Fig 2: Mechanisms of mTORC2 repression of mTORC1 activity in NK cells. a Intracellular flow cytometric analysis depicting the phosphorylation (p-) of Akt at Thr308 (p-AktThr308) in total splenic NK cells (CD3-CD19-NK1.1+). b The expression of p-S6ser235/236 in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO (set as the solvent control) or 2 µM AKTi-1/2 for 6 h ex vivo. c The abundance of CD122 expression on splenic NK cells (CD3-CD19-NK1.1+NKp46+) from control versus Rictor?/? mice, analyzed by flow cytometry. d Statistical quantification of p-STAT5Y694 in splenic NK cells (CD3-CD19?NK1.1+) with or without IL-15 stimulation for 4 h ex vivo. e–g, i Statistical quantification of p-S6ser235/236 (e), perforin and granzyme B (f), frequencies of CD107a+ NK cells (g), SLC7A5 expression (i) in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO or 100 µM STAT5-IN for 6 h ex vivo. h Statistical quantification of SLC7A5 expression on both total NK cells and the indicated subsets. j The expression of p-S6ser235/236 in splenic NK cells from control versus Rictor?/? mice after treatment with DMSO or 25 mM BCH for 6 h ex vivo. The MFI was relative to the littermates of control + DMSO group (b, e, f, i, j). Each dot represents one mouse, and all experiments were replicated 3 (a, b, e–j), 5 (d), 6 (c) times; error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed Student’s t test (a, c, h); one-way ANOVA (b, d–g, i, j)

Fig 3: CD38+ OT-I T cells do not intrinsically express MHC-II and acquire it via trogocytosis. C57BL/6 and MHC-II-/- mice (n = 4 or 5/group) received 104 OT-I T cells via the i.v. route 24 hours prior to infection with 104 PFU of X31-OVA. (a) Representative dot plots of CD38 and MHC-II presence on OT-I T cells in MLN, BAL and spleens 8 days p.i. (b) Bar graphs show the (i) number and (ii) frequency of CD38-MHC-II+and CD38+MHC-II+ cells within the MHC-II+ OT-I T cell pool in each organ. (c) Representative dot plots and histograms of CD11c and CD19 presence on CD38-MHC-II+ (blue) and CD38+MHC-II+ (green) OT-I T cells in C57BL/6 mice as well as d (i) their numbers and (ii) frequencies. The experiment was performed two times. Statistical analysis was performed using Sidak's multiple comparisons test.

Fig 4: Loss of POLE4 Leads to Failed Lymphoid Progenitor Maturation following p53 Activation(A) White blood cell (left), red blood cell (middle), and platelet (right) counts in Pole4+/+ and Pole4-/- mice. n = 26 Pole4+/+ and n = 41 Pole4-/-. Significance: t test, p < 0.0001 for white blood cells (WBCs) and red blood cells (RBCs), p < 0.05 for platelets.(B) White blood cell distribution. Note the lower number of lymphocytes in Pole4-/- mice.(C) Representative flow cytometry plots of Pole4+/+ and Pole4-/- mice spleen gated on T lymphocytes and demonstrating a significant reduction of CD4+ and CD8+ single-positive cells and an increase in CD4-CD8- double-negative population. At least three animals have been analyzed per condition, and analyses were done in triplicates. CD5 was used as T cell marker(D) Frequency of Pole4+/+ and Pole4-/- mice presenting abnormal thymus. Note the size of Pole4-/- thymus is greatly reduced compared to WT littermates. Significance: Fisher’s exact test, p = 0.026 for thymus absence, p < 0.0001 for thymus with medulla only. n = 36 Pole4+/+ and n = 36 Pole4-/-.(E) Representative flow cytometry plots of Pole4+/+ and Pole4-/- mice spleen gated on B lymphocytes and demonstrating a significant reduction of CD19+ cells and an increase in IgD- population. At least 3 animals have been analyzed per condition, and analyses were done in triplicates.(F) Top: representative picture of spleen from Pole4 mice. Note the reduction in size following deletion of Pole4. Bottom: frequency of Pole4+/+ and Pole4-/- mice presenting spleen with underdeveloped follicles. n = 36 Pole4+/+ and n = 36 Pole4-/-. Significance: Fisher’s exact test, p = 0.001.(G) Left: representative flow cytometry plots of Pole4+/+ and Pole4-/- mice bone marrow gated on Lin-Sca1+cKit+ hematopoietic stem cells (HSCs) and demonstrating an increase of HSCs in Pole4-/- mice. Right: Pole4+/+ and Pole4-/- mice bone marrow gated on CD34 and Flt3 illustrating a decrease of long-term and short-term HSCs and an increase of multipotent progenitors in Pole4-/- mice. At least three animals have been analyzed per conditions, and analyses were done in triplicates.(H) Western blot analysis of proliferation and apoptosis in Pole4+/+ and Pole4-/- embryos at 13.5 dpc. Note the increased expression of p53 and caspase 3 cleavage in Pole4-/- extracts. SMC1 was used as loading control.(I) Immunohistochemistry quantification of ?H2AX, cleaved caspase 3, and p53 in forebrain, and liver section of 13.5 dpc embryos. Significance: t test.

Fig 5: Superior recall capacity of CD38+MHC-II+ CD8+ T cells. (a) Naïve OT-I cells (104 per mouse) were transferred into C57BL/6 mice and the next day challenged with 104 PFU X31-OVA virus. OT-I cells (from MLN and spleen) were sorted based on CD38 and MHC-II presence on day 8 p.i. Sorted OT-I cells were adoptively transferred into new recipient C57BL/6 mice (5 × 103 cells per mice) and rested for 30 days. Mice were then challenged with 104 PFU X31-OVA virus and sacrificed on day 8 p.i. (b) Mice body weight were monitored over the course of 8 days following secondary viral challenge. (c) (i) Representative FACS plots gated on CD8+ T cells across different recipient mice groups and showing the frequency of OT-I cells in BAL and spleen. Numbers of OT-I cells in (ii) BAL and (iii) spleen. (d) (i) Representative FACS plot for CD38 and MHC-II presence on OT-I cells from different recipient mice groups. Percentage of phenotypes based on CD38 and MHC-II of OT-I cells in (ii) BAL and (iii) spleen. (e) (i) Representative FACS plot for CD19 and CD11c presence on OT-I cells from different recipient mice groups. Percentage of phenotypes based on CD19 and CD11c of OT-I cells in (ii) BAL and (iii) spleen. The experiment was performed two times. Statistics comparing original body weight were performed using the unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Statistics comparing OT-I cell numbers were performed using Sidak's multiple comparisons test.