![HPK1 inhibition augments the activation of T cells in the presence of CD3/CD28 or bispecific antibodies. Peripheral blood CLL cells were treated with the indicated concentration of HY-138568 or BGB15025 for 2 hours. Dimethyl sulfoxide treated cells served as controls (NT). (A-C). Following treatment, the cells were stimulated with Dynabeads Human T cell Activator CD3/CD28 for 24 hours at 37°C. After incubation, the cells were stained with Pacific Blue Anti-Human CD8 and PE Anti-Human CD69 or CD25. Samples were acquired by FACSCanto II (BD) and analyzed using BD FACSDiva software. (A) Flow cytometric dot-plots of CD8-Pacific Blue vs CD69-PE expression on samples of 1 representative CLL case. (B) Quantification of CD69 mean-fluorescence intensity (MFI) in CD8+ population (n = 7). (C) Quantification of CD25 MFI in CD8+ population (n = 5). (D-I) Following treatment, the cells were cultured in anti-CD3/CD28–coated plates for 48 hours at 37°C in a humidified 5% CO2 atmosphere. (D,G) IFN-γ secretion was measured from supernatants using enzyme-linked immunosorbent assay (ELISA)–based assay platform (n = 12). (E,H) IFN-γ levels in M-CLL samples (n = 6). (F,I) IFN-γ levels in UM-CLL samples (n = 6). (J-K) Following treatment, 10 μg/mL of CD20XCD3 bispecific antibody [epcoritamab (E)/glofitamab (G)/mosunetuzumab (M)] was added for 48 hours. (J) IFN-γ secretion was measured from supernatants using ELISA-based assay platform (n = 4). (K) IFN-γ secretion following treatment with HPK inhibitors and glofitamab (n = 7). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. M-CLL, mutated CLL; NT, no treatment; UM-CLL, unmutated CLL.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/12/10.1182_bloodadvances.2024015242/1/blooda_adv-2024-015242-gr1.jpeg?Expires=1752942021&Signature=bAxGsUuA0QeOd-4~-zYvusWVS8weGom9OU1fjJ-ENRbb6ufCdECPqh08RIjo1lgESuiooG7cOjlViBiu8oVuULPvzTzNMTFMhjl4AA0708I3I~O7FjjW5lAsgJOyHabB7Fm4y~DlnZauB-Q0N6nMBGSEsoIEIdu6TaQlVto73~TXfbAvkP~T~MzvhQ7FrCVcO-7PyMQVnpVcnjoE1bB6QR037n8IQhmKVPGa-MTdR04P46Gk723dDU4xuDgX4mY7reMkqocMG6ivSlXej3futaLTGyignRIZQI0u7D5tSLE1bBbizNCjr7Zb4dM8wyNa45da-h8qdLQNf2HiJMK3LQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
HPK1 inhibition augments the activation of T cells in the presence of CD3/CD28 or bispecific antibodies. Peripheral blood CLL cells were treated with the indicated concentration of HY-138568 or BGB15025 for 2 hours. Dimethyl sulfoxide treated cells served as controls (NT). (A-C). Following treatment, the cells were stimulated with Dynabeads Human T cell Activator CD3/CD28 for 24 hours at 37°C. After incubation, the cells were stained with Pacific Blue Anti-Human CD8 and PE Anti-Human CD69 or CD25. Samples were acquired by FACSCanto II (BD) and analyzed using BD FACSDiva software. (A) Flow cytometric dot-plots of CD8-Pacific Blue vs CD69-PE expression on samples of 1 representative CLL case. (B) Quantification of CD69 mean-fluorescence intensity (MFI) in CD8+ population (n = 7). (C) Quantification of CD25 MFI in CD8+ population (n = 5). (D-I) Following treatment, the cells were cultured in anti-CD3/CD28–coated plates for 48 hours at 37°C in a humidified 5% CO2 atmosphere. (D,G) IFN-γ secretion was measured from supernatants using enzyme-linked immunosorbent assay (ELISA)–based assay platform (n = 12). (E,H) IFN-γ levels in M-CLL samples (n = 6). (F,I) IFN-γ levels in UM-CLL samples (n = 6). (J-K) Following treatment, 10 μg/mL of CD20XCD3 bispecific antibody [epcoritamab (E)/glofitamab (G)/mosunetuzumab (M)] was added for 48 hours. (J) IFN-γ secretion was measured from supernatants using ELISA-based assay platform (n = 4). (K) IFN-γ secretion following treatment with HPK inhibitors and glofitamab (n = 7). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. M-CLL, mutated CLL; NT, no treatment; UM-CLL, unmutated CLL.
