Zhou P, Ma X, Scalia S, Toskic D, Wu X, Fogaren T, Lyons Nancy Coady, Del Pozo-Yauner Luis, Comenzo R L
Tufts Medicine Myeloma and Amyloid Program, USA.
Division of Hematology-Oncology, Department of Medicine, Tufts Medical Center, Boston, MA, USA.
Biochem Biophys Rep. 2025 Apr 23;42:102017. doi: 10.1016/j.bbrep.2025.102017. eCollection 2025 Jun.
Light chain research is hampered by lack of mammalian cell lines producing human light chains (FLC). Therefore, we used heterohybridoma (HH) technology to produce clones making FLC thereby providing tools to study light chain behavior.
Marrow CD138+ cells from patients with multiple myeloma (MM) and polyclonal gammopathy (PG) were selected, fused with B5-6 T cells and cultured in hypoxanthine-aminopterin-thymidine medium (HAT). HH clones were selected based on ELISA for human immunoglobulins and flow cytometry for intracellular (IC) FLC. We compared marrow cell counts and HH yields by diagnosis, evaluated clones making only FLC by flow and by dimer/monomer (D/M) ratios in vitro and in vivo, and sequenced FLC genes with RT-PCR.
Marrows from 13 patients with active disease, 10 MM and 3 PG, were no different in mononuclear or CD138-selected cell counts. HH FLC clones (7 λ, 1 κ) were obtained from 5/10 MM and 2/3 PG; one PG case produced 2 HH FLC clones (one λ and one κ). Of the 10 MM cases, 8 had high risk cytogenetic features and 4 of the 8 produced HH clones while of the 3 PG cases 2 had negative cytogenetics and 1 had loss of IgH identified and produced an HH clone. Mononuclear (MNC) and CD138-selected cell numbers were markedly higher in the samples that enabled productive fusions. Median MFI for the 8 HH clones by IC flow for FLC was 9849 (range, 5344-27451) and median percentage of cells IC positive was 88 % (69-95). Medians of in vitro and in vivo FLC production were 47 μg/mL (9-80) per million cells after 2 days of culture and 66.4 μg/mL (16-1100) in NOD-SCID γ (NSG) mice 14 days after intraperitoneal (IP) implants of 2 × 10 HH cells. Dimer/monomer ratio medians were 0.575 (0.08-0.939) in vitro and 0.91 (0.82-2.7) in vivo, values that were correlated (R = 0.565) by two-tailed paired -test with < 0.05.
B5-6 T HH producing human FLC were obtained from 50 % of MM and PG cases. High numbers of MNC and CD138+ cells enabled productive fusions. The HH clones produced FLC with easily appreciated dimers and monomers in vitro and in vivo. With IP in vivo implants after 2 weeks more dimers were seen than in short term cultures in vitro. These HH clones will be made available for study of FLC metabolism and testing of therapeutics designed to abrogate FLC production or enable FLC clearance in vivo.
由于缺乏能够产生人轻链（FLC）的哺乳动物细胞系，轻链研究受到阻碍。因此，我们利用异种杂交瘤（HH）技术来产生能够制造FLC的克隆，从而提供研究轻链行为的工具。
选取多发性骨髓瘤（MM）和多克隆丙种球蛋白病（PG）患者的骨髓CD138+细胞，与B5-6 T细胞融合，并在次黄嘌呤-氨基蝶呤-胸腺嘧啶核苷培养基（HAT）中培养。基于人免疫球蛋白的酶联免疫吸附测定（ELISA）和细胞内（IC）FLC的流式细胞术筛选HH克隆。我们通过诊断比较骨髓细胞计数和HH产量，通过流式细胞术以及体外和体内的二聚体/单体（D/M）比率评估仅产生FLC的克隆，并采用逆转录聚合酶链反应（RT-PCR）对FLC基因进行测序。
来自13例活动性疾病患者（10例MM和3例PG）的骨髓，其单核细胞或经CD138筛选的细胞计数无差异。从5/10例MM和2/3例PG中获得了HH FLC克隆（7个λ型，1个κ型）；1例PG患者产生了2个HH FLC克隆（1个λ型和1个κ型）。在10例MM病例中，8例具有高危细胞遗传学特征，其中4例产生了HH克隆，而在3例PG病例中，2例细胞遗传学为阴性，1例检测到免疫球蛋白重链（IgH）缺失并产生了1个HH克隆。能够实现有效融合的样本中的单核细胞（MNC）和经CD138筛选的细胞数量明显更高。8个HH克隆通过IC流式细胞术检测FLC的中位平均荧光强度（MFI）为9849（范围为5344 - 27451），细胞IC阳性的中位百分比为88%（69 - 95）。培养2天后，每百万细胞体外FLC产量的中位数为47μg/mL（9 - 80），在腹腔内（IP）植入2×10个HH细胞14天后，NOD-SCIDγ（NSG）小鼠体内FLC产量的中位数为66.4μg/mL（16 - 1100）。体外D/M比率中位数为0.575（0.08 - 0.939），体内为0.91（0.82 - 2.7），通过双尾配对检验，二者相关（R = 0.565），P < 0.05。
从50%的MM和PG病例中获得了产生人FLC的B5-6 T HH。大量的MNC和CD138+细胞实现了有效融合。HH克隆在体外和体内产生的FLC具有易于识别的二聚体和单体。与体外短期培养相比，体内IP植入2周后可见更多二聚体。这些HH克隆将可用于研究FLC代谢以及测试旨在消除FLC产生或促进体内FLC清除的治疗方法。
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