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We thank L. H. Park for direction, redirection, and support;
S. R. Vora for continual critical discussion and support;
W. Michaud for providing feeder cells and training for the
development of patient-derived cell lines; and N. Gray for
providing several kinase inhibitors. This study was funded by
support from the NIH R01CA137008 (J.A.E.), R01CA164273
(A. T.S. and J.A.E.), 1U54HG006097-01 (C.H.B.), the Wellcome
Trust (086357 and 102696, C.H.B.), the National Cancer
Institute Lung SPORE P50CA090578 (A.S.C., A.J.I., and J.A.E.),
the Department of Defense (L.V.S. and J.A.E.), Conquer Cancer
Foundation Young Investigator Award (A.S.C.), Uniting Against
Lung Cancer (A.S.C. and A. T.S.), Free to Breathe (A.S.C.),
Lungevity (L.V.S. and J.A.E.), National Foundation for Cancer
Research (A. T.S.), and Be a Piece of the Solution.
J.A.E. is a paid consultant for Novartis, Sanofi-Aventis,
AstraZeneca, Chugai, Amgen, Genentech, GSK, Merck, and
Pfizer, and he holds equity in Gatekeeper Pharmaceuticals,
which has a potential equity interest in T790M inhibitors.
J.A.E. also receives research support from Novartis. J.F.G. is a
paid consultant for Boehringer Ingelheim. A. T.S. is a paid
consultant for Pfizer, Novartis, Ariad, Chugai, Genentech,
Roche, and Ignyta. A.J.I. is a Senior Advisory Board member
and holds equity in Enzymatics Inc. For gene expression
analyses, the raw data are deposited in ArrayExpress
(accession number is E-MTAB-783). The normalized data are
available at www.cancerrxgene.org/downloads.
Materials and Methods
Figs. S1 to S16
Tables S1 to S8
Databases S1 to S8
14 April 2014; accepted 21 October 2014
Published online 13 November 2014;
MAVS, cGAS, and endogenous
retroviruses in T-independent
B cell responses
Ming Zeng,1 Zeping Hu,2 Xiaolei Shi,2 Xiaohong Li,1 Xiaoming Zhan,1 Xiao-Dong Li,1,4
Jianhui Wang,1,4 Jin Huk Choi,1 Kuan-wen Wang,1 Tiana Purrington,1 Miao Tang,1
Maggy Fina,1 Ralph J. DeBerardinis,2 Eva Marie Y. Moresco,1 Gabriel Pedersen,3
Gerald M. McInerney,3 Gunilla B. Karlsson Hedestam,3 Zhijian J. Chen,1,4 Bruce Beutler1†
Multivalent molecules with repetitive structures including bacterial capsular
polysaccharides and viral capsids elicit antibody responses through B cell receptor
(BCR) crosslinking in the absence of T cell help. We report that immunization with
these T cell–independent type 2 (TI-2) antigens causes up-regulation of endogenous
retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a
mitochondrial antiviral signaling protein (MAVS)–dependent RNA sensing pathway or
reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a
second, sustained wave of signaling that promotes specific immunoglobulin M production.
Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse
transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings
suggest that ERV and two innate sensing pathways that detect them are integral
components of the TI-2 B cell signaling apparatus.
Specific antibody production is a hallmark of the B cell response to antigens. T cell– dependent (TD) antibody responses typ- ically elicited by protein antigens require follicular helper T cells for full B cell activation, proliferation, and antibody production.
In contrast, T cell–independent (TI) antigens
stimulate antibody production in the absence
of major histocompatibility complex (MHC) class
II–restricted T cell help. TI antigens include the
TI type 1 (TI-1) antigens, which engage Toll-like
receptors (TLRs) in addition to the B cell receptor (BCR), and TI type 2 (TI-2) antigens, which
engage the BCR in a manner that induces extensive crosslinking, leading to BCR activation and
immunoglobulin M (IgM) production. TI-2 antigens are large multivalent molecules with highly
repetitive structures, such as bacterial capsular
polysaccharides and viral capsids (1).
B cell–intrinsic cytosolic DNA and RNA
sensing in the TI-2 antibody response
We tested the requirement for innate immune
sensing pathways in the antibody response to the
model TI-2 antigen 4-hydroxy-3-nitrophenylacetyl-
Ficoll (NP-Ficoll) by monitoring anti-NP IgM in the
serum of mice after immunization (2). C57BL/6J
mice mounted a robust NP-specific IgM response
by day 4.5 after immunization, which peaked
around day 14.5 after immunization (Fig. 1A and
fig. S1). Similarly, mice that could not signal via
NLRP3, TLR3, TLR7, TLR9, TLR2, TLR4, CD36,
MyD88, TICAM1, or IRAK4, all nucleic acid–sensing
TLRs (Unc93b13d/3d), or all TLRs (Ticam1Lps2/Lps2;
Irak4otiose/otiose) produced normal levels of NP-
specific IgM on day 4.5 after immunization (Fig.
1A). In contrast, Tmem173gt/gt mice and Mb21d1−/−
mice, deficient in the cytosolic DNA-sensing path-
way components stimulator of interferon gene
(STING) and cGMP-AMP synthase (cGAS), respec-
tively, exhibited suboptimal IgM responses to
NP-Ficoll on day 4.5 and for up to 30 days after
immunization (Fig. 1A and fig. S1). Mice lacking
mitochondrial antiviral signaling protein (MAVS),
an adaptor for the cytoplasmic RNA-sensing RIG-
I–like helicases, also produced diminished amounts
of NP-specific IgM (Fig. 1A and fig. S1). Antibody
responses to the TI-1 antigen NP-LPS (LPS, lipo-
polysaccharide) (Fig. 1B), and the TD antigen
b-galactosidase (b-gal) encoded by a nonrepli-
cating recombinant Semliki Forest virus (rSFV)
vector (3) (Fig. 1C), were normal in STING-,
cGAS-, and MAVS-deficient mice.
We evaluated marginal zone (MZ) and B-1 B
cell populations in STING-, cGAS-, and MAVS-deficient mice and found no deficiencies in frequencies or numbers (fig. S2 and supplementary
text), except in the NP-specific populations after
NP-Ficoll immunization (fig. S3). Also, NP-Ficoll
capture by MZ B cells and MZ macrophages was
normal in the mutant mice (fig. S4).
We performed adoptive transfer of C57BL/6J,
STING-, cGAS-, or MAVS-deficient splenic and
1Center for the Genetics of Host Defense, University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard,
Dallas, TX 75390-8502, USA. 2Department of Pediatrics and
Children's Medical Center Research Institute, and McDermott
Center for Human Growth and Development, University of
Texas Southwestern Medical Center, 5323 Harry Hines
Boulevard, Dallas, TX 75390-8502, USA. 3Department of
Microbiology, Tumor and Cell Biology, Karolinska Institutet,
Nobels väg 16, SE-171 77 Stockholm, Sweden. 4Howard
Hughes Medical Institute, Department of Molecular Biology,
University of Texas Southwestern Medical Center, 5323
Harry Hines Boulevard, Dallas, TX 75390-9148, USA.
*These authors contributed equally to this work. †Corresponding
author. E-mail: Bruce.Beutler@UTSouthwestern.edu