Fig 2: Adoptive transfer of OT-I CD8+ T cells stimulated by LmOVAPurified OT-1 CD8+ cells are adoptively transferred to CD45.1 mice and then the recipient mice are infected with LmOVA (Figure 5)(A) FACS gating strategy for splenic CD45.2+CD8+ T cells.(B) Proportion and number of CD45.2+ CD8+ T cells in the spleens of mice treated with PBS (-) or infected with LmOVA for 7 days in transfer adoptive model (Figure 5). All data are the mean ± s.e.m.; n = 5 independent wells per experiment. Two-tailed Student’s t test; **p < 0.01, ****p < 0.0001.

Fig 3: Kinetics of Early T Cell Differentiation in Murine ATOs(A) Schematic of the M-ATO system.(B) Representative kinetic analysis of T cell differentiation from bone marrow lineage-negative, Sca1+, cKit+ (LSK) isolated from C57BL/6 wild-type (WT) mice at the indicated time points, gated on CD45+ cells to exclude non-hematopoietic cells. Middle and bottom rows: cells are gated on CD11b-, CD11c-, Gr1-, CD19-, B220-, and NK1.1- (Lin neg) and TCR?d- cells. Bottom row: cells are further gated on double-negative (DN) cells CD8- CD4- to analyze DN subsets: DN1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44- CD25+), and DN4 (CD44- CD25-) (Figure S1C).(C) Fold increase and frequencies of cell populations in M-ATOs over time. Left graph shows fold increase in total cell number from LSK cells seeded in M-ATOs at day 0. Frequencies of DN cells (CD8-CD4-), immature single-positive CD8+ (ISP8) cells (CD8+CD4-CD3-), and double-positive (DP) cells (CD8+CD4+) are shown as percentage of total CD45+ TCR?d- Lin- cells. Error bar denotes ±SD (n = 6 independent experiments).(D) Representative kinetic analysis of early T cell development within the DN population during the first 2 weeks of culture in M-ATOs.(E) Frequencies of early T cell populations over the first 2 weeks in M-ATOs. Frequencies of early thymic progenitors (ETPs) (ckitHi, CD44+CD25-), DN2a (ckitHi, CD44+CD25+), DN2b (ckitmed, CD44+CD25+), DN3 (CD44-CD25+), and DN4 (CD44-CD25-) are shown as percentage of total DN cells. Error bar denotes ±SD (n = 3 independent experiments).

Fig 4: WNK1 is required for thymocytes to develop past the pre-TCR checkpoint.(A) Left: flow cytometric analysis of Thy1.2+Lineage- thymocytes separated by expression of CD44 and CD25, and then DN3 (CD44-CD25+) cells separated into DN3a (small, FSClowCD28-) and DN3b (large, FSChighCD28+) cells. Right: Mean ± SEM cell numbers in DN subsets. (B) Left: Flow cytometric analysis of thymocytes from Wnk1fl/+CD2-Cre and Wnk1fl/flCD2-Cre mice showing intracellular TCRß levels in DN2+DN3 and DN4 subsets. Numbers indicate the percentage of TCRß+ cells. Right: Mean ± SEM percentage of DN2+DN3 and DN4 cells that are intracellular TCRß+. (C) Mean ± SEM of CD3e or pre-Ta surface levels measured as mean fluorescence intensity (MFI) in flow cytometry of DN2+DN3 and DN4 thymocytes from Wnk1fl/+CD2-Cre and Wnk1fl/flCD2-Cre mice. (D) Left: flow cytometric analysis of CD4 and CD8 levels on thymocytes from Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/flRag1-/-CD2-Cre mice harvested 4 d after intraperitoneal injection with PBS or anti-CD3e antibody. Gate indicates DP thymocytes and number shows percentage of cells in the gate. Right: mean ± SEM number of DP thymocytes defined using the gates on the left. *0.01Sample sizes: six of each genotype (A, B), eight Wnk1fl/+CD2-Cre and ten Wnk1fl/flCD2-Cre mice (C), four Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/flRag1-/-CD2-Cre mice injected with PBS and five Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/flRag1-/-CD2-Cre mice injected with anti-CD3e (D). Data are pooled from two independent experiments.

Fig 5: Antitumor immunity induced by the sHDLs in vivo. (A) Flow cytometry qualification of CRT levels on tumor cell surface. (B) ELISA qualification of HMGB1 in the tumors. (C) Western-blot analysis of pTBK1 and pIRF3 levels in the tumors. (D) Flow cytometry qualification of the percentage of mature BMDCs (CD80+CD86+) in the DLNs collected 3 days after the administration of different sHDLs (n = 5). (E) ELISA qualification of TNF-a, IL12p40, and IFN-? secretion in the DLNs collected 3 days after the administration of different sHDLs (n = 3). In an additional experiment, the tumors were collected 7 days after the last treatment of a three-treatment regimen (n = 5). (F) Intratumoral densities of mature DCs. (G) ELISA qualification of IFN-a and IFN-ß in the tumors. Intratumoral densities of CD3+ T lymphocytes (H), CD8+ T cells (I), and CD8+IFN-?+ T cells (J). (K) CD8+ T cell-to-Treg (CD4+Foxp3+ T cells) ratio. (L) M1-to-M2 ratio. (M) The expression levels of PDL1 on tumor cells after different treatments. Statistical significance was calculated using a two-sided one-way ANOVA test. The data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.