TLR-9 and IL-15-Driven Clonal Expansion of B-CLL Cells
Authors: Mongini Patricia K. A. , Gupta Rashmi , Chu Charles C. , Stein Joanna , Stankovic Tatjana , Barrientos Jacqueline C. , Kolitz Jonathan E. , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) is linked to clonal growth within pseudo-follicles, typically within lymph nodes, bone marrow and spleen, and occasionally lungs and skin. Both the clone’s antigen receptor and stromal milieu appear to influence its growth rate. An involvement of TLR signals seems probable based on atypically elevated TLR-9 expression within B-CLL cells and the likelihood that the specificity of B-CLL antigen receptors (BCR) facilitates the internalization of molecules from apoptotic cells and/or microbes that are physically linked to CpG DNA. Nevertheless, recent findings that a large subset of B-CLL undergoes in vitro apoptosis upon stimulation with CpG-rich oligodeoxynucleotides (ODN) raised questions about a central role for TLR-9 signaling. Using a CFSE-based model for examining in vitro B-CLL clonal expansion/viability and a cohort consisting of 19 IGHV mutated (M) and 19 unmutated (U) B-CLL, we report that TLR-9 signaling is uniformly stimulatory when accompanied by signals from IL-15. Importantly, this cytokine is known to be constitutively produced by stromal cells in normal bone marrow, lymph nodes, and spleen and in a constitutive/inducible manner within skin and lungs. We show that B-CLL display reproducible inter-clonal differences in the number of division cycles attained and/or lymphoblast survival that were not linked to IGHV mutation status, but were statistically linked to whether the patient leukemic population contained subclones with trisomy-12 (p=0.0003) or contained subclones with both an ATM anomaly (11q22 del and/or ATM mutation) and 13q14 del (p=0.009). When all B-CLL clones were assessed, in vitro high-division or high-viability status in response to ODN + IL-15 was not statistically linked to clinical progression as determined by time to first treatment (TFT). Nonetheless, in vitro high-division status showed a statistically-significant direct linkage to patient survival (OS) (p=0.019 for OS within B-CLL manifesting > 50% cells with > 2 divisions versus B-CLL with < 50% cells with > 2 divisions). Subdivision of the total cohort into U-CLL and M-CLL subsets revealed that the link of high division status with overall survival is most characteristic of U-CLL. Immunohistological evidence of IL-15-producing cells within or proximal to Ki-67-positive pseudo-follicles in B-CLL-infiltrated spleen is consistent with a role for ODN + IL-15 signaling in promoting in vivo leukemic cell growth. Taken together, the findings from this study support the concept that in vivo B-CLL clonal expansion is dependent upon leukemic B-CLL homing to tissue sites where IL-15 is typically sequestered along with intrinsic properties of the B-CLL clone, e.g. cytogenetic anomalies within members of a B-CLL clone that heighten leukemic cell growth and/or survival and an expression of U-BCRs specific for apoptotic cell debris that increase the opportunity for TLR-9 signaling.

Disclosures
No relevant conflicts of interest to declare.