Center for Comparative Medicine
SVM: Anatomy, Physiology & Cell Biology
University of California, Davis
Research Interest: Gene regulation in the control of antibody
Antibody is an important component of immunity; however, it needs to be tightly controlled to prevent excessive immune-mediated damage. The specificity, quality and quantity of antibody are regulated by distinct mechanisms which are coordinated to ensure an effective immune response. Coordination depends upon transcriptional control of gene regulatory networks that both induce the expression of antibody secretory gene programs while simultaneously repressing alternative gene programs that function to diversify antibodies (and vice versa). Our lab focuses on a better understanding of the control of antibody by investigating transcription factor activity and the architecture of gene regulatory networks in which they are embedded.
IRF4 and affinity maturation
We have identified a gene regulatory network orchestrating Germinal Center (GC) B cell and low affinity plasma cell differentiation. The network is comprised of the transcription factor IRF4 that controls the expression of the Bcl6 and Blimp-1 transcription factors, determinants of GC B cells and plasma cells, respectively. This demonstrates, that IRF4 functions as a switch to control which cell fate trajectory an activated B cell will adopt. However, because Bcl6 and Blimp-1 antagonize each other's expression, we studied the dynamics of IRF4 activity to determine the conditions whereby IRF4 regulates cell fate trajectories and the expression of Bcl6 and Blimp-1. We found that low levels of IRF4 expression favor the differentiation of Bcl6-expressing GC B cells at the expense of Blimp-1-expressing plasma cells. In contrast, high levels of IRF4 antagonize the differentiation of Bcl6 expressing GC B cells in favor of Blimp-1-expressing plasma cells. Importantly, we found that the levels of IRF4 expression are dictated by the affinity and avidity of the B cell antigen receptor for antigen such that increasing signal strength results in more abundant IRF4 expression. We conclude that the Irf4 locus functions as a "sensor" of antigen receptor signal strength and the IRF4 protein functions as a "writer" of B cell fate trajectories.
Importantly, the process of affinity maturation involves orchestrating a diversification gene program in GC B cells that functions to diversify the antibody repertoire to obtain clones of that exhibit higher affinity towards antigen. Although it is clear that improvements to the affinity of the B cell antigen receptor is "tested" by its ability to capture, internalize, process and present pMHC to T follicular helper cells, the role of antigen receptor signaling in this process is unclear. We aim to gain insight into this process by investigating the role of IRF4, an immediate early gene of antigen receptor signaling. Relatedly, whether IRF4 plays a role to control the fate of exiting GC B cells will be researched.
Memory B cell fatesAllo-specific B cells and their antibody products are strongly predict with acute and chronic allograft rejection, especially in "sensitized" transplant recipients with pre-existing donor-specific antibodies (DSA). The source of antibody in the sensitized recipients derives from two distinct cellular pools: constitutive production of allo-specific antibody from the long lived plasma cell and de novo production from reactivated memory B cells in the recall response. While the long lived plasma cells can be viewed as a static population that produces a finite amount of antibody (life time times secretion rate), memory B cells represent a highly dynamic population that re- cycle indefinitely to produce bursts of antibody and to reseed the long lived plasma cell and memory B cell pools. Thus, we posit that understanding the roles of memory B cells under circumstances of transplantation, especially the mechanisms that regulate the dynamic behavior of allo-specific memory B cells, will ultimately prove to be important for controlling transplant rejection. Our work has uncovered a central regulator of B cell differentiation that controls the identity of differentiated B cells as a function of antigen affinity/avidity of the B cell antigen receptor (BCR). The Irf4 transcription factor controls the generation of plasma cells (PC) and Germinal Center B (GC B) cells by activating the expression of the rate limiting transcription factors important for those cell fates, Blimp-1 and Bcl6, respectively. We hypothesize that Irf4 plays a similarly critical role in controlling the generation of memory B cells as well as in controlling the dynamics of memory B cell reactivation. Furthermore, we have optimized a strategy to follow the fate of individual allogeneic MHC-specific B cells responding to transplants in mice. This technology has enabled us to quantify the proportions of PC, GC B, and memory B cells after primary and secondary immunizations. Therefore we propose to define the life cycle of allo-antigen specific memory B cells and how that may be altered by costimulation blockade, to identify the conditions with which memory B cells reactivate, and to determine the impact of memory B cells on mechanisms of humoral rejection.
IRF4 and T follicular helper cell fate decisions
Well executed cell fate choices of CD4+ T helper (Th) cells into effector Th1, Th2, or Th17 which control microbicidal actions of innate immune cells or into T follicular helper (Tfh) cells which control B cell and antibody responses are essential for vaccine-elicited memory responses and immune protection. Signal regulated transcription factor networks coordinate Th cell fate decisions. The proposed studies address the central hypothesis that TCR affinity-based induction of graded IRF4 expression functions to control alternate T helper cell fate choices. It has been shown that variations in TCR affinity for pMHC play a determining role in Th cell fate choices by somehow regulating the expression of the Blimp-1/Bcl6 transcription factors, important for T effector/Tfh, respectively. The challenges that remain are to identify the TCR-proximal molecular determinants and how they function to establish the distal pattern of Blimp-1 and Bcl6 expression. Important insight comes from our recent demonstration that, in B cells, expression of IRF4 functions as a switch to apportion the frequency of Bcl6-expressing Germinal Center (GC) B cells and Blimp-1-expressing plasma cells. This pivotal function of IRF4 is due to its ability to directly activate either side of the Bcl6-Blimp-1 negative feedback loop depending on whether IRF4 cellular concentrations are transient/low or whether they are sustained/high, respectively. Notably, IRF4 expression dynamics is set by the intensity of B cell antigen receptor (BCR) signaling such that increased antigen affinity/avidity augments IRF4 levels. Our findings in B cells as well as our evidence that a TCR ► IRF4 ► Bcl6/Blimp-1 axis may operate in Th cell fate gene regulatory networks raise the possibility that TCR signal strength sets forth a dynamic of IRF4 expression that in turn functions to control the proportions of Blimp-1-expressing effector Th1 cells or Bcl6-expressing Tfh cells. We will test this hypothesis by determining the relationship between TCR signal strength and IRF4 expression dynamics in vivo (Aim 1). In Aim 2, we will determine whether TCR-induced variations in IRF4 levels correlate with outcomes in Th cell fate choice. Importantly, to establish whether observed outcomes are dependent on IRF4 expression levels (as opposed to other factors controlled by the TCR) we will use an Irf4-inducible mouse model that we developed to modulate the timing and levels of IRF4 expression. Lastly, in Aim 3, we will determine how TCR signal strength dependent control of IRF4 expression levels are wired within Th cell fate gene regulatory networks to enable appropriate Th cell fate choices. Overall, we expect to establish whether TCR-regulated dynamics of IRF4 expression functions as a Th cell fate switch. Our studies seek to reveal cell intrinsic mechanisms of Th cell fate choice that could define unique targets and strategies to enhance the efficacy of future vaccines.