Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: Construction of CAR-WT1 specific for WT1(126–134) peptide presented by HLA-A2. A Schematic diagram of the third-generation CAR-WT1 construct harboring signal peptide, TCR-like scFv, CD8 hinge, CD28, 4-1BB, and CD3ζ, under EF-1α promoter. B Schematic illustration of the generation of CAR-WT1 T cells in primary T cells. C Flow cytometric analysis of surface CAR expression in CAR-WT1 T cells using F(ab’)2 antibody. D Flow cytometric gating strategy to specifically detect cell death in PKH67-labeled target tumor (T) cells in response to effector (E) cells, e.g., T or NK cells, using Annexin V/7-AAD assay. Debris was excluded from the analysis (see also Suppl. Fig. ). E Percentage of total tumor cell death, comprising Annexin V + and/or 7-AAD + cells, of WT1 + /HLA-A2 + Jeko-1 and THP-1 cells, as well as WT1 + /HLA-A2 – K562 and HL60 cells in response to CAR-WT1 or non-transduced (NTD) T cells at various E:T ratios and at the indicated time. Data are mean ± SD (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus NTD T cells under the same conditions; two-sided Student’s t -test

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Construct, Expressing, Labeling

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: CAR-WT1 alone fails to express on NK-92 cell surface. A Schematic illustration of the generation of CAR-WT1 NK-92 cells, which lack surface CAR expression and are designated as intracellular (ic)-CAR-WT1 NK-92 cells. B Flow cytometric analysis of surface CAR expression in ic-CAR-WT1 NK-92 cells after the third transduction. C Schematic diagram of the primer design for genomic CAR detection (primers #1 and #2) by PCR and for the detection of CD3ζ region of the CAR (CAR[ CD3Z ]; primer #3) by qPCR. D Gel electrophoresis shows the products of genomic CAR in ic-CAR-WT1 NK-92 cells, with bands at 669 bp and 788 bp for primer #1 and #2, respectively. E mRNA expression of CAR[ CD3Z ] in ic-CAR-WT1 NK-92 cells by qPCR. Data are mean ± SD (n = 3). *** P < 0.001 versus NTD NK-92 cells; two-sided Student’s t -test. F Flow cytometric analysis of intracellular CAR expression in ic-CAR-WT1 NK-92 cells. Expression frequency, determined as the percentage of total cells, is shown. Data are mean ± SD (n = 3). *** P < 0.001 versus NTD NK-92 cells; two-sided Student’s t -test

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Expressing, Transduction, Nucleic Acid Electrophoresis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: Human CD3 complex is required to enable surface CAR-WT1 in NK-92 cells. A Schematic illustration of the two-step generation of NK-92 cells with surface CAR-WT1, which required the presence of human CD3 complex, designated as CAR-WT1/CD3 NK-92 cells. B NK-92 cells were firstly transduced with human CD3 retroviral particles (RPs), and mRNA expression of CD3D , CD3E , and CD3Z (also known as CD247 ) was evaluated by qPCR. The established CD3 NK-92 cells were further used for CAR transduction. Data are mean ± SD (n = 3). *** P < 0.001 versus NTD NK-92 cells; two-sided Student’s t -test. C Flow cytometric analysis of surface CD3 in CD3 NK-92 cells. D CD3 NK-92 cells were subjected to multiple rounds of CAR-WT1 lentiviral transduction, followed by FACS enrichment of CAR-WT1 + cells with CD3 +/– (green box). Flow cytometric analysis of surface CAR and CD3 expression at each round of transduction/sorting is shown. E Flow cytometric analysis of surface CAR in stable CAR-WT1/CD3 NK-92 cells after the third transduction. F mRNA expression of CAR[ CD3Z ] in CAR-WT1/CD3 NK-92 cells by qPCR. Data are mean ± SD (n = 5). *** P < 0.001 versus NTD NK-92 cells; two-sided Student’s t -test. G mRNA expression of CD3D , CD3E , and CD3Z in CAR-WT1/CD3 NK-92 cells by qPCR. Data are mean ± SD (n = 6). *** P < 0.001 versus NTD NK-92 cells; two-sided Student’s t -test

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Transduction, Retroviral, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: CAR-WT1/CD3 NK-92 cells effectively induce cell death of WT1 + /HLA-A2 + target tumor cells. A Tumor cells, including WT1 + /HLA-A2 + Jeko-1 and THP-1 cells, as well as WT1 + /HLA-A2 – K562 and HL60 cells, were pre-labeled with PKH67 dye and NK cytotoxicity of CAR-WT1/CD3 and NTD NK-92 cells at various E:T ratios was evaluated by Annexin V/7-AAD assay at the indicated time using the same gating strategy shown in Suppl. Fig. . Data are mean ± SD (n = 3 or 5). * P < 0.05, ** P < 0.01, *** P < 0.001 versus NTD NK-92 cells under the same conditions; two-sided Student’s t -test. B The proportion of CAR-WT1/CD3 or NTD NK-92 cells forming binding doublets with Jeko-1 (left) or HL60 (right) cells, expressed as a percentage of all effector cells at various E:T ratios (see Suppl. Fig. for the gating strategy). Negative (Neg) controls were NK-92 and target cells at the E:T ratio of 1:1 incubated at 4 °C. Data are mean ± SD (n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 versus NTD NK-92 cells under the same conditions; two-sided Student’s t -test

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Labeling, Binding Assay, Incubation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: Activation and function of CAR-WT1/CD3 NK-92 cells in response to target tumor cells. CAR-WT1/CD3 NK-92 cells were exposed with or without WT1 + /HLA-A2 + Jeko-1 cells at the E:T ratio of 1:1 for 4 h. A Flow cytometric analysis of surface CD107a in CD56 + CAR-WT1/CD3 or NTD NK-92 cells (see Suppl. Fig. for the gating strategy and representative plots). Data are mean ± SD (n = 3). * P < 0.05 versus NTD NK-92 cells under the same conditions; two-sided Student’s t -test. B Quantitative measurement of IFN- γ and TNF-⍺ by ELISA in cell-free supernatant. Data are mean ± SD (n = 3). *** P < 0.001 versus NTD NK-92 cells under the same conditions; two-sided Student’s t -test. ND, not detectable. C Jeko-1 cells were pre-labeled with PKH67 dye before co-exposure with CAR-WT1/CD3 or NTD NK-92 cells. (upper) mRNA expression of genes involved in immune cell signaling was evaluated in PKH67 – NK-92 cells by qPCR, normalized to GAPDH , and represented in a heatmap. (lower) mRNA expression of certain genes, e.g., IFNA21 , IFNG , and TNF , are plotted. Data are mean ± SD (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 versus NTD NK-92 cells alone; # P < 0.05, ### P < 0.001 versus the indicated group; one-way ANOVA with Tukey’s post- test. NS, not significant

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Labeling, Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: Pathway analysis reveals activated immune cell signaling in CAR-WT1/CD3 NK-92 cells. A Top-ranked KEGG pathways generated from a list of DEGs between CAR-WT1/CD3 and NTD NK-92 cells without target exposure. Sankey and dot plots of enrichment analysis were shown. B Causal interactions of query proteins (encoded by DEGs) were visualized by SIGNOR to illustrate potential link to immune-related phenotypes/stimuli

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Generated

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Human CD3 complex is required for the generation of T-cell receptor-like chimeric antigen receptor targeting WT1 in natural killer cells

doi: 10.1007/s00262-026-04352-9

Figure Lengend Snippet: Surface CD3 is not required for the antitumor activity of CAR-WT1/CD3 NK-92 cells. A Gating strategy for FACS sorting of CAR-WT1/CD3 NK-92 cells according to its surface CD3, into: (i) mixed surface CD3 +/– ; (ii) surface CD3 – ; and (iii) surface CD3 + , all with surface CAR-WT1 + . B , C mRNA expression of CAR[ CD3Z ] (B) and CD3D , CD3E , and CD3Z (C) in different subgroups of sorted CAR-WT1 + NK-92 cells by qPCR. Data are mean ± SD (n = 3). *** P < 0.001 versus NTD NK-92 cells; # P < 0.05, ## P < 0.01, ### P < 0.001 versus the indicated group; one-way ANOVA with Tukey’s post-test. NS, not significant. D , E Different subgroups of sorted CAR-WT1 + NK-92 cells were exposed to Jeko-1 cells at the E:T ratio of 1:1 for 4 h. D Percentage of total Jeko-1 tumor cell death, as evaluated by Annexin V/7-AAD assay using the same gating strategy shown in Suppl. Fig. . E Percentage of surface CD107a in CD56 + NK-92 cells (see Suppl. Fig. for the gating strategy). For panels D and E, data are mean ± SD (n = 6). ** P < 0.01, *** P < 0.001 versus NTD NK-92 cells at the E:T ratio of 1:1; one-way ANOVA with Tukey’s post-test. NS, not significant

Article Snippet: The full-length third-generation CAR-WT1 construct recognizing HLA-restricted WT1(126–134) peptide, RMFPNAPYL, presented by HLA-A*02:01 was synthesized and cloned into a lentiviral vector with the EF1⍺ promoter by Creative Biolabs (Shirley, NY).

Techniques: Activity Assay, Expressing