The Many Faces of TRAF Molecules in Immune Regulation

When I started postdoctoral work in the laboratory of the late Geoffrey Haughton at the University of North Carolina in Chapel Hill, I began to pursue a fascination with how lymphocytes interact and collaborate in immune regulation that has lasted for decades. I was especially interested in learning about how T cells induce B cells to produce high-affinity, isotype-switched Ab responses, leading to both Ab production and the development of humoral memory. At that time, the most popular paradigm indicated that following Ag binding, the B cell presents processed peptides bound to class II MHC molecules to cognate-activated T cells. The T cell secretes lymphokines, which are both necessary and sufficient to produce a full humoral response. However, Geoff convinced me of his belief that contact-dependent T–B cell interactions were also key to B cell activation. Influenced by new and elegant work on T–B cell contact (1), we performed studies showing that fixed, activated T cells could synergize with Ag receptor signals to drive B cell activation in a contact-dependent manner (2). Subsequent work I performed with Jeffrey Frelinger revealed that B cell class II MHC molecules can deliver one of these T cell–mediated signals (3, 4). As a new Assistant Professor at the University of Iowa, my laboratory further characterized class II–mediated signaling pathways (5, 6–8), and many other laboratories also made important contributions to this topic (reviewed in Ref. 9).

In the early 1990s, my attention was caught by a newly revealed major player in T–B cell interactions— CD40. A member of the TNFR superfamily, CD40 was revealed as the key signal missing in the human immunodeficiency disease X-linked hyper IgM syndrome (reviewed in Ref. 10), and mouse models also emphasized its importance in both humoral and cellular immunity, as well as various immune-mediated diseases (11–15). However, although CD40 clearly delivers many potent signals to immune cells, its cytoplasmic (CY) domain doesn’t contain any of the well studied tyrosine kinase–binding motifs or domains that have been a major focus of studies of lymphocyte Ag receptors, by far the most well studied of immune cell receptors. Our laboratory was experienced in structure–function analysis, so we thus produced a group of CD40 molecules with CY mutations, stably expressed them in B cell lines, and examined the structural requirements for various upstream signals and B cell effector functions. We discovered multiple signaling determinants, controlling overlapping but distinct CD40 signals and functions (16–18). During this time, a number of groups demonstrated that the CD40 CY domain can bind cytoplasmic signaling molecules of the TNFR-associated factor (TRAF) family (19–22), and in fact the signaling determinants that we had identified corresponded well to the location of binding sites for various TRAFs. This led us on yet another journey, to study the roles of TRAF molecules in immune regulation.

A critical insight was provided to us by our parallel studies of a CD40-mimicking protein encoded by the EBV, called latent membrane protein 1 (LMP1). While LMP1 strikingly replicates CD40 functions in B cells (23), we and others found that it does so in a manner that delivers abnormally amplified and sustained signals, consistent with the implication of LMP1 in the pathogenesis of EBV-associated B cell malignancies, as well as exacerbation of certain autoimmune conditions (reviewed in Refs. 24–26). Although the CY domains of CD40 and LMP1 have little sequence homology, binding studies with exogenously overexpressed molecules in epithelial cells or fibroblasts showed that LMP1, like CD40, can bind TRAFs 1, 2, 3, 5 and 6 (27, 28). It was thus logical to assume that because LMP1 is a CD40 mimic, the two molecules use these TRAFs in the same manner. TRAFs 1, 2, 3, and 5 share an overlapping binding motif in CD40, and there is a similar although distinct motif in LMP1. Thus, discerning the specific roles for individual TRAF family members in signaling through receptors that have this motif was challenging. Overexpressing any of these TRAFs will inevitably alter the binding of the other TRAFs sharing the motif, and mutating the binding site will similarly affect binding of multiple TRAF family members. Thus, drawing clear conclusions from such approaches was problematic. Mice made completely deficient in TRAFs 2, 3, or 6 have an embryonic or neonatal lethality (29–31). Thus, to begin to dissect the roles of individual TRAF members for specific receptors, we developed a novel method for complete and specific removal of one or multiple TRAFs from B cell lines, using in vitro gene targeting by homologous recombination (32). This allowed us, for the first time, to directly compare CD40 versus LMP1 requirements for specific TRAFs in B cell activation. To our considerable surprise, we subsequently found that LMP1 utilizes each of the TRAFs in a manner quite distinct from the ways these same TRAFs are used by CD40 (33–38).

Perhaps most striking was the distinct way in which TRAF3 impacts CD40 versus LMP1 signaling to B cells. While TRAF3 serves as a negative regulator of CD40 signals, possibly via competition with TRAF2 (39, 40), we discovered that LMP1 instead utilizes TRAF3 as a positive mediator (37, 41). It was thus now clear that TRAFs serve highly receptor-specific roles in immune regulation. To determine if TRAF3 also has cell type–specific functions, we circumvented the early lethality of TRAF3^−/− mice by producing a conditionally deleted, TRAF3^flox/flox strain. We first bred these mice to a CD19^Cre/+ strain (42), to produce a mouse lacking TRAF3 specifically in all CD19⁺ B cells. This new mouse revealed another striking finding that could not have been discerned in cell lines—that TRAF3 in B cells plays a critical role in restraining B cell survival. B-TRAF3^−/− mice display highly increased B cell numbers, resulting in enlarged spleens and lymph nodes, as well as spontaneous germinal centers, elevated autoantibodies, immune complex deposition, and B cell infiltration of various organs (43), a phenotype reproduced in a similar mouse made by the Brink laboratory (44). This phenotype does not involve enhanced B cell proliferation, but results from enhanced BAFF-independent survival, as well as increased response to innate immune signals (43, 45). While this abnormal survival correlates with increased basal noncanonical NF-kB2 signaling, TRAF3^−/− T cells and dendritic cells also display constitutive NF-kB2 activation, without any increase in cell survival (45, 46). A current laboratory focus is thus a better understanding of precisely how TRAF3 regulates B cell–specific survival.

Breeding the TRAF3^flox/flox mouse to a CD4-Cre strain produced a mouse lacking TRAF3 in all mature T cells. The phenotype of this mouse clearly reveals the strongly cell-type specific roles of TRAF3. T-TRAF3^−/− mice have normal numbers of immune cells, including CD4 and CD8 conventional T cells, but highly defective T-dependent humoral responses, and marked impairment in both CD4 and CD8 T cell responses to infection with the intracellular pathogen, L. monocytogenes (46). We initially assumed these functional defects arose from defective signaling via TRAF3-binding T cell costimulatory receptors of the TNFR superfamily, such as OX40, 4-1BB, CD27, and CD30. We were thus surprised to discover that in vitro responses to engagement of the TCR complex are markedly defective in the absence of TRAF3, and that following TCR engagement, TRAF3 associates with the TCR complex (46). Activation of multiple kinases and adaptor proteins in early TCR signaling is present, but reduced by ≥50% in TRAF3^−/− T cells (46). Although this reduced TCR signal strength is apparently adequate for production of normal numbers of conventional T cells, we recently found that invariant NKT cells (iNKT) are greatly reduced (≥10-fold) in T-TRAF3^−/− mice. This results from a block between stages 2 and 3 of iNKT development, is a cell-intrinsic defect, and can be rescued either by reintroduction of TRAF3 or the transcriptional regulator T-bet (47). Current studies focus upon the specific mechanisms by which TRAF3 modulates the quality and functions of the TCR complex.

Thus, our studies and those of many others have revealed that TRAF molecules serve both different receptors and distinct cell types in diverse and highly context-dependent ways, an important principle to consider in designing and interpreting experiments involving this fascinating family of adapter proteins. My own winding path through the forest of lymphocyte activation has impressed upon me that the most interesting and important findings often result from overturned predictions. I have been tremendously fortunate to pursue the life of a scientist, in the company of excellent and talented mentors, colleagues, and trainees, and look forward to learning what new results await around the next bend in the road.