Fig. 6: LILRB1 CAR-T cells demonstrate target specificity and functionality after antigen stimulation.

A Cytotoxicity of LILRB1 CAR-T cells against B-ALL cells (697) genetically modified to overexpress LILRB1 was assessed by flow cytometry-based killing assay. CAR-T cells and CTV-labeled target cells were co-cultured for 24 h at 1:1 E:T ratio. The samples were then stained with PI, and the percentage of dead CTV⁺PI⁺ target cells was determined. Data shows mean ± SD from n = 3 donors, P values were calculated using two-way ANOVA with Tukey’s multiple comparisons test (MOCK vs. CAR-T cells in each group and CAR-T vs. control or modified cell line). B Cytotoxicity of LILRB1 CAR-T cells against 6 KMT2A-r B-ALL PDX cells was assessed by flow cytometry-based killing assay. CAR-T cells and CTV-labeled target cells were co-cultured for 24 h at 0.5:1 E:T ratio. The samples were then stained with PI, and the percentage of dead CTV⁺PI⁺ target cells was determined. Data shows mean ± SD from n = 2–3 donors, P values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test (MOCK vs. CAR-T and various CAR-T comparisons). C Cytotoxicity of LILRB1 CAR-T cells against B-ALL cells (RS4;11) with CD19 KOFootnote

Knockout

was assessed using a luciferase-based killing assay. CAR-T cells and luciferase-expressing target cells were co-cultured for 24 h at a 2:1 E:T ratio. The percentage of dead target cells was determined by measuring the decrease in luminescence signal. Data shows mean ± SD from n = 3 donors, P values were calculated using two-way ANOVA with Tukey’s multiple comparisons test (CAR-T vs. control or CD19 KO cell line). D Cytotoxicity of LILRB1 CAR-T cells against B-NHL cells (Ramos) with CD19 KO was assessed by flow cytometry-based killing assay. CAR-T cells and CTV-labeled target cells were co-cultured for 24 h at 2:1 E:T ratio. The samples were then stained with PI, and the percentage of dead CTV⁺PI⁺ target cells was determined. Data shows mean ± SD from n = 3 donors, P values were calculated using two-way ANOVA with Tukey’s multiple comparisons test (CAR-T vs. control or CD19 KO cell line). E Cytotoxicity of LILRB1 CAR-T cells against Raji cells resistant to CD19 CAR-T cells was assessed using a luciferase-based killing assay. CAR-T cells and luciferase-expressing target cells were co-cultured for 24 h at 5:1 E:T ratio. The percentage of dead target cells was determined by measuring the decrease in luminescence signal. Data shows means ± SD from n = 3 donors, P values were calculated using two-way ANOVA with Tukey’s multiple comparisons test (MOCK vs. CAR-T cells in each group and CAR-T vs. control or resistant cell line). F Degranulation of LILRB1 CAR-T cells was assessed by flow cytometry. CAR-T cells and target cells (SD-1) were co-incubated for 18 h at 1:2 E:T ratio, in the presence of anti-CD107a Ab. Next, the samples were stained with anti-CD3 Ab, and the percentage of CD3⁺CD107a⁺ T cells was determined. Data shows mean ± SD from n = 4 donors, P values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test (MOCK vs. CAR-T and CAR-T comparison). G. Cytokine release by LILRB1 CAR-T cells was evaluated using ELISAFootnote

Enzyme Linked Immunosorbent Assay

assay. CAR-T cells and target cells (SD-1) were co-incubated for 24 h at 1:2 E:T ratio. The concentration of IFNγFootnote

Interferon Gamma

and TNFαFootnote

Tumor Necrosis Factor Alpha

was then measured in the culture medium. Data shows mean ± SEMFootnote

Standard Error Of The Mean

from n = 3 donors, P values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (MOCK vs. CAR-T). H Cytotoxicity of LILRB1 CAR-T cells against normal PBMCFootnote

Primary Peripheral Blood Mononuclear Cell

was assessed by flow cytometry. CFSEFootnote

Cell trace™ Carboxyfluorescein Succinimidyl Ester

-labeled CAR-T cells were co-incubated with PBMCs for 24 h at 1:2 E:T ratio. The samples were then stained with anti-CD19, anti-CD56, anti-CD3, and anti-CD14 antibodies to determine the approximate percentages of B-cells (CD19⁺ cells), monocytes (CD14⁺ cells), T cells (CD3⁺ cells), and NK cells (CD56⁺ cells). Data shows mean ± SD from n = 4 donors, P values were calculated using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (MOCK vs. CAR-T). I CFU-EFootnote

Colony-Forming Unit–Erythroid

, BFU-EFootnote

Burst-Forming Unit–Erythroid

, CFU-GMFootnote

Colony-Forming Unit–Granulocyte, Macrophage

, and CFU-GEMMFootnote

Colony-Forming Unit–Granulocyte, Erythrocyte, Monocyte, Megakaryocyte

colonies were counted upon co-culture of 2.0 × 10⁵ BMNCFootnote

Bone Marrow Mononuclear Cell

with 2.0 × 10⁶ CAR-T cells respectively (E:T ratio of 10:1) for 6 h, followed by plating in methylcellulose and incubated for 14 days at 37 °C (n = 3; technical replicates).