FcγRIIB功能缺損導致生發中心反應異常使低抗原親和性抗體增加之研究

Abstract

後天免疫系統包含體液性和細胞性兩種，主要由淋巴球負責。體液性免疫最重要的目的在產生具抗原專一性的高親和性抗體。而抗體的製造則必須透過脾臟和淋巴結的生發中心(germinal center)對B細胞進行挑選和使之分化成漿細胞，專司抗體分泌的漿細胞則經由血流定居骨髓。在疫苗反應時，生發中心B細胞須經歷體細胞高突變(somatic hypermutation)、親和力成熟(affinity maturation)和免疫球蛋白轉換(class switching)。B細胞抗原受器(B-cell antigen receptor, BCR)在體細胞高突變反應後，使生發中心B細胞對抗原具有各式不同親和力，接著抗原針對這些B細胞進行抗原親和力成熟反應，最重要的作用是去除低抗原親和性的生發中心B細胞，以確保產生高效價的高抗原親和性抗體。FcγRIIB是B細胞上抑制BCR最重要的受器，負責調節體內抗體製造量。此外，它也能夠單獨活化使B細胞凋亡。由於研究指出生發中心B細胞的FcγRIIB表現在疫苗反應會上升，而FcγRIIB基因剔除鼠脾臟的生發中心會自發性地增加和變大，導致自體免疫抗體產生，終致紅斑性狼瘡。因此，我們推論FcγRIIB在生發中心進行親和力成熟反應時，可能扮演促使低抗原親和性生發中心B細胞凋亡的角色。我們採用功能低下的人類FcγRIIB-T232基因多型性突變鼠來研究這個問題。FcγRIIB-I232T突變是FcγRIIB的第232個胺基酸位置由蘇胺酸(threonine)取代原本的異白胺酸(isoleucine)，這種多型性變異在亞洲人紅斑性狼瘡佔高比例。我們採用4-hydroxy-3-nitrophenylacetyl (NP) hapten-chicken gamma globulin (CGG)做為疫苗原來探討FcγRIIB-T232突變鼠是否比正常鼠殘留較多的低抗原親和性生發中心B細胞？結果顯示在單次疫苗接種時，FcγRIIB-T232突變鼠血液中的低抗原親和性B細胞和漿細胞的數量均增加和伴隨著血清中低抗原親和性抗體的增加。當再次疫苗接種，FcγRIIB-T232突變鼠脾臟和骨髓中分泌低抗原親和性IgG的B細胞和低抗原親和性抗體均顯著增加。脾臟生發中心在FcγRIIB-T232突變鼠也是變大和B細胞數量增加，尤以位在執行親和力成熟的亮區(light zone)最顯著。此外，我們更發現FcγRIIB-T232突變鼠的低抗原親和性B細胞和抗體的增加確實是因為亮區的低抗原親和性生發中心B細胞的細胞凋亡(apoptosis)減少，未能被清除所致。有趣的是，FcγRIIB-T232突變鼠的低抗原親和性抗體對於同源類似抗原NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl)的交互反應性(cross-reactivity)亦比正常鼠增加。總結來說，本研究提供多項證據顯示FcγRIIB在生發中心進行親和力成熟反應時，扮演積極去除低抗原親和性B細胞的關鍵角色。在臨床應用上，則建議在疫苗接種時，應可藉由調控FcγRIIB的表達使產生的抗體對同源相近的病原菌具有交叉中和的保護效果。

Germinal center (GC) reaction, which undergoes somatic hypermutation, affinity maturation and class switching, is the hallmark of humoral immunity. One important function of it is to eliminate GC B cells that secrete low-affinity antibody (Ab) to the antigen (Ag) to ensure successful generation of effective neutralizing Abs through affinity maturation. FcγRIIB, a critical inhibitory Fcγ receptor (FcγR) that can mediate apoptosis in B cells, has been found to up-regulate on the GC B cells. Interestingly FcγRIIB-deficient mice spontaneously develop lupus accompanied by retention of somatic mutated BCRs as well as increased number and increased size of GCs in the spleen, indicative of a failure in negative selection of GC B cells. Moreover, people carrying a functionally impaired variant of FcγRIIB, of which isoleucine at position 232 was replaced by threonine, are susceptible to lupus particularly in Asians. Here, we generated FcγRIIB-T232 mutant mice to investigate the functional role of FcγRIIB in GC reaction. We hypothesized that to some extent the GC phenotype of FcγRIIB-T232 mice should recapitulate that of FcγRIIB knockout mice after immunization. To address this, we immunized FcγRIIB-I232 (wild-type) and FcγRIIB-T232 mice respectively with 4-hydroxy-3-nitrophenylacetyl (NP) hapten-chicken gamma globulin (CGG), a model Ag that allows distinction of low vs. high affinity Ag-specific B cells. Indeed, we found an increase of low-affinity NP-specific IgG-expressing B cells and plasma cells in circulation and a concomitant increase of serum Abs in FcγRIIB-T232 mice than those of FcγRIIB-I232 mice after primary immunization. After secondary immunization, we detected a significant increase of NP-specific B cells and plasma cells in the blood, spleen and bone marrow of FcγRIIB-T232 mice compared to that of FcγRIIB-I232 mice, accompanying an accumulation of low-affinity NP-specific IgG-secreting B cells in the spleen and bone marrow of FcγRIIB-T232 mice. Furthermore, FcγRIIB-T232 mice increased the formation of low-affinity NP-specific IgG in the circulation. Consistent with these findings, the spleen of FcγRIIB-T232 mice displayed an increased size of the GC of lymphoid follicles and had a concomitant increase of the number of GC B cells, especially the light zone GC B cells. Moreover, a decrease of apoptosis of GC B cells in the light zone of FcγRIIB-T232 mice correlated with their increased levels of low-affinity NP-specific B cells and Abs, supporting an active and essential role of FcγRIIB in GC reaction. Finally, we also found that the increased low-affinity antibodies in FcγRIIB-T232 mice resulted in the increase of cross-reactive antibodies. Taken together, these results support the notion that FcγRIIB plays an important role in negative selection of GC B cells. In addition, FcγRIIB may be considered as an ideal target for immunomodulation if the expression level of FcγRIIB on GC B cells holds the key to determine the stringency of Ab affinity as well as Ab repertoire to be generated during vaccination.