B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells Unresponsive to CD180 Ligation Fail to Respond to Anti-IgM Stimulation as Well

We have demonstrated that CD180, an orphan receptor of the Toll-like receptor family, is expressed heterogeneously on B-CLL cells, mainly on those with mutated IGVH genes. We further showed that specific ligation of CD180 with mAbs induced activation and cycling of only ~50% CD180⁺ B-CLL clones (“R”: responders), while CD180⁺ B-CLL cells unresponsive to CD180 ligation (“NR”: non-responders) or CD180⁻ B-CLL cells could not be activated through either CD40 or IL-4 suggesting anergy. Because CD180 has a short intracellular domain, it presumably, signals through pathways associated with other receptors, such as smIgM. Indeed, engagement of smIgM or CD180 induces Lyn and Syk phosphorylation. Here we compare activation, cycling and phosphorylation of intracellular protein kinases in R and NR and CD180⁻ B-CLL clones and B lymphocytes from healthy subjects upon ligation of smIgM.

B-CLL cells were analyzed for smCD180 and smIgM, and sm CD180⁺IgM⁺ B-CLL clones were categorized as R and NR by responsiveness to CD180 ligation. Leukemic clones from 15 smCD180⁺IgM⁺R, 14 smCD180⁺IgM⁺NR, 12 smCD180⁻IgM⁺ untreated B-CLL patients and 14 healthy age-matched individuals were stimulated with goat F(ab’)₂ anti-human IgM pAbs for 72h, and stained with PE~anti-CD86 mAbs, or fixed, permeabilized and stained with PE~anti-Ki-67 to assess B-cell activation and cycling, respectively. In order to study early intracellular signalling events, cells were stimulated with the same antibodies for 20 min, fixed, permeabilized and stained with Alexa Fluor~rabbit/mouse antibodies to phospho-Akt, phospho-ERK, phospho-p38MAPK, and phospho-ZAP70/Syk. Unstimulated cells in medium were used as controls. Results were assessed by flow cytometry and analyzed with the Mann-Whitney U test and paired t-test where appropriate.

ligation of sIgM on smCD180⁺IgM⁺R B-CLL cells resulted in a significant increase in CD86⁺ cells (66.3±21.7% vs 18.7±12.0%, p=0.00004) and Ki-67+ cells (38.9±10.5% vs 11.1±5.9%, p=0.0001) compared to medium controls; this was not different from the increase in activation and cycling of normal B cells (not shown). In contrast, smCD180⁺IgM⁺NR B-CLL cells failed to significantly upregulate CD86 in response to anti-IgM pAbs (20.6±13.8% vs 17.6±13.7%, p=0.334) and Ki-67 (8.4±4.6% vs 5.3±1.4%, p=0.063). Interestingly, smCD180⁻IgM⁺ B-CLL cells demonstrated diminished CD86 upregulation following sIgM ligation: 36.9±21.7% vs 11.0±4.7% in medium, p=0.058 (difference with smCD180⁺IgM⁺R B-CLL, p=0.0069). Cell cycling was also decreased: 9.7±4.1% vs 5.4±3.6% in medium, p=0.015 (difference with smCD180⁺IgM⁺R, p=0.0022). The proximal stages of anti-smIgM responses were further studied by intracellular signalling of protein kinases associated with the IgM-signalling pathway. While ligation of sIgM on control B cells and smCD180⁺IgM⁺R B-CLL cells resulted in phosphorylation of all four enzymes studied, smCD180⁺IgM⁺NR cells failed to signal downstream from ZAP70/Syk following sIgM ligation (Table 1), although there was a greater heterogeneity in smCD180⁺IgM⁺R B-CLL responses, compared to normal B cells. Importantly, smIgM ligation of smCD180⁻IgM⁺ B-CLL cells did not increase phosphorylation of Erk or p38MAPK, although some such clones responded to smIgM ligation by phosphorylation of ZAP70/Syk and Akt (data not shown).

B-CLL clones that are smCD180⁺IgM⁺ but unresponsive to CD180 ligation (~30% of all B-CLL cases) are also unresponsive (anergic) to smIgM ligation measured by intracellular signalling, cell activation and cycling. Meanwhile, smCD180⁻IgM⁺ B-CLL clones respond heterogeneously to IgM crosslinking.

No relevant conflicts of interest to declare.