Numbers Of IFN-gamma Secreting Cells Research Articles

Induction of myeloma-specific cytotoxic T lymphocytes ex vivo by CD40-activated B cells loaded with myeloma tumor antigens

We investigated to establish CD40-activated B cells (CD40-B cells) as alternative antigen-presenting cells (APCs) for the induction of myeloma-specific cytotoxic T lymphocytes (CTLs). To generate CD40-B cells, peripheral blood mononuclear cells were co-cultured with CD40L-transfected J558 cells in the presence of IL-4, insulin, transferrin, and cyclosporine for 14 days, and pulsed with myeloma lysates. The CD40-B cells consistently expressed high levels of CD80, CD86, CD54, CCR7, and HLA-DR. The CD40-B cells produced IL-12, IFN-gamma, and IL-6 during the culture period, but not IL-10. In addition, the CD40-B cells showed potent allogeneic T-cell stimulatory capacities that depended on the dose ratio and had the potential to polarize naïve T cells into Th1 subsets. The CD40-B cells loaded with tumor lysates induced strong target-specific CTLs, based on large numbers of IFN-gamma secreting cells and higher cytotoxic activity against target cells compared to the CD40-B cells without the tumor lysates. These results suggest that CD40-B cells loaded with myeloma lysates might provide alternative APCs for cellular immunotherapy in patients with myeloma.

Development of a Listeria monocytogenes based vaccine against prostate cancer.

Prostate specific antigen (PSA) is a likely immunotherapeutic target antigen for prostate cancer, the second leading cause of cancer-related death in American men. Previously, we demonstrated that attenuated strains of Listeria monocytogenes (Lm) can be used as effective vaccine vectors for delivery of tumor antigens causing regression of established tumors accompanied by strong immune responses toward these antigens in murine models of cancer. In the present study, we have developed and characterized a recombinant live attenuated L. monocytogenes/PSA (Lm-LLO-PSA) vaccine with potential use for the treatment of pCa. Human PSA gene was cloned into and expressed by an attenuated Lm strain. This recombinant bacterial vaccine, Lm-LLO-PSA was tested for stability, virulence, immunogenicity and anti-tumor effects in a murine model for pCa. Immunization with Lm-LLO-PSA was shown to lower the number of tumor infiltrating T regulatory cells and cause complete regression of over 80% of tumors formed by an implanted genetically modified mouse prostate adenocarcinoma cell line, which expressed human PSA. Lm-LLO-PSA was immunogenic in C57BL/6 mice and splenocytes from mice immunized with Lm-LLO-PSA showed significantly higher number of IFN-gamma secreting cells over that of the naïve animals in response to a PSA H2Db-specific peptide, as measured by both, ELISpot and intracellular cytokine staining. In addition, using a CTL assay we show that the T cells specific for PSA were able to recognize and lyse PSA-peptide pulsed target cells in vitro. In a comparison study with two other PSA-based vaccines (a pDNA and a vaccinia vaccine), Lm-LLO-PSA was shown to be more efficacious in regressing established tumors when used in a homologues prime/boost regimen. Together, these results indicate that Lm-LLO-PSA is a potential candidate for pCa immunotherapy and should be further developed.

Potent, persistent cellular immune responses elicited by sequential immunization of rhesus macaques with Ad5 host range mutant recombinants encoding SIV Rev and SIV Nef.

Vaccines incorporating multiple HIV components should elicit broad immunity and protection against a spectrum of HIV strains. Early regulatory and accessory gene products are attractive candidates for such vaccines. Here, immunogenicity studies on SIV Rev and Nef expressed in replication competent Adenovirus type 5 host range mutant vectors (Ad5hr) are summarized. Interferon-gamma (IFN-gamma)-secreting cells in response to Env and Rev peptides were enumerated by ELISPOT after two sequential immunizations of 55 macaques with Ad5hr-SIVenv/rev. Responses to SIV Nef were assessed in 16 macaques also immunized with Ad5hr-SIVnef. Potent cellular immunity to both Rev and Nef was induced following the second Ad-recombinant immunization and persisted for at least 30 weeks. Persistent cellular immunity to SIV Env was also seen, with a mean of 700 IFN-gamma-secreting cells per million PBMC. Rev and Env responses were positively correlated. While greater responses to early gene products occur in natural infection, as immunogens Rev and Nef elicited the same number of IFN-gamma secreting cells as Env, after adjusting for differences in protein size. The same percentage of macaques also responded to Rev, Nef, and Env: 59, 63, and 64%, respectively. Overall, Ad5hrSIVenv/rev and -SIVnef were highly effective immunogens. Their contribution to protective efficacy will be addressed in future studies.

VIP and PACAP induce shift to a Th2 response by upregulating B7.2 expression.

VIP and PACAP, two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunological functions. Although primarily anti-inflammatory in nature, VIP and PACAP also affect resting macrophages. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for antigen-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP upregulate B7.2, but not B7.1, MHC class II, or ICAM-1 expression, and activate macrophages to stimulate the proliferation of naïve T cells in response to soluble anti-CD3 or allogeneic stimulation. The stimulatory effect is mediated through the specific receptor VPAC1, and involves the cAMP/PKA pathway as second messengers. The enhancement in B7.2 expression occurs at both mRNA and protein levels, and correlates with the VIP/PACAP induced upregulation of the costimulatory activity of macrophages for antigen-primed CD4+ T cells. VIP/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5, and to reduce Th1-type cytokines such as IFN gamma and IL-2. In vivo administration of VIP or PACAP in antigen-immunized mice reduces the numbers of IFN gamma-secreting cells, enhances the numbers of IL-4-secreting cells, and affects the pattern of antigen-specific Ig isotypes. The preferential differentiation into Th2 effector cells induced by VIP/PACAP-treated macrophages is mediated through the upregulation of B7.2 expression. Since Th1-dominated responses are associated with some autoimmune diseases and inflammatory reactions typical of cell-mediated immunity, the VIP/PACAP inhibition of Th1 development may represent an additional mechanism for the general anti-inflammatory activity of the two neuropeptides.

Peripheral blood in Sjögren's syndrome does not contain increased levels of T lymphocytes reactive with the recombinant Ro/SS-A 52 kD and La/SS-B 48 kD autoantigens.

Patients with Sjögren's syndrome (SS) frequently have anti-Ro/SS-A and anti-La/SS-B autoantibodies. The aim of this study was to investigate if these patients have peripheral blood lymphocytes (PBL) secreting IFN-gamma after short-term cultivation in the presence of Ro/SS-A and La/SS-B antigens. The frequency of PBL secreting IFN-gamma was examined in 12 SS patients and 11 healthy controls. The enzyme-linked immunospot (ELISPOT) assay was performed after 48 hours cultivation of PBL in the presence of recombinant Ro 52 kD protein or recombinant La 48 kD protein. The number of unstimulated IFN-gamma secreting cells in the SS patient group was not significantly different from that of the control group. Moreover, no increase in the number of IFN-gamma secreting cells after Ro/SS-A and La/SS-B stimulation was detected in the two groups. Thus, T cells reactive with the recombinant Ro 52 kD and La 48 kD proteins do not occur with any increased frequency in peripheral blood of SS patients.

T cells with specificity for idiotypic determinants on human monoclonal autoantibodies in myasthenia gravis

The activation of T cells from 46 patients with myasthenia gravis and 28 healthy individuals by two human monoclonal autoantibodies was studied. B-cell clones were produced by transformation of peripheral lymphocytes from a patient using Epstein-Barr virus and subsequent cloning. Two myasthenia-specific autoantibodies, one anti-receptor-antibody and one antiidiotypic antibody, both carrying separate recurrent idiotopes, were used in this study. Single activated T cells were identified by their secretion of IL2 and IFN gamma using a cell enzyme-linked-immunosorbent technique. The idiotypic antibody activated T cells in patients but not in most of the controls at concentrations of 1 pg/ml and 10 pg/ml. High concentrations of antibody resulted in T-cell activation in both groups. A similar dose-response pattern was recorded using the antiidiotypic antibody. Incubation with the idiotypic antibody resulted in T-cell stimulation, measured as numbers of IFN gamma-secreting cells that exceeded the mean +2 SD of controls, in 78% of patients and in 7% of the healthy individuals (p less than 0.001). The antiidiotypic antibody activated T cells in 50% of patients and in 4% of the healthy individuals (p less than 0.001). T-cell activation measured as numbers of IL2-secreting cells showed a difference between patients and controls which was as significant as for IFN gamma secretion. The results demonstrate the presence of T cells with specificity for disease-specific determinants on idiotypic and antiidiotypic antibodies in myasthenia gravis.