Journal: International Immunology

Article Title: Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells

doi: 10.1093/intimm/dxac002

Figure Lengend Snippet: CS production and CD8 + T-cell infiltration are inversely correlated in colon cancer tissue samples. (A) Comparison of the SULT2B1 expression in 17 human cancer types. Data are from the Human Protein Atlas ( https://www.proteinatlas.org ) and are indicated as medians of the fragments per kilobase million (FPKM) values. (B) Kaplan–Meier curves showing survival of colon cancer patients with high ( n = 404) or low ( n = 193) SULT2B1 expression. The cut-off value was set at 5.68. P = 0.0077 (log-rank test). (C) Comparison of CS production between normal colon tissues and colon cancer tissues. Data are presented as the mean ± SD. ** P < 0.01 (two-tailed Mann–Whitney test). (D) Representative images for CS production and CD8 + T-cell infiltration in colon cancer tissue samples. Scale bars indicate 1 mm (the second column) and 250 µm (the third and fourth columns). The graph shows the comparison of the number of infiltrating CD8 + T cells between the CS-high and CS-low regions ( n = 10). Data are presented as the mean ± SD. ** P < 0.01 (two-tailed unpaired Student’s t -test).

Article Snippet: The following antibodies and reagents were used: anti-Fcg III/II receptor (2.4G2, 0.5 μg ml −1 ; TONBO Biosciences, San Diego, CA, USA, for blocking), APC-conjugated anti-mouse CD274 (PD-L1; 10F.9G2, 2 μg ml −1 ; Biolegend, San Diego, CA, USA), APC-conjugated Rat IgG2b (RTK4530, 2 μg ml −1 ; Biolegend), PE-conjugated anti-MHC II (M5/114.15.2, 0.2 μg ml −1 ; Invitrogen), PE-conjugated Rat IgG2b (A95-1; 0.2 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD3ε (145-2C11, 5 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD4 (RM4-5, 5 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD4 (RM4-5, 2 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD8a (53-6.7, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse CD8a (2.43, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse Vα2 (B20.1, 2 μg ml −1 ; BD Pharmingen), biotin-conjugated anti-mouse Vβ5 (MR9-4, 5 μg ml −1 ; BD Pharmingen), APC-streptavidin (0.4 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD366 (TIM-3; 5D12, 2 μg ml −1 ; BD Pharmingen), PerCP-Cy5.5-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend), APC-conjugated anti-mouse CD279 [programmed cell death 1 (PD-1); RMP1-30, 2 μg ml −1 ; Biolegend], biotin-conjugated anti-mouse F4/80 (BM8.1, 5 μg ml −1 ; TONBO Biosciences), PE-Cy7-conjugated anti-mouse CD3ε (145-2C11, 2 μg ml −1 ; BD Pharmingen) and PE-Cy7-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend).

Techniques: Comparison, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: International Immunology

Article Title: Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells

doi: 10.1093/intimm/dxac002

Figure Lengend Snippet: CS production in tumors inhibits infiltration of effector T cells. (A) CS production in cells ( n = 6) and culture supernatant ( n = 5) were compared between E0771-SULT and E0771-MOCK. Data are presented as the mean ± SD. ** P < 0.01 (two-tailed unpaired Student’s t- test). (B) Trans-cancer migration assays showing that the presence of E0771-SULT (SULT+) and CS-treated E0771-MOCK (SULT–) in Matrigel reduces CCL21-induced T-cell migration ( n = 5–7). Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01 (one-way ANOVA followed by Dunnett’s post hoc test). (C) In vitro growth was compared between E0771-MOCK and E0771-SULT ( n = 5). (D and E) After transplantation, tumor growth in wild-type (WT) or Sult2b1 –/– C57BL/6 mice was compared between E0771-MOCK and E0771-SULT ( n = 8–9). Data are presented as the mean + SD. * P < 0.05, ** P < 0.01 (two-tailed unpaired Student’s t -test). (F) viSNE plots highlighting the distribution of tumor-infiltrating lymphocytes in E0771-SULT and E0771-MOCK. (G) The percentages of CD8 + T cells and NK cells in infiltrating leukocytes and their expression of granzyme B were compared between E0771-SULT and E0771-MOCK ( n = 9). Data are presented as the mean ± SD. * P < 0.05 (two-tailed unpaired Student’s t -test). (H and I) After transplantation, tumor growth in BALB/c nude mice (H; n = 7) or DOCK2 –/– mice (I; n = 6) was compared between E0771-SULT and E0771-MOCK. (J) After transplantation, tumor growth in C57BL/6 mice was compared between Pan02-control and Pan02-ΔSULT ( n = 10, 8). Data are presented as the mean + SD. ** P < 0.01 (two-tailed Mann–Whitney test). (K) The percentages of CD8 + T cells in infiltrating leukocytes were compared between Pan02-control and Pan02-ΔSULT ( n = 9). Data are presented as the mean ± SD. ** P < 0.01 (two-tailed unpaired Student’s t -test).

Article Snippet: The following antibodies and reagents were used: anti-Fcg III/II receptor (2.4G2, 0.5 μg ml −1 ; TONBO Biosciences, San Diego, CA, USA, for blocking), APC-conjugated anti-mouse CD274 (PD-L1; 10F.9G2, 2 μg ml −1 ; Biolegend, San Diego, CA, USA), APC-conjugated Rat IgG2b (RTK4530, 2 μg ml −1 ; Biolegend), PE-conjugated anti-MHC II (M5/114.15.2, 0.2 μg ml −1 ; Invitrogen), PE-conjugated Rat IgG2b (A95-1; 0.2 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD3ε (145-2C11, 5 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD4 (RM4-5, 5 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD4 (RM4-5, 2 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD8a (53-6.7, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse CD8a (2.43, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse Vα2 (B20.1, 2 μg ml −1 ; BD Pharmingen), biotin-conjugated anti-mouse Vβ5 (MR9-4, 5 μg ml −1 ; BD Pharmingen), APC-streptavidin (0.4 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD366 (TIM-3; 5D12, 2 μg ml −1 ; BD Pharmingen), PerCP-Cy5.5-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend), APC-conjugated anti-mouse CD279 [programmed cell death 1 (PD-1); RMP1-30, 2 μg ml −1 ; Biolegend], biotin-conjugated anti-mouse F4/80 (BM8.1, 5 μg ml −1 ; TONBO Biosciences), PE-Cy7-conjugated anti-mouse CD3ε (145-2C11, 2 μg ml −1 ; BD Pharmingen) and PE-Cy7-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend).

Techniques: Two Tailed Test, Migration, In Vitro, Transplantation Assay, Expressing, Control, MANN-WHITNEY

Journal: International Immunology

Article Title: Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells

doi: 10.1093/intimm/dxac002

Figure Lengend Snippet: SULT2B1b-mediated CS production in tumors dampens anti-tumor T-cell responses. (A) The effect of OTI CD8 + T-cell transfer on tumor growth in C57BL/6 mice was compared between E.G7-OVA-MOCK ( n = 5) and E.G7-OVA-SULT ( n = 6). After the intravenous injection of activated Vα2 + Vβ5 + OTI T cells on day 10, the infiltration of OTI T cells was analyzed on day 15 ( n = 7). Data are presented as the mean + SD or ± SD. * P < 0.05, ** P < 0.01 (two-tailed Mann–Whitney test for both tumor growth and infiltration of OTI T cells). (B) The effect of OTI CD8 + T-cell transfer on tumor growth in C57BL/6 mice was compared between E0771-OVA-control ( n = 8) and E0771-OVA-SULT ( n = 8). After the intravenous injection of activated Vα2 + Vβ5 + OTI T cells on day 10, the infiltration of OTI T cells was analyzed on day 15 ( n = 7 or 8). Data are presented as the mean + SD or ± SD. * P < 0.05, ** P < 0.01 (two-tailed Mann–Whitney test for tumor growth and two-tailed unpaired Student’s t- test for infiltration of OTI T cells). (C) Schematic representation of the interaction between OTII CD4 + T cells and tumor cells expressing I-A b molecule covalently bound to OVA peptide. (D and E) The effect of OTII CD4 + T-cell transfer on tumor growth in C57BL/6 mice was compared between (D) E0771-control-I-A b /OVA ( n = 8) and E0771-SULT-I-A b /OVA ( n = 7) or (E) 3LL-control-I-A b /OVA ( n = 8) and 3LL-SULT-I-A b /OVA ( n = 8). After the intravenous injection of activated Vα2 + Vβ5 + OTII T cells on day 9, infiltration of OTII T cells was analyzed on day 14 (D, n = 9; E, n = 7). Data are presented as the mean + SD or ± SD. * P < 0.05, ** P < 0.01 (two-tailed unpaired Student’s t- test for tumor growth and two-tailed Mann–Whitney test for infiltration of OTII T cells). (F) Comparison of the effect of DOCK2 +/+ OTII CD4 + T cells and DOCK2 –/– OTII CD4 + T cells ( n = 7) on the growth of E0771-control-I-A b /OVA transplanted into C57BL/6 mice. Data are presented as the mean + SD. * P < 0.05 (two-tailed unpaired Student’s t- test).

Article Snippet: The following antibodies and reagents were used: anti-Fcg III/II receptor (2.4G2, 0.5 μg ml −1 ; TONBO Biosciences, San Diego, CA, USA, for blocking), APC-conjugated anti-mouse CD274 (PD-L1; 10F.9G2, 2 μg ml −1 ; Biolegend, San Diego, CA, USA), APC-conjugated Rat IgG2b (RTK4530, 2 μg ml −1 ; Biolegend), PE-conjugated anti-MHC II (M5/114.15.2, 0.2 μg ml −1 ; Invitrogen), PE-conjugated Rat IgG2b (A95-1; 0.2 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD3ε (145-2C11, 5 μg ml −1 ; BD Pharmingen), FITC-conjugated anti-mouse CD4 (RM4-5, 5 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD4 (RM4-5, 2 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD8a (53-6.7, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse CD8a (2.43, 2 μg ml −1 ; TONBO Biosciences), PerCP-Cy5.5-conjugated anti-mouse Vα2 (B20.1, 2 μg ml −1 ; BD Pharmingen), biotin-conjugated anti-mouse Vβ5 (MR9-4, 5 μg ml −1 ; BD Pharmingen), APC-streptavidin (0.4 μg ml −1 ; BD Pharmingen), PE-conjugated anti-mouse CD366 (TIM-3; 5D12, 2 μg ml −1 ; BD Pharmingen), PerCP-Cy5.5-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend), APC-conjugated anti-mouse CD279 [programmed cell death 1 (PD-1); RMP1-30, 2 μg ml −1 ; Biolegend], biotin-conjugated anti-mouse F4/80 (BM8.1, 5 μg ml −1 ; TONBO Biosciences), PE-Cy7-conjugated anti-mouse CD3ε (145-2C11, 2 μg ml −1 ; BD Pharmingen) and PE-Cy7-conjugated anti-mouse CD45 (30-F11, 2 μg ml −1 ; Biolegend).

Techniques: Injection, Two Tailed Test, MANN-WHITNEY, Control, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A – C ) WT or ATXN3-KO LLC1 cells were injected subcutaneously into C57BL/6 mice ( n = 10). Tumor growth curve ( A ), photograph ( B ), and weight ( C ) are shown. ( D ) MFI of surface PD-L1 on LLC1 tumors ( n = 5). ( E – G ) Quantification of CD4 + ( E ) and CD8 + T cell ( F ) and Treg ( G ) percentages ( n = 5–10). ( H and I ) Quantification of cell-surface PD-1 ( H ) and PD-L1 ( I ) MFI on CD8 + T cells ( n = 5). ( J and K ) Quantification of cell-surface CTLA-4 MFI ( J ) and Tim3 percentage ( K ) in CD8 + T cells ( n = 5). ( L ) MFI of cell-surface LAG3 and percentage in CD8 + T cells ( n = 5). ( M – O ) Intracellular staining of Blimp1 + CD8 + T cell ( M ), EOMES + CD8 + T cell ( N ), and T-bet + CD8 + T cell ( O ) percentage in LLC1 tumors ( n = 5-7). ( P ) Quantification of cell-surface CD44 + CD8 + T cell percentage from LLC1 tumors ( n = 5–10). ( Q ) Apoptotic CD8 + T cells in the tumors were analyzed ( n = 5–7). ( R and S ) Representative flow staining and quantification of intracellular cytokine staining of granzyme B + CD8 + and IFN-γ + CD8 + in CD45 + T cell populations from LLC1 tumors ( n = 5). ( T ) Tumor growth curve and tumor photograph of C57BL/6 mice injected subcutaneously with WT and ATXN3-KO LLC1 cells with or without treatment of anti-CD8 depleting antibodies ( n = 5). ( U ) Left: Tumor cell-surface PD-L1 expression. Right: Tumor growth of WT or ATXN3-KO LLC1 cells stably expressing PD-L1 (as shown in the left plot) in C57BL/6 mice ( n = 5). A and C – S : 2-tailed unpaired t test; T and U : ordinary 1-way ANOVA. * P < 0.05, ** P < 0.01,*** P < 0.001.

Article Snippet: For in vivo CD8 + T cell depletion, mice received intraperitoneal injection of anti–mouse CD8 antibody (200 μg; Bio X Cell, catalog 53-6.72) or rat IgG2a isotype control (200 μg; Bio X Cell, catalog BE0089) on day 5 after tumor injection and were treated every 3 days.

Techniques: Injection, Staining, Expressing, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A and B ) Scheme representing the experimental procedure ( A ) and tumor growth curves ( B ) of C57BL/6 mice injected subcutaneously with WT or ATXN3-KO LLC1 cells and treated with PD-1 antibody (25 μg per mouse, once every 2 days, n = 5). ( C – E ) Tumor photograph ( C ), tumor growth curves ( D ), and tumor burdens ( E ) for C57BL/6 mice bearing LLC1 tumors treated with PD-1 antibody (50 μg per mouse, once every 2 days, n = 10). ( F ) Representative flow staining of CD4 + T cells, CD8 + T cells, and IFN-γ + CD8 + T cells in CD45 + T cell populations from LLC1 tumors ( n = 10) as described in C and D . ( G – I ) Quantification of CD4 + T cell ( F ), CD8 + T cell ( G ), and IFN-γ + CD8 + T cell ( H ) percentage in CD45 + populations from LLC1 tumors ( n = 10) as described in C and D . ( J and K ) C57BL/6 mice (6–8 weeks) were injected subcutaneously with WT or ATXN3-KO LLC1 cells and treated with PD-1 antibody (100 μg per mouse, once every 2 days, n = 5). Tumor growth curve was measured every 2 days ( J ), and mouse tumors were weighed at the end of the experiment ( K ). B , D , E , and G – K : Ordinary 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For in vivo CD8 + T cell depletion, mice received intraperitoneal injection of anti–mouse CD8 antibody (200 μg; Bio X Cell, catalog 53-6.72) or rat IgG2a isotype control (200 μg; Bio X Cell, catalog BE0089) on day 5 after tumor injection and were treated every 3 days.

Techniques: Injection, Staining

Journal: Nature Communications

Article Title: Live-cell imaging reveals the relative contributions of antigen-presenting cell subsets to thymic central tolerance

doi: 10.1038/s41467-019-09727-4

Figure Lengend Snippet: Both conventional DC subsets induce negative selection of OT-I and OT-II SPs responding to RIP-mOVA and RIP-OVA hi TRAs. a Sequential gating of live CD45 + cells from a digested CD11c-EYFP thymus to detect the indicated APC subsets. Histograms show EYFP and MHC-II levels for the indicated APC subsets. b EYFP mean fluorescence intensity (MFI) of Sirpα + cDC2 and Sirpα − cDC1. Flow-cytometry data averaged from three CD11c-EYFP mice, stained independently. Data points represent mice, and bars show mean ± SEM. c 2PM volume from three perspectives, showing two activated OT-I CD8SPs (red mask, SP1 and SP2) interacting with cDC2 (yellow) and cDC1 (gray), respectively. Scale bar is 10 µm. d Frequency of activated OT-I CD8SP and OT-II CD4SP thymocytes contacting DCs, that interacted with cDC2 or cDC1 on RIP-mOVA or RIP-OVA hi thymic slices. Graph shows mean + SEM. Data are compiled from experiments analyzed in Fig. . Analyzed by t tests with multiple comparison correction, p -values: * < 0.05, *** < 0.001. ns not significant. e mTEC hi (green), cDC2 (red), and cDC1 (blue) were sorted from RIP-mOVA and RIP-OVA hi thymi and cultured with CFSE-labeled splenic OT-I CD8 + T cells. WT splenocytes ± OVAp served as positive and negative control APCs. Histograms show CFSE dilution in Vα2 + Vβ5 + CD8 + cells after incubation with APCs for 72 h, the gate shows the percent of cells that proliferated. Dashed line shows the CFSE profile for T cells cultured with WT splenocytes in the absence of OVAp, and gray shading shows that of T cells cultured with OVAp-pulsed WT splenocytes. Data are representative of two independent experiments per condition, and graphs depict mean ± SEM of the percent proliferation after incubation with indicated APCs for triplicate wells. Source data are provided as a Source Data file. See also Supplementary Figs. ,

Article Snippet: Antibodies directed against the following mouse markers were used: CD3 (145–2C11, Tonbo Biosciences 60–0031), CD4 (RM4–5, BioLegend 100559; GK1.5, BioXCell BE0003–1), CD8 (53–6.7, Tonbo Biosciences 75–0081; 53.6.72, BioXCell BE0004), CD11b (M1/70, BioLegend 101204; M1/70, BioXCell BE0007), CD11c (N418, BioLegend 117322), CD19 (6D5, BioLegend 115534), CD25 (PC61, BioLegend 102024; PC61.5.3, BioXCell BE0012), CD45 (30-F11, BioLegend 103138), CD45.1 (A20, BioLegend 110714), CD69 (H1.2F3, BioLegend 104504), CD80 (16–10A1, BioLegend 104724), B220 (RA3–6B2, BioLegend 103232; RA3.3A1/6.1, BioXCell BE0067), AIRE (5H12, eBioscience 53–5934–82), EpCAM (G8.8, BioLegend 118206), F4/80 (BM8, BioLegend 123116), Gr-1 (RB6–8C5, BioLegend 108410; RB6–8C5, BioXCell BE0075), I-A/I-E (M5/114.15.2, BioLegend 107628), NK1.1 (PK136, BioLegend 108716), PDCA1 (eBio927, eBioScience 12–3172–82), Sirpα (P84, BioLegend 144008), TER-119 (TER-119, BioLegend 116210; TER-119, BioXCell BE0183), Vα2 (B20.1, BioLegend 127818), Vβ5 (MR9–4, BioLegend 139504), and XCR1 (ZET, BioLegend 148212).

Techniques: Selection, Fluorescence, Flow Cytometry, Staining, Comparison, Cell Culture, Labeling, Negative Control, Incubation

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

Article Title: SUBSET-SPECIFIC REDUCTIONS IN LUNG LYMPHOCYTE ACCUMULATION FOLLOWING INTRATRACHEAL ANTIGEN CHALLENGE IN ENDOTHELIAL SELECTIN-DEFICIENT MICE

doi:

Figure Lengend Snippet: Peripheral blood analysis indicates a lymphocytosis in E-P- mice. Peripheral blood lymphocytes were analyzed by flow cytometry. Black bars, CD4 cells; light gray bars, CD8 cells; dark gray bars, CD19 cells; white bars, γ† cells. Data represent mean ± SEM of six mice per group (untreated) or 11-12 mice per group (day 7) assayed individually in at least two separate experiments. **, p<0.01, unpaired Student t test.

Article Snippet: mAbs The following epitopes were assessed by three-color flow cytometry analyses using directly-conjugated mAbs (PharMingen; San Diego, CA) (clone, isotype and fluorochrome are indicated in parentheses): CD4 (RM4-4; rat IgG2a; cychrome), CD8 (53-6.72; rat IgG2a; FITC), CD19 (1D3; rat IgG2a; phycoerythrin), CD45 (30-F-11; rat IgG2b; cychrome), γ/†-TCR (GL3; hamster IgG2; cychrome), Gr-1 (RB6-8C5; rat IgG2b; phycoerythrin), pan-NK cell (DX-5; rat IgM; FITC), CD14 (rmC5-3; rat IgG1; FITC), and Mac-3 (M3/84; rat IgG1; FITC).

Techniques: Flow Cytometry

Journal: Aging Cell

Article Title: Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

doi: 10.1111/acel.13309

Figure Lengend Snippet: Interleukin‐37 suppresses inflammaging, and decreased levels are observed in aged human monocytes. (a) The R2 Database was mined to determine IL‐37 gene expression levels in healthy donors between the ages of 1555 years of age. The gene expression levels are shown for young and middle‐aged donors. (b) Monocytes were purified from PBMCs of healthy donors using MACs selection. The gene expression levels are shown for young, middle‐aged, and old donors. (c) C57BL/6 wild‐type and IL‐37 transgenic mice were aged for 24 months and dissected to observe potential anatomical changes. The spleen appearance and weight are shown. (d) Young (2 months) and old (24 months) C57BL/6 mice were treated every other day for 2 weeks with control Ig or rIL‐37, and the ratio of CD4 + to CD8 + T‐cells was determined via flow cytometric analysis. Means ± SD are shown with ** p < 0.01 and *** p < 0.001 determined using a Student's t test relative to young donors in B or young Control Ig‐treated mice in (d). A one‐way ANOVA with Tukey's post‐test was used to determine significance in (c). For results presented in (c), 3 independent experiments were conducted ( n = 9 mice/group). In (d), data represent 5 mice/group

Article Snippet: T‐cells were depleted from aged (24 months) C57BL/6 mice using CD4 (anti‐CD4; clone GK1.5; purchased from Bio X Cell) and CD8 (anti‐CD8α; clone 53–6.72 and anti‐CD8β; clone 53–5.8; purchased from Bio X Cell) T‐cell depleting antibodies (i.p.; 0.5 mg/mouse/each antibody for two consecutive days).

Techniques: Gene Expression, Purification, Selection, Transgenic Assay, Control

Journal: Aging Cell

Article Title: Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

doi: 10.1111/acel.13309

Figure Lengend Snippet: Recombinant IL‐37 treatment reduces PD‐1 surface expression and improves the function of aged T‐cells. Aged (24 months old) C57BL/6 mice were treated with control immunoglobulin (Control Ig) or recombinant IL‐37 (rIL‐37) every other day for 2 weeks. Naïve CD4 + T‐cells and CD8 + T‐cells were purified from treated mice using MACs selection and stimulated in vitro with αCD3/αCD28. On day 3 post‐stimulation, (a–c) the mean surface expression of CD44 and PD1 (mean fluorescence intensity [MFI]) and (d–g) the percentage and MFI of IL‐2/IFN‐γ‐producing T‐cells were determined using flow cytometric analysis. Means ± SD are shown in (b, c, e, f, and g) with ** p < 0.01, *** p < 0.001, and **** p < 0.0001 determined using a Student's t test relative to aged T‐cell responses from Control Ig treated mice. n = 9 mice/group with 3 independent experiments conducted. The red boxes in (a) and (d) denote functional parameters of interest

Article Snippet: T‐cells were depleted from aged (24 months) C57BL/6 mice using CD4 (anti‐CD4; clone GK1.5; purchased from Bio X Cell) and CD8 (anti‐CD8α; clone 53–6.72 and anti‐CD8β; clone 53–5.8; purchased from Bio X Cell) T‐cell depleting antibodies (i.p.; 0.5 mg/mouse/each antibody for two consecutive days).

Techniques: Recombinant, Expressing, Control, Purification, Selection, In Vitro, Fluorescence, Functional Assay

Journal: Aging Cell

Article Title: Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

doi: 10.1111/acel.13309

Figure Lengend Snippet: Recombinant IL‐37 treatment opposes TNF‐α signaling in aged T‐cells. Naïve CD4 + and CD8 + T‐cells were purified from aged (24 months old) C57BL/6 mice via MACs selection and stimulated in vitro with αCD3/αCD28 in the presence of Control Ig, rTNF‐α, or rTNF‐α + rIL‐37. (a, c) After 10’ of stimulation, phospho‐flow cytometry was performed to determine NF‐κB activation. (b, d) After 3 days of stimulation, the surface expression of PD‐1 on aged T‐cells was determined using flow cytometric analysis. (e, f) Naïve T‐cells were purified as described above and stimulated with Control Ig or rIL‐37 for 4 h. After the stimulation period, qPCR analysis was performed to ascertain the expression levels of genes involved in T‐cell activation (IFN‐γ, TBK1, IRF3) and inhibition (TMEM16F, GM130, SHP2, and PD1). Importantly, the genes chosen for assessment are regulated by TNF‐α and PD‐1 signaling. Significance was determined using a Student's t test relative to αCD3/αCD28 + Control Ig (a–d) and media +Control Ig (e, f) treated groups. For a–d, means ± SD are shown with **** p < 0.0001. For (e) and (f), a =* p < 0.05, b =** p < 0.01, c =*** p < 0.001, and d =**** p < 0.0001 where gene expression levels observed in Control Ig‐treated aged T‐cells were used as the positive control for each gene tested. n = 9 mice/group with 3 independent experiments conducted

Article Snippet: T‐cells were depleted from aged (24 months) C57BL/6 mice using CD4 (anti‐CD4; clone GK1.5; purchased from Bio X Cell) and CD8 (anti‐CD8α; clone 53–6.72 and anti‐CD8β; clone 53–5.8; purchased from Bio X Cell) T‐cell depleting antibodies (i.p.; 0.5 mg/mouse/each antibody for two consecutive days).

Techniques: Recombinant, Purification, Selection, In Vitro, Control, Flow Cytometry, Activation Assay, Expressing, Inhibition, Gene Expression, Positive Control

Journal: Aging Cell

Article Title: Interleukin‐37 improves T‐cell‐mediated immunity and chimeric antigen receptor T‐cell therapy in aged backgrounds

doi: 10.1111/acel.13309

Figure Lengend Snippet: Recombinant IL‐37 treatment protects aged mice from B‐ALL pathogenesis in a T‐cell dependent manner. (a) Aged (24 months old) C57BL/6 mice were treated with T‐cell depleting antibodies (αCD4 and αCD8) 2 days prior to intravenous challenge with BCR‐ABL + Arf − / − murine B‐ALL cells (mB‐ALL). Mice were also treated with Control Ig or rIL‐37 1 day prior to the injection of mB‐ALL cells, and this treatment continued throughout the experiment. (b) Survival was monitored for over 3 months. Significance was determined using log‐rank test with **** p < 0.0001 indicating a significant extension of survival in aged mice treated with rIL‐37 relative to each experimental group tested. n = 5 mice/group

Article Snippet: T‐cells were depleted from aged (24 months) C57BL/6 mice using CD4 (anti‐CD4; clone GK1.5; purchased from Bio X Cell) and CD8 (anti‐CD8α; clone 53–6.72 and anti‐CD8β; clone 53–5.8; purchased from Bio X Cell) T‐cell depleting antibodies (i.p.; 0.5 mg/mouse/each antibody for two consecutive days).

Techniques: Recombinant, Control, Injection