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and counterintuitive hypothesis that alum pro-

motes antigen uptake without being internal-ized by antigen-presenting cells. These findings

increase our understanding of the biologicalattributes of alum and provide a previously

unknown mechanism for immune stimulationdriven by alum interactions with lipids rather

than protein receptors, providing insights thatmight facilitate vaccine design.

Flach et al.8used the sophisticated experi-

mental approach of atomic force microscopyto reveal strong and selective binding betweenalum and DCs. Further biochemical assays

revealed that specific membrane lipidssphingomyelin and cholesterolwere key

mediators of these interactions. The sustainedassociation of alum with DCs requires activ-

ity of the kinases Syk and phosphoinositide3-kinase (PI3K). The authors data suggest that

lipid sorting induced by alum binding of theplasma membrane of DCs induces clustering

of immunoreceptor tyrosine-based activationmotif (ITAM)-containing receptors, which in

Vaccines remain the most effective means toeradicate infectious diseases, and there are

ongoing efforts to apply active immunization

approaches to prevent and treat autoimmunediseases and cancer. Adjuvants potentiateantigen-specific immune responses and can

be a key element of vaccine effectiveness.Therefore, research to better understand adju-

vants mechanisms of action, thereby allowingrational approaches to adjuvant design and

optimization, has become increasingly crucialto 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 itsexcellent 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 poorunderstanding of its mechanism of action,

which has been a controversial subject. Earlywork suggested that the immune-boosting

capacity of alum was related to its ability toform a depot with the antigen at the injection

site that favors antigen uptake, processingand presentation. However, this hypothesis

is not consistent with recent experimentalobservations that alum injection sites can

be excised shortly after immunization withno impact on adjuvanticity2. In vitroexperi-ments have shown that alum increases anti-

gen uptake by dendritic cells (DCs), a keycell population involved in antigen presen-

tation and immune activation, suggestingthat, at least in part, the adjuvant activity

of alum may be explained by its antigen

delivery properties3.Given the established connection between

innate immune signaling and downstreamadaptive immune responses, researchers have

explored a potential link between the adju-vanticity of alum and its ability to trigger sig-

naling by innate immune receptors. However,unlike the effects of various agonists of Toll-

like receptors (TLRs), the adjuvant effects of

alum are independent of the TLR pathway4.Recent reports have implicated activationof the Nalp3 inflammasome pathway5,6 by

alum, but other reports7do not support a role

for the inflammasome as the primary targetof alum.

In this issue of Nature Medicine, Flach et al.8show that crystalline alum binds lipid moi-

eties on DCs, which promotes lipid sorting inthe DC plasma membrane. This then triggers

intracellular signal transduction pathways thatlead to the initiation of an immune response.

In addition, the data support the unexpected

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

Alums adjuvant action: grease is the wordM Lamine Mbow, Ennio De Gregorio & Jeffrey B Ulmer

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).

Vaccine administration site Draining lymph node

a b cAlum

Antigen

DC CD4+ T cell

ITAM

Syk

PI3K

ICAM-ILFA-1

TCRMHC II

CD80 CD28

Figure 1 Flach et al.8propose 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.

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understanding of the mechanisms underlyingthe immune-enhancing effects of adjuvants is

needed to enable the rational design of safe andeffective vaccines to meet unmet medical needs

in emerging infectious diseases, cancer andautoimmune diseases. The findings of Flachet al.8therefore represent an important step in

this direction.

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests:details accompany the full-text HTML version of thepaper at http://www.nature.com/naturemedicine/.

1. Glenny, A.T. BMJ2, 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. Today14,

541551 (2009).

DCs react to cell surface binding of alum byupregulating the expression of co-stimulatory

molecules (CD80 and CD86) and intercellularadhesion molecule-1 (ICAM-1), a key adhe-

sion molecule that promotes tight interactionsbetween DCs and CD4+T cells via its bind-

ing partner lymphocyte functionassociated

antigen-1 (LFA-1). These observations couldhelp to explain why alum is a strong enhancerof humoral immune responses, as inhibition

of DC-mediated phagocytosis could facili-tate endocytic delivery of soluble antigen

for processing and presentation by majorhistocompatibility complex class II (MHC

II) molecules on the surface of DCs and theconsequent promotion of B cell responses via

CD4+T cells.Despite its widespread success, alum is not a

universal solution for all vaccines, such as thoserequiring induction of potent T helper type 1

T cell responses and cytotoxic T lymphocytes,

for example, in treatment of cancer and chronicinfections. Hence, there is room to improvealum as an adjuvant. One successful approach

has been to include TLR agonists to providedirect innate immune stimulation as a comple-

ment to facilitated antigen delivery10. A better

turn activate Syk and PI3K pathways througha phosphorylation cascade8(Fig. 1).

The authors elegantly identified the role ofanother kinase, extracellular signalregulated

kinase (ERK), in the selective effects of alumon 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 macro-phages showed early or constant ERK phosphory-

lation, which rendered them refractory to theeffects of alum. Unexpectedly, although alum

crystals bind DCs strongly, they do not enterthem but instead mediate abortive phagocyto-

sis via the differential regulation of ERK in DCsversus other types of cells. Yet antigen uptake

is nevertheless facilitated by alum, suggestingthat antigens are delivered into DCs by a route

that does not involve phagocytosis. These datacontrast with a previous study showing that

alum-antigen complexes colocalize in intra-

cellular vesicles of mouse macrophages, andthat the presence of alum destabilizes thephagosomes, leading to inflammasome activa-

tion9. However, it is possible that macrophages

and DCs have a differential ability to internal-ize alum crystals.

trastuzumab also functions through induc-tion of antibody-dependent cell-mediated

cytotoxicity6. Although the relative contri-bution of these modes of action during treat-

ment response is not known, it is likely thatmultiple mechanisms of action are engaged

simultaneously during tumor inhibition.

The mechanisms that contribute to thefrequent development of resistance to trastu-zumab are only beginning to be understood

and are an active area of investigation7. In this

issue of Nature Medicine, Zhang et al.8showthat activation of the cytoplasmic tyrosine

kinase SRC is important during developmentof trastuzumab resistance. Breast cancer cells

that became spontaneously resistant andthose that were engineered to become resis-

tant through overexpression of insulin-likegrowth factor receptor (IGF-1R) or HER1,

or through phosphatase and tensin homolog

Human epidermal growth factor receptor-2(HER2) is one of the most dominant onco-

genes in breast cancer. It is overexpressed inapproximately 20% of human breast cancers

and is associated with poor clinical prognosisand patient survival1. HER2 belongs to a fam-

ily of four receptors including HER1, HER3

and HER4 that activates downstream signal-ing pathways by forming both homo- and het-erodimers2. A monoclonal antibody that binds

to the juxtamembrane domain of HER2, tras-

tuzumab, was the first anti-HER2 treatment

that was approved by the US Food and DrugAdministration for clinical use for people with

HER2-positive breast cancer3. Individuals withmetastatic, HER2-positive breast cancer treated

with trastuzumab as an adjuvant and in com-bination with chemotherapy show significant

clinical benefit3,4.

However, the majority of HER2-positiveindividuals possess de novoresistance to trastu-zumab or acquire resistance during treatment,

highlighting the need for identifying betterways to control HER2-positive breast cancers.

Trastuzumab uses multiple mechanisms toinhibit tumor growth, which include inhibi-

tion of downstream signaling by blockingHER2 homodimers5and ligand-independentHER2 heterodimers. Trastuzumab also inhibits

HER2 activation by blocking cleavage of theextracellular domain of HER2, which leads

to activation of HER2 receptor. In addition,

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

Trastuzumab resistance: all roads lead to SRCSenthil 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 vitroand in mouse tumor models, suggesting a new way to tackle drug resistance

in breast tumors.