Fig 2: CD8+ T cell depletion abrogates CXCL14-mediated tumor suppression.Wildtype B6 mice (n = 10 per group) were depleted of NK and CD8+ T cells using anti-NK1.1 and anti-CD8 antibodies, respectively, with biweekly intraperitoneal (i.p.) injections. Corresponding isotype antibodies were used as controls. Two days after the first antibody injection, MOE/E6E7Vector or MOE/E6E7CXCL14 cells (5 × 105 cells/mouse) were subcutaneously (s.c.) injected into the left flank of each mouse (A). NK and CD8+ T cell depletion was validated using peripheral blood by flow cytometry (B). Tumor volume was measured twice per week in mice injected with either MOE/E6E7Vector (C) or MOE/E6E7CXCL14 (D) cells. Survival rates of mice injected with MOE/E6E7CXCL14 cells were analyzed using a Kaplan-Meier estimator (E). The time to event was determined for each group (isotype, NK, and CD8+ T cell depletion) with the event defined as a tumor burden larger than 2,500 mm3. Deaths not associated with tumor were censored. P values were determined by the log rank test (E). Values that were not significantly different (n.s.) are also shown. Shown are individual tumor growth curves injected with MOE/E6E7Vector (F-H) or MOE/E6E7CXCL14 (I-K) cells. P values of NK or CD8+ T cell depleted mice compared to isotype injected mice were determined for tumor growth (C and D) and survival (E) by two-way ANOVA analysis. *p < 0.001; n.s., not significant. Shown are representative of two independent experiments.

Fig 3: Histopathological analysis of spinal cord in EAE mice. (a) Representative spinal cord sections from wild-type (WT), Asic1-/- , and Asic2-/- mice at 45 days after immunization. Sections of the spinal cord anterior horn were stained with hematoxylin and eosin (E&H) to assess inflammation and immunostained for MCHII, CD4+, CD8+ infiltrating cells. (b) Graphs show the quantification of the extent of inflammation and infiltrating MCHII, CD4+ and CD8+ cells in the anterior horn of the spinal cord. Values are means + SEM (n = 3 animals for each group). *p = 0.014 for CD4+ cells/mm2 vs. WT mice; ** p = 0.008 for CD4+ cells/mm2 vs. WT mice; p < 0.001 for % white matter with CD4+ immunoreactivity vs. WT mice; p < 0.001 for CD8+ cells/mm2 vs. WT mice; p < 0.001 for % white matter with CD8+ immunoreactivity vs. WT mice; p = 0.091 for MHCII cells/mm2 vs. WT mice; p < 0.002 for % white matter with MHCII immunoreactivity vs. WT mice (One-way ANOVA + Fisher's LSD). [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 4: CXCL14 mediates CD8+ T cell infiltration into the tumor microenvironment.Representative image of MOE/E6E7Vector (A) and MOE/E6E7CXCL14 (B) tumor sections immunostained with anti-CD8a antibody (red), anti-cytokeratin antibody (green) and DAPI (blue) to identify CD8+ T cells and tumor tissues. CD8+ T cells were quantified as a number of CD8a-positive cells per total number of DAPI-positive cells across 5 representative images (C). P values were calculated using Student’s t-test. *p < 0.05. Scale bars are 50 µm.

Fig 5: Secreted tumor antigen induces PD-1 expression in cytotoxic T lymphocytes (CTLs) and induce mTGO apoptosis when treated with PD-L1IFlow cytometric histograms showing the expression of CD8 and PD-1 on CTLs co-cultured with DCs pulsed (+pulsed DC) with either (A) mGOsCM or, (B) mTGOsCM. The middle and right panels are sub-gated to the CD8+ cells from the left panels. Quantification of PD-1, IFN? and IL-2-expressing CTLs co-cultured with DCs pulsed with (C) mFGOsCM,, or (D) mTGOCM. *P < 0.05 compared to CTLs exposed to unpulsed DCs, n = 4 individual organoid/immune cell co-cultures. (E) Representative Immunofluorescence image of mTGO/immune cell co-culture stained for the expression of CD11c (green) and E cadherin (Ecad, red). (F) TUNEL stain of organoid/immune cell co-cultures of: 1) mTGOs/DCs/CTLs, 2) mTGOs alone with PD-L1I, 3) mTGOs/pulsed DCs and CTLs, and 4) mTGOs/pulsed DCs/CTLs culture treated with PD-L1I. (G) Quantification of apoptotic cells in co-culture conditions 1–4. *P < 0.05 compared to condition 1, n = 4 individual organoid/immune cell co-cultures.