Research ArticleImmunologyOncology Open Access | 10.1172/JCI180080

Lei Zhou,1,2 Maria Velegraki,1 Yi Wang,1,3 J K Mandula,1 Yuzhou Chang,1,4 Weiwei Liu,1,3 No-Joon Song,1 Hyunwoo Kwon,1,5 Tong Xiao,1,3 Chelsea Bolyard,1 Feng Hong,1,6 Gang Xin,1,7 Qin Ma,1,4 Mark P. Rubinstein,1,6 Haitao Wen,1,7 and Zihai Li1,6

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Zhou, L. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Velegraki, M. in: JCI | PubMed | Google Scholar |

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Wang, Y. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Mandula, J. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Chang, Y. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Liu, W. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Song, N. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Kwon, H. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Xiao, T. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Bolyard, C. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Hong, F. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Xin, G. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Ma, Q. in: JCI | PubMed | Google Scholar |

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Rubinstein, M. in: JCI | PubMed | Google Scholar

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Wen, H. in: JCI | PubMed | Google Scholar |

1Pelotonia Institute for Immuno-Oncology (PIIO), The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC), Columbus, Ohio, USA.

2Department of Anesthesiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.

3Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio, USA.

4Department of Biomedical Informatics,

5Department of Internal Medicine, Ohio State University College of Medicine, Columbus, USA.

6Division of Medical Oncology, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, Columbus, USA.

7Department of Microbial Infection and Immunity, Ohio State University College of Medicine, Columbus, USA.

Address correspondence to: Zihai Li, Pelotonia Institute for Immuno-Oncology, Biomedical Research Tower, Room 580, 460 W 12th Avenue, Columbus, Ohio 43210, USA. Phone: 614.293.9966; Email: Zihai.Li@osumc.edu.

Find articles by Li, Z. in: JCI | PubMed | Google Scholar |

Published May 24, 2024 - More info

Published in Volume 134, Issue 14 on July 15, 2024
J Clin Invest. 2024;134(14):e180080. https://doi.org/10.1172/JCI180080.
© 2024 Zhou et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published May 24, 2024 - Version history
Received: February 7, 2024; Accepted: May 17, 2024 View PDF 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.

Graphical Abstract Introduction

Regulatory T cells (Tregs) are characterized by high expression of IL-2 receptor α chain (CD25) and the transcription factor Foxp3, and their function in suppressing the effector immune response against self-antigens (1–3) and inhibiting antitumor immunity (4). Tregs represent a major barrier in cancer immunotherapy, given their ability to accumulate in the tumor microenvironment (TME) and suppress antitumor immune effector cells (5, 6). A reduction of intratumoural Tregs strongly correlates with clinical benefit in diverse cancer types, and Treg-directed therapy has been postulated as a promising anticancer therapy (7–10).

gp96/GRP94 is an endoplasmic reticulum (ER) master chaperone for various proteins including TLRs, integrins, and glycoprotein A repetitions predominant (GARP) (11). Our previous work demonstrated that gp96 is a critical mediator of Treg lineage stability, as its deletion resulted in downregulated Foxp3 expression and impaired Treg function (11, 12). However, it is unclear whether targeting Tregs via gp96 can reprogram the adaptive immunity against cancer to enhance immunotherapy without systemic toxicities.

Using mice with KO of tamoxifen-inducible, Treg-specific Hsp90b1 (encoding gp96), we discovered that gp96 was required for Treg infiltration into the tumor. Treg-specific gp96 deletion abolishes Treg infiltration into the tumor and eradicates cancer without evidence of autoimmunity. Mechanistically, we demonstrated that gp96 and its client αL integrin (LFA-1) were indispensable for Treg migration into the TME. gp96 deletion also inhibited Treg activation, as evidenced by reduced levels of CD25, Foxp3, and other activation markers, attributed primarily to the downregulation of the IL-2/phosphorylated STAT5 (IL-2/p-STAT5) signaling pathway. Importantly, by genetically and pharmacologically targeting the gp96/LFA-1 axis, we found that loss of infiltrating Tregs in the TME potentiated the antitumor CD8+ T cell effector response and prevented Treg functional exhaustion in an IL-2-dependent manner. Collectively, these findings suggest that targeting gp96/LFA-1 axis–mediated Treg infiltration into the TME is a powerful strategy to augment anti–PD-1 cancer immunotherapy by overcoming CD8+ T cell exhaustion without inducing autoimmune diseases.

Results

Treg-specific gp96 deletion results in tumor regression and prolonged survival without disturbing immune homeostasis. Tregs play a critical role in suppressing antitumor immunity, and targeting Tregs to enhance cancer immunotherapy holds great promise (13). We previously discovered that gp96 is critical for maintaining Treg homeostasis, as genetic deletion of Hsp90b1 in Tregs in nonobese diabetic (NOD) mice leads to rapid, fatal inflammatory disease (11). We found that gp96-deficient Tregs downregulated Foxp3 expression and ultimately converted to IFN-γ–producing “ex-Tregs” (11). To investigate the effect of Treg-specific gp96 deletion on tumor control in nonautoimmune-prone mice, we generated tamoxifen-inducible, Treg-specific gp96-KO mice (Foxp3eGFP-Cre-ERT2 Hsp90b1fl/fl) on a C57BL/6 background and challenged them with several syngeneic tumor models including MC38/colon (Figure 1, A and B, and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/JCI180080DS1); MB49/bladder (Figure 1C and Supplemental Table 1); and immune checkpoint blockade–resistant (ICB-resistant) B16-F10/melanoma (Figure 1D and Supplemental Table 1). Both WT (Foxp3eGFP-Cre-ERT2 Hsp90b1WT/WT) and gp96-KO mice were treated with 75 mg/kg tamoxifen for 10 days, which led to effective gp96 deletion in Tregs from KO mice for at least 20 days (Supplemental Figure 1). Following tamoxifen treatment (days –10 to 0), mice were implanted s.c. with tumor cells (day 0) and followed for tumor growth and overall survival. Tumors grew progressively in the WT mice but were rejected completely in the KO mice. The rejection of B16-F10 tumors in the KO mice was especially noteworthy because this model is notoriously poorly immunogenic. To evaluate the generation of immunologic memory, the KO mice were rechallenged with tumor cells on day 60 in the absence of tamoxifen treatment; these mice remained completely protected (Figure 1, B–D, and Supplemental Table 1). In both the primary tumor cell implantations and tumor cell rechallenge, all KO mice had prolonged survival (100%), whereas WT mice succumbed to the tumors (Figure 1, E–G).

Figure 1

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

Targeting Tregs for cancer immunotherapy confers the risk of eliciting systemic autoimmune diseases (14). Both humans and mice can develop various forms of autoimmune diseases upon genetic or pharmacologic inhibition of Tregs (4, 5, 15–17). Unexpectedly, we found that deletion of gp96 from Tregs in adult mice did not result in overt inflammation or autoimmune diseases for at least 3 months (Figure 2 and Supplemental Figure 2). We characterized the immune phenotype of effector T cells (Teffs) from the spleens (SPLs) and peripheral lymph nodes (pLNs) of these long-surviving mice (Figure 2, A–G). The total numbers of lymphocytes in both the SPL and pLNs were comparable between WT and KO mice (Figure 2B), however, their cellularity was distinct in the SPL. Upon gp96 deletion, splenic Tregs expanded, but the non–Treg T cell population dropped in both frequency and absolute number (Figure 2, C, D, and F). Hence, although the relative frequencies of activated CD44hiCD62LloCD4+Teffs and CD44hiCD62LloCD8+ T cells increased in the KO mice (Supplemental Figure 2, A and B), the absolute numbers of both subsets remained the same (Figure 2, E and G). In addition, KO mice did not have abnormal levels of systemic cytokines such as IL-10, IL-6, and IFN-γ (Figure 2H). To rule out subclinical organ inflammation in KO mice, we sacrificed KO mice and performed necropsy; this demonstrated no obvious infiltration by neutrophils or lymphocytes in any of the organs examined (Figure 2I). The comparable body weights between WT and KO mice also indicated that Treg-specific gp96 deletion did not lead to the development of subclinical autoimmune diseases (Supplemental Figure 2C). Collectively, these data suggest that Treg-specific gp96 deletion in adult mice results in vigorous eradication of various tumors and extends survival without disturbing immune homeostasis, indicating that gp96 is a promising candidate for Treg-targeted therapy against cancers.

Figure 2

Treg-specific gp96 deletion preserves immune homeostasis in mice. Both WT and KO mice received a 10-day tamoxifen treatment (days –10 to 0). Ninety days later (D90), the mice were euthanized and specified tissues were collected for analysis. (A) Representative flow plots illustrate the distribution of CD3+ T cell subsets in murine SPLs and pLNs from both groups (n = 5/group), with values indicating the percentages of the specified subsets in total CD3+ T cells. (B–G) Absolute numbers of total lymphocytes (total LN cells) (B), CD4+Foxp3+ Tregs (C), CD4+Foxp3– Teffs (D), CD44hiCD62LloCD4+ Teffs (E), CD8+ T cells (F), and CD44hiCD62LloCD8+ T cells (G) in SPLs and pLNs from mice of both groups. (H) Serum levels of IL-6, IFN-γ, and IL-10 were measured on day 90 following tamoxifen administration (days –10 to 0) in WT and KO mice using ELISA. n = 6–9/group. CTRL, positive control. (I) Representative H&E-stained images of the indicated organs from WT and KO mice (day 90; n = 5–8/group). Scale bars: 100 μm. Results are representative of more than 3 independent experiments. Data are shown as the mean ± SEM. *P < 0.05, ***P < 0.001, and ****P < 0.0001 (KO vs. WT). For statistical analyses, a 2-tailed, unpaired Student’s t test was performed (B–H).

The Gp96/LFA-1 axis is required for Treg infiltration into the TME. Next, we collected tumor-infiltrating lymphocytes (TILs) from MC38 and MB49 cells grown in WT and KO mice to examine the underlying mechanism. Strikingly, we found very few Tregs in the MC38 or MB49 TME, even at very early stages following gp96 deletion (Figure 3A and Supplemental Figure 3), suggesting that gp96 deletion limited either Treg survival or TME recruitment. We considered whether gp96-null Tregs retained the ability to migrate into the tumor but converted into so-called Foxp3– “ex-Tregs” in the TME. To evaluate this possibility, we crossed the Treg-specific gp96-KO mice with Ai14 reporter mice that express tdTomato following Cre recombination (R26STOP-tdTomato), generating mice that produce tdTomato-labeled gp96-KO Tregs upon tamoxifen treatment (R26STOP-tdTomato Foxp3eGFP-Cre-ERT2 Hsp90b1fl/fl, referred to herein as TdTomato-KO mice). Similar to gp96-KO mice, these TdTomato-KO mice, after a 10-day tamoxifen treatment, showed complete rejection of MC38 (Supplemental Figure 4, A and B, and Supplemental Table 2). However, we detected very few Foxp3–TdTomato+ or Foxp3+TdTomato+ Tregs in the MC38 TME (Supplemental Figure 4, C and D), suggesting that gp96-null Tregs did not become ex-Tregs, but lost their ability to migrate into the tumor. To confirm this possibility, we performed an adoptive T cell transfer and fate-mapping experiment. Tamoxifen was administrated for 10 days to TdTomato-WT (R26STOP-tdTomato Foxp3eGFP-Cre-ERT2 Hsp90b1WT/WT) and TdTomato-KO donor mice. Tregs were isolated from SPLs, preactivated, and adoptively transferred into MC38-bearing Rag2–/– recipient mice. On day 10, we found that TdTomato+ Treg frequencies in the SPL were comparable between the WT and KO groups, but tdTomato+ gp96-KO Tregs were significantly reduced in the TILs (Figure 3B). These findings suggest that gp96 is indispensable for Treg migration into the TME.

Figure 3

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

gp96 is known for the folding and cell-surface expression of selected integrins including integrins αl, β2, α4, and αV (18). Since integrins facilitate immune cell adhesion and transmigration into tissues (19), as well as mediate Treg function in controlling colitis (20), we hypothesized that Treg TME migration is also controlled by integrins. Using flow cytometry, we profiled in WT and KO Tregs the expression of several paired integrins, including CD11a(αL)/CD18(β2), CD49d(α4)/CD29(β1), CD51(αv)/CD61(β3), and CD103(αe)/integrin β7. As expected, since CD29 is not a client of gp96, we saw no reduction of its expression on the cell surface of KO Tregs (Figure 3D). Other integrins, including CD11a, CD18, CD49d, CD51, CD61, CD103, and integrin β7, began to decrease by day –6 and were nearly absent on day –4 during tamoxifen treatment (Figure 3, C–F, and Supplemental Figure 5). Chemokine receptors such as CCR4 are involved in the trafficking and recruitment of Tregs (21, 22). However, we found that gp96-KO Tregs expressed similar levels of CCR7, CCR6, CCR2, CX3CR1, and CXCR5 and even higher levels of CCR4, CCR9, and CXCR3 (Supplemental Figure 6), suggesting that gp96 controls Treg migration primarily through integrins rather than chemokine receptors.

To determine which integrins play a role in modulating Treg TME infiltration, we next genetically deleted various integrins from in vitro–differentiated induced Tregs (iTregs) via CRISPR/Cas9, followed by adoptive transfer into Rag2–/– mice bearing MC38 tumors. We confirmed the efficiency of the deletion of integrins before transfer (>80%; Supplemental Figure 7). On day 10, we found that infiltration of Tregs into the TME was almost completely abolished after deletion of CD18/CD11a (also called LFA-1) (Figure 3, G–I). By comparison, Treg TME infiltration was either not significantly affected or substantially reduced by deleting other integrins, including CD51 (αV), CD103 (αE), CD29 (β1), CD61 (β3), or integrin β7. To confirm that LFA-1 is required for Treg TME infiltration, we performed an in vivo experiment using an anti–LFA-1–blocking Ab (Figure 4A). Following systemic LFA-1 blockade, we noted a significant decrease in intratumoral Tregs on day-9 and day-16 MC38 tumors (Figure 4, B–I). Furthermore, CD8+ T cells and NK cells were also decreased in frequency at both time points, whereas anti-LFA-1 treatment induced minimal change in the frequencies of CD4+ Teffs, macrophages, neutrophils, and B cells. Reduced CD8+ T cells and NK cells in the TME could have been due to inactivation and inhibited proliferation upon LFA-1 blockade (23–25). Taken together, we conclude that gp96 promoted Treg trafficking into the TME largely through LFA-1. Although LFA-1 expression was previously shown to mediate gut tolerance by Tregs (26), to our knowledge, our study is the first to demonstrate important roles of LFA-1 in mediating Treg infiltration into the tumors.

Figure 4

LFA-1 blockade prevents Treg infiltration into the TME. (A) Experimental scheme illustrates the process of LFA-1 blockade using anti–LFA-1 or IgG2a isotype-matched control Abs in C57BL/6 mice implanted with MC38 tumor cells. Anti–LFA-1 or isotype Abs were administered every 2 days starting from day 4 after MC38 tumor cell implantation on day 0; TIL analysis was conducted on day 9 (n = 7/group) and day 16 (n = 7/group). (B–D) Spectral flow cytometric analysis of CD45+ TILs from day-9 MC38 tumors treated with anti–LFA-1 or isotype Abs. LFA-1 blockade significantly reduced the frequency of cluster 1 (NK cells), cluster 2 (including CD3+CD4+Foxp3– non-Tregs and CD3+CD4+Foxp3+ Tregs), and cluster 12 (CD8+ T cells) (highlighted in blue). As a subset of CD4+ T cells, Tregs expressed high levels of Foxp3 and were located at the bottom of cluster 2. (C) UMAP visualization shows the distribution of the indicated markers. (D) edgeR analysis indicating CD45+ TILs clusters with significant changes to frequency following treatment with anti–LFA-1 (left) versus isotype Abs (right). (E) Representative flow cytometric plots and graph depict the percentages of Foxp3+ Tregs in CD45+ TILs from day-9 MC38 tumors. (F–H) Spectral flow cytometric analysis of CD45+ TILs from day-16