Fig 2: HSP70 deficiency increases caspase-1 activation in myeloid-derived suppressor cells (MDSCs). (A, B) Splenic cells from tumor-bearing mice were treated in vitro with indicated 5-FU concentrations for 24 hours. MDSC viability (CD11b+, Ly6C+, Ly6G+, FVD-) (A) and proportion of MDSCs with active caspase-1 (CD11b+, Ly6C+, Ly6G+, FVD-, FLICA1+) (B) were evaluated by flow cytometry. (C–E) Spleen and tumors from tumor-bearing wild-type (WT) or Hsp70-/- C57BL/6 mice treated or not with 5-FU (50 mg/kg, intraperitoneally) were harvested, dissociated, stained with FLICA1 and then with CD11b, Ly6C and Ly6G antibodies, FVD and analyzed by flow cytometry. (C) The proportion of MDSCs (Ly6C+ and Ly6G+ in CD11b+, FVD-, cells) within spleen or tumor cells was evaluated. (D, E) The proportion of MDSCs with active caspase-1 (CD11b+, Ly6C+, Ly6G+, FVD-, FLICA1+) (D) and the MFI of FLICA1+ MDSCs (E) were determined. Data represent the mean±SEM. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns, not significant.

Fig 3: Inflammatory response in Wlsfl/fl LysM-Cre+ mice remain unperturbed after wounding. (A) Schematic of the experimental timeline demonstrating the Wlsfl/flLysM-Cre wound healing time course. Two 6mm wounds were created on the murine dorsal skin using punch biopsy at day 0. Granulation tissues were collected at day 1, 3, 5, and 7 for flow cytometry and immunofluorescence analysis. (B) Representative flow plots showing the gating strategy used to identified granulocytes (gate i), CD11b+Ly6Gneg (gate ii) cells which are further subdivided into monocytes (gate iii), monocyte/M? (gate iv), M? (gate v) and Ly6CnegMHCIIneg cells (gate vi). (C) Flow cytometry analysis comparing granulation of Wlsfl/flLysM-Cre+and Wlsfl/flLysM-Creneg mice enriched in (i) granulocytes and (ii) CD11b+Ly6Gneg myeloid cells, (iii) monocytes, (iv) monocytes/M?, (v) M?, and (vi) Ly6CnegMHCIIneg cells at all time points post wounding. Highest number of monocytes were recorded at day 1 and its percentage reduces progressively between day 3 and day 7. Monocytes/M? peaked at day 3. Wlsfl/flLysM-Cre+ has slightly higher percentage of M? at day 5 (p = 0.08). Ly6CnegMHCIIneg peaked at day 3 during the inflammatory stage of the wound healing with no difference seen between the Wlsfl/flLysM-Cre+and Wlsfl/flLysM-Creneg mice. Data are represented as mean ± SD. Statistical analysis was performed using unpaired Student t-tests. ns, not significant. Data are representative of 3-4 individual animal samples per time point.

Fig 4: Immuno-profiling revealed an overall immune active Xkr8-/- PSin TME(A and B) Flow cytometry analysis of TILs at the endpoint of experiment in Figure 4 (day 16). TAMs were marked by CD45+CD11b+Ly6G-Ly6C-F4/80+ (B), n = 4.(C and D) gMFI of CD80 (C) and CD163 (D) of (B).(E) RNA-seq analysis of sorted TAMs in WT or PSin TMEs. Shown were the log2 fold change of gene expression (PSin versus WT). Shown are genes associated with M1 polarization, suggesting an overall M1 toward TAMs in PSin TMEs. n = 2 for each PSin independent clone and WT mice.(F) Flow cytometry analysis of tumor-infiltrating CD8+ T cells.(G–I) gMFI of CD69 (G), granzyme B (H), and Ki67 (I) in CD8+T cells.(J) Flow cytometry analysis of tumor-infiltrating CD4+ T cells.(K–M) gMFI of CD69 (K), granzyme B (L), and Ki67 (M) in CD4+T cells.(N) Flow cytometry analysis of tumor-infiltrating NK cells.(O–Q) gMFI of CD69 (O), granzyme B (P), and Ki67 (Q) in NK cells.(R–T) Flow cytometry analysis of CD69 (R), granzyme B (S), and Ki67 (T) in NK cells.(U) RNA-seq analysis of sorted NK cells in WT or PSin TMEs. Shown are the Z score of gene expression. n = 4 for each group, PSin #2 was used in this analysis. Data were analyzed with unpaired t test. Data are representative of at least two independent experiments (except RNA-seq). Data represent mean ± SEM. ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5: Immuno-profiling revealed an overall immune-suppressive TME in Tmem30-/- (PSout) tumors(A–G) Flow cytometry quantitative analysis of TILs at the endpoint (day 16). Shown are no infiltrating differences in CD45+CD11b-CD3+CD4+ T cells (B), CD45+CD11b-CD3+CD8+ T cells (C), CD45+CD11b-CD3+CD4+CD25+ Foxp3+ Tregs (D), and CD45+CD11b+ Ly6G-Ly6C+ Mo-MDSC (F); but more infiltrating cells in CD45+CD11b+Ly6G-Ly6C-F4/80 TAMs (E) and CD45+CD11b+Ly6C-Ly6G+ PMN-MDSC (G).(H) Representative flow plots of MHC II expression in TAMs.(I) Flow cytometry analysis of MHC II+ and MHC II- TAM distribution (n = 5).(J and K) Flow cytometry quantitative analysis of MHC II+ and MHC II- TAMs (n = 5).(L) RNA-seq analysis of sorted TAMs in WT or PSout (Tmem30-/-) TMEs. Shown is the Z score of gene expression (n = 4 for PSout and n = 3 for WT).(M and N) Geometric mean fluorescence intensity (gMFI) of CD163 (M) and CD206 (N) shows higher expression on TAMs (n = 5).(O–Q) qPCR analysis of MHC II, CD163, and CD206 in MHC II+ and MHC II- TAMs from both MC38-WT and Tmem30a-/- TMEs (n = 4).(R and S) gMFI of PD-1 (R) and Tim-3 (S) shows lower expression on CD8+T cells (n = 5).(T) Flow cytometry analysis of PD-1 and TIM-3 CD8+ T cell distribution (n = 5).(U) Representative flow plots and analysis of tetramer+ in CD8+ T cells (n = 5). Data were representative of at least two independent experiments (except RNAseq). Data represent mean ± SEM and were analyzed with unpaired t test. Ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001. Tmem30a-/- clone #1 cells were used for these figures.