1049 - Impact of T-cell co-stimulatory molecule CLECL1 expressing CLL cells on CLL disease progression and transformation to Richter's syndrome

Disease progression of chronic lymphocytic leukemia (CLL) is highly dependent on elements in secondary lymphoid tissues. In these niches, stromal cells produce chemokines to recruit T cells and CLL cells, and their crosstalk produces factors such as IL-4 that promote tumor survival and growth. CLECL1 (C-type lectin-like protein 1) is a T-cell costimulatory molecule that instructs T cells to produce IL-4 . We found CLECL1 expression on the surface of CLL cells. Importantly, CLECL1+ but not CLECL1- cells express AID, an enzyme that drives aberrant somatic hypermutation associated with Richter’s transformation. While the level of CLECL1 is associated with first time to treatment in CLL, patients with Richter’s and accelerated CLL have significantly higher levels of membrane CLECL1. Thus, here we investigated the role of CLECL1 in CLL and Richter’s transformation.

Bulk RNAseq analyses of CLL cells suggest that the expression of CLECL1 correlates with IL-4-related gene expression and associates with genes expressed in G2/M. Indeed, flow cytometry analyses confirm that CLECL1+ cells are mainly in the G2/M phase of the cell cycle, whereas CLECL1- cells are chiefly in G1/S or undergoing apoptosis. CLECL1+ CLL cells are more activated, expressing higher levels of cell interaction molecules and activation markers, consistent with CLECL1+ CLL cells being generated from CLECL1- cells. Indeed, in vitro stimulation of CLECL1- cells with anti-IgM+IL-4 converts CLECL1- to CLECL1+ cells. Similar results were also seen when CLECL1- cells are stimulated with soluble anti-CD3 antibody activated autologous T cells; however, T-cell induced CLECL1 expression is only seen in U-CLL but not in M-CLL patients, emphasizing a role of CLECL1 in more aggressive CLL.

Immunohistochemistry staining of patient lymph nodes shows co-localization of CLECL1+ B cells, Ki67-expressing cells, and T cells. Thus, we investigated the function of CLECL1 in T-cell growth and cytokine production. We first identified direct binding of recombinant CLECL1 (rCLECL1) protein to both naïve (Tn) and memory (Tmem) T cells in CLL patients. When Tn and Tmem cells were incubated with rCLECL1, both subsets responded with enhanced proliferation and IL-4 production. Similar results were seen when we cocultured CLECL1+ but not CLECL1- CLL B cells with unseparated T cells or each subset of T cells.

When the same experiment was carried out with samples taken from two CLL patients before and after transformation into Richter’s syndrome, we found significant better ability of CLECL1+ Richter’s patient cells to induce IL-4 production but not the proliferation of T cells, compared to their counterparts at the CLL stage. To mimic tissue niches, this experiment was repeated in the presence of lymph node stromal cells obtained from normal donors. Interestingly, both CLL and Richter’s cells attached to stromal cells and reduced CXCR4 expression, potentially in response to CXCL12 chemokines produced by lymphatic stromal cells. Importantly, we also observed enhanced CLECL1 expression in malignant B cells co-cultured with stromal cells; these B cells were also better at inducing IL-4 producing T cells.

Finally, to see the key function of CLECL1 in CLL B cells, we sorted CLECL1+ and CLECL1- populations from MEC-1, OSU and RAJI cells line, and cultured these cells separately for 3 days. We found significantly better growth of the CLECL1+ than CLECL1- fractions in all three cell lines, supporting the findings that CLECL1 levels is associated with the activation and survival of B cells. To further understand whether separating out CLECL1 expressing cells can achieve better clinical outcome in vivo, we used patient derived xenografts. Injection of NSG mice with autologous CD3+ T cells plus CLECL1- PF cells led to significantly reduced numbers of CLL B and T cells in the spleens of recipient mice. In this model, Th2 cells outgrew Th1 T cells, and removing CLECL1-expressing cells significantly reduced the number of Th2 T cells in the spleens. We are currently repeating this experiment in Richter’s patients.

Overall, this study suggests that CLECL1-expressing CLL cells are a key subset that drives CLL survival and proliferation and shapes a Th2-biased tumor microenvironment, although whether the CLECL1+ fraction is the source of Richter’s transformation remains elucidated. Overall, these results strongly suggest that CLECL1+ cells in the PF of CLL clones are potentially key targets for therapy.