Spatial and functional targeting of intratumoral Tregs reverses CD8+ T cell exhaustion and promotes cancer immunotherapy
Lei Zhou, Maria Velegraki, Yi Wang, J K Mandula, Yuzhou Chang, Weiwei Liu, No-Joon Song, Hyunwoo Kwon, Tong Xiao, Chelsea Bolyard, Feng Hong, Gang Xin, Qin Ma, Mark P. Rubinstein, Haitao Wen, Zihai Li
Lei Zhou, Maria Velegraki, Yi Wang, J K Mandula, Yuzhou Chang, Weiwei Liu, No-Joon Song, Hyunwoo Kwon, Tong Xiao, Chelsea Bolyard, Feng Hong, Gang Xin, Qin Ma, Mark P. Rubinstein, Haitao Wen, Zihai Li
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Research Article Immunology Oncology

Spatial and functional targeting of intratumoral Tregs reverses CD8+ T cell exhaustion and promotes cancer immunotherapy

Abstract

Intratumoral Tregs are key mediators of cancer immunotherapy resistance, including anti–programmed cell death (ligand) 1 [anti–PD-(L)1] immune checkpoint blockade (ICB). The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8+ T cell exhaustion remain elusive. Here, we report that heat shock protein gp96 (also known as GRP94) was indispensable for Treg tumor infiltration, primarily through the roles of gp96 in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin), and not αV, CD103 (αE), or β7 integrin, was required for Treg tumor homing. Loss of Treg infiltration into the TME by genetic deletion of gp96/LFA-1 potently induced rejection of tumors in multiple ICB-resistant murine cancer models in a CD8+ T cell–dependent manner, without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/STAT5 signaling, which also contributed to tumor regression. By competing for intratumoral IL-2, Tregs prevented the activation of CD8+ tumor-infiltrating lymphocytes, drove thymocyte selection-associated high mobility group box protein (TOX) induction, and induced bona fide CD8+ T cell exhaustion. By contrast, Treg ablation led to striking CD8+ T cell activation without TOX induction, demonstrating clear uncoupling of the 2 processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and suggests that Treg-specific gp96/LFA-1 targeting represents a valuable strategy for cancer immunotherapy without inflicting autoinflammatory conditions.

Authors

Lei Zhou, Maria Velegraki, Yi Wang, J K Mandula, Yuzhou Chang, Weiwei Liu, No-Joon Song, Hyunwoo Kwon, Tong Xiao, Chelsea Bolyard, Feng Hong, Gang Xin, Qin Ma, Mark P. Rubinstein, Haitao Wen, Zihai Li

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Figure 1

Treg-specific gp96 deletion results in tumor regression and prolonged survival in mice.

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Treg-specific gp96 deletion results in tumor regression and prolonged su...
(A) Experimental schema for primary implantation and rechallenge of MC38 tumor cells in Foxp3eGFP-Cre-ERT2 Hsp90b1WT/WT (WT) and Foxp3eGFP-Cre-ERT2 Hsp90b1fl/fl (KO) mice. For primary implantation, WT or KO mice (8–10 weeks old; n = 9/group) received tamoxifen for 10 days (75 mg/kg, i.p.; days –10 to 0), followed by a single s.c. injection of MC38 tumor cells (2 × 106 cells/mouse; day 0) into their right flank. Tumor volumes were measured daily or every 2 days (length × width in mm) using a digital caliper, starting from day 5 after tumor cell implantation. For rechallenge, all tumor-regressed KO mice and age-matched tumor-naive WT mice were rechallenged s.c. on the opposite flank with 2 × 106 MC38 tumor cells 60 days after primary tumor cell implantation. Tumor growth was monitored as described. (B–D) Growth curves depict primary implantation and rechallenge with 2 × 106 MC38 (B), 1 × 106 MB49 (C), and 2.5 × 105 B16-F10 (D) tumor cells in WT and KO mice. MB49 and B16F10 tumors were implanted following the same scheme as that used for MC38 tumor cells. n = 6–10/group. (E–G) Survival curves following primary inoculation and rechallenge with MC38 (E), MB49 (F), and B16-F10 (G) tumor cells in WT and KO mice. Mice were euthanized when tumors reached more than 16 mm in diameter. n = 6–10/group. Results are representative of more than 3 independent experiments. Tumor growth curves were analyzed by repeated-measures, 2-way ANOVA (B–D); survival incidence analysis was performed by log-rank (Mantel-Cox) test (E–G); ****P < 0.0001 (KO vs. WT).