rabbit anti human il 21  (Bio-Rad)

Journal: Cell Death & Disease

Article Title: Single-cell profiling of immune cells after neoadjuvant pembrolizumab and chemotherapy in IIIA non-small cell lung cancer (NSCLC)

doi: 10.1038/s41419-022-05057-4

Figure Lengend Snippet: A The heatmap showing GSEA enrichment of the CXCL13, Tfh cell, and 12-chemokine signatures in treatment-naive, non-MPR, and MPR tumor tissues. P adj > 0.05 in the enrichment pathway was adjusted to 0 to draw the heat map. B The higher TLS density in neoadjuvant tumor lesions verified by mIHC staining in a single-cell sequencing cohort. C Higher intratumoral C2-CD4-IL7R and C4-CD4-TCF7 cluster proportions in neoadjuvant tumor lesions in scRNA profiles. D IL21 expression in different tissue types tested with Wilcoxon in scRNA profiles showing that IL21 was most prevalent in tumor tissues. **** p < 0.0001. E Go biological function analysis of the IL21 expression cells by clusterprofiler in scRNA profiles. F Flow cytometry of IL-21 in CD4 + T cells in tumor tissues. Data presented as mean ± SEM. Data were summarized from n = 8 treatment-naive, n = 5 non-MPR and n = 15 MPR tumor samples. P values were determined by ordinary one-way ANOVA. * p < 0.05, ** p < 0.01, ns: not significant. G Comparison of paired plasma IL-21 before and after neoadjuvant chemoimmunotherapy. P values were determined by Wilcoxon matched-pairs signed-rank test, two-tailed. * p < 0.05. H , I MIHC staining showing CD4 + IL21 + and IgG-positive cells were much abundant in MPR tumor tissues (image under 10× and 20×). Data presented as mean ± SEM. Data were summarized from n = 6 treatment-naive, n = 9 non-MPR and n = 18 MPR tumor samples. P values were determined by ordinary one-way ANOVA. * p < 0.05, ns: not significant.

Article Snippet: The primary antibodies and IHC metrics were: rabbit monoclonal anti-human CD4 antibody (Abcam, Cat# ab133616, diluted at 1:1000), rabbit monoclonal anti-human CD20 antibody (Abcam, Cat# ab78237, diluted at 1:2000), mouse monoclonal anti-human IgG antibody (Abcam, Cat# ab200699, diluted at 1:800), rabbit polyclonal anti-human IL-21 antibody (Invitrogen, Cat# PA5-34801, diluted at 1:2000), mouse monoclonal anti-human CD21 antibody (Invitrogen, Cat# MA5-11417, diluted at 1:200), mouse monoclonal anti-human FoxP3 antibody (Abcam, Cat# ab20034, diluted at 1:500), rabbit monoclonal anti-human IgG1 antibody (Abcam, Cat# ab108969, diluted at 1:1000), rabbit monoclonal anti-human IgG3 antibody (Abcam, Cat# ab193172, diluted at 1:800), recombinant rabbit monoclonal anti-human IgA antibody (Invitrogen, Cat# MA5-32575, diluted at 1:800).

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

Journal: Cell Death & Disease

Article Title: Single-cell profiling of immune cells after neoadjuvant pembrolizumab and chemotherapy in IIIA non-small cell lung cancer (NSCLC)

doi: 10.1038/s41419-022-05057-4

Figure Lengend Snippet: A Validation cohort: Surgical resected tumor tissues from 26 treatment-naive IIIA/IIIB NSCLC and 30 resectable IIIA/IIIB NSCLC who have received two cycles of neoadjuvant pembrolizumab and chemotherapy before surgery. B Neoadjuvant chemoimmunotherapy promoted more TLSs formation in non-MPR and MPR tumor lesions in our validation cohort. Data presented as mean ± SEM. Data were summarized from n = 26 treatment-naive, n = 10 non-MPR and n = 20 MPR tumor samples. P values were determined by ordinary one-way ANOVA. * p < 0.05, *** p < 0.001, ns: not significant. C , D MIHC staining of B cell isotypes, CD4 and IL-21 showing that CD20, IgG1, IgG3, CD4, and IL-21 were significantly higher in MPR tumor lesions, whereas IgA was much lower in MPR tumor lesions. Data presented as mean ± SEM. Data were summarized from n = 26 treatment-naive, n = 10 non-MPR and n = 20 MPR tumor samples. P values were determined by ordinary one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. E Pearson correlation coefficient analysis found that IgG1 and IgG3 were positively correlated with IL-21 within tumor lesions. F Plasma IL-21 was significantly higher in MPR patients than that in treatment-naive and non-MPR patients in our validation cohort. Data presented as mean ± SEM. Data were summarized from n = 18 treatment-naive, n = 5 non-MPR and n = 10 MPR tumor samples. P values were determined by ordinary one-way ANOVA. * p < 0.05, ns: not significant.

Article Snippet: The primary antibodies and IHC metrics were: rabbit monoclonal anti-human CD4 antibody (Abcam, Cat# ab133616, diluted at 1:1000), rabbit monoclonal anti-human CD20 antibody (Abcam, Cat# ab78237, diluted at 1:2000), mouse monoclonal anti-human IgG antibody (Abcam, Cat# ab200699, diluted at 1:800), rabbit polyclonal anti-human IL-21 antibody (Invitrogen, Cat# PA5-34801, diluted at 1:2000), mouse monoclonal anti-human CD21 antibody (Invitrogen, Cat# MA5-11417, diluted at 1:200), mouse monoclonal anti-human FoxP3 antibody (Abcam, Cat# ab20034, diluted at 1:500), rabbit monoclonal anti-human IgG1 antibody (Abcam, Cat# ab108969, diluted at 1:1000), rabbit monoclonal anti-human IgG3 antibody (Abcam, Cat# ab193172, diluted at 1:800), recombinant rabbit monoclonal anti-human IgA antibody (Invitrogen, Cat# MA5-32575, diluted at 1:800).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Single-cell profiling of immune cells after neoadjuvant pembrolizumab and chemotherapy in IIIA non-small cell lung cancer (NSCLC)

doi: 10.1038/s41419-022-05057-4

Figure Lengend Snippet: Neoadjuvant chemoimmunotherapy promotes LAMP3 + DCs aggregation in tumor lesions, and the increased LAMP3 + DCs acquire tumor antigen, migrate and interact with lymphocytes, and participate in the activation, recruitment, and regulation of T/B lymphocytes through L-R interactions. Anti-PD-1 therapy reinvigorates the expansion of intratumoral CD4 + T cells and peripheral cytotoxic CD8 + T clones and promoted CD8 + T cells migration to tumor tissues to exert an anti-tumor effect. Neoadjuvant chemoimmunotherapy promotes more TLSs formation in NSCLC tumor tissues. In tumor-associated TLSs, IL-21 secreted by Tfh cells promotes B cells class switching to IgG1 and IgG3 but not IgA to mediate anti-tumor response. IgG1 and IgG3 antibodies bind to Fcγ receptor (FcγR) and trigger ADCC and antibody-mediated phagocytosis and mediate complement-based cytotoxicity. In addition, the decrease of immunosuppressive IgA + plasma cells and TNFRSF4 + Tregs lead to the repression of immunosuppression during chemoimmunotherapy.

Article Snippet: The primary antibodies and IHC metrics were: rabbit monoclonal anti-human CD4 antibody (Abcam, Cat# ab133616, diluted at 1:1000), rabbit monoclonal anti-human CD20 antibody (Abcam, Cat# ab78237, diluted at 1:2000), mouse monoclonal anti-human IgG antibody (Abcam, Cat# ab200699, diluted at 1:800), rabbit polyclonal anti-human IL-21 antibody (Invitrogen, Cat# PA5-34801, diluted at 1:2000), mouse monoclonal anti-human CD21 antibody (Invitrogen, Cat# MA5-11417, diluted at 1:200), mouse monoclonal anti-human FoxP3 antibody (Abcam, Cat# ab20034, diluted at 1:500), rabbit monoclonal anti-human IgG1 antibody (Abcam, Cat# ab108969, diluted at 1:1000), rabbit monoclonal anti-human IgG3 antibody (Abcam, Cat# ab193172, diluted at 1:800), recombinant rabbit monoclonal anti-human IgA antibody (Invitrogen, Cat# MA5-32575, diluted at 1:800).

Techniques: Activation Assay, Clone Assay, Migration