CLECL1⁺ CLL Cells: A Key Subset That Drives CLL Survival and Proliferation and Shapes a Th2-Biased Tumor Microenvironment

Chronic lymphocytic leukemia (CLL) cells depend on elements in the tissue microenvironment (TME) that produce factors such as IL-4 to promote leukemic B cell survival and growth. CLECL1, a C-type lectin-like protein, is a T cell co-stimulatory molecule originally identified on dendritic cells, but also expressed on normal B and myeloid cells. Interactions of CLECL1 with naïve T cells (Tn) lead to Th2 cell development and production of IL-4. While IL-4 can have pro- and anti- tumor activities, a role for CLECL1 in tumorigenesis has not been tested. Here we studied the role of CLECL1 in CLL.

Flow cytometry analyses of CD19⁺CD5⁺ cells from 7 normal people and 77 patients with CLL revealed significantly higher CLECL1 levels on CLL cells (CLL vs Normal = 10.1% vs 3.9%, P = 0.01). CLECL1 was found most in the fraction of leukemic clones enriched in recently divided cells (PF; CXCR4^DimCD5^Bright) and least in the resting fraction (RF; CXCR4^BrightCD5^Dim) (PF vs RF = 20% vs 0.4%, P < 0.001). Importantly, high levels of CLECL1 in the PF correlated with shorter time to first treatment (P = 0.01). Notably, effective treatment with ibrutinib significantly reduced CLECL1⁺ cells (pre vs post treatment: 9.1% vs 1.8%, P = 0.01), and these levels increased when patients developed ibrutinib resistance (sensitive vs resistance = 1.8% vs 7.5%, P < 0.05).

Flow cytometry analyses also indicated that, compared to CLECL1^- cells, CLECL1⁺ CLL cells expressed higher levels of other cell interaction molecules such as CD40 and CD80, as well as activation markers, CD23, CD27, CD69, CD71 and CD72. Consistent with this, CLECL1⁺ PF cells were mainly in the G2/M phase of the cell cycle and express AID. In contrast, CLECL1^- PF cells were AID^- and chiefly in G1/S phases or undergoing apoptosis. Single cell RNAseq analyses of CLL PF versus RF cells corroborated that the expression of CLECL1 is [1] restricted to the PF, [2] associated with genes expressed in G2/M, and [3] correlated with IL-4-related gene expression. Together, these data suggest CLECL1⁺ CLL cells are activated cells generated from CLECL1^- cells. Indeed, stimulation of CLECL1^- cells in vitro revealed that anti-IgM+IL-4 converted CLECL1^- to CLECL1⁺ cells.

In CLL patient lymph nodes, CLECL1⁺ cells expressed Ki67 and co-localized with T cells. Thus, we questioned the abilities of CLECL1⁺ and CLECL1^- cells to interact with T cells and to affect T cell growth and cytokine production. Co-culture of unseparated autologous T cells with CLECL1⁺, but not CLECL1^- CLL cells significantly enhanced proliferation of T cells stimulated through T-cell receptors (CLECL1⁺ vs CLECL1^-: 12.9% vs 2.8%, P = 0.02) and induced increased production of IL-4 (CLECL1⁺ vs CLECL1^-: 15% vs 7%, P < 0.05). Contrary to published results that recombinant CLECL1 (rCLECL1) only binds to healthy Tn, we identified direct binding of rCLECL1 to both Tn and T memory (Tmem) cells in CLL patients. Moreover, when Tn and Tmem cells were incubated with rCLECL1, both T cell subsets responded with enhanced proliferation and IL-4 production.

Subsequently, we tried to recapitulate the above in vitro findings in vivo, using patient derived xenografts. Injection of NSG mice with autologous CD3⁺ T cells plus CLECL1⁺ or CLECL1^- PF cells led to significantly higher numbers of CLL B and T cells in the spleens of recipient mice (CLECL1⁺ vs CLECL1^- for B cells: 508,800 vs 37,000; P = 0.03; for T cells: 273,700 vs 47,000; P = 0.03). Additionally, Th2 cells outgrew Th1 T cells (CLECL1⁺ vs CLECL1^- for Th2 cells: 60.1% vs 12%; P < 0.05; for Th1 cells: 20.1% vs 1.8%; N.S). The same results were seen when we injected CLECL1⁺ or CLECL1^- CLL PF cells plus autologous Tn or Tmem cells into NSG mice, as only CLECL1⁺ PF cells stimulated Tn or Tmem cells to expand and produce IL-4. In turn, these CLECL1 stimulated T cells facilitated the growth of CLL cells in recipient spleen and bone marrow.

Overall, these ex vivo, in vitro, and in vivo results indicate that CLL cells that express CLECL1, specifically those in the recently-divided fraction, engage both Tn and Tmem cells to promote T cell growth and IL-4 production. Consequently, the CLECL1-shaped Th2 TME facilitates the survival and proliferation of tumor cells. Therefore, despite the relatively small numbers, CLECL1⁺ cells in the PF of CLL clones are potential key targets for therapy. Eliminating CLECL1⁺ CLL cells or blocking their promotion of IL-4 production could improve treatment, especially for those patients with CLL resistant to ibrutinib.

Rhodes:Genmab: Consultancy; Beigene: Consultancy; Morphosys: Consultancy; Janssen: Consultancy, Research Funding; SeaGen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Oncternal: Research Funding; Abbive: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Velosbios: Research Funding; Loxo Oncology: Research Funding; Epizyme: Research Funding.