DataSheet_3_A Novel Image Analysis Approach Reveals a Role for Complement Receptors 1 and 2 in Follicular Dendritic Cell Organization in Germinal Centers.pdf

Follicular dendritic cells (FDCs) are rare and enigmatic cells that mainly reside in germinal centers (GCs). They are capable of capturing immune complexes, via their Fc (FcRs) and complement receptors (CRs) and storing them for long periods in non-degradative vesicles. Presentation of ICs on FDCs to B cells is believed to drive affinity maturation. CR1 and CR2 are expressed on B cells and FDCs. Cr2 knock out (KO) mice, lacking both receptors, have impaired antibody and GC responses. Utilizing a novel ImageJ macro to analyze confocal fluorescence microscopy images of spleen sections, we here investigate how FDCs in wild type (WT) and Cr2 KO mice behave during the first two weeks after immunization with sheep red blood cells (SRBC). Mice were immunized with SRBC i.v. and spleen and serum samples harvested at various time points. As expected, antibody and GC responses in Cr2 KO mice were impaired in comparison to WT mice. Fewer FDCs were identified in Cr2 KO mice, and these exhibited differential localization and organization in comparison to WT mice. WT FDCs were primarily located within GCs at the light zone/dark zone border. FDCs from WT but not Cr2 KO mice were actively dispersed in GCs, i.e. tended to move away from each other, presumably to increase their surface area for B cell interaction. FDCs from Cr2 KO mice were more often found on follicles outside of the GCs and those within the GCs were closer to the periphery in comparison to WT FDCs. Expression of CR1 and CR2, FcγRIIB, and FcµR increased in FDCs from WT mice during the course of immunization. The results suggest that decreased ability to capture ICs by FDCs lacking CR1 and CR2 may not be the only explanation for the impaired GC and antibody responses in Cr2 KO mice. Poor FDC organization in GCs and failure to increase receptor expression after immunization may further contribute to the inefficient immune responses observed.

滤泡树突状细胞（Follicular dendritic cells, FDCs）是一类罕见且功能尚不明确的细胞，主要定植于生发中心（germinal centers, GCs）。它们可通过Fc受体（Fc receptors, FcRs）与补体受体（complement receptors, CRs）捕获免疫复合物，并将其储存在非降解性囊泡中长达较长时间。学界普遍认为，滤泡树突状细胞呈递免疫复合物给B细胞的过程可驱动B细胞的亲和力成熟。CR1与CR2在B细胞和滤泡树突状细胞表面均有表达。敲除Cr2基因（Cr2 knock out, Cr2 KO）的小鼠会缺失这两种受体，其抗体应答与生发中心反应均存在缺陷。本研究利用一款新型ImageJ宏脚本分析脾脏组织的共聚焦荧光显微镜图像，探究野生型（wild type, WT）与Cr2 KO小鼠的滤泡树突状细胞在经绵羊红细胞（sheep red blood cells, SRBC）免疫后的前两周内的动态变化。实验中，小鼠经静脉注射（intravenous, i.v.）绵羊红细胞免疫，并在不同时间点采集脾脏与血清样本。正如预期，与野生型小鼠相比，Cr2 KO小鼠的抗体应答与生发中心反应确实存在缺陷。Cr2 KO小鼠体内的滤泡树突状细胞数量更少，且相较于野生型小鼠，其定位与分布模式存在显著差异。野生型小鼠的滤泡树突状细胞主要定植于生发中心的明区-暗区交界区域。野生型小鼠的滤泡树突状细胞可在生发中心内主动分散，即细胞间彼此远离，推测此举可扩大其与B细胞相互作用的表面积；而Cr2 KO小鼠的滤泡树突状细胞则无此现象。Cr2 KO小鼠的滤泡树突状细胞更多分布于生发中心外的淋巴滤泡中，即便位于生发中心内，其位置也更靠近外周，与野生型小鼠的滤泡树突状细胞不同。免疫进程中，野生型小鼠滤泡树突状细胞表面的CR1、CR2、FcγRIIB以及FcµR的表达水平均有所升高。本研究结果提示，缺失CR1与CR2的滤泡树突状细胞捕获免疫复合物的能力下降，或许并非Cr2 KO小鼠生发中心与抗体应答缺陷的唯一原因。生发中心内滤泡树突状细胞的分布异常，以及免疫后受体表达水平未能上调，可能进一步加剧了所观察到的低效免疫应答。