ZAP-70 Expression in Non Tumoral B Cells: Role in B Tolerance Breakdown?

Abnormal expression of the tyrosine kinase ZAP-70 by tumoral B cells in chronic lymphocytic leukemia (CLL) is associated with bad prognosis, related to B cell receptor (BCR) hypersignalling, clonal expansion and autoimmune cytopenia (AIC) occurrence, these latter being mostly induced by polyclonal IgG from the residual non tumoral B cells. We previously shown that ZAP-70 is expressed by these non tumoral B cells in CLL, positively associated with its expression in CLL B cells and with AIC occurrence (Ghergus et al. Poster ASH 2017). Here, we show for the first time a potential role of ZAP-70 expression in tolerance breakdown in CLL and in an original knock in mouse model overexpressing ZAP-70 conditionally in B cells.

First, to assess a potential molecular link between ZAP-70+ CLL and non tumoral B cells, an analysis of their BCR repertoire has performed on FACS-sorting CD19+CD5-IgM-IgD- (non tumoral) and CD19+CD5+IgM-IgD- (tumoral) single B cells from blood samples of CLL patients with AIC. ZAP-70 positivity was screened by RT-PCR, and variable regions of heavy (IGVH) and light (IGVK/VL) immunoglobulin genes amplified by RT-PCR on ZAP-70+ cells. To date, analysis of 24 BCR sequences from 7 patients showed that non tumoral ZAP-70+ B cells were polyclonal, without stereotypy, using different V(D)J and CDR3 in comparison with those of the corresponding CLL B cells. IGVH of non tumoral ZAP-70+ B were mostly mutated, of replacement type, suggesting antigenic contact, contrary to CLL B cells.

To determine potential autoreactivity of the non-tumoral ZAP-70+ B cells, IGVH and corresponding IGVK/VL were amplified for production of recombinant antibodies (rAb). To date, among 17 rAB from 7 different patients, 2/13 (15.4%) have an antinuclear autoreactivity on HEp-2 cells and 4/17 (23.5%) were polyreactive on ELISA (DNA, lipopolysaccharide, insulin), compared respectively to 6% and 4,3% of control B cells (Wardemann et al., Science 2003). Production of 7 additional rAb and tests for anti-erythrocytes and anti-platelets reactivity are in process.

To study functional consequences of early ZAP-70 expression in B cells in vivo, we generated a knock in Zap-70⁺/Mb1-Cre⁺mouse model (KI ZAP), to induce conditional expression of ZAP-70 in the B cell compartment from the proB stage, with KI Zap-70⁺/Mb1-Cre^-mice as controls (CTRL). The ZAP-70 mRNAs levels in B cells from KI ZAP mice were on average 20 times higher than that in CTRL B cells.

Up to 20 months-old, KI ZAP mice did not develop signs of lymphoproliferation. KI ZAP mice had hypo-IgG since 16 weeks-old (p<0.001) together with hypo-IgM from 14 months-old (p<0.01). Immunophenotyping revealed a reduction in mature naive, mature switched as well as in germinal center B cells (p<0.001, p=0.002 and p<0.01 respectively) and a trend for plasma cells (p=0.07). Microarrays showed enrichment in circulating IgG and IgM autoantibodies against various antigens in KI ZAP mice. These mice had reduced apoptosis rates of proB (p<0.01), preB (p=0.02), and immatures B cells (p=0.03), together with enrichment in marginal zone (p=0.01), trend for transitional T2/T3, and reduction in B1a cells (p<0.01).

After immunization by ovalbumin + Freund's adjuvant, a reduced production of specific IgG and IgM was observed (p=0.01 and p=0.03 respectively) with a trend in decreased number of antibody-secreting cells (p=0.07). KI ZAP B cells shown increased spontaneous activation and proliferation levels holding after BCR stimulation (p<0.01), as well as an increased intracellular calcic flow (p<0.001). Preliminary data suggested a reduced SYK phosphorylation after BCR stimulation in KI ZAP B cells.

Our findings highlight for the first time that non tumoral B cells ZAP-70+ are distinct from CLL cells at cellular level, but probably enriched in autoreactive cells. Moreover, we shown that early ZAP-70 expression in normal B cells in vivois associated with autoimmune characteristics, together with partial block in B cells peripheral maturation, and a conversely early increased activation and proliferation status. ZAP-70 could interfere early with SYK leading to an altered BCR signaling responsible for defect in normal B maturation promoting emergence of autoreactive B cells. Mechanistic role of ZAP-70 in BCR signaling has to be further analyzed but our data open new opportunities involving ZAP-70 in the understanding of B cell development and physiopathology of tolerance breakdown.