Myeloid Derived Dendritic Cells Inhibit CD8+ T Cell Responses by Down-Modulating NKG2D and 4–1BB Expression.
Authors: Kim Young-June , Broxmeyer Hal E. 
Published: October 2019

Abstract
NKG2D is constitutively expressed on naïve human CD8+ T cells (CTLs) and is involved in antitumor and antiviral responses as a costimulatory receptor. Down-modulating NKG2D expression from the surface of CTLs may allow tumors to evade anti-tumor host immune reactions. Thus, stable NKG2D expression on CTLs is a potential clinical strategy to counteract tumor-induced immune evasion in cancer patients. Recently, evidence suggests that tolerogenic myeloid derived dendritic cells (MDCs) recruited to the tumor microenvironment are involved in immune evasion against anti-tumor CTL functions. In this study, we investigated the possibility that tolerogenic MDCs may control CTL activities by modulating NKG2D expression. To test this possibility, we developed MDCs in vitro from CD14+ monocytes isolated from human cord blood through a long term culture with M-CSF and IL-4 (Li et al. J Immunol. 174: 4706, 2005 and Blood in press, 2007) and subsequent activation with LPS. We found that the MDCs generated were able to dramatically inhibit CTL killing activity to the target C1R B lymphoma cell line that expressed MHC class I related chains (MIC), a ligand for NKG2D. Most importantly, the CTLs co-cultured with the allogenic MDCs lost expression of NKG2D, which was necessary to induce 4–1BB, another costimulatory receptor known to be important for effector/memory CTL responses. MDCs generated without IL-4 were less potent in down-regulating NKG2D. In contrast to MDCs, prototype DCs developed with GM-CSF and IL-4 rather increased both NKG2D and 4-1BB expression indicating that capacity for down-modulating NKG2D and 4-1BB on CTLs was unique for MDCs. CTLs co-cultured with MDCs became tolerogenic because they failed to respond to CD3 stimulation. A majority of the cells stayed in a Go cell cycle stage. Neither NKG2D nor cytotoxic activity could be restored in the presence of CD3/CD28 stimulation. We next investigated whether tolerization of CTLs induced by MDCs was correlated with lack of NKG2D and 4-1BB expression. To test this possibility, we generated CTLs of which NKG2D and 4–1BB expression were similarly down-modulated in the absence of MDCs by culturing naïve human cord blood CD8+ T cells with TGF-b1 and IL-4. These CTLs were tolerized similarly as seen in those co-cultured with MDCs, suggesting that lack of both NKG2D and 4–1BB expression on CTLs was a major contributor to the tolerization induced by MDCs. To further verify the importance of NKG2D and 4–1BB expression in CTL functions, we generated CTLs expressing both NKG2D and 4–1BB in vitro from naïve CD8+ T cells by costimulating consecutively with agonistic anti-NKG2D and anti-4–1BB in the presence of anti-CD3. These CTLs were not susceptible to down-modulation of NKG2D by MDCs and exhibited high levels of cytotoxic activity. They expressed CD45RO and CD44 and promptly produced IFN-g in response to IL-12, which are characteristic phenotypes of effector/memory CTL. In conclusion, MDCs developed in vitro from human cord blood CD14+ monocytes showed tolerizing capacity for CTLs by down-modulating NKG2D as well as 4-1BB expression. Because expression of NKG2D and 4–1BB was inter-dependent, CTLs in which NKG2D and 4–1BB expression was down-modulated by MDCs were not able to differentiate into NKG2D+4–1BB+ effector/memory CTLs. Our results suggest that tolerogenic MDCs recruited to the tumor microenvironment might allow evasion of immune tumor responses by down-modulating NKG2D and 4–1BB expression on tumor specific CTLs.