Fig 2: Deletion of COX-2 in both CD11c+ and Lyz2+ cells further reduces PGE2 production.Indicated strains of mice were infected with 107 LADD L. monocytogenes. 12hpi spleens were harvested and assessed for PGE2 by mass spectrometry. Data was normalized to d-PGE2 levels and percent change is compared to L. monocytogenes-infected controls (A). Indicated strains of mice were infected with 107 LADD L. monocytogenes. 7dpi splenocytes were examined for B8R-specific CD8+ T-cell responses. %IFNγ (B) or number IFNγ (C) per spleen was assessed. Data shown are representative of two independent experiments of 3–5 mice per group. Significance was determined by a one-way ANOVA with Bonferroni’s correction (A) or a Mann-Whitney U test (B-C). *p < 0.05, **p < 0.01.

Fig 3: PAM-primed BMDMs and BMDCs express PGE2 after cytosolic infection with L. monocytogenes.Wild-type or COX-2-/- BMDMs or BMDCs were infected with indicated strains of L. monocytogenes at an MOI of 10 +/- the TLR2 agonist PAM3CSK4 and assessed 6hpi for expression of Ptgs2 (encoding COX-2) and Ptges (encoding mPGES-1) by qRT PCR (A-B) or COX-2 protein by western blot (C). Supernatant was harvested and assessed for PGE2 by mass spectrometry (D). Mass spectrometry data was normalized to d-PGE2 and fold change is relative to PBS treated controls. Data are a combination of at least two independent experiments (A,B, D, and western blot quantification), or a representative of at least two independent experiments (western blot image). Significance was determined by a one-way ANOVA with Bonferroni’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: The beneficial effect of indo on ACT is independent of its COX2/PGE2 inhibitory activity and does not result in improved T cell phenotype. (A) A20 tumor cells express minimal levels of COX2. A20 tumor cells were cultured to confluence and then stained for intracellular COX2. CT26 cells, which are known to express COX2, were used as a positive control. Representative dot plots are shown. Numbers denote % of COX2-positive cells. (B) Paucity of PGE2 released by A20 cells. Grown to confluence, A20 and CT26 cell culture supernatants were collected for PGE2 measurement by ELISA. Each sample was measured in triplicate and results are shown as mean±SEM Data shown are representative of two independent experiments with similar results. (C) Measurement of PGE2 in tumor tissues by LC/MS. A20HA-bearing mice received the indicated treatments. Preactivated HA-specific CD4+ T cells were used for adoptive transfer in this experiment following the procedure described in figure 1C. Ten days after T cell transfer, tumor tissues were collected from all mice and homogenized to measure PGE2. For comparison, CT26 tumor tissues from untreated and indo-treated mice were collected for PGE2 measurement at the same time as A20HA tumor samples. (D) Measurement of indo in A20HA tumor tissues. Selected A20HA tumor tissues were used for indo detection by LC/MS. The results are summarized in the bar graphs shown as mean±SEM of three samples per group. (E) The schema depicts the experimental procedures. Mice with established A20HA tumors received CTX-conditioning followed by adoptive transfer of pre-activated HA-specific CD4+ T cells, and a cohort of mice received additional indo administration. On day 11, tumor tissues were collected and processed into single cell suspensions for analysis. Donor T cells in tumor were identified as CD4+Thy1.1+ cells. Cells were stained with a panel of markers to evaluate the frequency, activation status and functionality of the donor T cells. The results are summarized in bar graphs and shown as mean±SEM of six samples per condition. (F) Frequencies of donor T cells in tumor (left), and fraction of cells exhibiting naïve (CD62L+CD44-), effector memory (CD62L-CD44+), and central memory (CD62L+CD44+) markers within the donor T cell population (right). (G) Levels of T cell activation/exhaustion markers PD1 and Tim3 in donor T cells. (H) Cytokine profile of donor T cells. T cells were stimulated with PMA/ionomycin in the presence of GolgiPlug for 3.5 hours before intracellular cytokine staining for the indicated cytokines. (I) Frequencies of Tregs and MDSCs. The frequencies of Tregs in donor T cell population and endogenous CD4+ T cells were determined by Foxp3 staining. The frequencies of MDSCs in tumor were determined by % CD11b+Gr1+ cells. ns: non-significant. (J) Migration and retention of infused HA-specific CD4+ T cells in tumor. Following the procedures depicted in the schema, luciferase-transduced HA-specific CD4+ T cells were transferred to CTX-conditioned A20HA-bearing mice followed by indo administration to a cohort of mice for 28 consecutive days. BLI was conducted periodically to visualize luciferase-expressing donor CD4+ T cells in vivo. Representative images of mice in each group are shown. Results of intratumoral donor T cell luciferase signal intensity quantified as mean±SEM are summarized in (K). Each symbol in the plot represents one mouse. Statistics: (B, F, G, H, I) Unpaired t-test, (C, D) One-way ANOVA followed by Tukey’s multiple comparison test. *P<0.05, ***p<0.001, ****p<0.0001, ns: non-significant. ACT, adoptive cell therapy; ANOVA, analysis of variance;