Production of Bystander Dendritic Cells but Not IL-12 Newly Activated

Newly Activated T Cells Promote Maturation of Bystander Dendritic Cells but Not IL-12 Production

This information is current as Roman Spörri and Caetano Reis e Sousa of September 25, 2021. J Immunol 2003; 171:6406-6413; ; doi: 10.4049/jimmunol.171.12.6406 http://www.jimmunol.org/content/171/12/6406 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Newly Activated T Cells Promote Maturation of Bystander Dendritic Cells but Not IL-12 Production1

Roman Spo¨rri and Caetano Reis e Sousa2

The activation of dendritic cells (DC) leads to increased costimulatory activity (termed DC maturation) and, in some instances, production of immunomodulatory cytokines such as IL-12. Both innate and T cell-derived signals can promote DC activation but it is unclear to what extent the two classes of stimuli are interchangeable or regulate distinct aspects of DC function. In this study, we show that signals from newly activated CD4؉ T cells cannot initiate IL-12 synthesis although they can amplify secretion of bioactive IL-12 p70 by DC exposed to an appropriate innate stimulus. This occurs exclusively in cis and does not inﬂuence IL-12 synthesis by bystander DC that do not present Ag. In marked contrast, signals from newly activated CD4؉ T cells can induce an increase in DC costimulatory activity in the absence of any innate priming. This occurs both in cis and in trans, affecting all DC

in the microenvironment, including those that do not bear specific Ag. Consistent with the latter, we show that newly activated Downloaded from CD4؉ T cells in vivo can deliver “help” in trans, effectively lowering the number of MHC/peptide complexes required for proliferation of third-party naive CD4؉ T cells recognizing Ag on bystander DC. These results demonstrate that DC maturation and cytokine production are regulated distinctly by innate stimuli vs signals from CD4؉ T cells and reveal a process of trans activation of DC without secretion of polarizing cytokines that takes place during T cell priming and may be involved in amplifying immune responses. The Journal of Immunology, 2003, 171: 6406–6413. http://www.jimmunol.org/ endritic cells (DC)3 are the major class of APC regulat- arising from innate recognition and signals emanating from cells of ing adaptive immunity (1, 2). Resting DC in vivo present the adaptive immune system. D low amounts of Ag and lack the capacity to promote a “DC activation” is used loosely to refer to any one of multiple functional T cell immune response (1, 2). However, upon activa- changes in DC phenotype and function. These include, among oth- tion, DC mature into potent immunostimulatory APC that can ers, “DC maturation” (increase in DC expression of MHC, co- drive T cell clonal expansion and, through production of immu- stimulatory and adhesion molecules resulting in an increased abil- nomodulatory cytokines, direct the differentiation of Th precursors ity to stimulate T cell proliferation), migration, endocytic pathway into type 1 or type 2 effectors (1, 2). The signals that initiate and remodeling, and production of immunomodulatory cytokines (re- regulate DC activation are, therefore, critical for eliciting appro- viewed in Refs. 1 and 2). It is becoming increasingly clear that not by guest on September 25, 2021 priate immune responses. Microbial infection is thought to be the all aspects of DC activation are regulated concordantly and that primary stimulus for DC activation in vivo (3). DC express pat- they can be differentially induced by innate vs adaptive signals. tern-recognition receptors such as members of the Toll-like recep- For example, innate stimuli can promote production of cytokines tor (TLR) family, which recognize unique molecular features of such as IL-12, IL-10, or IFN-␣ whereas CD40 ligation cannot microbes and signal directly for activation (4, 5). DC can also be initiate cytokine synthesis but can amplify that which has been activated indirectly by cytokines produced by infected cells, by initiated by microbial signals (14, 15). However, it is not clear stress induced by an infection, or by signals from other innate cells whether other aspects of DC activation are similarly dependent on such as NK cells, NKT cells, and ␥␦ T cells (reviewed in Ref. 6). microbial priming and whether T cell-derived signals act exclu- In addition to infection-related signals, T cells themselves can be sively on Ag-bearing DC or also regulate the function of bystander potent activators of DC (7, 8). For example, CD40 ligand (CD40L) cells. In this study, we focus on the interplay between innate and up-regulated on CD4ϩ T cells after exposure to Ag is an important T cell-derived signals in the regulation of two distinct aspects of stimulus for DC activation and plays a unique role in licensing murine DC activation. We use naive T cells and splenic DC to APC for CTL priming (9–11). Similarly, signals from CD8ϩ T show that amplification of IL-12 synthesis by T cell signals occurs cells have also been shown to activate DC (12, 13). Thus, DC- strictly in cis and does not affect bystander DC in trans. In contrast, activating stimuli appear to fall broadly into two classes: signals we show that T cell-driven DC maturation occurs both in cis and in trans and is independent of microbial priming. Our results suggest that naive CD4ϩ T cells may help each other via DC and Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lon- don, United Kingdom reveal the complex interactions between innate and adaptive signals that modulate DC function. Received for publication May 23, 2003. Accepted for publication October 9, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Reagents 1 This study was supported by Cancer Research UK. CpG-containing phosphorothioate-linked oligonucleotide 1668 (CpG) 5Ј- 2 Address correspondence and reprint requests to Dr. Caetano Reis e Sousa, Immu- TCCATGACGTTCCTGATGCT-3Ј (16) was synthesized by the Cancer nobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Research UK oligonucleotide synthesis service (Clare Hall, South Mimms, Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. E-mail U.K.). Peptides derived from chicken OVA (ISQAVHAAHAEINEAGR, address: [email protected] residues 323–339; pOVA) and hen egg lysozyme (NTDGSTDY 3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; GILQINSR, residues 46–61; pHEL) were synthesized and HPLC-purified CD40L, CD40 ligand; HEL, hen egg lysozyme; MFI, median fluorescence intensity. by the Cancer Research UK peptide synthesis service. Throughout this

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6407 paper, pOVA and pHEL are used to refer to the peptides and not the ster IgG), I-Ad (AMS-32.1, mouse IgG2b), I-Ek (14-4-4S, mouse IgG2a), proteins. All reagents were free of endotoxin as determined using a Limulus Fc␥RIII/II (2.4G2, rat IgG2b), CD11b (M1/70, rat IgG2b), Gr-1 (RB6- Amebocyte Lysate test (BioWhittaker, Walkersville, MD). 8C5, rat IgG2b), B220 (RA3-6B2, rat IgG2a), pan-NK cells (DX5, rat IgM), ␥␦TCR (GL3, hamster IgG2), CD8␣ (53-6.7, rat IgG2a), and CD69 Mice (H1.2F3, hamster IgG1), washed and incubated with magnetic streptavidin- DO11.10 mice (17) on a BALB/c-scid background carrying a transgenic beads (Miltenyi Biotec). Labeled cells were passed through a MACS col- V␣2/V␤8 TCR specific for pOVA presented on I-Ad were originally a gift umn and the flow-through fraction was collected. Resulting cell prepara- from Dr. P. Garside (University of Glasgow, Glasgow, U.K.). 3A9 mice tions were found to be free of APC in functional assays. Serum-free buffers (18) bearing a transgenic V␣3/V␤8 TCR specific for hen egg lysozyme were used throughout when cells were used for adoptive transfer. k 46–61 (pHEL) presented on I-A were purchased from The Jackson Lab- Adoptive transfers oratory (Bar Harbor, ME) and bred onto a RAG-2-deficient B10.BR background. 3A9 and DO11.10 T cells were tested and found not to be allo- APC-depleted CD4ϩ DO11.10 T cells (5 ϫ 106) were transferred into reactive against BALB/c (H-2d) or B10.BR (H-2k), respectively (data not naive host animals by i.v. injection 1 day before immunization. In some shown). Mice genetically deficient in IL-12 p40 (19) on a BALB/c back- experiments, 1 ϫ 107 purified CD4ϩ DO11.10 T cells were cotransferred Ϫ Ϫ ground (IL-12 / BALB/c) were purchased from The Jackson Laboratory. with 2 ϫ 106 purified CD4ϩ 3A9 T cells that had been labeled with 2 ␮M All of the above mice, as well as wild-type B10.BR (H-2k), BALB/c (H- CFSE (Molecular Probes; 12 min at 37°C). d ϫ dϫk ϫ dϫk 2 ), BALB/c B10.BR F1 (H-2 ), and DO11.10 B10.BR F1 (H-2 ) were bred and kept at the animal facility of Cancer Research UK under Ex vivo DC stimulatory capacity assay specific pathogen-free conditions, and used at 6–12 wk of age, sex- and ϫ (DO11.10 ϫ B10.BR)F mice (H-2d k) were immunized by i.v. injection age-matched within experiments. 1 of 25 ␮g of pOVA and/or 25 ␮g of CpG. Twelve hours later, splenic DC For generation of chimeric mice, bone marrow cells harvested from from these mice were FACS-sorted and fixed with 1% paraformaldehyde femurs and tibias were depleted of T cells using anti-CD90 magnetic beads for 15 min at room temperature. The fixed DC were then washed twice and Downloaded from (Miltenyi Biotec, Bisley, U.K.). Four-week-old B10.BR mice were gam- the residual paraformaldehyde quenched by incubation in 0.1 mM glycine ma-irradiated with 2 ϫ 5 Gy and reconstituted with 2 ϫ 106 bone-marrow for 30 min at room temperature. After further washing, 1 ϫ 104 fixed DC cells from B10.BR mice mixed with an equal number of cells derived from Ϫ Ϫ ϩ ϩ were cocultured in triplicate in 200 ␮l of complete medium (RMPI 1640 either IL-12 / BALB/c or IL-12 / BALB/c mice. Six to 8 wk after supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 reconstitution, mice were tested for chimerism. Host mice were only used ␮g/ml streptomycin, 2 mM glutamine, and 50 ␮M 2-ME) in round-bottom in experiments if FACS analysis of blood leukocytes showed cells of both ϩ 96-well plates together with 5 ϫ 104 APC-depleted CD4 T cells purified host (H-2Kk) and donor (H-2Dd) origin at a ratio close to 1:1. All animal from naive 3A9 mice, and graded doses of pHEL peptide. After 48 h, IL-2 procedures and husbandry were in accordance with U.K. governmental http://www.jimmunol.org/ levels in the culture supernatants were determined by sandwich ELISA. regulations and institutional policies. Flow cytometry In vitro DC activation assays Splenic DC (5 ϫ 105) from BALB/c mice were cocultured in 24-well plates Cell suspensions were stained in ice-cold PBS supplemented with 2 mM ϩ with 1 ϫ 106 purified CD4 DO11.10 T cells and graded doses of pOVA EDTA, 1% FCS, and 0.02% sodium azide. mAbs were purchased from BD in the presence or absence of 0.5 ␮g/ml CpG in 2 ml of complete medium. PharMingen (Oxford, U.K.) preconjugated to various fluorochromes or to In experiments addressing trans feedback to nonpresenting DC, an addi- biotin and were directed against: CD11c (clone HL3, hamster IgG), B7-2 tional 5 ϫ 105 splenic DC from B10.BR mice were added to the wells. (GL1, rat IgG2a), CD40 (3/23, rat IgG2a), H-2Dd (34-2-12, mouse IgG2a), Twelve hours later, supernatants were harvested and IL-12 p70 was mea- H-2Kk (36-7-5, mouse IgG2a), CD4 (RM4-5, rat IgG2a) and V␤8 TCR ␥ sured. Surface expression of B7-2 and CD40 on DC of BALB/c or B10.BR

(MR5-2, mouse IgG2a). Purified 2.4G2 (anti-Fc RIII/II, rat IgG2b; used to ϩ Ϫ Ϫ ϩ by guest on September 25, 2021 origin (CD11chighH-2Dd H-2Kk or CD11chighH-2Dd H-2Kk , respec- block unspecific Ab binding), and 1G12 (anti-3A9 clonotypic mAb (Ref. tively) was determined by FACS analysis. Alternatively, in experiments 20, mouse IgG1; a kind gift from Dr. E. Unanue, Washington University, addressing cis vs trans feedback for the production of IL-12 p70, 2 ϫ 104 St. Louis, MO) were from the Cancer Research UK Ab production service. Ϫ Ϫ ϩ ϩ DC from IL-12 / BALB/c or IL-12 / BALB/c, respectively, were 1G12 was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, ϩ cocultured with 2 ϫ 104 B10.BR DC and 5 ϫ 104 APC-depleted CD4 Rockford, IL) according to the manufacturer’s instructions. Streptavidin DO11.10 T cells and graded doses of pOVA peptide in the presence of 0.5 conjugates (CyChrome, allophycocyanin) were from BD PharMingen. ␮g/ml CpG. Twelve hours later, IL-12 p70 levels in the supernatant of Where applicable, TO-PRO3, a DNA-binding dye for live-dead discrimi- triplicate cultures were determined by sandwich ELISA. In transwell ex- nation (Molecular Probes, Leiden, The Netherlands), was added to the periments designed to study the role of soluble T cell-derived factors in DC samples immediately before data acquisition. Data were collected on a maturation, splenic DC from BALB/c mice were incubated with 0.5 ␮g/ml FACSCalibur (BD Biosciences, Oxford, U.K.) and analyzed using FlowJo CpGfor1hat37°C, washed and fixed as described above. APC-depleted software (Treestar, San Carlos, CA). ϩ CD4 DO11.10 T cells (1 ϫ 106) were cocultured in 24-well plates with Cell purification 5 ϫ 105 fixed or fresh live DC Ϯ 1 ␮M pOVA. Alternatively, the T cells were cultured in wells coated with 5 ␮g/ml anti-CD3⑀ (2C11, hamster DC were purified from freshly isolated spleens injected with serum-free IgG1; Cancer Research UK) and 5 ␮g/ml anti-CD28 (37.51, hamster IgG2; medium supplemented with Liberase CI (1.7 Wu¨nsch-U/ml; Roche Diag- kind gift from Dr. J. P. Allison (University of California, Berkeley, CA); nostics, Lewes, U.K.) and DNase I (0.2 mg/ml; Roche Diagnostics) (14). produced at Cancer Research UK). Cell culture inserts with a permeable After 20 min at 37°C, digested spleens were strained through a 40-␮m cell membrane (0.4-␮m pore size, “transwell”) were inserted into all wells and sieve and washed once with ice-cold PBS supplemented with 2 mM EDTA additional aliquots of 5 ϫ 105 fresh BALB/c DC were added. After 12 h of and 1% FCS. Splenocyte suspensions were labeled with anti-CD11c coculture in a total volume of 2 ml of complete medium, the expression of MACS beads (clone N418; Miltenyi Biotec, Bisley, U.K.) for 10 min at costimulatory molecules on DC in the transwells was analyzed by flow 4°C, followed by washing with chilled PBS and positive selection using LS cytometry. magnetic columns (Miltenyi Biotec), according to the manufacturer’s instructions. Resulting cells were routinely Ͼ90% pure, viable and free of T Cytokine ELISA cells (CD11chigh, MHC class IIϩ, TCR␤Ϫ, TO-PRO3negative). In some experiments, DC were further purified by FACS sorting. Briefly, pre-enriched Concentrations of IL-12 p70 and IL-2 in culture supernatants and sera were DC were stained with sterile-filtered fluorescently labeled anti-CD11c Ab determined using standard sandwich ELISA using the following Ab pairs and, where applicable, with anti-H-2Dd and anti-H-2Kk for 30 min at 4°C (capture, detection), at the concentrations recommended by the manufac- and washed once with chilled PBS. CD11chigh H-2DdϩH-2Kk Ϫor turer (BD PharMingen): 9A5, C17.8 (biotinylated) for IL-12 p70 and JES6- CD11chighH-2DdϪH-2Kkϩ cells were then sorted on a MoFlo cytometer 1A12, JES6-5H4 (biotinylated) for IL-2. (Cytomation, Fort Collins, CO). Resulting cell preparations were routinely Quantitative RT-PCR Ͼ99% pure and viable (TO-PRO3negative, CD11chigh, H-2DdϩH-2KkϪ or H-2DdϪH-2Kkϩ, respectively). Total RNA from FACS-sorted DC was isolated using the RNeasy Mini kit T cells from naive TCR-transgenic mice were purified by negative se- (Qiagen, Crawley, U.K.) according to the manufacturer’s instructions. Re- lection using magnetic beads. Briefly, single cell suspensions were pre- sidual genomic DNA was digested using RNase-free DNase I (Qiagen). pared by forcing lymph nodes and spleens through 40-␮m cell sieves with cDNA was synthesized using Life Technologies reagents (Invitrogen, CA). a syringe plunger. Cells were labeled with a mixture of sterile-filtered For relative quantitation of IL-12 p35 and IL-12 p40 mRNA, amplification biotinylated mAbs (all from BD PharMingen) against CD11c (HL3, ham- of sample cDNA was monitored with the fluorescent DNA-binding dye 6408 DC ACTIVATION BY T CELLS IN cis AND trans

FAM in combination with the ABI PRISM 7700 detection system (Applied transfer of naive DO11.10 T cells into BALB/c mice, immuniza- Biosystems, Warrington, U.K.), according to the manufacturer’s instruc- tion with pOVA323–339 peptide (pOVA) alone did not induce mea- tions. IL-12 p40- and IL-12 p35-specific primers were purchased as pre- surable levels of IL-12 p70 in the circulation (Fig. 1A). However, developed TaqMan assay reagents (Applied Biosystems). Specific message levels were normalized to GAPDH as recommended by the manufacturer. the combination of a microbial stimulus such as a CpG-containing oligonucleotide (mimic of bacterial DNA; CpG) and pOVA in- Statistical analysis duced higher levels of the cytokine than CpG alone, confirming The statistical significance of differences between experimental samples that signals from newly activated T cells can contribute to IL-12 was determined using the Student t test. p70 production after microbial priming in vivo (Fig. 1A). Simi- larly, when splenic DC were used to stimulate naive DO11.10 T Results cells in vitro, there was little accumulation of IL-12 p70 in super- Signals from newly activated T cells are sufficient to promote natants unless CpG was added to the culture (data not shown; see DC maturation but not IL-12 p70 synthesis in the absence of Refs. 14 and 15). microbial priming In marked contrast, activation of DO11.10 T cells in vivo by We have previously reported that T cell feedback signals, includ- pOVA in the absence of CpG was sufficient to induce an increase ing CD40L, cannot initiate IL-12 production by splenic DC but in DC expression of B7-2 and CD40 (Fig. 1B), as well as B7-1 and markedly amplify the levels of IL-12 p70 elicited by a microbial MHC class II (not shown). This increase was not as marked as that stimulus (14). Consistent with those data, following adoptive elicited by CpG or the combination of CpG ϩ pOVA but it was Downloaded from http://www.jimmunol.org/

FIGURE 1. Activation of naive CD4ϩ T cells in- duces DC maturation but not IL-12 production in the absence of innate stimuli. A, Purified DO11.10 T cells were adoptively transferred into naive BALB/c mice. The next day, recipient mice were immunized with CpG Ϯ pOVA. Levels of IL-12p70 in pooled by guest on September 25, 2021 sera (two mice per group) were measured 12 h later. Error bars are shown and indicate one SD from the mean of triplicate determinations. One experiment representative of three is shown. B, Mice were treated as in A and splenic DC (TO-PRO3negative, CD11chigh) were analyzed by flow cytometry for surface expression of B7-2 (left) and CD40 (right). Upper panels, Representative staining profiles in one experiment of DC pooled from two mice in each group. Lower panels, The average across three experiments of the fold-increase in median fluorescence intensities (MFI) compared with the control group. Error bars are shown and represent one SD. Differences between the control and experimental groups were statistically significant (p Յ 0.008). C, DO11.10 ϫ B10.BR (H-2dϫk) were immunized as in A. Twelve hours later, CD11chigh splenic cells from three mice per group were purified, fixed, and cocultured with APC-depleted naive 3A9 CD4ϩ T cells with the indicated graded doses of pHEL peptide. IL-2 in the supernatants was measured after 48 h. Error bars indicate one SD from the mean of triplicate cultures. Results are representative of two independent experiments. The Journal of Immunology 6409 consistently seen in multiple experiments, both in vivo and in vitro, using different Ags and transgenic T cells (data not shown). Increases in B7 and CD40 expression by DC were unimodal, affecting all DC in the population (Fig. 1B). pOVA injection into naive BALB/c mice that had not received DO11.10 T cells did not induce any changes in DC phenotype, demonstrating that the peptide was not contaminated with a microbial activator of DC such as LPS (data not shown). To determine whether the observed increases in B7 and CD40 correlated with an increase in the ability of DC to stimulate naive T cells, we designed a system in which DC exposed to signals from newly activated T cells in vivo were subsequently tested for their ability to stimulate naive T cells of a different specificity ex vivo. We used DO11.10 transgenic mice on a mixed H-2kϫd background to ensure the presence of a sufficient number of pOVA-specific T cells without the need for adoptive transfer. These mice were immunized with CpG Ϯ pOVA and, 12 h later, splenic DC were purified by cell sorting, fixed in paraformaldehyde to prevent further functional and phenotypic changes, and used as stimulators for naive 3A9 T cells (specific for Downloaded from k pHEL46–61 peptide (pHEL) in the context of I-A ). Not surprisingly, resting fixed DC were poor stimulators whereas those isolated from mice that had received both CpG and pOVA displayed high stimulatory capacity (Fig. 1C). Remarkably, DC purified from mice that had received pOVA alone were just as potent stimulators

as CpG-activated DC, although not as powerful as DC from mice http://www.jimmunol.org/ treated with CpG ϩ pOVA (Fig. 1C). There was a good correlation between the relative stimulatory capacity of DC in each group and their level of expression of maturation markers such as B7-1, B7-2, and CD40 (Fig. 1, B and C). These results demonstrate that 1) FIGURE 2. Newly activated CD4ϩ T cells in vitro amplify IL-12 pro- exposure to T cell-derived signals alone can lead to phenotypic and duction by DC in cis but can induce DC maturation in trans via soluble functional maturation of DC in vivo but does not induce cytokine factors. A, Puriﬁed B10.BR DC (H-2k) were mixed with equal numbers of Ϫ/Ϫ d d production in the absence of microbial priming and 2) the combi- DC from IL-12 BALB/c (H-2 ) or wild-type BALB/c (H-2 ) mice and nation of microbial and T cell-derived stimuli leads to optimal DC cocultured with puriﬁed DO11.10 T cells in the presence of CpG and

graded doses of pOVA. IL-12 p70 in culture supernatants was measured by guest on September 25, 2021 activation whether assessed at the level of maturation or cytokine after 12 h. Error bars indicate one SD from the mean of triplicate cultures. production. Results are representative of three independent experiments. B, Equal num- d k Signals from newly activated T cells do not increase IL-12 p70 bers of DC purified from BALB/c (H-2 ) or B10.BR (H-2 ) mice were cocultured with purified DO11.10 T cells and the indicated doses of pOVA production by bystander nonpresenting DC primed by microbial in the absence of CpG. After 12 h, the surface expression of B7-2 on H-2k stimulation or H-2d DC was assessed by flow cytometry. Data represent MFI values The contribution of T cell-derived signals to DC maturation and and are representative of three independent experiments. C, DO11.10 T cytokine production was further dissected in vitro and in vivo. We cells were cocultured with fresh or fixed BALB/c DC ϫ pOVA. Alterna- first determined whether the amplification of IL-12 production by tively, DO11.10 T cells were cultured on tissue culture plates coated with ⑀ T cell signals requires a cognate interaction between the T cell and anti-CD3 /anti-CD28 Abs (anti-CD3, no DC). As indicated schematically, additional aliquots of fresh BALB/c DC were added to transwells placed the DC or whether amplification signals can be delivered in trans d into the tissue culture plates. After 12 h of coculture, the expression of to nonpresenting DC. H-2 splenic DC were purified from IL- CD40 on TO-PRO3negative DC in the transwells was analyzed by flow ϩ/ϩ Ϫ/Ϫ 12 or IL-12 BALB/c mice and were used to stimulate cytometry. Bars represent the normalized MFI values averaged from two k Samples are significantly different ,ء .DO11.10 T cells in the presence of bystander B10.BR H-2 DC. independent experiments Ϯ one SD The latter can produce IL-12 but cannot present pOVA and, there- from the control (p Յ 0.004). fore, can only receive feedback signals from T cells in trans. In the presence of CpG, pOVA led to a dose-dependent accumulation of IL-12 p70 in cultures containing IL-12ϩ/ϩ H-2d DC, as expected with chimeras made using a mixture of IL-12Ϫ/Ϫ H-2d and IL- (Fig. 2A). In contrast, little IL-12 p70 accumulated in wells con- 12ϩ/ϩ H-2k bone marrow (Fig. 3A), despite the presence in those taining IL-12Ϫ/Ϫ H-2d DC (Fig. 2A) despite the fact that the latter mice of H-2k APC capable of producing IL-12. This result indi- stimulated T cell proliferation to the same extent as IL-12ϩ/ϩ H-2d cates that in vivo, like in vitro, T cells deliver signals that augment DC (data not shown). These results suggested that T cell feedback IL-12 p70 production by Ag-bearing cells but not bystander APC. signals for IL-12 p70 production are delivered exclusively to cells The above experiment relied on serum levels of cytokine and presenting Ag and do not affect bystander nonpresenting DC in did not show regulation of IL-12 production at the level of DC. close contact. To determine whether this is also true in vivo, we Therefore, DC were purified from spleens of the H-2k/H-2d chi- constructed different types of mixed H-2k/H-2d bone marrow chimeric DO11.10 recipients and were sorted into cells of H-2k or meric mice and used them as adoptive recipients for naive H-2d origin (Fig. 3B). Each of these DC populations was analyzed DO11.10 T cells. Injection of recipients bearing wild-type H-2d for expression levels of mRNA for the IL-12 p40 and IL-12 p35 and H-2k bone marrow with CpG ϩ pOVA led to an increase in subunits. Feedback from T cells for IL-12 p70 secretion can mark- serum IL-12 p70 over the levels elicited by CpG alone (Fig. 3A), edly increase synthesis of the IL-12 p35 subunit, which is poorly indicating a role for T cell feedback. No such increase was seen induced by microbial stimulation alone (14). Consistent with this 6410 DC ACTIVATION BY T CELLS IN cis AND trans

FIGURE 3. Newly activated CD4ϩ T cells in vivo amplify IL-12 production in cis but not in trans. A, Puriﬁed DO11.10 T cells were adoptively transferred into IL-12Ϫ/Ϫ BALB/c ϩ B10.BR3B10.BR (left panel) or IL-12ϩ/ϩ BALB/c ϩ B10.BR3B10.BR (right panel) mixed radiation chimeras. The next day, these mice were injected i.v. with CpG Ϯ pOVA. IL-12 p70 levels in pooled sera from three mice per group were measured 9 h later. Error bars indicate one SD from the mean of tripli- Downloaded from cate cytokine measurements. B, Pooled splenocytes from some of the experimental groups in A (from IL-12ϩ/ϩ BALB/c ϩ B10.BR3B10.BR mice) were separated into DC of BALB/c (H-2d) or B10.BR (H- 2k) origin. Proﬁles of the sorted cells are shown in the upper panels (TO-PRO3negative events). cDNA

prepared from sorted DC from the groups treated http://www.jimmunol.org/ with PBS or CpG ϩ pOVA was used to determine relative message levels for IL-12 p40 and IL-12 p35 by quantitative PCR. Results are representative of two independent experiments. by guest on September 25, 2021

notion, there was marked up-regulation of IL-12 p35 message in presence of Ag (Fig. 2B and data not shown). Indeed, after taking H-2d DC puriﬁed from mice immunized with CpG ϩ pOVA (Fig. into account the starting levels of the two markers, up-regulation 3B). Importantly, IL-12 p35 mRNA was completely absent from looked identical for both the presenting (H-2d) and nonpresenting nonpresenting H-2k DC, indicating that they had not received ap- (H-2k) DC (Fig. 2B). Similar results were seen in vivo in H-2k/ propriate T cell signals (Fig. 3B). In contrast, IL-12 p40 message H-2d mixed chimeras given DO11.10 T cells and immunized with was found in both H-2k and H-2d DC taken from chimeric mice CpG Ϯ pOVA: CD40 and B7-2 were up-regulated on both H-2d given CpG ϩ pOVA (Fig. 3B) or CpG alone (not shown), consis- and H-2k DC in mice receiving pOVA compared with PBS or CpG tent with the fact that the IL-12 p40 subunit is primarily regulated ϩ pOVA compared with CpG alone (Fig. 4 and data not shown). by microbial stimulation (14) and acting as a positive control for This was true independent of whether the H-2d bone marrow was the ability of H-2k DC to produce IL-12. These results demonstrate of IL-12ϩ/ϩ or IL-12Ϫ/Ϫ origin (data not shown). In vitro exper- that signals from newly activated naive T cells in vivo can amplify iments further established that up-regulation of B7-2 and CD40 on microbial induction of IL-12 by regulating the level of IL-12 p35 DC by T cell-derived signals could be triggered across a transwell message on DC but that this form of T cell feedback occurs strictly or by supernatants from anti-CD3⑀-activated T cell cultures, in a cognate fashion (cis) and does not affect in trans bystander DC thereby implicating a soluble factor of T cell origin (Fig. 2C). We also primed by microbial exposure. conclude that factors produced by newly activated T cells can lead to maturation of DC in either cis or trans. Signals from newly activated T cells increase expression of costimulatory molecules on bystander nonpresenting DC Newly activated T cells help to promote the activation of other To determine whether other parameters of DC activation were sim- T cells both in cis and in trans ilarly regulated strictly in cis by T cell-derived signals, we exam- The ability of newly activated T cells to promote maturation of DC ined the up-regulation of maturation markers in both the presenting in vivo might facilitate activation of other T cells. To test this and nonpresenting DC used in the above experiments. In contrast hypothesis, CFSE-labeled 3A9 and unlabeled DO11.10 T cells k kϫd to IL-12 p70, both B7-2 and CD40 were up-regulated on H-2 DC were cotransferred adoptively into H-2 F1 mice, which were that had been cocultured with H-2d DC and DO11.10 T cells in the then immunized with pHEL together with CpG and/or pOVA. 3A9 The Journal of Immunology 6411

FIGURE 4. Newly activated CD4ϩ T cells in vivo induce DC maturation in cis and in trans. IL-12ϩ/ϩ BALB/c ϩ B10.BR3B10.BR chimeric mice were used as adoptive recipients for DO11.10 T cells and were subsequently treated as in Fig. 3. Splenic DC (pooled from three mice per Downloaded from group) of BALB/c origin (H-2d) and B10.BR origin (H-2k) were analyzed at 9 h posttreatment for surface expression of CD40. Bars display the normalized MFI Ϯ one SD averaged from two independent experiments. Experimental groups were significantly different from the respective con- trols (H-2d: p Յ 0.008; H-2k: p Յ 0.01). http://www.jimmunol.org/ T cells were subsequently identified using the gating procedure shown in Fig. 5A and analyzed for CFSE content as a measure of proliferation. pOVA coadministration significantly lowered the amount of pHEL required for 3A9 proliferation (Fig. 5B): whereas 1 ␮g of pHEL alone failed to induce measurable CFSE dilution, a fraction of the 3A9 T cells in mice immunized with 1 ␮gof pHEL ϩ pOVA underwent one cell division (Fig. 5B, arrows). Similarly, pOVA augmented 3A9 proliferation at the 5 ␮gof pHEL dose (Fig. 5B, arrows). Like pOVA, CpG, a bona fide ad- by guest on September 25, 2021 juvant, also increased 3A9 proliferation at both doses of pHEL and the combination of CpG and pOVA was additive and promoted the ϩ ϩ greatest expansion of the 3A9 T cells (Fig. 5B). FIGURE 5. CD4 help for CD4 T cells is effective in cis or in trans. If DO11.10 “help” for 3A9 proliferation involved DC matura- DO11.10 T cells and CFSE-labeled 3A9 T cells were adoptively transferred into (BALB/c ϫ B10.BR)F mice (H-2dϫk) or BALB/c ϩ tion, it should act in trans as well as in cis, consistent with the 1 B10.BR3B10.BR chimeric (H-2d ϩ H-2k3H-2k) mice. The next day, all observations on regulation of B7-2 and CD40. To test this possi- animals were immunized with CpG Ϯ pOVA, in combination with the bility, 3A9 and DO11.10 T cells were cotransferred adoptively into indicated doses of pHEL and the CFSE content of 3A9 T cells was deter- k d mixed H-2 /H-2 bone marrow chimeric mice. As shown in Fig. mined 72 h later. A, Gating procedure used to identify 3A9 T cells 5C, 3A9 T cells proliferated at lower doses of pHEL peptide when (CD4ϩV␤8 TCRϩ1G12ϩ lymphocytes). B, CFSE profiles of 3A9 T cells dϫk d ϩ k3 k pOVA was coinjected even though the two T cells recognized their in H-2 F1 recipients. C,AsforB but in H-2 H-2 H-2 chimeric respective Ags on separate APC. We conclude that newly activated recipients. Results are representative of three (B) and two (C) independent T cells can help lower the Ag dose threshold for activation of other experiments. T cells both in cis and in trans.

Discussion classes of DC activating signals are not functionally equivalent. Understanding the signals that initiate and regulate DC activation IL-12 p70 production is strictly dependent on DC presensitization is critical to our ability to manipulate the immune system for vac- by an innate stimulus and requires signals delivered in cis by cination and therapy. Both innate stimuli and T cell-derived signals newly activated T cells. In contrast, DC maturation can be induced contribute to DC activation and regulate adaptive immune re- by the same T cells in cis or in trans independent of microbial sponses (21, 22). However, it is not clear whether the two types of priming. This unexpected dichotomy in the ability of T cells to DC-activating signals are functionally equivalent and whether they regulate different aspects of the activation of presenting vs non- act synergistically or have differential effects on DC phenotype and presenting DC may have important consequences, as discussed function. In this study, we try to mimic some of the innate and below. adaptive signals that may regulate DC function at the onset of a The fact that T cell signals do not initiate IL-12 production by primary immune response to infection, namely signals from the themselves makes teleological sense as it ensures that T cell prim- pathogen and signals from newly activated naive CD4ϩ T cells ing does not lead to Th1 differentiation by default. In fact, we have that respond to it. We show that TLR signaling and feedback from previously shown that T cell feedback or CD40 ligation are “neu- newly activated T cells synergize to give maximal up-regulation of tral” signals that simply reveal a pattern of cytokine synthesis pre- costimulatory molecules and T cell stimulatory potential, as well determined by pattern recognition (14, 15, 23). In this study, we as high levels of bioactive IL-12 p70 secretion. However, the two use IL-12 p70 synthesis as a model to explore in more detail how 6412 DC ACTIVATION BY T CELLS IN cis AND trans innate and adaptive signals regulate cytokine synthesis by DC. terminant required for immunity in many models (39–41). Indeed, Surprisingly, we find that T cell feedback for IL-12 production is in their pioneering work on the DO11.10 adoptive transfer system, delivered strictly in cis and does not affect DC that do not present Jenkins and colleagues (42) showed that pOVA injection led to T Ag. This is seen both in vivo and in vitro, in conditions in which cell deletion, in line with the notion that administration of Ag in T cells are in very close proximity to nonpresenting DC. These the absence of adjuvant favors tolerance rather than immunity. findings have two implications. First, they shed light on a contro- Tolerance induction in that system involved initial T cell expan- versial aspect of DC biology, namely the capacity of DC to acquire sion followed by death and a reduction in frequency to below the MHC/peptide complexes from other cell types (24–27). The fact starting level (42). This implies that, despite the abnormally high that we fail to see any feedback in trans for IL-12 p35 synthesis in precursor frequency of Ag-specific DO11.10 T cells, which prob- our chimeras (Fig. 3) suggests that such transfer in vivo may be ably provides a supraphysiological increase in DC costimulatory inefficient. Second, our findings suggest that T cell feedback sig- potential, the latter is not sufficient as signal 2 to prevent tolerance nals for cytokine production are delivered in a very polarized fash- induction (although it may contribute to the initial proliferation). In ion toward the presenting cell, perhaps in the context of an immu- contrast, injection of pOVA together with LPS rescues some nological synapse (28). This polarization has been described DO11.10 T cells from death and promotes immunity (42). Given during T-B cell interactions (29, 30) and would be analogous on the dogma that adjuvants act via APC, this suggests that adjuvants the T cell side to the recently described polarized exocytosis of induce a signal 2 that is qualitatively distinct from costimulation. MHC class II molecules by DC toward the DC-T cell interface (31, This signal might correspond to DC-derived cytokines which, if 32). Studies are in place to identify the T cell-derived factors re- regulated like IL-12, cannot be elicited by T cell signals alone. sponsible for IL-12 amplification and whether they show any po- Such cytokines could counteract the functions of regulatory T cells Downloaded from larization toward the APC. These include CD40L, which contrib- and/or act on clonally expanded T cells to sustain survival (43– utes to ϳ50% of the IL-12 p70 levels seen in our experiments (data 46). Alternatively, adjuvants may simply have a quantitative effect, not shown), as well as other signals yet to be identified. The latter synergizing with T cell feedback to push DC costimulatory poten- might arise from the MHC class II molecule itself upon TCR en- tial past a critical threshold required to sustain T cell clonal gagement, consistent with the strict cis dependence seen here. In- expansion.

deed, certain Abs against MHC class II promote IL-12 secretion by In line with the experiments showing DC maturation in response http://www.jimmunol.org/ DC (33) and, like T cells, can regulate expression of the p35 sub- to T cell signals, we show that activation of naive DO11.10 T cells unit (34). In addition, CD8ϩ T cells or polarized CD4ϩ T cells lowers the Ag dose required for proliferation of naive 3A9 T cells produce cytokines such as IFN-␥, IL-4, or GM-CSF, which po- in vivo (Fig. 5). Although we cannot formally exclude that this tentiate IL-12 p70 production by DC (35, 36). However, such cy- effect is a consequence of direct T-T interactions, DC exposed to tokines should be effective in trans and are not thought to be pro- T cell feedback in vivo display a higher stimulatory potential for T duced by naive CD4ϩ T cells such as the DO11.10 cells used in cells ex vivo (Fig. 1C), suggesting that CD4ϩ T cell help for other our experiments (37). Consistent with this notion, supernatants CD4ϩ T cells can certainly occur via the APC. In addition, the fact from newly activated DO11.10 T cells do not synergize with TLR that T-T help in vivo can take place in trans (Fig. 5), like DC ligation to promote IL-12 p70 synthesis by DC (data not shown). maturation (Fig. 4), further suggests that the two effects may be by guest on September 25, 2021 It remains possible that the strict cis effect of T cell feedback is linked. Trans-activation of DC could be prevalent during infection, relaxed when dealing with cytokine-secreting effector or memory when the T cell precursor frequency for any given foreign Ag and T cells, thereby promoting IL-12 production by bystander DC. the multiplicity of available Ags may allow the simultaneous ac- This may explain why trans feedback for IL-12 production can be tivation of many clones of T cells. What, then, might be the role of seen with T cell hybridomas in vitro (33). trans-activated DC in the immune response? There are two sce- In contrast to IL-12, induction of DC maturation by newly ac- narios to consider. One is the trans-maturation of DC that have tivated CD4ϩ T cells did not require microbial priming and oc- captured Ags from the infectious agent in question and, therefore, curred in trans as well as in cis. DC maturation was monitored by have also, in all likelihood, been exposed to innate signals derived the up-regulation of B7-2 and CD40 and correlated with an in- from that agent. Trans-maturation of those DC will increase their creased ability of DC to activate naive T cells (Fig. 1). The ability stimulatory potential beyond that gained by exposure to the innate of CD4ϩ T cells to promote DC maturation in vivo has been pre- stimulus alone and may also improve Ag processing and presen- viously described by Muraille et al. (8) who showed that injection tation (47). The net result is that such DC may now be better able of superantigens into T cell-sufficient, but not T cell-deficient, to stimulate lower affinity T cells. Once they have been activated, mice leads to up-regulation of B7-2, CD40 and, to a lesser extent, these T cells can then sustain DC maturation through cis effects B7-1 on splenic DC. Similarly, Ruedl et al. (12) showed that ac- and amplify cytokine production. This form of T-T help would tivation of naive CD8ϩ T cells in vivo leads to DC maturation and, effectively result in clonal diversification and/or epitope spreading like us, found that this could take place in both cis and trans.In early in the immune response. A second scenario is the trans- addition, we have previously shown that T cell feedback in vivo is maturation of lymph node DC that have not been in contact with sufficient to promote increased presentation of protein Ags by DC, the infectious agent or infected cells and, as a consequence, have another aspect of DC maturation (7). The signals that mediate mat- not acquired Ag or been primed by innate recognition. Those cells uration of DC are likely to be soluble factors made by the newly will, presumably, present only self-peptides and their role is more activated T cells themselves (Fig. 2C). The molecular nature of intriguing. One possibility is that trans-activated DC presenting such factors is not known but Ruedl et al. (12) and Muraille et al. self Ags may, nevertheless, contribute to the clonal expansion of T (8) have excluded CD28, CD40L, receptor activator of NF-␬B cells specific for foreign Ag. Newly activated T cells enter a tran- ligand, TNF, IL-1, IL-4, IL-6, IL-17, IFN␣␤, and IFN-␥. In con- sient state in which they are able to proliferate in response to mul- trast, human DC maturation induced by CD8ϩ or ␥␦ T cells is tiple unrelated Ags (R. N. Germain, personal communication) and largely mediated by IFN-␥ and/or TNF, respectively (13, 38). there is some evidence that self recognition can contribute to re- The ability of DC to mature in response to T cell signals in the sponses against foreign Ags in some cases (48), although not in absence of adjuvant questions the notion of whether costimulatory others (49). In line with this hypothesis, MHC-bearing endogenous potential corresponds to “signal 2,” the critical APC-derived de- DC amplify responses elicited by DC vaccines, even if this was The Journal of Immunology 6413 originally interpreted to mean that peptide was transferred from fection switches non-plasmacytoid dendritic cells into high interferon producers. donor to recipient MHC molecules (50). Other possibilities are that Nature 424:324. 24. Bedford, P., K. Garner, and S. C. Knight. 1999. MHC class II molecules trans- trans-matured self-presenting DC restimulate memory T cells in a ferred between allogeneic dendritic cells stimulate primary mixed leukocyte re- manner analogous to the mechanism recently proposed for main- actions. Int. Immunol. 11:1739. 25. Smith, A. L., and B. Fazekas de St. Groth. 1999. Antigen-pulsed CD8␣ϩ den- tenance of B cell memory (51). All of these hypotheses remain dritic cells generate an immune response after subcutaneous injection without speculative at present; any experiment to look at the long-term homing to the draining lymph node. J. Exp. Med. 189:593. effects of trans-matured DC on T cell responses will have to dif- 26. Harshyne, L. A., S. C. Watkins, A. Gambotto, and S. M. Barratt-Boyes. 2001. Dendritic cells acquire antigens from live cells for cross-presentation to CTL. ferentiate between the effects of initial trans activation vs subse- J. Immunol. 166:3717. quent cis interactions with readout T cells. 27. Thery, C., L. Duban, E. Segura, P. Veron, O. Lantz, and S. Amigorena. 2002. Indirect activation of naive CD4ϩ T cells by dendritic cell-derived exosomes. Nat. Immunol. 3:1156. Acknowledgments 28. Davis, S. J., and P. A. van der Merwe. 2001. The immunological synapse: re- We thank Gary Warnes and Cathy Simpson for cell sorting and Facundo quired for T cell receptor signalling or directing T cell effector function? Curr. Batista, Ron Germain, Alan Sher, and members of the Immunobiology Lab Biol. 11:R289. 29. Kupfer, A., S. L. Swain, C. A. Janeway, Jr., and S. J. Singer. 1986. The specific for discussions and critical reading of the manuscript. direct interaction of helper T cells and antigen-presenting B cells. Proc. Natl. Acad. Sci. USA 83:6080. References 30. Poo, W. J., L. Conrad, and C. A. Janeway, Jr. 1988. Receptor-directed focusing 1. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. of lymphokine release by helper T cells. Nature 332:378. Annu. Rev. Immunol. 9:271. 31. Boes, M., J. Cerny, R. Massol, M. Op Den Brouw, T. Kirchhausen, J. Chen, and 2. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, H. L. Ploegh. 2002. T-cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature 418:983.

B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Downloaded from Rev. Immunol. 18:767. 32. Chow, A., D. Toomre, W. Garrett, and I. Mellman. 2002. Dendritic cell matu- 3. Reis e Sousa, C. 2001. Dendritic cells as sensors of infection. Immunity 14:495. ration triggers retrograde MHC class II transport from lysosomes to the plasma 4. Akira, S. 2003. Mammalian Toll-like receptors. Curr. Opin. Immunol. 15:5. membrane. Nature 418:988. 5. Reis e Sousa, C. Toll-like receptors and dendritic cells: for whom the bug tolls. 33. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, Semin. Immunol. In press. and G. Schuler. 1996. High level IL-12 production by murine dendritic cells: 6. Reis e Sousa, C. Activation of dendritic cells: translating innate into adaptive upregulation via MHC class II and CD40 molecules and downregulation by IL-4 immunity. Curr. Opin. Immunol. In press. and IL-10. J. Exp. Med. 184:741. 7. Manickasingham, S., and C. Reis e Sousa. 2000. Microbial and T cell-derived 34. Yamane, H., T. Kato, and H. Nariuchi. 1999. Effective stimulation for IL-12 p35 stimuli regulate antigen presentation by dendritic cells in vivo. J. Immunol. mRNA accumulation and bioactive IL- 12 production of antigen-presenting cells http://www.jimmunol.org/ 165:5027. interacted with Th cells. J. Immunol. 162:6433. 8. Muraille, E., C. De Trez, B. Pajak, M. Brait, J. Urbain, and O. Leo. 2002. T 35. Hochrein, H., M. O’Keeffe, T. Luft, S. Vandenabeele, R. J. Grumont, cell-dependent maturation of dendritic cells in response to bacterial superanti- E. Maraskovsky, and K. Shortman. 2000. Interleukin (IL)-4 is a major regulatory gens. J. Immunol. 168:4352. cytokine governing bioactive IL-12 production by mouse and human dendritic 9. Ridge, J. P., F. Di Rosa, and P. Matzinger. 1998. A conditioned dendritic cell can cells. J. Exp. Med. 192:823. be a temporal bridge between a CD4ϩ T-helper and a T-killer cell. Nature 36. Kalinski, P., H. H. Smits, J. H. Schuitemaker, P. L. Vieira, M. van Eijk, 393:474. E. C. de Jong, E. A. Wierenga, and M. L. Kapsenberg. 2000. IL-4 is a mediator 10. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, and of IL-12p70 induction by human Th2 cells: reversal of polarized Th2 phenotype W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 by dendritic cells. J. Immunol. 165:1877. signalling. Nature 393:478. 37. Hsieh, C.-S., A. B. Heimberger, J. S. Gold, A. O’Garra, and K. M. Murphy. 1992. 11. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, and C. J. Melief. Differential regulation of T helper phenotype development by interleukins 4 and 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L 10 in an ␣␤ T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA by guest on September 25, 2021 interactions. Nature 393:480. 89:6065. 12. Ruedl, C., M. Kopf, and M. F. Bachmann. 1999. CD8ϩ T cells mediate CD40- 38. Leslie, D. S., M. S. Vincent, F. M. Spada, H. Das, M. Sugita, C. T. Morita, and independent maturation of dendritic cells In vivo. J. Exp. Med. 189:1875. M. B. Brenner. 2002. CD1-mediated ␥/␦ T cell maturation of dendritic cells. 13. Mailliard, R. B., S. Egawa, Q. Cai, A. Kalinska, S. N. Bykovskaya, M. T. Lotze, J. Exp. Med. 196:1575. M. L. Kapsenberg, W. J. Storkus, and P. Kalinski. 2002. Complementary den- 39. Lafferty, K. J., and A. J. Cunningham. 1975. A new analysis of allogeneic in- dritic cell-activating function of CD8ϩ and CD4ϩ T cells: helper role of CD8ϩ teractions. Aust. J. Exp. Biol. Med. Sci. 53:27. T cells in the development of T helper type 1 responses. J. Exp. Med. 195:473. 40. Janeway, Jr., C. A. 1989. Approaching the asymptote: evolution and revolution 14. Schulz, O., A. D. Edwards, M. Schito, J. Aliberti, S. Manickasingham, A. Sher, in immunology. Cold Spring Harbor Symp. Quant. Biol. 54:1. and C. Reis e Sousa. 2000. CD40 triggering of heterodimeric IL-12 p70 produc- 41. Matzinger, P. 1994. Tolerance, danger, and the extended family. Annu. Rev. tion by dendritic cells in vivo requires a microbial priming signal. Immunity Immunol. 12:991. 13:453. 42. Kearney, E. R., K. A. Pape, D. Y. Loh, and M. K. Jenkins. 1994. Visualization 15. Edwards, A. D., S. P. Manickasingham, R. Spo¨rri, S. S. Diebold, O. Schulz, of peptide-specific T cell immunity and peripheral tolerance induction in vivo. A. Sher, T. Kaisho, S. Akira, and C. Reis e Sousa. 2002. Microbial recognition Immunity 1:327. via Toll-like receptor-dependent and -independent pathways determines the cy- 43. Pasare, C., and R. Medzhitov. 2003. Toll pathway-dependent blockade of ϩ ϩ tokine response of murine dendritic cell subsets to CD40 triggering. J. Immunol. CD4 CD25 T cell-mediated suppression by dendritic cells. Science 299:1033. 169:3652. 44. Vella, A. T., T. Mitchell, B. Groth, P. S. Linsley, J. M. Green, C. B. Thompson, 16. Krieg, A. M., A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, J. W. Kappler, and P. Marrack. 1997. CD28 engagement and proinflammatory G. A. Koretzky, and D. M. Klinman. 1995. CpG motifs in bacterial DNA trigger cytokines contribute to T cell expansion and long-term survival in vivo. J. Im- direct B-cell activation. Nature 374:546. munol. 158:4714. 17. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of 45. Pape, K. A., A. Khoruts, A. Mondino, and M. K. Jenkins. 1997. Inflammatory intrathymic apoptosis of CD4ϩCD8ϩTCRlo thymocytes in vivo. Science cytokines enhance the in vivo clonal expansion and differentiation of antigen- ϩ 250:1720. activated CD4 T cells. J. Immunol. 159:591. 18. Ho, W. Y., M. P. Cooke, C. C. Goodnow, and M. M. Davis. 1994. Resting and 46. Mitchell, T. C., D. Hildeman, R. M. Kedl, T. K. Teague, B. C. Schaefer, J. White, anergic B cells are defective in CD28-dependent costimulation of naive CD4ϩ T Y. Zhu, J. Kappler, and P. Marrack. 2001. Immunological adjuvants promote cells. J. Exp. Med. 179:1539. activated T cell survival via induction of Bcl-3. Nat. Immunol. 2:397. 19. Magram, J., J. Sfarra, S. Connaughton, D. Faherty, R. Warrier, D. Carvajal, 47. Manickasingham, S. P., and C. Reis e Sousa. 2001. Mature T cell seeks antigen C. Y. Wu, C. Stewart, U. Sarmiento, and M. K. Gately. 1996. IL-12-deficient for meaningful relationship in lymph node. Immunology 102:1. mice are defective but not devoid of type 1 cytokine responses. Ann. NY Acad. 48. Wulfing, C., C. Sumen, M. D. Sjaastad, L. C. Wu, M. L. Dustin, and Sci. 795:60. M. M. Davis. 2002. Costimulation and endogenous MHC ligands contribute to T 20. Peterson, D. A., R. J. DiPaolo, O. Kanagawa, and E. R. Unanue. 1999. Quanti- cell recognition. Nat. Immunol. 3:42. tative analysis of the T cell repertoire that escapes negative selection. Immunity 49. Spo¨rri, R., and C. Reis e Sousa. 2002. Self peptide/MHC class I complexes have 11:453. a negligible effect on the response of CD8ϩ T cells to foreign antigen. Eur. 21. McLellan, A. D., E. B. Brocker, and E. Kampgen. 2000. Dendritic cell activation J. Immunol. 32:3161. by danger and antigen-specific T-cell signalling. Exp. Dermatol. 9:313. 50. Kleindienst, P., and T. Brocker. 2003. Endogenous dendritic cells are required for 22. Reis e Sousa, C., S. S. Diebold, A. D. Edwards, S. P. Manickasingham, amplification of T cell responses induced by dendritic cell vaccines in vivo. N. Rogers, O. Schulz, and R. Spo¨rri. 2003. Regulation of dendritic cell function J. Immunol. 170:2817. by microbial stimuli. Pathol. Biol. 51:67. 51. Bernasconi, N. L., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of se- 23. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. Haswell, rological memory by polyclonal activation of human memory B cells. Science A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa. 2003. Viral in- 298:2199.