Inactivation of tumor‐specific CD8⁺ CTLs by tumor‐infiltrating tolerogenic dendritic cells

Abstract

Cancer immunosurveillance failure is largely attributed to the insufficient activation of tumor-specific class I major histocompatibility complex (MHC) molecule (MHC-I)-restricted CD8+ cytotoxic T lymphocytes (CTLs). DEC-205+ dendritic cells (DCs), having the ability to cross-present, can present captured tumor antigens on MHC-I alongside costimulatory molecules, inducing the priming and activation of tumor-specific CD8+ CTLs. It has been suggested that reduced levels of costimulatory molecules on DCs may be a cause of impaired CTL induction and that some tumors may induce the downregulation of costimulatory molecules on tolerogenic DCs. To examine such possibilities, we established two distinct types of murine hepatoma cell lines, named Hepa1-6-1 and Hepa1-6-2 (derived from Hepa1-6 cells), and confirmed that they display similar antigenicities, as well as identical surface expression of MHC-I. We found that Hepa1-6-1 had the ability to grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and did not prime CD8+ CTLs. In contrast, Hepa1-6-2 cells, which display reduced levels of adhesion molecules, such as Intercellular Adhesion Molecule 1 (ICAM-1), failed to grow in vivo and efficiently primed CTLs. Moreover, Hepa1-6-1-derived factors, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP), converted CD11chigh MHC-IIhigh DEC-205+ DC subsets into tolerogenic cells, displaying downregulated costimulatory molecules and having impaired cross-presenting capacities. These immunosuppressive tolerogenic DCs appeared to inhibit the induction of tumor-specific CD8+ CTLs and suppress their cytotoxic functions within the tumor. Together, the findings presented here provide a new method of cancer immunotherapy using the selective suppression, depletion or alteration of immunosuppressive tolerogenic DCs within tumors.

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC‐205+ dendritic cells by sequential administration of α‐galactosylceramide in vivo

“…We confirmed that these cells showed similar antigenicity as well as identical surface class I MHC molecule restriction 21. The original Hepa1‐6‐1 cells showed continuous growth after s.c. implantation into syngeneic B6 mice and did not prime CD8 + CTLs, whereas the Hepa1‐6‐2 cells failed to grow in vivo by Hepa1‐6‐2‐specific CD8 + CTLs, which also efficiently eliminated Hepa1‐6‐1 cells in vitro 11. Therefore, we first established Hepa1‐6‐2‐specific CD8 + CTLs through the repetitive stimulation of Hepa1‐6‐2‐primed spleen cells with Hepa1‐6‐2 tumour cells in vitro and confirmed that these cells specifically killed both Hepa1‐6‐1 and Hepa1‐6‐2 cells in vitro (Fig.…”

“…The murine hepatoma cell lines Hepa1‐6‐1 and Hepa1‐6‐2 were established from Hepa1‐6 11. Briefly, Hepa1‐6‐1 cells were obtained from the purified cells of a collagenase‐digested and surgically removed tumour mass that was grown for approximately 3 months after the s.c. implantation of Hepa1‐6 cells into syngeneic B6 mice and were maintained in D‐10 medium.…”

“…For estimating the volume of the growing tumour mass, the diameters for both the length ( a ) and the width ( b ) were measured every day until day 24 after implantation, and the tumour volume ( V ) was calculated according to the formula V = ab 2 /2 as previously described 11. When the longer axis of each tumour was > 20 mm in diameter, the mice were anaesthetized and killed according to the NIH guidelines.…”

“…Moreover, the priming and activation of antigen‐specific CD8 + CTLs required co‐stimulatory signalling via CD80 and CD86 10. Hence, CD8 + CTLs, which are tumour‐specific, can be primed and activated by tumour‐antigen‐loaded activated DEC‐205 + DCs expressing co‐stimulatory molecules 11, 12…”

“…After subcutaneous (s.c.) implantation into syngeneic C57BL/6 (B6) mice, Hepa1‐6‐1 cells showed continuous growth and did not prime CD8 + CTLs, whereas Hepa1‐6‐2 cells failed to grow in vivo and efficiently primed the CTLs 11. Through a careful examination of the cells within these two distinct tumours, among tumour‐infiltrating DCs (TIDCs), we found that the DEC‐205 + tolerogenic DCs had reduced levels of co‐stimulatory molecules as well as impaired cross‐presenting capacities in the Hepa1‐6‐1‐derived tumour mass but not within the Hepa1‐6‐2‐derived tumour mass, and we concluded that the tolerogenic DCs may be a cause of the impaired CTL induction 11…”