Monoclonal B lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL) are both characterized by the accumulation of clonal B lymphocytes with a mature phenotype and an abnormal BCR signaling and function in peripheral blood, bone marrow or secondary lymphoid tissues (1, 2). CLL-like MBL shares immunophenotypic, biological, and molecular features with CLL, its distinction from CLL being mostly based on the monoclonal B lymphocyte count (< 5000 cells/μL) in the absence of signs or symptoms of a B cell lymphoproliferative disorder (1, 3, 4). Based on the clonal B cell count, MBL can be further categorized as low-count (LC) (<500 cells/μL) or high-count (HC) MBL (>500 cells/μL). HC-MBL is thought to be a pre-malignant state that precedes CLL and it is associated with an annual rate of progression to CLL of 1-2% (3–6). Up to now, a few tumor cell features have been associated with progression from MBL to CLL, including an increase in total lymphocyte count in blood and an unmutated somatic hypermutation (SHM) status of the immunoglobulin heavy chain variable region (IGHV) gene (3, 6–9).

For decades now, it is well known that the expansion of malignant B cells is accompanied by numerical and phenotypic changes in T cells, including clonal T cell expansions, with preferential usage of restricted TCR Vβ gene families, a higher frequency of regulatory T cells (Tregs), reduced naïve CD4+ T cell counts and an enrichment in memory CD4+ T cells, together with a higher frequency of follicular, Th1, Th17, and CD8+ T cells. In addition, functional exhaustion of CD8+ T cells has been claimed to be responsible for an impaired cytotoxic response against tumor cells, which might favor the growth and expansion of malignant B cells (3, 10–14).

Recently, a new subpopulation of T cells, characterized by the expression of CD20 (a membrane protein expressed by mature B cells) has been identified (15). The origin of CD20+ T cells remains uncertain, trogocytosis being identified as one of the potential mechanisms for the acquisition of this B-cell associated receptor by T cells, though the presence of CD20 mRNA in these cells contradicts this hypothesis (15–18). From the functional point of view, CD20+ T cells have been reported to be involved in the pathophysiology of autoimmune diseases, particularly in multiple sclerosis, as well as in cancer, where numerical, phenotypic and functional alterations of these cells have been detected (18–24). Studies in autoimmune diseases suggest a pathogenic behavior of CD20+ T cells by producing IL-17 and TNF-α (17, 20, 22, 25). In contrast, other studies suggest a protective role for these cells in cancer. Independently of their specific pathogenic role, CD20+ T cells exhibit a predominantly Tc1 effector memory phenotype, suggesting an antitumor activity (15, 18, 24, 25).

This study aims to contribute to a better understanding of the role of CD20+ T cells, in LC-MBL, HC-MBL and CLL, through their enumeration and both phenotypic and functional characterization in these conditions, compared to a group of age-matched healthy subjects.

Cooperation between B cells and T cells is crucial for a robust and efficient immune response, as these cells are the cornerstone of adaptive immunity and, together with NK cells, critical cells for immune surveillance. At present, it is well-established that B cell neoplasms are characterized by a prominent and broad dysregulation of the immune response. Indeed, evidences have been described in CLL and CLL-like MBL that confirm a marked disruption of the normal immune homeostasis due to the interaction between pathological B cells (CD5+) and T cells (3, 11, 28).

In recent years, CD20+ T cells have been described in the literature to be involved in the pathophysiology of autoimmune diseases and cancer, where numerical, phenotypic, and functional alterations have been detected in this subset of T cells (18–24), fully in line with our findings in this study for CD20+ T cells in subjects presenting LC-MBL, HC-MBL, and CLL patients.

Despite the origin of CD20+ T cells is poorly understood, several studies have described trogocytosis as the process responsible for the transfer of the CD20 molecule from B cells to T cells as a result of the close interaction between both groups of cells. Based on this hypothesis, trogocytosis would possibly occur when B cells activate T cells during the immune response (22, 29). Here, we found decreased numbers of CD20+ T cells in peripheral blood of LC-MBL and CLL patients, even when the percentage of CD20+ T cells in blood was calculated after excluding the neoplastic B cells. These findings suggest that trogocytosis might not explain the decrease in the frequency of CD20+ T cells in MBL and CLL, when compared to non-MBL healthy individuals, as in both conditions an accumulation of B cells exists, which could facilitate the contact and interaction with T cells. However, neoplastic B cells from MBL and CLL display low expression of CD20, which corresponds to a lower amount of CD20 protein per cell (30, 31), and the accumulation of neoplastic B cells in these conditions may hamper the interaction between T cells and normal B cells in CLL patients and, to a lesser extent, in MBL.

Among all T cell subsets, CD20+ T cells were more represented in the CD8+ T cell compartment in our cohort, in line with previous studies (15, 17, 18), in the absence of significant differences between the studied and control groups. However, when we investigated the different functional compartments of CD4+ CD20+ and CD8+ CD20+ T cells, the majority of them presented a memory (central and effector) Th1 or Tc1 phenotype (15, 17, 18, 32). Cytotoxic CD8+ T cells play an important role in antitumor immunity since, together with NK cells, they are the main effector cells responsible for eliminating neoplastic cells (33). Furthermore, the predominantly memory phenotype suggests that the majority of CD20+ T cells are antigen experienced cells (15, 34).

An overall decrease in naïve CD4+ T cells, accompanied by an increase in the memory cell compartments, had been reported in CLL compared to healthy individuals (3, 12, 35). Overall, we could not confirm these alterations within the CD4+ CD20+ T cell compartment, as no differences between CLL and healthy controls were observed, despite an increase in CD4+ CD20+ central memory cells accompanied by a decrease in effector memory cells was found in CLL vs HC-MBL. However, an increase in CD4+ CD20+ TFH cells compared to healthy individuals was observed in patients with LC-MBL, HC-MBL and CLL. Of note, these cells mainly displayed a Th1-like TFH phenotype. These results are consistent, at least in part, with previous studies which described an increased frequency of TFH cells in CLL (36, 37), but contradicted the data reported by Wu et al., 2021 (38), who did not observe an increase in TFH cells in MBL. Overall, these results also suggest that the expansion of CD20+ TFH cells parallels the expansion of the B-CLL clone, which would indirectly support the notion that TFH cells might provide survival and proliferation signals to pathological B cells in both MBL and CLL (38, 39). In our study, an expansion of CD4+ CD20+ Th1 cells was also observed in the blood of patients with HC-MBL. Th1 cells have been shown to mediate the immune response against tumor cells through secretion of IFN-γ, and in CLL, IFN-γ is associated with the inhibition of apoptosis of B-CLL clones (40, 41). An increased percentage of activated (CD25+) CD20+ T cells, predominantly within the CD4+ T cell compartment, has been described by Roessner & Seiffert, 2020 (10) in CLL; such activated T cells in patients with CLL contribute to the proliferation and survival of B-CLL cell clones (10, 42). Here, we detected an increased percentage of activated (CD25+) CD4+ CD20+ T cells from LC-MBL patients, but not in HC-MBL or CLL.

When we analyzed the distribution of CD8+ CD20+ T cells according to the maturation-associated T cell compartments of naïve, central memory, effector memory, and terminal effector cells in the control group, we found most cells to be memory cells. As for other functional T cell subsets, CD8+ CD20+ T cells also exhibited here a predominantly Tc1 phenotype (18, 32). However, phenotypic analysis of CD8+ CD20+ T cells revealed an increased percentage of CXCR5+ cells in CLL compared to the control group, and an expansion of CD8+ CD20+ Tc1 cells that was consistent across all patient groups. The cytotoxic function of CD8+ T cells, particularly of Tc1 cells, has been associated with a key role in mediating T cell antitumoral responses (18, 24, 43, 44).

Several alterations of different T cell compartments have been described as relevant factors in the dysregulation of the immune response in the tumor microenvironment in CLL, resulting in an impaired antitumor immunity (3, 12). In CLL, this has been associated with a restricted usage in the T cell receptor (TCR) gene repertoire (3, 13, 45). Here, we further characterized the TCR-Vβ gene family usage of CD4+ CD20- T and CD8+ CD20- T cells compared to their CD4+ CD20+ T and CD8+ CD20+ T cell counterparts, respectively. Of note, among CD4+ CD20+ T cells from CLL patients, a relative expansion of T cells expressing the TCR Vβ 5.1 gene family was observed in all CLL patients analyzed. Interestingly, bystander expansions of TCR Vβ 5.1 have been reported in the literature for CLL, but its meaning remains to be understood (13, 45, 46).

Altogether, the above findings suggest that in CLL and its precursor states of MBL, the expansion of neoplastic B cells is accompanied by phenotypic and functional changes in the T cell compartment of CD20+ T cells. The abnormal distribution of T cell subpopulations, and in particular the decreased number of CD20+ T cells with a cytotoxic phenotype, might contribute to the dysregulation of the immune system in MBL and CLL, potentially reflecting a decreased antitumor activity and the inability of T cells to complete an effective immune response against neoplastic B cells which may acquire additional genetic abnormalities (47–51). Determination of the mutational status of the IGHV gene revealed that about half of all CLL cases displayed mutated IGHV gene, at a (slightly) lower percentage than found in HC-MBL, although these differences did not reach statistical significance, in line with previous studies (5, 7, 52). Analysis of the specific IGHV gene usage by purified neoplastic B cells showed that HC-MBL shares the IGHV3-23, IGHV1-69, and IGHV4-34 gene repertoire with CLL, although a greater prevalence of the IGHV4-34 and IGHV3-23 gene in HC-MBL was observed, particularly among the mutated clones, in line with previous studies (7, 9, 53, 54). In contrast, IGHV1-69 gene, that was typical in CLL, has been associated with an unmutated profile and adverse features in CLL (3, 5, 7, 49, 52). Of note, greater CD20+ T cells numbers were found in this study, associated with mutated IGHV gene, which suggests a protective role of CD20+ T cells for both in MBL and CLL. Further studies, in larger cohorts of MBL and CLL patients, are needed in order to determine whether CD20+ T cells are in fact associated with a greater risk of progression of MBL to CLL.

In summary, here we describe a decreased in CD20+ T cells in blood of both MBL and CLL patients consistent with a predominantly Tc1 memory cell phenotype, particularly among IGHV unmutated cases. These results suggest that CD20+ T cells might have a pathogenic role in MBL and CLL as tumor-suppressive T cell population, which decreases on frequency in parallel to the expansion of the neoplastic B cells. Further studies in larger cohorts of patients with long follow-up are needed to clarify whether CD20+ T cells might be in fact associated with the risk of progression from MBL to CLL.

The data presented in the study are deposited in the https://www.ebi.ac.uk/eva/?eva-study=PRJEB73349, accession number PRJEB73349.

The studies involving humans were approved by Ethics Committee of Faculty of Medicine, University of Coimbra. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

CR: Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. PL: Formal analysis, Validation, Visualization, Writing – review & editing. APi: Validation, Writing – review & editing. IS: Investigation, Writing – review & editing. SS: Investigation, Writing – review & editing. MC: Investigation, Methodology, Writing – review & editing. ACO: Investigation, Writing – review & editing. AS: Investigation, Writing – review & editing. ID: Investigation, Resources, Writing – review & editing. HS: Resources, Writing – review & editing. MU: Investigation, Resources, Writing – review & editing. AS: Resources, Writing – review & editing. CG: Resources, Writing – review & editing. MD: Supervision, Writing – review & editing. JA: Writing – review & editing. IC: Writing – review & editing. AO: Visualization, Writing – review & editing. APa: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.