Fig 1: IDR promoted CAR condensation on the T cell surface.A) Schematics of the CD19 CAR used in this study. It contained an scFv targeting CD19 (FMC63), a CD8 hinge and transmembrane domain, an intracellular signaling domain composed of CD28, 41BB, and CD3ζ, an intrinsically disordered region (IDR), and superfolder GFP.B) Expression of IDR CARs. Human pan primary T cells were transduced with lentivirus encoding the control or IDR CAR. The expression level of CAR on the T cell surface was measured via flow cytometry using an anti-FMC63 antibody. A GFP tag was fused on the C-terminus of each CAR to monitor the total expression level of CAR.C) Condensation of CAR on the plasma membrane. TIRF microscopy revealed condensation of the control or IDR CAR on the T cell membrane. GFP was tagged on the C-terminus of CAR for visualizing CAR. The plasma membrane was labeled by a CellMask Deep Red dye.D) Quantification of CAR clustering by normalized variance. N=48–50 cells. Shown are mean ± std. All comparisons in this and following figures were made between individual IDR CARs and the control CAR (n.s. p>=0.05, * p<0.05, ** p<0.01, *** p<0.001).E) CAR-T cell proliferation with or without IL-2. Quantification of the absolute cell number in the culture with or without IL-2. N=3 independent experiments. Shown are mean ± std.
Fig 2: The balance between anti-virus immunity and anti-tumor immunity of NK cells in combination therapy (n = 5 per group). a Treatment protocol for HCT116 tumor-bearing NPG mice (by Figdraw). In group 1, NK infusion was started on day 3 after rAd.DCN injection, and in group 2, NK infusion was started on day 6 after rAd.DCN injection. b Tumor growth curves and images of tumor tissues from sacrificed mice at day 24 (n = 3 for the control group or n = 5 for rAd.DCN + NK group). Tumor volumes were subjected to analysis using a two-way repeated-measure ANOVA. c Body weights of mice during the 24-day course of treatment (n = 3 for the control group or n = 5 for rAd.DCN + NK group). d Analysis of NK cell counts in the peripheral blood of mice using flow cytometry (n = 3 for the control group or n = 5 for rAd.DCN + NK group). One-way ANOVA was used for comparisons between multiple groups. e HE staining of tumor tissue at the experimental endpoint. Scale bar = 50 µm. f TUNEL staining of tumor tissues. Scale bar = 200 µm. Immunohistochemistry staining for g adenovirus, h Decorin, and i CD56 distribution in the tumor tissues. Scale bar = 50 µm. j Statistical results of positive area ratio for adenovirus, k Decorin and l CD56 (n = 9). Comparisons between the two groups were made using an unpaired t-test. m qPCR detection of Decorin, IL-2, and IFN-γ expression in tumor tissues (n = 3). One-way ANOVA was used for comparisons between multiple groups. Data are presented as mean ± SD. *p < 0.05; ***p < 0.001
Fig 3: rAd.DCN enhances cytotoxic activity of NK cells in vitro. a NK cells were pre-stained with CFSE (5 µM) and then infected with Oncolytic Viruses (OVs) at MOIs of 0.1 and 1 for 72 h. Flow cytometry was utilized to analyze the proliferation of NK cells. b NK cells were co-incubated with rAd.Null or rAd.DCN (50 MOI) pretreated HCT116 cells or LoVo cells for 4 h, and a cytotoxicity assay was performed. The percentage of specific cell lysis of HCT116 and LoVo cells was analyzed. c NK cells were co-cultured with rAd.Null or rAd.DCN (50 MOI) pretreated HCT116 or LoVo cells for 4 h, and flow cytometry was used to detect the percentage of CD69 and CD107 positive NK cells. d Co-incubated supernatants were collected and assayed for perforin, IFN, sFasL, and GZMA secretion using the LEGENDplex™ Human CD8/NK Panel Kit. e NK cells were treated with the supernatant of control or OVs-infected HCT116 or LoVo cells for 48 h, and the expression of granzyme B, perforin, TNF-α, and TGF-β of NK cells was evaluated by qPCR assay. f HCT116 or LoVo cells pretreated with rAd.Null or rAd.DCN (50 MOI) were labelled using CFSE, and after co-culturing with NK cells for 4 h, the cells were collected and labelled with 7AAD, and apoptosis rate of target cells was detected by flow cytometry. One-way ANOVA was used for comparisons between multiple groups. All results are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01, ***P < 0.001, ns means no significance
Fig 4: Compared to IL-2 alone, cultivation with IL-2+7+15 resulted in an increase in the stem cell memory CD4+ CAR-T cell population and enhanced Tscm-associated gene expression. (A) Representative contour plot of the memory phenotype of CD4+ CAR-T cells on day 8 and day 15 after initial anti-CD3/CD28 activation. Tscm: CD62L+CD45RA+CD95+; Tcm: CD62L+CD45RA−; Tem: CD62L−CD45RA−; Temra: CD62L−CD45RA+. (B) Statistical evaluation of memory phenotype of HER2-CAR-T cells based on CD62L and CD45RA expression on day 8 and day 15 after initial anti-CD3/CD28 activation. (C) Volcano plot showing log-transformed gene expression comparison at mRNA level of HER2-CAR-T cells cultured with IL-2 alone or with IL-2+7+15 on day 8 (n = 3). Key genes upregulated or downregulated in IL-2+7+15-treated cells were marked as red or blue, respectively. The statistical analysis in (B) was done by matched ANOVA by donors. **: p < 0.01; ***: p < 0.001.
Fig 5: Enhancement of NK Cell degranulation and cytokine secretion by Fc-optimized CD276 antibody. PBMCs from healthy donors were co-cultured with CRC cell lines at an E:T ratio of 2.5:1 in the presence or absence of the Fc-optimized 8H8_SDIE antibody or isotype control MOPC_SDIE (both at 1 µg/mL). NK cell degranulation and cytokine responses were evaluated as follows: (A) Representative flow cytometric plots showing CD107a expression in NK cells co-cultured with CaCo2 cells after 4 h. (B) Individual and pooled flow cytometric data showing CD107a expression in NK cells co-cultured with CRC cell lines and PBMCs from healthy donors (n = 4). (C) Intracellular expression of IFNγ (upper panel) and TNF (lower panel) in NK cells identified by CD3−CD56+ counterstaining and analyzed using flow cytometry after 4 h of co-culture. (D+E) Supernatants were analyzed after 24 h for (D) immunoregulatory cytokines TNF and IFNγ, and (E) effector molecules, such as granzyme A, granzyme B, perforin, and granulysin, as assessed by the Legendplex multiplex assay. Heatmaps showing individual results for CRC cell lines and PBMCs donors (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001.
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