Abstract A074: Immune dysregulation and B-cell fitness driven by PLCγ2 R665W: Insights from a novel conditional B-cell mouse model

Abstract PLCγ2 (phospholipase C gamma 2) functions as a critical signaling molecule in immune cells, particularly B-cells, and is associated with immune dysregulation. PLCγ2 mutations drive immunodeficiency syndromes (PLCγ2-associated Antibody deficiency and Immune Dysregulation (PLAID) and Autoinflammatory PLCγ2-associated Antibody deficiency and Immune Dysregulation (APLAID)). PLCγ2 mutations are also seen in Bruton tyrosine kinase (BTK) inhibitor-resistant chronic lymphocytic leukemia (CLL). The recurrent PLCγ2 R665W is the most frequently occurring BTKi resistance mutation in 45% of cases, yet it’s in-vivo immunologic consequences remain poorly understood. To characterize PLCγ2 function in-vivo in B-cells, both in and out of a cancer context, we generated conditional B-cell specific Mb1-Cre+ PLCγ2 R665W heterozygous mice with and without crossing to the CLL mouse model Eμ-TCL1. First, C57BL/6 PLCγ2 R665W mice were generated and crossed to C57BL/6 Mb1-Cre to produce B-cell restricted Mb1-Cre+ PLCγ2 R665W heterozygous mice. R665W heterozygous mice without Cre expression were used as controls. The point mutation was confirmed using Sanger sequencing. Beginning at week 4, and at subsequent 4-week intervals, the mice were weighed, bled, and flow cytometry performed to analyze blood cell types (n≥10, 5M and 5F). These mice were followed till end-of-life and a Kaplan-Meier survival curve generated. To study B-cell development by B-cell subtyping, other cohorts of mice (n≥9) was euthanized at week 12 and 48, and blood, spleen and bone marrow analyzed. Subsequently, scRNAseq was performed on week 12 splenocytes (n=5). Mb1-Cre+ PLCγ2 R665W mice were crossed to Eμ-TCL1 to study R665W in a CLL context. B-cell–restricted PLCγ2 R665W expression did not affect overall survival or weight but caused a sustained reduction in total white blood cells and B-cells, with the most pronounced differences at week 12 and after week 32, suggesting a possible developmental or selection disruption. B-cell subtyping at week 12 and 48 showed decreased peripheral blood T2 transitional B-cells and decreased spleen follicular II B-cells in experimental vs control mice. scRNAseq on week 12 splenocytes revealed decreased B-cell receptor signaling through ERK1/2 and NFκB in almost all mutant B-cell subtype clusters. Ongoing studies following antigenic immunization suggest altered activation potential in mutant B-cells. In the CLL context, Eμ-TCL1 Mb1-Cre+ PLCγ2 R665W mice exhibited significantly reduced total B-cells and a non-significant trend toward fewer CD19+CD5+ leukemic cells, without changes in survival or body weight. Due to variability in the development of CLL, a transplant model is being generated for further studies. These results indicate that PLCγ2 R665W impairs B-cell development and attenuates BCR signaling in-vivo, reshaping immune homeostasis and modifying leukemic B-cell expansion. This model provides a versatile platform for dissecting how PLCγ2-driven immune alterations influence resistance to targeted therapy and antitumor immunity. Citation Format: Vindhya Nawaratne, Jacob Jahn, Alexandra Chirino, Wasif N. Khan, David G. Coffey, Justin Taylor. Immune dysregulation and B-cell fitness driven by PLCγ2 R665W: Insights from a novel conditional B-cell mouse model [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A074.