Fate Decisions During B-Lymphocyte Development and Activation

Understanding B-lymphocyte fate specification during B-cell development and humoral responses is important for developing vaccines and tools for studying B cells.

In the first study, I elucidated major factors influencing the fate of 2F5 B-cell receptor (BCR) knockin B cells as they traverse immune tolerance checkpoints. 2F5 is an HIV-1 broadly neutralizing antibody (bnAb) whose neutralizing activity is linked to its ability to bind two autoantigens: host-derived viral membrane phospholipids and the ELDKWA epitope found in both the viral envelope (Env) protein and in the host enzyme, kynureninase (KYNU). The development of 2F5-like B cells is proscribed by immune tolerance controls, but it is unknown whether tolerizing counterselection is driven primarily by lipid- or by KYNU-specificity (or by both equally). It is also unclear whether BCR editing purges the 2F5 BCR from the peripheral repertoire of 2F5 knockin mice. Answering these questions is important for evaluating potential HIV-1 vaccine strategies.

I sampled thousands of B cells from before and after the first and second tolerance checkpoints in 2F5 BCR knockin mice. After culturing individual B cells in an in vitro system that supports B-cell proliferation and differentiation into Ab-secreting cells, I determined the specificities of the secreted clonal IgGs, and recovered the V(D)J rearrangements encoding these Abs. I found that in 2F5 knockin mice, nearly all (pre-tolerance) small pre-B cells express the knockin heavy chain and light chain (LC), and avidly bind Env, KYNU, and a model lipid, cardiolipin. In contrast, extensive LC editing completely purges the (post-tolerance) mature B cell compartment of Env- and KYNU-reactivity, although cardiolipin-reactivity remains mostly intact. Thus, I conclude that tolerization of 2F5 B cells is driven primarily by KYNU-reactivity, and occurs in large part due to LC editing. Remarkably, peripheral anergic B cells (B220+IgM-IgDhi) are enriched for Env- and KYNU-reactivity, and express a restricted repertoire that partially overlaps with that of mature B cells, indicating that peripheral fate determination is at least partly stochastic. The data support that activation of anergic B cells may be a viable route for HIV-1 vaccination.

In the second study, I tested whether transcriptional re-programming can force activated mature B cells to sustain B-cell identity and block terminal differentiation into plasma cells (PCs). Controlling B-cell terminal fate commitment has numerous potential applications in science and medicine, but the means to do so have remained elusive. PC differentiation is governed by a transcription factor network comprising Pax5, Bcl6, and Bach2 – which promote B-cell identity and antagonize PC differentiation – and IRF4 and Blimp1, which cooperate to extinguish B-cell identity and coordinate PC differentiation.

I generated mouse primary B cells harboring gain-of-function in Pax5, Bach2, or Bcl6, or loss-of-function in IRF4 or Blimp1, and then continuously stimulated these B cells with CD154 and IL-21 in vitro. I found that transgenic expression of Bach2 or Bcl6 prohibits PC commitment, maintains markers of B-cell identity, and endows B cells with extraordinary growth potential in response to T-cell help signals. Long-term Bach2-transgenic B cell lines have genetically stable BCRs, express high levels of MHCII and molecules for co-stimulation of T cells, and transduce intracellular signals when incubated with BCR ligands. Silencing the Bach2 transgene in an established cell line causes the cells to secrete large quantities of immunoglobulin. These results provide insight into molecular control over activated B-cell fate, and suggest that enforced expression of Bach2 in vivo may augment germinal center (GC) B cell or memory B cell (MBC) differentiation at the expense of PC commitment. Additionally, culturing Bach2-transgenic B cells has potential applications in monoclonal Ab production, BCR signaling studies, T-cell epitope-mapping studies, and more.

Next, I generated a new knockin mouse harboring a doxycycline-inducible Bach2 transgene and a fluorescent reporter protein, and I used this mouse strain to determine how enforced expression of Bach2 influences the fate of B cells activated in vivo. Constitutive Bach2 expression had no impact on the total number of GC B cells or MBCs in the draining lymph node at 12 days post-immunization, but reduced local PC numbers by ~10-fold. Interestingly, B cells expressing transgenic Bach2 were ~2-fold enriched among GC B cells and MBCs relative to their frequency among mature follicular B cells. Additionally, populations of GC B and MBCs expressing transgenic Bach2 contained significantly higher frequencies of cells brightly stained by fluorescent antigen. As these antigen-bright cells are presumed to have the highest-affinity BCRs, which would normally pre-dispose them toward PC differentiation, I tentatively conclude that enforced expression of Bach2 prevents GC B cells from differentiating into PCs, and instead causes them to remain in GCs or join the MBC pool.

The inducible Bach2-knockin mouse also made it practical to attempt high-throughput isolation of long-lived, monoclonal, antigen-specific B cell lines. By isolating and culturing individual follicular and GC B cells constitutively expressing transgenic Bach2, I generated >20 long-lived cell lines, of which two expressed immunogen-specific BCRs. On withdrawing doxycycline and silencing the Bach2 transgene, all of the cell lines rapidly differentiated into PCs and secreted large quantities of Ig. Thus, inducible Bach2 mice may be an attractive tool for cloning antigen-specific B-cell lines.