Intratumoral CD4 T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer
Authors: Eric Chow
Maxwell V Meng
Serghei Mangul
Elizabeth McCarthy
Matthew H Spitzer
Sima Porten
Arielle Ilano
Dvir Aran
Serena S Kwek
Arun Burra
Yang Sun
Kathryn Allaire
Chun Jimmie Ye
Siddharth S Raju
David Y Oh
Terence W Friedlander
Chien-Chun Steven Pai
Tony Li
Lawrence Fong
Chiara Rancan
Journal: Cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Bladder cancer; PD-1 blockade; anti-PD-L1; checkpoint inhibition; cytotoxic CD4(+) T cells
Affiliations: Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA., Division of Rheumatology, Department of Medicine; Department of Epidemiology and Biostatistics; and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Otolaryngology - Head and Neck Surgery, University of California, San Francisco, San Francisco, CA 94143, USA., Division of Rheumatology, Department of Medicine; Department of Epidemiology and Biostatistics; and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA., Department of Biochemistry and Biophysics, Center for Advanced Technologies, University of California, San Francisco, San Francisco, CA 94143, USA., Bakar Computational Health Sciences Institute, University of California, San Francisco, San Francisco, CA 94143, USA., Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Otolaryngology - Head and Neck Surgery, University of California, San Francisco, San Francisco, CA 94143, USA., Department of Computer Science, University of California, Los Angeles, Los Angeles, CA 90095, USA., Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA., Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA; Division of Rheumatology, Department of Medicine; Department of Epidemiology and Biostatistics; and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; Bakar Computational Health Sciences Institute, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub, San Francisco, CA, USA. Electronic address: ., Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: .

Abstract:  Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1. 

Retracted: No

PubPeer comments:
 For Figure 1E 2F, did the authors used a matched-pair exact permutation test? In the method part, it was not described whether the test is for matched-pair and there are also no lines linking paired samples in the bar chart. And also, the authors mentioned that there are no statistically significant differences in 1E but yes for 2F, which does not seem to be consistent with the messages from the two figures (Actually, it seems that both CD4 and CD8 clusters have some statistically significant differences).  Surprisingly, although the frequency of CD8ENTPD1 cells was higher in tumors, none of the CD8+ states displayed statistically significant differences in frequency between the tumor and non-malignant bladder (exact permutation test; Figure 1E; density plots in Figure 1F) , 
 In the supplementary table, there are muliple  NA for the column of CD8+ T cell from adjacent normal tissues, does it means that those samples are not available? Is it the cause of lack of statistical significance for compaing CD8 subtypes between tumor and adjacent nomal? Why didn t the authors address this issue in the paper? , 
 For the cytotoxic CD4 signature, how can the authors make sure that this is specific for CD4 T cells based on only CD8 and CD4 data? For example, some genes may overlap with signatures of NK cells. On the other hand, why the authors claim that this signature  predicts responses to ICB without showing any prediction model or prediction results? , 
 For the two Treg states, what s the significance of separating two populations of IL2RA-high and IL2RA-low? For any particular cell types, we can certainly cluster it into any number of clusters based on clustering parameters. Since the two populations do not show discrete differences, is it possible that this is due to over-clustering? , 
 The T subtypes seem to be not well resolved in the UMAP plot (i.e. some subtypes seem to overlap in the UMAP space). What is the cause of this and how would this affect the results? ,