DataSheet_1_Age-related changes in antigen-specific natural antibodies are influenced by sex.docx

IntroductionNatural antibody (NAb) derived from CD5+ B-1 cells maintains tissue homeostasis, controls inflammation, aids in establishing long-term protective responses against pathogens, and provides immediate protection from infection. CD5+ B-1 cell NAbs recognize evolutionarily fixed epitopes, such as phosphatidylcholine (PtC), found on bacteria and senescent red blood cells. Anti-PtC antibodies are essential in protection against bacterial sepsis. CD5+ B-1 cell-derived NAbs have a unique germline-like structure that lacks N-additions, a feature critical for providing protection against infection. Previously, we demonstrated the repertoire and germline status of PtC+CD5+ B-1 cell IgM obtained from male mice changes with age depending on the anatomical location of the B-1 cells. More recently, we demonstrated serum antibody from aged female mice maintains protection against pneumococcal infection, whereas serum antibody from male mice does not provide protection.
ResultsHere, we show that aged female mice have significantly more splenic PtC+CD5+ B-1 cells and more PtC specific serum IgM than aged male mice. Furthermore, we find both age and biological sex related repertoire differences when comparing B cell receptor (BCR) sequencing results of PtC+CD5+ B-1 cells. While BCR germline status of PtC+CD5+ B-1 cells from aged male and female mice is similar in the peritoneal cavity, it differs significantly in the spleen, where aged females retain germline configuration and aged males do not. Nucleic acid sensing toll-like receptors are critical in the maintenance of PtC+ B-1 cells; therefore, to begin to understand the mechanism of differences observed between the male and female PtC+CD5+ B-1 cell repertoire, we analyzed levels of cell-free nucleic acids and found increases in aged females.
ConclusionOur results suggest the antigenic milieu differs between aged males and females, leading to differential selection of antigen-specific B-1 cells over time. Further elucidation of how biological sex differences influence the maintenance of B-1 cells within the aging environment will be essential to understand sex and age-related disparities in the susceptibility to bacterial infection and will aid in the development of more effective vaccination and/or therapeutic strategies specific for males and females.

## 引言
源自CD5阳性B-1细胞的天然抗体（Natural antibody, NAb）可维持组织稳态、调控炎症反应、协助建立针对病原体的长期保护性免疫应答，并为机体提供即时的感染防护能力。CD5阳性B-1细胞来源的天然抗体能够识别进化保守的表位，例如存在于细菌与衰老红细胞表面的磷脂酰胆碱（phosphatidylcholine, PtC）。抗PtC抗体对于抵御细菌性脓毒症至关重要。CD5阳性B-1细胞衍生的天然抗体具有独特的胚系样结构，不带有N端插入序列，这一特征对于其发挥抗感染防护功能至关重要。此前我们的研究显示，从雄性小鼠体内分离得到的PtC阳性CD5阳性B-1细胞的IgM抗体库及其胚系状态，会随年龄增长发生变化，且变化情况取决于B-1细胞的解剖位置。近期我们的研究还发现，老年雌性小鼠的血清抗体仍可抵御肺炎链球菌感染，而老年雄性小鼠的血清抗体则丧失了这一防护能力。

## 结果
本研究结果显示，与老年雄性小鼠相比，老年雌性小鼠的脾脏中PtC阳性CD5阳性B-1细胞数量更多，其血清中PtC特异性IgM水平也更高。此外，通过对PtC阳性CD5阳性B-1细胞的B细胞受体（B cell receptor, BCR）测序结果进行分析，我们发现抗体库的差异同时与年龄和生理性别相关。尽管老年雌雄小鼠体内PtC阳性CD5阳性B-1细胞的BCR胚系状态在腹膜腔中并无显著差异，但在脾脏中二者却存在明显区别：老年雌性小鼠的B细胞仍保留胚系构型，而老年雄性小鼠则不再具备这一特征。感知核酸的Toll样受体（Toll-like receptor, TLR）对于维持PtC阳性B-1细胞的存活至关重要。因此，为了探究雌雄小鼠PtC阳性CD5阳性B-1细胞抗体库差异的潜在机制，我们对游离核酸水平进行了检测，结果发现老年雌性小鼠体内的游离核酸水平有所升高。

## 结论
本研究结果表明，老年雌雄小鼠的抗原微环境存在差异，这会随时间推移对抗原特异性B-1细胞产生差异化的选择压力。进一步阐明生理性别差异如何在衰老环境中影响B-1细胞的维持，对于理解细菌性感染易感性的性别与年龄相关差异至关重要，同时也将有助于开发针对不同性别、更为高效的疫苗接种与治疗策略。