CD4+ T cells induce rejection of urothelial tumors after immune checkpoint blockade
Yuji Sato, Jennifer K. Bolzenius, Abdallah M. Eteleeb, Xinming Su, Christopher A. Maher, Jennifer K. Sehn, Vivek K. Arora
Yuji Sato, Jennifer K. Bolzenius, Abdallah M. Eteleeb, Xinming Su, Christopher A. Maher, Jennifer K. Sehn, Vivek K. Arora
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Research Article Immunology Oncology

CD4+ T cells induce rejection of urothelial tumors after immune checkpoint blockade

Abstract

Immune checkpoint blockade (ICB) provides clinical benefit to a minority of patients with urothelial carcinoma (UC). The role of CD4+ T cells in ICB-induced antitumor activity is not well defined; however, CD4+ T cells are speculated to play a supportive role in the development of CD8+ T cells that kill tumor cells after recognition of tumor antigens presented by MHC class I. To investigate the mechanisms of ICB-induced activity against UC, we developed mouse organoid-based transplantable models that have histologic and genetic similarity to human bladder cancer. We found that ICB can induce tumor rejection and protective immunity with these systems in a manner dependent on CD4+ T cells but not reliant on CD8+ T cells. Evaluation of tumor infiltrates and draining lymph nodes after ICB revealed expansion of IFN-γ–producing CD4+ T cells. Tumor cells in this system express MHC class I, MHC class II, and the IFN-γ receptor (Ifngr1), but none were necessary for ICB-induced tumor rejection. IFN-γ neutralization blocked ICB activity, and, in mice depleted of CD4+ T cells, IFN-γ ectopically expressed in the tumor microenvironment was sufficient to inhibit growth of tumors in which the epithelial compartment lacked Ifngr1. Our findings suggest unappreciated CD4+ T cell–dependent mechanisms of ICB activity, principally mediated through IFN-γ effects on the microenvironment.

Authors

Yuji Sato, Jennifer K. Bolzenius, Abdallah M. Eteleeb, Xinming Su, Christopher A. Maher, Jennifer K. Sehn, Vivek K. Arora

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Figure 5

IFN-γ mediates ICB activity and is sufficient to inhibit tumor growth.

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IFN-γ mediates ICB activity and is sufficient to inhibit tumor growth. (...
(A) αPD-1 and αCTLA-4 combination treatment from day 9 to 24 coadministered with IFN-γ–neutralizing antibodies administered i.p. every 3 days from day 8 to 23. Tumor sizes were compared for an additional 9 days after the last IFN-γ neutralization, a time frame within the reported half-life of the neutralizing antibody. Data represent mean tumor diameter ± SEM. n = 5 per group. (B) Quantification of Ck5 staining of MCB6C tumor sections obtained 5 days after initiation of combination ICB with and without IFN-γ neutralization. IFN-γ neutralization antibody was administered on days 8 and 11 after MCB6C injection. Quantification was performed using images at an original magnification of ×20. For each tumor, percentage Ck5 positivity was averaged from 4 independent fields and quantified using ImageJ software. The graph shows mean ± SD of 9 individual tumors from each treatment group. (C) Representative images used for B at low and high magnification. Scale bars: 1 mm (top); 200 μM (bottom). (D) MCB6C Infgr1-KO organoids constitutively expressing recombinant IFN-γ (rIFN-γ) were injected to mice. For all groups, mice were subjected to CD4+ T cell depletion that was started at day –1 and continued weekly throughout the duration of the experiment. IFN-γ neutralization or control treatments were also started at day –1 and continued weekly for the duration of the experiment. The low IFN-γ group was maintained on regular chow. The high IFN-γ group was initiated on doxycycline-containing chow at day 8. Constitutive low and high ectopic IFN-γ expression in tumor epithelial cells was confirmed by flow cytometry (see Supplemental Figure 7B). Data are plotted as mean ± SEM of n = 6–7 mice per group. (E) Mass of tumors described in D at day 34. (F) Ck5 staining and quantification as described in B. Representative images used for Ck5 quantification. Scale bars: 500 μM. Three tumors from each IFN-γ neutralization groups were scored for Ck5 positivity, and the remainder were utilized for flow cytometric analysis (See Supplemental Figure 7B). Comparisons for growth curves are by 2-way ANOVA for repeated measures and for column data are by Student’s t test. NS > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.