Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Single-cell sequencing analysis of biopsies from non-small cell lung cancer (NSCLC) patients. Panels indicate the expression of PDCD1 , LAG3 and CBLB and CBLC analyzed from the single-cell lung cancer extended atlas (LuCA) (Salcher et al, ) repository as indicated. ( B ) Dot plot with the percentage of CD4 and CD8 T-cells that co-express PD-1 and LAG-3 after ex vivo activation, from healthy donors ( n = 8) and NSCLC patients ( n = 10). Statistical comparisons were performed by the Mann–Whitney test. Error bars correspond to ±SD ( C ) CBL-B expression by mean fluorescent intensities in CD4 and CD8 T-cells from a sample of non-responder NSCLC patients ( n = 4), activated ex vivo in the presence of the indicated treatments. Shown data from total CD4 and CD8 gated populations. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( D ) Same as ( C ) but for C-CBL expression. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( E ) Percentage of proliferating CD4 T cells (left) and CD8 T cells (right) from a sample of high PD-1/LAG-3 co-expression patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests ( n = 5). Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( F ) Flow cytometry histograms of SATB1, Phospho SMAD 2/3, LCK and ZAP70 expression. Gates were established according to unstained controls in T-cells from a sample of non-responder NSCLC patients. Percentage of expression and Mean Fluorescence Intensity values are indicated. Data information: Statistical comparisons are shown in the graph as indicated in Methods. Briefly, for ( B ) statistical comparisons were performed by the Mann–Whitney test. For ( C – E ), statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Error bars correspond to ±SD. **, ***, ****, indicate P < 0.01, P < 0.001 and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: Sequencing, Expressing, Ex Vivo, Activation Assay, MANN-WHITNEY, Flow Cytometry, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Schematic design of the experiment. BALB/c female mice were randomly allocated and subcutaneously injected with 2 × 10 6 Lung adenocarcinoma (Lacun3) cells per animal. 100 µg of anti-PD-1, 100 µg of anti-LAG3, 30 mg/kg of CBL-Bi and the corresponding depletion antibodies were administered intraperitoneally at days 0, 2, 6, 9, 13 and 15 as indicated in the figure. NK, CD4, and CD8 T‐cell depletions were carried out by intraperitoneal administration of 100 μg of anti‐mouse CD8a, CD4 or NK1.1 antibody. Mice were humanely sacrificed when tumor size reached ~150–200 mm 2 , or when tumor ulceration or discomfort were observed. ( B ) Kaplan–Meier survival plot of mice under the indicated treatments or depletion (percent). Statistical significance was tested with the Log-rank test. ( C ) Evolution of mean tumor size following the indicated treatments (left). Tumor volumes 9 days after treatment initiation (right). Error bars correspond to ±SEM (left) and box and whiskers with min to max values (right), computing the minimum, maximum, median and quartiles for 25th and 75th percentiles. The whiskers go down to the smallest value and up to the largest ( n = 6 mice per group). Data information: Statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. ( D ) Tumor growth of individual mice in the indicated treatment groups ( n = 6 mice per group). Statistical comparisons are shown in the graph as indicated in Methods. Data information: Briefly, for ( B ), survival was represented by Kaplan–Meier plots and analyzed by log-rank test. For ( C ), statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. *, **, ****, indicate P < 0.05, P < 0.01, and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: Injection

Journal: The Journal of investigative dermatology

Article Title: ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

doi: 10.1016/j.jid.2021.02.753

Figure Lengend Snippet: Figure 2. Human cSCCs are enriched for ENTPD1D memory T cells. ENTPD1 expression among (a) CD8þ, (b) CD4þ FoxP3e, and (c, d, e) CD4þ FoxP3þ T cells isolated from human SCC, perilesional skin (NL), and blood (n ¼ 13). Percentage-positive cells are shown by flow cytometry in a‒c and e and MFI by flow cytometry is shown in d. Percentage of CD45ROþ cells among ENTPD1þ populations of (f) CD4þ and (g) CD8þ T cells isolated from human SCC, perilesional skin (NL), and blood (n ¼ 7). (h) CD45ROþENTPD1þ cells are enriched among CD4þFoxP3þ T cells in cSCC compared with those in NL skin and blood (n ¼ 7). (i, j) Representative flow cytometry analysis of CD45RO, FoxP3, and ENTPD1 expression in cells isolated from human SCC, perilesional skin (NL), and blood. P-values were determined by Tukey’s multiple comparisons test. cSCC, cutaneous squamous cell carcinoma; ENTPD1, ectonucleotidase triphosphate diphosphodydrolase 1; MFI, mean fluorescence intensity; NL, nonlesional; SCC, squamous cell carcinoma.

Article Snippet: Antibodies to human CD45, CD3, CD4, CD8, CD45R0, CLA, ENTPD1, CD73, CD11b, CD11c, CD206, DC-SIGN, PD-L1, PD-L2, PD-1, gdTCR, FoxP3, and IL17A were purchased from Tonbo Biotechnologies, eBioscience, Bio- Legend, R&D Systems (Minneapolis, MN), and BD Biosciences.

Techniques: Expressing, Isolation, Cytometry

Journal: The Journal of investigative dermatology

Article Title: ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

doi: 10.1016/j.jid.2021.02.753

Figure Lengend Snippet: Figure 3. ENTPD1D T cells express CLA. (a) Representative flow cytometry of analysis of total T cells isolated from human cSCC showing CLA expression within the ENTPD1þ compared with that within the ENTPD1e populations. CLA expression among (b) CD4þ ENTPD1þ and (c) CD8þENTPD1þ T cells isolated from human SCC, perilesional skin (NL), and blood (n ¼ 12). P-values were determined by Tukey’s multiple comparisons test. CLA, cutaneous lymphocyte antigen; cSCC, cutaneous squamous cell carcinoma; ENTPD1, ectonucleotidase triphosphate diphosphodydrolase 1; FSC, forward scatter; NL, nonlesional; SCC, squamous cell carcinoma.

Article Snippet: Antibodies to human CD45, CD3, CD4, CD8, CD45R0, CLA, ENTPD1, CD73, CD11b, CD11c, CD206, DC-SIGN, PD-L1, PD-L2, PD-1, gdTCR, FoxP3, and IL17A were purchased from Tonbo Biotechnologies, eBioscience, Bio- Legend, R&D Systems (Minneapolis, MN), and BD Biosciences.

Techniques: Cytometry, Isolation, Expressing

Journal: Cell reports

Article Title: Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism

doi: 10.1016/j.celrep.2024.114245

Figure Lengend Snippet: Female mice ( n = 4 per group) were given 200 μL PBS vehicle control, 10 ng D270N, or 10 ng TcdB2 in PBS by the s.c. route. Seven days post treatment, RNA was purified from axillary and inguinal lymph nodes (aLNs and iLNs). Gene expression was quantified using the Nanostring nCounter SPRINT profiler platform. (A) Differentially expressed genes (DEGs) comparing TcdB2 to PBS (left), TcdB2 to D270N (center), or D270N to PBS (right). (B) Summary of the log2 fold change, raw p values, and adjusted p values (Benjamin-Yekutieli method) for each two-way comparison in the experiment. Values for cxcr4, cxcr5, ccr7 , and their ligands are depicted. A full list of chemokines and their receptors is shown in . (C) Relative expression of cxcr4, cxcr5 , and ccr7 in isolated B cells as determined by qPCR. Graphs show the increase in expression relative to vehicle-treated control mice and are normalized to gapdh expression using the ΔΔC T method. Data show mean ± SD for 5 mice per group. (D and E) B cell (D) and CD4 + T cell (E) CXCR4 and CXCR5 expression was measured by flow cytometry. Flow plots show CXCR4 versus CXCR5 expression, while graphs depict the mean ± SD percentage of each cell type expressing high levels of CXCR4. Data in are pooled from 3 experiments ( n = 9 per group). Statistical significance was determined by one-way ANOVA with Kruskal-Wallace post-test (** p < 0.01, *** p < 0.001).

Article Snippet: V450 anti-mouse CD4, clone GK1.5 , Tonbo , Cat # 75–0041 RRID: AB_2621927.

Techniques: Control, Purification, Gene Expression, Comparison, Expressing, Isolation, Flow Cytometry

Journal: Cell reports

Article Title: Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism

doi: 10.1016/j.celrep.2024.114245

Figure Lengend Snippet: (A) Female B6 mice ( n = 4 per group) were given 1 ng TcdB2, 1 ng D270N, or PBS vehicle control by the s.c. route. After 48 h, splenocytes, B cells, or CD4 + T cells were isolated (B and CD4 + T cells by magnetic separation) and seeded into the top of a Transwell. The bottom of the Transwell contained serum-free medium with or without CXCR12. Cells were incubated for 6 h, and then migratory cells were fixed and stained with crystal violet and counted. (B) Quantification of migratory splenocytes averaged from 4 fields of view from each Transwell membrane (mean ± SD, n = 4 per group). Data are representative of two independent experiments. (C) Representative flow cytometry plots for isolated B cells and CD4 + T cells. Graphs depict quantification of migratory B cells and CD4 + T cells (mean ± SD, n = 4 per group). Data are representative of two independent experiments. (D) Isolated B cells from vehicle-, D270N-, and TcdB2-treated mice were stimulated in vitro with ligands for CXCR4, CXCR5, and CCR7 (CXCL12, CCL19/CCL21, and CXCL13, respectively). Data are pooled from two independent experiments (mean ± SD, n = 8 per group). (E) Isolated splenic B cells were cultured with vehicle, D270N, or TcdB2 for 6 h ( n = 3). Graph shows mean ± SD, and data are representative of 2 similar experiments. Statistical significance was determined by one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: V450 anti-mouse CD4, clone GK1.5 , Tonbo , Cat # 75–0041 RRID: AB_2621927.

Techniques: Control, Isolation, Incubation, Staining, Membrane, Flow Cytometry, In Vitro, Cell Culture

Journal: Cell reports

Article Title: Clostridioides difficile toxin B subverts germinal center and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism

doi: 10.1016/j.celrep.2024.114245

Figure Lengend Snippet:

Article Snippet: V450 anti-mouse CD4, clone GK1.5 , Tonbo , Cat # 75–0041 RRID: AB_2621927.

Techniques: Control, Virus, Recombinant, Expressing, Suspension, Protease Inhibitor, SYBR Green Assay, Cell Isolation, Binding Assay, Bradford Protein Assay, Cell Counting, Enzyme-linked Immunosorbent Assay, Gene Expression, Software, Simple Western, Protein Extraction