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Alums adjuvant action: grease is the word

M Lamine Mbow, Ennio De Gregorio & Jeffrey B Ulmer

2011 Nature America, Inc. All rights reserved.

Alum is the most widely used vaccine adjuvant, but its mechanism of action remains largely unknown. A recent
study shows that alum interacts directly with membrane lipids on the surface of dendritic cells, triggering
signaling cascades that promote CD4+ T cell activation and humoral immune responses (pages 479487).
Vaccines remain the most effective means to
eradicate infectious diseases, and there are
ongoing efforts to apply active immunization
approaches to prevent and treat autoimmune
diseases and cancer. Adjuvants potentiate
antigen-specific immune responses and can
be a key element of vaccine effectiveness.
Therefore, research to better understand adjuvants mechanisms of action, thereby allowing
rational approaches to adjuvant design and
optimization, has become increasingly crucial
to exploit the full potential of vaccinology for
infectious diseases and beyond.
Aluminum salts (alum) have been widely
used as vaccine adjuvants since 1926 (ref. 1).
Alum is the most common adjuvant used in
approved prophylactic vaccines because of its
excellent safety profile and ability to enhance
protective humoral immune responses.
However, the long history behind the use of
alum as an adjuvant contrasts with our poor
understanding of its mechanism of action,
which has been a controversial subject. Early
work suggested that the immune-boosting
capacity of alum was related to its ability to
form a depot with the antigen at the injection
site that favors antigen uptake, processing
and presentation. However, this hypothesis
is not consistent with recent experimental
observations that alum injection sites can
be excised shortly after immunization with
no impact on adjuvanticity2. In vitro experiments have shown that alum increases antigen uptake by dendritic cells (DCs), a key
cell population involved in antigen presentation and immune activation, suggesting
that, at least in part, the adjuvant activity
M. Lamine Mbow and Jeffrey B. Ulmer are at
Novartis Vaccines & Diagnostics, Cambridge,
Massachusetts, USA, and Ennio De Gregorio is at
Novartis Vaccines & Diagnostics, Siena, Italy.
e-mail: jeffrey.ulmer@novartis.com

Vaccine administration site

Draining lymph node

Alum

Antigen

ITAM

ICAM-I

Syk
PI3K

DC

LFA-1

MHC II

TCR

CD80

CD28

CD4+ T cell

Figure 1 Flach et al.8 propose a new mechanism by which alum acts as an adjuvant. (a) Interaction of
alum crystals with sphingomyelin and cholesterol lipids on the plasma membrane of DCs at the vaccine
administration site induces clustering of ITAM-containing receptors and downstream Syk and PI3K
signaling. (b) Antigens are internalized by DCs in the absence of alum uptake. At the same time, DCs
upregulate the expression of MHC II, ICAM-1 and co-stimulatory molecules such as CD80 on their cell
surface. (c) Activated, antigen-loaded DCs dissociate from alum crystals and migrate to lymph nodes,
where they activate antigen-specific CD4+ T cells, which then promote humoral immune responses.
TCR, T cell receptor.

of alum may be explained by its antigen

delivery properties3.
Given the established connection between
innate immune signaling and downstream
adaptive immune responses, researchers have
explored a potential link between the adjuvanticity of alum and its ability to trigger signaling by innate immune receptors. However,
unlike the effects of various agonists of Tolllike receptors (TLRs), the adjuvant effects of
alum are independent of the TLR pathway4.
Recent reports have implicated activation
of the Nalp3 inflammasome pathway5,6 by
alum, but other reports7 do not support a role
for the inflammasome as the primary target
of alum.
In this issue of Nature Medicine, Flach et al.8
show that crystalline alum binds lipid moieties on DCs, which promotes lipid sorting in
the DC plasma membrane. This then triggers
intracellular signal transduction pathways that
lead to the initiation of an immune response.
In addition, the data support the unexpected

nature medicine volume 17 | number 4 | APrIl 2011

and counterintuitive hypothesis that alum promotes antigen uptake without being internalized by antigen-presenting cells. These findings
increase our understanding of the biological
attributes of alum and provide a previously
unknown mechanism for immune stimulation
driven by alum interactions with lipids rather
than protein receptors, providing insights that
might facilitate vaccine design.
Flach et al.8 used the sophisticated experimental approach of atomic force microscopy
to reveal strong and selective binding between
alum and DCs. Further biochemical assays
revealed that specific membrane lipids
sphingomyelin and cholesterolwere key
mediators of these interactions. The sustained
association of alum with DCs requires activity of the kinases Syk and phosphoinositide
3-kinase (PI3K). The authors data suggest that
lipid sorting induced by alum binding of the
plasma membrane of DCs induces clustering
of immunoreceptor tyrosine-based activation
motif (ITAM)-containing receptors, which in
415

 2011 Nature America, Inc. All rights reserved.

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turn activate Syk and PI3K pathways through
a phosphorylation cascade8 (Fig. 1).
The authors elegantly identified the role of
another kinase, extracellular signalregulated
kinase (ERK), in the selective effects of alum
on DCs8. ERK phosphorylation was delayed in
DCs after alum treatment, thereby enabling a
productive interaction between DCs and alum.
In contrast, other immune cells such as macrophages showed early or constant ERK phosphorylation, which rendered them refractory to the
effects of alum. Unexpectedly, although alum
crystals bind DCs strongly, they do not enter
them but instead mediate abortive phagocytosis via the differential regulation of ERK in DCs
versus other types of cells. Yet antigen uptake
is nevertheless facilitated by alum, suggesting
that antigens are delivered into DCs by a route
that does not involve phagocytosis. These data
contrast with a previous study showing that
alum-antigen complexes colocalize in intracellular vesicles of mouse macrophages, and
that the presence of alum destabilizes the
phagosomes, leading to inflammasome activation9. However, it is possible that macrophages
and DCs have a differential ability to internalize alum crystals.

DCs react to cell surface binding of alum by

upregulating the expression of co-stimulatory
molecules (CD80 and CD86) and intercellular
adhesion molecule-1 (ICAM-1), a key adhesion molecule that promotes tight interactions
between DCs and CD4+ T cells via its binding partner lymphocyte functionassociated
antigen-1 (LFA-1). These observations could
help to explain why alum is a strong enhancer
of humoral immune responses, as inhibition
of DC-mediated phagocytosis could facilitate endocytic delivery of soluble antigen
for processing and presentation by major
histocompatibility complex class II (MHC
II) molecules on the surface of DCs and the
consequent promotion of B cell responses via
CD4+ T cells.
Despite its widespread success, alum is not a
universal solution for all vaccines, such as those
requiring induction of potent T helper type 1
T cell responses and cytotoxic T lymphocytes,
for example, in treatment of cancer and chronic
infections. Hence, there is room to improve
alum as an adjuvant. One successful approach
has been to include TLR agonists to provide
direct innate immune stimulation as a complement to facilitated antigen delivery10. A better

understanding of the mechanisms underlying

the immune-enhancing effects of adjuvants is
needed to enable the rational design of safe and
effective vaccines to meet unmet medical needs
in emerging infectious diseases, cancer and
autoimmune diseases. The findings of Flach
et al.8 therefore represent an important step in
this direction.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests:
details accompany the full-text HTML version of the
paper at http://www.nature.com/naturemedicine/.
1. Glenny, A.T. BMJ 2, 244245 (1930).
2. Marrack, P., McKee, A.S. & Munks, M.W. Nat. Rev.
Immunol. 9, 287293 (2009).
3. Morefield, G.L. et al. Vaccine 23, 15881595
(2005).
4. Gavin, A.L. et al. Science 314, 19361938
(2006).
5. Eisenbarth, S.C., Colegio, O.R., OConnor, W.,
Sutterwala, F.S. & Flavell, R.A. Nature 453,
11221126 (2008).
6. Kool, M. et al. J. Immunol. 181, 37553759
(2008).
7. Franchi, L. & Nunez, G. Eur. J. Immunol. 38,
20852089 (2008).
8. Flach, T.L. et al. Nat. Med. 17, 479487 (2011).
9. Hornung, V. et al. Nat. Immunol. 9, 847856
(2008).
10. OHagan, D.T. & De Gregorio, E. Drug Discov. Today 14,
541551 (2009).

Trastuzumab resistance: all roads lead to SRC

Senthil K Muthuswamy
A new study shows how SRC, a nonmembrane tyrosine kinase, is a common signaling node in trastuzumab
resistance caused by different mechanisms in HER2-positive breast cancers (pages 461469). A SRC inhibitor
restored trastuzumab sensitivity in vitro and in mouse tumor models, suggesting a new way to tackle drug resistance
in breast tumors.
Human epidermal growth factor receptor-2
(HER2) is one of the most dominant oncogenes in breast cancer. It is overexpressed in
approximately 20% of human breast cancers
and is associated with poor clinical prognosis
and patient survival1. HER2 belongs to a family of four receptors including HER1, HER3
and HER4 that activates downstream signaling pathways by forming both homo- and heterodimers2. A monoclonal antibody that binds
to the juxtamembrane domain of HER2, trastuzumab, was the first anti-HER2 treatment

Senthil K. Muthuswamy is at the Ontario Cancer

Institute, Campbell Family Breast Cancer Research
Institute, University of Toronto, Toronto, Ontario,
Canada, and at Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York, USA.
e-mail: s.muthuswamy@utoronto.ca

416

that was approved by the US Food and Drug

Administration for clinical use for people with
HER2-positive breast cancer3. Individuals with
metastatic, HER2-positive breast cancer treated
with trastuzumab as an adjuvant and in combination with chemotherapy show significant
clinical benefit3,4.
However, the majority of HER2-positive
individuals possess de novo resistance to trastuzumab or acquire resistance during treatment,
highlighting the need for identifying better
ways to control HER2-positive breast cancers.
Trastuzumab uses multiple mechanisms to
inhibit tumor growth, which include inhibition of downstream signaling by blocking
HER2 homodimers5 and ligand-independent
HER2 heterodimers. Trastuzumab also inhibits
HER2 activation by blocking cleavage of the
extracellular domain of HER2, which leads
to activation of HER2 receptor. In addition,

trastuzumab also functions through induction of antibody-dependent cell-mediated

cytotoxicity6. Although the relative contribution of these modes of action during treatment response is not known, it is likely that
multiple mechanisms of action are engaged
simultaneously during tumor inhibition.
The mechanisms that contribute to the
frequent development of resistance to trastuzumab are only beginning to be understood
and are an active area of investigation7. In this
issue of Nature Medicine, Zhang et al.8 show
that activation of the cytoplasmic tyrosine
kinase SRC is important during development
of trastuzumab resistance. Breast cancer cells
that became spontaneously resistant and
those that were engineered to become resistant through overexpression of insulin-like
growth factor receptor (IGF-1R) or HER1,
or through phosphatase and tensin homolog

volume 17 | number 4 | APrIl 2011 nature medicine