Differential Functional Outcomes After Stimulation Via Innate Immunity Receptors In Chronic Lymphocytic Leukemia Subtypes Defined by the Molecular Features of the Immunoglobulin Receptor
Authors: Ntoufa Stavroula , Vardi Anna , Papakonstantinou Nikos , Anagnostopoulos Achilles , Aleporou-Marinou Vassiliki , Caligaris-Cappio Federico , Belessi Chrysoula , Ghia Paolo , Muzio Marta , Stamatopoulos Kostas 
Published: October 2019

Abstract
Abstract 374
Mature B cells recognize antigens through the B cell receptor (BCR) in a specific way and through innate immunity receptors, such as the Toll-like receptors (TLRs) and Nod-like receptors (NLRs), in a costimulatory manner. Signaling through the BCR appears to be a major contributing factor in CLL development and, perhaps, evolution. However, the timing, duration and, most importantly, the nature of antigenic exposures remain unclear. Although emerging data suggest that innate immunity receptors are relevant for CLL pathogenesis, the functionality of TLRs and NLRs in CLL remains largely unexplored. We investigated TLR and NLR function within CLL subgroups defined by IGHV gene usage, IGHV gene mutational status or BCR stereotypy. To this end, we studied a series of 67 patients, with an intentional bias to cases expressing stereotyped BCRs assigned to subsets #1 (IGHV1/5/7-IGKV1(D)-39) and #4 (IGHV4-34/IGKV2-30), which can be considered as prototypes of “unmutated/adverse prognosis” and “mutated/good prognosis” subsets, respectively. Negatively isolated CLL cells were stimulated with specific ligands for all TLRs/NLRs expressed in B cells. The functional outcome was assessed after 24 hours by flow cytometric determination of CD25 and CD86 expression as a measure of cell activation. At cohort level, TLR1/2, TLR2/6, TLR9, and NOD2 were responsive to stimulation with their respective ligands in most cases, yet in a heterogeneous fashion. TLR7 stimulation with Imiquimod, a small synthetic antiviral molecule, induced CD25 and/or CD86 in ∼50% of cases; in contrast, loxoribine, a naturally occurring TLR7 agonist, had no effects. Markedly different qualitative responses were observed when comparing mutated vs. unmutated CLL cases, with TLR9 upregulating CD86 expression in mutated CLL cases, while TLR7 upregulating CD25 expression in unmutated CLL cases (p<0.05 for all comparisons). Different patterns of functional responses to TLR and NOD2 stimulation were also identified in subsets with stereotyped BCRs. In particular, stimulation of TLR9, TLR1/2, TLR2/6, and NOD2 with the respective ligands strongly upregulated CD86 expression in subset #4 cases, whereas the same treatment had a significantly less pronounced effect on subset #1 cases (p<0.05 for all comparisons). Opposite results were obtained after stimulation of the TLR7 with imiquimod, in that subset #1 cases exhibited stronger up-regulation of CD25 expression compared to subset #4 cases (p<0.05). Our study also included three cases each belonging to stereotyped subsets #8 (IGHV4-39/IGKV1(D)-39, unmutated) and #16 (IGHV4-34/IGKV3-20, mutated). Differential, subset-biased profiles were also identified when limiting the comparisons to subsets with similar mutational status (i.e. subset #1 vs. #8 and subset #4 vs #16), underscoring the idea of “subset-biased” innate immunity responses independently of IGHV gene usage and/or mutational status. To further corroborate this concept, we focused on cases utilizing the IGHV4-34 gene, which were intentionally over-represented in the cohort. By this approach, we identified significantly (p<0.05) different responses after stimulation of TLR1/2, TLR2/6, TLR9 and NOD2 in subset #4 vs. non subset#4/16 IGHV4-34 cases (in terms of induction of either CD25 or CD86 or both); subset-biased profiles were also noted for subset #16 vs. other IGHV4-34 cases. In conclusion, we demonstrate that TLRs and NLRs are functional in CLL cells, yet in a heterogeneous fashion. Distinct patterns of TLR/NLR functionality and/or TLR tolerance can be recognized among CLL subgroups defined by specific molecular characteristics of the clonotypic BCRs with potential implications about both the nature of the relevant antigen(s) and the eventual implementation of targeted therapies.

Disclosures:
No relevant conflicts of interest to declare.