REGULATION OF THE IMMUNE RESPONSE TO POLYSACCHARIDES AND POLYSACCHARIDE CONJUGATE

对多糖（PS）抗原的免疫反应受到高度调控，并具有几个显著特征，包括限制亚类、可变区域基因使用、良好的特异性和亲切性。未与蛋白结合的简单PS（如Levan细菌（BL）、C组脑膜炎奈瑟菌（MCPS））可引起胸腺非依赖性（TI）反应。另一方面，与蛋白质结合的PS（如MCPS与破伤风类毒素（MCPS- tt）结合）会引起一种不同类型的反应，称为胸腺依赖性（TD）。我们早期的抗bl抗体分析显示，菊粉（In）分支决定因子对种系有初级反应。然而，两次注射BL引发了具有体细胞突变和更高亲和力的IgM单抗。我们现在已经对种系抗体进行了定点诱变，并确定了导致更高或更低亲和力的特定位点。通过分析抗- in反应的多样性调控，我们绘制了Sr1多样性基因图谱。我们之前的数据表明Sr1与小鼠14号染色体上的标记之间存在联系，这已经得到CXB重组自交系（RI）研究和最近（BALB/c x B6.C-H8）F1小鼠表型分析的支持。我们正在完成更多BALB/c x (BALB/c x C57BL/6)F1回交小鼠的表型和基因型分析。此前对抗mcps和抗mcps TT单抗的分析表明，VH基因家族主要由VHJ558使用。从3个板中分离IgG、IgA和IgM单克隆抗体，进行亲和度检测。用MCPS- tt偶联物2X（C2）免疫和MCPS- tt免疫加MCPS（CP）增强的小鼠单克隆抗体的50%结合浓度一般比抗-MCPS单克隆抗体低2个数量级，表明TD单克隆抗体的亲和力显著高于抗-MCPS单克隆抗体（高10-100倍）。正在进行的序列分析将确定是体细胞突变还是J558或其他家族的不同VH基因使用导致了发病率的增加。我们实验室早期的小鼠模型研究表明，即使将MCPS作为TD MCPS- tt结合物施用，对MCPS的免疫反应也会出现发育迟缓。因此，研究人员进行了研究，以确定新生小鼠对TD偶联反应的延迟是否由于抗原呈递缺陷。我们发现成人B细胞在向T细胞呈递TT或MCPS-TT时比脾脏细胞或巨噬细胞更有效，成人树突状细胞是该系统中最有效的抗原呈递细胞，效率约为B细胞的2倍。值得注意的是，新生儿B细胞和新生儿树突状细胞在呈递抗原的能力上都存在缺陷。数据表明，提高新生儿细胞呈递抗原能力的因素可能有助于刺激新生儿对结合疫苗的反应。

The immune response to polysaccharide (PS) antigens is highly regulated and has several distinguishing features including restricted subclass, variable region gene usage, fine specificity, and avidity. Simple PS not conjugated to protein (such as bacterial Levan (BL, Neisseria meningitidis group C (MCPS)) elicit a thymus-independent (TI) response. PS conjugated to proteins (such as MCPS coupled to tetanus toxoid, (MCPS-TT)), on the other hand, elicit a different type of response, termed thymus-dependent (TD). Our earlier analysis of anti-BL antibodies had shown a germline primary response to the inulin (In) branch determinants. Two injections of BL, however, elicited IgM mAb with somatic mutations and higher affinity. We have now performed site-directed mutagenesis of the germline antibodies and identified specific sites resulting in higher or lower affinity. Analysis of the regulation of diversity in the anti-In response has led us to map the Sr1 diversity gene. Our previous data indicated linkage between Sr1 and markers on mouse chromosome 14 and this has been supported by studies using CXB recombinant inbred (RI) lines and more recently by phenotype analysis of (BALB/c x B6.C-H8)F1 mice. We are in the process of completing phenotype and genotype analysis of additional BALB/c x (BALB/c x C57BL/6)F1 backcross mice. Previous analyses of anti-MCPS and anti-MCPS TT mAb reveal that VH gene family usage is dominated by VHJ558. IgG, IgA and IgM mAbs from 3 panels were purified and tested for avidity. MAbs from mice immunized with MCPS-TT conjugate 2X(C2) and immunized with MCPS-TT and boosted with MCPS(CP), in general, had 2 orders of magnitude lower concentrations for 50% binding than anti _MCPS mAb, indicating that the TD mAb were of significantly higher avidity (10-100 fold higher) than the anti-MCPS. Sequence analysis in progress will determine if somatic mutation or different VH gene usage with in J558 or other families accounts for the increased avidity. Earlier mouse model studies in our laboratory indicated a developmental delay in the immune response to MCPS even when it was administered as TD MCPS-TT conjugates. Therefore, studies were undertaken to examine whether the delay in response TD conjugates in neonatal mice is due to defective antigen presentation. we have found that adult B cells were much more effective in presenting TT or MCPS-TT to T cells than total spleen cells or macrophages and that adult dendritic cells were the most effective antigen presenting cells in this system,the efficiency being about 2-fold higher than B cells. Of significance, both neonatal B cells and neonatal dendritic cells were defective in their ability to present antigen. The data suggest that factors that inprove the ability of neonatal cells to present antigen might be useful in stimulating neonatal responses to conjugate vaccines.