Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands; and.
Department of Internal Medicine, BovenIJ Hospital, 1034 CS Amsterdam, the Netherlands.
J Immunol. 2014 Aug 1;193(3):1071-9. doi: 10.4049/jimmunol.1400706. Epub 2014 Jun 25.
B cell memory to T cell-dependent (TD) Ags are considered to largely reside in class-switched CD27(+) cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D(+) erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid-specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27(-) B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27(+)IgG(+) B cells was observed. Next, B cells were enriched with D(+) erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27(-)IgM(+) and CD27(+)IgM(+) B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27(-)IgM(+), CD27(+)IgM(+), and CD27(+)IgG(+) B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM(+) B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27(+) B cells from Ag-experienced precursors.
B 细胞记忆对 T 细胞依赖性 (TD) 抗原的记忆主要存在于类别转换的 CD27(+) 细胞中。然而，我们之前观察到，从两个用 D(+)红细胞高免疫的供体中克隆的抗 RhD (D) 抗体主要是 IgM 同种型。因此，我们在这项研究中通过在辐照的 CD40L 表达的 EL4.B5 细胞上进行有限稀释培养 B 细胞，并测试培养上清液，分析了 D 和破伤风类毒素特异性 B 细胞的表型和频率。我们发现，对于两种 TD 抗原，大多数 Ag 特异性 B 细胞都存在于 IgM 表达的 B 细胞中，包括 CD27(-)B 细胞，在两个高免疫供体和非高免疫志愿者中都是如此。仅在免疫后不久，就观察到 Ag 特异性 CD27(+)IgG(+)B 细胞急剧增加。接下来，用 D(+)红细胞 ghost 对 B 细胞进行富集，并作为单细胞进行分选。从这些 D 特异性 B 细胞克隆中对 IGHV、IGLV、IGKV 和 BCL6 基因进行测序表明，CD27(-)IgM(+)和 CD27(+)IgM(+)B 细胞都存在体细胞突变，证明了它们的 Ag 选择性质。此外，测序揭示了 CD27(-)IgM(+)、CD27(+)IgM(+)和 CD27(+)IgG(+)B 细胞亚群之间的克隆关系。这些数据强烈支持了最近在小鼠中描述的 TD 抗原的多个层次的记忆 B 细胞，其中 IgM(+)B 细胞代表一个记忆库，可以重新进入生发中心，并确保从有经验的前体中补充类别转换的记忆 CD27(+)B 细胞。
