Fig 2: Normal T-cell development and homeostasis upon deletion of the miR-497/195 cluster. (A) Representative FACS plot illustrating the gating of double-negative, double-positive, and single-positive CD4/CD8 T cells within the thymus. (B) Bar graphs show the percentage of developing T cells within the lin- population in the thymus of miR-497/195+/+ Vav-Cre control (n = 8) and miR-497/195fl/fl Vav-Cre (n = 7) mice as gated in A. (C) Gating strategy for the identification of different T-cell subsets in the spleen. TCRbeta+ CD4+ and CD8+ T cells were divided into naive (CD62L+ CD44-), central memory (CD62L+ CD44+), and effector memory cells (CD62L- CD44+). (D) Bar graphs indicate the mean percentages of the indicated populations in miR-497/195+/+ Vav-Cre control (n = 8) and miR-497/195fl/fl Vav-Cre (n = 8) mice for CD4+ (upper row) and CD8+ (bottom row) T cells. Error bars depict the standard deviation of the mean. *P < 0.05.

Fig 3: Circulating memory CD4+ and CD8+ T cells are crucial in the protection induced by S. Typhi porins. C57BL/6J mice were injected intradermally in the ear with vehicle (Veh), proteinase K-digested porins (PorK), porins (PorID), or intraperitoneally with porins (PorIP) or heat-inactivated S. Typhi (HIS) at day 0 with a homologous boost at day 14. PorID immunized mice were divided into 4 conditions: anti-CD4 (a-CD4), anti-CD8 (a-CD8), isotype control (Isotype) and no treatment (PorID). At days 25, 28 and 31, CD4+ and CD8+ T cells were depleted with GK1.5 and TIB-210 monoclonal antibodies, respectively, in a-CD4 and a-CD8 groups (A). Representative dot-plot of the depletion of circulating CD4+ and CD8+ T cells at day 28 (challenge day) (B). IgM (C) and IgG (D) serum titers were measure before challenge at day 28. Mice were challenged with 108 S. Typhi CFU and bacterial burden in spleen (E) and liver (F) was quantified at 5 days post-challenge (mice that succumbed during challenge are marked in black). Mouse survival (G) and clinical scores (H) were recorded once daily for 5 days post-challenge (n = 10; 3 independent experiments; significant difference for a Kruskal–Wallis test ** p < 0.05 with Dunn’s multiple comparison).

Fig 4: Only circulating memory but not resident memory CD4+ and CD8+ T cells are depleted using GK1.5 and TIB-210 antibodies. C57BL/6J mice were injected with porins intradermally at day 0 and with a homologous boost at day 14. Mice were divided into 4 conditions: anti-CD4 (a-CD4), anti-CD8 (a-CD8), isotype control (Isotype) and no treatment (PorID). At days 25, 28 and 31, CD4+ and CD8+ cells were depleted with GK1.5 and TIB-210 monoclonal antibodies, respectively, in a-CD4 and a-CD8 groups. Total number of CD4+ (A) and CD8+ (B) T cells per microliter of blood were evaluated; CD4+ (C) and CD8+ (D) T cell numbers in skin were also measured (n = 10; 3 independent experiments; ** significant difference for a Kruskal–Wallis test ** p < 0.05 * p < 0.1 with Dunn’s multiple comparison).

Fig 5: Representative FACS contour plots showing Live CD45+CD3+TCRß+ cells and the consequences of excessive concentration of Dispase used for gut digestion on the CD4 markerIdentical protocols were used to isolate intestinal immune cells from mouse #1 and #2 except for the concentration of Dispase as indicated. Lung and mesenteric lymph nodes were used as controls. Anti-CD45 BV786 (30-F11, BD Bioscience, CAT# 564225, 1/1000), anti-KLRG1 BUV395 (2F1, BD Bioscience, CAT# 740279, 1/200), anti-CD3 APC-eF780 (17A2, eBioscience, CAT# 47-0032-82, 1/200), anti-TCRß PerCP-Cy5.5 (H57-597, BioLegend, CAT# 109228, 1/200), and anti-CD4 APC (GK1.5, eBioscience, CAT# 17-0041-83, 1/200) antibodies together with the fixable viability dye eF506 (eBioscience, CAT# 65-0866-14, 1/800) were used.