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Biomedical subjects

F Manca

Publications and source records attributed to F Manca.

At least 37 records · Page 2Linked to original sources
PubMed1998

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 Vbeta 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.

Antigen Presentation↗
PubMed1998

Repertoire breadth of human CD4+ T cells specific for HIV gp120 and p66 (primary antigens) or for PPD and tetanus toxoid (secondary antigens)

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Amino Acid Sequence↗
PubMed1998

Requirement for different presenting cells and for different processing mechanisms by human CD4 T helper clones specific for M. tuberculosis antigens.

Human T helper cells specific for mycobacterial antigens have been extensively investigated. Differences have been detected according to antigen specificity and to fine epitope specificity. In this work we have analyzed two additional parameters that allow discrimination among antigen specific T helper cells: requirement for certain types of antigen presenting cells (APC) and requirement for protease-sensitive antigen processing pathways. We used T cell clones from peripheral blood or from pleural exudates, and specific for different antigenic fractions of M. tuberculosis. APC were autologous peripheral blood mononuclear cells, adherent monocytes, adherent pleural monocytes, EBV transformed B lymphocytes and dendritic cells. Seven clones out of twelve were stimulated by all APC irrespective of their specificity, whereas other clones had more selective requirements. When protease inhibitors were used during antigen pulsing of APC, the production of certain epitopes, and thus T cell activation, was impaired with six clones out of sixteen. These results demonstrate that the human T helper repertoire specific for mycobacterial antigens is highly diverse also according to APC populations needed for presentation and to processing mechanisms required for production of the relevant T epitopes.

Antigen Presentation↗
PubMed1998

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.

Adult↗
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↗
PubMed1997

Anti-HIV genetic treatment of antigen-specific human CD4 lymphocytes for adoptive immunotherapy of opportunistic infections in AIDS.

HIV-1 infection results in the loss of CD4+ T helper lymphocytes which make up the immune repertoire. This leads to opportunistic infections that define AIDS. Here, we show that CD4 T cell lines from normal donors with specificity for different antigens can be rendered resistant to HIV-1 replication by retroviral transduction with an antisense vector directed to the HIV-1 tat gene. The genetic treatment did not affect the properties of antigen-specific CD4 lymphocytes such as proliferative response, lymphokine production and phenotypic markers. The HIV-1 challenge dose that resulted in productive infection was two to four logs higher for transduced cells as compared with control cells. Resistance was shown with the HXB2 strain, whose tat sequence was used to design the antisense gene, and with the SF2 strain, whose targeted tat sequence carries five nucleotide mismatches. Retroviral transduction was also performed on a Candida-specific T cell line from a seropositive individual. This line, derived from T cells infected in vivo, produced infectious virus when stimulated in vitro with antigen, but was no longer productive after transduction. In addition, a four log higher HIV-1 challenge dose was needed for a productive superinfection of this T cell line. The production of antigen-specific CD4 T cells resistant to HIV-1 replication to be used in adoptive immunotherapy of opportunistic infections may represent a new form of gene therapy of AIDS.

AIDS-Related Opportunistic Infections↗
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↗