LTβR signaling in dendritic cells induces a type I IFNresponse that is required for optimal clonal expansionof CD8+ T cellsLeslie Summers deLuca1, Dennis Ng1, Yunfei Gao1, Michael E. Wortzman, Tania H. Watts, and Jennifer L. Gommerman2

Department of Immunology, University of Toronto, Toronto, ON, Canada M5S 1A8

Edited* by Tak Wah Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University HealthNetwork, Toronto, ON, Canada, and approved December 17, 2010 (received for review September 21, 2010)

During an immune response, antigen-bearing dendritic cells (DCs)migrate to the local draining lymph node and present antigen toCD4+ helper T cells. Antigen-activated CD4+ T cells then up-regulateTNF superfamily members including CD40 ligand and lymphotoxin(LT)αβ. Although it is well-accepted that CD40 stimulation on DCsis required for DC licensing and cross-priming of CD8+ T-cellresponses, it is likely that other signals are integrated into a com-prehensive DC activation program. Here we show that a cognateinteractionbetweenLTαβonCD4+ helper T cells and LTβ receptor onDCs results in unique signals that are necessary for optimal CD8+

T-cell expansion via a type I IFN-dependent mechanism. In con-trast, CD40 signaling appears to be more critical for CD8+ T-cellIFNγ production. Therefore, different TNF family members provideintegrative signals that shape the licensing potential of antigen-presenting DCs.

CD8+ T-cell responses are crucial for host responses to viralinfection and are also involved in allograft rejection and

tumor immunity. In some settings, provision of T-cell help isrequired to adequately license dendritic cells (DCs) for cross-priming of CD8+ T-cell responses (1). However, the nature ofthese help signals remains incompletely characterized, and it isunclear how these signals integrate into a program of DC mat-uration. Manipulation of such signals represents a promisingtherapeutic approach for promoting tumor immunity or forquieting autoimmune disease.Tumor necrosis factor (TNF) family members including CD40

ligand (CD40L), RANK ligand, LIGHT, and lymphotoxin-αβ(LTαβ) are rapidly up-regulated on antigen (Ag)-activated CD4+helper T cells (2, 3). During the immune response, DC/T-cellinteractions result in the ligation of CD40, CD70, and RANK onDCs, and this has been shown to promote DC cross-priming ca-pacity and survival (4–8). We have recently shown that LTαβ ex-pression on Ag-specific CD4+ T cells is also critical for DC func-tion in the context of protein Ag (3). These studies provoke thequestion of whether different TNF family pathways are redundant,or somehow act cooperatively in the context of DC maturation.Previous studies have hinted at a role for the LT pathway in T-

cell function (9). Focusing on cases where CD8+ T-cell respon-ses rely on T-cell help, such as CD8+ T-cell responses to allo-Ag(10, 11) and tumor-Ag (12), LTβ-receptor (LTβR) signaling hasa significant effect on CD8+ T-cell activation and clonal ex-pansion. To resolve how this pathway may impact the maturationof a CD8+ T-cell response in vivo, we used different approacheswhereby we selectively inhibited LTβR signaling on the hema-topoietic compartment, or specifically on DCs, to evaluateeffects of LTβR signaling on DC-mediated CD8+ T-cell cross-priming. Our data revealed that the LT pathway was importantfor CD8+ T-cell clonal expansion but not effector function,whereas the CD40 pathway was necessary for CD8+ T-cell func-tion but was dispensable for T-cell expansion in response to cell-associated Ag. LTβR stimulation on DCs was found to provokea type I interferon (IFN) response even in the absence of addedToll-like receptor (TLR) agonist, and exogenous IFN could recover

CD8+ T-cell proliferation, suggesting a mechanism for the effectsof this pathway on CD8+ T-cell priming. Therefore, the LT path-way provides necessary and nonredundantDC-intrinsic signals thatprovoke optimal CD8+ T-cell clonal expansion.

ResultsLTβR Signaling Cooperates with CD40-Derived Signals for Priming ofCD8+ T Cells in Vivo. We previously demonstrated that the ex-pression of LTαβ ligand on Ag-specific CD4+ T cells in vivo isrequired for DC function ex vivo (3). However, a hallmark of DClicensing is the ability to cross-prime a cytotoxic T-cell response.To ascertain the relevance of LTβR signaling on DC licensing,we assessed whether LTβR signaling was required for priming ofa CD8+ T-cell response to cell-associated Ag in vivo. To achievea relatively CD4+ T-cell help-dependent system, spleen andlymph node (LN) cells from bm1 mice were used as syngeneicvehicles for Ag (OVA) delivery so that bm1 cells could not di-rectly present Ag to OTI T cells in bm1xB6 F1 recipient hosts(13). bm1 cells were hypotonically loaded with OVA protein (4)and were used to immunize mice that had received congenicallylabeled OTI T cells 1 d prior. We first assessed the consequencesof global inhibition of LTβR signaling by treating recipient micewith a decoy fusion protein, LTβR-Ig, which was administeredthe day before immunization with bm1-OVA. We found that,compared with the control treatment group, OTI T-cell expan-sion was significantly impaired in LTβR-Ig–treated recipientmice (Fig. 1A). Reduced frequency of OTI T cells was observedthroughout the immune response from day 3 to 21 in the spleen(see Fig. S1 for representative FACS) and the blood (Fig. 1A).However, despite this observed decrease in clonal expansion,OTI T cells exhibited no defect in IFNγ production at any timepoint, with equivalent proportions of IFNγ-producing OTI Tcells in both control and LTβR-Ig–treated mice (Fig. 1B; see Fig.S1 for representative FACS). Because the homeostasis of DCs innaïve mice is perturbed in LTβR−/− animals (14–16), we con-firmed that short-term treatment with the LTβR-Ig agent did notresult in a reduction in DC numbers, an alteration in DC phe-notype, or an inability to acquire and process OVA protein forpresentation on MHC class I (Fig. S2). Therefore, LTβR-Igtreatment results in suboptimal clonal expansion, but not effec-tor function, of CD8+ T cells in response to cell-associated Ag.CD40 signaling is a potent maturation cue during DC activa-

tion and can also induce CD86 expression on DCs (17). How-

Author contributions: L.S.-d., D.N., Y.G., and J.L.G. designed research; L.S.-d., D.N., and Y.G.performed research; M.E.W. and T.H.W. contributed new reagents/analytic tools; L.S.-d.,D.N., Y.G., and J.L.G. analyzed data; and L.S.-d. and J.L.G. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1L.S.-d., D.N., and Y.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: jen.gommerman@utoronto.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1014188108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1014188108 PNAS Early Edition | 1 of 6

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

ever, these data indicate that in the context of abrogated LTβRsignaling, physiological CD40 signaling cannot compensate tomaintain DC stimulatory function, and it is unclear whethersignals derived from both CD40 and LTβR act additively orsynergistically to license DCs for CD8+ T-cell cross-priming oralternatively whether these two pathways contribute somethingdistinct to DC maturation. To resolve this, we generatedWT→WT and LTβR−/−→WT chimeric mice, and at 8 wk post-reconstitution these mice were injected with OTI T cells andimmunized with cell-associated OVA as in Fig. 1A. At day 3,which in our system is the peak of the CD8 (OTI) response (seeFig. 1A for kinetics), we observed robust expansion of OTI Tcells in WT→WT chimeras; however, in mice lacking LTβR ex-pression on Ag-presenting cells, we observed a statistically sig-nificant reduction in OTI T-cell accumulation (Fig. 1C, whitebars). Not unlike in LTβR-Ig–treated mice, OTI T cells primedin LTβR−/−→WT mice exhibited no defect in IFNγ production,with equivalent proportions of IFNγ-producing OTI T cells inboth groups (Fig. 1D, white bars). We also confirmed that DCsfrom LTβR−/− mice expressed normal levels of maturationmarkers and could acquire and process OVA protein for pre-sentation on MHC class I in a manner that was comparable toDCs from WT mice (Fig. S3). Therefore, under circumstanceswhere the organization of splenic stroma is normal (whichdepends on LTβR expression in radio-resistant host cells), ex-pression of LTβR in radio-sensitive hematopoietic cells is re-quired for full clonal expansion of OTI T cells.

To determine whether this CD8+ T-cell defect would be ex-acerbated by the additional absence of CD40 licensing cues,WT→WT and LTβR−/−→WT bone marrow (BM) chimeric micewere treated with α-CD40L-blocking Ab. In contrast to theLTβR−/−→WT BM chimeric mice, we observed no defect in OTIT-cell expansion in WT→WT mice in which CD40 signaling wasprevented, and there was a minimal compound defect beyondthe impaired OTI expansion in the absence of LTβR signalingwhen both CD40 and LTβR signaling were simultaneously ab-rogated (Fig. 1C, gray bars). Whereas secretion of IFNγ by OTIT cells was uncompromised in the absence of LTβR licensing, itwas grossly impaired in α-CD40L–treated mice (Fig. 1D, graybars). Together, these results identify unique roles for LTβR andCD40 signaling in promoting CD8+ T-cell responses to cell-associated Ag, the former in regulating CD8+ T-cell expansionand the latter in instructing CD8+ T-cell effector function.

DC-Intrinsic LTβR Signaling Is Required for Optimal CD8+ T-Cell ClonalExpansion.We next addressed whether DCs, which express LTβR(15), are the relevant LTβR+ hematopoietically derived cell re-quired for cross-priming CD8+ T cells. We therefore generatedmixed BM chimeras using CD11c-DTR/GFP donor BM alongwith either WT or LTβR−/− donor BM transferred into lethallyirradiated WT hosts, and reconstitution of BM-derived cells wasconfirmed using GFP and CD45 congenic markers. CD11c-DTRmice express a diphtheria toxin (DT) receptor (DTR) under thecontrol of the CD11c promoter, and treatment of these micewith DT results in thorough but temporary depletion of DCs(18). Treatment of LTβR−/− + CD11c-DTR→WT chimeric micewith DT would therefore specifically deplete WT CD11c+ cellswhile preserving LTβR−/− DCs. At 12 wk postreconstitution,chimeric mice were given an adoptive transfer (A/T) of re-sponder OTI T cells, and the following day were immunized withOVA-loaded bm1 splenocytes. Chimeric mice were treated withDT on day 0 and day 1 postimmunization, and depletion ofCD11c-DTR+ (GFP+) DCs in the blood, spleen, and LN wasconfirmed. At the peak of the CD8+ response, expansion of OTIT cells was significantly impaired in LTβR−/− + CD11c-DTRchimeras in terms of frequency (P < 0.001; Fig. 1E) and also inthe case of total numbers of OTI (P < 0.05; Fig. S4), whereasIFNγ production remained intact (Fig. 1F), recapitulating thedefect observed in LTβR−/−→WT chimeras. Consistent with thesedata, mice that received LTβ-deficient helper T cells also exhibiteda significant reduction in the peak expansion of splenic OTI CD8+

T cells (Fig. S5A) but not IFNγ production (Fig. S5B) followingimmunization with OVA-loaded bm1 cells, suggesting that cross-talk between LTαβ-expressing Ag-specific T cells and LTβR+ DCsis required for optimal clonal expansion of CD8+ T cells in re-sponse to cell-associated Ag. Collectively, these data identify DC-intrinsic LTβR signaling as a requirement for CD8+ T-cell clonalexpansion but not for CD8+ T-cell-derived IFNγ production.

LTβR Signaling Is Required for Full Activation and Cell-CycleProgression of Ag-Specific CD8+ T Cells. Given the reduction in theclonal burst of Ag-specific CD8+ T cells in immunized LT-inhibited mice, we asked whether the activation, proliferation, orpersistence of OVA-specific CD8+ T cells was impaired. At 2 dpostimmunization, we measured cell division/activation, andnoted a lag in carboxyfluorescein succinimidyl ester (CFSE) di-lution as well as a significant reduction in CD25 up-regulation onOTI T cells derived fromLTβR−/−→WTmice compared withOTIT cells derived from WT→WT mice (Fig. 2 A and B). The re-duction in CFSE dilution suggested that in the absence of LTβR-derived DC licensing, OTI T cells were either not dividing or weredividing but failing to survive. We therefore measured the cell-cycle status of CFSE-labeled OTI from OVA-bm1–immunizedchimeric mice. Interestingly, a significant increase in the percent ofcycling, CFSEint OTI in LTβR−/−→WT compared with WT→WT

*

***

* *

NS

A E

B D F

C

Fig. 1. DC-derived LTβR and CD40 signals contribute distinctly to CD8+ T-cellcross-priming in vivo. (A and B) WT bm1xB6 F1 mice were given an A/T ofresponder CD45.1 or Thy1.1 OTI T cells, treated with either control humanIgG (huIgG) or LTβR-Ig, and immunized the next day with OVA-loaded bm1cells. At multiple days postimmunization, OTI expansion (A) and IFNγ pro-duction (B) were measured. Data are representative of six (A) or two (B)independent experiments (n = 3–6 per experiment). (C and D) WT→WT orLTβR−/−→WT chimeric mice were given an A/T of responder CD45.1 or Thy1.1OTI T cells, treated with either control Ab or α-CD40L, and immunized withOVA-loaded bm1 cells, and OTI expansion (C) and IFNγ production (D) weremeasured at day 3. These experiments were performed four times withsimilar results, and results shown represent the average of four mice pergroup. NS, nonsignificant. (E and F) Mixed chimeric WT + CD11c-DTR→WT orLTβR−/− + CD11c-DTR→WT mice were given an A/T of responder CD45.1 OTIT cells, treated with diphtheria toxin, and immunized with OVA-loadedbm1 cells, and OTI expansion (E) and IFNγ production (F) were measured atday 3. Data are representative of two experiments (n = 4 per experiment).Empty circles, control Ig-treated mice; filled circles, LTβR-Ig–treated mice;white bars, control treated mice; gray bars, α-CD40L–treated mice. *P < 0.05,***P < 0.0001 (two-way ANOVA for A).

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1014188108 Summers deLuca et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

chimeric mice was observed (Fig. 2C; P < 0.01), and this was ac-companied by a significant reduction in the percent of resting/G1CFSEneg (fully divided) OTI in LTβR−/−→WT versus WT→WTchimericmice (P<0.005). Staining for cleavedcaspase3 leadingupto and at the peak of the OTI response showed no differences inapoptotic OTI in WT→WT and LTβR−/−→WT mice; however,analysis of dying cells in vivo is unreliable because they are quicklyphagocytosed, and although apoptotic demise is common, thereexist additional death pathways that may not be captured by tech-niques designed to measure apoptotic cell death (19). It thereforeseems likely that OTI T cells primed in LTβR−/−→WT mice arecycling in equivalent proportion to those primed inWT→WTmice,but are dying before reaching a terminally divided resting state.

Stimulation of LTβR on DCs Results in the Production of Type I IFN. Todetermine the mechanism whereby DC-intrinsic LTβR signalingcontributes to the clonal expansion of CD8+ T cells in vivo, weinvestigated the possibility that production of type I IFN wasdownstream of LTβR activation in DCs for several reasons. First,LTβR signaling has been shown to induce type I IFN in radio-resistant stromal cells independently of TLR-derived signals(20). Second, type I IFN has been shown to induce CD25 ex-pression on T cells (21), is required for the optimal clonal ex-pansion of CD8+ T cells (22, 23), and can exert theseproproliferation effects independently of CD40/CD40L activity(24). Finally, type I IFN induces the expression of CD86 on DCs(25), and we have observed a transient decrease in CD86 ex-pression on DCs post–OVA immunization in vivo that is re-covered in the presence of WT DCs (Fig. S6), suggesting a factorthat acts in trans can rescue CD86 expression. Therefore, wereasoned that the poor OTI expansion, the reduced expression ofCD25 on OTI T cells, and the failure to up-regulate CD86 onLTβR−/− DCs could all reflect a defect in type I IFN production.To assess whether LTβR signaling induces type I IFN in con-ventional DCs, we generated bone marrow–derived DCs(BMDCs) and cocultured WT versus LTβR−/− BMDCs withOVA-specific CD4+ helper OTII T cells as a source of licensingsignals (CD40L, LTαβ). These cocultures were then supple-mented with either LPS alone or LPS + OVA323–339 to inducethe expression of LTαβ/CD40L on OTII (3). Exposure to LPSalone induced a baseline level of IFNβ and IFNα5 mRNA, whichdid not differ between WT and LTβR−/− DCs (open versusclosed circles, Fig. 3 A and B). However, the addition of theOTII cognate peptide OVA323–339 to the DC/T-cell cocultures,which activates OTII CD4+ T cells and stimulates them to ex-

press LTαβ/CD40L (3), resulted in a robust induction of bothIFNβ and IFNα5 mRNA that was blunted in the absence ofLTβR expression on DCs (open versus closed circles, Fig. 3 Cand D). Indeed, the induction of IFNβ and IFNα5 mRNA fromLTβR−/− DCs in response to LPS + OVA323–339 was not anydifferent from what was observed with LPS alone. The reductionin IFNβ and IFNα5 mRNA from LTβR−/− DCs was not dueto any developmentally associated DC-intrinsic defect becauseLTβR−/− BMDCs were similar to WT BMDCs in terms of DCsurface marker expression and their capacity to make IL-12 (Fig.S7). Moreover, blockade of LTβR/LTαβ interactions between Tcells and WT BMDCs with LTβR-Ig in vitro recapitulated thereduction in IFNβ and IFNα5 expression (Fig. S8). These dataindicate that DC-intrinsic LTβR ligation induces a necessary andunique signal(s) that collaborates with TLR4 stimulation to pro-voke optimal induction of IFNβ and IFNα5 gene expression.We next assessed whether LTβR signals could provoke type I

IFN expression independently of TLR signaling by coculturingWT versus LTβR−/− BMDCs with recombinant LTαβ ligand orwith agonist Abs directed at LTβR. Interestingly, in the absenceof any added TLR signal, we detected a modest induction ofIFNβ and IFNα5 mRNA in response to LTβR ligation inBMDCs (open versus closed bars, Fig. 4 A and B; P < 0.001 andP < 0.01 for IFNβ and IFNα5, respectively, between WT andLTβR−/− BMDCs). IFNβ and IFNα5 induction was specific tothe LT pathway, as LTβR−/− BMDCs failed to induce IFNα/βunder these conditions. Furthermore, IFNα/β was not signifi-cantly induced following stimulation with α-CD40 (Fig. 4 A andB), even though α-CD40 (but not α-LTβR) Abs readily provokedIL-12 secretion from BMDCs (Fig. S7).Because the amount of IFNα/β expression was relatively

modest in response to LTαβ ligand or agonist Abs directed atLTβR, we evaluated whether signals through TLR4 and LTβRcould collaborate to increase type I IFN expression. Indeed,BMDCs pretreated with LPS showed evidence of further up-regulation of IFNα/β expression when subsequently treated withanti-LTβR agonist Ab, and this effect was not observed forLTβR−/− BMDCs (Fig. 4 C and D). Thus, we postulate thatpathogen/danger-associated molecular patterns (PAMPs/DAMPs)precondition DCs to receive signals through the LTβR which fur-ther augment IFNα/β expression.

LTβR−/− DCs Fail to Support OTI Proliferation in Vitro but ProliferationCan Be Rescued by Exogenous IFNα. To determine whether reducedtype I IFN production contributes to the failure of LTβR−/− DCs

**

**

**

A

B Ci ii

iii iv

Fig. 2. LTβR signaling is required for normal CD8+ T-cell activation and cell-cycle completion. Chimeric mice(WT→WT or LTβR−/−→WT) were given an A/T of CFSE-labeled responder OTI T cells, and then were either leftunimmunized or immunized 1 d later with OVA-loadedbm1 cells. At day 2 postimmunization, CFSE dilution andCD25 up-regulation were evaluated (A), and the extent ofCD25 up-regulation on divided versus undivided OTI cellswas measured (B). Data are representative of three in-dependent experiments (n = 3–5 mice per experiment).**P < 0.01. (C) To assess cell cycling in the described invivo experiment from A and B, CFSE-stained OTI T cellswere gated and analyzed with DyeCycle Violet to mea-sure cell-cycle status, with a representative FACS plotshown (i). Enumeration of OTI that had divided [CFSEmed

(ii)], had “terminally divided” [CFSE− (iii)], or had re-mained undivided [CFSEhigh (iv)] was performed. Dataare representative of two experiments (in one case per-formed at day 2, and another at day 3, n = 4 per experi-ment). Note that in the presence of DyeCycle Violet, CFSEintensity appears different from without DyeCycle Violet(A versus C). **P < 0.01.

Summers deLuca et al. PNAS Early Edition | 3 of 6

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

to induce full CD8+ T-cell activation and expansion in vivo, weestablished an in vitro system for measuring OTI T-cell pro-liferation. Given the flexible nature of such a system, we wereable to carefully titrate the amount of LPS and the ratio of DCsto T cells such that OTI T-cell proliferation was rendered help-dependent and, indeed, in the absence of OTII CD4+ T cells, weobserved minimal OTI proliferation which was equivalent towhat was observed in the absence of any OVA Ag (Fig. 5A, lefttwo panels). Although this in vitro system is an artificial system,we observed a similar defect in OTI proliferation when OVA-bm1 cells were introduced into the cultures (Fig. S9). However,because the OTI proliferation observed with bm1-OVA stimu-lation in vitro was less robust and somewhat delayed comparedwith LPS + OVA (Fig. 5A), we focused on the LPS version ofthe in vitro assay. Interestingly, using the LPS/OVA system, in thepresence of OTII CD4+ T cells, we noted more than a twofoldreduction in OTI CFSE dilution when OTI T cells were culturedwith LTβR−/− versus WT DCs, confirming a requirement for DC-intrinsic LTβR signaling for optimal OTI expansion in vitro (Fig.5A versus Fig. 5B) and recapitulating the nature andmagnitude ofour OTI expansion defect that was observed in vivo (Fig. 1).Similar defects in CFSE dilution were observed with LTβR−/−

BMDCs cocultured with bm1-OVA in vitro (Fig. S9), and alsowhen WT OTII helper T cells were compared with LTβ−/− OTIICD4+ T cells (Fig. S5). These data demonstrate that LTαβ-LTβRDC licensing signals are required for stimulating OTI expansionin vitro.To determine whether type I IFN could rescue the pro-

liferation of OTI T cells primed by LTβR−/− DCs, we addedIFNα into our WT or LTβR−/− DC-OTII cocultures and mea-sured OTI proliferation by CFSE dilution. Exogenous IFNα re-stored proliferation of OTI T cells stimulated with LTβR−/−

DCs, but had a minimal effect on proliferation of OTI stimulatedby WT DCs (Fig. 5A versus Fig. 5B), where maximal type I IFNinduction would have been achieved through the combination ofLPS and LTβR ligation. Furthermore, although we noted thatCD25 levels on OTI T cells from LTβR−/− DC-OTII cocultures

were reduced to 65% of the normal CD25 levels observed in WTDC-OTII cocultures, addition of IFNα restored CD25 levels onOTI T cells cocultured with LTβR−/− DCs to levels equivalent tothose observed in WT DC-OTII cocultures (94% of normal).Therefore, production of type I IFN is a critical mediatordownstream of LTβR signaling in DCs, and recovery of type IIFN levels can rescue the otherwise impaired proliferation ofCD8+ T cells primed by LTβR−/− DCs. These data providea mechanism for the unique function of LTβR in DC-mediatedT-cell priming.

DiscussionT-cell help is required in many cases for optimal cross-priming ofCD8+ T-cell responses to cell-associated Ag (1). The nature ofthese help signals, however, remains incompletely characterized.Here we show that DC-intrinsic LTβR signaling is required forex vivo DC function and for optimal cross-priming of a CD8+

T-cell response in vivo. Our study identifies a nonredundantfunction for LTβR signaling alongside CD40 stimulation in pro-moting DC maturation and identifies LTβR-dependent type IIFN production as a unique contribution from this TNF familymember in shaping an effective CD8+ T-cell response.We have previously shown that LTαβ is rapidly up-regulated

on OVA-specific CD4+ helper T cells in response to immuni-zation with Ag, and the kinetics of LTαβ expression resemblethose for CD69 expression (3). Such kinetics are very similar tothe induction of CD40L on CD4+ helper T cells, and representsa form of “help” for DC conditioning in vivo via interaction withLTβR on DCs. Although our studies indicate that LTαβ on cellsother than Ag-specific CD4+ T cells (B cells, for example) is in-sufficient to trigger LTβR on DCs to prime CD8+ T cells, it ispossible that LIGHT expression on CD4+ helper T cells may alsocontribute to LTβR signaling in DCs to facilitate CD8+ T-cellresponses to cell-associated Ag, and indeed the defect we observe

5

5A B

C D

Fig. 3. LTβR synergizes with TLR4 to maximize type I IFN production inBMDCs. WT (open circles) and LTβR−/− (gray circles) BMDCs were coincubatedwith OVA-specific CD4+ T cells (OTII) and LPS (A and B). In some cases, thesecocultures were also supplemented with OVA323–339 (C and D). DCs wereisolated at indicated time points and the expression of IFNβ (A and C) andIFNα5 (B and D) was measured by real-time RT-PCR. This experiment wasperformed three times with similar results. *P < 0.05.

5

5

A B

C D

Fig. 4. LTβR signaling can mediate type I IFN production independently ofTLR activation in BMDCs. BMDCs were stimulated with anti-LTβR, LTαβ, oranti-CD40 in the absence of any added LPS. Expression of IFNβ (A) and IFNα5(B) was measured by real-time RT-PCR at 6 and 18 h poststimulation of WT(open bars) and LTβR−/− (gray bars) BMDCs. These experiments were per-formed at least three times with similar results. *P < 0.05, **P < 0.01, ***P <0.001. BMDCs were also preincubated with LPS for 2 h, washed, and thenstimulated with anti-LTβR. Expression of IFNβ (C) and IFNα5 (D) was mea-sured by real-time RT-PCR at 4, 5, and 6 h poststimulation of WT (open bars)and LTβR−/− (gray bars) BMDCs. This experiment was performed three timeswith similar results. *P < 0.05 for data in (C).

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1014188108 Summers deLuca et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

with LTβR−/− DCs is typically larger than what is observed whenwe transfer LTβ−/− OTII (Fig. S5). Nevertheless, the expression ofLTαβ on CD4+ helper T cells is likely strictly regulated becausewe observe very little expression on resting CD4+ helper T cells(3) and, indeed, the expression of LTαβ on Th1 and Th17 cells hasbeen exploited for depletion strategies in the context of rodentmodels of autoimmunity (26).One possible explanation for our findings is that DC-intrinsic

LTβR signaling is required for their homeostatic maintenance inthe spleen (14–16). However, we have confirmed that the re-duction in OTI expansion is due to impaired DC function andnot reduced DC numbers in three ways. First, in WT mice whosesplenic DC populations are intact, the absence of LTβ expressionon a small population of adoptively transferred helper CD4+ Tcells recapitulates the defect in OTI clonal expansion observed inLTβR−/− settings. Second, treatment of WT mice with an LTβRsignaling inhibitor, LTβR-Ig, 1 d before immunization againresults in a twofold reduction in the peak OTI expansion withoutany impact on DC numbers. Finally, the defect of OTI expansionwas confirmed using LTβR−/− BMDCs (as well as WT DC in thepresence of LTβR-Ig) in vitro. Therefore, in scenarios in whichDC numbers remain equivalent but the LTαβ-LTβR signal isabsent, peak OTI expansion is significantly and comparably re-duced. Thus, the LTβR licensing requirement for DC stimulatoryfunction is independent of its role in DC homeostasis.The discrepant capacity of CD40 and LTβR licensing signals to

induce IFNα/β may explain, at least in part, the inability of physi-ological CD40 signals to compensate for the absence of DC-intrinsic LTβR signaling. The exception to this scenario is in caseswhere anti-CD40 agonist Abs are added in vivo, which we havepreviously shown can compensate for the absence of LTαβ onhelperOTIIT cells (3).However, anti-CD40Abs have been shownto provoke robust and sustained up-regulation of LTαβ on B cells(up to 11 d)which could conceivably have overcome the absence ofLTαβ on helper T cells under those circumstances (27). In anycase, our finding that CD40 signaling was dispensable for CD8+T-cell clonal expansion contradicts other reports that suggest thesufficiency of CD40 ligation for full DC licensing (4, 5) and forearly CD8+ T-cell expansion (28–31). Indeed, the evidence forDC-derived CD40 signals supporting CD8+ T-cell expansion ismixed (32, 33). The variable requirement forCD40 inCD8+T-cellcross-priming could be explained by the recent identification of

CD40L expression on DCs which may drive CD8+ T-cell prim-ing in more help-independent systems (34). Furthermore, CD70has been shown to play a more predominant role in CD40-independent CD8+ T-cell responses (35). Given that the defectswe observe in CD8+T-cell clonal expansion are significant but notabsolute (i.e., residual CD8+ T-cell proliferation is observed),a model of complex interplay of multiple TNF family receptors,rather thanCD40 alone, inmediatingDC licensing seems likely. Inaddition, the relative importance of each of these molecules willlikely depend on the stimulation conditions.We found that IFNα/β mRNA was synthesized in response to

LTβR stimulation even in the absence of TLR costimulation(Figs. 3 and 4). Interestingly, IFNα/β is a potent inducer of cos-timulatory molecule expression on DCs, including CD86 (36, 37).Consistent with a role for IFNα/β in regulating CD86 expression,we observed a transient decrease in CD86 expression on DCsfrom LTβR−/− mice. Moreover, this defect was rescued by thepresence of WT DCs, indicating that a soluble mediator such asIFNα/βmay stimulate the expression of CD86 on LTβR−/−DCs intrans (Fig. S6). IFNα/β is also induced by TLR ligation, and ad-ditionally the IFNα/β receptor (IFNAR) has been shown to becritical for MyD88-independent DC maturation in response toSalmonella infection (37). Thus, it is likely that collaborationbetween PAMPs/DAMPs and LTβR signaling is required foroptimal IFNα/β production, and indeed we found that treatmentof LPS-stimulated DCs with anti-LTβR augmented type I IFNexpression in vitro.IFNAR expression on CD8+ T cells is critically required for

CD8+ T-cell priming and expansion (24) and prolongs expres-sion of genes involved in T-cell programming by modulatingchromatin accessibility (38). As in the LT-deficient scenarios,Ag-specific IFNAR−/− CD8+ T cells fail to expand followingpriming, and overzealous cell cycling followed by defective per-sistence of IFNAR−/− CD8+ T cells has been reported (23),suggesting that normal proliferation and subsequent loss ofCD8+ T cells in the absence of LTβR signaling may be the resultof a suboptimal type I IFN response. Consistent with our findingthat LT-derived signals are required for optimal CD25 expres-sion, IFNα can also induce a dramatic up-regulation of CD25 onCD8+ T cells in vivo (21). Because add-back of IFNα to LTβR−/−

BMDC cultures rescued poor CD8+ T-cell proliferation in vitro,such collaboration between innate signals and LTβR signals maybe required for optimal CD8+ T-cell proliferation in vivo to cell-associated Ag such as auto-Ag or tumor-Ag.Although the role of LTβR signaling in CD8+ responses

during infectious disease has been mixed (9, 39, 40), this couldreflect varying levels of help dependency in different systemswhere there may have been robust type I IFN production elicitedby PAMPs/DAMPs. Our study focuses on the role of DC-intrinsic LTβR signaling in provoking a CD8+ T-cell response tocell-associated Ag, and this may have functional significance tohelp-dependent situations such as tumor eradication and graftrejections, scenarios where LTβR signaling has been implicated(10–12, 41). The evaluation of LTβR signaling in DCs duringautoimmunity remains to be fully elucidated and would providetherapeutic insight into the potential value of LTβR inhibitionfor treatment of such chronic diseases.

Materials and MethodsIn Vivo Help-Dependent CD8+ T-Cell Responses. OTI (1 × 106) T cells werepurified (as above) and adoptively transferred into C57BL/6 mice treatedwith LTβR-Ig or control Ig or alternatively into WT→WT and LTβR−/−→WTBM chimeric mice. In some cases, 3 × 106 of either WT OTII or LTβ−/− OTIIcells were transferred into mice. On the following day, these mice wereprimed with 25 × 106 OVA-loaded bm1 splenocytes. OVA-loaded spleno-cytes were prepared by osmotic shock. Briefly, 20 × 107 bm1 splenocyteswere resuspended in 1 mL of hypertonic solution (0.5 M sucrose, 10%polyethylene glycol 1000, and 10 mM Hepes in RPMI 1640) containing 10mg/mL OVA protein for 10 min at 37 °C. Fourteen milliliters of prewarmed

A

B

Fig. 5. LTβR−/− BMDCs do not support full OTI proliferation in vitro, and thiscan be rescued with type I IFN. WT (A) and LTβR−/− (B) BMDCs were pre-incubated with LPS and OVA protein for 18 h, washed, and then plated withCFSE-labeled OVA-specific CD8+ T cells (OTI) with or without OTII CD4+

T cells. Seventy-two hours later, OTI T cells were gated based on CD45.1expression and/or CD8 expression, and then assessed for CD25 expressionand CFSE dilution. In some cases, 25 U/mL of IFNα was added to the culturesat 48 h. The experiment is a representative example of three independentexperiments.

Summers deLuca et al. PNAS Early Edition | 5 of 6

IMMUNOLO

GY

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

hypotonic solution (40% H2O, 60% RPMI 1640) was added, and the cellswere incubated for an additional 2 min at 37 °C. The cells were spun im-mediately after the incubation, washed five times with PBS, and injectedinto mice. The OTI response was evaluated at day 3, 7, 14, and 21 in theblood and/or spleen.

In Vitro Help-Dependent CD8+ T-Cell Responses. Day 10 BMDC cultures weretreated with LPS (100 ng/mL) or LPS with OVA (50 μg/mL) overnight. OTIand OTII T cells were purified as described in SI Materials and Methods,and OTI T cells were CFSE-labeled (1 μM). Activated BMDCs were cocul-tured with OTII and OTI T cells at a ratio of 1:10:10. Cultures were trea-ted with or without IFNα (25 U/mL) in rescue experiments. Similar resultswere obtained when OVA-loaded bm1 splenocytes were substituted forLPS/OVA.

In Vitro BMDC Stimulation. Stimulations were performed in 96-well plateswith cell density at 1 × 106 cells/mL in complete RPMI medium 1640 (Sigma-Aldrich). Plates were coated for 1 h at 21 °C with anti-Armenian hamsterantibody (BD Biosciences) (4 μg/mL) in 0.05 M NaHCO3 (pH 9.6), and agonisticanti-mLTβR antibody AFH6 (20 μg/mL), recombinant LTαβ (100 ng/mL), oranti-mCD40 antibody FGK4/5 (10 μg/mL) was added overnight in PBS at 4 °C.Supernatant was removed before adding DCs. In some cases, DCs were

pretreated with 100 ng/mL LPS for 2 h, washed, and then transferred toplates coated with agonistic anti-mLTβR Ab AFH6. In coculture experiments,OVA-specific OTII CD4+ T cells were purified using negative bead isolation asabove and then rested overnight. T cells were then incubated with BMDCs ata ratio of 2:1 and the mixed cultures were stimulated with either LPS alone(100 ng/mL) or LPS with OVA peptide 323–339 (5 μg/mL) for the indicatedtime points. In some cases, LPS/OVA was substituted with 50,000 bm1 cellshypotonically loaded with OVA. BMDCs were then isolated from T cells byCD11c-based positive selection as described in SI Materials and Methods.Isolated DC fractions were homogenized in TRIzol for cDNA preparation andquantitative PCR analysis.

More details can be found in SI Materials and Methods.

ACKNOWLEDGMENTS. We acknowledge Dr. Klaus Pfeffer for generatingLTβR−/− mice and Dr. Rodney Newberry for shipping LTβR−/− mice, as well asDr. Jeff Browning for provision of LTβR-Ig. We thank Dionne White, man-ager of the flow cytometry facility in the Faculty of Medicine, University ofToronto. We thank Dr. James Carlyle for B3Z cells and technical advice andDoug McCarthy, Dr. Pam Ohashi, and Dr. Jeff Browning for critical reading ofthe manuscript. This work was supported by a Multiple Sclerosis Society ofCanada postdoctoral fellowship to Y.G., a Canadian Institutes of Health Re-search (CIHR) doctoral award to L.S.-d., a CIHR New Investigator award toJ.L.G., and a CIHR operating grant to J.L.G. (MOP 67157).

1. Bevan MJ (2004) Helping the CD8(+) T-cell response. Nat Rev Immunol 4:595–602.2. Hochweller K, Anderton SM (2005) Kinetics of costimulatory molecule expression by T

cells and dendritic cells during the induction of tolerance versus immunity in vivo. EurJ Immunol 35:1086–1096.

3. Summers-deLuca LE, et al. (2007) Expression of lymphotoxin-αβ on antigen-specific Tcells is required for DC function. J Exp Med 204:1071–1081.

4. Bennett SR, et al. (1998) Help for cytotoxic-T-cell responses is mediated by CD40signalling. Nature 393:478–480.

5. Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ (1998) T-cell help forcytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480–483.

6. Ridge JP, Di Rosa F, Matzinger P (1998) A conditioned dendritic cell can be a temporalbridge between a CD4+ T-helper and a T-killer cell. Nature 393:474–478.

7. Wong BR, Josien R, Choi Y (1999) TRANCE is a TNF family member that regulatesdendritic cell and osteoclast function. J Leukoc Biol 65:715–724.

8. Wong BR, et al. (1997) TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, isa dendritic cell-specific survival factor. J Exp Med 186:2075–2080.

9. Puglielli MT, et al. (1999) Reversal of virus-induced systemic shock and respiratoryfailure by blockade of the lymphotoxin pathway. Nat Med 5:1370–1374.

10. Guo Z, et al. (2001) Cutting edge: Membrane lymphotoxin regulates CD8(+) T cell-mediated intestinal allograft rejection. J Immunol 167:4796–4800.

11. Tamada K, et al. (2002) Blockade of LIGHT/LTβ and CD40 signaling induces allospecificT cell anergy, preventing graft-versus-host disease. J Clin Invest 109:549–557.

12. Kanodia S, et al. (2010) Expression of LIGHT/TNFSF14 combined with vaccinationagainst human papillomavirus type 16 E7 induces significant tumor regression. CancerRes 70:3955–3964.

13. Bennett SR, Carbone FR, Karamalis F, Miller JF, Heath WR (1997) Induction of a CD8+

cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help.J Exp Med 186:65–70.

14. De Trez C, et al. (2008) The inhibitory HVEM-BTLA pathway counter regulateslymphotoxin receptor signaling to achieve homeostasis of dendritic cells. J Immunol180:238–248.

15. Kabashima K, et al. (2005) Intrinsic lymphotoxin-β receptor requirement forhomeostasis of lymphoid tissue dendritic cells. Immunity 22:439–450.

16. Wang YG, Kim KD, Wang J, Yu P, Fu YX (2005) Stimulating lymphotoxin β receptor onthe dendritic cells is critical for their homeostasis and expansion. J Immunol 175:6997–7002.

17. Caux C, et al. (1994) Activation of human dendritic cells through CD40 cross-linking.J Exp Med 180:1263–1272.

18. Probst HC, et al. (2005) Histological analysis of CD11c-DTR/GFP mice after in vivodepletion of dendritic cells. Clin Exp Immunol 141:398–404.

19. Driessens G, et al. (2003) Micronuclei to detect in vivo chemotherapy damage in a p53mutated solid tumour. Br J Cancer 89:727–729.

20. Schneider K, et al. (2008) Lymphotoxin-mediated crosstalk between B cells and splenicstroma promotes the initial type I interferon response to cytomegalovirus. Cell HostMicrobe 3:67–76.

21. Le Bon A, et al. (2006) Direct stimulation of T cells by type I IFN enhances the CD8+ Tcell response during cross-priming. J Immunol 176:4682–4689.

22. Curtsinger JM, Valenzuela JO, Agarwal P, Lins D, Mescher MF (2005) Type I IFNsprovide a third signal to CD8 T cells to stimulate clonal expansion and differentiation.J Immunol 174:4465–4469.

23. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K (2005) Type Iinterferons act directly on CD8 T cells to allow clonal expansion and memoryformation in response to viral infection. J Exp Med 202:637–650.

24. Le Bon A, et al. (2003) Cross-priming of CD8+ T cells stimulated by virus-induced type Iinterferon. Nat Immunol 4:1009–1015.

25. Luft T, et al. (1998) Type I IFNs enhance the terminal differentiation of dendritic cells.J Immunol 161:1947–1953.

26. Chiang EY, et al. (2009) Targeted depletion of lymphotoxin-α-expressing TH1 andTH17 cells inhibits autoimmune disease. Nat Med 15:766–773.

27. Vu F, Dianzani U, Ware CF, Mak T, Gommerman JL (2008) ICOS, CD40, andlymphotoxin β receptors signal sequentially and interdependently to initiatea germinal center reaction. J Immunol 180:2284–2293.

28. Ahonen CL, et al. (2004) Combined TLR and CD40 triggering induces potent CD8+ Tcell expansion with variable dependence on type I IFN. J Exp Med 199:775–784.

29. Lefrançois L, Altman JD, Williams K, Olson S (2000) Soluble antigen and CD40triggering are sufficient to induce primary and memory cytotoxic T cells. J Immunol164:725–732.

30. Matthews KE, et al. (2007) Increasing the survival of dendritic cells in vivo does notreplace the requirement for CD4+ T cell help during primary CD8+ T cell responses.J Immunol 179:5738–5747.

31. Maxwell JR, Ruby C, Kerkvliet NI, Vella AT (2002) Contrasting the roles ofcostimulation and the natural adjuvant lipopolysaccharide during the induction of Tcell immunity. J Immunol 168:4372–4381.

32. Bachmann MF, et al. (2004) Cutting edge: Distinct roles for T help and CD40/CD40ligand in regulating differentiation of proliferation-competent memory CD8+ T cells.J Immunol 173:2217–2221.

33. Hernandez MG, Shen L, Rock KL (2008) CD40 on APCs is needed for optimalprogramming, maintenance, and recall of CD8+ T cell memory even in the absence ofCD4+ T cell help. J Immunol 180:4382–4390.

34. Johnson S, et al. (2009) Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells.Immunity 30:218–227.

35. Van Deusen KE, Rajapakse R, Bullock TN (2010) CD70 expression by dendritic cellsplays a critical role in the immunogenicity of CD40-independent, CD4+ T cell-dependent, licensed CD8+ T cell responses. J Leukoc Biol 87:477–485.

36. Hoshino K, Kaisho T, Iwabe T, Takeuchi O, Akira S (2002) Differential involvement ofIFN-β in Toll-like receptor-stimulated dendritic cell activation. Int Immunol 14:1225–1231.

37. Tam MA, Sundquist M, Wick MJ (2008) MyD88 and IFN-αβ differentially controlmaturation of bystander but not Salmonella-associated dendritic cells orCD11cintCD11b+ cells during infection. Cell Microbiol 10:1517–1529.

38. Agarwal P, et al. (2009) Gene regulation and chromatin remodeling by IL-12 and typeI IFN in programming for CD8 T cell effector function and memory. J Immunol 183:1695–1704.

39. Kumaraguru U, Davis IA, Deshpande S, Tevethia SS, Rouse BT (2001) Lymphotoxin α−/−

mice develop functionally impaired CD8+ T cell responses and fail to contain virusinfection of the central nervous system. J Immunol 166:1066–1074.

40. Lund FE, et al. (2002) Lymphotoxin-α-deficient mice make delayed, but effective, Tand B cell responses to influenza. J Immunol 169:5236–5243.

41. Lukashev M, et al. (2006) Targeting the lymphotoxin-β receptor with agonistantibodies as a potential cancer therapy. Cancer Res 66:9617–9624.

6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1014188108 Summers deLuca et al.

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021