Jennifer R. Knapp

Negative Altered cytoskeletal gene expression Actg1, Tuba1b, Tubb4b Supplemental Table 1: Cluster identity determined by transcriptional differences between each cluster. Each cluster was given a brief description based on the genes that were the most significantly upregulated (or downregulated where noted) relative to either other tetramer-positive or tetramer-negative clusters. Purple clusters are predominantly tetramer-positive, blue clusters are a mix of tetramer-positive and tetramer-negative cells and gray clusters are predominantly tetramer-negative cells. Clusters are numbered based on the number of cells occupying each cluster, with cluster 0 having the most cells and cluster 13 the least. Tetramer binding was based on if cells within the cluster came predominantly from a tetramer-positive or tetramer-negative library, the majority of clusters had minimal mixing of these groups. 1.

Differential gene expression between all clusters

Differential gene expression between Tet-Positive or Tet-Negative clusters

Intra cluster differential gene expression between Tet-High and Tet-Low cells

Flow cytometry panels, gating strategy and comparison of TILs to other gene signatures.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell–mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation in vivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

Intra cluster differential gene expression between Tet-High and Tet-Low cells

Negative Altered cytoskeletal gene expression Actg1, Tuba1b, Tubb4b Supplemental Table 1: Cluster identity determined by transcriptional differences between each cluster. Each cluster was given a brief description based on the genes that were the most significantly upregulated (or downregulated where noted) relative to either other tetramer-positive or tetramer-negative clusters. Purple clusters are predominantly tetramer-positive, blue clusters are a mix of tetramer-positive and tetramer-negative cells and gray clusters are predominantly tetramer-negative cells. Clusters are numbered based on the number of cells occupying each cluster, with cluster 0 having the most cells and cluster 13 the least. Tetramer binding was based on if cells within the cluster came predominantly from a tetramer-positive or tetramer-negative library, the majority of clusters had minimal mixing of these groups. 1.

Negative Altered cytoskeletal gene expression Actg1, Tuba1b, Tubb4b Supplemental Table 1: Cluster identity determined by transcriptional differences between each cluster. Each cluster was given a brief description based on the genes that were the most significantly upregulated (or downregulated where noted) relative to either other tetramer-positive or tetramer-negative clusters. Purple clusters are predominantly tetramer-positive, blue clusters are a mix of tetramer-positive and tetramer-negative cells and gray clusters are predominantly tetramer-negative cells. Clusters are numbered based on the number of cells occupying each cluster, with cluster 0 having the most cells and cluster 13 the least. Tetramer binding was based on if cells within the cluster came predominantly from a tetramer-positive or tetramer-negative library, the majority of clusters had minimal mixing of these groups. 1.