Deciphering the Immune Microenvironment in Waldenstrom's Macroglobulinemia
Authors: Sacco Antonio , Desantis Vanessa , Celay Jon , Giustini Viviana , Rigali Fabio , Savino Francesco , Cea Michele , Soncini Debora , Cagnetta Antonia , Solimando Antonio Giovanni , D'Aliberti Deborah , Spinelli Silvia , Ramazzotti Daniele , Almici Camillo , Todoerti Katia , Neri Antonino , Anastasia Antonella , Tucci Alessandra , Motta Marina , Chiarini Marco , Kawano Yawara , Martinez-Climent Jose A , Piazza Rocco , Roccaro Aldo 
Published: December 2023

Recent studies have demonstrated the occurrence of somatic mutations of MYD88 and CXCR4 as key players in Waldenstrom's Macroglobulinemia (WM) pathogenesis and disease progression. Despite the significant improvement in the knowledge of the molecular mechanisms supporting WM biology whether immunosuppressive mechanisms could contribute to WM pathogenesis remains unexplored.
We interrogated the transcriptome signatures of the bone marrow (BM) microenvironment of WM patients (n:22) as compared to healthy individuals (n:10), we found a significant enrichment for a Treg-signature, and for CD40/CD40L signaling-related genes (FDR &lt;0.001; P &lt;0.01). These findings were confirmed using a transgenic murine lymphoplasmacytic/WM model, where the BM immune microenvironment showed an increased number of infiltrating CD4+ T lymphocytes over CD8+ T cells. Further characterization of the T-cell compartment revealed the presence of abundant CD4+CD25+FoxP3+ Treg cells, with respect to control mice. Murine WM cells recruited a significantly higher number of more abundant Ki67+Treg cells, as compared to B lymphocytes from healthy mice. Collectively, these results suggest that immunosuppressive Treg cells may play in supporting WM disease biology.
These findings prompted us to focus on Tregs and their role in supporting WM pathogenesis. We performed bulk RNA sequencing of Tregs isolated from both WM patients (n:14) and healthy donors/HD (n:8); showing a peculiar transcriptome signature characterizing WM-derived Tregs as compared to their normal cellular counterpart. WM patient-derived Tregs presented with transcriptome profiling enriched for FOXP3-target genes and to Treg induction, as compared to healthy donor-derived Tregs; with WM-Tregs showing a significant enrichment for genes related to interferon- and TNFα-related genes via NF-kB.
The functional impact of Treg in WM biology was next studied. We observed a significantly higher ability of WM cells to favor both induction and expansion of CD4+CD25+FoxP3+ Tregs, as compared to HD-derived CD19+ B-cells; paralleled by a significantly higher increase of Ki67-positive Treg induced by WM cells as compared to HD-derived CD19+ B-cells. Same results were obtained using WM primary cells. Data were further corroborated by demonstrating significant enrichment of several pro-proliferative- and pro-survival pathways in WM-Tregs, versus HD-counterpart, including MAPK- and PI3K/AKT-related genes.
To gain insight into the potential molecular mechanisms responsible for the observed Treg induction in WM, we performed scRNAseq using the whole cell population harvested at the end of the induction assay. We evaluated the B-T-cell interactions at single cell level, adopting a B-&gt;T cross-talk model; and identified a set of four high-priority genes (CD40, TNF, TNFSF14, ICAM2): although all of them were expressed in B cells, only the expression of CD40 was maximal and virtually restricted to the B clusters, with the other three genes showing a more diffuse pattern. Importantly, CD40-ligand was shown to be the interactor expressed within the Treg cell compartment.
Given the identified CD40/CD40L axis as a potential regulator of the WM cell/Treg cross-talk, we used the CD40/CD40L inhibitor DRI-C21045; and found a significant decrease in Treg induction and Treg proliferation. Halting CD40/CD40L interaction inhibited Treg induction and growth, also within the context of CXCR4-mutated WM, supported by inhibition of p-AKT and p-ERK in Treg cells. CD40/CD40L blockade led to inhibition of WM cell growth.
Overall our studies have demonstrated the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.