction of apoptosis. Exposure to the specific antigen alone was able to trigger apoptosis in a significant fraction of cells, this effect was potentiated by pretreatment with gp120. Apoptosis was characterized by the typical morphological changes and by the expression of ‘tissue’ Transglutaminase (tTG), one of the few characterized effector elements of programmed cell death. Interestingly, the tTG protein induction was detectable within the first 24 hours following the gp120 treatment and preceded the appearance of the typical apoptotic phenotype. Noteworthy, CsA treatment prevented the gp120‐dependent induction ofapoptosis by blocking the activation of the Ca2+‐dependent effector elements such as tTG.

Epstein-Barr virus-transformed B cells process and present Mycobacterium tuberculosis particulate antigens to T-cell clones.

We have analyzed the presentation of mycobacterial antigens by Epstein-Barr virus-transformed human B (EBV-B) cells to mycobacteria-specific T-cell clones and lines, and to purified resting T cells. EBV-B cells were able to process and present not only soluble forms of antigen, such as PPD and the expressate preparation of M. tuberculosis strain H37Rv, but also particulate forms of antigen, such as whole mycobacterial H37Rv or M. bovis organisms. Electron microscopy studies demonstrated the capacity of EBV-B cells to phagocytose mycobacterial cells in 18 hr and pulsing experiments confirmed that an 18-hr of incubation is required for an efficient processing and presentation of mycobacterial determinants to T cells. The processing of whole-H37Rv particulate antigen by EBV-B cells was inhibited by the lysosomotrophic compound chloroquine and by high doses of irradiation. Finally, the analysis of the presentation of soluble and particulate mycobacterial antigens by PPD-positive and PPD-negative EBV-B cell clones has shown a preferential presentation of both forms of antigen by PPD-positive EBV-B clones.

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.

Identification of a 35-kilodalton Mycobacterium tuberculosis protein containing B- and T-cell epitopes.

Screening of a Mycobacterium tuberculosis genomic DNA library in the lambda gt11 expression vector was carried out by using, as probes, sera from tuberculous patients and murine monoclonal antibody H61.3 recognizing a mycobacterial 35-kilodalton protein present only on the M. tuberculosis complex. The recombinant beta-galactosidase-fused protein present in the crude lysate induced the proliferation of T lymphocytes from patients with tuberculous pleuritis. As the recombinant insert contains an internal EcoRI restriction site, it was possible to identify two fragments, one proximal to the lacZ gene and 1.7 kilobases (kb) in size and the other distal to the lacZ gene and 2.2 kb in size. Southern blot analysis showed that both of them hybridized with the genomic DNA from M. tuberculosis and M. bovis but not with the DNA from other mycobacterial species. To perform extensive immunological studies, the amount of beta-galactosidase-fused protein being very low, we fused the 1.7-kb fragment to the N-terminal part of the gene coding for the DNA polymerase of bacteriophage MS2 in the expression vector pEx34. The fusion protein was partially purified, and subsequent Western blotting (immunoblotting) and T-cell proliferation experiments confirmed the presence of B- and T-cell mycobacterial epitopes. Furthermore, to isolate the chromosomal region containing the 35-kilodalton gene, we constructed another mycobacterial genomic library in the lambda 2001 vector by cloning 15 to 20 kb of foreign DNA. Screening of this library was carried out by using 1.7- and 2.2-kb recombinant fragments as probes. Restriction maps of some clones isolated were determined.

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.

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes

The contributions of individual amino acids in the polymorphic beta chain and the conserved alpha chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the beta 1 domain and 11 positions in the alpha 1 domain. The beta 1-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR alpha 1 domain were based on the sequences of other human and mouse class II alpha chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the beta 1-domain alpha-helix caused substantial reduction in the responses of all of the clones. Substitutions at multiple positions in the beta 1-domain floor and in the alpha 1 domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the beta 1 domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules.

Limiting dilution analysis of T cell unresponsiveness to mycobacteria in advanced disseminated tuberculosis

Peripheral blood mononuclear cells from patients with advanced disseminated tuberculosis (Dis-TB) do not respond to purified protein derivative (PPD) measured as cell proliferation, lymphokine production and interleukin (IL)-2 receptor (Tac antigen) expression. Limiting dilution analysis revealed “multi-hit” curves and low frequencies of PPD-reactive T cells in cultures of Dis-TB, and “single-hit” curves and high frequencies of PPD-reactive T cells in cultures of patients with localized form of pulmonary tuberculosis. Moreover, a strict relationship between Tac antigen expression and ability of exogenous IL-2 to enhance bulk culture cell proliferation was observed in Dis-TB patients.

Immunology of tuberculosis: New directions in research

SummaryTuberculosis is still one of the major health problems in almost all over the world. Thus, new directions in basic and applied research on tuberculosis are under investigation. In this review we have provided recent data obtained in our laboratories on three main aspects of the immunology of tuberculosis, namely:i. the role of B lymphocytes in the processing and presentation ofMycobacterium tuberculosis antigens to T cells;ii. the activation and characterization of mycobacterial-specific T cell clones;iii. the T cell regulation of the immune response toM. tuberculosis. The analysis of the antigenic determinants ofM. tuberculosis relevant in the antimycobacterial immunity is the major goal of the WHO programme on the immunology of tuberculosis. In fact, the attempt to develop a second generation vaccine against this microorganism is now possible by analyzing recombinant genomic DNA libraries ofM. tuberculosis with monoclonal antibodies and T cell clones. In the near future, the identification of epitopes recognized by mycobacterial-specific T cells with helper, cytotoxic and suppressor functions will allow the preparation of recombinant and synthetic vaccines effective in the control of this disease.

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.

Measurement of the cytotoxicity of human lymphocytes using HeLa cells as target

Abstract A simple and convenient method is described for the determination of spontaneous, PHA-induced, and antigen-induced cytotoxic activities of human peripheral blood lymphocytes. It involves measuring [ 3 H]thymidine incorporation in HeLa cells as target. This methodology presents some advantages over the more commonly used 51 Cr-release test.