Inflammatory mediators are insufficient for fulldendritic cell activation and promote expansionof CD4+ T cell populations lacking helper function

Roman Sporri1 & Caetano Reis e Sousa

Dendritic cells (DCs) can be activated directly by triggering of receptors for pathogens or, indirectly, by exposure to inflammatory

signals. It remains unclear, however, whether the two pathways result in qualitatively similar DCs or lead to equivalent adaptive

immune responses. Here we report that indirect activation by inflammatory mediators generated DCs that supported CD4+ T cell

clonal expansion but failed to direct T helper cell differentiation. In contrast, exposure to pathogen components resulted in fully

activated DCs that promoted T helper responses. These results indicate that inflammation cannot substitute for contact with

pathogen components in DC activation and suggest that the function of pattern recognition by DCs is to couple the quality

of the adaptive immune response to the nature of the pathogen.

Dendritic cells (DCs) are the main antigen-presenting cells (APCs)regulating T cell responses1. It is now generally accepted that, in theabsence of external stimuli, DCs exist in a resting state in which theyhave only a limited ability to prime naive T cells. However, in responseto infection, DCs undergo many phenotypic and functional changes,which coordinately result in an improved ability to interact withT cells and promote T cell clonal expansion and differentiation. Thisconversion of resting DCs into effector APCs is often loosely termedDC activation2. However, it is becoming increasingly clear thatactivation is not a linear process and that the nature, intensity and/or duration of the activation signal are important in determining theeffector function of DCs3. Therefore, defining DC-activating signals iscritical for understanding and manipulating adaptive immunity.

Many signals have been shown to induce at least some aspects ofDC activation2,3. Among the most powerful of these are microbial andviral products (pathogen-associated molecular patterns (PAMPs)4),which are directly recognized by pattern-recognition receptors (PRRs),including members of the Toll-like receptor (TLR) family5,6. PRRscontrol the expression of many innate response genes and can directlysignal for DC activation. In addition, PRR signaling in both immuneand nonimmune cells often leads to the synthesis of inflammatorycytokines such as interferon-a (IFN-a) or IFN-b, tumor necrosisfactor (TNF) and interleukin 1 (IL-1), which can also promote DCactivation indirectly7–13. Indeed, many features of DC activationinduced by direct PRR triggering are due to secondary effects ofcytokines made by the DCs themselves. This is the case for the TLR-induced upregulation of costimulatory molecules that in many casescan be ascribed to autocrine and paracrine effects of interferons and

IL-15 produced by the cells14–17. Thus, inflammatory cytokines couldrepresent the actual mediators of DC activation in vivo and may serveto trigger adaptive immune reactions in response to pathogens thatfail to engage DC-expressed PRRs. In addition, TLR signaling pro-motes the synthesis of inflammatory mediators other than cytokines,including soluble molecules (such as chemokines, prostaglandins andleukotrienes) and membrane-bound molecules (such as ligands for theactivatory receptor NKG2D18), all of which may potentially lead to DCactivation and thereby act as links between innate and adaptiveimmunity2. However, it is not known whether inflammatory media-tors produced in physiological amounts in vivo can indeed substitutefor direct PAMP recognition in APC activation and T cell priming.Here we address this issue by comparing direct and indirect activationinduced by microbial stimuli in mixtures of TLR-sufficient and TLR-deficient DCs. TLR signaling resulted in activated DCs that primed aneffective T helper type 1 (TH1) response, whereas indirect activationby inflammatory mediators alone induced DCs that supported CD4+

T cell clonal expansion but did not promote TH1 or TH2 effectordifferentiation. These findings indicate that direct recognition ofPAMPs by DCs is critical for priming an appropriate T cell responseand suggest that inflammatory mediators can amplify but not initiateadaptive immune responses.

RESULTS

Indirect DC maturation without IL-12 production

To address the relative contribution of direct TLR signaling versusparacrine signals to DC activation in vitro, we designed a coculturesystem using DCs differentiated from bone marrow progenitors. Half

Published online 16 January 2005; doi:10.1038/ni1162

Immunobiology Laboratory, Cancer Research UK, London Research Institute, London WC2A 3PX, UK. 1Present address: Institute for Microbiology, Swiss Federal Instituteof Technology, ETH Honggerberg, 8093 Zurich, Switzerland. Correspondence should be addressed to C.R.S. (caetano@cancer.org.uk).

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the cells were from B6.SJL mice, expressed a functional TLR4 gene(Tlr4) and responded directly to lipopolysaccharide (LPS; activatorDCs: wild-type, H-2b and CD45.1). The other half were fromC3H/HeJ mice, expressed a mutant TLR4 (test DCs: Tlr40/0, H-2k

and CD45.2) and could respond to LPS only indirectly. As controls,we set up parallel cultures with DCs expressing functionalTLR4 (control DCs from C3H/HeN mice; Tlr4+/+, H-2k andCD45.2) similarly mixed with activator DCs. We monitoredLPS-dependent DC activation from a ‘T cell perspective’ by assessingthe ability of DCs to provide T cells with T cell receptor (TCR)ligands (signal 1), costimulation (signal 2) and signals that direct Thelper polarization (signal 3)19. In practice, this involved measuringincreased major histocompatibility complex (MHC) class II expres-sion and presentation of the model antigen hen egg lysozyme (HEL),upregulation of CD86 and CD40, and production of the IL-12p40 subunit. Use of the CD45 allotype or the MHC haplotype

enabled us to discriminate between the test (or control) DCs and theactivator cells.

We used the C4H3 antibody specific for the HEL peptide consistingof amino acids 46–61 (HEL(46–61)) in the context of H-2 I-Ak tomeasure the ability of test and control DCs to process and presentHEL protein. LPS-free HEL alone was presented poorly by eitherTlr4+/+ control DCs or Tlr40/0 test DCs. However, as reported20, therewas a notable increase in the C4H3 signal when HEL was addedtogether with LPS to cultures of Tlr4+/+ control bone marrow–derivedDCs (Fig. 1a, left). As expected, we found no such increase withcultures of Tlr40/0 test DCs (Fig. 1a, left). We noted a similar LPS- andTLR4-dependent pattern for the increase in surface expression ofMHC I-Ak (Fig. 1a, right) and for the upregulation of CD86 andCD40 expression (Fig. 1b). In contrast, when we added wild-typeTLR4-competent activator DCs to the culture, the bystander test DCs(Tlr40/0 CD45.2) showed C4H3 staining comparable to that of the

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Figure 1 Indirect DC activation by inflammatory signals in vitro. (a) Antigen processing and presentation, (b) expression of costimulatory molecules and

(c) IL-12 production by test or control DCs after LPS treatment for 12 h. (a,b) ‘fold increase’ in normalized (Norm.) MFI for C4H3 (a, left), MFI for I-Ak

(a, right), CD86 (b, left) and CD40 (b, right) on gated CD11chiCD45.2+H-2Kk live (TO-PRO3—) cells. (c) CD45.2 versus intracellular IL-12 p40 on gated

CD11chi cells. WT, wild-type. Results are representative of three independent experiments.

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Figure 2 Indirect DC activation by inflammatory signals

in vivo. (a) Experimental design. i.v., intravenously; BM1

and BM2, bone marrow 1 and 2, respectively. (b) Surface

expression of B7-2, CD40 and I-Ab or (c) production of IL-12

p40 by splenic DCs for mice treated with CpG 12 h before.

(b) MFI values of staining for I-Ab (left), CD86 (middle) or

CD40 (right) on gated CD11chi, live (TO-PRO3—) cells and,

where appropriate, further gated on the CD45.2 marker.

(c) CD45.2 versus intracellular IL-12 p40 after gating

on CD11chi cells. Results are representative of three

independent experiments.

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corresponding control DCs (Tlr4+/+ CD45.2) (Fig. 1a, left). Similarly,the presence of TLR4-competent activator DCs enabled the Tlr40/0

CD45.2 DCs to upregulate MHC I-Ak, CD86 and CD40 (Fig. 1a,b).This effect of activator cells was not dependent on cell contact, ascocultures across a transwell produced similar results (data notshown). Thus, soluble signals produced by DCs in response to TLRstimulation in vitro are sufficient to activate other DCs in trans toupregulate MHC presentation and costimulation.

IL-12 production by DCs is strongly linked to TLR triggering,which induces transcription of the IL-12 p40 subunit6. To determinewhether factors produced by LPS-stimulated activator cells could alsoinduce IL-12 production by Tlr40/0 DCs in trans, we stained the cellsfor intracellular IL-12 p40. In the absence of LPS, no IL-12 p40 wasdetectable in test, control or activator cells (Fig. 1c). As expected, bothactivator and bystander control DCs stained for the cytokine whenLPS was added (Fig. 1c). In contrast, bystander test Tlr40/0 DCs failedto stain for IL-12 p40 in the same conditions (Fig. 1c), although theydid so in response to CpG-containing DNA (CpG), a TLR9 ligand,demonstrating that they were not intrinsically defective in IL-12production (data not shown). Thus, inflammatory mediators pro-duced by activated DCs in vitro cannot substitute for direct TLRtriggering in IL-12 induction.

We next tested whether these findings also applied in vivo, wheremany cell types besides DCs express relevant TLRs and synthesizeinflammatory mediators. We made mixed bone marrow radiationchimeras in which the host and half the hematopoietic cells weredeficient in the adaptor molecule MyD88 (Myd88�/�) and weretherefore unresponsive to TLR9 signaling5, while the remaining cellswere wild-type and fully responsive (Fig. 2). In such chimeras,injection of CpG can activate Myd88�/� DCs only indirectly, throughmediators made by Myd88+/+ leukocytes. As a positive control, weinjected CpG into nonchimeric wild-type mice and induced upregula-tion of MHC class II, CD86 and CD40 on splenic DCs, as well asproduction of IL-12 p40 (Fig. 2b,c). In contrast, all signs of DCsactivation were abrogated in Myd88�/� nonchimeric negative controlmice (Fig. 2b,c). However, in chimeric mice, both Myd88�/� andMyd88+/+ DCs upregulated surface markers to a similar extent,although in line with the results obtained in vitro, only theMyd88+/+ DCs produced IL-12 p40 (Fig. 2b,c). We obtained identicalresults with a MyD88-dependent DC activator derived fromToxoplasma gondii21 (data not shown). The failure of Myd88�/� DCsin the chimeras to make IL-12 in response to CpG was not due totheir inability to respond to IL-1 or IL-18 (ref. 22), which also signalthrough MyD88-coupled receptors, as we obtained identical datain chimeras made with mixtures of Tlr9+/+ and Tlr9�/� bone marrowin which IL-1 and IL-18 signaling were unimpaired (discussed below).Likewise, Myd88�/� DCs upregulated surface markers but did notproduce IL-12, even when chimeras were made using Myd88+/+ hosts(discussed below), indicating that even inflammatory mediatorsproduced by nonhematopoietic cells and/or radioresistant leukocytessuch as Langerhans cells23 cannot compensate for direct TLR trigger-ing in inducing IL-12 production by DCs in vivo.

Indirect activation results in functional DC maturation

The abundant expression of CD86, CD40 and MHC class II moleculesseen in TLR-deficient DCs activated in trans by inflammatory signalsresembled the phenotype of mature DCs, originally defined as cellsthat have an increased capacity to stimulate T cell proliferation24.However, trans-activated DCs could still express an altered overallrepertoire of costimulatory molecules, making them inferior tocis-activated DCs in stimulating T cells. To test this, we purified

splenic DCs from mixed chimeras injected with CpG, fixed them withparaformaldehyde to prevent further functional changes and usedthem as stimulators for TCR-transgenic naive CD4+ T cells in thepresence of the appropriate peptide. DCs purified from CpG-treatedwild-type nonchimeric mice were far superior stimulators than wereDCs from PBS-injected mice, demonstrating that they had undergonematuration in vivo (Fig. 3). Tlr9+/+ and Myd88+/+ DCs also underwentmaturation in chimeric mice injected with CpG, as indicated by thefact that they were nearly as potent as the corresponding cells fromnonchimeric mice at stimulating the transgenic CD4+ cells (Fig. 3).Notably, the same was true of the Tlr9�/� or Myd88�/� DCs in thechimeras, which could only have responded to CpG in trans (Fig. 3).In contrast, Tlr9�/� or Myd88�/� DCs purified from nonchimericCpG-treated mice were poor stimulators of naive CD4+ T cells andacted identically to DCs from PBS-injected groups, confirming thatsuch mice cannot respond to CpG (Fig. 3). These results show thattrans-activated DCs are equivalent to their cis-activated counterpartsin stimulating T cell proliferation, indicating that they have similaroverall costimulatory potential. We conclude that indirect activationof DCs in vivo by inflammatory signals does not lead to IL-12production but manifests itself in upregulation of MHC and costi-mulatory molecules, as well as in functional maturation, allowing forenhanced proliferation of naive CD4+ T cells.

Indirectly activated DC drive T cell clonal expansion

Given this split between maturation and IL-12 production, we soughtto determine whether DCs matured in trans by inflammatory med-iators are able to prime a CD4+ T cell response in vivo. We tookadvantage of the known MHC restriction of TCR transgenic T cells tomake mixed bone marrow chimeras in which the only APCs capableof presenting a given model antigen to the T cell in question wereunable to respond directly to the TLR ligand used as the adjuvant

from test chimeraTlr9 –/– or Myd88 –/– DCs Tlr9 –/– or Myd88 –/– DCs

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Figure 3 Ex vivo T cell stimulatory capacity of directly versus indirectly

activated DCs. Nonchimeric mice (CD45.2 wild-type, Tlr9�/� or Myd88�/�),

test chimeric mice (CD45.2 Tlr9�/� or Myd88�/� + CD45.1 wild-type -

CD45.2 Tlr9�/� or Myd88�/�) or control chimeric mice (CD45.2

wild-type + CD45.1 wild-type - CD45.2 Tlr9�/� or Myd88�/�) were

injected with PBS or CpG intravenously. Then, 12 h later, CD45.2 splenic

DCs were purified by cell sorting, fixed with paraformaldehyde and used

as stimulators for naive OT-II cells. Results represent [3H]thymidineincorporation at 48 h and are representative of two independent experiments

for each chimera combination.

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(Fig. 4a). We then compared the T cell responses induced by the APCsin these test chimeras with those induced in control chimeras in whichthe APCs could both present antigen and respond directly to adjuvant(Fig. 4a). The combinations of chimeras, antigens, adjuvants andresponding CD4+ T cells used are described in Table 1.

Independently of the combination tested, in all cases adoptivelytransferred CD4+ T cell populations expanded to the same degreewhether primed on APCs that had been activated directly or indirectly.For example, inclusion of CpG as an adjuvant for ovalbumin (OVA)increased the frequencies of OVA-responsive DO-11.10 (Fig. 4b) orOT-II (Supplementary Fig. 1 online) T cells in the draining lymphnodes of both test and control chimeras. Analysis of carboxyfluor-escein diacetate succinimidyl ester (CFSE) dilution profiles furthershowed that the adoptively transferred CD4+ T cells underwent asimilar number of divisions in the test and control chimeras immu-nized with OVA and CpG (Fig. 4c and Supplementary Fig. 1 online).In addition, the transgenic T cells accumulated and persisted to asimilar extent in the draining lymph nodes of both test and controlchimeras, as determined by analysis of absolute cell numbers at thepeak of expansion (day 7 after immunization; Table 1) or at a timewhen the expanded T cell pool was already contracting (day 14 afterimmunization; data not shown). Thus, exposure to physiologicalconcentrations of inflammatory signals in vivo is sufficient to generatetrans-activated APCs that support T cell clonal expansion to the sameextent as their directly activated counterparts.

Indirectly activated DCs do not drive helper responses

Many TLR ligands drive TH1 responses5. Indeed, when we analyzedthe CD4+ T cells from control chimeras immunized with OVA plusadjuvant, a substantial fraction stained positive for IFN-g, whereas

almost none showed this phenotype in the absence of TLR ligand(Fig. 4c and Supplementary Fig. 2 online). In contrast, IFN-g+CD4+

cells were absent in test chimeras, regardless of whether TLR ligand wasused for the immunization (Fig. 4c and Supplementary Fig. 2 online).These T cells also did not stain for IL-4, indicating that they had notdifferentiated into TH2 cells (data not shown; discussed below). Thefailure of the T cells in test chimeras to differentiate into IFN-gproducers was not due to an insufficient number of cell divisions25;although IFN-g staining in the control chimeras was present exclusivelyin a small fraction (10%) of those T cells that had undergone multipledivision cycles (representing about 58% of total transgenic cells;Fig. 4c), test chimeras had a similar frequency (61%; Fig. 4c) of highlydivided cells (Fig. 4c and Supplementary Fig. 1 online). We concludethat APCs activated indirectly in vivo by inflammatory mediators cansupport CD4+ T cell clonal expansion but do not direct TH1 effectorcell differentiation.

As a further assessment of the ability of indirectly activated APCs toprime an adaptive immune response in vivo, we measured antibodyproduction in the chimeras. TH1 cells help B cells switch to immu-noglobulin G2a (IgG2a) production, whereas TH2 cells are associatedwith the induction of IgG1 (ref. 26). The immunoglobulin switch isstrictly dependent on cognate interactions between antigen-specificT and B cells. Therefore, in the case of ‘combination 1’ test chimeras,only B cells of BALB/c origin (H-2d) are able to receive help from theCD4+ T cells primed by indirectly activated (H-2d) APCs (Table 1).We used the fact that BALB/c B cells produce immunoglobulins ofthe a allotype, whereas B cells of B10.BR origin produce the b allotype,to measure the OVA-specific antibody (anti-OVA) response madeby H-2d B cells in combination 1 test versus control chimeras. Serafrom control chimeras immunized with OVA plus CpG showed

BM1: generatesAPCs that can present

antigen and eithercan (control) or

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Figure 4 CD4+ T cells recognizing antigen on indirectly activated APCs clonally expand but do not become IFN-g-producing effectors. (a) Experimental

design. 7, with or without. (b) DO-11.10 T cells were adoptively transferred into mixed radiation chimeras as described in Table 1 (Combination 1). Then,

1 d later, mice were immunized in the hind footpads with OVA with or without CpG. Expansion of DO-11.10 cell populations in popliteal lymph nodes was

assessed by flow cytometry after 3 d. Contour plots represent staining for CD4 and KJ1-26 of cells in a lymphocyte scatter gate. Numbers beside outlined

areas indicate percent of cells in that area. (c) As described in b, but with CFSE-labeled DO-11.10 CD4+ T cells. At 3 d after immunization, IFN-g productionand proliferation of DO-11.10 T cells in the popliteal lymph nodes were assessed after in vivo restimulation with OVA peptide. Data represent CFSE profiles

versus IFN-g staining after gating on CD4+ KJ1-26+ lymphocytes. Numbers in boxes indicate the percentage of IFN-g+ DO-11.10 cells. Numbers above

horizontal lines indicate the percentage of highly divided DO-11.10 cells. Results are representative of three (b) or two (c) independent experiments.

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considerable titers of OVA-specific IgMa and IgG2aa but almost noIgG1a (Fig. 5a), indicating that they contained H-2d-restricted OVA-specific TH1 cells. In contrast, although anti-OVA IgMa was present,anti-OVA IgG2aa was completely absent from the sera taken from testchimeras, consistent with the failure to detect IFN-g+ DO-11.10 cellsin those mice (Fig. 5a). The same sera also contained no OVA-specificIgG1a (Fig. 5a), suggesting that the T cells in these mice had notbecome TH2 effectors. As a positive control for the latter, whenflagellin was used as adjuvant27,28, anti-OVA IgG1a became readilydetectable (Supplementary Fig. 3 online).

A possible caveat for the experiment presented above comes fromthe fact that the BALB/c-derived B cell compartment in the testchimeras was unresponsive to TLR9 triggering (Table 1) and that

direct signaling by CpG could be required for B cells to switch toIgG2a29. To show that MyD88-deficient OVA-specific B cells are notintrinsically incapable of switching to IgG2a, we adoptively transferredOT-II T cells into Myd88�/� mice and immunized them with OVAplus CpG plus wild-type bone marrow–derived DCs. The rationalebehind this experiment was that the injected wild-type DCs shouldprime a robust TH1 response and lead to IgG2a production by OVA-specific B cells, unless the latter require a direct CpG signal to switch.Indeed, administration of wild-type DCs together with the antigenand adjuvant ‘rescued’ the deficiency of Myd88�/� mice in generatingantigen-specific IgG2a (Fig. 5b). Thus, direct CpG triggering of B cellsis not essential for IgG2a switching in the face of a strong TH1response and, therefore, the failure to find IgG2a production in test

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Figure 5 Indirectly activated APCs do not support the priming of helper

T cells. (a) Combination 1 chimeric mice (Table 1) were immunized in the

hind footpads with OVA plus CpG. On day 7, sera were tested for the

presence of OVA-specific IgMa, IgG1a and IgG2aa by ELISA. (b) OT-II T cells

were adoptively transferred into Myd88�/� or wild-type H-2b mice. Then,

1 d later, mice were immunized in the hind footpads with CpG and OVA.

Some groups also received 1.5 � 106 wild-type bone marrow–derived DCs

(+ WT-DC). Then, 7 d later, OVA-specific IgG2a in sera was measured byELISA. Results are presented as mean 7 s.d. of triplicate determinations

of endpoint dilution titers and are representative of (a) three or (b) two

independent experiments. a, anti-.

Table 1 Experiment summary

Cell combinations Experiments

Combination BM1 BM2 Host T cells Restriction Antigen Adjuvant Expt. Chimera Total cells Fold expansion

1 Test: Myd88�/�, H-2d (BALB/c) WT, H-2k (B10.BR) B10.BR DO-11.10 I-Ad OVA CpG 1 Test: 26,921 10.2

Control: WT, H-2d (BALB/c) Control: 28,008 10.6

2 Test: 60,118 17.9

Control: 58,138 17.3

3 Test: 42,371 18.9

Control: 44,217 19.7

2 Test: Myd88�/�, H-2b (C57BL/6) WT, H-2k (B10.BR) B10.BR OT-II I-Ab OVA CpG 1 Test: 78,696 11.7

Control: WT, H-2b (C57BL/6) Controla: 91,923 13.7

2 Test: 68,157 14.1

Control: 69,418 14.4

3 Test: Tlr9�/�, H-2b (C57BL/6) WT, H-2k (B10.BR) B10.BR OT-II I-Ab OVA CpG 1 Test: 94,655 14.1

Control: WT, H-2b (C57BL/6) Controla: 91,923 13.7

2 Test: 72,675 15.0

Controla: 69,418 14.4

4 Test: Tlr40/0, H-2k (C3H/HeJ) WT, H-2b (C57BL/6) C57BL/6 3A9 I-Ak HEL LPS 1 Test: 51,088 10.8

Control: WT, H-2k (C3H/HeN) Control: 54,387 11.5

2 Test: 61,726 11.7

Control: 58,973 11.2

Data represent the average number of transgenic T cells recovered from the popliteal lymph nodes and their population expansion relative to that of unimmunized controls 7 d afterinjection of antigen plus adjuvant in the hind footpads.aExperiments including combinations 2 and 3 were set up in parallel and the same wild-type chimeric control mice were used for both.

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chimeras may be attributed to the failure of trans-activated APCs toprime appropriate TH1 effectors. Consistent with this conclusion,OVA-specific delayed-type hypersensitivity, another TH1-dependentresponse, was also impaired in the test chimeras (data not shown). Weconclude that indirect activation by inflammatory signals in vivoresults in mature DCs that can support CD4+ T cell clonal expansionbut do not prime an effector response.

DISCUSSION

The signals that lead to DC activation and priming of an adaptiveimmune response remain mostly unidentified. Although microbialand viral patterns have emerged as important determinants, it remainsunclear to what extent they need to act directly on DCs versus simplypromoting inflammation and alerting the cells to the presence ofinfection2. Here we have shown that DCs that have been activatedindirectly by inflammatory mediators are able to upregulate MHCmolecules and costimulation and to drive T cell proliferation andclonal expansion. However, such DCs lack the ability to produce IL-12p40 and this correlates with an inability to drive differentiation ofCD4+ T cells into TH1 effectors in vivo. These observations haveimportant implications for the present models of T cell priming. Theysuggest that models centered on induction of costimulation by APCas the critical parameter in T cell priming do not adequately explainimmunity. They also suggest that the main purpose of PRR expressionby APCs is to not regulate expression of costimulatory molecules4

but that of T helper–polarizing signals19. Teleologically, this ideamakes sense, because inflammation could be induced by either avirus or a helminth and thus inflammatory mediators cannot conveyinformation about the nature of the pathogen. Therefore, APCs mayuse inflammation to sense the presence of danger30,31 but requiredirect PAMP recognition to obtain information about the quality ofthat danger.

Interferons, IL-15, TNF and IL-1 have been proposed to act assignals for DCs activation and T cell priming9–11,13,14. Indeed, signalingthrough the IFN-a/b receptor seems to be critical for mediating the‘immuno-potentiating’ effects of several adjuvants in vivo11. Our datado not challenge the idea of the necessity for inflammatory cytokinesin DC activation and T cell priming. Instead, we have shown that thosemediators produced in response to TLR engagement in vivo are not perse sufficient for inducing CD4+ T cell effector responses. In contrast,coadministration of IFN-a and antigen led to T cell priming in adelayed-type hypersensitivity model9 and to enhancement of cytotoxicT lymphocyte cross-priming and IgG2a responses in other studies11,12.We cannot comment on the cytotoxic T lymphocyte cross-primingdata, as we restricted our investigation to the study of CD4+ T cellresponses. However, given that both CpG and LPS induce IFN-a/b5, itis difficult to reconcile the published delayed-type hypersensitivity andIgG2a data with our failure to detect TH1 priming in our test chimeras.It is possible that the biodistribution or amounts of IFN-a/b inducedby TLR ligation in vivo are different from those achieved by cytokineinjection, or that TLR ligands also induce factors that counteractIFN-a/b action. Alternatively, it is conceivable that some of theproteins used in the previous studies contained traces of microbialcontaminants. If so, interferons and other cytokines suggested to relaydanger30,31 may act mainly as amplifiers rather than initiators of APCactivation. This does not exclude the possibility that other ‘dangersignals’ (such as uric acid32 and kinins33) may, like microbial products,act as true DC activators and initiators of immunity30.

‘DC activation’ is a poorly defined term that can encompass a widevariety of parameters. Here we have defined it as the process thatconverts resting DCs into effector DCs capable of priming TH1 or TH2

cells. Using this criterion, we have found that DCs can adopt what isoften called a ‘mature’ DC phenotype (high expression of MHC,CD86 and CD40 and superior ability to drive T cell clonal expansion)without necessarily becoming effector DCs. This split between DCmaturation and T helper priming ability is reminiscent of publisheddata showing that mature or semimature DCs can induce T celltolerance34,35 and is in line with a report demonstrating a defect inCD4+ T cell priming by DC partially activated by LPS, which produceIFN-b but not other cytokines36. The idea that incomplete DCactivation can follow exposure of DCs to inflammatory mediatorsin the absence of direct PRR triggering is unexpected and has severalimplications. It explains why DC subsets that lack expression ofcertain TLRs can nevertheless become mature in response to ligandsfor that TLR in vivo. For example, maturation of the CD8a+ DCsubset without IL-12 production, identical to the trans-activationphenotype reported here, has been seen after injection of TLR7 ligandsinto mice37 despite the fact that CD8a+ DCs do not express TLR7(ref. 38). Moreover, if inflammation cannot fully substitute for TLRsignaling, it cannot normalize for differential TLR expression amongleukocyte subsets. Thus, differences in PRR expression among APCsubtypes are likely to be of physiological importance6. Our data alsochallenge the idea that mature DCs prime a TH2 response by defaultunless exposed to a TH1-promoting PAMP39. Indeed, trans-activatedDCs in our chimeras failed to promote the development of IL-4-producing T cells (data not shown) or switching of B cells to IgG1despite inducing notable T cell population expansion. This finding isin line with recent data showing lack of default TH2 priming in IL-12p40–deficient mice40 and fits the suggestion that TH2-promotingPAMPs are not simply maturation stimuli but instead act on APCsto drive expression of a TH2-inducing signal 3 such as the notch ligandjagged41. Finally, our data also suggest that IL-12 and other TH1-promoting cytokines must be provided to newly activated T cells in cis(that is, by the presenting DCs) and cannot be replaced by cytokinessecreted by nonpresenting cells in the microenvironment. This restric-tion may be explained by the previous observation that, in addition toTLR triggering, production of bioactive IL-12 p70 requires a secondfeedback signal from the T cell, which acts strictly in cis on thepresenting cell42. Nevertheless, the lack of TH1 differentiation in ourtest chimeras may not stem solely from the lack of synthesis of IL-12(or IL-23, which shares the IL-12 p40 subunit) by the presenting DCs.TLR triggering is essential for the synthesis of other TH1-promotingcytokines and is necessary for the secretion of cytokines such as IL-6,which render responding T cells refractory to suppression43. Therefore,our results indicate more generally a cis requirement of at least oneAPC-derived factor without which TH1 priming cannot occur in vivo.

Our experimental system required the use of adoptively transferredTCR transgenic T cells because the endogenous T cell repertoire isrestricted by the MHC haplotype of the host mouse, which differsfrom that of the APCs we sought to test. Using a single clone ofresponder T cells allowed us to be certain that we were dealing withactual differences in T cell priming and not with differences inrepertoire selection or maintenance in the different chimeras. Trackingan individual clone additionally allowed us to accurately monitorexpansion and cell division in response to antigen. However, the fateof the expanded T cell populations that failed to become effector cellsin our test chimeras remains unclear. Those cell populations under-went greater expansion and persisted at higher frequencies than didcells primed in the absence of adjuvant, suggesting that they were notbeing deleted. Thus during an infection T cells might be exposed to amixture of directly and indirectly activated DCs and the balance couldhave a regulatory effect on the immune response. Then again, most

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indirectly activated DCs are likely to present self, not foreign, antigens;any DC that presents pathogen antigens will most likely have been incontact with pathogen components and therefore will have beendirectly activated by engagement of PRRs. In a physiological context,inflammatory mediators draining from a site of infection through theafferent lymph would lead to indirect activation of resident lymphnode DCs. Efficient self presentation by such DCs could restimulatememory T cells, similar to the proposal made for the maintenance ofB cell memory44, or could act to augment the clonal expansion ofT cells responding to limiting amounts of foreign antigen45. Manyindirectly activated DCs presenting self antigens in an inflamed lymphnode could also act to trap any low-affinity self-reactive T cells thatescaped central and peripheral tolerance. Because such T cell popula-tions would expand without acquiring typical T helper effectoractivity, indirectly activated resident lymph node DCs could maintainself-tolerance while allowing directly activated DCs immigrating fromthe site of infection to promote priming of pathogen-specific T helpercell responses.

METHODSReagents. CpG oligonucleotide 1668 (CpG)46 was synthesized at Cancer

Research UK and was used in vivo at a dose of 0.5 mg/footpad or 5 mg/mouse

for intravenous injections. OVA peptide (residues 323–339; pOVA) was

synthesized and purified by high-performance liquid chromatography at

Cancer Research UK. OVA and HEL protein antigens were from Interchim

and Belovo, respectively. Residual endotoxin contamination in the protein

preparations was removed with Detoxi-Gel (Pierce) according to the manu-

facturer’s instructions. OVA and HEL were given in vivo at a dose of

25 mg/footpad. All reagents were free of endotoxin as determined by the

Limulus Amebocyte Lysate Test (BioWhittaker). LPS from Salmonella abortus

equi and flagellin from Salmonella typhimurium were from Apotech and were

used in vivo at doses of 1 mg/footpad and 0.5 mg/footpad, respectively.

Mice. All animal procedures and husbandry were carried out under the

authority of a project license, in accordance with UK governmental regulations

(Animals Scientific Procedures Act 1986), and were approved by the Cancer

Research UK Animal Ethics Committee. DO-11.10 mice on a BALB/c-SCID

background carrying a transgenic TCR specific for pOVA–I-Ad were originally a

gift from P. Garside (University of Glasgow, Glasgow, UK). The 3A9 mice

bearing a transgenic TCR specific for HEL(46–61)–I-Ak were originally

purchased from The Jackson Laboratory and were bred onto a recombination

activating gene 2–deficient B10.BR background. OT-II mice expressing a

transgenic Va2 Vb5 TCR specific for pOVA–I-Ab were a gift from W. Heath

(Walter and Eliza Hall Institute, Melbourne, Australia). The 3A9, DO-11.10

and OT-II T cells were found not to be alloreactive against BALB/c (H-2d) and

C57BL/6 (H-2b), B10.BR (H-2k) or C3H/He (H-2k), respectively (data not

shown). Mice genetically deficient in MyD88 or TLR9 on an H-2b background

were gifts from S. Akira (Osaka University, Osaka, Japan). All the mice

described above, as well as wild-type B10.BR (H-2k), BALB/c (H-2d),

C57BL/6 (H-2b) and B6.SJL (H-2b, CD45.1) mice, were bred at Cancer

Research UK in specific pathogen–free conditions. C3H/HeJ and C3H/HeN

mice (both H-2k) were purchased from Harlan. Bone marrow from Myd88�/�

mice on a BALB/c background was a gift from T. Kaisho (Osaka University,

Osaka, Japan). All mice were used at 6–12 weeks of age and were sex-matched

and age-matched within experiments.

For the generation of chimeras, recipient mice were g-irradiated twice with

5 Gy and were reconstituted with 2 � 106 T cell–depleted bone marrow cells of

each relevant donor strain. At 6–8 weeks after reconstitution, mice were tested

for chimerism. Chimeras were used for subsequent experiments only if analysis

of blood leukocytes showed the presence of cells of host and donor origin at a

ratio close to 1:1.

Flow cytometry. Cell suspensions were stained in ice-cold PBS supplemented

with 2 mM EDTA, 1% heat-inactivated FCS and 0.02% sodium azide (FACS

buffer). For intracellular staining, saponin was added to the FACS buffer to

0.1% final concentration. The monoclonal antibodies (conjugated to various

fluorochromes or biotin) and fluorescence-labeled streptavidin were from

PharMingen or CALTAG, unless stated otherwise. Purified 2.4G2 (anti-

FcgRIII/II; used to block unspecific antibody binding), C4H3 (anti-I-Ak/

HEL(46–61); ref. 47), and monoclonal antibody to 3A9 clonotypic (1G1248;

a gift from E. Unanue (Washington University, St. Louis, MO, USA)) were

produced at Cancer Research UK. C4H3 and 1G12 were biotinylated with

EZ-Link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer’s

instructions. Data were collected on FACSCalibur or LSR II cytometers

(Becton Dickinson) and were analyzed with FlowJo software (Treestar). Where

applicable, TO-PRO3, a DNA-binding dye for live versus dead discrimination

(Molecular Probes), was added to the samples immediately before data

acquisition. C4H3 median fluorescent intensity (MFI) was normalized by

division by the corresponding I-Ak MFI and is expressed as a percentage of

the control, as described49.

Cell purification. DCs were purified from freshly isolated spleens after

Liberase CI/DNase digestion (Roche Diagnostics) by immunomagnetic

sorting with anti-CD11c MACS beads (Miltenyi Biotec). Splenic DCs were

further purified by flow cytometry sorting (CD11chiCD45.1+CD45.2� or

CD11chiCD45.1�CD45.2+) with a MoFlo cytometer (Cytomation). Resulting

cell preparations were routinely more than 99% pure and viable.

Bone marrow–derived DCs were grown, following a standard protocol,

in complete medium (RMPI 1640 medium with 10% heat-inactivated

FCS, 100 units/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine

and 50 mM 2-mercaptoethanol) supplemented with granulocyte-macrophage

colony-stimulating factor at a concentration of about 1 mg/ml.

Naive CD4+ T cells from spleens and lymph nodes of TCR-transgenic donor

mice were purified by negative selection with MACS beads (Miltenyi Biotec).

Resulting cell preparations were found to be free of APCs in functional assays

(data not shown). When cells were used for adoptive transfer into host mice,

serum-free buffers were used throughout.

Adoptive transfers. APC-depleted CD4+ DO-11.10, OT-II or 3A9 T cell popu-

lations (5 � 106 cells) were transferred into host mice by intravenous injection

1 d before immunization. In some experiments, purified CD4+ T cells were

labeled with 2 mM CFSE (Molecular Probes) for 12 min at 37 1C before transfer.

In vivo restimulation. For the detection of IFN-g synthesis by primed transgenic

T cells, mice were injected intravenously with 100 mg pOVA 2 h before the

removal of popliteal lymph nodes and processing, essentially as described50.

In vitro DC activation assay. Bone marrow–derived DCs (5 � 105) from

C3H/HeJ (Tlr40/0 H-2k CD45.2) or C3H/HeN (Tlr4+/+ H-2k CD45.2) mice

were cultured together for 12 h in 24-well plates with or without an equal

number of bone marrow–derived DCs from B6.SJL (wild-type H-2b CD45.1)

with 1 mg/ml of HEL with or without 1 mg/ml of LPS in complete medium.

Presentation of HEL(46–61)–I-Ak, surface expression of I-Ak, CD86 and CD40,

and production of IL-12 p40 by DCs of C3H/HeJ or C3H/HeN origin

(CD11chiH-2KkCD45.2+) were determined by flow cytometry.

Ex vivo DC stimulatory capacity assay. Chimeric mice (Myd88�/� C57BL/6

(B6) CD45.2+ + wild-type B6.SJL CD45.1+ - Myd88�/� B6 CD45.2+; or

Tlr9�/� B6 CD45.2+ + wild-type B6.SJL CD45.1+ - Tlr9�/� B6 CD45.2+; or

wild-type B6 CD45.2+ + wild-type B6.SJL CD45.1+ - wild-type B6 CD45.2+)

or nonchimeric wild-type B6.SJL, Myd88�/� and Tlr9�/� mice were immu-

nized by intravenous injection with CpG. Then, 12 h later, CD45.2+ splenic

DCs from these mice were sorted by flow cytometry and were fixed for 15 min

at 25 1C with 1% paraformaldehyde. The fixed DCs were then washed and

residual paraformaldehyde was ‘quenched’ by incubation for 30 min at 25 1C in

0.1 mM glycine. Fixed and washed DCs (1 � 104) were cultured in triplicate in

round-bottom 96-well plates together with CD4+ T cells (5 � 104) purified

from naive OT-II mice, plus graded doses of pOVA. OT-II T cell proliferation

was assessed by measurement of [3H]thymidine incorporation at 48 h.

Antigen-specific immunoglobulin isotype enzyme-linked immunosorbent

assay (ELISA). Relative titers of OVA-specific IgG1 and IgG2a in sera from

experimental mice were determined by ELISA with anti-mouse IgG1 (A85-1) or

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anti-mouse IgG2a (R19-15) to detect immunoglobulin bound to the OVA-

coated plates. In some experiments, allotype-specific antibodies were used to

detect IgG1a and IgG2aa (10.9 and 8.3, respectively). All antibodies were

purchased from PharMingen.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank A. Eddaoudi for FACSorting services; C. Watkins for animalhusbandry; K. Rowan for secretarial support; and M. Albert (Institut Pasteur,Paris, France), M. Bachmann (Cytos Biotechnology, Zurich, Switzerland),F. Batista (Cancer Research UK, London, UK), P. Matzinger (NationalInstitutes of Health, Bethesda, Maryland), A. Oxenius (Swiss Federal Instituteof Technology, Zurich, Switzerland) and members of the ImmunobiologyLaboratory, Cancer Research UK, for advice and critical review of themanuscript. Supported by Cancer Research UK.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 15 October; accepted 20 December 2004

Published online at http://www.nature.com/natureimmunology/

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