A Percentage of Normal B Cells from CLL Patients Express ZAP-70, Which Is Associated with Autoimmunity and Could be Responsible for Arising of Malignant Clone

Introduction

Chronic lymphocytic leukemia (CLL) has the particularity of a dysregulated immune system that favors the development of autoimmunity (AI). The mechanisms that lead to AI in CLL are not clear, but the great majority of pathogenic autoantibodies are polyclonal IgG synthesized by non-malignant B cells (Kipps and Carson 1993). ZAP-70 is the most discriminative marker between CLL patients with or without AI (Zanotti et al. 2010). We hypothesized that in CLL ZAP-70 expression is not restricted to malignant B cells, but could rather be an earlier phenomenon, predisposing to malignancy and AI.

Methods

Expression of ZAP-70 in CLL, normal B and T cells was analyzed in 63 CLL patients and in 36 controls by flow cytometry and RT-qPCR. Then, single normal B cells were FACS-sorted, the mRNA was reverse-transcribed, and amplified for Ig heavy and light chain (IgH/L) as well as ZAP-70 genes. We cloned and expressed in vitro recombinant antibodies (rAb) from normal ZAP-70⁺ B cells and studied their reactivity in ELISA and on HEp-2 cells.

Results

We clearly demonstrated that a percentage of normal B cells from CLL patients express ZAP-70, as compared to normal B cells from controls, at the protein level as shown by flow cytometry and at mRNA level by RT-qPCR experiments, although the later was not statistically significant (Fig 1A). This was definitely proven by sequence analysis of ZAP-70 amplified from single normal B cells mRNA (Fig 1B). These cells could not represent a contamination of normal B cells with a contingent of the CLL clone because they express a high level of CD19 and surface IgL and a normal k/λ ratio. The polyclonality of these cells was further confirmed by the analysis of IgH/L sequences rearranged variable regions (V) genes amplified from ZAP-70⁺ normal single B cells.

ZAP-70⁺ normal B cells do not belong to a specific B-cell subset and do not express activation markers as compared with normal B cells from the same patient. Moreover, we observed a clear correlation between the expression levels of ZAP-70 in malignant, normal B and T cells in each CLL patient.

Importantly, we found a percentage of ZAP-70⁺ normal B cells in all of the AI-associated CLL cases in our cohort. To study the role of these cells in the pathogenesis of autoimmunity, they were single-cell sorted and the IgH/L V genes as well as ZAP-70 were amplified by RT-qPCR. After sequencing of PCR products we found that in each patient a proportion of normal B cells indeed actively transcribe ZAP-70. This is the first time that ZAP-70 mRNA can be detected in single normal B cells, at the same time as IgH/L V transcripts.

We cloned and expressed in vitro 13 rAb from normal ZAP-70⁺ IgD^- class switched B cells and we found that 4 of the rAb reacted with HEp-2 cells and 2 were polyreactive (Fig 1C and D). This was not different from the class-switched normal B cells described in the literature (Tiller et al. 2007; Schickel et al. 2017), but the reactivity of more rAb is ongoing. Also, we plan to test them in MAIPA and Coombs tests to see if they could be at the origin of AI in CLL patients.

We then compared IgH/L V genes sequenced from single cells between ZAP-70⁺ and ZAP-70^- normal B cells and CLL B cells from 7 patients with CLL and AI. CDR3 from clonal CLL cells tend to be longer than those from normal B cells in line with the hypothesis that clonal CLL cells originate from autoreactive B cells which have undergone receptor editing (Gacia-Munoz et al. 2012), and VH replacement (Zhang et al. 2004). This is the case for one autoreactive ZAP-70⁺ normal B cell (HIT7) that expresses the same light chain as CLL clone, but a different and shorter IgH CDR3. In addition, some cells share the same IgH but display different IgL, therefore suggesting receptor editing.

Conclusions

We describe a novel normal B-cell population that expresses ZAP-70. There is a good correlation in the level of ZAP-70 expression between normal B cells and CLL B cells. The advent of AI in CLL is associated with the expression of ZAP-70 by clonal CLL cells as well as by normal residual B cells as we found a significant percentage of ZAP-70⁺ normal B cells in all AI-associated CLL cases. The reactivity of rAb produced from these cells is currently under evaluation. The presence of ZAP-70 in the naïve B-cell subset suggests that this is an early process, which probably occurs before malignant transformation. Analyzis of Ig sequences suggests a clonal relationship between ZAP-70⁺ normal B cells and CLL cells.

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