Fig 2: Perk regulates Chop expression in primed CD8+ T cells and CD8+ TILs. a Upper panel: Time-dependent induction of Chop in murine (left) and human (right) T cells primed in vitro. T cells were stimulated with anti-CD3/CD28 and collected at the indicated time points (0–72 h). Lower panel: Densitometry quantitation of immunoblots (n = 3). b Carboxyfluorescein succinimidyl ester-labeled Pmel CD8+ T cells (CD90.1+) were transferred into wild-type mice (CD90.2+), followed or not by vaccination with gp10025–33 plus IFA. On day 4, Chop levels by mean fluorescence intensity (MFI) (left) and percentage of Chop+ cells (right) were established by fluorescence-activated cell sorter (FACS) in gated CD8+ CD90.1+ T cells from lymph nodes of vaccinated vs. non-vaccinated mice (n = 3). c Left: Tauroursodeoxycholic acid (0.5 mM, at time 0) or Thapsigargin (100 nM, after 48 h) were added to CD8+ T cells stimulated with plate-bond anti-CD3/CD28. Protein extracts were collected 72 h post-activation. Right: Quantitation of immunoblots (n = 3). d Unfolded protein response mediators Perk, Ire-1a, and Atf4 in stimulated murine (left) and human (right) T cells (0–72 h). Representative Immunoblot from n = 3. e CD8+ T cells from Eif2ak3flox or Eif2ak3T cell-KO mice were primed with anti-CD3/CD28 for 0–72 h. Upper panel: Chop and Perk detected by immunoblotting; lower panel: Densitometry quantitation of immunoblots (n = 3). f Chop MFI levels in CD8+ TILs from B16-bearing Eif2ak3flox and Eif2ak3T cell-KO mice (n = 3). Chop was detected in viable CD45+CD8+ TILs by FACS (left) and MFI values compiled (right). g Representative reactive oxygen species levels in murine and human DHE-labeled CD8+ T cells activated with anti-CD3/CD28 and treated for 24 h with 5% cell-free ovarian ascites obtained from mice bearing ID8-Defb29/Vegf-a ovarian tumors or 5% primary ascites from patients with ovarian cancer, respectively (n = 4). h Murine CD8+ T cells were primed with anti-CD3/CD28 for 48 h and then cultured with or without ID8-Defb29/Vegf-a cell-free ovarian tumors ascites for 24 h (n = 3). i CD8+ T cells treated as in h were cultured in the presence or the absence of 2 mM L-NAC during the ascites treatment (n = 3). Bar graphs represent mean ± s.e.m., *p < 0.05 was calculated using two-tailed unpaired Student’s t test

Fig 3: ILC2s are the predominant source of IL-5 in dystrophic muscle(A) Gating strategy used to interrogate IL-5 (Red5)-expressing cells in mdx muscle.(B) Quantification of muscle IL-5+ cells using flow cytometry. n = 7–9.(C) Percent of stroma (CD45-), Lin+ (Lin+CD4-), CD4+ (Lin+CD4+), and ILC2s (Lin-Thy1+KLRG1+CD127+) that express IL-5 in mdx muscle. n = 9. Statistics are compared to CD45-.(D) Representative histograms showing the expression of common ILC2 markers on muscle CD45+Thy1+Lin- cells in WT and mdx mice. Iso, isotype control. n = 5–6.(E) Gating strategy used to interrogate muscle ILCs.(F–H) The number of muscle CD45+Thy1+NK1.1+ (F), CD45+Thy1+Lin-KLRG1+ (G), and CD45+Thy1+Lin-ROR?t+ (H) cells. n = 4.(I and J) The frequency (I) and number (J) of IL-13+ muscle ILC2s.(K and L) Representative histogram (K) and the average geometric mean fluorescence intensity (MFI) (L) of IL-13 expression in CD45+Thy1+Lin-KLRG1+ muscle ILC2s.n = 3–4 (I–L). 4-wk-old mice were analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using an unpaired Welch’s t test (B, F–H, I, J, and L) or one-way ANOVA with Bonferroni correction (C).

Fig 4: Increase of B and T cells proportion in Male NOD.H-2b mice with age. CD45+ immune cells were analyzed by flow cytometry of LGs from BALB/c (B) and NOD.H.2b (N) mice. (A, A’) Comparison of B cells (CD19+) and T cells (CD3+) between LGs of NOD.H-2b male and female have been performed at 2 and 6M within immune population (CD45+) 2M old BALB/c have been used as control (empty bar). A representative plot at 2M is shown for male and female (A’). (B) Histogram showing proportion of CD4+ and CD8+ cells. A representative plot at 2M is shown for male and female (B’). (A, B) Values are shown as mean ± SD. Statistical significance assessed with ANOVA test. Adjusted P-value significance: *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; ns: non-significant. (C, D) H&E staining of LGs from NOD.H-2b female (C) and male (D) showed immune infiltrates (black arrows). (E) Histogram representing focus score of LGs between male and female at 4 months Plot shows mean ± SD. Statistical significance assessed with an unpaired t-test (**: p=0.0021).

Fig 5: Depletion of CTGF partially abolishes pancreatic inflammation and fibrosis in Lats1/2 double knockout mice.(A) Ctgf mRNA expression in P and PL mice (n = 3). **P < 0.01. (B) Western blot of CTGF protein levels in P, PL, and PLTY mice. Tubulin served as the internal control. (C) Schematic diagram of experimental design (n = 6 per group). Mice without TAM injection served as control group. FG-3019: CTGF-neutralizing antibody. (D) Top: histological examination by HE staining; center: immunofluorescent staining of αSMA (Green); bottom: immunofluorescent staining of CD45 (Red). Nuclei stained with DAPI (blue). (E) Quantitative analysis of FG-3019 treatment effects on Lats1/2 deletion-induced ADM, PSC activation, and immune cell infiltration in the mouse pancreas (n = 6); *P < 0.05. Underlying numerical values can be found in S1 Data. αSMA, α-smooth muscle actin; ADM, acinar-to-ductal metaplasia; CD45, ; CTGF, connective tissue growth factor; HE, hematoxylin–eosin; IgG, human Immunoglobulin G control; LATS1, large tumor suppressor 1; P, control; PL, double knockout; PLTY, quadruple deletions of Lats1/2 and YAP1/TAZ; PSC, pancreatic stellate cell; TAM, tamoxifen; TAZ, transcriptional coactivator with PDZ binding motif; YAP1, yes-associated protein 1.