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D Fenoglio

Publications and source records attributed to D Fenoglio.

At least 37 records · Page 2Linked to original sources
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

A restricted T cell response to myelin basic protein (MBP) is stable in multiple sclerosis (MS) patients.

The close resemblance of MS to the animal model experimental autoimmune encephalomyelitis (EAE) has provided compelling data sustaining a pathogenic role of circulating T cells reactive against MBP. T cell antigen receptor (TCR) usage in EAE is commonly considered restricted; nevertheless, dynamic changes of TCR usage correlate with the course of EAE, resulting in a limited repertoire during early stages of disease activity followed by the recruitment of other T cells reactive against new determinants. Although a broader TCR repertoire mediates the response to MBP in humans, a restricted intraindividual heterogeneity may occur in some MS patients. In the present study we characterize the response to MBP in MS subjects with relapsing remitting disease from two sampling time points 12 months apart. MBP-specific T cell lines (TCL) were first generated from eight MS individuals and two healthy subjects. New TCL were obtained after 12 months from one control and three MS patients whose response, at the first time point, was directed against a single epitope. Interestingly, these three subjects had a stable and mild disease. Few TCL obtained at two time points from the MS individuals recognized the same immunodominant epitope and shared identical TCR Vbeta sequences. In the control we could not detect a restriction of the repertoire. These findings suggest that in some MS patients with benign disease a predominant T cell response to a single determinant may be detectable at different moments and is mediated by clonally expanded populations.

Adult↗
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

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

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↗
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

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

Intravenous immunoglobulin, plasmalymphocytapheresis and azathioprine in chronic progressive multiple sclerosis.

Five patients with a severe form of chronic progressive multiple sclerosis no longer responsive to steroid therapy were treated for six months with high-dose intravenous immunoglobulin associated with plasmalymphocytapheresis and azathioprine. In spite of an apparent initial stabilization of the course of the disease, EDSS assessment after six and twelve months of therapy revealed progressive disability in all patients.

Adult↗
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

Enhanced activation of human T cell clones specific for virus-like particles expressing the HIV V3 loop in the presence of HIV V3 loop-specific polyclonal antibodies.

Recombinant virus-like particles (VLP), formed by the yeast Ty p1 protein, carrying the HIV gp120 V3 loop on their surface (V3-VLP) have been tested in vitro for immunogenicity and antigenicity by using VLP p1-specific human CD4+ T cell lines and clones. VLP-specific human T cell lines and clones were generated from normal individuals, indicating that VLP-specific precursor cells present in the peripheral lymphocyte pool can be induced to expand clonally upon antigen challenge in vitro, in the absence of previous immunization. It was also shown that V3-specific polyclonal antibodies enhance V3-VLP-induced activation of VLP-specific T cell clones. Antibody-dependent potentiation has been shown previously in other antigen systems, and it depends on enhanced uptake of complexed antigen by Fc receptor-positive antigen-presenting cells. Since in this case antigen is internalized by presenting cells as a complex, it can be inferred that a similar event of antibody-mediated antigen uptake can take place with V3-specific B cells, resulting in presentation by the B cells of T helper epitopes derived from processing of the VLP p1 moiety. This suggests that T helper cells specific for the carrier VLP p1 protein can be activated to provide help to V3-specific B cells in the presence of the appropriate antigen construct.

Amino Acid Sequence↗
PubMed1994

Quantitative analysis of peripheral allergen-specific B lymphocytes in asthma.

We have recently shown that the number of peripheral allergen-specific T cells can, in part, predict, together with methacholine hyperresponsiveness, the bronchial response to inhaled allergen in asthmatic patients. This study was designed to explore the role of blood B cells committed to produce allergen-specific IgE in asthma. Twenty-three asthmatic patients sensitized to Dermatophagoides pteronyssinus and 11 control subjects were studied. Peripheral blood B cells, committed to produce allergen-specific IgE, were enumerated by limiting dilution microcultures of Epstein-Barr virus (EBV)-transformed B cells. An allergen inhalation challenge was performed in all asthmatic subjects. No difference was found in the frequency of B cells committed to produce allergen-specific IgE either between asthmatic patients and controls or between asthmatic patients with or without late-phase bronchial response to allergen. No correlation was found between the frequency of B cells committed to produce allergen-specific IgE and the bronchial response to the allergen inhalation challenge. We conclude that, in quantitative terms, peripheral allergen-specific B cells are not as relevant as T cells to the development of the asthmatic response in the model of provoked asthma.

Administration, Inhalation↗
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↗