Fig 2: Blocking the PD-1 pathway re-engages T-bet transcriptional circuits during chronic LCMV infection(A) Heatmaps display the raw expression values of chronic-only T-bet (left) or Eomes (right) gene neighbors from clone-13-infected mice following PBS (TEX) or aPD-L1 treatment. Genes were clustered by K-means.(B) Gene Ontology pathway analysis was performed on the gene clusters from T-bet neighbors (top, cluster 3) or Eomes neighbors (bottom, cluster 4).(C) Histograms from a representative clone-13-infected control (shaded gray) or aPD-L1-treated (green line) mouse show the MFI of candidate proteins in CD44+ PD-1+ CD8 T cells. Expression of candidate proteins in naive CD8 T cells are shown (dashed line). Histograms are representative of 5 mice per experimental condition.

Fig 3: Enforced ATX expression in tumor cells does not affect induction of T cell responses by vaccination(A) Experimental set-up in the anti-cancer vaccination model. Mice were injected s.c. with wild-type (TC-1WT) or ATX-expressing (TC-1ATX) tumor cells on day 0, vaccinated on days 8, 11, and 14 and were either sacrificed on day 18, or monitored until day 70. Tumor cells were injected into one flank and the vaccine DNA was “tattooed” into the depilated skin of the opposing flank. Data are from one experiment representative of two experiments.(B) The DNA vaccine encodes HPV-E7 protein together with tumor-unrelated helper epitopes. The CD8+ T cells that have a TCR specific for the immunodominant E749–57 peptide presented in H-2Db can be detected with MHC class I (MHC-I) tetramers. A tetramer is made by folding E749–57 peptide with MHC-I monomer, conjugating this to biotin and multimerizing it with fluorochrome-conjugated streptavidin.(C–E) Monitoring of the T cell response to vaccination in peripheral blood by flow cytometry in TC-1WT (n = 6) and TC-1ATX (n = 5) tumor-bearing mice.(C) Frequency of H-2Db/E749–57 tetramer positive (Tet+) cells among total CD8+ T cells. (D and E) Frequency of cells with a CD44+CD62L- effector phenotype among total CD8+ T cells (D) or total CD4+ T cells (E).(F–J) Analysis of the CD8+ T cell response in spleen (F–J) and tumor (H and J) on day 18 in TC-1WT (n = 5) and TC-1ATX (n = 6) tumor-bearing mice.(F) Absolute number of tetramer positive (Tet+) CD8+ T cells in spleen.(G) Frequency of granzyme B (GZB)+ and IFN?+ cells among Tet+ CD8+ T cells in spleen. IFN? was measured after ex vivo PMA/ionomycin stimulation. The dotted line indicates IFN? signal in unstimulated cells.(H) Frequency among CD45+ hematopoietic cells (left) and absolute number (#, right) of Tet+ CD8+ T cells in TC-1WT and TC-1ATX tumors.(I) Representative flow cytometry plots indicating the percentage of Tet+ cells among total CD8+ T cells in TC-1WT and TC-1ATX tumors after vaccination and in TC-1WT tumors of non-vaccinated (untreated) mice.(J) Mean fluorescence intensity (MFI) of GZB+ and IFN?+ cells within Tet+ CD8+ T cells in TC-1WT and TC-1ATX tumors. IFN? was measured as in (G).(C–H and J) Data are expressed as mean ± SD; *p < 0.05, **p < 0.01 (Mann-Whitney U test).

Fig 4: M-ATOs Recapitulate T Cell Differentiation from Different Hematopoietic Subsets(A–C) Different subsets in the hematopoietic hierarchy from HSCs to T cell progenitors were isolated from the bone marrow and the thymus of C57BL/6 WT mice and seeded into M-ATOs. From the bone marrow: HSC (hematopoietic stem cell) (Lin-Sca1+cKit+CD48 CD150+IL7R-); MPP (multi-potent progenitor) (Lin-Sca1+c-Kit+CD48-CD150-IL7R-); LMPP (lymphoid-primed multi-potent progenitor) (Lin-Sca1+c-Kit+IL7R+Flk2+); and CLP (common lymphoid progenitor) (Lin-Sca1Loc-KitLo IL7R+Flk2+). From the thymus: ETP (Lin-CD4-CD8-c-Kithi CD44hiCD25-); DN2 (Lin-CD4-CD8-c-Kithi CD44hi CD25+); and DN3 (Lin-CD4-CD8-CD44-CD25+). Representative phenotypes of M-ATO-derived cells are shown at weeks 1 (A), 2 (B), and 6 (C). Data are representative of three biological replicates.(D) Frequencies of T cell populations shown as percentage of total CD45+ TCR?d- Lin- cells initiated from the different hematopoietic subsets in week 1, week 2, and week 6 M-ATOs. Error bars denote ± SD (n = 3 independent experiments).

Fig 5: 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).