Comparative Analysis of Induced CD4+ Tregs (Itreg) In Response to CD8+ CTL Induction From PBL By MHC-I Bound Mart-1 Peptide Pulsed DC or Autologous Tumor Cells with Freshly Isolated TIL from Human Melanoma.

Abstract

CD4 + CD25 + thymus derived Tregs (tTregs) and peripherally induced Tregs (pTreg) are known to control auto-destruction that needs stimulation through major histocompatibility complex-II (MHC-II). For cellular immunotherapy of cancer, tumor associated antigen (TAA, these are also self-antigens) specific in vivo induced CD8+ Cytotoxic T Lymphocytes (CTL) are rapidly eliminated. Under ex vivo condition TAA specific CTL generated with purified CD8 + cells continued to grow and remain active for months. Ex vivo induced CD8+ CTL with un-fractionated Peripheral Blood Lymphocytes (PBL) stimulated with Mart-1 27-35 A2 peptide pulsed autologous dendritic cells (DC), died within three weeks. Whereas Influenza (Flu 58-66) peptide antigen specific CTL induced identically, continued to expand and remain functional beyond four weeks. No significant difference between binding affinity of Mart-1 27-35 peptide and Flu 58-66 peptide to HLA A2 was observed. Upon activation CD8 + cells from the cultures expressed MHC-II molecules along with other activation and homing receptors. Expanding CD4 + cells were isolated from In vitro Co-cultures (IVC) with PBL for Mart-1 peptide and Flu peptide. CD4+ cells isolated from Mart-I IVC secreted significantly higher amount of IL-10 and IL-4 upon re-stimulation with autologous DC and/or anti-CD3 antibody, than CD4 + cells isolated from Flu PBL IVC. In a similar fashion, CD4 + cells isolated from PBL IVC with autologous Tumor or from freshly obtained tumor tissues were found to secrete significant amount of IL-10, subsequently CD8+ CTLs in such cultures did not survive for long. This finding suggests that CD4 + CD25 + iTreg (or pTreg for in vivo) cells induced in vivo / in vitro, negatively regulate effect or immune response via CD8+ CTL against tumor by elaborating IL-10. In this process activated CD8 + CTL against different antigens might provide differential additional signals via newly expressed MHC-II molecules to the CD4 + cells as needed for different antigens.