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Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells (reviewed in Ref. 6). In addition to infection-related signals, T cells themselves can be potent activators of DC (7, 8). For example, CD40 ligand (CD40L) up-regulated on CD4+ T cells after exposure to Ag is an important stimulus for DC activation and plays a unique role in licensing APC for CTL priming (9, 10, 11). Similarly, signals from CD8+ T cells have also been shown to activate DC (12, 13). Thus, DC-activating stimuli appear to fall broadly into two classes: signals arising from innate recognition and signals emanating from cells of the adaptive immune system.
"DC activation" is used loosely to refer to any one of multiple changes in DC phenotype and function. These include, among others, "DC maturation" (increase in DC expression of MHC, costimulatory and adhesion molecules resulting in an increased ability to stimulate T cell proliferation), migration, endocytic pathway remodeling, and production of immunomodulatory cytokines (reviewed in Refs.1 and 2). It is becoming increasingly clear that not all aspects of DC activation are regulated concordantly and that they can be differentially induced by innate vs adaptive signals. For example, innate stimuli can promote production of cytokines such as IL-12, IL-10, or IFN-
whereas CD40 ligation cannot initiate cytokine synthesis but can amplify that which has been initiated by microbial signals (14, 15). However, it is not clear whether other aspects of DC activation are similarly dependent on microbial priming and whether T cell-derived signals act exclusively on Ag-bearing DC or also regulate the function of bystander cells. In this study, we focus on the interplay between innate and T cell-derived signals in the regulation of two distinct aspects of murine DC activation. We use naive T cells and splenic DC to show that amplification of IL-12 synthesis by T cell signals occurs strictly in cis and does not affect bystander DC in trans. In contrast, 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 CD4+ T cells may help each other via DC and reveal the complex interactions between innate and adaptive signals that modulate DC function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CpG-containing phosphorothioate-linked oligonucleotide 1668 (CpG) 5'-TCCATGACGTTCCTGATGCT-3' (16) was synthesized by the Cancer Research UK oligonucleotide synthesis service (Clare Hall, South Mimms, U.K.). Peptides derived from chicken OVA (ISQAVHAAHAEINEAGR, residues 323–339; pOVA) and hen egg lysozyme (NTDGSTDYGILQINSR, residues 46–61; pHEL) were synthesized and HPLC-purified by the Cancer Research UK peptide synthesis service. Throughout this paper, pOVA and pHEL are used to refer to the peptides and not the proteins. All reagents were free of endotoxin as determined using a Limulus Amebocyte Lysate test (BioWhittaker, Walkersville, MD).
Mice
DO11.10 mice (17) on a BALB/c-scid background carrying a transgenic V2/V
8 TCR specific for pOVA presented on I-Ad were originally a gift from Dr. P. Garside (University of Glasgow, Glasgow, U.K.). 3A9 mice (18) bearing a transgenic V3/V8 TCR specific for hen egg lysozyme 46–61 (pHEL) presented on I-Ak were purchased from The Jackson Laboratory (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 alloreactive against BALB/c (H-2d) or B10.BR (H-2k), respectively (data not shown). Mice genetically deficient in IL-12 p40 (19) on a BALB/c background (IL-12-/- BALB/c) were purchased from The Jackson Laboratory. All of the above mice, as well as wild-type B10.BR (H-2k), BALB/c (H-2d), BALB/c x B10.BR F1 (H-2dxk), and DO11.10 x B10.BR F1 (H-2dxk) were bred and kept at the animal facility of Cancer Research UK under specific pathogen-free conditions, and used at 6–12 wk of age, sex- and age-matched within experiments.
For generation of chimeric mice, bone marrow cells harvested from femurs and tibias were depleted of T cells using anti-CD90 magnetic beads (Miltenyi Biotec, Bisley, U.K.). Four-week-old B10.BR mice were gamma-irradiated with 2 x 5 Gy and reconstituted with 2 x 106 bone-marrow cells from B10.BR mice mixed with an equal number of cells derived from either IL-12-/- BALB/c or IL-12+/+ BALB/c mice. Six to 8 wk after reconstitution, mice were tested for chimerism. Host mice were only used in experiments if FACS analysis of blood leukocytes showed cells of both host (H-2Kk) and donor (H-2Dd) origin at a ratio close to 1:1. All animal procedures and husbandry were in accordance with U.K. governmental regulations and institutional policies.
Flow cytometry
Cell suspensions were stained in ice-cold PBS supplemented with 2 mM EDTA, 1% FCS, and 0.02% sodium azide. mAbs were purchased from BD PharMingen (Oxford, U.K.) preconjugated to various fluorochromes or to biotin and were directed against: CD11c (clone HL3, hamster IgG), B7-2 (GL1, rat IgG2a), CD40 (3/23, rat IgG2a), H-2Dd (34-2-12, mouse IgG2a), H-2Kk (36-7-5, mouse IgG2a), CD4 (RM4-5, rat IgG2a) and V8 TCR (MR5-2, mouse IgG2a). Purified 2.4G2 (anti-FcRIII/II, rat IgG2b; used to block unspecific Ab binding), and 1G12 (anti-3A9 clonotypic mAb (Ref. 20 , mouse IgG1; a kind gift from Dr. E. Unanue, Washington University, St. Louis, MO) were from the Cancer Research UK Ab production service. 1G12 was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacturer’s instructions. Streptavidin conjugates (CyChrome, allophycocyanin) were from BD PharMingen. Where applicable, TO-PRO3, a DNA-binding dye for live-dead discrimination (Molecular Probes, Leiden, The Netherlands), was added to the samples immediately before data acquisition. Data were collected on a FACSCalibur (BD Biosciences, Oxford, U.K.) and analyzed using FlowJo software (Treestar, San Carlos, CA).
Cell purification
DC were purified from freshly isolated spleens injected with serum-free medium supplemented with Liberase CI (1.7 Wünsch-U/ml; Roche Diagnostics, Lewes, U.K.) and DNase I (0.2 mg/ml; Roche Diagnostics) (14). After 20 min at 37°C, digested spleens were strained through a 40-µm cell sieve and washed once with ice-cold PBS supplemented with 2 mM EDTA and 1% FCS. Splenocyte suspensions were labeled with anti-CD11c MACS beads (clone N418; Miltenyi Biotec, Bisley, U.K.) for 10 min at 4°C, followed by washing with chilled PBS and positive selection using LS magnetic columns (Miltenyi Biotec), according to the manufacturer’s instructions. Resulting cells were routinely >90% pure, viable and free of T cells (CD11chigh, MHC class II+, TCR-, TO-PRO3negative). In some experiments, DC were further purified by FACS sorting. Briefly, pre-enriched DC were stained with sterile-filtered fluorescently labeled anti-CD11c Ab and, where applicable, with anti-H-2Dd and anti-H-2Kk for 30 min at 4°C and washed once with chilled PBS. CD11chigh H-2Dd+H-2Kk -or CD11chighH-2Dd-H-2Kk+ cells were then sorted on a MoFlo cytometer (Cytomation, Fort Collins, CO). Resulting cell preparations were routinely >99% pure and viable (TO-PRO3negative, CD11chigh, H-2Dd+H-2Kk- or H-2Dd-H-2Kk+, respectively).
T cells from naive TCR-transgenic mice were purified by negative selection using magnetic beads. Briefly, single cell suspensions were prepared by forcing lymph nodes and spleens through 40-µm cell sieves with a syringe plunger. Cells were labeled with a mixture of sterile-filtered biotinylated mAbs (all from BD PharMingen) against CD11c (HL3, hamster IgG), I-Ad (AMS-32.1, mouse IgG2b), I-Ek (14-4-4S, mouse IgG2a), FcRIII/II (2.4G2, rat IgG2b), CD11b (M1/70, rat IgG2b), Gr-1 (RB6-8C5, 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 (H1.2F3, hamster IgG1), washed and incubated with magnetic streptavidin-beads (Miltenyi Biotec). Labeled cells were passed through a MACS column and the flow-through fraction was collected. Resulting cell preparations were found to be free of APC in functional assays. Serum-free buffers were used throughout when cells were used for adoptive transfer.
Adoptive transfers
APC-depleted CD4+ DO11.10 T cells (5 x 106) were transferred into naive host animals by i.v. injection 1 day before immunization. In some experiments, 1 x 107 purified CD4+ DO11.10 T cells were cotransferred with 2 x 106 purified CD4+ 3A9 T cells that had been labeled with 2 µM CFSE (Molecular Probes; 12 min at 37°C).
Ex vivo DC stimulatory capacity assay
(DO11.10 x B10.BR)F1 mice (H-2dxk) were immunized by i.v. injection of 25 µg of pOVA and/or 25 µg of CpG. Twelve hours later, splenic DC from these mice were FACS-sorted and fixed with 1% paraformaldehyde for 15 min at room temperature. The fixed DC were then washed twice and the residual paraformaldehyde quenched by incubation in 0.1 mM glycine for 30 min at room temperature. After further washing, 1 x 104 fixed DC were cocultured in triplicate in 200 µl of complete medium (RMPI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 50 µM 2-ME) in round-bottom 96-well plates together with 5 x 104 APC-depleted CD4+ T cells purified from naive 3A9 mice, and graded doses of pHEL peptide. After 48 h, IL-2 levels in the culture supernatants were determined by sandwich ELISA.
In vitro DC activation assays
Splenic DC (5 x 105) from BALB/c mice were cocultured in 24-well plates with 1 x 106 purified CD4+ DO11.10 T cells and graded doses of pOVA in the presence or absence of 0.5 µg/ml CpG in 2 ml of complete medium. In experiments addressing trans feedback to nonpresenting DC, an additional 5 x 105 splenic DC from B10.BR mice were added to the wells. Twelve hours later, supernatants were harvested and IL-12 p70 was measured. Surface expression of B7-2 and CD40 on DC of BALB/c or B10.BR origin (CD11chighH-2Dd+H-2Kk- or CD11chighH-2Dd-H-2Kk+, respectively) was determined by FACS analysis. Alternatively, in experiments addressing cis vs trans feedback for the production of IL-12 p70, 2 x 104 DC from IL-12-/- BALB/c or IL-12+/+ BALB/c, respectively, were cocultured with 2 x 104 B10.BR DC and 5 x 104 APC-depleted CD4+ DO11.10 T cells and graded doses of pOVA peptide in the presence of 0.5 µg/ml CpG. Twelve hours later, IL-12 p70 levels in the supernatant of triplicate cultures were determined by sandwich ELISA. In transwell experiments designed to study the role of soluble T cell-derived factors in DC maturation, splenic DC from BALB/c mice were incubated with 0.5 µg/ml CpG for 1 h at 37°C, washed and fixed as described above. APC-depleted CD4+ DO11.10 T cells (1 x 106) were cocultured in 24-well plates with 5 x 105 fixed or fresh live DC ± 1 µM pOVA. Alternatively, the T cells were cultured in wells coated with 5 µg/ml anti-CD3
(2C11, hamster IgG1; Cancer Research UK) and 5 µg/ml anti-CD28 (37.51, hamster IgG2; kind gift from Dr. J. P. Allison (University of California, Berkeley, CA); produced at Cancer Research UK). Cell culture inserts with a permeable membrane (0.4-µm pore size, "transwell") were inserted into all wells and additional aliquots of 5 x 105 fresh BALB/c DC were added. After 12 h of coculture in a total volume of 2 ml of complete medium, the expression of costimulatory molecules on DC in the transwells was analyzed by flow cytometry.
Cytokine ELISA
Concentrations of IL-12 p70 and IL-2 in culture supernatants and sera were determined using standard sandwich ELISA using the following Ab pairs (capture, detection), at the concentrations recommended by the manufacturer (BD PharMingen): 9A5, C17.8 (biotinylated) for IL-12 p70 and JES6-1A12, JES6-5H4 (biotinylated) for IL-2.
Quantitative RT-PCR
Total RNA from FACS-sorted DC was isolated using the RNeasy Mini kit (Qiagen, Crawley, U.K.) according to the manufacturer’s instructions. Residual genomic DNA was digested using RNase-free DNase I (Qiagen). cDNA was synthesized using Life Technologies reagents (Invitrogen, CA). For relative quantitation of IL-12 p35 and IL-12 p40 mRNA, amplification of sample cDNA was monitored with the fluorescent DNA-binding dye FAM in combination with the ABI PRISM 7700 detection system (Applied Biosystems, Warrington, U.K.), according to the manufacturer’s instructions. IL-12 p40- and IL-12 p35-specific primers were purchased as predeveloped TaqMan assay reagents (Applied Biosystems). Specific message levels were normalized to GAPDH as recommended by the manufacturer.
Statistical analysis
The statistical significance of differences between experimental samples was determined using the Student t test.
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Results |
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We have previously reported that T cell feedback signals, including CD40L, cannot initiate IL-12 production by splenic DC but markedly amplify the levels of IL-12 p70 elicited by a microbial stimulus (14). Consistent with those data, following adoptive transfer of naive DO11.10 T cells into BALB/c mice, immunization with pOVA323–339 peptide (pOVA) alone did not induce measurable levels of IL-12 p70 in the circulation (Fig. 1A). However, the combination of a microbial stimulus such as a CpG-containing oligonucleotide (mimic of bacterial DNA; CpG) and pOVA induced higher levels of the cytokine than CpG alone, confirming that signals from newly activated T cells can contribute to IL-12 p70 production after microbial priming in vivo (Fig. 1A). Similarly, when splenic DC were used to stimulate naive DO11.10 T cells in vitro, there was little accumulation of IL-12 p70 in supernatants unless CpG was added to the culture (data not shown; see Refs.14 and 15).
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Signals from newly activated T cells do not increase IL-12 p70 production by bystander nonpresenting DC primed by microbial stimulation
The contribution of T cell-derived signals to DC maturation and cytokine production was further dissected in vitro and in vivo. We first determined whether the amplification of IL-12 production by T cell signals requires a cognate interaction between the T cell and the DC or whether amplification signals can be delivered in trans to nonpresenting DC. H-2d splenic DC were purified from IL-12+/+ or IL-12-/- BALB/c mice and were used to stimulate DO11.10 T cells in the presence of bystander B10.BR H-2k DC. The latter can produce IL-12 but cannot present pOVA and, therefore, 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-12+/+ H-2d DC, as expected (Fig. 2A). In contrast, little IL-12 p70 accumulated in wells containing IL-12-/- H-2d DC (Fig. 2A) despite the fact that the latter stimulated T cell proliferation to the same extent as IL-12+/+ H-2d DC (data not shown). These results suggested that T cell feedback signals for IL-12 p70 production are delivered exclusively to cells presenting Ag and do not affect bystander nonpresenting DC in close contact. To determine whether this is also true in vivo, we constructed different types of mixed H-2k/H-2d bone marrow chimeric mice and used them as adoptive recipients for naive DO11.10 T cells. Injection of recipients bearing wild-type H-2d and H-2k bone marrow with CpG + pOVA led to an increase in serum IL-12 p70 over the levels elicited by CpG alone (Fig. 3A), indicating a role for T cell feedback. No such increase was seen with chimeras made using a mixture of IL-12-/- H-2d and IL-12+/+ H-2k bone marrow (Fig. 3A), despite the presence in those mice of H-2k APC capable of producing IL-12. This result indicates that in vivo, like in vitro, T cells deliver signals that augment IL-12 p70 production by Ag-bearing cells but not bystander APC.
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Signals from newly activated T cells increase expression of costimulatory molecules on bystander nonpresenting DC
To determine whether other parameters of DC activation were similarly regulated strictly in cis by T cell-derived signals, we examined the up-regulation of maturation markers in both the presenting and nonpresenting DC used in the above experiments. In contrast to IL-12 p70, both B7-2 and CD40 were up-regulated on H-2k DC that had been cocultured with H-2d DC and DO11.10 T cells in the presence of Ag (Fig. 2B and data not shown). Indeed, after taking into account the starting levels of the two markers, up-regulation looked identical for both the presenting (H-2d) and nonpresenting (H-2k) DC (Fig. 2B). Similar results were seen in vivo in H-2k/H-2d mixed chimeras given DO11.10 T cells and immunized with CpG ± pOVA: CD40 and B7-2 were up-regulated on both H-2d and H-2k DC in mice receiving pOVA compared with PBS or CpG + pOVA compared with CpG alone (Fig. 4 and data not shown). This was true independent of whether the H-2d bone marrow was of IL-12+/+ or IL-12-/- origin (data not shown). In vitro experiments further established that up-regulation of B7-2 and CD40 on DC by T cell-derived signals could be triggered across a transwell or by supernatants from anti-CD3-activated T cell cultures, thereby implicating a soluble factor of T cell origin (Fig. 2C). We conclude that factors produced by newly activated T cells can lead to maturation of DC in either cis or trans.
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The ability of newly activated T cells to promote maturation of DC in vivo might facilitate activation of other T cells. To test this hypothesis, CFSE-labeled 3A9 and unlabeled DO11.10 T cells were cotransferred adoptively into H-2kxd F1 mice, which were then immunized with pHEL together with CpG and/or pOVA. 3A9 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 µg of pHEL alone failed to induce measurable CFSE dilution, a fraction of the 3A9 T cells in mice immunized with 1 µg of pHEL + pOVA underwent one cell division (Fig. 5B, arrows). Similarly, pOVA augmented 3A9 proliferation at the 5 µg of pHEL dose (Fig. 5B, arrows). Like pOVA, CpG, a bona fide adjuvant, also increased 3A9 proliferation at both doses of pHEL and the combination of CpG and pOVA was additive and promoted the greatest expansion of the 3A9 T cells (Fig. 5B).
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Discussion |
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The fact that T cell signals do not initiate IL-12 production by themselves makes teleological sense as it ensures that T cell priming does not lead to Th1 differentiation by default. In fact, we have previously shown that T cell feedback or CD40 ligation are "neutral" signals that simply reveal a pattern of cytokine synthesis predetermined by pattern recognition (14, 15, 23). In this study, we use IL-12 p70 synthesis as a model to explore in more detail how innate and adaptive signals regulate cytokine synthesis by DC. Surprisingly, we find that T cell feedback for IL-12 production is delivered strictly in cis and does not affect DC that do not present Ag. This is seen both in vivo and in vitro, in conditions in which T cells are in very close proximity to nonpresenting DC. These findings have two implications. First, they shed light on a controversial aspect of DC biology, namely the capacity of DC to acquire MHC/peptide complexes from other cell types (24, 25, 26, 27). The fact that we fail to see any feedback in trans for IL-12 p35 synthesis in our chimeras (Fig. 3) suggests that such transfer in vivo may be inefficient. Second, our findings suggest that T cell feedback signals for cytokine production are delivered in a very polarized fashion toward the presenting cell, perhaps in the context of an immunological synapse (28). This polarization has been described during T-B cell interactions (29, 30) and would be analogous on the T cell side to the recently described polarized exocytosis of MHC class II molecules by DC toward the DC-T cell interface (31, 32). Studies are in place to identify the T cell-derived factors responsible for IL-12 amplification and whether they show any polarization toward the APC. These include CD40L, which contributes to 
50% of the IL-12 p70 levels seen in our experiments (data not shown), as well as other signals yet to be identified. The latter might arise from the MHC class II molecule itself upon TCR engagement, consistent with the strict cis dependence seen here. Indeed, certain Abs against MHC class II promote IL-12 secretion by DC (33) and, like T cells, can regulate expression of the p35 subunit (34). In addition, CD8+ T cells or polarized CD4+ T cells produce cytokines such as IFN-, IL-4, or GM-CSF, which potentiate IL-12 p70 production by DC (35, 36). However, such cytokines should be effective in trans and are not thought to be produced by naive CD4+ T cells such as the DO11.10 cells used in our experiments (37). Consistent with this notion, supernatants from newly activated DO11.10 T cells do not synergize with TLR ligation to promote IL-12 p70 synthesis by DC (data not shown). It remains possible that the strict cis effect of T cell feedback is relaxed when dealing with cytokine-secreting effector or memory T cells, thereby promoting IL-12 production by bystander DC. This may explain why trans feedback for IL-12 production can be seen with T cell hybridomas in vitro (33).
In contrast to IL-12, induction of DC maturation by newly activated CD4+ T cells did not require microbial priming and occurred in trans as well as in cis. DC maturation was monitored by the up-regulation of B7-2 and CD40 and correlated with an increased ability of DC to activate naive T cells (Fig. 1). The ability of CD4+ T cells to promote DC maturation in vivo has been previously described by Muraille et al. (8) who showed that injection of superantigens into T cell-sufficient, but not T cell-deficient, mice leads to up-regulation of B7-2, CD40 and, to a lesser extent, B7-1 on splenic DC. Similarly, Ruedl et al. (12) showed that activation of naive CD8+ T cells in vivo leads to DC maturation and, like us, found that this could take place in both cis and trans. In addition, we have previously shown that T cell feedback in vivo is sufficient to promote increased presentation of protein Ags by DC, another aspect of DC maturation (7). The signals that mediate maturation of DC are likely to be soluble factors made by the newly activated T cells themselves (Fig. 2C). The molecular nature of such factors is not known but Ruedl et al. (12) and Muraille et al. (8) have excluded CD28, CD40L, receptor activator of NF-
B ligand, TNF, IL-1, IL-4, IL-6, IL-17, IFN, and IFN-. In contrast, human DC maturation induced by CD8+ or T cells is largely mediated by IFN- and/or TNF, respectively (13, 38).
The ability of DC to mature in response to T cell signals in the absence of adjuvant questions the notion of whether costimulatory potential corresponds to "signal 2," the critical APC-derived determinant required for immunity in many models (39, 40, 41). Indeed, in their pioneering work on the DO11.10 adoptive transfer system, Jenkins and colleagues (42) showed that pOVA injection led to T cell deletion, in line with the notion that administration of Ag in the absence of adjuvant favors tolerance rather than immunity. Tolerance induction in that system involved initial T cell expansion followed by death and a reduction in frequency to below the starting level (42). This implies that, despite the abnormally high precursor frequency of Ag-specific DO11.10 T cells, which probably provides a supraphysiological increase in DC costimulatory potential, the latter is not sufficient as signal 2 to prevent tolerance induction (although it may contribute to the initial proliferation). In contrast, injection of pOVA together with LPS rescues some DO11.10 T cells from death and promotes immunity (42). Given the dogma that adjuvants act via APC, this suggests that adjuvants induce a signal 2 that is qualitatively distinct from costimulation. This signal might correspond to DC-derived cytokines which, if regulated like IL-12, cannot be elicited by T cell signals alone. Such cytokines could counteract the functions of regulatory T cells and/or act on clonally expanded T cells to sustain survival (43, 44, 45, 46). Alternatively, adjuvants may simply have a quantitative effect, synergizing with T cell feedback to push DC costimulatory potential past a critical threshold required to sustain T cell clonal expansion.
In line with the experiments showing DC maturation in response to T cell signals, we show that activation of naive DO11.10 T cells lowers the Ag dose required for proliferation of naive 3A9 T cells in vivo (Fig. 5). Although we cannot formally exclude that this effect is a consequence of direct T-T interactions, DC exposed to T cell feedback in vivo display a higher stimulatory potential for T cells ex vivo (Fig. 1C), suggesting that CD4+ T cell help for other CD4+ T cells can certainly occur via the APC. In addition, the fact that T-T help in vivo can take place in trans (Fig. 5), like DC maturation (Fig. 4), further suggests that the two effects may be linked. Trans-activation of DC could be prevalent during infection, when the T cell precursor frequency for any given foreign Ag and the multiplicity of available Ags may allow the simultaneous activation of many clones of T cells. What, then, might be the role of trans-activated DC in the immune response? There are two scenarios to consider. One is the trans-maturation of DC that have captured Ags from the infectious agent in question and, therefore, have also, in all likelihood, been exposed to innate signals derived from that agent. Trans-maturation of those DC will increase their stimulatory potential beyond that gained by exposure to the innate stimulus alone and may also improve Ag processing and presentation (47). The net result is that such DC may now be better able to stimulate lower affinity T cells. Once they have been activated, these T cells can then sustain DC maturation through cis effects and amplify cytokine production. This form of T-T help would effectively result in clonal diversification and/or epitope spreading early in the immune response. A second scenario is the trans-maturation of lymph node DC that have not been in contact with the infectious agent or infected cells and, as a consequence, have not acquired Ag or been primed by innate recognition. Those cells will, presumably, present only self-peptides and their role is more intriguing. One possibility is that trans-activated DC presenting self Ags may, nevertheless, contribute to the clonal expansion of T cells specific for foreign Ag. Newly activated T cells enter a transient state in which they are able to proliferate in response to multiple unrelated Ags (R. N. Germain, personal communication) and there is some evidence that self recognition can contribute to responses against foreign Ags in some cases (48), although not in others (49). In line with this hypothesis, MHC-bearing endogenous DC amplify responses elicited by DC vaccines, even if this was originally interpreted to mean that peptide was transferred from donor to recipient MHC molecules (50). Other possibilities are that trans-matured self-presenting DC restimulate memory T cells in a manner analogous to the mechanism recently proposed for maintenance of B cell memory (51). All of these hypotheses remain speculative at present; any experiment to look at the long-term effects of trans-matured DC on T cell responses will have to differentiate between the effects of initial trans activation vs subsequent cis interactions with readout T cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Caetano Reis e Sousa, Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. E-mail address: caetano{at}cancer.org.uk
3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; CD40L, CD40 ligand; HEL, hen egg lysozyme; MFI, median fluorescence intensity.
Received for publication May 23, 2003. Accepted for publication October 9, 2003.
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References |
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+ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node. J. Exp. Med. 189:593. T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065./ T cell maturation of dendritic cells. J. Exp. Med. 196:1575.This article has been cited by other articles:
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F. Miro, C. Nobile, N. Blanchard, M. Lind, O. Filipe-Santos, C. Fieschi, A. Chapgier, G. Vogt, L. de Beaucoudrey, D. S. Kumararatne, et al. T Cell-Dependent Activation of Dendritic Cells Requires IL-12 and IFN-{gamma} Signaling in T Cells J. Immunol., September 15, 2006; 177(6): 3625 - 3634. [Abstract] [Full Text] [PDF]
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M.-C. Devilder, S. Maillet, I. Bouyge-Moreau, E. Donnadieu, M. Bonneville, and E. Scotet Potentiation of Antigen-Stimulated V{gamma}9V{delta}2 T Cell Cytokine Production by Immature Dendritic Cells (DC) and Reciprocal Effect on DC Maturation J. Immunol., February 1, 2006; 176(3): 1386 - 1393. [Abstract] [Full Text] [PDF]
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R. Mallone, S. A. Kochik, H. Reijonen, B. Carson, S. F. Ziegler, W. W. Kwok, and G. T. Nepom Functional avidity directs T-cell fate in autoreactive CD4+ T cells Blood, October 15, 2005; 106(8): 2798 - 2805. [Abstract] [Full Text] [PDF]
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E. Segura, C. Nicco, B. Lombard, P. Veron, G. Raposo, F. Batteux, S. Amigorena, and C. Thery ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming Blood, July 1, 2005; 106(1): 216 - 223. [Abstract] [Full Text] [PDF]
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N. Misra, J. Bayry, S. Lacroix-Desmazes, M. D. Kazatchkine, and S. V. Kaveri Cutting Edge: Human CD4+CD25+ T Cells Restrain the Maturation and Antigen-Presenting Function of Dendritic Cells J. Immunol., April 15, 2004; 172(8): 4676 - 4680. [Abstract] [Full Text] [PDF]
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0.008). C, DO11.10 x B10.BR (H-2dxk) 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 CD4+ 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.
B10.BR (left panel) or IL-12+/+ BALB/c + B10.BR
