Spatial and functional targeting of intratumoral Tregs reverses CD8+ T cell exhaustion and promotes cancer immunotherapy
Lei Zhou, … , Haitao Wen, Zihai Li
Lei Zhou, … , Haitao Wen, Zihai Li
Published May 24, 2024
Citation Information: J Clin Invest. 2024;134(14):e180080. https://doi.org/10.1172/JCI180080.
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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 3

Gp96 regulates CD11a/CD18 (LFA-1) integrin expression in Tregs and facilitates their infiltration into the TME.

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Gp96 regulates CD11a/CD18 (LFA-1) integrin expression in Tregs and facil...
(A) WT and KO mice (n = 5–8/group) were pretreated with tamoxifen (days –10 to 0) and s.c. implanted with MC38 tumors on day 0. TILs were harvested on days 7, 9, and 11. Representative flow cytometric plots and summary graphs show the percentages of CD4+Foxp3+ Tregs in specified tissues at these time points. (B) Rag2–/– recipient mice (n = 5/group) were implanted s.c. with 2 × 106 MC38 cells on day 0. TdTomato-expressing Tregs from SPLs of R26STOP-tdTomato Foxp3eGFP-Cre-ERT2 Hsp90b1WT/WT (TdTomato-WT) or R26STOP-tdTomato Foxp3eGFP-Cre-ERT2 Hsp90b1fl/fl (TdTomato-KO) donor mice were collected, preactivated, and transferred (2 × 106 cells/mouse; n = 5/group) into recipient mice on day 3. On day 10, SPLs and tumors were harvested for flow cytometry. Representative flow cytometric plots and summary graphs indicate the percentages of infiltrating TdTomato+Foxp3+ Tregs among CD45+ cells. (C–F) WT and KO mice (n = 3/group) received tamoxifen (days -10 to 0), and splenic Tregs’ integrin expression was assessed using flow cytometry at designated time points (D–8, day –8; D–7, day –7; D–6, day –6; D–4, day –4; D10, day 10). Representative flow cytometric plots (day 10) and summary graphs (time course) show frequencies of indicated surface integrins on splenic Foxp3+ Tregs in WT and KO mice. (G–I) Naive CD4+ T cells from SPLs of C57BL/6 mice were differentiated into iTregs under Treg-skewed conditions for 2 days (days –2 to 0) followed by CRISPR/Cas9 KO of indicated integrins on day 0; cells were cultured for 3 more days (days 0–3). MC38 tumor–bearing Rag2–/– mice (n = 3–6/group) received specific integrin-KO or nontargeting control iTregs on day 3 post-tumor implantation; SPLs and tumors were collected on day 10 for flow cytometry. Representative flow cytometric plots (G) and summary graph (H) show relative number of Foxp3+ Tregs (in CD45+ cells total). (I) Absolute numbers of Tregs in SPLs and TILs. Results represent 3 independent experiments. Data indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (KO vs. WT), by 2-tailed Student’s t test used for comparisons of different experimental groups (A–F) and 1-way ANOVA with Dunnett’s multiple-comparison test for multiple-comparison analyses (H and I).