BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer
Mathieu Rouanne, … , Laurence Zitvogel, Aurélien Marabelle
Mathieu Rouanne, … , Laurence Zitvogel, Aurélien Marabelle
Published May 3, 2022
Citation Information: J Clin Invest. 2022;132(12):e145666. https://doi.org/10.1172/JCI145666.
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Research Article Immunology Oncology

BCG therapy downregulates HLA-I on malignant cells to subvert antitumor immune responses in bladder cancer

Abstract

Patients with high-risk, nonmuscle-invasive bladder cancer (NMIBC) frequently relapse after standard intravesical bacillus Calmette-Guérin (BCG) therapy and may have a dismal outcome. The mechanisms of resistance to such immunotherapy remain poorly understood. Here, using cancer cell lines, freshly resected human bladder tumors, and samples from cohorts of patients with bladder cancer before and after BCG therapy, we demonstrate 2 distinct patterns of immune subversion upon BCG relapse. In the first pattern, intracellular BCG infection of cancer cells induced a posttranscriptional downregulation of HLA-I membrane expression via inhibition of autophagy flux. Patients with HLA-I–deficient cancer cells following BCG therapy had a myeloid immunosuppressive tumor microenvironment (TME) with epithelial-mesenchymal transition (EMT) characteristics and dismal outcomes. Conversely, patients with HLA-I–proficient cancer cells after BCG therapy presented with CD8+ T cell tumor infiltrates, upregulation of inflammatory cytokines, and immune checkpoint–inhibitory molecules. The latter patients had a very favorable outcome. We surmise that HLA-I expression in bladder cancers at relapse following BCG does not result from immunoediting but rather from an immune subversion process directly induced by BCG on cancer cells, which predicts a dismal prognosis. HLA-I scoring of cancer cells by IHC staining can be easily implemented by pathologists in routine practice to stratify future treatment strategies for patients with urothelial cancer.

Authors

Mathieu Rouanne, Julien Adam, Camélia Radulescu, Diane Letourneur, Delphine Bredel, Séverine Mouraud, Anne-Gaëlle Goubet, Marion Leduc, Noah Chen, Tuan Zea Tan, Nicolas Signolle, Amélie Bigorgne, Michael Dussiot, Lambros Tselikas, Sandrine Susini, François-Xavier Danlos, Anna K. Schneider, Roman Chabanon, Sophie Vacher, Ivan Bièche, Thierry Lebret, Yves Allory, Jean-Charles Soria, Nicholas Arpaia, Guido Kroemer, Oliver Kepp, Jean Paul Thiery, Laurence Zitvogel, Aurélien Marabelle

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Figure 5

BCG induces posttranscriptional downregulation of HLA-I associated with inhibition of autophagy flux.

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BCG induces posttranscriptional downregulation of HLA-I associated with ...
(A) HLA-IA/-B/-C mRNA expression in HLA-I– and HLA-I+ cancer cells following BCG exposure for 24 hours (unpaired, 2-tailed Student’s t test). (B) Gene expression in tumors that became HLA-I– upon BCG therapy. Top: differentially expressed genes in paired human bladder tumors (n = 8; significantly up-/downregulated genes are identified by color dots; P < 0.05). Bottom: Gene set analysis showing enrichment of autophagy pathway genes (n = 8). (C) Before and after BCG therapy, paired analysis of autophagy-related gene expression levels in bladder tumors with decreasing (red) or increasing (blue) levels of HLA-I expression after BCG (paired, 2-tailed Student’s t test). (D) U2OS cells were engineered to express LC3 (autophagosome marker) and were fused to GFP with or without RFP. LC3-GFP cells, with or without RFP U2OS cells, were seeded overnight before adding BCG and cocultured for 6 hours, 12 hours, and 24 hours at different BCG MOI. Torin (1 μM; autophagy inducer) was used as a positive control and media as a negative control. (E) LC3-GFP U2OS cells were cultured for 6 hours, 12 hours, and 24 hours with BCG (MOI of 1:10, 1:30, 1:100, and 1:300), torin (1 μM), or media. Live cells counts (left) and GFP-LC3 puncta surface (right). Statistical analysis was determined by Kruskal-Wallis ANOVA and Dunn’s multiple-comparison test with media as the control. Data are from 1 of 2 independent experiments and are shown as the mean ± SEM of 4 technical replicates. (F) LC3-GFP-RFP U2OS cells were cocultured for 6 hours, 12 hours, and 24 hours with BCG (MOI of 1:10, 1:30, 1:100, and 1:300), torin (1 μM), or media. The linear regression curve is between the total surface of yellow (red plus green) and red (autophagosomes plus autophagolysosomes) dots. The negative control (media) is shown in green and the positive control (torin) in red. The gray dots indicate wells with BCG. (G) Quantification of the flux inhibition (iFlux) score. Kruskal-Wallis ANOVA and Dunn’s multiple-comparison test were performed, with media as the control. Data from 1 experiment are shown as the mean ± SEM of 4 technical replicates. *P < 0.05 and **P < 0.005.