The clinical relevance of combined chemotherapy and immunotherapy in mouse models of TMBLo and TMBHi ovarian cancer. A and B, Bar plots showing single-nucleotide variants positions (SNVs) (A) and the somatic mutations prevalence (mutations per megabase) (B) in ID8 (n = 3) and Brca1−/−Trp53−/−/Myc/Hras SO1 (n = 3) C57BL/6 syngeneic mouse ovarian cancer cell lines. Mean and SEM are shown. Statistical significance was calculated by multiple t test. P values are indicated. C, Experimental design for the analysis of TLS aggregates development and efficacy of combined chemotherapy and aPD1 and/or aTIM-3 therapy in TMBLo ID8 and TMBHi SO1 experimental syngeneic mouse models. D and E, Representative immunostaining for CD4, CD8, CD20, and CD21 (D) and a box plot showing density of TLS aggregates within chemo-naïve (n = 5) and treated TMBHi SO1 (n = 9) ovarian tumors (E). Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. F–H, Representative dot plot (F) and flow cytometry analyses for percentages of CD62L+CD44+ central memory (CM) and CD62L−CD44− terminally differentiated CD8+ T cells (TEMRA) (G) and TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells (H) in tumor samples of the TMBHi SO1 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. I and J, Overall survival (OS) of TMBHi SO1 experimental model (I) and flow cytometry analyses for percentage of TCF1+PD1+CD8+ T cells after NACT, aPD1, aTIM-3 and combined therapy (J). Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. K and L, Representative immunostaining for CD4, CD8, CD20, and CD21 (K) and a box plot showing density of TLS aggregates within chemo-naïve (n = 8) and treated TMBLo ID8 (n = 8) (L) ovarian tumors. Scale bars, 100 µm and 2.5 mm. Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. M, Flow cytometry analyses for TCF1+PD1+CD8+ and TIM-3+PD1+CD8+ T cells in tumor samples of the TMBLo ID8 experimental model in the presence or absence of carboplatin and taxane chemotherapy (NACT). Box plots: lower quartile, median, upper quartile; whiskers, minimum, maximum. Statistical significance was calculated by two-sided Mann–Whitney test. P values are indicated. N, Overall survival (OS) of TMBLo ID8 experimental model after NACT, anti-PD1, and combined therapy. Survival curves were estimated by the Kaplan–Meier method, and differences between groups were evaluated using log-rank test. P values are indicated. (Panel C created with BioRender.com.)