Fabrizio Manca

HIV envelope glycoprotein, antigen specific T-cell responses, and soluble CD4

Specific T cells stimulated by antigen presenting cells (APC) pulsed with antigen in the presence of HIV were no longer detectable with a functional assay, which suggests that HIV has been transferred from APC to the specific activated T cell via an antigen-dependent mechanism to exert its cytopathic effect on activated T cells. In contrast soluble gp120 inhibited antigen-driven proliferation, but this action was reversible and could be blocked by soluble CD4. Thus the chief mechanism of HIV pathogenesis may be gp120/CD4 interaction and HIV may be pathogenic mainly as a producer of gp120.

The role of host immune responses in determining the outcome of HIV infection

The progression of disease following infection with human immunodeficiency virus (HIV) correlates with an activated immune system and would appear to depend to some degree on the immunogenetics of the host. Here, Michael Westby, Fabrizio Manca and Angus Dalgleish discuss the evidence for HLA determination of clinical outcome and the potential implications of a restricted T-cell receptor repertoire for pathogenesis.

Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii

Our genetic system for expression of heterologous proteins on the surface of the Gram-positive bacterium Streptococcus gordonii was used to express a human T-helper epitope of HIV-1 envelope glycoprotein gp120. In previous work on the naive repertoire of human T-helper cells, it was shown that a 15-amino acid synthetic peptide of the HIV-1 gp120 sequence contained an immunodominant T-helper epitope. Synthetic DNA coding for this peptide was cloned in frame within the gene for the streptococcal surface protein M6, and the gene fusion was integrated by transformation into the chromosome of S. gordonii. The expected M6-gp120 fusion protein was found to be expressed on the surface of the recombinant streptococci. To test whether the T epitope could be recognized by T cells when expressed on the bacterial surface within the context of M6, recombinant bacteria were used as antigen in proliferation assays to stimulate the 15-amino acid-specific human T-helper clone, in the presence of autologous antigen-presenting cells. Bacteria expressing the T epitope were efficiently recognized by the T cells in culture. In proliferation assays, 10(6)-10(7) bacteria induced responses comparable to those obtained by standard amounts of synthetic peptide (0.02-0.2 micrograms). Recombinant S. gordonii, a candidate for a live vaccine vector, appeared suitable for delivering T epitopes to the immune system.

.An enzymatically active antigen-antibody probe to measure circulating immune complexes by competition. I. Use of Escherichia coli beta-galactosidase in the probe and of bovine conglutinin as the complex-binding reagent.

Abstract An antigen-antibody complex obtained by simple mixing of specific rabbit antiserum and Escherichia coli β-galactosidase is used as a “probe” to determine immune complexes in biological fluids in a novel competitive assay. The complex-recognizing unit, immobilized on plastic beads, is bovine conglutinin, a large protein with affinity for C3bi bound to the complex. The intrinsic enzyme activity of the probe provides an ecologically acceptable signal with high resolution. The new assay has been tested in clinical trials and, by using a battery of recognition units, it should provide a tool for “finger-printing” complexes found in different diseases according to their structures and functional characteristics.

Attenuated Listeria monocytogenes carrier strains can deliver an HIV-1 gp120 T helper epitope to MHC class II-restricted human CD4+ T cells.

Listeria monocytogenes is a facultative intracellular pathogen which, following uptake by macrophages, escapes from the phagosome and replicates in the cytoplasm. This property has been exploited using recombinant L. monocytogenes as a carrier for the intracytoplasmic expression of antigens when MHC class I‐restricted cytotoxic T lymphocyte responses are required. Much less is known of the ability of these bacteria to trigger MHC class II‐restricted responses. Here, we demonstrate that after ingestion of L. monocytogenes expressing a T helper epitope from the gp120 envelope glycoprotein of HIV, human adherent macrophages and dendritic cells can process and present the epitope to a specific CD4+ T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR Vβ 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well‐established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

Inhibition of the accessory function of murine macrophages in vitro by cyclosporine

The accessory function of macrophages, which is strictly related to the induction of T cell activation, has been studied to determine whether it is affected by cyclosporine (CsA). Irradiated spleen cells, used as a source of macrophages, were pulsed overnight with β-galactosidase (GZ) in the presence of CsA. After washing of the pulsed macrophages, cells from a GZ-specific T cell line were added to cultures and 3H-thymidine incorporation was measured 72 hr later. We found that 500 ng/ml CsA present during macrophage pulsing with GZ reduced T cell proliferation to 5%. On the other hand, 100 ng/ml CsA almost completely abrogated the proliferative response when present for the duration of the culture. Similar results were also obtained using antigen-pulsed peritoneal-adherent macrophages to stimulate the T cell line to proliferate, or a T hybridoma clone to produce interleukin-2 (IL-2). The possibility that CsA actually affects interleukin-1 (IL-1) production by macrophages by inhibiting uninvolved T cells could be ruled out. We conclude that CsA-induced inhibition of T cell functions (proliferation and IL-2 production) is partially due to the effect of the drug on the accessory function of macrophages. This immunosuppressive mechanism of action of CsA on macrophages has not been previously described.

Recognition of Antigenic Clusters of Candida albicans by T Lymphocytes from Human Immunodeficiency Virus-Infected Persons

The fine specificity of the cellular immune response to Candida albicans (i.e., recognition of different antigenic components) between normal controls and human immunodeficiency virus-infected patients in various stages of disease was compared. C. albicans-specific T cells, enriched by antigen stimulation and interleukin-2 expansion, were challenged with antigenic fractions of different molecular weight obtained by SDS-gel fractionation of C. albicans extracts in the presence of autologous mononuclear cells as antigen-presenting cells. Proliferative responses showed similar patterns of reactivity between controls and category A and B seropositive subjects. Category C patients with concurrent C. albicans infections did not give rise to C. albicans-specific T cell lines, confirming the T cell defect. Patients without clinically evident C. albicans infection had a low but broad reactivity pattern of C. albicans-specific T cells. These results suggest that depletion of C. albicans-specific T cells, independent of their fine specificity, occurs along with disease progression.

Generation of Cytomegalovirus (CMV)—Specific CD4 T Cell Lines Devoid of Alloreactivity, by Use of a Mixture of CMV—Phosphoprotein 65 Peptides for Reconstitution of the T Helper Repertoire

BACKGROUND CD8 cells specific for cytomegalovirus (CMV) provide valuable support in immunocompromised patients. Recent studies have focused on the generation of cytotoxic T lymphocyte (CTL) lines by use of new biotechnological techniques. Yet CD4 cells have been neglected, even though they contribute to the persistence of adoptively transferred CTLs. METHODS We identified novel T helper (Th) peptides recognized by CD4 cells on the immunodominant protein pp65. These peptides were used as a mixture to generate CD4 cell lines. RESULTS The peptide library, which, theoretically, is recognized by 85% of white individuals on the basis of the frequency of the relevant human leukocyte antigen class II alleles, was stimulatory for 82% of pp65 responders. T cell lines generated by use of the pool recognized protein antigens. Selection for CMV-specific cells resulted in rapid depletion of allospecific T cells. Selection with individual peptides allowed further selection against potential cross-alloreactivity. Cultured CD4 cells showed no signs of functional senescence. CD4 cells activated by use of a Th peptide helped expand CMV-specific CTLs. CONCLUSIONS By use of a simple procedure, an immunodominant peptide library can generate T cell lines from allodonors, for the perspective reconstitution of the Th repertoire in immunocompromised hemopoietic stem-cell transplant recipients.

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis

Expression of HLA and CD1b molecules was investigated in the THP‐1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2 – 3 days, cell surface expression of HLA class II and CD1b was drastically down‐modulated, whereas HLA class I expression was up‐modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down‐modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down‐modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN‐γ, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP‐1 cells to process and present mycobacterial antigens to HLA‐DR‐restricted antigen‐specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen‐presenting molecules.