Impact of Protein Kinase C Activation and Monoclonal Antibodies on Immune Checkpoint Regulation and B Cell Function in Patients with Chronic Lymphocytic Leukemia

Abstract

Background: Chronic lymphocytic leukemia (CLL) is characterized by the proliferation of dysfunctional B cells, resulting in significant immune dysregulation. Patients with CLL exhibit varied responses to B cell receptor (BCR) targeted therapies, emphasizing the need for tailored immunotherapy approaches. This study investigated B cell function in untreated patients with CLL, and we further explored the effects of ex vivo protein kinase C activation on immune checkpoint expression and B cell profiles. Methods: Peripheral blood samples were collected from 21 untreated patients with CLL at King Edward Hospital in South Africa, between 2019 and 2022. B cells were stimulated with phorbol myristate acetate (PMA) and ionomycin. Using flow cytometry, the study explored the levels of B cell subsets and immune checkpoint proteins programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), programmed cell death-ligand 2 (PD-L2) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) expression on various B cell subsets. Results: PMA and ionomycin B cell stimulation upregulated PD-1, CTLA-4 and PD-L2 expression on B cell subsets (p < 0.01). As expected, monoclonal antibodies targeting PD-1, PD-L1 and CTLA-4 significantly downregulated the CTLA-4 expression of B cell subsets (p < 0.05), while PD-L2 exhibited varied responses in different B cell subsets. Moreover, PD-1 and PD-L1 expression on total B cells significantly declined following their blockage (p < 0.01). In addition, these monoclonal antibodies increased the levels of CD19⁺CD27⁺ B cells (p < 0.0128) and activated CD19⁺CD27⁺ B cells (p < 0.01). Conclusions: Protein kinase C activation on B cells stimulates immune checkpoint expression. The use of monoclonal antibodies on B cells plays a critical role in the B cell function through the reduction in CD38 expressing activated B cells and upregulation of CD19⁺CD27⁺ B cells. Moreover, the monoclonal antibody targeting PD-1, PD-L1 and CTLA-4 are effective in reducing the expression of CTLA-4 on B cell subsets, while PD-1 and PD-L1 blockage may be effective in reducing the expression of these immune checkpoints on total B cells.

Keywords: B cell subsets; chronic lymphocytic leukemia; immune checkpoint inhibitor; ionomycin; phorbol myristate acetate.

Figure 1

Gating strategy. B cells were isolated using magnetic bead sorting. (A) illustrates the gates applied to distinguish between viable and non-viable B cells based on a Zombie Aqua dye. (B) illustrates the gating on B cells based on side scatter (SSC) and CD19 expression. (C) illustrates the gating of activated B cells defined as CD19⁺CD38⁺CD27⁻ events, memory B cells defined as CD19⁺CD27⁺CD38⁻ events and activated memory B cells defined as CD19⁺CD27⁺CD38⁺ events, respectively. (D–G) illustrates the gating of CTLA-4, PD-1, PD-L1 and PD-L2, respectively. Histograms were used to demonstrate the levels of CTLA-4 (H), PD-1 (I), PD-L1 (J) and PD-L2 expression (K) on various co-culture conditions.

Figure 2

B Cell Subsets in patients with CLL upon B cell stimulation and immune checkpoint inhibition. (A) illustrates the expression levels of activated B cells, (B) memory B cells, and (C) activated memory B cells. The data are presented as the median ± interquartile range (IQR). *, **, *** and **** shows the level of significance between groups, ns: not significant.

Figure 3

Immune checkpoint expression on B Cell subsets under different treatment conditions. The figure illustrates the levels of PD-L2 and CTLA-4 expression on (A) total B cells, (B) activated B cells and (C) memory B cells, respectively. The data are presented as the median ± interquartile range (IQR). *, **, *** and **** shows the level of significance between groups, ns: not significant.