Fig 2: Analysis of TCF-1 and TOX expression in tumor-infiltrating exhausted CD8+ T cells(A) Histograms showing TCF-1 expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells.(B) Histograms showing TOX expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells.

Fig 3: Impact of immune checkpoint blockade on tumor-infiltrating CD8+ T cells(A–H) Representative images of flow cytometry analysis of tumor-infiltrating CD8+ T cells (A and E), PD-1+ CD8+ T cells (B and F), SLAMF6+TIM3- stem-like and SLAMF6-TIM3+ terminally exhausted CD8+ T cells (C and G) and Ki67high SLAMF6+TIM3- stem-like CD8+ T cells (D and H) in the MCAprog tumor model following treatment with Isotype control (Iso) or combined anti-PD-1 and anti-CTLA-4 antibodies (ICB).

Fig 4: Hypoxia induces dysfunction and terminal exhaustion of human T cells.a Schematic graph demonstrating the coculture model. b Representative flow cytograms (upper panel) gated from human pan-T cell culture and quantification (lower panel, n = 3) of differentiated CD8+ T cell subtypes: Tn (naïve T cells), Tcm (central memory T cells), Tem (effector memory T cells), Teff (effector T cells). c Schematic graph demonstrating the normoxia (20% O2) and hypoxia (1% O2) culture condition of T cells coculturing with human TNBC cell line. d Heatmap of the differentially expressed genes (DEGs) in hypoxic cultured human T cells compared to normoxia group. DEGs were identified in edgeR (|logFC| > 1, adjusted P < 0.01). P values were adjusted using Benjamini–Hochberg method in edgeR. DEGs identified in the indicated GO gene clusters are marked in the heatmap. e GSEA analysis of human T cells in hypoxic versus normoxic conditions. Analysis was based on ranked logFC from edgeR. FDR and adjusted p value are shown in the graph. P values were adjusted using Benjamini–Hochberg method in GSEA analysis. f Flow cytometry quantifications of immune effector molecules and exhaustion markers in CD8+ T cells gated from human pan-T cells cultured under the indicated conditions (n = 4). g Representative flow cytograms of PD-1 and TIM-3 expression in CD8+ T cells gated from human pan-T cells culture. h Flow cytometric quantification of terminally exhausted T cells (PD-1+ TIM-3+) in CD8+ T cells gated from human pan-T cells culture (n = 3). i Flow cytometric quantification of proliferating cells (Ki76+) in CD8+ and CD4+ T cells gated from human T cells cocultured with TNBC (n = 3). All flow cytometry data (b, f, h, and i) are presented as the mean ± SD of samples from three to four donors. For all flow cytometry data, P values were determined by one-way ANOVA (f, h) or two-way ANOVA (b) with Turkey’s test, or paired two-tailed t-test (i). Raw RNA-seq data is available in the GEO database with accession number GSE179885. For the remaining data, source data are provided in Source Data file.

Fig 5: Pharmacological inhibition of PSGL-1 promotes decreased T cell exhaustion and functional T cell responses(A) Histograms of CD162/PSGL-1 expression on activated CD8+ T cells from 3 healthy donors and 3 patients with melanoma.(B) Relative expression of PSGL-1 on CD8+ T cells from healthy donors and patients with melanoma.(C–E) PBMCs from healthy donors were assessed for transcription factor expression or restimulated to assess cytokine production.(C) Flow cytometry plots showing IFN? and TNF-a production by CD8+ T cells from two different healthy donor PBMCs after iTEFF or iTEX culture and restimulated on day 9; pre-gated on live, CD8+CD45RO+ cells.(D) Top: frequencies of IFN? and TNF-a double-producing CD8+CD45RO+ cells cultured under iTEFF, iTEFF + PSGL-1 agonist, or iTEX conditions. Lines are connecting data from the same donor under the different culture conditions. Bottom: relative reduction of IFN? and TNF-a double-producing CD8+CD45RO+ cells upon PSGL-1 ligation under iTEFF conditions (4 out of 5 donors).(E) EOMES/T-bet expression ratio in CD8+CD45RO+ cells from donors in (D).(A–E) Each dot represents a unique donor. Experiments were performed 2× (A and B) or 3× (C–E). Data are parametric data; unpaired t tests were performed. Error bars are SEM.(F) Growth (volume) of AE17 mesothelioma tumors in C57BL/6 and PSGL-1-/- mice.(G) Top: splenic NP(396–404)-specific CD8+ T cells of control- and recombinant PSGL-1-human Fc protein (rPSGL-1 Fc)-treated LCMV Cl13-infected mice assessed on 8 dpi. Bottom: PD-1 and TIM-3 expression on NP(396–404)-specific CD8+ T cells.(H) Co-expression of PD-1, LAG3, and TIM-3 on NP(396–404) specific CD8+ T cells assessed via Boolean gating.(I) Growth of YUMM1.5 tumors in control C57BL/6 mice and mice treated with rPSGL-1 Fc beginning on day 0.(J) Representative H&E histology with anti-CD3 staining of YUMM1.5 tumor sections collected on day 21. (K) YUMM1.5 tumor volumes following therapeutic treatment with rPSGL-1 Fc beginning on day 14.(F–J) Experiments were performed 2× with >3 mice/group per experiment.See also Figure S7.