Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Association between gut bacterial composition and response to CAR T-cell therapy. A, Schematic representation of the prospective longitudinal collection of biological samples (fecal material, blood, tumor tissues, and BM aspirations) from patients with B-cell malignancies treated with commercial anti-CD19 CAR T cells. Feces collection occurred at three time points: visit 1 (prior to lymphodepleting chemotherapy), visit 2 (7–15 days after CAR T-cell infusion), and visit 3 (3 months after CAR T-cell infusion). B, α-Diversity analysis using the Shannon index (left) and the species richness index (right) across visits 1, 2, and 3. Data are presented as means ± SEM, with statistical significance evaluated using Wilcoxon signed-rank tests. C, Comparison of bacterial alpha diversity between ORR + and ORR − at visits 1, 2, and 3, calculated using the Shannon index (left) and the species richness index (right). Results are shown as means ± SEM, with statistical analysis by Wilcoxon signed-rank tests. D, β-Diversity analysis using the Bray–Curtis metric to measure microbial community distances, with PERMANOVA tests performed at visits 1, 2, and 3. E, Comparison of bacterial β-diversity between ORR + and ORR − at visits 1, 2, and 3 using PERMANOVA tests. F, PFS curves comparing patients classified as SIG2 + or SIG1 + according to the TOPOSCORE. G, Serum levels of sMAdCAM-1 (ng/mL) in patients with B-cell lymphoma across longitudinal analyses (left) and according to 6-month response status (right). Results are shown as means ± SEM, analyzed using Mann–Whitney tests. H, LEfSe graph generated from baseline (visit 1) metagenomic data, highlighting the most discriminant species between ORR + and ORR − based on linear discriminant analysis scores. MDS, multidimensional scaling.

Article Snippet: Briefly, after obtaining single-cell suspensions, tumors were stained for 30 minutes at 4°C with a mix of antibodies containing anti-CAR T-cell CD19 (Cat. No. 130-127-342, clone REA1297, Miltenyi), anti-CCR6 (Cat. No. 566130, clone 11A9, BD), anti-CCR7 (Cat. No. 566437, clone 3D12 RUO, BD), anti-CD11b (Cat. No. 301325, clone ICRF44), anti-CD11c (Cat. No. 130-128-247, clone REA618, Miltenyi), anti-CD123 (Cat. No. 130-115-272, clone REA918, Miltenyi), anti-CD127 (Cat. No. 351363, clone A019D5, BioLegend), anti-CD141 (Cat. No. 344113, clone M80, BioLegend), anti-CD16 (Cat. No. 130-113-395, clone REA423, Miltenyi), anti-CD163 (Cat. No. 333631, clone GHI/61, BioLegend), anti-CD19 (Cat. No. 612916, clone SJ25C1, BD), anti-CD1c (Cat. No. 331537, clone L161, BioLegend), anti-CD20 (Cat. No. 612906, clone 2H7, BD), anti-CD206 (Cat. No. 130-126-623, clone DCN228, Miltenyi), anti-CD25 (Cat. No. 356145, clone M-A251, BioLegend), anti-CD3 (Cat. No. 130-113-142, clone REA613, Miltenyi), anti-CD38 (Cat. No. 303549, clone HIT2, BioLegend), anti-CD39 (Cat. No. 749967, clone TU66, BD), anti-CD4 (Cat. No. 344669, clone SK3, BioLegend), anti-CD45 (Cat. No. 130-113-120, clone 5B1, Miltenyi), CD45RA (Cat. No. 740298, clone HI100, BD), anti-CD56 (Cat. No. 362513, clone 5.1H11, BioLegend), anti-CD66b (Cat. No. 305111, clone G10F5, BioLegend), CD79b (Cat. No. 612814, clone CB3-1, BD), anti-CD8 (Cat. No. 344759, clone SK1, BioLegend), anti-CXCR3 (Cat. No. 353755, clone G025H7, BioLegend), anti-CXCR5 (Cat. No. 356941, clone J252D4, BioLegend), anti-FOLR2 (Cat. No. 391705, clone 94b/FOLR2, BioLegend), anti–HLA-DR (Cat. No. 307683, clone L243, BioLegend), anti–PD-1 (Cat. No. 329927, clone EH12.2H7, BioLegend), anti-TCF1 (Cat. No. 905115, Cell Signaling Technology), anti–TIM-3 (Cat. No. 345027, clone F38-2E2, BioLegend), anti-TREM2 (Cat. No. FAB17291G, clone 237920, RD), and Viability (Cat. No. 423105, BioLegend).

Techniques: Comparison, MANN-WHITNEY, Generated

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Akkermansia spp. supplementation improves CD19/CD28-ζ CAR T-cell efficacy against B-cell lymphoma. A, Schematic representation of the experimental design for the CD19/CD28-ζ CAR T-cell mouse model. B and C, Tumor growth kinetics and animal survival following CAR T-cell infusion with or without oral live biotherapeutics. B, Tumor sizes monitored by caliper, presented as means ± SEM. A representative experiment from three independent trials is shown, with 15 mice per treatment group. Tumor sizes were analyzed using one-way ANOVA, with specific time points compared using Mann–Whitney tests. C, Survival analysis was performed using the Kaplan–Meier estimator and log-rank test. D, Flow cytometry analysis of the blood compartment on day 14 after CAR T-cell injection. Evaluation of CAR T-cell expansion rate (% CD45.1 EGFR + cells) and percentage of persistent target cells (% CD19 + cells). Data are presented as means ± SEM, with statistical analysis by Mann–Whitney tests. E, Flow cytometry analysis of the tumor compartment on day 24. Representative contour plots and absolute numbers/percentages of viable CAR T cells across treatment groups. F, Phenotype of tumor-infiltrating CAR T cells, including CD4/CD8 ratio and percentage of IFNγ + CD8 + CAR T cells. Data are shown as means ± SEM, with statistical analysis by Mann–Whitney tests.

Article Snippet: Briefly, after obtaining single-cell suspensions, tumors were stained for 30 minutes at 4°C with a mix of antibodies containing anti-CAR T-cell CD19 (Cat. No. 130-127-342, clone REA1297, Miltenyi), anti-CCR6 (Cat. No. 566130, clone 11A9, BD), anti-CCR7 (Cat. No. 566437, clone 3D12 RUO, BD), anti-CD11b (Cat. No. 301325, clone ICRF44), anti-CD11c (Cat. No. 130-128-247, clone REA618, Miltenyi), anti-CD123 (Cat. No. 130-115-272, clone REA918, Miltenyi), anti-CD127 (Cat. No. 351363, clone A019D5, BioLegend), anti-CD141 (Cat. No. 344113, clone M80, BioLegend), anti-CD16 (Cat. No. 130-113-395, clone REA423, Miltenyi), anti-CD163 (Cat. No. 333631, clone GHI/61, BioLegend), anti-CD19 (Cat. No. 612916, clone SJ25C1, BD), anti-CD1c (Cat. No. 331537, clone L161, BioLegend), anti-CD20 (Cat. No. 612906, clone 2H7, BD), anti-CD206 (Cat. No. 130-126-623, clone DCN228, Miltenyi), anti-CD25 (Cat. No. 356145, clone M-A251, BioLegend), anti-CD3 (Cat. No. 130-113-142, clone REA613, Miltenyi), anti-CD38 (Cat. No. 303549, clone HIT2, BioLegend), anti-CD39 (Cat. No. 749967, clone TU66, BD), anti-CD4 (Cat. No. 344669, clone SK3, BioLegend), anti-CD45 (Cat. No. 130-113-120, clone 5B1, Miltenyi), CD45RA (Cat. No. 740298, clone HI100, BD), anti-CD56 (Cat. No. 362513, clone 5.1H11, BioLegend), anti-CD66b (Cat. No. 305111, clone G10F5, BioLegend), CD79b (Cat. No. 612814, clone CB3-1, BD), anti-CD8 (Cat. No. 344759, clone SK1, BioLegend), anti-CXCR3 (Cat. No. 353755, clone G025H7, BioLegend), anti-CXCR5 (Cat. No. 356941, clone J252D4, BioLegend), anti-FOLR2 (Cat. No. 391705, clone 94b/FOLR2, BioLegend), anti–HLA-DR (Cat. No. 307683, clone L243, BioLegend), anti–PD-1 (Cat. No. 329927, clone EH12.2H7, BioLegend), anti-TCF1 (Cat. No. 905115, Cell Signaling Technology), anti–TIM-3 (Cat. No. 345027, clone F38-2E2, BioLegend), anti-TREM2 (Cat. No. FAB17291G, clone 237920, RD), and Viability (Cat. No. 423105, BioLegend).

Techniques: MANN-WHITNEY, Flow Cytometry, Injection

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Prevalence of intestinal Akkermansia spp. and lymphoma TME. A, CAR T-cell expansion in the peripheral blood of patients receiving anti-CD19 CAR T cells, stratified by the presence of Akkermansia spp. based on MGS data ( n = 6 Akk + ; n = 17 Akk − ). CAR T-cell expansion is represented as a percentage (left) and absolute numbers per mL (right). Data are shown as means ± SEM, with statistical significance determined by Mann–Whitney tests. B, Schematic representation of the experimental workflow. Tumor biopsies obtained shortly after CAR T-cell infusion were analyzed by spectral flow cytometry in patients with available MGS data ( n = 3 Akk + ; n = 3 Akk − ). Tumor supernatants were further analyzed using Meso Scale Discovery (MSD) multiplex assays. C, t-Distributed stochastic neighbor embedding (t-SNE) visualization of unsupervised spectral flow cytometry analysis. D, Comparisons of CAR T cells and T-cell frequencies between Akk + and Akk − patients. Data are presented as means ± SEM, with statistical comparisons performed using Mann–Whitney tests. E, Heatmap representation of expression levels [mean fluorescence intensity (MFI)] of CD39, CD38, and PD-1 within the T-cell population. F, Tumor secretome analysis in patients with available metagenomic data ( n = 6), focusing on levels of GM-CSF, IFNγ, and LAG-3. Results are shown as means ± SEM, with statistical significance assessed by Mann–Whitney tests.

Article Snippet: Briefly, after obtaining single-cell suspensions, tumors were stained for 30 minutes at 4°C with a mix of antibodies containing anti-CAR T-cell CD19 (Cat. No. 130-127-342, clone REA1297, Miltenyi), anti-CCR6 (Cat. No. 566130, clone 11A9, BD), anti-CCR7 (Cat. No. 566437, clone 3D12 RUO, BD), anti-CD11b (Cat. No. 301325, clone ICRF44), anti-CD11c (Cat. No. 130-128-247, clone REA618, Miltenyi), anti-CD123 (Cat. No. 130-115-272, clone REA918, Miltenyi), anti-CD127 (Cat. No. 351363, clone A019D5, BioLegend), anti-CD141 (Cat. No. 344113, clone M80, BioLegend), anti-CD16 (Cat. No. 130-113-395, clone REA423, Miltenyi), anti-CD163 (Cat. No. 333631, clone GHI/61, BioLegend), anti-CD19 (Cat. No. 612916, clone SJ25C1, BD), anti-CD1c (Cat. No. 331537, clone L161, BioLegend), anti-CD20 (Cat. No. 612906, clone 2H7, BD), anti-CD206 (Cat. No. 130-126-623, clone DCN228, Miltenyi), anti-CD25 (Cat. No. 356145, clone M-A251, BioLegend), anti-CD3 (Cat. No. 130-113-142, clone REA613, Miltenyi), anti-CD38 (Cat. No. 303549, clone HIT2, BioLegend), anti-CD39 (Cat. No. 749967, clone TU66, BD), anti-CD4 (Cat. No. 344669, clone SK3, BioLegend), anti-CD45 (Cat. No. 130-113-120, clone 5B1, Miltenyi), CD45RA (Cat. No. 740298, clone HI100, BD), anti-CD56 (Cat. No. 362513, clone 5.1H11, BioLegend), anti-CD66b (Cat. No. 305111, clone G10F5, BioLegend), CD79b (Cat. No. 612814, clone CB3-1, BD), anti-CD8 (Cat. No. 344759, clone SK1, BioLegend), anti-CXCR3 (Cat. No. 353755, clone G025H7, BioLegend), anti-CXCR5 (Cat. No. 356941, clone J252D4, BioLegend), anti-FOLR2 (Cat. No. 391705, clone 94b/FOLR2, BioLegend), anti–HLA-DR (Cat. No. 307683, clone L243, BioLegend), anti–PD-1 (Cat. No. 329927, clone EH12.2H7, BioLegend), anti-TCF1 (Cat. No. 905115, Cell Signaling Technology), anti–TIM-3 (Cat. No. 345027, clone F38-2E2, BioLegend), anti-TREM2 (Cat. No. FAB17291G, clone 237920, RD), and Viability (Cat. No. 423105, BioLegend).

Techniques: MANN-WHITNEY, Flow Cytometry, Multiplex Assay, Expressing, Fluorescence

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Akkermansia spp. systemically releases immunogenic indole metabolites, increasing CAR T-cell efficacy via AhR activation. A, Targeted mass spectrometry-based metabolomic analyses of plasma from patients receiving anti-CD19 CAR T cells at three visits ( n = 43 for visit 1; n = 38 for visit 2; n = 29 for visit 3). Heatmaps represent the log 2 fold change of normalized metabolite values. B, Correlation between plasma levels of IPA and Akkermansia spp. presence in patients with available MGS data, analyzed longitudinally. Results are shown as means of log 2 fold change values ±SEM, with Wilcoxon signed-rank tests applied. C, Spearman correlation between bacteria with the highest relative abundance in ORR + or ORR − patients and metabolites from the indole pathway. Statistical significance was determined using Benjamini–Hochberg FDR–corrected P values for each microorganism across all indoles. D, Schematic representation of CAR T-cell experimental design comparing wild-type and AhR-deficient CAR T cells with or without Akk. p2261 supplementation. E and F, Tumor growth kinetics ( E ) and cross-sectional tumor size comparisons between treatment arms at day 13 ( F ). G, Phenotypic characterization comparison of wild-type and AhR-deficient CAR T cells supplemented with supernatant from anaerobic cultures of Akkermansia spp. at 10% (or control media). H, Tumor growth kinetics of lymphoma following CAR T-cell infusion with or without daily oral gavage administration of IPA( n = 10 mice per group). One-way ANOVA was used for tumor growth curve analyses. Data are presented as mean ± SEM; Mann–Whitney tests were performed for specific comparisons. KO, knockout.

Article Snippet: Briefly, after obtaining single-cell suspensions, tumors were stained for 30 minutes at 4°C with a mix of antibodies containing anti-CAR T-cell CD19 (Cat. No. 130-127-342, clone REA1297, Miltenyi), anti-CCR6 (Cat. No. 566130, clone 11A9, BD), anti-CCR7 (Cat. No. 566437, clone 3D12 RUO, BD), anti-CD11b (Cat. No. 301325, clone ICRF44), anti-CD11c (Cat. No. 130-128-247, clone REA618, Miltenyi), anti-CD123 (Cat. No. 130-115-272, clone REA918, Miltenyi), anti-CD127 (Cat. No. 351363, clone A019D5, BioLegend), anti-CD141 (Cat. No. 344113, clone M80, BioLegend), anti-CD16 (Cat. No. 130-113-395, clone REA423, Miltenyi), anti-CD163 (Cat. No. 333631, clone GHI/61, BioLegend), anti-CD19 (Cat. No. 612916, clone SJ25C1, BD), anti-CD1c (Cat. No. 331537, clone L161, BioLegend), anti-CD20 (Cat. No. 612906, clone 2H7, BD), anti-CD206 (Cat. No. 130-126-623, clone DCN228, Miltenyi), anti-CD25 (Cat. No. 356145, clone M-A251, BioLegend), anti-CD3 (Cat. No. 130-113-142, clone REA613, Miltenyi), anti-CD38 (Cat. No. 303549, clone HIT2, BioLegend), anti-CD39 (Cat. No. 749967, clone TU66, BD), anti-CD4 (Cat. No. 344669, clone SK3, BioLegend), anti-CD45 (Cat. No. 130-113-120, clone 5B1, Miltenyi), CD45RA (Cat. No. 740298, clone HI100, BD), anti-CD56 (Cat. No. 362513, clone 5.1H11, BioLegend), anti-CD66b (Cat. No. 305111, clone G10F5, BioLegend), CD79b (Cat. No. 612814, clone CB3-1, BD), anti-CD8 (Cat. No. 344759, clone SK1, BioLegend), anti-CXCR3 (Cat. No. 353755, clone G025H7, BioLegend), anti-CXCR5 (Cat. No. 356941, clone J252D4, BioLegend), anti-FOLR2 (Cat. No. 391705, clone 94b/FOLR2, BioLegend), anti–HLA-DR (Cat. No. 307683, clone L243, BioLegend), anti–PD-1 (Cat. No. 329927, clone EH12.2H7, BioLegend), anti-TCF1 (Cat. No. 905115, Cell Signaling Technology), anti–TIM-3 (Cat. No. 345027, clone F38-2E2, BioLegend), anti-TREM2 (Cat. No. FAB17291G, clone 237920, RD), and Viability (Cat. No. 423105, BioLegend).

Techniques: Activation Assay, Mass Spectrometry, Clinical Proteomics, Bacteria, Comparison, Control, MANN-WHITNEY, Knock-Out

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Association between gut bacterial composition and response to CAR T-cell therapy. A, Schematic representation of the prospective longitudinal collection of biological samples (fecal material, blood, tumor tissues, and BM aspirations) from patients with B-cell malignancies treated with commercial anti-CD19 CAR T cells. Feces collection occurred at three time points: visit 1 (prior to lymphodepleting chemotherapy), visit 2 (7–15 days after CAR T-cell infusion), and visit 3 (3 months after CAR T-cell infusion). B, α-Diversity analysis using the Shannon index (left) and the species richness index (right) across visits 1, 2, and 3. Data are presented as means ± SEM, with statistical significance evaluated using Wilcoxon signed-rank tests. C, Comparison of bacterial alpha diversity between ORR + and ORR − at visits 1, 2, and 3, calculated using the Shannon index (left) and the species richness index (right). Results are shown as means ± SEM, with statistical analysis by Wilcoxon signed-rank tests. D, β-Diversity analysis using the Bray–Curtis metric to measure microbial community distances, with PERMANOVA tests performed at visits 1, 2, and 3. E, Comparison of bacterial β-diversity between ORR + and ORR − at visits 1, 2, and 3 using PERMANOVA tests. F, PFS curves comparing patients classified as SIG2 + or SIG1 + according to the TOPOSCORE. G, Serum levels of sMAdCAM-1 (ng/mL) in patients with B-cell lymphoma across longitudinal analyses (left) and according to 6-month response status (right). Results are shown as means ± SEM, analyzed using Mann–Whitney tests. H, LEfSe graph generated from baseline (visit 1) metagenomic data, highlighting the most discriminant species between ORR + and ORR − based on linear discriminant analysis scores. MDS, multidimensional scaling.

Article Snippet: After obtaining single-cell suspensions, BM mononuclear cells were stained with a mix of antibodies for 30 minutes at 4°C, comprising CD223 antibody, anti-human, REAfinity (Cat. No. 130-118-549, Miltenyi); BUV395 Mouse Anti-Human CD41a (Cat. No. 740295, BD); BUV496 Mouse Anti-Human CD90 (Cat. No. 741160, BD); BUV661 Mouse Anti-Human CD39 (Cat. No. 749967, BD); BUV737 Mouse Anti-Human CD7 (Cat. No. 753774, BD); Brilliant Violet 421 anti-human CD34 Antibody (Cat. No. 343610, BioLegend); Brilliant Violet 570 anti-human CD3 Antibody (Cat. No. 300436, BioLegend); BV605 Mouse Anti-Human CD56 (Cat. No. 562779, BD); Brilliant Violet 650 anti-human CD16 Antibody (Cat. No. 302042, BioLegend); BV786 Mouse Anti-Human CD33 (Cat. No. 740974, BD); PerCP anti-human CD45 Antibody (Cat. No. 368506, BioLegend); PE/Fire 640 anti-human CD19 Antibody (BioLegend); CD19 CAR FMC63 Idiotype Antibody, REAfinity (Cat. No. 130-127-342, Miltenyi); BUV615 Rat Anti-Human CD115 (CSF-1R; Cat. No. 751279, BD); BB515 Armenian Hamster Anti-ICOS (CD278; Cat. No. 565880, BD); BUV805 Mouse Anti-Human CD38 (Cat. No. 742074, BD); and BV510 Mouse Anti-Human CD279 (PD-1; Cat. No. 563076, BD).

Techniques: Comparison, MANN-WHITNEY, Generated

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Akkermansia spp. supplementation improves CD19/CD28-ζ CAR T-cell efficacy against B-cell lymphoma. A, Schematic representation of the experimental design for the CD19/CD28-ζ CAR T-cell mouse model. B and C, Tumor growth kinetics and animal survival following CAR T-cell infusion with or without oral live biotherapeutics. B, Tumor sizes monitored by caliper, presented as means ± SEM. A representative experiment from three independent trials is shown, with 15 mice per treatment group. Tumor sizes were analyzed using one-way ANOVA, with specific time points compared using Mann–Whitney tests. C, Survival analysis was performed using the Kaplan–Meier estimator and log-rank test. D, Flow cytometry analysis of the blood compartment on day 14 after CAR T-cell injection. Evaluation of CAR T-cell expansion rate (% CD45.1 EGFR + cells) and percentage of persistent target cells (% CD19 + cells). Data are presented as means ± SEM, with statistical analysis by Mann–Whitney tests. E, Flow cytometry analysis of the tumor compartment on day 24. Representative contour plots and absolute numbers/percentages of viable CAR T cells across treatment groups. F, Phenotype of tumor-infiltrating CAR T cells, including CD4/CD8 ratio and percentage of IFNγ + CD8 + CAR T cells. Data are shown as means ± SEM, with statistical analysis by Mann–Whitney tests.

Article Snippet: After obtaining single-cell suspensions, BM mononuclear cells were stained with a mix of antibodies for 30 minutes at 4°C, comprising CD223 antibody, anti-human, REAfinity (Cat. No. 130-118-549, Miltenyi); BUV395 Mouse Anti-Human CD41a (Cat. No. 740295, BD); BUV496 Mouse Anti-Human CD90 (Cat. No. 741160, BD); BUV661 Mouse Anti-Human CD39 (Cat. No. 749967, BD); BUV737 Mouse Anti-Human CD7 (Cat. No. 753774, BD); Brilliant Violet 421 anti-human CD34 Antibody (Cat. No. 343610, BioLegend); Brilliant Violet 570 anti-human CD3 Antibody (Cat. No. 300436, BioLegend); BV605 Mouse Anti-Human CD56 (Cat. No. 562779, BD); Brilliant Violet 650 anti-human CD16 Antibody (Cat. No. 302042, BioLegend); BV786 Mouse Anti-Human CD33 (Cat. No. 740974, BD); PerCP anti-human CD45 Antibody (Cat. No. 368506, BioLegend); PE/Fire 640 anti-human CD19 Antibody (BioLegend); CD19 CAR FMC63 Idiotype Antibody, REAfinity (Cat. No. 130-127-342, Miltenyi); BUV615 Rat Anti-Human CD115 (CSF-1R; Cat. No. 751279, BD); BB515 Armenian Hamster Anti-ICOS (CD278; Cat. No. 565880, BD); BUV805 Mouse Anti-Human CD38 (Cat. No. 742074, BD); and BV510 Mouse Anti-Human CD279 (PD-1; Cat. No. 563076, BD).

Techniques: MANN-WHITNEY, Flow Cytometry, Injection

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Prevalence of intestinal Akkermansia spp. and lymphoma TME. A, CAR T-cell expansion in the peripheral blood of patients receiving anti-CD19 CAR T cells, stratified by the presence of Akkermansia spp. based on MGS data ( n = 6 Akk + ; n = 17 Akk − ). CAR T-cell expansion is represented as a percentage (left) and absolute numbers per mL (right). Data are shown as means ± SEM, with statistical significance determined by Mann–Whitney tests. B, Schematic representation of the experimental workflow. Tumor biopsies obtained shortly after CAR T-cell infusion were analyzed by spectral flow cytometry in patients with available MGS data ( n = 3 Akk + ; n = 3 Akk − ). Tumor supernatants were further analyzed using Meso Scale Discovery (MSD) multiplex assays. C, t-Distributed stochastic neighbor embedding (t-SNE) visualization of unsupervised spectral flow cytometry analysis. D, Comparisons of CAR T cells and T-cell frequencies between Akk + and Akk − patients. Data are presented as means ± SEM, with statistical comparisons performed using Mann–Whitney tests. E, Heatmap representation of expression levels [mean fluorescence intensity (MFI)] of CD39, CD38, and PD-1 within the T-cell population. F, Tumor secretome analysis in patients with available metagenomic data ( n = 6), focusing on levels of GM-CSF, IFNγ, and LAG-3. Results are shown as means ± SEM, with statistical significance assessed by Mann–Whitney tests.

Article Snippet: After obtaining single-cell suspensions, BM mononuclear cells were stained with a mix of antibodies for 30 minutes at 4°C, comprising CD223 antibody, anti-human, REAfinity (Cat. No. 130-118-549, Miltenyi); BUV395 Mouse Anti-Human CD41a (Cat. No. 740295, BD); BUV496 Mouse Anti-Human CD90 (Cat. No. 741160, BD); BUV661 Mouse Anti-Human CD39 (Cat. No. 749967, BD); BUV737 Mouse Anti-Human CD7 (Cat. No. 753774, BD); Brilliant Violet 421 anti-human CD34 Antibody (Cat. No. 343610, BioLegend); Brilliant Violet 570 anti-human CD3 Antibody (Cat. No. 300436, BioLegend); BV605 Mouse Anti-Human CD56 (Cat. No. 562779, BD); Brilliant Violet 650 anti-human CD16 Antibody (Cat. No. 302042, BioLegend); BV786 Mouse Anti-Human CD33 (Cat. No. 740974, BD); PerCP anti-human CD45 Antibody (Cat. No. 368506, BioLegend); PE/Fire 640 anti-human CD19 Antibody (BioLegend); CD19 CAR FMC63 Idiotype Antibody, REAfinity (Cat. No. 130-127-342, Miltenyi); BUV615 Rat Anti-Human CD115 (CSF-1R; Cat. No. 751279, BD); BB515 Armenian Hamster Anti-ICOS (CD278; Cat. No. 565880, BD); BUV805 Mouse Anti-Human CD38 (Cat. No. 742074, BD); and BV510 Mouse Anti-Human CD279 (PD-1; Cat. No. 563076, BD).

Techniques: MANN-WHITNEY, Flow Cytometry, Multiplex Assay, Expressing, Fluorescence

Journal: Cancer Discovery

Article Title: Gut Microbiota Modulation through Akkermansia spp . Supplementation Increases CAR T-cell Potency

doi: 10.1158/2159-8290.CD-24-1230

Figure Lengend Snippet: Akkermansia spp. systemically releases immunogenic indole metabolites, increasing CAR T-cell efficacy via AhR activation. A, Targeted mass spectrometry-based metabolomic analyses of plasma from patients receiving anti-CD19 CAR T cells at three visits ( n = 43 for visit 1; n = 38 for visit 2; n = 29 for visit 3). Heatmaps represent the log 2 fold change of normalized metabolite values. B, Correlation between plasma levels of IPA and Akkermansia spp. presence in patients with available MGS data, analyzed longitudinally. Results are shown as means of log 2 fold change values ±SEM, with Wilcoxon signed-rank tests applied. C, Spearman correlation between bacteria with the highest relative abundance in ORR + or ORR − patients and metabolites from the indole pathway. Statistical significance was determined using Benjamini–Hochberg FDR–corrected P values for each microorganism across all indoles. D, Schematic representation of CAR T-cell experimental design comparing wild-type and AhR-deficient CAR T cells with or without Akk. p2261 supplementation. E and F, Tumor growth kinetics ( E ) and cross-sectional tumor size comparisons between treatment arms at day 13 ( F ). G, Phenotypic characterization comparison of wild-type and AhR-deficient CAR T cells supplemented with supernatant from anaerobic cultures of Akkermansia spp. at 10% (or control media). H, Tumor growth kinetics of lymphoma following CAR T-cell infusion with or without daily oral gavage administration of IPA( n = 10 mice per group). One-way ANOVA was used for tumor growth curve analyses. Data are presented as mean ± SEM; Mann–Whitney tests were performed for specific comparisons. KO, knockout.

Article Snippet: After obtaining single-cell suspensions, BM mononuclear cells were stained with a mix of antibodies for 30 minutes at 4°C, comprising CD223 antibody, anti-human, REAfinity (Cat. No. 130-118-549, Miltenyi); BUV395 Mouse Anti-Human CD41a (Cat. No. 740295, BD); BUV496 Mouse Anti-Human CD90 (Cat. No. 741160, BD); BUV661 Mouse Anti-Human CD39 (Cat. No. 749967, BD); BUV737 Mouse Anti-Human CD7 (Cat. No. 753774, BD); Brilliant Violet 421 anti-human CD34 Antibody (Cat. No. 343610, BioLegend); Brilliant Violet 570 anti-human CD3 Antibody (Cat. No. 300436, BioLegend); BV605 Mouse Anti-Human CD56 (Cat. No. 562779, BD); Brilliant Violet 650 anti-human CD16 Antibody (Cat. No. 302042, BioLegend); BV786 Mouse Anti-Human CD33 (Cat. No. 740974, BD); PerCP anti-human CD45 Antibody (Cat. No. 368506, BioLegend); PE/Fire 640 anti-human CD19 Antibody (BioLegend); CD19 CAR FMC63 Idiotype Antibody, REAfinity (Cat. No. 130-127-342, Miltenyi); BUV615 Rat Anti-Human CD115 (CSF-1R; Cat. No. 751279, BD); BB515 Armenian Hamster Anti-ICOS (CD278; Cat. No. 565880, BD); BUV805 Mouse Anti-Human CD38 (Cat. No. 742074, BD); and BV510 Mouse Anti-Human CD279 (PD-1; Cat. No. 563076, BD).

Techniques: Activation Assay, Mass Spectrometry, Clinical Proteomics, Bacteria, Comparison, Control, MANN-WHITNEY, Knock-Out