M. T. Valle

Phenotypic and functional analysis of T cells homing into the CSF of subjects with inflammatory diseases of the CNS.

The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon‐γ‐producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7− CD27− compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1‐polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.

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.

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‐γ) up‐regulated MHC class II and intercellular adhesion molecule‐I (ICAM‐I) 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.

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.

Primary human mesothelioma cells express class II MHC, ICAM-1 and B7-2 and can present recall antigens to autologous blood lymphocytes.

Mesothelioma cells (MMc) are considered to be weakly immunogenic and the experimental approaches attempting to induce an immune response against these cells have been disappointing. Our aim was to investigate whether MMc possess the surface accessory molecules involved in antigen presentation and whether these cells are capable of presenting recall antigens to autologous blood lymphocytes. Four primary MMc cultures were generated from malignant effusions and examined to assess whether the accessory molecules required for antigen presentation were present on their surfaces. Intercellular adhesion molecule‐1 (ICAM‐1; CD54); class I and class II major histocompatibility complex‐DR (MHCI and MHCII‐DR); B7–1 (CD80.3); and B7–2 (CD86) expression by MMc was studied by immunocytochemical and/or FACScan analysis. MMc were pulsed with purified protein derivative (PPD), Tetanus toxoid (TT) and Candida albicans (CA) bodies, and incubated with autologous lymphocytes. Lymphocyte proliferation was estimated by radionucleotide incorporation. Phenotypic analysis showed the presence of MHCII‐DR, ICAM‐1 and B7–2 on primary MMc cultures, whereas the phenotypic evaluation of 2 established MMc lines did not show the presence of the B7–1 and B7–2 molecules. In addition, MHCII‐DR was detectable only after interferon gamma (IFN‐γ) stimulation. Primary MMc cultures acquired the capability to induce lymphocyte proliferation after pulse with the recall antigens. To achieve characterization of these lymphocytes, we generated a PPD‐specific CD4+ T‐cell clone. PPD‐pulsed MMc were shown to specifically induce T‐cell clone proliferation through a MHCII‐DR‐mediated process. We conclude that primary MMc possess the surface molecules required for antigen presentation and can present recall antigens to CD4+ lymphocytes. Int. J. Cancer 78:740–749, 1998.

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91–108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91–108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross‐reactive peptide behaved as quasi‐universal. The analysis of TCR‐BV gene usage by cell lines showed that most Ag85‐specific T cells correspond to 91–108‐specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon‐gamma (IFN‐γ)‐IL‐4 double‐positive cells, whereas antigen stimulation resulted in production of IFN‐γ only. The data show that peptide 91–108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.

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.

HIV-1 Tat mutants in the cysteine-rich region downregulate HLA class II expression in T lymphocytic and macrophage cell lines.

Human macrophage and T cell lines were stably transfected with HIV‐1 wild‐type Tat or Tat mutants in the cysteine‐rich region displaying trans‐dominant negative effects on HIV‐1 life cycle. The expression of HLA class I and class II molecules was not affected by wild‐type Tat. Tat mutants, instead, profoundly down‐regulated in a dose‐dependent fashion the expression of class II, but not of class I, in both cell types by acting at the transcriptional level. Down‐regulation was manifested on constitutive and IFN‐γ‐induced class II gene expression and did not correlate with reduced transcription of the AIR‐1 gene product CIITA, the major transcriptional activator of class II genes, indicating that Tat mutants did not act by inhibiting AIR‐1 gene expression. Class II down‐modulation had important functional implications in macrophages, as both antigen processing and presenting capacity were inhibited. These results represent the first evidence that a modified HIV‐1 Tat product can act as a potent immunosuppressor by inhibiting the HLA class II expression necessary for triggering both cellular and humoral responses against pathogens. The use of these HIV‐1 Tat mutants also discloses new opportunities to investigate the molecular mechanisms underlying the coordinate HLA class II gene transcription.

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.

HIV‐induced deletion of antigen‐specific T cell function is MHC restricted

When antigen‐specific T cells are pulsed by antigen‐presenting cells (APC) in the presence of HIV they are functionally deleted following subsequent exposure to syngeneic APC in the absence of HIV. Recombinant soluble HIV envelope (gp 120) is able to induce a similar effect which, unlike that induced by HIV, is reversible. Neither HIV nor gp120 affect the ability to respond to IL‐2. Thus it is only antigen‐specific responses involving the T cell receptor pathways and CD4/MHC class II interaction that appear to be inhibited by HIV‐1 and gp120. Furthermore, the functional impairment caused by HIV‐1 is specific to the T cells that respond to the antigen in co‐culture with HIV, as there is no apparent effect on ‘bystander’‐activated T cells specific for another antigen. Antigen‐specific T cell lines may be deleted by A signalling mechanism which involves molecules other than gp120/CD4 but still requires MHC class II restriction.