Multiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38⁺CD138⁺ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.

Multiple myeloma (MM) is a bone marrow plasma cells malignancy. Despite impressive advancements of treatments, MM remains incurable due to complex clonality. Here, we present a novel 14-color flow cytometry (FACS) protocol for robust detection and visualization of MM clonality. We developed a single-tube 14-color FACS protocol to analyze fresh bone marrow samples, tested on 8 MM patients. A premixed, stable antibody cocktail was used to enhance reproducibility and streamline workflow. Dimensionality reduction technique (t-SNE) was applied to visualize plasma cell clonality. Our panel shown CD38⁺ CD138⁺ plasma cells, and their clonality. As expected, MM patients had unique clonal patterns with significant heterogeneity at primary diagnosis. Advanced t-SNE analysis provides a convenient visualization of multi-parameter heterogeneity in a unified 2-D plot. Importantly, our data presents some similarities among patients, and further differences between primary analysis and secondary relapse. Taken together, our protocol provides a robust and efficient method for improving MM diagnosis using commercially available antibodies and a standard FACS machine. Concise visualization of multi-parameter FACS will help therapeutic decisions and advance the personalized treatment strategies with emerging antigen-specific drugs.