Krembil Research Institute, University Health Network, Toronto, Ontario, Canada.
Department of Medicine and Immunology, University of Toronto, Toronto, Ontario, Canada.
PLoS One. 2020 Jul 28;15(7):e0236664. doi: 10.1371/journal.pone.0236664. eCollection 2020.
Systemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population.
Flow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy.
c1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice.
Anergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production.
系统性红斑狼疮（SLE）是一种严重的自身免疫性疾病，其中免疫耐受缺陷导致致病性抗核自身抗体的产生。由于自身反应性和在 SLE 患者中的过度表达，无反应性 B 细胞被认为是这些自身抗体的潜在来源。狼疮易感小鼠的研究表明，介导自身免疫的遗传缺陷可以突破 B 细胞无反应性，但就内源性核抗原而言，这种突破是如何发生的尚不清楚。我们研究了源自狼疮易感新西兰黑色品系的同源小鼠（c1）中的 B 和 T 细胞缺陷是否可以在具有产生 ssDNA 反应性、无反应性 B 细胞群体的基因敲入基因（Vκ8/3H9；dKI）的情况下突破对核自身抗原的耐受。
使用流式细胞术评估 8 月龄 c1 dKI 小鼠脾脏中的 B 和 T 细胞，并通过 ELISA 测量血清自身抗体。通过检测 CFSE 衰减和 CD86，评估体外用抗 IgM 刺激的 dKI B 细胞的增殖和激活。通过用 PMA 和离子霉素培养 dKI 脾细胞，通过流式细胞术鉴定细胞因子产生的 T 细胞。将 6-8 周龄的 dKI B 细胞过继转移到 4 月龄野生型受体中，并在 7 天后通过流式细胞术和免疫荧光显微镜进行评估。
c1 dKI 小鼠表现出 B 细胞增殖，表明无反应性受损，但与野生型同窝小鼠相比，自身抗体和生发中心减弱。这种衰减似乎源于 dKI 株中 PD-1hi T 辅助细胞的减少，因为 c1 dKI B 细胞在过继转移到 c1 野生型小鼠时被招募到生发中心。
只有在支持高亲和力浆细胞产生的 T 细胞亚群同时存在缺陷的情况下，无反应性、DNA 特异性自身反应性 B 细胞似乎才会导致严重的自身免疫。
