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successive exposure to the same influ-
enza vaccine in humans are recalled
from the same B cell clonotypes (Andrews
et al., 2015). It is possible that this
discrepancy is due to prior antigen expe-
rience in humans and the early GC
response being dominated by memory B
cells, which have faster response kinetics
than the naive B cells. It is not clear how
this would alter the GC response, but
these results suggest that there is a point
where repeated antigen exposure no
longer results in increased diversity.
Perhaps future research, taking into ac-
count these new findings, will finally
reveal how to properly harness the hu-
moral immune response in the develop-
ment of broadly protective vaccines.
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Silence of the ROS

Judith Agudo1 and Brian D. Brown1,2,*
1Genetics and Genomic Sciences Department, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
2Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
*Correspondence: brian.brown@mssm.edu
http://dx.doi.org/10.1016/j.immuni.2016.02.027

Reactive oxygen species (ROS) are generated during T cell activation and serve a signaling function but can
also be damaging. In this issue of Immunity, Zhang et al. (2016) show that miR-23a prevents ROS-elicited
necrosis by suppressing cyclophilin D (PPIF), a regulator of ROS escape from mitochondria.

T cell activation sets off a signaling
cascade that leads to proliferation and
production of cytokines, which are key
to the effector phase of the immune
response. T cell activation also causes
the accumulation of intracellular reactive
oxygen species (ROS) (Devadas et al.,
2002). ROS are highly reactive molecules
derived from oxygen (O2) and mostly
include superoxide (O2
) and hydrogen
peroxide (H2O2). The majority of intracel-
lular ROS are produced either by NAPDH
oxydases (NOX) or by the electron trans-
port chain (ETC). NOX enzymes produce
ROS in the cytoplasm by transferring
one electron to oxygen, forming O2
,
which can further react to form H2O2.
ROS are also formed in the mitochondria
as a by-product of ETC. These ROS accu-
mulate inside the mitochondria and can
be released to the cytoplasm through
the permeability transition pore (PTP).
The increased ROS amounts in activated
T cells are believed to be due to pro-
duction by NOX enzymes and the conse-
quence of a more active metabolic cell
state.
ROS are now known to be important for
intracellular signaling through their ability
to mediate reversible oxidation of pro-
teins by reacting with the thiol groups
of cysteine residues (Sena and Chandel,
2012). The ROS produced during acti-
vation affects T cell function by modifying
phosphatases, such as Shp1, as well
as other proteins (Simeoni and Bogeski,
2015). These changes contribute to
increased cytokine production and prolif-
eration. However, ROS can also damage
cell components through oxidative stress
and ultimately lead to cell death. Thus,
although ROS have an important role in
T cell activation and proliferation, they
can also be harmful to the T cells. T cells
must somehow balance ROS amounts
within the cell to enable ROS-mediated
signaling while preventing ROS-induced
pathological damage. How this is done
is not well understood.
In this issue of Immunity, Zhang et al.
(2016) set out to better understand what
role microRNA (miRNA) have in the CD4+
T cell response to infection. miRNAs are
small non-coding RNAs that complex
with Argonaute proteins to form RNA-
induced silencing complexes (RISC). The
miRNA guides RISC to sequences en-
coded on mRNAs that are partially com-
plementary to the miRNA (Pasquinelli,
2012). miRISC binding prevents transla-
tion of the mRNA and can also accelerate
decay of the transcript. The outcome of
miRNA regulation is decreased protein
translation of its target genes. There are
more than 1,000 miRNAs encoded in
the mammalian genome, many of which
are completely conserved from mouse
to humans. miRNAs have been found
to regulate different aspects of T cell

520 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.

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generally occurs subsequent to initial

T cell expansion, and thus the early death
of the activated Mir23afl/fl-cre CD4+
T cells was unexpected. Further analysis
indicated that the cells were dying not
by apoptosis but by necrosis associated
with mitochondrial swelling and rupture.
This appeared to be caused by excessive
ROS, because overexpression of the
ROS scavenger GPX1 rescued activated
Mir23afl/fl-cre CD4+ T cells from prema-
ture necrotic death. These findings indi-
cate that early after T cell activation, mito-
chondrial ROS rise to a threshold that
can precipitate necrosis, but that this is
controlled by the concomitant increase
in miR-23a. This helps explain why
Listeria infection of the Mir23afl/fl-cre
Figure 1. miR-23a Regulates ROS Leakage from the Mitochondria of Activated T Cells mice led to liver pathology and sys-
The amount of reactive oxygen species (ROS) in the mitochondria increases upon CD4+ T cell activation
due to Ca2+ influx and increased metabolism. ROS can leak out through the mitochondria permeability temic inflammation because the activated
transition pore (PTP), which is opened by peptidylprolyl isomerase F (PPIF or Cyclophilin D) (right). Mir23afl/fl-cre T cells were unable to pre-
Excessive ROS release can lead to cell necrosis. To prevent this, T cells increase miR-23a concentration vent ROS-induced necrosis.
during activation, which then suppresses PPIF by binding to PPIF’s 30 UTR and preventing translation. The
reduction in PPIF keeps the mitonchondria PTP closed and reduces the escape of ROS (left), which
Because miRNAs regulate mRNA, and
maintains CD4+ T cell survival during the early hypermetabolic and inflammatory state that accompanies not ROS, Zhang et al. (2016) looked for
activation. predicted targets of miR-23a that are
known to have a role in necrosis. They
biology, including T cell development, expression of miR-27a or miR-24. Loss of found two conserved target sites for
proliferation, and cytokine production, miR-23a did not affect T cell development miR-23a in the 30 UTR of peptidylprolyl
but the role of most miRNAs expressed or homeostatic maintenance, as shown isomerase F (PPIF or Cyclophilin D), which
in T cells is still unknown. by the facts that T cell numbers were is a vital pore-opening regulator of
Zhang et al. (2016) started by profiling normal in the Mir23afl/fl-cre mice and that the PTP that controls ROS flow from
miRNA expression in Listeria-specific the cells proliferated similar to wild-type the mitochondria (Baines et al., 2005).
CD4+ T cells (LLO118 TCR trans- T cells in response to activation. Quite They confirmed that the PPIF mRNA
genic) at different time points after infec- strikingly, the Mir23afl/fl-cre mice died was a direct target of miR-23a and
tion of mice with Listeria monocyto- within 4–7 days of being infected with a that the PPIF protein was significantly
genes. They found that there was a dose of Listeria that was relatively well upregulated in miR-23a-deficient cells.
group of 45 miRNAs whose expres- tolerated by wild-type mice. Surprisingly, Importantly, when they reversed this
sion gradually increased during the early mortality was not the consequence of upregulation by silencing the expression
expansion phase of activation, then increased bacterial loads; the Listeria of PPIF in Mir23afl/fl-cre cells, they re-
rapidly decreased during contraction, colonies were actually lower in Mir23afl/fl- balanced the intracellular ROS amounts
and increased again as the effector cre mice compared to wild-type mice. and prevented the activated T cells from
T cells transitioned to memory. Among Instead, Zhang et al. (2016) found that prematurely dying. Thus, it appears that
this group was miR-23a, which is en- there were large areas of necrosis in the PPIF, and by extension mitochondrial
coded in the genome as a part of a miRNA liver and elevations of the inflammatory release of ROS, is the main regulatory
cluster that also contains Mir27a and cytokines IL-6 and TNF in the serum of target of miR-23a in preventing T cell ne-
Mir24 and has a paralog cluster en- the Mir23afl/fl-cre mice infected with Liste- crosis during the early effector phase of
coded elsewhere in the genome that is ria, which suggested that the cause of activation (Figure 1). This helps to explain
comprised of Mir23b, Mir27b, and Mir24. death was due to a severe inflammatory how T cells handle the burden of rapid
The three miRNAs in each cluster are response. Closer analysis indicated that ROS production during the high meta-
co-expressed on a polycistronic tran- miR-23a-deficient CD4+ T cells died at a bolic demands of activation and prolifera-
script, but each has a different sequence substantially higher frequency than wild- tion. It will be interesting to explore the
and thus target different sequences. type cells, and as quickly as 6 hr after acti- relevance of miR-23a in CD4+ T cell sur-
To determine whether miR-23a had any vation. Thus, they found that miR-23a was vival in additional contexts associated
function in CD4+ T cells, Zhang et al. (2016) important for the survival of activated with high amounts of ROS, such as radia-
generated mice in which Mir23a was spe- T cells. tion therapy and cancer.
cifically ablated in CD4+ cells by floxing T cell death is a normal and pro- Recently, two different groups have
Mir23a and crossing with Cd4-cre mice grammed consequence of T cell activa- shown that deletion of the entire Mir23a-
(Mir23afl/fl-cre). The gene for miR-23a tion that occurs by apoptosis. However, Mir27a-Mir24-2 cluster alters the commit-
was floxed in a manner that did not disrupt apoptotic activation-induced cell death ment of CD4+ T cells to become T helper

Immunity 44, March 15, 2016 ª2016 Elsevier Inc. 521

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2 (Th2) cells and affects airway allergic
reactions (Cho et al., 2016; Pua et al.,
2016). The effects were predominantly
dependent upon miR-24 and miR-27,
which were found to directly and indirectly
regulate IL-4 and Gata3, and no increase in
activated CD4+ T cell death was reported.
The results are not necessarily contradic-
tory with Zhang et al. (2016) because the
aforementioned studies focused on Th2
cell responses and used OVA sensitization
and did not involve a pathogen such as
Listeria, which might induce higher ROS
production in CD4+ T cells. There might
also be competing effects of the miRNAs
that are lost when all three were deleted.
The findings of these three new studies
highlight the breadth of control mediated
by the miR-23a cluster on CD4+ T cell
biology and suggest context-dependent
functions, which will need to be further
explored. It will be equally important to
determine whether miR-23a, or the other
miRNAs in the cluster, has a similar role in
humans and whether their dysregulation
contributes to pathology.
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Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, N.S.,
Osinska, H., Hambleton, M.A., Brunskill, E.W.,
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Cho, S., Wu, C.-J., Yasuda, T., Cruz, L.O.,
Khan, A.A., Lin, L.-L., Nguyen, D.T., Miller, M.,
Lee, H.-M., Kuo, M.-L., et al. (2016). J. Exp.
Med. 213, 235–249, http://dx.doi.org/10.1084/
jem.20150990.
Devadas, S., Zaritskaya, L., Rhee, S.G., Oberley,
L., and Williams, M.S. (2002). J. Exp. Med. 195,
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Pasquinelli, A.E. (2012). Nat. Rev. Genet. 13,
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Pua, H.H., Steiner, D.F., Patel, S., Gonzalez, J.R.,
Ortiz-Carpena, J.F., Kageyama, R., Chiou, N.-T.,
Gallman, A., de Kouchkovsky, D., Jeker, L.T.,
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issue.

Chitosan: An Adjuvant with an Unanticipated STING

Nicolas Riteau1 and Alan Sher1,*
1Immunobiology Section, Laboratory of Parasitic Diseases, National Institute for Allergy and Infectious Diseases, National Institutes of Health,

Bethesda, MD 20892, USA

*Correspondence: asher@niaid.nih.gov
http://dx.doi.org/10.1016/j.immuni.2016.03.002

Adjuvants promote adaptive immunity through the triggering of innate signals that are largely poorly under-
stood. In this issue of Immunity, Lavelle and colleagues describe an unexpected role for the DNA sensing
cGAS-STING pathway in the mechanism of action of the Th1 cell-promoting polysaccharide adjuvant chitosan.

Adjuvants are an important gateway to
better vaccines. A superb illustration of
this point is the experimental recombinant
RTS,S vaccine used for vaccination
against malaria that became significantly
protective in humans only after formula-
tion with the appropriate lipid-based adju-
vants (Hoffman et al., 2015). Nevertheless,
the science of adjuvant development re-
mains a backwater of translational immu-
nology because of our poor understanding
of how to selectively trigger and boost
different classes of immune responses.
It is generally thought that adjuvants
work by linking innate and adaptive immu-
nity and, indeed, Charles Janeway was
clearly thinking of adjuvants when he
coined this mechanism as the ‘‘dirty little
secret’’ of the immune system (Janeway,
1989). The discovery of pattern-recogni-
tion receptors (PRRs) and their corre-
sponding microbial PAMPs has helped
define the molecular basis of innate im-
mune recognition in adjuvant function
and has led to the development of new
adjuvant products. However, the mecha-
nism of action of many natural adjuvants
remains poorly defined, a situation that
might reflect the underappreciated contri-
bution of host-derived ‘‘danger signals.’’
In this issue of Immunity, Carroll et al.
(2016) dissect the pathway by which a
natural adjuvant, chitosan, induces den-
dritic cell (DC) activation to promote T
helper 1 (Th1) cell immune responses. In
so doing, they uncover an unanticipated
role for the cytosolic DNA sensing cGAS-
STING pathway in this process. Impor-
tantly, they present intriguing evidence
that the chitosan-induced trigger of this
response is host mitochondrial DNA that
is likely recognized as a danger-associ-
ated molecular pattern (DAMP).
Chitosan is a polysaccharide derived
from the partial deacetylation of chitin,
the second most abundant natural poly-
mer. There are a vast variety of chitosan
products that differ in their biochemical
and physical characteristics. The chitosan
preparation utilized in this study was pre-
viously shown by the authors to promote
peritoneal Th1 and Th17 cell responses
to ovalbumin. In the current study, they
demonstrate that the same polymer
triggers highly polarized Th1 cell cyto-
kine and immunoglobulin G2c (IgG2c)
antibody responses to a recombinant

522 Immunity 44, March 15, 2016 ª2016 Elsevier Inc.