Durable humoral immunity regulated by intrinsic CD28 function in plasma cells

描述（由申请人提供）：持续的长期抗体水平是保护性免疫的基石，但这种滴度持久维持的机制尚不清楚。普遍的范式是，这是通过在骨髓（BM）中有限数量的特定生态位中存活的长寿命浆细胞（LLPC）子集连续产生抗原不依赖抗体发生的。然而，该范式的核心原则，即BM LLPC是由本质上不同的生物学定义的一个单独的子集，并且它们实际上有助于长期抗体滴度，尚未得到直接证明。我们最近报道（Rozanski et al, JEM July, 2011），长期体液免疫依赖于CD28的浆细胞内在功能（最好的特征是原型T细胞共刺激受体），它选择性地支持BM LLPC的生存，而不是脾短期PC （SLPC）。尽管LLPC和SLPC都表达CD28，但CD28信号传导和生存增强仅发生在LLPC中（与T细胞不同，它不需要外源信号1）。在体内，即使有足够的T细胞帮助，浆细胞中CD28的丢失（或CD28配体CD80或CD86的全局丢失）也会导致LLPC群体的显著损失，LLPC在BM中的生存半衰期从426天减少到63天，并且无法长期维持抗体滴度，而对SLPC群体没有任何影响。在微环境/生态位方面，我们发现CD80+树突状细胞（DC）与BM内的LLPC直接接触，骨髓DC在体外以cd28依赖的方式支持LLPC的长期存活，并且通过CD80和CD86连接的“反向信号传导”诱导DC产生促pc存活的细胞因子IL-6，这对于维持体外免疫球蛋白（Ig）的产生是意想不到的必要条件。总之，这些发现确立了独特的BM LLPC亚群的存在，这是维持抗体滴度所必需的，并揭示了pc固有CD28功能在浆细胞寿命和体液免疫中的一个以前未被认识但重要的作用。虽然先前的研究已经明确定义了CD28在保护性和致病性（如自身免疫）抗体反应中的作用，但这几乎完全归因于辅助性T细胞活化的共刺激。重新考虑这些文献提出了一个重要的问题，即在T细胞和浆细胞中，有多少体液反应是由CD28功能引起的。因此，我们认为pc固有的CD28功能在浆细胞生物学和持久体液免疫的产生中起着核心作用，但在此背景下几乎完全没有被表征。本提案的总体目标是确定CD28激活调节PC存活、功能和体液免疫反应发展的具体机制。本建议的目的是：1)。明确CD28激活下游调节PC存活和功能的信号转导途径和分子靶点，2)。表征CD80/CD86+ LLPC BM基质生态位；在T细胞或B细胞谱系中条件敲除CD28的小鼠体液免疫应答中，表征PC与T细胞固有CD28功能的作用。

DESCRIPTION (provided by applicant): Sustained long-term antibody levels are the cornerstone of protective immunity, yet the mechanism(s) by which such titers are durably maintained remains unclear. The prevailing paradigm is this occurs via continuous antigen-independent antibody production by a subset of long-lived plasma cells (LLPC) that survive within a limited number of specific niches in the bone marrow (BM). The paradigm's central tenets however, that BM LLPC are a separate subset defined by intrinsically distinct biology and that they in fact contribute to long-term antibody titers, have not been directly demonstrated. We have recently reported (Rozanski et al, JEM July, 2011) that long-term humoral immunity is dependent on plasma cell-intrinsic function of CD28 (best characterized as the prototypic T cell costimulatory receptor), which selectively supports the survival of BM LLPC but not splenic short-lived PC (SLPC). Although LLPC and SLPC both expressed CD28, CD28 signaling and enhanced survival only occurred in the LLPC (and unlike T cells did not require an exogenous signal 1). In vivo, even with sufficient T cell help the loss of CD28 in the plasma cells (or the global loss of the CD28 ligands CD80 or CD86) caused significant loss of the LLPC population, reduction of the half-life of LLPC survival in the BM from 426 to 63 days, and inability to maintain antibody titers long-term - without having any effect on SLPC populations. On the microenvironment/niche side, we found that CD80+ dendritic cells (DC) were in direct contact with LLPC within the BM, that myeloid DC supported long-term LLPC survival in a CD28-dependent fashion in vitro, and that "backsignaling" via CD80 and CD86 ligation induced the DC to produce the pro-PC survival cytokine IL-6 that was unexpectedly essential for sustaining immunoglobulin (Ig) production in vitro. Altogether, these findings establish the existence of the distinct BM LLPC subset that is necessary to sustain antibody titers, and uncover a previously unrecognized but essential role for PC-intrinsic CD28 function in the longevity of plasma cells and humoral immunity. While previous studies have clearly defined a role for CD28 in both protective and pathogenic (e.g. autoimmune) antibody responses, this has been almost entirely attributed to costimulation of helper T cell activation. Reconsideration of this literature raises he important question of how much of the humoral response is due to CD28 function in T cells vs. plasma cells. Thus we believe that PC-intrinsic CD28 function plays a central role in plasma cell biology and the generation of durable humoral immunity, but has been almost entirely uncharacterized in this context. The overall goal of this proposal is to define the specific mechanisms by which CD28 activation regulates PC survival, function, and the development of humoral immune responses. The Aims of this proposal are to: 1). Define the signal transduction pathways and molecular targets downstream of CD28 activation that modulate PC survival and function, 2). Characterize the CD80/CD86+ LLPC BM stromal niche, and 3). Characterize the role of PC vs. T cell intrinsic CD28 function in humoral immune responses in mice conditionally knocked out for CD28 in the T or B cell lineage.