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 (T_FR) 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 (T_FR) cells, suppressing B cells at different steps during the B-cell differentiation process. Sage and Sharp hypothesize that the T_FR 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.

浆细胞 (PCs) 来源于活化的 B 细胞，这些细胞在生发中心 (GCs) 中经历了克隆扩增、体细胞超突变 (SHM)、选择和类别转换重组 (CSR)。在 SHM 或 CSR 过程中，可能会发生起源于恶性 B 细胞的异常遗传事件。然后这些转化的 B 细胞回到骨髓 (BM) 并分化成克隆 PC，这些 PC 通过与 BM 微环境的相互作用被刺激增殖，从而产生单克隆丙种球蛋白病 (MGs)。MGs 包括一组疾病，例如意义未明的癌前单克隆丙种球蛋白病 (MGUS)、冒烟型多发性骨髓瘤的中期和有症状的多发性骨髓瘤，它们使 PC 呈现表型异常 (Tian et al.,  2021 ; Wang) &林, 2019 年）。

T 细胞在 B 细胞激活、选择和进一步分化为记忆 B 细胞或 PC，特别是具有滤泡表型的 T 细胞中起着至关重要的作用，这是 GC 维持和 GC B 细胞选择所必需的。最近，已经鉴定出具有滤泡和调节特性的 T 细胞亚群，称为滤泡调节性 T (T _{FR ) 细胞，它能够调节 GC 反应。}我们建议研究不同的 T 细胞群，包括 CD4 ⁺、CD8 ⁺、γδ ⁺、双阴性 (DN) αβ ⁺和双阳性 (DP) T 细胞及其在 BM 中具有滤泡样表型的亚群单克隆丙种球蛋白病。

在这项研究中，EDTA 抗凝 BM 样本来自 6 名 BM 正常个体（2 名女性和 4 名男性，平均年龄：60 ± 16）、14 名 MGUS 患者（7 名女性和 7 名男性，平均年龄：72 ± 11）和 27分析了新诊断的未经治疗的多发性骨髓瘤 (MM) 患者（7 名冒烟型 MM，2 名女性和 5 名男性，平均年龄：68 ± 19；20 名有症状的 MM，9 名女性和 11 名男性，平均年龄：77 ± 10）。为了排除 BM 抽吸物的血液稀释，我们评估了成红细胞和 PC 的频率（对照：11.80 ± 4.15、0.26 ± 0.10；MGUS：14.36 ± 8.08、0.83 ± 0.61；MM：分别为 12.44 ± 7.96、12.05 ± 13.99）。

执行染色-裂解-洗涤方案。为此，将 100 μl BM 用以下单克隆抗体染色：CD3-PerCP-Cy5.5（克隆 SK7，Becton Dickinson Biosciences [BD]，San Jose，USA）；CD4-PB（克隆 RPA-T4，BD Pharmingen，圣地亚哥，美国）；CD8-APC-H7（克隆 SK1、BD）；TCRγ/δ-PE-Cy7（克隆 11F2，BD）；CXCR5-APC（克隆 51,505，R&D Systems，美国明尼阿波利斯）；CD25-PE（克隆 2A3，BD）；CD127-BV510（克隆 HIL-7R-M21，BD）；HLA-DR-FITC（克隆 L243，BD）。

使用 FACSDiva 软件 (BD) 在 FACSCanto II 流式细胞仪 (BD) 中进行数据采集。对于数据分析，使用 Infinicyt™ 软件 V.1.7 (Cytognos SL, Salamanca, Spain)。

表 1 描述了不同患者组中不同 T 细胞亚群的分布。该研究表明，与对照组和 MGUS 患者相比，MM 组中 HLA-DR 激活的 CD4 ⁺ Treg 滤泡样细胞减少（图 1(a-ii)）。几项研究报告了 CD4 ⁺ Treg 滤泡 (T _FR ) 细胞的抑制功能，在 B 细胞分化过程的不同步骤中抑制 B 细胞。Sage 和 Sharp 假设对 PC 施加的 T _FR抑制可能会限制这些细胞产生抗体（Sage 等人，  2016）。滤泡调节性 T 细胞主要存在于人类血液和淋巴结中，据我们所知，我们的工作是首次在 BM 中鉴定出具有滤泡样表型的调节性 T 细胞。^{与之前的观察结果一致，γδ +}滤泡样 T 细胞（表 1 和图 1(b-ii)）和 HLA-DR 激活的 CD4 ⁺滤泡样 T 细胞的频率显着降低（图 1(bi)）与 MGUS 组相比，在 MM 组中观察到。MM 组的CD25 ⁺活化 DN αβ ⁺滤泡样 T 细胞也低于对照组和 MGUS 患者（图 1(b-iii)）。

此外，我们还研究了没有滤泡表型的 Treg 细胞。与对照组和 MGUS 患者相比，在 MM 组中发现HLA-DR 激活的 CD4 ⁺ Treg 细胞的频率明显更高，这可能导致免疫抑制状态。

总之，我们的研究结果表明，在多发性骨髓瘤患者中，CD4 ^{+ Treg 滤泡样细胞在调节骨髓瘤细胞产生抗体方面可能受损，并且由于 HLA-DR 激活的 CD4 +} Treg 细胞增加而导致免疫抑制状态。但是，必须考虑到我们的研究人群相对较小的事实。