陈立功/刘万里合作发文揭示MCT1转运蛋白在IgG抗体类别转换中功能

点评｜孟飞龙（中国科学院分子细胞科学卓越创新中心研究员）、Tiffany Horng（上海科技大学生命科学与技术学院教授）

生物体持续受到外界环境的刺激，为了应对这些刺激而产生的免疫反应需要消耗机体大量的能量。生发中心（Germinal Center，GC）是一个在周围淋巴器官中形成的动态结构，它为B细胞的高频突变和抗体类别转换提供了一个独特的微环境。生发中心的正常形成对于高质量IgG抗体的产生至关重要。B细胞在受到免疫刺激激活后，会经历一个广泛的代谢重塑过程。乳酸既是糖酵解的产物，又可以作为能源为细胞提供能量，而在大部分细胞中乳酸主要是通过乳酸转运蛋白MCT1进行运输。因此，阐述MCT1在IgG抗体类别转换中的功能至关重要。

2024年1月2日，清华大学药学院陈立功课题组及生命学院刘万里课题组合作以MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation为题在Nature Communications杂志在线发表研究论文，该研究报道了单羧酸转运蛋白MCT1通过丙酮酸代谢调控B淋巴细胞（B细胞）免疫活化，免疫分化和IgG型抗体的产生；发现系统性红斑狼疮患者的MCT1表达水平明显上调，且其表达缺陷或特异性抑制可以有效缓解小鼠系统性红斑狼疮的症状，揭示MCT1可能成为治疗自身免疫性疾病系统性红斑狼疮的潜在靶点。

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为了探索MCT1转运蛋白在B细胞中的功能，在该研究中，作者通过CRISPR-Cas9技术构建了B细胞中特异性敲除MCT1小鼠，系统性研究了MCT1在B细胞中的作用和潜在机制。研究发现在抗原免疫刺激条件下，MCT1缺失小鼠血清中的抗原特异性的IgG1抗体含量显著下降。进一步分析发现，抗体产生受阻主要是由于生发中心B细胞抗体类别转换（Class switch recombination,CSR）严重受损导致。

为了研究乳酸和丙酮酸在MCT1缺陷B细胞中的去向，作者采用了葡萄糖同位素示踪（U-13C6葡萄糖）来研究进行CSR的B细胞中的葡萄糖代谢动态。示踪实验表明，MCT1缺失的B细胞发生代谢重塑，糖酵解代谢下降，氧化磷酸化代谢显著上调。

另一方面，通过RNA-seq分析发现，调控B细胞抗体类别转换的重要蛋白，激活诱导胞嘧啶脱氨酶（Activation-induced cytidine deaminase,AID）的表达显著下调，同时作者发现脂溶性的甲基丙酮酸可以有效恢复MCT1缺失B细胞中AID的表达，上调表达IgG1-BCR B细胞的比例。通过ATAC-seq和13C3丙酮酸对组蛋白乙酰化位点标记的定量蛋白组学分析发现，丙酮酸可以调控组蛋白H3K27乙酰化修饰。同时结合B细胞的Chip-seq分析发现，H3K27乙酰化修饰可以直接调控AID的表达。

在此基础上，作者也探索了此发现的临床意义。系统性红斑狼疮（SLE）是一种自身免疫性疾病，其特征是自身耐受性缺失，导致过度活化的B细胞异常扩增。作者发现系统性红斑狼疮患者的MCT1表达水平明显上调。也在bm12诱导系统性红斑狼疮小鼠模型中，发现敲除MCT1可以有效缓解SLE小鼠的症状，降低抗dsDNA抗体的产生以及IgG抗体在肾小球的聚集。

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综上所述，该研究发现MCT1缺失引起B细胞发生代谢重塑，消耗丙酮酸进行氧化磷酸化代谢，导致组蛋白H3K27乙酰化修饰降低，进而抑制AID蛋白的表达，最终造成B细胞抗体类别转换受损，减少抗体的分泌。在该研究中，甲基丙酮酸在体外实验中可以有效恢复MCT1缺失B细胞中的抗体类别转换过程，这为未来增强疫苗效果提供了新的研究思路，比如丙酮酸是否可以作为佐剂的有效成分，值得我们进一步思考和探索。更加令人感兴趣的是，MCT1转运蛋白抑制剂也被证明可以缓解系统性红斑狼疮小鼠炎症反应的症状，因此MCT1转运蛋白在B细胞中的功能研究也为系统性红斑狼疮治疗提供了新的治疗策略。

清华大学药学院陈立功研究员和清华大学生命学院刘万里教授为本文通讯作者，清华大学药学院已毕业博士生池文娜、生命学院博士后康娜、药学院博士生盛琳琳以及生命学院已毕业硕士生刘思辰为该论文共同第一作者。

专家点评

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孟飞龙（中国科学院分子细胞科学卓越创新中心研究员）

抗体类型转换是适应性免疫应答中重要生物学过程。在受到抗原刺激后，激活态的B细胞能够特异表达胞苷脱氨酶AID，在抗体重链基因区产生程序性DNA损伤，引起体细胞基因组DNA的重组，从而实现抗体从IgM到IgG等类型的转换。这一转换的过程在体内受到各个层次的精细调控，从而避免免疫失调的发生。陈立功与刘万里研究团队的合作成果，惊喜地向我们展示了细胞内糖代谢过程调控抗体类型转换的一面。

作者从MCT1小鼠模型入手，发现激活态B细胞中抗体类型转换受到了严重影响。从分子、细胞、个体等多个角度，这篇文章证明这种缺陷并不是间接的影响。MCT1缺陷的小鼠B细胞中，抗体类型转换失调与糖代谢紊乱直接相关。进一步，糖代谢如何影响细胞核内DNA水平上的生物学事件成为解决这一谜题的关键。我相信研究者在这一步做了大量的尝试和摸索的工作，发现糖代谢紊乱的B细胞中，AID编码基因Aicda的表达受到了影响。最后，通过分析数据库和临床队列，作者发现在SLE患者的B细胞中，MCT1表达水平较高。与健康对照组相比，SLE患者的IgG类转换记忆B细胞和活化的初始B细胞中MCT1的表达显著上调。进一步作者通过构建SLE小鼠模型证实了MCT1在SLE发病机制和治疗中的作用。这些研究结果表明，MCT1可能在SLE的治疗中具有重要的潜力。

Aicda基因上具有丰富的表达调控元件。早期来自于Tasuku Honjo实验室的研究曾发现调控Aicda基因的5个增强子；近年，Angela Rao实验室也发现了TET蛋白对于Aicda基因表达的表观调控机制。本篇文章从组蛋白乙酰基化入手，发现了一条“糖代谢-组蛋白乙酰基化-免疫关键基因表达-动物免疫表型”的通路。这一工作从分子机制、表型分析、免疫功能测定等方面全面地解析了这一全新的过程，为我们理解抗体类型转换这一经典生物学过程开启了一个新的角度。同时，这一工作也为这一的领域工作提出了新的启示：糖代谢失调是否通过类似机制引起病人的免疫失调？糖代谢失调是否会引起某种形式的原发性免疫缺陷？未来，我们非常期待通过代谢干预实现免疫功能的调控应用。

专家点评

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Tiffany Horng（上海科技大学生命科学与技术学院教授）

Antibody memory is the basis for nearly all successful vaccines. B lymphocytes are responsible for the protective antibody responses arising from the recognition of the invaded pathological antigens by the surface expressed B cell receptor (BCR). Upon the first encounter with an antigen, the IgM-BCR expressing naïve B cells generate low-titered primary antibody responses. Instead, memory B cells that express class-switched IgG-BCR are mainly responsible for memory antibody response, leading to high-titered IgG antibody responses shortly upon antigen recall. This is fundamentally different from the strategy that has been used by all other immune cells including T cells as there is no such a class-switch recombination (CSR) event. However, there is an unmet basic need to investigate the metabolic basis of CSR in B cells. A recent study published in Nature Communications through a joint effort by Chen Lab (metabolism biology) and Liu Lab (B cell biology) from Tsinghua University reveals that the monocarboxylate carrier MCT1 promotes pyruvate oxidation and histone acetylation to support CSR, the process by which B cells make antibodies of different isotypes with unique effector functions.

Upon immune activation, B cells must shift rapidly from a metabolically quiescent state to a high energy-consumption state. This metabolic shift is absolutely required for B cells to rapidly undergo clonal expansion, and differentiate into germinal center B cells (GCBCs) and plasma cells that produce high levels of antibodies. Despite the rapid rise of the immunometabolism field in recent years, how cellular metabolism supports the functions of activated B cells, including CSR and the production of high affinity IgG antibodies, has significantly lagged behind that of other activated immune cells like T cells and macrophages. MCTs transport monocarboxylates (including lactate and pyruvate), and have been shown to be important in supporting aerobic glycolysis in rapidly proliferating cells like tumor cells and activated CD8 T cells. In this paper, the authors started their study by comparing expression of various MCTs in activated B cells, and found that MCT1 was expressed at the highest level. Using newly generated mice in which MCT1 is specifically deleted in the B cell lineage, the authors found that B cell development in the bone marrow, and B cell homeostasis in the periphery, were unimpaired. However, upon challenge with a T cell-dependent model antigen, the authors found that MCT1-deficient B cells mounted a normal antigen specific IgM response, but produced antigen-specific IgG that was of lower quantity and affinity. GCBC differentiation and proliferation were also reduced in the absence of MCT1. Together these findings indicated a defect of CSR in MCT1-deficient B cells.

The MCT1 substrates lactate and pyruvate are intermediates in aerobic glycolysis, prompting the authors to examine glucose metabolism. Metabolic analyses indicated that activated MCT1-deficient B cells downregulated aerobic glycolysis, but upregulated pyruvate oxidation and oxidative metabolism, relative to WT counterparts. Presumably the absence of MCT1 forced a shunting of pyruvate towards oxidation in the TCA cycle. By tracing glucose and pyruvate into glycolysis, TCA cycle, and ultimately into histones, the authors nicely showed that in WT activated B cells, pyruvate acted as a metabolic substrate for histone acetylation to support a global increase in histone acetylation. Such an increase in histone acetylation drove enhanced expression of Aicda, the gene encoding activation-induced cytidine deaminase (AID), which mediates CSR and affinity maturation. Of note, the precise mechanism of pyruvate redistribution between the nuclear and non-nuclear compartments would be important for the epigenetic modification and energy production. Intriguingly, relative to WT counterparts, activated MCT1-deficient B cells had reduced histone acetylation at the Aicda promoter, despite enhanced pyruvate oxidation. Although the mechanistic basis remained unclear, the findings nevertheless implicated dysregulated pyruvate metabolism and histone acetylation at Aicda as the basis of attenuated CSR in MCT1-deficient B cells.

Finally, the study addressed disease relevance of their basic findings in genetically modified murine model. Although B cells are protective against pathogens, they contribute to disease pathogenesis in autoimmune diseases like SLE, where autoimmune antibodies are known to be positively correlated to the clinical manifestation of SLE. First, by analyzing both existing and newly-established RNA-seq datasets from multi-sources, the authors found that MCT1 levels were increased in switched memory B cells from SLE patients, relative to healthy controls. Next, the authors showed that mice with MCT1-deficient B cells were protected in a mouse model of SLE, displaying reduced splenomegaly, numbers of GCBCs and IgG1+ B cells, and titers of autoreactive, anti-dsDNA antibodies. Finally, administration of a MCT1 inhibitor in this model reduced titers of anti-dsDNA antibodies. Intriguingly, the authors also revealed that a clinically-approved SLE drug, dexamethasone, was particularly effective in decreasing the expression levels of cellular MCT1 from SLE patients in vitro. It is likely of genuine interest to investigate the biological mechanism of these clinical findings in the future endeavors of the authors.

In summary, this comprehensive study uncovered an important role for MCT1 in regulating pyruvate metabolism to modulate AID expression and CSR in activated B cells. The findings have relevance for our basic understanding of how metabolism influences humoral immunity in settings of health and disease, and may pave the way towards opening new therapeutic windows in SLE.

https://www.nature.com/articles/s41467-023-44540-0

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