Fig 2: Non-classical CD8+ T Cells Participate during Acute MCMV Infection in KbDb-/- Mice(A) Representative staining of CD8+ T cells in the spleen and liver of KbDb-/- mice on day 0 and day 7 post-MCMV infection.(B) Frequency (black) and number (gray) of CD8+ T cells in the spleen and liver of KbDb-/- mice on indicated days post-MCMV infection (n = 9). Numbers indicate fold change of cell number compared to day zero.(C) Frequency of CD8+ TEFF cells (KLRG1+CD127-) in the spleen (—) and liver (- -) of KbDb-/- mice on indicated days post-MCMV infection (n = 9). Data are pooled from three independent experiments and represent mean ± SEM.See also Figures S1 and S2.

Fig 3: Blocking 6PGD induces an effector T cell phenotype(A and B) CD8+ T cells were isolated from C57BL/6 mice and were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 in the presence of either 6-aminonicotinamide (6-AN) (10 µM), DHEA (20 µM), or vehicle control (DMSO) (A). IFN-? expression was assessed by intracellular staining and flow cytometry, and (B) viability was calculated as percentage of total. The plot shown is representative of three independent experiments with n = 4 per experiment.(C and D) Thymic T cells were collected from 6PGD-/- and 6PGDfl/fl mice, and fractions of CD4+ and CD8+ T cells as well as expression of CD44 and CD25 on gated double-negative (DN) T cells were assessed by flow cytometry. Results are representative of three independent experiments.(E and F) Percentage of CD4+ and CD8+ T cells in spleen (E) and lymph nodes (F) of 6PGD-/- and 6PGDfl/fl mice was examined by flow cytometry.(G and H) Absolute numbers of CD4+ (G) and CD8+ (H) T cells in thymus, lymph nodes, and spleen of 6PGD-/- and 6PGDfl/fl mice were calculated from three replicates.(I–K) Splenocytes from 6PGD-/- and 6PGDfl/fl mice were analyzed for expression of CD44 and CD62L (I), KLRG1 and CD127 (J), and CD69 (K) by flow cytometry. Results are representative of five independent experiments with n = 4 mice per group. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: Non-classical CD8+ T Cells Form Memory Populations and Are Sufficient to Protect against MCMV-Induced Lethality(A and B) Frequency (A) and number (B) of non-classical CD8+ T cells in the SMG from long-term infected KbDb-/- and C57BL/6 mice (n = 12).(C) Frequency of CD8+ TRM cells (CD103+CD69+) in the SMG from long-term infected KbDb-/- and C57BL/6 mice (n = 12).(D) Representative expansion and activation of donor non-classical CD8+ T cells (CD45.2+) transferred into KbDb-/-.SJL (CD45.1+) recipients, on day 7 post-MCMV infection. Donor CD8+ TM (KLRG1-CD27+) cells were sorted from long-term infected KbDb-/- mice.(E) Frequency of donor and recipient non-classical CD8+ TEFF cells (KLRG1+CD127-) on day 7 post-infection (n = 7).(F) Fold change of donor CD8+ T cell number, assuming 100% engraftment in indicated organ on day 7 post-infection (n = 7).(G) Frequency of donor and recipient non-classical CD8+ TEFF cells (KLRG1+CD127) on day 7 post-infection (n = 8); KLRG1CD27+ donor cells were sorted from naive KbDb-/- mice prior to infection.(H) Fold change of donor CD8+ T cell number, assuming 100% engraftment in indicated organ, on day 7 post-infection (n = 8).(I) Survival of RAG1-/-KbDb-/- mice with (—) or without (- -) adoptive transfer of 5 × 104 sorted CD8+ TM cells (KLRG1CD27+) from long-term infected KbDb-/- mice (n = 6). Statistical analysis determined by Mantel-Cox test.(J) Fold-change of donor cells in the spleen (n = 12) and liver (n = 6) of RAG1-/-KbDb-/- recipients upon euthanization between 10 to 16 weeks post-infection.Data represent mean ± SEM and are pooled (A–C, E–H, and I) or representative (D and I) of at least two independent experiments. **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figure S3.

Fig 5: ß2-Integrins Regulate Macrophage Production of IL-1ß in Response to C. rodentium Infection(A) ILCs (CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD90+CD127+CD25+KLRG1-) were sort-purified from the intestines of naive CD18+/- or CD18-/- littermates. Sorted ILCs were cultured in 96-well plates in the presence of 20 ng/mL IL-1ß, 20 ng/mL IL-23, both IL-1ß and IL-23, or in medium only for 72 h. The release of IL-22 (left) and IL-17 (right) in culture supernatants was determined by ELISA.(B and C) Eight-week-old CD18-/- mice and CD18+/- littermate controls were orally inoculated with C. rodentium. On day 5, colons were harvested, and production of the indicated cytokines was determined by ELISA of (B) colonic tissue homogenates and (C) supernatants from ex vivo cultures of total large intestinal lamina propria mononuclear cells (LPMCs).(D) Bone marrow-derived macrophages (BMDMs) were generated from CD18+/- and CD18-/- mice and stimulated with C. rodentium (C. rod) (MOI 20:1) for 1 h without antibiotics and then cultured for another 17 h with gentamicin (50 µg/mL).(E) Eight-week-old CD18-/- mice and CD18+/- littermate controls were orally inoculated with C. rodentium. On day 5 post-infection, LPMCs were isolated from the large intestine and analyzed by flow cytometry; shown are representative FACS plots (left) and absolute numbers (right) of F4/80+CX3CR1+ macrophages (gated on CD45+CD3-B220-NK1.1-Ly6G-).(F)ILCs (CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD90+CD127+CD25+KLRG1-) were sort-purified from the intestine of naive CD18-/- mice. Sorted ILCs and BMDMs from naive CD18+/- or CD18-/- mice were cultured alone or co-cultured, with or without stimulation with C. rodentium (MOI 20:1) for 24 h, in the presence of neutralizing antibody to IL-1ß or isotype control (IC). The indicated cytokines were measured by ELISA of culture supernatants.Data are means ± SDs and are pooled from 3 independent experiments performed in duplicate, for a total of 6 replicates/group (A); are from 3 independent experiments with 2 mice/group in 2 experiments and 3 mice/group in another, for a total of 7 mice/group (B); are from 2 independent experiments with 3–4 mice/group, for a total of 6–8 mice/group (C); are from 2 independent experiments with 3 mice/group in one experiment and 4 mice/group in the other experiment, for a total of 7 mice/group (E); or are from 3 independent experiments performed in triplicate, for a total of 9 mice/group (D and F). **p < 0.01 and ***p < 0.001 (2-tailed Student’s t test). NS, non-significant.