Fig 2: Cardiolipin Synthesis Supports CD8+ TM Cell Development, Metabolism, and Function(A) Schematic of CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h and then cultured ± AD for 72 h during IL-15 TM cell development.(B) Quantification of total CL content in IL-15 TM cells cultured as in (A).(C) OCR of IL-15 TM cells cultured as in (A) at baseline and after exposure to aCD3/aCD28 coated beads, Oligo, FCCP, and Rot/Ant.(D) IL-15 TM cells were cultured as in (A) and then restimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells, and IFN-? MFI are shown.(E) Schematic of the activation of CD8+ T cells isolated from Ptpmt1flox/floxErt2-Cre-/- (PTPMT1 iWT) Ptpmt1flox/floxErt2-Cre+/- (PTPMT1 i?T) mice and 4-OHT treatment.(F) CL content in Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E).(G) OCR of Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) at baseline and after exposure to PMA/Ionomycin, Oligo, FCCP, and Rot/Ant.(H) Ptpmt1 iWT and Ptpmt1 i?T CD8+ T cells cultured as in (E) and stimulated with aCD3/aCD28 for 20 h. Percentage of producing IFN-? cells and IFN-? MFI are shown.(I) Schematic of WT CD8+ T cells activated with aCD3/aCD28 + IL-2 for 48 h, cultured additional 24 h in IL-2 before performing Ctrl/Ptpmt1 CRISPR. 24 h after CRISPR, equal number of CRISPR Ctrl/Ptpmt1 KO cells were transferred in congenic recipient mice and donor cell frequencies followed for 21 days before LmOVA WT infection.(J) Percentage of CD8+ CD45.2+ Tet+ CRISPR Ctrl/Ptpmt1 KO cells treated as in (I), shown as mean ± SEM.(K) Bacterial burden shown as CFU per µg of liver isolated from mice treated as in (I) 3 days post-LmOVA challenge. Dots represent individual mice.(L) Schematic of WT CD8+ T cells activated and cultured in IL-2 in the presence of BSA-conjugated 50 µM PG(18:1)2 or 50 µM PG(18:2)2 for 48 h.(M) Quantification of CL species in IL-2 TE CD8+ T cell cultured ± PG(18:2)2 for 48 h.(N) Maximal cristae width measured in IL-2 TE CD8+ T cell cultured as in (M).(O) SRC of IL-2 TE CD8+ T cells cultured as in (M) stimulated with aCD3/aCD28 coated beads.(P) Cytokine production (normalized to untreated cells) measured in IL-2 TE CD8+ T cells activated as in (M) and stimulated for 20 h with aCD3/aCD28 + IL-2.(Q) Schematic of adoptive transfer strategy of OT-I IL-2 CD8+ TE cells—pre-treated as in (L)— into congenic recipient mice. Twenty-one days post-transfer, mice were infected i.v. with 1 × 106 CFU of LmOVA ?Acta.(R) Survival of donor cells 21 days post-transfer, expressed as number of donor cells in the spleen. Dots represent individual mice.(S) Percentage of circulating donor cells 21 days post-transfer. Dot represent individual mice.(T) Number of donor cells recovered from spleens of recipient mice 5 days after infection with LmOVA ?Acta. Dots represent individual mice.Data shown as mean ± SEM of =3 independent experiments unless indicated. Statistical comparisons for 2 groups calculated by unpaired two-tailed Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S4.

Fig 3: SSC improves quality of clustering analyses(A–C) Section from FFPE inflamed gut tissue was stained with 18 markers (CD45, CD45RA, CD45RO, CD3, CD4, CD8, CD14, CD68, CD20, CD25, Foxp3, Gata3, pan-cytokeratin, Ki-67, PD-1, PD-L1, vimentin, SMA). Protein expression, by means of staining fluorescence intensity, was used as input to perform neighborhood embedding. (A) UMAP neighborhood embeddings showing distribution of CD8, CD4, CD3, CD68, CD14, and CD45 markers in presence or not of SSC. (B) Depiction of Leiden clustering (resolution = 0.3) according to the neighborhood embedding. Each number represents one cluster. (C) Silhouette score for the Leiden clustering was calculated based on the UMAP coordinates for clustering resolutions ranging from 0 to 10 in steps of 0.1.

Fig 4: Establishment of protocols for staining of FFPE tissue sections by ChipCytometry in healthy human colon tissues(A) Schematic representation of tissue preparation and iterative staining-acquisition-bleaching cycles for highly multiplexed ChipCytometry. For FFPE tissue section, a pre-treatment for antigen retrieval is necessary before loading into the microfluidic chip.(B) Representative staining of CD3, CD45, and pan-cytokeratin according to different antigen retrieval conditions.(C) Quantification of signal-to-noise ratio for the markers shown in (B). Data are depicted as interquartile ranges, with whiskers extending to 10% and 90% and outliers plotted as dots. Individual crypts (pan-cytokeratin staining) or cells (CD3/CD45 staining) were quantified for eight positions per section.(D) Hematoxylin-eosin staining of consecutive tissue sections after different antigen retrieval treatments. Zoom in (marked area in the upper row) is shown at higher magnification in the lower panel. Numbers indicate regions of mucosa (1), submucosa (2), and muscularis (3).(E) Scoring of tissue loss (0 = no tissue integrity loss, 3 = complete loss of the tissue). Data are shown as mean ± standard deviation. In (C) and (E), significances are calculated using Tukey's test followed by Dunn's multiple comparisons test (*p < 0.05; **p < 0.01; ****p < 0.0001). A minimum of three donors in two or three independent experiments were used. See also Figures S1–S3.