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The Many Faces of TRAF Molecules in
Immune Regulation
Gail A. Bishop
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References
J Immunol 2013; 191:3483-3485; ;
doi: 10.4049/jimmunol.1390048
http://www.jimmunol.org/content/191/7/3483
The Many Faces of TRAF Molecules in Immune Regulation
Gail A. Bishop
W
Department of Microbiology, University of Iowa, Iowa City, IA 52242
Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of
Microbiology, University of Iowa, 2193B MERF, 375 Newton Road, Iowa City, IA
52242. Email address: [email protected]
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1390048
Gail A. Bishop
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
Abbreviations used in this article: CY, cytoplasmic; iNKT, invariant NKT cell; LMP1,
latent membrane protein 1; TRAF, TNFR-associated factor.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
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hen 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
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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 TRAF32/2 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-TRAF32/2 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.
Disclosures
The author has no financial conflicts of interest.
References
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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 TRAF32/2 mice
by producing a conditionally deleted, TRAF3flox/flox strain. We
first bred these mice to a CD19Cre/1 strain (42), to produce
a mouse lacking TRAF3 specifically in all CD191 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-TRAF32/2 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,
TRAF32/2 T cells and dendritic cells also display constitutive NFkB2 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 TRAF3flox/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-TRAF32/2 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
PRESIDENT’S ADDRESS
The Journal of Immunology
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