Chronic Lymphocytic Leukemia Shares a Common Cellular Origin with Regulatory B10 Cells

The cell of origin of CLL is unknown. Researchers have proposed various B cell subsets as the normal counterparts based on surface marker similarities or Ig gene utilization comparisons of normal and CLL cells. Regulatory B lymphocytes (“B10” cells), with the capacity to produce IL-10, negatively regulate T cell, B cell, and mononuclear phagocyte function. CLL patients are immunosuppressed with abnormalities in both humoral and cellular immunity. B10 cells have a phenotype similar to CLL cells (CD24^hiCD27⁺CD5⁺CD19⁺). B10 cells are increased in autoimmune mice and in humans with autoimmune diseases—situations in which these cells negatively regulate immune-mediated inflammation. Since CLL cells and B10 cells may share common phenotypes and immunosuppressive mechanisms, we sought to determine if mouse and human CLL cells share common cellular origins and regulatory properties.

Mouse spleen, lymph node, and bone marrow cell, and human blood B lymphocyte and CLL cell preparation and culture; IL-10, TNF, IGHV determinations; and flow cytometry were done as we have reported before (Blood 109:1559, 2007; Blood 117:530–541, 2010; Immunity 28:639–650, 2008). After culture for 5 hours with LPS and PMA+ionomycin+brefeldin A (PIB), or CpG+PIB, we assessed for intracellular IL-10 by flow cytometry. We term these IL-10 producing cells “B10” cells. Alternately, cells were cultured 48 hours with CD40 ligand+LPS or CD40L+CpG, and then PIB was added during the last 5 hours, after which cells were assessed for intracellular IL-10. We term these IL-10 positive cells “B10+B10pro” cells.

We examined CLL cells from 54 CLL patients. Most had low-risk disease: 90% were either Rai stage 0 or stage 1, 89% were CD38 negative, 46% were Zap70 negative, and 70% had a mutated IGHV. Twenty percent had normal cytogenetics, 48% del13q, 20% trisomy 12, 4% del17p, 4% del11q, and 4% with complex abnormalites. Patients with CLL as compared to healthy controls had higher numbers of B10pro cells compared to those of normal controls (7.7±0.9% and 3.2±0.3%, respectively; p<0.0001). CLL cells had a CD24^hiCD27⁺ memory B cell phenotype similar to normal human B10 cells, and CLL cells secreted IL-10 when treated in vitro with CpG or CD40L/CpG, as do normal human B10 cells. CLL cell TCL-1 protein levels (immunoblot) correlated directly with CLL B10pro percentages (p=0.001) and absolute numbers (p=0.01). CLL patients' plasma IL-10 levels were 1.5 fold higher than those of age-matched healthy controls (p=0.008), and these levels correlated directly with the absolute numbers of CLL cells that were competent to produce IL-10 after 48 hours stimulation with CD40L/CpG.

To validate the precursor/product relationship between B10 cells and CLL, we studied the TCL-1 transgenic mouse model of CLL. TCL-1 transgenic mice had an age-dependent expansion of splenic CD5⁺B220^int cells, and these leukemic cells were IL-10-competent. Likewise, aged TCL-1 mice had increased numbers of B10 cells in the bone marrow, lymph nodes, and peritoneal cavity. The TCL-1 CLL cells were similar in phenotype (IgM⁺CD11B^hiCD23^lowCD43^hiCD19⁺) to mouse regulatory B10 cells (CD1d^hiIgM^hiIgD^lowCD19^hiCD23^lowCD24^hiCD43^±) that we have previously reported. TCL-1 CLL cells produced IL-10 in vitro and in vivo, and depressed mouse macrophage TNF production. This TCL-1 CLL cell-mediated inhibition of mouse TNF production was blocked by anti-IL-10 antibody. Plasma IL-10 increased with age and with development of overt leukemia in TCL-1 mice.

We demonstrate for the first time that human CLL cells and CLL-like cells from TCL-1 mice share a common origin with regulatory B10 and B10pro cells. Both CLL cells and B10 cells can produce the immuno-inhibitory cytokine IL-10 in vitro and in vivo, and they can suppress mononuclear phagocyte activation in vitro through IL-10-dependent pathways. The immunophenotype of CLL cells matches that of human B10, and B10pro cells. It is likely that IL-10 competent CLL cells derive directly from either regulatory B10pro or B10 cells. B10 cell-derived IL-10 may contribute to the immunosuppression noted in mice and humans with CLL. Future studies may lead to new and better treatments that take advantage of links between B10 cells, IL-10, and CLL.

Lanasa:GlaxoSmithKline: Consultancy, Speakers Bureau. Tedder:Angelica: Consultancy, Share holder; Takeda Therapeutics: Consultancy.