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Biomedical subjects

F Manca

Publications and source records attributed to F Manca.

At least 19 recordsLinked to original sources
PubMed1997

Priming of human CD4+ antigen-specific T cells to undergo apoptosis by HIV-infected monocytes. A two-step mechanism involving the gp120 molecule.

The study of the pathology of HIV-1 infection in chimpanzees supports the idea of the crucial role of HIV-infected monocytes in the pathogenesis of AIDS, although viral mechanisms that lead to T cell dysfunction and deletion during HIV infection are still unclear. We show here that HIV-1-infected antigen-presenting monocytes (APCs) are able to prime in vitro non-HIV-infected antigen-specific CD4+ T cell lines or peripheral blood CD4+ T cells to undergo apoptosis after antigen-specific restimulation. The priming of T cells for apoptosis occurs in the absence of HIV replication in the T cells. Priming for apoptosis required two concomitant signals present on the same APC, an antigenic stimulus and a second signal provided by the HIV gp120 protein as demonstrated by the use as APCs of EBV-LCLs infected with different recombinant deleted proviruses or transfected with different HIV proteins. These results provide a mechanism for the priming for apoptosis of T cells in HIV-infected patients, implicating a role for HIV-infected APCs in the induction of T cell dysfunction and depletion in AIDS.

Antigen-Presenting Cells↗
PubMed1997

Epitope context and reshaping of activated T helper cell repertoire.

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST-peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor V beta usage and fine specificities of selected T cells.

Antigen Presentation↗
PubMed1996

Antigenicity of HIV-derived T helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection.

T helper (Th) epitopes can be included in a recombinant protein with B and CTL epitopes to create more effective immunogens. To determine whether the antigenicity of HIV Th epitopes is preserved in this altered molecular context, human Th clones specific for peptides of HIV gp120 and reverse transcriptase p66 were challenged with recombinant proteins carrying the antigenic epitopes in different sites. We found that a given epitope was recognized by a specific T cell clone only when it was inserted in a particular position of the carrier. However, the permissive position was not the same for all epitopes. Enzymatic excision from a nonpermissive context or insertion of a polyserine spacer between the epitope and the carrier restored antigenicity. Nevertheless, antigenicity was not abolished in a synthetic peptide encompassing the epitope and the neighboring residues from the nonpermissive location. These data suggest that, in this case, the primary sequence of the chimeric protein flanking the HIV peptide is not responsible for loss of antigenicity. Furthermore, constructs carrying the epitope in a given position were recognized by peptide-specific Th clones raised from some individuals, but not from others. We show that this is due neither to individual modes of processing nor to the use of distinct major histocompatibility complex MHC class II restriction elements, but rather that it is related to the fine specificity of the clones. To study the effect of epitope context on selection of T cell repertoire in a naive individual, T cell lines were generated in vitro by stimulation with different peptide constructs. This resulted in the induction of diverse clonotypes defined by the pattern of recognition of different constructs, by T cell receptor V beta gene usage and by fine epitope mapping.

AIDS Vaccines↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity.

The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vbeta gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the V beta gene usage were determined by comparing the TCR Vbeta gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro. Diverse Vbeta genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro. Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vbeta gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo, the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development.

Base Sequence↗
PubMed1996

Cultured human Langerhans' cells are superior to fresh cells at presenting native HIV-1 protein antigens to specific CD4+ T-cell lines.

Cultured Langerhans' cells (CLC) exhibit enhanced antigen-presenting function compared to freshly isolated LC (FLC), but they are commonly believed to be inefficient at processing intact proteins. In this study, FLC and CLC from normal, human immunodeficiency virus (HIV) seronegative volunteers were compared for their ability to present the HIV-1 envelope glycoprotein gp120 or reverse transcriptase (p66) antigens to autologous, specific CD4+ T cell lines. Epidermal cell suspensions enriched for LC were prepared from suction blister roofs. FLC stimulated T cells at lower antigen concentrations compared to unfractionated peripheral blood mononuclear cells (PBMC). CLC were more potent on a per cell basis than FLC, PBMC or adherent monocytes at presenting native gp120, native p66 or immunogenic peptides. CLC were also more efficient than FLC or PBMC in terms of the amount of antigen required for T-cell activation. Chloroquine and leupeptin inhibited presentation of intact p66, but not of an immunodominant peptide, by FLC or CLC, thus indicating that both cells utilize antigen-processing mechanisms that are based on intracellular acidification and protease activity. Incubation of CLC with monoclonal antibodies against HLA-DR, CD11b, CD18, CD50, CD54, CD58 or CD80, but not anti-major histocompatibility complex class I (MHC-I), inhibited antigen-specific T-cell proliferation to varying degrees. We conclude that human CLC retain the ability to process and present protein antigens potently to CD4+ T cells. Thus, CLC have the capacity to participate actively in the generation and maintenance of T-helper cell immunity to viral antigens during HIV-1 infection.

Antigen Presentation↗
PubMed1995

Human CD4+ T cells can discriminate the molecular and structural context of T epitopes of HIV gp120 and HIV p66.

CD4+ T cell lines and clones specific for human immunodeficiency virus (HIV) antigens have been generated from peripheral lymphocytes of naive individuals by priming with the envelope protein gp120, the enzyme reverse transcriptase (p66), and their synthetic peptides. T cells were tested for proliferation to proteins, to peptides, and to HIV virions. Different patterns of reaction were identified. T cells primed in vitro with the whole antigen responded to the protein, but recognition of overlapping peptides occurred with a fraction of the lines or clones. The virus was recognized by some, but not all, of the gp120- and p66-specific T cells, with an efficiency 2 logs higher than the recombinant soluble proteins on a molar basis. One T cell line specific for gp120 responded to virions presented by B cells, but not by monocytes. In contrast, T cells induced with peptides did not always respond to the proteins. Generation of T cell lines from naive individuals may be an in vitro model for T cell immunization, and the response patterns may have implications for the design of vaccines aimed at inducing a T helper response. In fact our in vitro data suggest that (a) immunization with peptides does not always induce T cells recognizing the whole protein, (b) immunization with proteins does not always induce T cells recognizing the protein in the context of the HIV virus, and (c) recognition of gp120 in the context of HIV may be dictated by the type of presenting cells.

Amino Acid Sequence↗
PubMed1995

Recognition of human T-leukemia virus (HTLV-1) envelope by human CD4+ T-cell lines from HTLV-1 seronegative individuals: specificity and clonal heterogeneity.

Because T-helper cells are critical for immune responses in retroviral infections, CD4+ T-cell lines specific for the human T-leukemia virus type 1 (HTLV-1) envelope have been generated from peripheral T lymphocytes of nonimmune donors to study their naive repertoire. Recombinant fragments (RE1, amino acids [aa] 26-200; RE3, aa 165-307; RE5, aa 308-401; and RE6, aa 165-401) of HTLV-1 envelope, whole envelope glycoprotein, and synthetic peptides were used to induce T-cell lines. CD4+ T-cell lines specific for one or more fragments were obtained from seven of eight individuals tested. T-cell lines generated against envelope glycoprotein from five of five donors did not cross-react with the RE fragments and vice versa. The lines specific for RE and env were mapped with overlapping peptides. The lines with single peptide (narrow) specificity contained a variety of clones that used different T-cell receptor V beta genes. These data (1) suggest that most of the normal individuals carry T-helper precursors specific for epitopes on HTLV-1 envelope; (2) indicate that heterogeneity of HTLV-1 envelope-specific T cells can be detected in the naive repertoire; and (3) define optimal antigenic preparations to be used to assess cellular immunity in HTLV-1-infected individuals.

CD4-Positive T-Lymphocytes↗
PubMed1995

Human T helper cells specific for HIV reverse transcriptase: possible role in intrastructural help for HIV envelope-specific antibodies.

Cooperation between B cells specific for an antigen exposed on a viral structure and T helper (Th) cells specific for an internal antigen, as demonstrated with influenza, hepatitis B and rabies viruses, has been termed intrastructural help. Th cells specific for internal proteins of HIV, which are much less mutated than its exposed antigens, may be valuable in vaccine design against this virus. We investigated the human Th repertoire specific for the core HIV antigen reverse transcriptase (p66), and determined whether these cells could be candidate intrastructural T helpers. CD4+ T lines and clones were generated from non-immune individuals by stimulation with p66-pulsed antigen-presenting cells (APC). Specific lines were obtained with p66 from 19 out of 21 (90%) of these individuals, vs. 7 out of 29 (24%) with gp120. Diverse epitopes were recognized by different individuals, and various V beta genes were used by these clones. Clones using the same V beta genes were of diverse origin, according to VDJ region sequence. Of these lines 45% responded to p66 in the context of HIV virions. Moreover, p66-specific clones could respond to APC that had internalized HIV complexed with envelope-specific monoclonal antibodies, suggesting that p66-specific Th cells may participate in intrastructural help. These studies indicate that p66-specific Th cells are detectable in vitro in most naive individuals and exhibit clonal heterogeneity, and that the majority recognize an HIV conserved antigen. They respond to p66 following processing of whole virions and are clearly candidates for intrastructural help. If confirmed in vivo, p66 should be included among vaccine candidates investigated to optimize the anti-HIV Th response.

Amino Acid Sequence↗
PubMed1995

Establishment and characterization of human immunodeficiency virus type 1 (HIV-1) envelope-specific CD4+ T lymphocyte lines from HIV-1-seropositive patients.

Human immunodeficiency virus type 1 (HIV-1) gp160-, gp120-, and tetanus toxoid-specific CD4+ T lymphocyte lines were developed from 11 HIV-1-seropositive volunteers enrolled in a vaccine therapy trial. Of the 20 HIV-1 envelope-specific T cell lines, 9 were challenged with a panel of overlapping peptides spanning the gp120LAI sequence. The most frequently recognized regions were amino acids 74-105 in the C1 region and 306-328 in the V3 region. When tested against a panel of divergent HIV-1 envelopes, 55% of the envelope-specific lines were able to recognize gp120MN, while only 22% recognized gp120SF2. Cytotoxicity testing with HIV-1 envelope antigen or peptides demonstrated killing by all 3 envelope-specific lines tested. Supernatants from 2 of 9 lines had high titers of p24 gag antigen, which did not seem to interfere with functional properties.

Amino Acid Sequence↗
PubMed1995

Antigen-presenting function of human peritoneum mesothelial cells.

Mesothelial cells (MC) from human peritoneal omentum fragments obtained during surgical insertion of peritoneal catheters for continuous peritoneal dialysis in end stage renal failure (ESRF) patients were cultured in vitro. MC exhibited a phenotype different from macrophages, but MHC class II molecules were well expressed. Therefore MC lines were tested for antigen-presenting capacity by pulsing with soluble antigens (tetanus toxoid and purified protein derivative (PPD)) or with a corpusculate antigen (Candida albicans bodies). Autologous peripheral blood mononuclear cells (PBMC) depleted of adherent monocytes and cloned T cells generated from an individual matched for the MHC class II antigen DR2 were used to test antigen-presenting function. MC effectively presented the soluble and corpusculate antigens to autologous and MHC-compatible allogeneic lymphocytes, indicating that they are endowed with both endocytic/phagocytic activity and with processing/presenting capacity. Preincubation of MC with human recombinant interferon-gamma (IFN-gamma) up-regulated MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression, but the effect on antigen-presenting function was not consistent. Since MC are an important component of the peritoneal environment, they may participate, along with macrophages, in activation of specific T cells and in the generation of local cell-mediated immunity to various pathogens.

Adolescent↗
PubMed1994

Isolation of lysozyme-specific T cell clones that discriminate between native and denatured antigen.

Hen egg white lysozyme (HEL)-specific T cell lines and clones were generated from B6 and BDF1 mice. A variety of clonotypes were found among clones generated at an early stage (1 month) whereas fewer clonotypes were detected after several weeks of culture. Furthermore, a bulk line switched from its initial fine peptide specificity pattern (positive for fragment L2--aa. 13-105--and negative for fragment NC--aa. 1-17:Cys 6-Cys 127:120-129) to the opposite pattern (negative for L2 and positive for NC), indicating that in bulk lines, besides selection toward oligo- or monospecificity, clones previously silent can emerge after a period of time. Irrespective of early or late cloning, T cell clones could be isolated from three independent T cell lines from different mouse strains that were stimulated by either native or denatured HEL, but not both. Furthermore, 1 clone of 20 from a B6 line, 3 clones of 25 from a BDF1 line, and 1 T hybridoma clone of 10 of B10.A origin lost their capacity to respond to native HEL, yet continued to respond to reduced, carboxymethylated HEL or cyanogen bromide-cleaved, unreduced HEL. These results suggest that T cells may produce activation signals for efficient processing of native antigen.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Dendritic cells are potent antigen-presenting cells for in vitro induction of primary human CD4+ T-cell lines specific for HIV gp120.

Activation of T helper cells specific for viral antigens is critical for antibody production and for generation of cytotoxic cells during the immune response to HIV. Since T-cell activation depends on antigen-presenting cells (APCs), it is important to define the cells that have a role in presentation of HIV antigens in general and of gp120 in particular. Peripheral blood mononuclear cells (PBMCs), adherent monocytes (AMs), dendritic cells (DCs) and Epstein Barr virus-transformed B-cell lines (LCLs) were tested for the capacity to present gp120 to a specific T-cell clone. DCs proved to be the most effective APC. Primary T-cell lines were generated from uninfected and unprimed individuals by using different APCs in the presence of gp120 or an immunodominant peptide. T-cell lines specific for gp120 were obtained with PBMCs or DCs as APCs, but not with AMs or LCLs. The data showed that (a) DCs are the most effective APCs for presentation of gp120 to specific T cells and (b) DCs are necessary for in vitro induction of primary T-cell lines specific for gp120.

Antigen Presentation↗
PubMed1994

Detection of T cell CD4 epitopes in HIV-infected individuals.

HIV antigens can be detected in the circulation of HIV-infected patients and are associated with active virus production. Free virions and shedded gp120 bind CD4 with high affinity. We have studied the expression of Leu3a and OKT4 epitopes on a CD4+ T cell line (HPB-ALL), pretreated with HIV rgp120, and on CD4+ pheripheral blood T lymphocytes of HIV-infected patients. The associated determination of these epitopes (the Leu3a mapping at the gp120 binding site of CD4 and the OKT4 mapping at a site independent of gp120 binding) allowed to monitor binding of gp120 to surface CD4 and maintenance of CD4 expression. The comparison of MFI of gp120-treated versus untreated Leu3a+ HPB-ALL cells suggested that the Leu3a epitope was masked by treatment with 20 micrograms/ml rgp120, while with 1 microgram/ml rgp120 masking was undetectable, although gp120 was bound to cells. The determination of the Leu3a and OKT4 epitopes in 105 HIV-infected individuals and in 50 normal controls, showed that the Leu3a epitope is detected equally well in HIV-infected and in normal subjects, provided the anti-Leu3a is used at saturation. Therefore the binding to epitopes distinct from the gp120-binding site does not seem to be a requisite for the selection of anti-CD4 mAbs for immunophenotyping. To optimize the detection of CD4 masking, a limiting amount of conjugated anti-Leu3a has to be used. Measurements of CD4 binding by gp120 in terms of gp120-free CD4 molecules, as detected by reactivity with anti-Leu3a, may be used to monitor disease progression in HIV-infected subjects.

AIDS-Related Complex↗