Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral STING agonist reverses immune evasion in PD-(L)1-refractory Merkel cell carcinoma: mechanistic insights from detailed biomarker analyses

doi: 10.1136/jitc-2024-009803

Figure Lengend Snippet: Merkel cell carcinoma cancer cells are deficient in STING signaling. ( A ) Multiplexed immunohistochemistry of post-ADU-S100 treated tumor. Areas of CD56 positivity (representing MCC cancer cells) are non-overlapping with STING positivity, which is primarily expressed in stromal and immune tissues. ( B ) Quantification of STING expression. Each point represents 1 of 88 unique tumor specimens on a tissue microarray. STING is universally absent in MCC cancer cells. Cancer cells defined as CD56+, CD45−, CD8 T cells defined as CD8+, Treg cells defined as CD4+, FoxP3+, macrophages defined as CD68+ or CD163+, other immune cells defined as CD45+ cells which did not fall into prior categories, stromal cells defined as CD45−, CD56− cells. Statistical comparison with one-way ANOVA compared with tumor expression. *p<0.05, **p<0.01, ****p<0.0001, NS (not significant). ( C ) MCC cell lines do not produce interferon-beta in response to ADU-S100 treatment. WaGa, MKL-2, MKL-1, MS1 MCC cells and THP1 cells (control human monocytic cells) were all treated with decreasing doses of ADU-S100. None of the MCC cell lines produced detectable interferon-beta at any tested ADU-S100 concentration. These cells were all also deficient in STING protein (western blot, below). ANOVA, analysis of variance; 4',6-diamidino-2-phenylindole (DAPI); Mac, macrophage; MCC, Merkel cell carcinoma; STING, stimulator of interferon genes; T-Reg, Regulatory T cell.

Article Snippet: Antibodies against the following targets were used for staining: CD56 (clone 123C3.D5; Bio SB), CD8 (clone C8/144B; DAKO), STING (clone SP338; Abcam) CD45 LCA (clone 2B11+PD7/26; DAKO), FoxP3 (clone 236A/E7; eBioscience), CD163 (clone ERP324; BioSB), CD68 (clone PG-M1; DAKO).

Techniques: Immunohistochemistry, Expressing, Microarray, Comparison, Control, Produced, Concentration Assay, Western Blot

Journal: British Journal of Cancer

Article Title: Immune activation by combination human lymphokine-activated killer and dendritic cell therapy

doi: 10.1038/bjc.2011.290

Figure Lengend Snippet: Phenotype of clinical grade LAKs and DCs. Phenotype analysis was performed on CD14− PBMCs before and after culture for 5 days in IL-2 to generate LAKs. ( A ) Relative populations of NK (CD3− CD56+), NKT (CD3+CD56+) and T cells (CD3+CD56−) are shown as representative plots from one donor (of n =10); ( B ) mean % of total cells (PBMCs grey bars, LAKs black bars)±s.e.m (of n =10 donors; * P <0.05). Expression of activation markers on LAKs are shown as representative plots from one donor (of n =5): ( C ) % positive expression and ( D ) mean fluorescence intensity of LFA-1 ( * P <0.05). DCs were generated by culture of CD14+ PBMCs with GMCSF and IL-4 for 5 days. ( E ) Phenotype analysis is shown as representative FACS plots from one donor (of n =5; DCs: black; isotype controls: grey).

Article Snippet: LAK ( CD3+CD56−, CD3-CD56+ and CD3+CD56+ ): anti-human CD3-PerCP, CD56-FITC, CD8-FITC, CD16-FITC, CD69-FITC, NKG2D-PE, CCR7-PE, DNAM-1-PE, NKp30-PE, NKp44-PE, LFA-1(CD18)-PE, CD40 L(CD154)-PE (BD Biosciences), CD56-PE (Serotec, Oxford, UK) and NKp46-PE (Miltenyi).

Techniques: Expressing, Activation Assay, Fluorescence, Generated

Journal: British Journal of Cancer

Article Title: Immune activation by combination human lymphokine-activated killer and dendritic cell therapy

doi: 10.1038/bjc.2011.290

Figure Lengend Snippet: DCs mediated activation of NK-LAK. LAKs and DCs were co-cultured at 10 : 1 ratio for 48 h before NK-LAK (CD3-CD56+) phenotype analysis. Upper panels: representative histograms from one donor showing expression of cell surface markers on NK-LAK cultured in the absence (black) or presence (grey) of DCs; isotype controls are shaded. Lower panels: Line graphs denoting data from all six donors tested ( * P <0.05).

Article Snippet: LAK ( CD3+CD56−, CD3-CD56+ and CD3+CD56+ ): anti-human CD3-PerCP, CD56-FITC, CD8-FITC, CD16-FITC, CD69-FITC, NKG2D-PE, CCR7-PE, DNAM-1-PE, NKp30-PE, NKp44-PE, LFA-1(CD18)-PE, CD40 L(CD154)-PE (BD Biosciences), CD56-PE (Serotec, Oxford, UK) and NKp46-PE (Miltenyi).

Techniques: Activation Assay, Cell Culture, Expressing

Journal: Nature Communications

Article Title: Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy

doi: 10.1038/s41467-020-20475-8

Figure Lengend Snippet: a Cord blood non-naive (Supplementary Fig. ) CD4 T cell frequency in exposed (red, n = 98) and unexposed (blue, n = 68) infants. b Cord blood T reg frequency (CD25 high FoxP3 + CD127 lo ) in exposed (red, n = 98) and unexposed (blue, n = 68) infants. c Myeloid (Lin-2 – HLA-DR + CD11c + CD123 - ) and plasmacytoid (Lin-2 – HLA-DR + CD11c − CD123 + ) dendritic cell counts in whole cord blood, in exposed (red, n = 142) and unexposed (blue, n = 96) infants. d Cord blood plasma cytokine levels (unadjusted p values: TNF-RI ( p < 0.0001), IL-2 ( p < 0.0001), IL-4 ( p = 0.037), IL-5 ( p < 0.0001), IL-7 ( p < 0.0001), MDC ( p < 0.0001), TNF-RI ( p < 0.0001), IP-10 ( p = 0.0009), and MIP-1α ( p = 0.01), ran in duplicate) in exposed (red, n = 114) and unexposed (blue, n = 73) infants. Following Bonferroni adjustment for all cytokines and chemokines measured, all associations indicated above remained significant with the exception of IL-4 and MIP-1α. All comparisons were made by Wilcoxon rank-sum test. Bars indicate median with interquartile range. Two-sided p values were calculated for all test statistics.

Article Snippet: Dendritic cell staining was performed on 238 cord blood samples using FITC Lin-2 (clone MφP9/SJ25C1/SK7/L27/NCAM16.2, anti-CD3, CD14, CD19, CD20, CD56), PE CD123 (clone 9F5), PerCP HLA-DR (clone L243), and APC CD11c (clone S-HCL-3, BD Pharmingen).

Techniques:

Journal: Leukemia

Article Title: CD133 is a positive marker for a distinct class of primitive human cord blood-derived CD34-negative hematopoietic stem cells

doi: 10.1038/leu.2013.326

Figure Lengend Snippet: Long-term multilineage reconstitution abilities of CD34 + CD133 + and CD34 − CD133 + SRCs. First, the R1 gate was set on the total murine BM cells obtained from these two representative NOG mice that received ( a ) CD34 + CD133 + SRCs (upper column) and ( b ) CD34 − CD133 + SRCs (lower column), 20 weeks after transplantation. Thereafter, the human CD45 + cells were gated as R3 (solid line) from the R2-gated 7-AAD − cells. The expression of surface markers, including CD34, CD33, CD19, CD41, CD14 and CD11b, on the R3-gated cells was analyzed by six-color FCM. Only the expression of CD235a was analyzed using the R4 gate (dotted line) containing CD45 +/− cells. The percentages of positive cells in each scattergram (CD45, CD34, CD33, CD19, CD41, CD14, CD11b and CD235a) are presented in the indicated squares. The expression of CD3, CD4 and CD8 on the human CD45 + cells in the thymus, and the expression of CD56 on the human CD45 + cells in the spleen was also analyzed and presented. The percentages of positive/negative cells in each scattergram are indicated.

Article Snippet: We used fluorescein isothiocyanate-conjugated 18Lin mAbs against CD2, CD16, CD24 and CD235a (Dako, Kyoto, Japan); CD3, CD7, CD10, CD11b, CD20, CD41 and CD66c (Beckman Coulter, Fullerton, CA, USA); CD19 and CD56 (BD Biosciences, San Jose, CA, USA); CD4, CD14, CD33 and CD127 (eBioscience, San Diego, CA, USA); CD45RA (Southern Biotech, Birmingham, AL, USA); a Pacific Blue-conjugated anti-CD45 mAb (BioLegend, San Diego, CA, USA); an apophycocyanin (APC)-conjugated anti-CD34 mAb (BD Biosciences) and a phycoerythrin (PE)-conjugated anti-CD133 mAb (Miltenyi Biotec, Bergish Gladbach, Germany).

Techniques: Transplantation Assay, Expressing