Expansion Of Antigen-specific CD8 Research Articles

Depletion of CD25+CD4+T cells (Tregs) enhances the HBV-specific CD8+T cell response primed by DNA immunization

Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8(+) T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25(+)CD4(+) regulatory T (Treg) cells might be involved in a inhibition of CD8(+) T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8(+) T cell response primed by DNA immunization. B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 microg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25(+) cells. Induction of HBV-specific CD8(+) T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-gamma staining. DNA immunization induced HBV-specific CD8(+) T cells. At the peak T cell response (d 10), 7.1+/-2.0% of CD8(+) T cells were HBV-specific after DNA immunization, whereas 12.7+/-3.2% of CD8(+) T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8(+) T cells in the absence of CD25(+) Treg cells (n = 6, P<0.05). Similarly, fewer HBV-specific memory T cells were detected in the presence of these cells (1.3+/-0.4%) in comparison to Treg-depleted mice (2.6+/-0.9%) on d 30 after DNA immunization (n = 6, P<0.01). Both IFN-gamma production and the avidity of the HBV-specific CD8(+) T cell response to antigen were higher in HBV-specific CD8(+) T cells induced in the absence of Treg cells. CD25(+) Treg cells suppress priming and/or expansion of antigen-specific CD8(+) T cells during DNA immunization and the peak CD8(+) T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8(+) T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.

4-1BB (CD137) or CD40 Signaling Fails To Improve the Expansion of Antigen Specific T Cells Demonstrated with Engagement of TCR, CD28 and CD83 Ligand.

Following the engagement of the T cell receptor by HLA class I and antigenic peptide, naïve CD8+ T cells are primed to receive one or more costimulatory signals. Some of these signals, which are upregulated on and delivered by mature dendritic cells, include members of the immunoglobulin superfamily such as CD80 and CD83. Using a K562 derived artificial antigen presenting cell (aAPC) that expresses HLA-A2, CD80, and CD83, we have shown that the coengagement of CD83 ligand:CD83 and CD28:CD80 induces prolonged and preferential expansion of antigen specific CD8+ T cells. Furthermore, we have found that CD28:CD80 signaling is required for the induction of CD83 ligand expression on peripheral T cells. In order to identify additional immunoaccessory molecules that can augment this response, we have developed a system to efficiently transfer any chosen molecule into aAPC. This provides an excellent platform for studying a potentially immunogenic molecule given the relative lack of immunoaccessory molecules expressed by K562 (i.e. no expression of CD40, CD40 ligand, CD83, CD86, 4-1BB, 4-1BB ligand, OX40, OX40 ligand, HLA class I, or HLA class II). Following the transduction of a candidate molecule under study, the stimulatory capacity of a supertransduced aAPC can be compared to parental aAPC. Attractive candidates include members of the TNF superfamily since they have been shown to deliver important costimulatory signals to T cells. It has been suggested that 4-1BB signaling supports the survival of newly generated effector CD8+ T cells and that CD40 signaling confers “CD4+ T cell-like” help directly to CD8+ T cells. However, the impact of each of these molecules on the stimulation and expansion of antigen specific T cells has not been exhaustively studied. In this report, we transfected aAPC with either 4-1BB ligand or CD40 ligand, allowing us to compare the stimulatory capacity of aAPC/CD40 ligand, aAPC/4-1BB ligand and parental aAPC. We stimulated HLA-A2 positive CD8+ T cells from healthy donors three times at weekly intervals with A2-restricted MART1 peptide pulsed onto either irradiated aAPC/CD40 ligand, aAPC/4-1BB ligand or parental aAPC. Between the stimulations, cells were treated with IL2 and IL15 every three days. When MART1 peptide pulsed aAPC/CD40 ligand were used as stimulators, the total number of CD8+ T cells and number of MART1 specific CD8+ T cells was slightly smaller. IFN-γ ELISPOT analysis revealed that functional avidity of T cell receptors on MART1 specific CD8+ T cells was similar whether they were stimulated by aAPC/CD40 ligand or parental aAPC. These results indicate that CD40 ligand, at least in the human setting, does not directly provide “CD4+ T cell-like” help to antigen-specific CD8+ T cells. In contrast, stimulation with peptide pulsed aAPC/4-1BB ligand did generate a larger total number of CD8+ T cells. Surprisingly, however, most of these T cells were not antigen specific. In fact, significantly fewer MART1 specific CD8+ T cells were generated by aAPC/4-1BB ligand compared to aAPC alone. These results suggest that, unlike CD80 and CD83, 4-1BB ligand delivers a costimulatory signal resulting in the non-specific expansion of CD8+ T cells. This work demonstrates the versatility of our system to dissect the function of particular immunoaccessory molecules and determine the optimal conditions in the stimulation and expansion of antigen-specific human CD8+ T cells ex vivo.

Transfection of Dendritic Cells with In Vitro Transcribed RNA Induces Polyclonal CMV-Specific CD8+ and CD4+ Mediated T Cell Responses.

Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.

Antioxidant treatment reduces expansion and contraction of antigen-specific CD8+ T cells during primary but not secondary viral infection.

During many viral infections, antigen-specific CD8(+) T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8(+) T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis. The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8(+) T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8(+)-T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAP-treated mice than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8(+) T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8(+) memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect on the expansion of antigen-specific CD8(+) secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction of antigen-specific CD8(+) T cells during primary, but not secondary, viral infection.

Differential tissue-specific regulation of antiviral CD8+ T-cell immune responses during chronic viral infection.

The hallmarks of the immune response to viral infections are the expansion of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) after they encounter antigen-presenting cells in the lymphoid tissues and their subsequent redistribution to nonlymphoid tissues to deal with the pathogen. Control mechanisms exist within CTL activation pathways to prevent inappropriate CTL responses against disseminating infections with a broad distribution of pathogen in host tissues. This is demonstrated during overwhelming infection with the noncytolytic murine lymphocytic choriomeningitis virus, in which clonal exhaustion (anergy and/or deletion) of CTLs prevents immune-mediated pathology but allows persistence of the virus. The mechanism by which the immune system determines whether or not to mount a full response to such infections is unknown. Here we present data showing that the initial encounter of specific CTLs with infected cells in lymphoid tissues is critical for this decision. Whether the course of the viral infection is acute or persistent for life primarily depends on the degree and kinetics of CTL exhaustion in infected lymphoid tissues. Virus-driven CTL expansion in lymphoid tissues resulted in the migration of large quantities of CTLs to nonlymphoid tissues, where they persisted at stable levels. Surprisingly, although virus-specific CTLs were rapidly clonally exhausted in lymphoid tissues under conditions of chronic infection, a substantial number of them migrated to nonlymphoid tissues, where they retained an effector phenotype for a long time. However, these cells were unable to control the infection and progressively lost their antiviral capacities (cytotoxicity and cytokine secretion) in a hierarchical manner before their eventual physical elimination. These results illustrate the differential tissue-specific regulation of antiviral T-cell responses during chronic infections and may help us to understand the dynamic relationship between antigen and T-cell populations in many persistent infections in humans.

Efficient generation and expansion of antigen-specific CD4+ T cells by recombinant influenza viruses.

Adoptive transfer of in vitro generated antigen-specific T cells has been successfully used to treat viral infections in immunodeficient patients. Therefore, methods for the rapid in vitro expansion of antigen-specific T cells are needed. Influenza virus efficiently infects dendritic cells, and peptides derived from viral proteins are processed and presented to CD8(+) cytotoxic T cells. However, both, CD4(+) and CD8(+) T cells are necessary for the efficient control of viral infections, and it is becoming increasingly clear that a T helper cell response is very important for the maintenance and strength of the immune response. Here we show that recombinant influenza virus efficiently infects a wide range of professional antigen-presenting cells and does not interfere with antigen presentation pathways. Using T cell clones for three different MHC class II-restricted antigens we demonstrate that peptides derived from these antigens are efficiently presented on MHC class II molecules. Importantly, it was possible to generate and expand antigen-specific CD4(+) T cells following in vitro infection of professional antigen-presenting cells with recombinant influenza virus. These findings support the notion that recombinant influenza virus is a valuable tool for the expansion of antigen-specific CD4(+) T cells in vitro.

Ex vivo enhancement of antigen-presenting function of dendritic cells and its application for DC-based immunotherapy.

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.

In vivo activation of melanoma-specific CD8+ T cells by endogenous tumor antigen and peptide vaccines. A comparison to virus-specific T cells

Many new types of vaccines against infectious or malignant diseases are currently being proposed. Careful characterization of the induced immune response is required in assessing their efficiency. While in most studies human tumor antigen-specific T cells are analyzed after in vitro re-stimulation, we investigated these T cells directly ex vivo using fluorescent tetramers. In peripheral blood lymphocytes from untreated melanoma patients with advanced disease, a fraction of tumor antigen (Melan-A/MART-1)-specific T cells were non-naive, thus revealing tumor-driven immune activation. After immunotherapy with synthetic peptides plus adjuvant, we detected tumor antigen-specific T cells that proliferated and differentiated to memory cells in vivo in some melanoma patients. However, these cells did not present the features of effector cells as found in cytomegalovirus specific T cells analyzed in parallel. Thus, peptide plus adjuvant vaccines can lead to activation and expansion of antigen specific CD8(+) T cells in PBL. Differentiation to protective CD8(+) effector cells may, however, require additional vaccine components that stimulate T cells more efficiently, a major challenge for the development of future immunotherapy.

Role of CD28 for the generation and expansion of antigen-specific CD8(+) T lymphocytes during infection with Listeria monocytogenes.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity. Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice. In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions. Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis. However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.

T-cell response

One of the key features in the immune reaction to primary virus infection is usually a CD8+ T-cell response. Limiting dilution analysis for precursor cytotoxic T cell frequencies has shown only a minor proportion of these CD8+ T cells to be virus specific. The remainder were thought to be activated as bystanders by cytokines induced by the infection. Recent technical developments, including staining of antigen-specific T cell receptors with tetrameric major histocompatibility complex (MHC) class I and peptide, intracellular cytokine staining and ELISPOT assays for interferon γ production, have shown that, in fact, as much as 50% of the CD8+ cells in the spleen of mice acutely infected with lymphocytic choriomeningitis virus (LCMV) are virus specific (Butz and Bevan[1xMassive expansion of antigen-specific CD8+ T cells during an acute virus infection. Butz, E.A. and Bevan, M.J. Immunity. 1998; 8: 167–175Abstract | Full Text | Full Text PDF | PubMed | Scopus (599)See all References[1]; Gallimore et al.[2xInduction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatiblity complex class I–peptide complexes. Gallimore, A. et al. J. Exp. Med. 1998; 187: 1383–1393Crossref | PubMed | Scopus (414)See all References[2]; Murali-Krishna et al.[3xCounting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Murali-Krishna, K. et al. Immunity. 1998; 8: 177–187Abstract | Full Text | Full Text PDF | PubMed | Scopus (1540)See all References[3]). In humans suffering from acute infectious mononucleosis (Epstein–Barr virus), similar levels of antigen-specific CD8+ T cells are seen in the peripheral blood (Callan et al.[4xDirect visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein–Barr virus in vivo. Callan, M.F.C. et al. J. Exp. Med. 1998; 187: 1395–1402Crossref | PubMed | Scopus (654)See all References[4]). Similarly, in persistent virus infections, such as HIV (Altman et al.[5xPhenotypic analysis of antigen-specific T lymphocytes. Altman, J.D. et al. Science. 1996; 274: 94–96Crossref | PubMed | Scopus (2680)See all References[5]) or simian immunodeficiency virus (Kuroda et al.[6xAnalysis of gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I–peptide. Kuroda, M.J. et al. J. Exp. Med. 1998; 187: 1373–1381Crossref | PubMed | Scopus (237)See all References[6]), a significant proportion of peripheral blood CD8+ T cells are virus specific. These findings have helped to confirm that CD8+ T cells are an extremely important effector cell in the immune response to most virus infections.