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. 2021 Sep 6;12:5303. doi: 10.1038/s41467-021-25645-w

Fig. 7. Ly6C+ CD8+ Tn cells preferentially home to lymph nodes and show improved homeostatic properties.

Fig. 7

A CD8+ T cells were magnetically enriched isolated LN and spleen of C57BL/6 mice (N = 6), Ly6C+ and Ly6C− Tn cells were sorted by FACS. Ly6C+ cells were labeled with CFSE and Ly6C− with CTV and 4 × 105 cells were adoptively transferred into RAG-1−/− hosts Four days later, the proliferation of donor cells was analyzed by FACS (Color schemed: blue-undivided cells; red-divided cells) in the LN and spleen of host mice (n = 4 recipient mice, data presented as mean ± sd, *p = 0.001, ***p < 0.001). B MFI of IL-7R (CD127) was measured on CD8+ Tn cells from C57/BL6 mice (n = 15 mice, data presented as mean ± sd). C CD62LhiCD44lo CD8+ T cells were magnetically enriched from pooled secondary lymphoid tissues (n = 5 mice) and FACS sorted by Ly6C expression. Ly6C ± cells were labeled with CFSE and stimulated with 500 ng/ml rIL-7, 100 ng/mL rIL-15, or both. 7 days letter proliferation was measured on live cells (Color scheme: red-Ly6C− cells; blue-Ly6C+ cells). D C57BL/6 mice were treated with IL-7/Anti-IL-7Ab (M25) complexes (1.5 µg/mouse every 48 h). On day 6 after the start of treatment mice were bled retroorbitally and expression of Ki67 was measured on CD8+ Tn cells (n = 5 mice per group, data presented as mean ± sd, *p = 0.02, ****p < 0.0001). E We transferred 2 × 106 of magnetically enriched Tn CD8+s from CD45.1 control mice (CTV labeled), from IFN-α treated mice (48 h prior to harvest; not labeled) and IFN-α treated mice blocked with anti-Ly6C Ab (clone HK1.4; CFSE-labeled) to 5 recipient C57BL/6 mice. 20 min later spleen and LN pools (inguinal, brachial, cervical) were harvested and the proportion of three transferred cell populations was measured (n = 10 recipient mice, data presented as mean ± sd). A, C, D data are representative of two independent experiments. B, E data are pooled from two experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). A, D, E, one-way ANOVA, with Sidak post hoc correction; B paired two-tailed Student’s test.