Abstract
Germinal center (GC) B cell differentiation is critical for the production of affinity-matured pathogen-specific antibodies, the dysregulation of which may lead to humoral immunodeficiency or autoimmunity. The development of an in vivo screening system for factors regulating GC B cell differentiation has been a challenge. Here we describe a small-scale in vivo screening system with NP-specific B1-8hi cells and a retroviral shRNA library targeting 78 candidate genes to search for B cell-intrinsic factors that specifically regulate GC B cell differentiation. Zdhhc2, a gene encoding palmitoyltransferase ZDHHC2 and highly expressed in GC B cells, is identified as a strong positive regulator of GC B cell differentiation. B1-8hi cells transduced with Zdhhc2-shRNA are severely compromised in differentiating into GC B cells. A further analysis of in vitro differentiated B cells transduced with Zdhhc2-shRNA shows that Zdhhc2 is critical for the proliferation and the survival of B cells stimulated by CD40L, BAFF, and IL-21 and consequently impacts on their differentiation into GC B cells and post-GC B cells. These studies not only identify Zdhhc2 as a novel regulator of GC B cell differentiation but also represent a proof of concept of in vivo screen for regulators of GC B cell differentiation.
Highlights
The germinal center (GC) B cell response is a fundamental process in humoral immunity that enables the rapid evolution and selection of B cells, so B cells that express pathogen-specific affinity-matured B cell receptors (BCRs) with reduced autoreactivity can be selected for antibody production (1, 2)
While the empty vector-transduced cells were present in both non-Germinal center (GC) and GC B cell compartments, the Bcl6–shRNA transduced cells were almost completely absent from the GC B cell compartment, but not in the non-GC B cell compartment, which led to a significantly reduced GC/non-GC ratio for Bcl6– shRNA transduced cells (Figure 1D). While these results are consistent with the previous report that Bcl[6] is essential for GC B cell differentiation (28), they provide the basis for the use of retroviral shRNA transduced B1-8hi B cells for in vivo screen
To screen for genes that impact on GC B cell differentiation, we focused on shRNA constructs that consistently recovered in either the non-GC or the GC B cell compartment
Summary
The germinal center (GC) B cell response is a fundamental process in humoral immunity that enables the rapid evolution and selection of B cells, so B cells that express pathogen-specific affinity-matured B cell receptors (BCRs) with reduced autoreactivity can be selected for antibody production (1, 2). GC B cell differentiation is a T cell-dependent process in which antigen-specific B cells present antigens to cognate T cells, which in turn provide reciprocal activating signals for the differentiation for both GC B cells and follicular T helper (TFH) cells. Antigen-specific B/T cell interactions result in the activation and the proliferation of both B and T cells and their chemokine-dependent migration to the B cell follicles to form the GCs (3–7). The expanded and BCR-diversified B cells migrate to the light zone of GCs and compete for capturing antigens and T cell help (6, 9–13). It has been proposed that high-affinity B cells can capture and present more antigens to TFH cells and acquire sufficient signals from TFH cells for their further differentiation into plasmablasts and memory B cells (14–16).
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