Bone marrow follicular-like T cells in monoclonal gammopathies.

Abstract

Plasma cells (PCs) originate from activated B cells which underwent clonal expansion, somatic hypermutations (SHM), selection and class switch recombination (CSR) in the germinal centers (GCs). During the SHM or CSR processes, aberrant genetic events can occur which originate malignant B cells. These transformed B cells then home to the bone marrow (BM) and differentiate into clonal PCs which, through interactions with the BM microenvironment, are stimulated to proliferate, giving rise to monoclonal gammopathies (MGs). MGs comprise a group of disorders, such as the pre-malignant monoclonal gammopathy of undetermined significance (MGUS), the intermediate stage of smoldering multiple myeloma and the symptomatic multiple myeloma, which present PCs with phenotypic aberrancies (Tian et al., 2021; Wang & Lin, 2019). T cells play a crucial role in B-cell activation, selection and further differentiation into either memory B cells or PCs, particularly, T cells with follicular phenotype, which are required for GC maintenance and GC B-cell selection. Recently, a T cell subset with both follicular and regulatory properties, designated by follicular regulatory T (TFR) cells, has been identified, which is capable of regulating GC reactions. We propose to study different T cell populations, including CD4+, CD8+, γδ+, double-negative (DN) αβ+ and double-positive (DP) T cells and its subpopulations with follicular-like phenotype in the BM of patients with monoclonal gammopathies. In this study, EDTA-anticoagulated BM samples from six individuals with normal BM (two women and four men, average age: 60 ± 16), 14 patients with MGUS (seven women and seven men, average age: 72 ± 11) and 27 newly diagnosed untreated multiple myeloma (MM) patients (seven smoldering MM, two women, and five men, average age: 68 ± 19; 20 symptomatic MM, nine women and 11 men, average age: 77 ± 10) were analyzed. In order to exclude hemodilution of BM aspirates we evaluated the frequency of erythroblasts and PCs (controls: 11.80 ± 4.15, 0.26 ± 0.10; MGUS: 14.36 ± 8.08, 0.83 ± 0.61; MM: 12.44 ± 7.96, 12.05 ± 13.99, respectively). A stain-lyse-wash protocol was performed. For that, 100 μl of BM was stained with the following monoclonal antibodies: CD3-PerCP-Cy5.5 (clone SK7, Becton Dickinson Biosciences [BD], San Jose, USA); CD4-PB (clone RPA-T4, BD Pharmingen, San Diego, USA); CD8-APC-H7 (clone SK1, BD); TCRγ/δ-PE-Cy7 (clone 11F2, BD); CXCR5-APC (clone 51,505, R&D Systems, Minneapolis, USA); CD25-PE (clone 2A3, BD); CD127-BV510 (clone HIL-7R-M21, BD); HLA-DR-FITC (clone L243, BD). Data acquisition was performed in a FACSCanto II flow cytometer (BD) using the FACSDiva software (BD). For data analysis the Infinicyt™ software, V.1.7 (Cytognos SL, Salamanca, Spain) was used. Table 1 describes the distrubution of disticnt T cell subpopulations in the different patient groups. This study showed that HLA-DR activated CD4+ Treg follicular-like cells were decreased in the MM group in comparison to controls and MGUS patients (Figure 1(a-ii)). Several studies have reported a suppressive function for CD4+ Treg follicular (TFR) cells, suppressing B cells at different steps during the B-cell differentiation process. Sage and Sharp hypothesize that the TFR suppression exerted on PCs may restrict antibody production by these cells (Sage et al., 2016). T follicular regulatory cells have been mainly reported on blood and lymph nodes in humans and to our knowledge, our work is the first identifying regulatory T cells with a follicular-like phenotype in the BM. In line with the previous observation, a significant decrease in the frequency of γδ+ follicular-like (Table 1 and Figure 1(b-ii)) and HLA-DR activated CD4+ follicular-like T cells (Figure 1(b-i)) was observed in the MM group when compared with the MGUS group. CD25+ activated DN αβ+ follicular-like T cells were also lower in the MM group than in controls and MGUS patients (Figure 1(b-iii)). Additionally, we also studied Treg cells without follicular phenotype. HLA-DR activated CD4+ Treg cells were found at a significantly higher frequency in the MM group in comparison to controls and MGUS patients, which could contribute to an immunosuppressive state. In summary, our findings point to a possible impairment of CD4+ Treg follicular-like cells in regulating antibody production by myeloma cells and to an immunosuppressive state due to the increase in HLA-DR activated CD4+ Treg cells, in patients with multiple myeloma. However, the fact that our study population is relatively small must be taken into consideration.