Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12,Journal of Clinical Immunology

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Inherited CD19 Deficiency Does Not Impair Plasma Cell Formation or Response to CXCL12
Journal of Clinical Immunology ( IF 7.2 ) Pub Date : 2023-05-29 , DOI: 10.1007/s10875-023-01511-w
Kieran Walker ₁ , Anoop Mistry ₂ , Christopher M Watson _{1,

3} , Fatima Nadat ₂ , Eleanor O'Callaghan ₁ , Matthew Care ₁ , Laura A Crinnion _{1,

3} , Gururaj Arumugakani ₂ , David T Bonthron _{1,

4} , Clive Carter ₂ , Gina M Doody ₁ , Sinisa Savic _{2,

5}

Affiliation

Background

The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear.

Objective

Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.

Methods

Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells.

Results

Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation.

Conclusion

CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

中文翻译：

遗传性 CD19 缺陷不会损害浆细胞形成或对 CXCL12 的反应

背景

人 CD19 抗原在整个 B 细胞个体发育过程中表达，但肿瘤性浆细胞和正常浆细胞的子集除外。 CD19 在传播来自 B 细胞受体和其他受体（例如成熟 B 细胞中的 CXCR4）的信号方面发挥着作用。对 CD19 缺陷患者的研究证实了其在 B 细胞激活和记忆 B 细胞生成的初始阶段的功能；然而，其在 B 细胞分化后期的作用尚不清楚。

客观的

使用来自新鉴定的 CD19 缺陷个体的 B 细胞，我们使用体外分化模型研究了 CD19 在浆细胞的生成和功能中的作用。

方法

使用流式细胞术和使用位点特异性长程扩增产物的长读长纳米孔测序来筛查疑似原发性免疫缺陷的患者。使用 CD40L、IL-21、IL-2 和抗 Ig 激活来自患者和健康对照的纯化 B 细胞，然后转移至不同的细胞因子条件以诱导浆细胞分化。随后，用 CXCL12 刺激细胞，以通过 CXCR4 诱导信号传导。通过蛋白质印迹评估包括 ERK 和 AKT 在内的关键下游蛋白的磷酸化。 RNA-seq 也在体外分化细胞上进行。

结果

长读长纳米孔测序鉴定出纯合致病性突变 c.622del (p.Ser208Profs*19)，该突变通过缺乏 CD19 细胞表面染色得到证实。主要是幼稚的 CD19 缺陷 B 细胞产生表型正常的浆细胞，具有预期的分化相关基因模式和正常水平的 CXCR4。分化的 CD19 缺陷细胞能够对 CXCL12 做出反应；然而，与总 B 细胞产生的浆细胞相比，源自幼稚 B 细胞的浆细胞（无论 CD19 缺陷还是充足）的信号传导相对减弱。此外，正常浆细胞上的 CD19 连接会导致 AKT 磷酸化。