Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 2 Q-2361 dose-dependently rescues mouse CD8 T-cell function in the presence of tacrolimus. Mouse T cells were labeled with CellTrace Violet and cocultured with CD3/CD28 Dynabeads. For the first hour, they were incubated with 0.6 ng/ mL of tacrolimus (left panels) or vehicle control (central and right panels) alone. Subsequently, the indicated concentrations of (A) VX-710 or (B) Q-2361 were added. After 3 days, CD8 T-cell proliferation was determined by flow cytometry through the measurement of CellTrace Violet dilution (left and central panels), and cell viability was determined using the 7AAD live/dead discrimination dye (right panels). Results in (A) and (B) show representative data from two experiments each with similar results. One-way analysis of variance followed by Tukey’s multiple comparison test, **p<0.01; ***p<0.001; ****p<0.0001. n=3/group. TAC, tacrolimus.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Cell Function Assay, Labeling, Incubation, Control, Flow Cytometry, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 4 Regression of tacrolimus-dependent squamous cell carcinoma (SCC) growth. (A–D) Description of model and experimental set up. (A) Creation of a transplantable SCC cell line. (B) Growth characteristics of SCC in immune-competent mice receiving normal or tacrolimus-containing diet. (C) Research concept for local Q-2361 treatment in tacrolimus-suppressed mice, and (D) experimental outline. (E) Left: Tumor volume following intratumoral (IT) injection of Q-2361 or vehicle solution. Groups were further subdivided by receipt of anti-CD8β antibody (mAb) to deplete CD8 T cells, or receipt of an isotype control mAb. Right: Confirmation of CD8 T-cell depletion in the blood on day 10 (IT Vehicle+Isotype mAb, IT Vehicle+anti-CD8β, IT Q-2361+anti-CD8β; CD45+TCR-β+CD8α+ T cells as a percentage of all CD45+TCR-β+ T cells). Mice with poorly established tumors<0.07 cm3 on day 14 and/or fold growth was <1.15 from day 9 to 14 were excluded from the study; excluded data not included in analysis. Dotted line in (E) represents start of two times per day injections on day 11. Error bars represent SEM, n=8–12; (F) Body weight on day 25. One-way analysis of variance on day 32 (E); Left, or day 10 (E); Right, p<0.0001 (****), or day 25 (F) followed by Tukey’s multiple comparison test.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Injection, Control, Comparison

Journal: Journal for immunotherapy of cancer

Article Title: Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

doi: 10.1136/jitc-2023-006783

Figure Lengend Snippet: Figure 5 Q-2361 treatment increases CD8 T-cell activation and effector function within squamous cell carcinoma (SCC) tumors. (A) Impact of systemic tacrolimus withdrawal on SCC tumor growth. (B) Impact of local Q-2361 treatment on SCC tumor growth in immunosuppressed mice. In (A), mice with poorly established tumors<0.06 cm3 on day 12 and/or fold growth was <1 from day 12 to 19 were excluded from the study, and in (B) mice with poorly established tumors<0.07 cm3 on day 13 and/or fold growth was <1.15 from day 9 to 13 were excluded from the study; excluded data not included in analysis. Dotted line in (A) represents TAC diet removal on day 14 and in (B) represents start of two times per day injections on day 8. (C,D) Analysis of tumor weight and flow cytometry analysis of intratumoral T-cell abundance, activation, and effector molecule production in tumors harvested from (A) and (B), respectively. Data for T cells, CD8 T cells, and CD69+CD8 T cells represent number of cells per gram of tumor. Error bars represent SEM, (A/C) n=6, (B/D) n=10. (A/B) One-way analysis of variance on day 26 (A) or day 19 (B) followed by Tukey’s multiple comparison test. (C/D) Unpaired student’s t-test, p<0.05 (*), p<0.01 (**), p<0.001 (***), and p<0.0001 (****). IT, intratumoral; ns, not significant; TAC, tacrolimus.

Article Snippet: In experiments involving CD8 T- cell depletion, mice were injected with 250 μg of anti- CD8β (clone 53–5.8, Bio X Cell) antibody or isotype control antibody (clone HRPN, Bio X Cell) intraperitoneal on days 8 and 15 post tumor challenge, and 100 μg of antibody on day 22 post tumor challenge.

Techniques: Activation Assay, Flow Cytometry, Comparison

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: CD8–IL2 selectively and potently activates CD8 + T cells in human tumor fragments. A, Overview of the PDTF platform and analysis strategy (created with BioRender.com ). B, Representative flow cytometry plots displaying markers of proliferation (Ki-67), cytotoxicity (granzyme B), and activation (PD-1, CD137) in intratumoral CD8 + T cells from PDTFs that were left untreated or treated with ex vivo CD8–IL2 (RE098). C, Quantification of activation markers on total intratumoral CD8 + T cells in untreated or CD8–IL2-treated PDTFs measured by flow cytometry ( n = 23). D, Quantification of intracellular IFNγ in total intratumoral CD8 + T cells in untreated or CD8–IL2-treated tumor digests measured by flow cytometry ( n = 5). E, Representative gating for PD-1 + CD39 + (late dysfunctional) and PD-1 + CD39 − (early dysfunctional cells) CD8 + T cells. F, Same analysis as in C but separated for PD-1 + CD39 + (late dysfunctional) and PD-1 + CD39 − (early dysfunctional cells). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 by two-tailed Wilcoxon test ( C, D ). ***, P < 0.001; **, P < 0.01 by the Friedman test corrected for multiple comparisons ( F ). Only significant comparisons are shown.

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Flow Cytometry, Activation Assay, Ex Vivo, Two Tailed Test

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: CD8–IL2 induces immunologic responses in a subset of tumors. A, Heat map displaying normalized delta values (CD8–IL2 condition − untreated condition) of 24 soluble mediators secreted by PDTFs, ordered according to unsupervised hierarchical clustering ( n = 23). B, Examples of T-cell effector cytokines (IFNγ, TNFα), cytotoxic mediators (granzyme B), and chemokines (CXCL9, CXCL10, and CXCL11) in unstimulated and CD8–IL2 stimulated PDTFs in ex vivo responding (R) and nonresponding (NR) tumors. C, Separation of tumors by cumulative z-scores of soluble mediators in CD8–IL2 R and NR tumors. D, Baseline infiltration of dysfunctional (PD-1 + CD39 + CD103 + ) and memory (PD-1 + IL7R + ) CD8 + T cells and CXCL13 expression in CD8–IL2-R and NR tumors measured by flow cytometry. E, CD8 + T-cell activation markers measured by flow cytometry separately plotted for R and NR tumors. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05 by two-tailed Wilcoxon test ( B – E ). Only significant comparisons are shown.

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Ex Vivo, Expressing, Flow Cytometry, Activation Assay, Two Tailed Test

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: TCR signaling is required for a functional immune response to CD8–IL2. A, Schematic overview of Lck inhibition (LCKi) in the context of CD8–IL2 treatment (created with BioRender.com ). B, Normalized delta values (CD8–IL2 or CD8–IL2 + LCKi condition − untreated condition) of soluble mediators secreted by PDTFs. Tumor samples were selected based on prior response to CD8–IL2 (as shown in ) and material availability ( n = 8). C, Cumulative z-scores of soluble mediators in CD8–IL2-responding tumors when treated with either CD8–IL2 or CD8–IL2 + LCKi. D, Correlation of log 2 fold changes (LOG2FC) of soluble mediators induced by either CD8–IL2 and CD8–IL2 + LCKi versus the untreated condition. E, Intratumoral CD8 + T-cell activation of CD8–IL2-responding tumors upon CD8–IL2 and CD8–IL2 + LCKi treatment measured by flow cytometry. F, Intracellular expression of ranzyme B and ( G ) secreted soluble Granzyme B and IFNγ in human PBMCs upon 5-day incubation with decreasing concentrations of anti-CD3 in the absence and presence of CD8–IL2 ( n = 4). **, P < 0.01 by two-tailed Mann–Whitney U test (C). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 by the Friedman test corrected for multiple comparisons ( E ).

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Functional Assay, Inhibition, Activation Assay, Flow Cytometry, Expressing, Incubation, Two Tailed Test, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: CD8–IL2 elicits broad transcriptional rewiring of the intratumoral CD8 + T-cell landscape. A, UMAP visualization of all intratumoral CD8 + T cells (from untreated and all treated conditions) in human tumor fragments from five tumors ( n = 8,712 cells) identifying 13 different clusters. B, Normalized expression of a selected set of previously published gene signatures for effector cells , memory cells , cycling cells , and T-cell exhaustion . C, Annotated canonical marker gene expression in the different CD8 + T-cell clusters. D, Cluster fractions of CD8 + T-cell states derived from untreated or CD8–IL2 treated PDTFs. E, Quantification of the distribution of the number of cell labels transferred from the Cycl_c1 and Cycl_c2 clusters into the rest of the cell populations. (This analysis was not performed for the Cycl_c3 cluster due to limited cell numbers.) F, UMAP visualization of CD8 + T displaying the cells in diffusion pseudotime (DPT). G, Cell order along DPT for each CD8 + T cell cluster. H, UMAP visualization of the T-cell clonal size and ( I ) per CD8 + T-cell cluster. Clonotypes are ranked based on frequency ranges: “single” = found in 1 cell, “small” = in >1 and <5 cells, “medium” = in >5 and <10 cells, “large” = >10 and <20 cells, “hyperexpanded” = in >20 and <100 cells. J, Clonal overlap between CD8 + T-cell clusters calculated by the Jaccard index.

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Expressing, Marker, Derivative Assay, Diffusion-based Assay

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: CD8–IL2 activates the dysfunctional tumor-specific CD8 + T-cell pool. A, UMAPs of intratumoral CD8 + T cells displaying the expression of MKI67, GZMB, TNFRSF9, and IFNG ( n = 5 tumors). B, UMAP representation of CD8 + T-cell density in the untreated, CD8–IL2-treated, and CD8–IL2 + LCKi conditions. C, Paired fractions of CD8 + T-cell clusters in untreated, CD8–IL2-treated, and CD8–IL2 + LCKi conditions for the five clusters that increased or formed in response to CD8–IL2 treatment. The one CD8–IL2-NR tumor (RE027) is marked by a square, and the four responders are depicted as circles. D, Heat map displaying normalized expression of genes discriminating the distinct transcriptional CD8 + T-cell states present in untreated PDTFs. E, Gating strategy for flow-cytometric sorting of dysfunctional, memory, effector, and transitional CD8 + T cells. F, Tumor reactivity coculture of expanded TILs from sorted dysfunctional, memory, effector, and transitional CD8 + T cells with autologous tumor digest in the presence or absence of CD8–IL2. Reactivity was measured by assessing CD137, CD107a, and IFNγ/TNFα expression by flow cytometry. Note that due to differences in expansion, not all populations reached sufficient cell numbers for all experiments. *, P < 0.05 by the Kruskal–Wallis test. G, Heat map showing the difference in marker expression between TIL + digest or TIL + digest + CD8–IL2 versus TIL alone as measured in F . Values are averaged for each marker between the two tumors for which data for all conditions were available (RE088, OV054-1).

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Expressing, Flow Cytometry, Marker

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: CD8 + T cells acquire distinct transcriptional programs following CD8–IL2 treatment dependent on antigen recognition. A, IL2 program (123 genes) and TCR program (66 genes) scores in CD8 + T cells from the untreated, CD8–IL2 and CD8–IL2 + LCKi conditions. B, UMAP displaying the expression of the IL2 program and TCR program. C, Expression of the IL2 program and TCR program for CD8 + T cells order along DPT in the untreated, CD8–IL2 and CD8–IL2 + LCKi conditions. D, Heat maps displaying expression of the genes in the IL2 program and TCR program, respectively, in the different CD8 + T-cell clusters. E, Violin plots displaying IL2 program, TCR program, and “better effector” signature expression in cells separated for the 13 different intratumoral CD8+ T-cell clusters. F and G, Quantification of activation and maturation markers in CD8 + T cells ( F ) and distinct immune populations ( G ) in untreated and CD8–IL2-treated PDTFs measured by flow cytometry ( n = 6). *, P < 0.05 by a two-tailed Wilcoxon test. H, DGE analysis of non-T immune cells between the untreated and CD8–IL2-treated condition in the 4 CD8–IL2-R tumors. I, IFN-downstream response signature in non-T immune (top) and B cells (bottom) from untreated and CD8–IL2-treated PDTFs. J, Quantification of CD137 and granzyme B expression on total CD8 + T cells in PDTFs that were untreated or treated with CD8–IL2 and CD8–IL2 + anti-IFNγR1, respectively ( n = 6). K, Correlation of log 2 fold changes (LOG2FC) of soluble mediators induced by either CD8–IL2 and CD8–IL2 + anti-IFNγR1 versus the untreated condition. Parameters with fold change >2 between stimulated conditions are marked by a large dot. P values were calculated by a two-tailed Wilcoxon test ( A, F–I ). P values were adjusted by Bonferroni correction ( H ).

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Expressing, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: Cancer Discovery

Article Title: CD8-Targeted IL2 Unleashes Tumor-Specific Immunity in Human Cancer Tissue by Reviving the Dysfunctional T-cell Pool

doi: 10.1158/2159-8290.CD-23-1263

Figure Lengend Snippet: Reinvigoration of tumor-reactive T cells by CD8–IL2 is superior to PD-1 blockade. A, Normalized delta values (CD8–IL2 condition, anti–PD-1 condition, or anti–PD-1 + CD8–IL2 condition − untreated condition) of soluble mediators secreted by PDTFs, ordered based on unsupervised hierarchical clustering. B, Percentage of tumors displaying an ex vivo response (positive cumulative z-score) to anti–PD-1 ( n = 4/13), to CD8–IL2 ( n = 6/13), and to anti–PD-1 + CD8–IL2 ( n = 8/13). C, Correlation of log 2 fold changes (LOG2FC) of concentrations of soluble mediators upon CD8–IL2 or anti–PD-1 treatment versus the untreated condition. D, UMAP representation of CD8 + T-cell density from the untreated, CD8–IL2, anti–PD-1, and anti–PD-1 + CD8–IL2 conditions for the three tumors responsive to ex vivo anti–PD-1. E, CD8 + T-cell cluster distribution in PDTFs left untreated or treated with CD8–IL2, anti–PD-1, and anti–PD-1 + CD8–IL2, respectively. F, Circos plots displaying clonal sharing of the different clusters in the untreated and anti–PD-1 conditions, and in the untreated and CD8–IL2 conditions, respectively. All clonotypes are included in the analysis. G, Neoantigen-specific gene signature score and Neo_TCR_8 score for CD8 + T cells from ex vivo anti–PD-1 responsive tumors color-coded by their clonal expansion. H, Cluster distribution of tumor-reactive cells, defined in G as scoring top 20% (0%–20%), intermediate (21%–40%), or low (41%–100%) for both tumor-reactive gene signatures in the untreated, anti–PD-1 and CD8–IL2 conditions. I, TOX gene-expression levels in cells scoring as top 20% for the tumor reactivity signatures in untreated, anti–PD-1 and CD8–IL2-treated PDTFs. P values were calculated by the two-tailed Wilcoxon test.

Article Snippet: To treat the PDTFs ex vivo , tumor medium was supplemented with either CD8–IL2 (CD8-specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L for comparison with PD1–IL2, 1 nmol/L for all other experiments,), PD1–IL2 (PD-1–specific cis-targeting not-alpha attenuated-beta IL2, Asher Biotherapeutics, 10 nmol/L), IL2v (not-alpha IL2, Asher Biotherapeutics, 1 nmol/L), anti-CD8β (clone 97/47, Asher Biotherapeutics, 1 nmol/L), recombinant human IL2 (PeproTech, see titration), recombinant human IL15 (Preprotech, 1 mmol/L), anti–PD-1 (Nivolumab, Bristol-Myers Squibb, 10 μg/mL), or anti-CD3 (OKT3, BioLegend, 0.5 μg/mL).

Techniques: Ex Vivo, Expressing, Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Anti-CD8 antibodies can trigger CD8 + T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

doi: 10.4049/jimmunol.1003941

Figure Lengend Snippet: (A) 5 × 104 naïve mouse transgenic F5 T-cells were either mock treated or incubated with 100 μg/ml CT-CD8a, 100 μg/ml 53.6.7, 100 μg/ml CT-CD8b or 100 μg/ml KT112 at 4°C for 25 minutes, then stained with cognate PE-conjugated H-2Db tetramer (25μg/ml) as described in the Materials & Methods. No staining was observed under any of the conditions shown with a control H-2Db tetramer folded around the lymphocytic choriomeningitis virus GP1-derived epitope KAVYNFATC (residues 33-41). Data were acquired using a modified FACSAriaII flow cytometer and analyzed with FlowJo software. Results were obtained by gating on the CD3+CD4− population. (B) 3×104 naïve mouse transgenic F5 T-cells were incubated at 37°C for 18 hours with either 100 μg/ml CT-CD8a, 100 μg/ml 53.6.7, 100 μg/ml CT-CD8b, 100 μg/ml KT112 or PMA (50 ng/ml) and ionomycin (1 μg/ml). Supernatants were harvested and assayed for MIP1β by ELISA. Error bars represent standard deviations.

Article Snippet: For experiments with mouse cells, the following unconjugated anti-mouse CD8 antibodies were used: (i) anti-CD8α clone CT-CD8a (Caltag-Medsystems, Buckingham, UK); (ii) anti-CD8α clone 53.6.7 (Biolegend, Cambridge, UK); (iii) anti-CD8β clone KT112 (hybridoma kindly provided by Prof. Rose Zamoyska); and, (iv) anti-CD8β clone CT-CD8b (Caltag-Medsystems).

Techniques: Transgenic Assay, Incubation, Staining, Derivative Assay, Modification, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Anti-CD8 antibodies can trigger CD8 + T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining

doi: 10.4049/jimmunol.1003941

Figure Lengend Snippet: The heterogeneity of anti-mouse CD8 antibodies Summary of the effects exerted by anti-mouse CD8 antibodies on pMHCI tetramer binding and CD8 + T-cell activation in the absence of TCR engagement.

Article Snippet: For experiments with mouse cells, the following unconjugated anti-mouse CD8 antibodies were used: (i) anti-CD8α clone CT-CD8a (Caltag-Medsystems, Buckingham, UK); (ii) anti-CD8α clone 53.6.7 (Biolegend, Cambridge, UK); (iii) anti-CD8β clone KT112 (hybridoma kindly provided by Prof. Rose Zamoyska); and, (iv) anti-CD8β clone CT-CD8b (Caltag-Medsystems).

Techniques: Binding Assay, Activation Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme for characterizing the effect of BBM on primary CD3+ T cells. b Fold changes in cell number and c viability determined using the standard trypan blue method. Error bars indicate SD from six donors. d Representative FACS plots illustrating the expression of CCR7 and CD45RA in CD4+ cells (top) and CD8β+ cells (bottom) on day 14. e Estimated numbers of CD4+CCR7+CD45RA+ cells (left) and CD8β+CCR7+CD45RA+ cells (right) in cultured CD3+ T cells calculated from the total cell number and frequencies of the populations that are PI- on day 14. f , g Results of the CFSE assay in CD4+ ( f ) and CD8β+ ( g ) fractions within CD3+ cells on day 3. Error bars indicate SD from six ( b , c ) or four donors ( e – g ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Expressing, Cell Culture, CFSE Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Experimental scheme designed to assess the in vivo persistence of BBM-treated primary CD8+ T cells. b Gating strategy employed to determine the expression of CCR7 and CD45RA in the CD45+CD8β+ fractions. c Frequencies of injected CD45+CD8β+ cells in the spleen (left), peripheral blood (center), and bone marrow (right) on day 14. Each data point represents one injected mouse and its corresponding donors: circle; donor A, triangle; donor B, square; donor C. All error bars indicate SD. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (unpaired t -test). d Representative flow cytometry plots of CCR7 and CD45RA in CD45+CD8β+ cells in the spleen samples.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: In Vivo, Expressing, Injection, Flow Cytometry

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Gating strategy for FACS to purify the naïve and memory T cell fractions from healthy donor-derived CD8+ cells. b , c Gene ontology analysis of DEGs in BBM-treated T SCM cells. Upregulated ( b ) or downregulated ( c ) genes from RNA-sequencing results on day 12. Genes were queried in the DAVID functional annotation database. d Transcripts per million (TPM) values of cell cycle-related and effector function-related genes. Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 ( n = 4, paired t -test). T SCM stem cell memory T, T CM central memory T, T EM effector memory T.

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, RNA Sequencing, Functional Assay

Journal: Communications Biology

Article Title: A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism

doi: 10.1038/s42003-024-06297-0

Figure Lengend Snippet: a Metabolomics of BBM-treated T SCM cells from healthy donor-derived CD8+ cells. Lipids commonly upregulated or downregulated in all three donors are shown. b Representative flow cytometry plots of Phosflow assay in BBM-treated CD3+ cells on day 10. Phosphorylated proteins in CD8+ T cells are shown. c Mean fluorescence intensity (MFI) of phosphorylated p38 in CD8+ T cells. Error bars indicate SD from four donors. d Oxygen consumption rate (OCR) across time for BBM-treated CD3+ T cells. e Spare respiratory capacity (SRC) calculated by OCRmax-OCRbasal. f Extracellular acidification rates (ECAR) across time for BBM-treated CD3+ T cells. Error bars indicate the standard error of the mean from six donors ( d – f ). Statistical significance is denoted as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001 (paired t -test) ( c , e ).

Article Snippet: Cultured CD3+ T cells (as described in “Primary T cell culture”) were stained with anti-CD3-APC (3 uL/sample), anti-CD4-FITC (2 uL/sample), and anti-CD8β-PE (4 uL/sample) antibodies and sorted using a SONY MA900 cell sorter at 9 d after stimulation.

Techniques: Derivative Assay, Flow Cytometry, Fluorescence