In vitro selection of HIV and CMV specific T-lymphocytes.

CMV and HIV produce life-long infections. During CMV infection, cellular responses mediated by virus specific CD8 and CD4 lymphocytes are effective, while during HIV infection cellular responses are ineffective in the long run. In recent years, much work has been carried out to better characterize such responses by using different methodologies to define the fine epitope specificity, the frequency and the function of specific T-cells. These studies have diagnostic and therapeutic implications. In fact, monitoring of specific lymphocytes may help define the immune status of the patients for therapeutic interventions. Identification of CD8 and CD4 epitopes allows the use of relevant peptides for lymphocyte stimulation or for vaccine development. Enumeration of specific cells permits a quantitative estimate of the immune response. In vitro selection provides large numbers of virus specific T-cells for studies on clonal composition, on epitope mapping and on HLA restriction as well as for therapeutic immunoreconstitution with ex vivo expanded T-cells.

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals.

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

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-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma 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.

Natural analogue peptides of an HIV-1 GP120 T-helper epitope antagonize response of GP120-specific human CD4 T-cell clones.

Neutralizing antibodies and specific cytotoxic T lymphocytes (CTL) may contribute to controlling viral spread, and ideally, to virus clearance in HIV infection. Both effector mechanisms depend on specific CD4 T-helper (Th) cells. Nevertheless, HIV hypervariability facilitates appearance of escape mutants for antibodies and for CTL responses. Here we also show that natural mutations (i.e., from sequences of different HIV strains) in an immunodominant Th epitope recognized by human CD4 clones specific for the envelope glycoprotein gp120 escape CD4 T-cell recognition. Furthermore, several natural analogue peptides exert an antagonistic function by inhibiting proliferative response of T cells specific to gp120 with a wild-type sequence. If similar events occur in vivo, they may represent an additional escape mechanism for HIV. In fact, antagonism for CD4 Th response may occur during superinfection with a different strain, or with the appearance of a variant carrying a mutated antagonistic sequence. In both cases, impaired Th cell function could lead to reduced immune control of HIV infection by interfering with CTL and antibody response.

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.

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.