Supplementary Figure 7: The impact of TIMs and TINs on ICB therapy.

a. Tumour growth curves show responses of two tumour models to ICB therapy (anti-PD1+anti-CTLA4) with escalated dose. Treatment was initiated Day 1 post-tumour implantation and continued every other day till end-point. Number in parentheses show the specific n values of biologically independent mice. Dotted lines indicate the time point at which tumour sizes were compared between control and treatment group. P value was computed by two-sided Student’s t-test. b. Tumour growth curves show responses of tumour tissue-derived cell lines to ICB therapy. PyMT-M exhibited enhanced response to ICB (compared to Fig. 7a). Number in parentheses show the specific n values of biologically independent mice. Dotted lines indicate the time point at which tumour sizes were compared between control and treatment group. P value was computed by two-sided Student’s t-test. c. Flow cytometric quantification of tumour-infiltrating CD8+ cytotoxic T cells across eight untreated breast tumour models. Data are shown as mean ± S.D. d. Flow cytometric quantification of PDL1⁺ cells assorted by total leukocytes (identified by CD45⁺) and non-immune cells (identified by CD45^-) across eight untreated breast tumour models. Data are shown as mean ± S.D. e. Dot plot shows the quantification of total (left) and PD1⁺ (right) tumour-infiltrating CD8+ cytotoxic T cells upon ICB therapy (anti-PD1+anti-CTLA4). Number in parentheses show the specific n values of biologically independent mice per group denoted by different colour. Data are shown as mean ± S.D. P value was determined by two-sided Student’s t-test. f. Kaplan-Meier curves show the progression-free survival of CCR2 KO animals bearing E0771 or PyMT-M tumour cell line treated with ICB therapy (anti-PD1+anti-CTLA4). Number in parentheses show the specific n values of biologically independent mice. P value was computed by two-sided log rank test. g. Tumour growth kinetics of NES tumours (PyMT-N and 2208L) treated with combined anti-CXCR2+anti-Ly6G and ICB therapy (anti-PD1+anti-CTLA4). Data are shown as mean ± S.D at each measured time point. Number in parentheses show the specific n values of biologically independent mice. h. In vitro immunosuppression assay by co-culturing splenic Ly6c+ monocytes harvested from neutrophil-depleted (treated with anti-CXCR2+anti-Ly6G) PyMT-N tumour-bearing animals with T cells. Proliferation of T cells was determined based on CFSE intensity as measured by FACS. A left-shift of CFSE intensity histogram indicates dilution of signals by proliferation. Data are shown as mean ± S.D of three biological replicates (monocytes from three different mice). P value was determined by two-sided Student’s t-test. i. Flow cytometric quantification of total and PD1+ tumour-infiltrating CD4+ and CD8+ T cells in PyMT-N tumours treated with anti-CXCR2+anti-Ly6G. Number in parentheses show the specific n values of biologically independent mice. Data are shown as mean ± S.D. P value was determined by two-sided Student’s t-test.