Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from blood and spleen tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final dose injection. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (C) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype and function of CNS innate immune cells in EAE mice at first day post‐injection. Single‐cell suspensions were obtained from CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated EAE mice at first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Flow cytometry gating strategy. (B) CD11b + CD45 lo/neg resident microglia and (C) CNS CD11b + CD45 hi infiltrating macrophages and were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. CD11b + cells were isolated from CNS tissue of anti‐CD52‐Ab or vehicle‐treated mice at first day after the final treatment dose. (D) Respiratory burst activity of CD11b + ‐isolated cells. (E) Phagocytosis of FITC‐coated beads by CD11b + isolated cells. Unpaired two‐tailed Student's t test. (F) Cells were left unstimulated or were stimulated with LPS or Poly(I:C)(PIC) for 24 h and cytokine production determined by ELISA. Two‐way ANOVA with Sidak multiple comparisons test, * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test, Isolation, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the phenotype of periphery and CNS innate immune cells in EAE mice at three weeks post‐injection. Single‐cell suspensions were obtained from blood, spleen and CNS (brain and spinal cord) tissue of anti‐CD52‐Ab or vehicle‐treated mice three weeks after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II ( n = 9), CD40 ( n = 5), CD80 ( n = 5) and CD86 ( n = 9 blood; n = 5 spleen). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II ( n = 8), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5), CD38 ( n = 5) and CD206 ( n = 9 blood; n = 7 spleen). (C) CNS CD11b + CD45 hi ‐infiltrating macrophages and CD11b + CD45 lo/neg resident microglia were analysed for expression of MHC‐II ( n = 10), CD40 ( n = 5), CD80 ( n = 5), CD86 ( n = 5 microglia; n = 9 macrophages) and CD206 ( n = 7 microglia; n = 5 macrophages). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Immunology

Article Title: Anti‐CD52 antibody treatment in murine experimental autoimmune encephalomyelitis induces dynamic and differential modulation of innate immune cells in peripheral immune and central nervous systems

doi: 10.1111/imm.13437

Figure Lengend Snippet: Effect of anti‐CD52‐Ab treatment on the function of periphery and CNS innate immune cells in SCID mice at first day post‐injection. SCID mice were treated with either vehicle or 10 mg/kg murine anti‐CD52‐Ab‐Ab for five consecutive days. Single‐cell suspensions were then obtained from blood and spleen tissues first day after the final treatment dose. Cells were stained for various surface markers and analysed by flow cytometry. (A) Blood and spleen CD11c + cells were analysed for expression of MHC‐II, CD40, CD80 and CD86 (all n = 7). (B) Blood and spleen CD11b + cells were analysed for expression of MHC‐II, CD40, CD80, CD86, and CD206 (all n = 7). Unpaired two‐tailed Student's t test. * p < 0·05, ** p < 0·01, *** p < 0·001

Article Snippet: To block non‐specific Fc receptors, cells were resuspended in anti‐mouse CD16/CD32 Fc block (eBioscience) for 10 min before incubation with the appropriate antibodies against: CD11b (#130‐113‐236), CD11c (#130‐102‐545), CD38 (#130‐109‐257), CD40 (#130‐105‐376 or #130‐102‐533), CD80 (#130‐116‐465), CD86 (#130‐105‐137), MHC‐II (#130‐107‐942), all were purchased from MACS Miltenyi Biotec; and CD45 (#11‐0451‐82) and CD206 (#12‐2061‐82), all were purchased from Thermo Fisher Scientific.

Techniques: Injection, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Clinical Cancer Research

Article Title: A Novel Chemoimmunomodulating Property of Docetaxel: Suppression of Myeloid-Derived Suppressor Cells in Tumor Bearers

doi: 10.1158/1078-0432.ccr-10-0733

Figure Lengend Snippet: Fig. 4. A, MDSCs from docetaxel-treated mice express M1 markers. MDSCs were affinity column purified from three mice per group representing naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. They were immunostained for CCR7 and iNOS to represent M1 markers or MR to represent a specific M2 marker before flow cytometric analysis. A, histogram representation of MR, CCR7, and iNOS levels in naive mice, docetaxel-treated naive mice, tumor bearers, and docetaxel-treated tumor bearers. B, columns, mean fluorescence intensity units of CCR7, MR, and iNOS from three independent experiments; bars, SE. C, in vivo docetaxel-treated MDSCs have increased differentiation markers. MACS column– purified MDSCs from the above treatment groups were analyzed by flow cytometry for MHC class II, CD11c, CD40, CD80, and CD85 expression. Gray filled histograms are isotype controls, dotted lines represent the docetaxel-treated MDSCs, and black solid lines represent untreated MDSCs.

Article Snippet: Anti-Ly6G and anti-Ly6C ylated antibodies, FITC-conjugated anti-LY6C, APCgated anti-LY6C, PE-conjugated anti-Ly6G, FITCgated anti-mouse CD40, PE-conjugated anti-mouse , FITC-conjugated anti-mouse CD80, APC-conjugated ouse CD11c, and PE-conjugated anti–mouse I Ad purchased from BD Biosciences; rat anti-mouse MR (CD206) was from AbD Serotec; and streptavidin beads were purchased from Miltenyi Biotec.

Techniques: Affinity Column, Purification, Marker, Fluorescence, In Vivo, Flow Cytometry, Expressing

Journal: Cancer immunology research

Article Title: A multikinase and DNA-PK inhibitor combination immunomodulates melanomas, suppresses tumor progression, and enhances immunotherapies

doi: 10.1158/2326-6066.CIR-17-0009

Figure Lengend Snippet: (A–B) B16-F1 tumor-bearing mice were treated with Reg (4 mg/kg) with or without a combination of anti-CD40 (40) and c-di-GMP (STING agonist, S) and tumor volume (A) and survival (B) were monitored (n = 5–6/group). Differences for tumor volumes were determined on days 16–28 by one-way ANOVA with Tukey post-test comparing vehicle to all groups, and comparing 40/S or Reg to 40/S+Reg. All survival comparisons except Reg vs. 40/S were significant according to log-rank tests. On day 21, intratumoral CD45+ leukocytes (C), CD4+ T cells (D–E) and CD8+ T cells (F–G) were measured. Intratumoral CD4+ (H) and CD8+ (I) T cells were stimulated with PMA and ionomycin and evaluated for cytokine production. Differences in C–I were assessed by one-way ANOVA. (J–K) B16-F1 tumor-bearing mice were treated with anti-CD8 to deplete CD8+ T cells, Reg (5 mg/kg), anti-CD40, and c-di-GMP and tumor volume (J) and survival (K) were monitored (n = 5–7/group). Reg and 40/S+Reg groups were significantly different compared to associated anti-CD8-treated mice on days 19–29 by one-way ANOVA with Tukey post-test. Log-rank tests were used to evaluate survival comparisons. A–G are representative of two independent experiments; H–K were performed once. All results show mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Some mice received 100 μg of anti-CD40 (clone FGK4.5, BE0016-2, BioXCell) i.p and 50 μg c-di-GMP (tlrl-nacdg, InvivoGen) peritumorally on days 10, 15, 20, and 25.

Techniques: