IL-21 Gene Modified T Antigen Presenting Cells Enhance the Generation of Tumor-Specific Cytotoxic T Lymphocytes with a Memory Phenotype.

IL-21 is a potent cytokine that augments the proliferation and effector function of NK cells and acts in synergy with other γ-chain cytokines to enhance the cytotoxicity of T lymphocytes. IL-21 is transiently produced by activated CD4+ T cells and may facilitate the generation of effector and memory T cells. Recently, T cells have been shown to be effective antigen presenting cells (TAPC) and we hypothesized that this characteristic may be enhanced through overexpression of IL-21 following genetic modification of TAPC. We demonstrate here that transduction of TAPC with IL-21 significantly enhances the generation of MART-1-specific CD8+ T cells suggesting a potential use for IL-21 in tumor immunotherapy protocols. IL-21 was cloned from CD3/CD28-activated CD4+ T cells and inserted into the SFG retroviral vector. To generate IL-21-producing T-APC, CD8-selected T cells from healthy, HLA-A2 donors were stimulated on αCD3/αCD28-coated plates in the presence of IL-2. After 2 days, activated cells were harvested and transduced on Retronectin-coated plates with IL-21 retroviral supernatant. On day 5, TAPC were washed and expanded in growth media supplemented by IL-2. Prior to use as APCs, TAPCs were CD4-depleted by MACS to eliminate residual IL-21 production by CD4+ T cells. IL-21-transduced and non-transduced (NT) CD8+ TAPC pulsed with MART-1 HLA-A2-restricted peptide (ELAGIGILTV) were irradiated and cocultured with autologous CD8+ peripheral blood T cells in media supplemented with IL-7 and IL-12. On day 7, responder T cell cultures were restimulated with peptide-loaded IL-21 or NT CD8+ TAPCs in the presence of IL-2 to induce expansion. Responder T cell cultures were then analyzed for MART-1 specificity by pentamer, ELISPOT and cytotoxicity assays and for their memory phenotype using monoclonal antibodies to CD27, CD28, CD62L, CD45RA, CD45RO, CD127 and CCR7. TAPC were efficiently expanded (>100-fold expansion) and transduced by retrovirus encoding IL-21 (>50% as measured by GFP). Gene modification of TAPC with IL-21 had minimal effect on MHC class I, II, CD80, CD83 and CD86 levels when compared to NT TAPC. However, there was increased expression of CD27, CD28 and CD62L, suggesting that IL-21 was biologically active. Seven days after stimulation with MART-1/ELA peptide-pulsed IL-21-TAPC and NT-TAPC, we observed a substantial increase (10±5-fold) in ELA-specific T cells in cultures stimulated with IL-21-TAPC compared to NT-TAPC when analyzed by FACS using ELA pentamers. Subsequent stimulation with IL-21-TAPCs amplified this effect, resulting in >50-fold increase in absolute ELA-specific T cell numbers when compared to NT-TAPC. ELA-specific CTL generated from IL-21-TAPC stimulation were functional as determined by IFN-γ ELISPOT and cytotoxicity assays. ELA-specific CTL generated from IL-21-TAPC exhibited a unique phenotype (CD45RA−, CD27^high, CD28^high, CD62L^high) as compared to CTL generated form NT-TAPC (CD45RA−, CD27^low, CD28^low, CD62L^low) suggesting that IL-21 may play a role in the development of T cell memory. In summary, IL-21 enhances the generation of tumor-specific CD8+ T cells which exhibit a central/effector memory phenotype. Our results indicate that IL-21 improves proliferation of antigen-specific T cells, possibly by maintaining CD28 expression allowing costimulation upon secondary antigen encounter.