Gene expression was normalized by log2 transformation, and an average score across patient samples was calculated per gene

Gene expression was normalized by log2 transformation, and an average score across patient samples was calculated per gene. gene expression, adding significant functional information to our phenotypic data. The total naive/central memory (T_Tn/Tcm) cluster was enriched in markers of more naive T cell phenotypes (= 33) exhibited a good prognosis compared with those with depletion (= 14, 0.05) (Figure 2A). Although stage Ostarine (MK-2866, GTx-024) influenced survival as expected (Supplemental Figure 3A), our disaggregated data demonstrated that patients with Stage II and Stage III tumors who also had T_Tcyto1 enrichment exhibited improved survival compared with those with depletion (Figure 2A). Thus, our data suggest that enrichment of T_Tcyto1 gene signatures within tumors correlates with improved survival independent of stage. However, there was no statistically significant difference in survival between T_Tcyto2 gene setCenriched (= 36) and Cdepleted patients (= 22, = 0.2) (Figure 2B). T_Tcyto2 enrichment or depletion did not correlate with differences in survival in those patients with Stage II Ostarine (MK-2866, GTx-024) and Stage III tumors (Figure 2B), in contrast to T_Tcyto1. These Ostarine (MK-2866, GTx-024) results suggest that T_Tcyto1 and T_Tcyto2 clusters contain T cells with functionally distinct roles in CRC. Open in a separate window Figure 2 Clinical outcomes associated with single T cell subtypes in CRC.(ACD) Kaplan-Meier curves of overall survival in the CRC TCGA cohort for patients enriched or depleted for the following gene sets T_Tcyto1 (A), T_Tcyto2 (B), T_Treg (C), T_Tex (D). Kaplan-Meier curves from enriched or depleted patients with all stages (upper) or only those patients with Stage II and III tumors (lower). * 0.05; ** 0.02; *** 0.001 (log-rank test). See detailed values in Supplemental Table 6. (E) Bar graph depicting relative proportion of patients by stage that are enriched or depleted for T_Tcyto1, T_Tcyto2, or T_Treg gene sets. Average stage of patients is indicated in table. Since a cytotoxic T cell type with a similar phenotype has been reported to correlate with a positive outcome in melanoma (36), we queried gene sets from the T_Tcyto1 and T_Tcyto2 clusters in gene expression data from the TCGA cohort of melanoma (Supplemental Table 5 and Methods). In the melanoma cohort, positive clinical outcomes were associated with both T_Tcyto1 ( 0.05) and T_Tcyto2 ( 0.05) gene sets, and there was higher overlap between gene setCenriched patients (86 %, T_Tcyto1; 78 %, T_Tcyto2) than the CRC cohort, likely reflecting differences in cytotoxic T cell populations between these 2 cancers Rabbit Polyclonal to EDG4 (Supplemental Figure 3, BCD). Accordingly, resident memory phenotype CD8+ T cells have been correlated with positive prognosis in melanoma but not CRC (37). Enrichment of T_Tcyto1 or T_Tcyto2 gene signatures in melanoma patients was not likely to be dependent on stage (Supplemental Figure 3, A and E). T_Treg cluster gene setCenriched patients showed a significantly better prognosis than the Cdepleted patients ( 0.05) in CRC, although this cluster did not show any prognostic significance in melanoma (Figure 2C and Supplemental Figure 3F). These findings are consistent with the preponderance of histologic studies that concluded that Tregs are associated with favorable outcomes in CRC (21, 22, 38, 39). Enrichment of T_Treg genes was relatively higher in patients with early-stage cancer, indicating that the improved prognosis may be related to stage (Figure 2, C and E). Analysis of the prognostic value of the T_Tex gene set did not reach statistical significance in CRC (Figure 2D). Distinct prognostic effector memory and nonprognostic resident memory CD8+ T cell infiltrates. To define T cells with improved granularity, we segregated CD8+ and CD4+ T cells based on antibody-derived tag (ADT) signal and transcriptome, and we analyzed them separately (Supplemental Figure 4A). Within 12,642 single CD8+ T cells, Ostarine (MK-2866, GTx-024) unsupervised clustering based on transcriptome identified 13 distinct clusters consisting of 7 distinct nonsimulated populations and stimulated CD8+ T cells (Figure 3A). Those clusters populated by unstimulated Ostarine (MK-2866, GTx-024) T cells were identified by their canonical marker expression (Figure 3A). Based on 21 ADT signals, we were able to link nonstimulated clusters to cognate-stimulated clusters (Figure 3B). Overall, all CD8 subtypes were observed within tumors, while cells from normal tissue were largely confined to clusters CD8_Trm, CD8_IEL, and CD8_Tact (Figure 3C). Open in a.