Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet: Transcriptional profiling identifies therapy-responsive T cell subsets in the blood circulation (A) Schematic of the immune checkpoint therapy (ICT) regimen strategy. Mice were challenged s.c. with MC-38 or HCmel12 syngenic tumors and treated with different ICTs. (B) MC-38 and HCmel12 tumor growth (mean ± SEM) and survival curves of untreated and anti-OX40/CpG-, anti-PD-L1-, and anti-OX40/CpG plus anti-PD-L1 (PDOX)-treated wild-type mice. Data were combined from two replicate experiments (n = 8–16 mice per group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) tSNE scRNA-seq plot visualizing six transcriptional clusters of blood T cells from day 18 tumor-bearing mice that were untreated or received ICT. (D) Combined t-SNE scRNA-seq plot of blood T cells color coded for the untreated and ICT groups. (E) tSNE scRNA-seq plots of blood T cells color coded for the untreated group and each ICT group individually. (F) Heatmap displaying scaled expression values of discriminative genes per cluster. (G) tSNE scRNA-seq plots displaying gene expression of Cd4 , Cd8 , Id2 , Lgals1 , Klrg1 , Klrc1 , Klrk1 , Cxcr3 , Gzma , Gzmk , Gzmb , and Ly6a . (H) Stacked bar graphs representing the percentage of cells from the untreated and ICT groups present in the six transcriptional clusters. (I) Volcano plots showing significant gene expression related to Id2 expression in CD4 + and CD8 + T cells. Stacked bar graphs indicate the percentage of the cell origin according to their treatment. See also Figure S1 .

Article Snippet: Anti-mouse KLRG1 Pe-Cy7 (clone 2F1) , Thermo Fisher , Cat# 25-5893-82; RRID: AB_1518768.

Techniques: Expressing, Gene Expression

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet: Circulating therapy-responsive T cell subsets display effector cell properties with increased cytotoxic and migratory capacity (A) Representative histogram plots of NKG2A, NKG2D, KLRG1, and CD43 1B11 expression on gated ID2 − CD8 + or ID2 + CD8 + and gated ID2 − CD4 + or ID2 + CD4 + T cell populations residing in the blood circulation of MC-38-challenged PDOX-treated mice. Numbers indicate average mean fluorescence intensity. (B) Percentage of CD43 1B11+ cells within the total CD8 + and CD4 + T cell population in the blood of untreated and ICT-treated groups. (C) tSNE plots of flow cytometric data visualizing NKG2A, NKG2D, KLRG1, and CD43 1B11 expression (red) on CD8 + and CD4 + T cells in the blood from untreated and PDOX-treated groups. The blue/red tSNE plot indicates cell origin for CD8 + and CD4 + T cells of the untreated and PDOX-treated group, respectively. (D) Representative histograms (left) and quantification of fluorescence intensity (right) of granzyme B expression in blood circulating CD43 1B11− CD8 + and CD43 1B11+ CD8 + and CD43 1B11− CD4 + and CD43 1B11+ CD4 + T cell populations of MC-38-challenged PDOX-treated mice. (E) Percentage of Adpgk-specific CD8 + T cells in the blood of untreated and ICT-treated groups. (F) Heatmaps of RNA-seq data of sorted CD43 1B11− CD8 + and CD43 1B11+ CD8 + and CD43 1B11− CD4 + and CD43 1B11+ CD4 + T cells (n = 2 individual mice per subset) from spleens isolated from wild-type mice challenged with MC-38 and treated with PDOX. Scaled expression values of discriminating genes are displayed. Data (A)–(F) are were collected from mice on day 18 post tumor challenge. The p values in (B) and (E) were calculated by ANOVA and in (D) by unpaired Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data in (B) and (E) are presented as mean ± SEM, and each dot in (B), (D), and (E) represents an individual mouse. Data (A)–(E) are representative of 2–3 independent experiments. See also Figure S2 .

Article Snippet: Anti-mouse KLRG1 Pe-Cy7 (clone 2F1) , Thermo Fisher , Cat# 25-5893-82; RRID: AB_1518768.

Techniques: Expressing, Fluorescence, RNA Sequencing, Isolation

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet: Dynamic induction of therapy-responsive T cell subsets in the blood circulation (A and B) Kinetics of the CD43 1B11+ , NKG2A + , and KLRG1 + cells of CD8 + T cells (A) and CD43 1B11+ cells of CD4 + T cells (B) in the blood circulation after challenge with MC-38 tumor cells and treated or not treated with different ICTs (anti-PD-L1, anti-OX40, or PDOX). (C) Kinetics of the CD43 1B11+ cells of CD8 + and CD4 + T cells after mock challenge (saline) and treated similarly as in (A) and (B). (D) Ranking of the percentage CD43 1B11+ cells of CD8 + T cells in blood (on day 13 post tumor challenge) for each individual MC-38-bearing mouse (left panel) or HCmel12-bearing mouse (right panel). An asterisk indicates correlation with tumor-free mice. The p values in (A–C) were calculated by ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data in (A)–(C) are presented as mean ± SEM Data shown in (A)–(D) are representative of 2–3 independent experiments.

Article Snippet: Anti-mouse KLRG1 Pe-Cy7 (clone 2F1) , Thermo Fisher , Cat# 25-5893-82; RRID: AB_1518768.

Techniques: Saline

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet: Expansion of the therapy-responsive CD8 + T cell subset in the blood circulation, TME, and draining lymph nodes (A) TME: percentage of leukocytes, CD8 + T cells, M8-specific CD8 + T cells, FOXP3 − CD4 + T cells, and FOXP3 + CD4 + Treg cell among live cells; CD8 + T cell/Treg cell ratio and percentage of Treg cells among total CD4 + T cells in the MC-38 tumor microenvironment (TME) of untreated and PDOX treated mice. Blood circulation: percentage of FoxP3 + CD4 + Treg cells among total CD4 + T cells in the blood of untreated and PDOX-treated mice. (B) Representative immunofluorescence images of MC-38 tumor-infiltrating CD8 + T cells (red) of untreated and PDOX-treated mice. The bar graph indicates absolute CD8 + T cell count per square millimeter. (C) Ki-67 expression versus CD43 1B11 , KLRG1, or NKG2A of CD8 + T cells in the TME and blood of untreated and PDOX-treated mice. Numbers indicate the average percentage of double-positive cells. (D) Left: representative flow cytometry plots indicating CD43 1B11 versus granzyme B expression of MC-38 tumor-infiltrating CD8 + and CD4 + T cells. Numbers indicate the fluorescence intensity of granzyme B expression in CD43 1B11+ T cells. Right: median fluorescence intensity of granzyme B expression in CD43 1B11+ CD8 + and CD43 1B11+ CD4 + T cells of untreated and PDOX-treated animals. (E) The proportion of single-, double-, and triple-cytokine-producing cells within the tumor-infiltrating CD8 + T cells of untreated and PDOX MC-38 tumor-challenged mice treated or not treated with PDOX. (F) Total numbers of CD43 1B11 and KLRG1-positive CD8 + T cells in non-draining lymph nodes (ndLNs; closed circles) and tumor-draining lymph nodes (tdLNs; open squares). Lines connect ndLNs and tdLNs from the same mouse. (G) MC-38 tumor growth of untreated and PDOX-treated wild-type mice receiving CD8-depleting (yellow/green) and CD4-depleting (blue/purple) antibodies or mock. Data shown in (A)–(F) were collected from mice on day 20 post tumor challenge (PDOX treatment started on day 10). The p values in (A), (B), and (D)–(F) were calculated by unpaired Student’s t test and in (G) by ANOVA; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data in (A), (B), (D), (E), and (G) are presented as mean ± SEM, and each dot in (A), (B), and (D) represents an individual mouse. Data shown are representative of 2 independent experiments. See also Figure S6 .

Article Snippet: Anti-mouse KLRG1 Pe-Cy7 (clone 2F1) , Thermo Fisher , Cat# 25-5893-82; RRID: AB_1518768.

Techniques: Immunofluorescence, Cell Counting, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Reports Medicine

Article Title: OX40 agonism enhances PD-L1 checkpoint blockade by shifting the cytotoxic T cell differentiation spectrum

doi: 10.1016/j.xcrm.2023.100939

Figure Lengend Snippet:

Article Snippet: Anti-mouse KLRG1 Pe-Cy7 (clone 2F1) , Thermo Fisher , Cat# 25-5893-82; RRID: AB_1518768.

Techniques: Recombinant, Purification, Staining, Cell Isolation, Transgenic Assay, Software, Gene Expression

Journal: Frontiers in Immunology

Article Title: Distinctive phenotype for HLA-E- versus HLA-A2-restricted memory CD8 αβT cells in the course of HCMV infection discloses features shared with NKG2C + CD57 + NK and δ2 - γδT cell subsets

doi: 10.3389/fimmu.2022.1063690

Figure Lengend Snippet: Immunophenotype of HLA-E UL40 and HLA-A2 pp65 memory CD8 T cells after a primary HCMV infection, a reactivation in KTR and during chronic infection in HCMV+ HV. After immunostaining and fluorescence acquisition, CD8Tcell populations were defined using a dedicated gating strategy as reported in the Materials and methods section and in Figure S3 in order to identify pMHC tetramer- CD8Tcells (tet-CD8T), HLA-E UL40 (E/UL40+) and HLA-A2 pp65 (A2/pp65+) CD8Tcells. Box plots with median and interquartile values showing the specific expression for Tim-3 (A) , Lag-3 (B) , 4-1BB (C) , 2B4 (D) , Tigit (E) and KLRG1 (F) . Results are expressed as percents of positive cells in the different CD8 T cell subsets. (A–F) Box plots with median and interquartile values for analyses performed on HLA-E UL40 CD8T cells from KTR with a primary infection (n=5), a reactivation (n=7), from HV with chronic infection/latency (HV; n=9) and on HLA-A2 pp65 CD8T cells from KTR with a primary infection (n=7), a reactivation (n=7), from HV with chronic infection/latency (n=10) and the respective tet - CD8T cells. Each point corresponds to an individual CD8T response. Statistical analysis was performed by Mann-Whitney U -test. P values: *for p < 0.05, **for p<0.01, ***for p<0.001 and ****for p<0.0001.

Article Snippet: Antibody included: BB700-anti-CD45RA (clone 5H9, BD Optibuild™, BD Biosciences), BV786-anti-CD45RO (clone UCHL1, BD Horizon™, BD Biosciences), PE-Cy7 anti-CCR7 (CD197, clone 3D12, BD Pharmingen™, BD Biosciences) BB515-anti-CD27 (clone M-T271, BD Horizon™), PE-anti-CD28 (clone CD28.2, BD Pharmingen™) and PE-anti-CD57 (clone NK1), BB700-anti-CD56 (clone B159), BB700-anti-HLA-DR (clone 46-6), BB515-anti-CD38 (clone HIT2), PE-anti-2B4 (CD244, clone 2-69), BV786-anti-4-1BB (CD137, clone 4B4-1), BB700-anti-PD-1 (CD279, clone EH12.1), PE-Cy7-anti-TIGIT (clone A15153G), BB515-anti-Tim-3 (CD366, clone 7D3), BB515-or FITC- anti-Lag-3 (CD223, clone T47-530), PE-Cy7-anti-KLRG1 (clone 2f1/KLRG1), BV786-anti-CX3CR1 (clone 2A9-1) and PE-anti-CD62L (clone DREG-56) all from BD Biosciences.

Techniques: Infection, Immunostaining, Fluorescence, Expressing, MANN-WHITNEY