cell-DC Interactions Research Articles

Sublingual immune cell clusters and dendritic cell distribution in the oral cavity.

The oral mucosa is the first line of defense against pathogenic bacteria and plays a vital role in maintaining tolerance to food antigens and commensal bacteria. We used CD11c reporter mice to visualize dendritic cells (DCs), a key immune cell population, in the oral cavity. We identified differences in DC density in each oral tissue region. Sublingual immune cell clusters (SLICs) extended from the lamina propria to the epithelium, where DCs and T cells resided in close contact with each other and innate lymphoid cells. Targeted in situ photolabeling revealed that the SLICs comprised mostly CD11c+CD11b+ DCs and were enriched for cDC1s and Langerhans cells. Although the frequency of T cell subsets was similar within and outside the SLICs, tissue-resident memory T cells were significantly enriched within the clusters and cluster size increased in response to inflammation. Collectively, we found that SLICs form a unique microenvironment that facilitates T cell-DC interactions in the steady state and during inflammation. Since the oral mucosa is an important target for needle-free vaccination and sublingual immunotherapy to induce tolerogenic responses, the insight into the localized immunoregulation provided in this study may accelerate the development of these approaches.

Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common β-chain of the β2-integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. Patients with LAD-1 are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ Treg are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity, we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here, we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell-DC interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extralymphatic organs, but it resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.

A novel lipidic peptide with potential to promote balanced effector-regulatory T cell responses

T cell-dendritic cell (DC) interactions contribute to reciprocal stimulation leading to DC maturation that results in production of interleukin-12 (IL-12) and interferon-gamma (IFN-γ). Both cytokines have been implicated in autoimmune diseases while being necessary for effective immune responses against foreign antigens. We describe a lipidic peptide, designated IK14004, that modifies crosstalk between T cells and DCs resulting in suppression of IL-12p40/IFN-γ production. T cell production of interleukin-2 (IL-2) and IFN-γ is uncoupled and IL-12p70 production is enhanced. IK14004 induces expression of activating co-receptors in CD8+ T cells and increases the proportion of Foxp3-expressing CD4+ T regulatory cells. The potential for IK14004 to impact on signalling pathways required to achieve a balanced immune response upon stimulation of DCs and T cells is highlighted. This novel compound provides an opportunity to gain further insights into the complexity of T cell-DC interactions relevant to autoimmunity associated with malignancies and may have therapeutic benefit.

Rapid video-based deep learning of cognate versus non-cognate T cell-dendritic cell interactions

Identification of cognate interactions between antigen-specific T cells and dendritic cells (DCs) is essential to understanding immunity and tolerance, and for developing therapies for cancer and autoimmune diseases. Conventional techniques for selecting antigen-specific T cells are time-consuming and limited to pre-defined antigenic peptide sequences. Here, we demonstrate the ability to use deep learning to rapidly classify videos of antigen-specific CD8+ T cells. The trained model distinguishes distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and DCs over 20 to 80 min. The model classified high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalized well to other types of high and low affinity CD8+ T cells. The classification accuracy achieved by the model was consistently higher than simple image analysis techniques, and conventional metrics used to differentiate between cognate and non-cognate T cells, such as speed. Also, we demonstrated that experimental addition of anti-CD40 antibodies improved model prediction. Overall, this method demonstrates the potential of video-based deep learning to rapidly classify cognate T cell-DC interactions, which may also be potentially integrated into high-throughput methods for selecting antigen-specific T cells in the future.

The lymphoid microenvironment controls duration of T cell - DC interactions to promote accelerated effector cell generation

Abstract Naïve T cells and dendritic cells (DCs) are drawn to lymphoid tissue by the chemokine receptor CCR7, where its ligands CCL19 and CCL21 are secreted by lymphoid stroma. There, CCR7 fuels the fast migration of naïve T cells to expedite encounters with DCs presenting cognate pMHC, leading to the formation of long-lasting contacts. Direct in situ observations in mouse models have shown that after approximately 12–24 h, activated T cells detach from DCs before undergoing cell division and effector T cell differentiation. Yet, the mechanisms underlying T cell release from DCs, and hence termination of TCR signal integration, are not well understood. Using a reporter system for TCR signaling, we show here that CD8+T cell detachment from DCs does not occur efficiently in the absence of CCR7 ligands, suggesting that T cells do not intrinsically cease TCR signal integration. In contrast, after a short period of unresponsiveness, ex vivo stimulated T cells increasingly separate from DCs in response to CCR7 ligands in line with in vivo kinetics of detachment. Using ex vivo and in vivo imaging, we identify a critical role for phosphoinositide-3-kinase and the Rac activator DOCK2 in calibrating CCR7-dependent duration of T cell-DC interactions. To assess the impact of enforced T cell-DC separation, we examined effector differentiation kinetics as a function of interaction duration with DCs. Environmentally triggered CD8+T cell detachment from DCs positively correlated with their entry into proliferation and granzyme B production while limiting PD-1 expression. In sum, our data assign a novel function to CCR7 ligands secreted by lymphoid stroma to limit the duration of CD8+T cell - DC interactions for accelerated effector T cell generation.

Tertiary lymphoid organs in renal chronic allograft rejection

Abstract Chronic allograft rejection remains a major obstacle to long-term allograft survival. The immunologic role of tertiary lymphoid organs (TLO) in allograft rejection is unclear. Here, we employed a chronic renal allograft rejection model in mice and intravital 2-photon microscopy to investigate the function of TLO in transplant rejection. CB6F1 (F1) RIP-LTα (preformed TLO) or F1 (no TLO) kidney grafts were transplanted to WT B6 recipients and survival monitored. To investigate immunologic function of TLO, we adoptively transferred B6-RIPLTα CD11c-YFP mice with 10m naïve dsRed OT-I T cells or 10m CTR-labeled NP-specific B cells + 10m CFP+ OT-II cells and immunized with NP-OVA + alum. Intravital 2P imaging of renal TLO was performed at time points 0, 3, 6, 24 or 72 hours after immunization. 4D image analysis was performed and mean speed, displacement, arrest coefficient (AC) and contact times (CT) with DC were calculated for OT-I, OT-II and NP-B cells. F1 RIP-LTα grafts rejected significantly faster (MST= 54) than F1 grafts (MST= 225), demonstrating that TLO contribute to allograft rejection. F1 RIP-LTα grafts contained similar numbers of, but larger TLO than F1 grafts as demonstrated by histology. Mean speed and displacement of OT-I and OT-II cells significantly decreased over time after immunization while AC and mean CT significantly increased. B cell mean speed, displacement and AC increased after immunization. These data are consistent with B cell activation and productive T cell-DC interactions and mirror previously reported data in secondary lymphoid organs. We provide first evidence that TLO provide a local structure for T and B cell activation that propagates anti-graft immune responses in the setting of chronic rejection.

Dissecting cellular crosstalk by sequencing physically interacting cells.

Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.

Initial Viral Inoculum Determines Kinapse-and Synapse-Like T Cell Motility in Reactive Lymph Nodes

T cell activation in lymphoid tissue occurs through interactions with cognate peptide-major histocompatibility complex (pMHC)-presenting dendritic cells (DCs). Intravital imaging studies using ex vivo peptide-pulsed DCs have uncovered that cognate pMHC levels imprint a wide range of dynamic contacts between these two cell types. T cell—DC interactions vary between transient, “kinapse-like” contacts at low to moderate pMHC levels to immediate “synapse-like” arrest at DCs displaying high pMHC levels. To date, it remains unclear whether this pattern is recapitulated when the immune system faces a replicative agent, such as a virus, at low and high inoculum. Here, we locally administered low and high inoculum of lymphocytic choriomeningitis virus (LCMV) in mice to follow activation parameters of Ag-specific CD4+ and CD8+ T cells in draining lymph nodes (LNs) during the first 72 h post infection. We correlated these data with kinapse- and synapse-like motility patterns of Ag-specific T cells obtained by intravital imaging of draining LNs. Our data show that initial viral inoculum controls immediate synapse-like T cell arrest vs. continuous kinapse-like motility. This remains the case when the viral inoculum and thus the inflammatory microenvironment in draining LNs remains identical but cognate pMHC levels vary. Our data imply that the Ag-processing capacity of draining LNs is equipped to rapidly present high levels of cognate pMHC when antigenic material is abundant. Our findings further suggest that widespread T cell arrest during the first 72 h of an antimicrobial immune responses is not required to trigger proliferation. In sum, T cells adapt their scanning behavior according to available antigen levels during viral infections, with dynamic changes in motility occurring before detectable expression of early activation markers.

Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions

Abstract The recognition of peptide-MHC class II complexes (pMHCII) presented by dendritic cells (DCs) is critical for the activation of CD4+ T cells, and proper detection of such complexes is essential for understanding T cell-DC interactions. However, current detection methods for pMHCII complexes using antibodies may block cellular interactions, limiting their use, while conventional tags, such as GFP, are too large to be loaded onto MHCII without disrupting its function. To probe pMHCII complexes, we constructed a variant of the ovalbumin peptide (OVA323–339) linked to a tetracysteine tag (CCPGCC), which can be treated with organoarsenic compounds to induce fluorescence. This modified peptide retained its functionality as it was able to stimulate T cells to the same degree as the unmodified peptide. We also analyzed signal specificity both at the pMHCII level, by using MHCII−/− APCs, and at a broader level by quenching endogenous tetracysteine expression, and confirmed that our fluorescent signal was derived from the peptide modification. We then used microscopy to visualize pMHCII movement after interaction of pulsed DCs with OVA-specific T cells in vitro and subsequently in vivo, and detected a gradual increase of pMHCII complexes at the synapse. Interestingly, we also observed a transfer of fluorescent signal from DCs to antigen-specific T cells, but not to polyclonal T cells, indicating acquisition of pMHCII, which we were able to quantify. Taken together, our results show a non-disruptive approach to assay pMHCII, and provide a window to observe intricate T cell-DC interactions in real-time.

Regulatory T cells perform antigen specific suppression by depleting cognate peptide-MHC class II via trogocytosis

Abstract Tregs are professional suppressors of the immune response, yet their mechanism of action in vivo remains unclear. Using electron microscopy and dynamic intravital two-photon microscopy, we revealed that unlike effector T cells, Tregs displayed a distinct morphology with membrane projections that resemble filopodia at the DC binding site increasing the surface area of the contact dramatically. Despite the intense contact with DC, Tregs only abolished the interactions of effector T cells with DC when the effectors shared the same antigen specificity with Tregs. However, this cessation of the effector T cell interactions was not due to a physical hindrance created by the superior binding ability of Treg since the antigen specific inhibition of T cell-DC interaction was still observed after Tregs are removed from the environment. Instead, the antigen-specific inhibition was secondary to the removal of cognate peptide-MHC Class II (pMHCII) from the surface of DC as evidenced by the strictly antigen-specific depletion of the pMHCII from double antigen pulsed DC. pMHCII capture was only performed by antigen specific Treg, not by polyclonal Treg and it was not secondary to the interaction of CTLA-4 with B7 molecules suggesting the functional importance of TCR in Treg suppression. Prominent members of the B7 family, that are not de novo expressed in Tregs, such as CD86, ICOSL and PD-L2 were acquired alongside the TCR, suggestive of a process that includes a membrane transfer event called trogocytosis. Altogether, we propose antigen specific depletion of peptide-MHCII complexes via trogocytosis as a new mechanism for Treg-mediated suppression.

RNA Aptamers Recognizing Murine CCL17 Inhibit T Cell Chemotaxis and Reduce Contact Hypersensitivity InVivo.

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases.

Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy.

Dendritic cells are targeted by regulatory T (T reg) cells, in a manner that operates as an indirect mode of T cell suppression. In this study, using a combination of single-cell force spectroscopy and structured illumination microscopy, we analyze individual T reg cell-DC interaction events and show that T reg cells exhibit strong intrinsic adhesiveness to DCs. This increased DC adhesion reduces the ability of contacted DCs to engage other antigen-specific cells. We show that this unusually strong LFA-1-dependent adhesiveness of T reg cells is caused in part by their low calpain activities, which normally release integrin-cytoskeleton linkage, and thereby reduce adhesion. Super resolution imaging reveals that such T reg cell adhesion causes sequestration of Fascin-1, an actin-bundling protein essential for immunological synapse formation, and skews Fascin-1-dependent actin polarization in DCs toward the T reg cell adhesion zone. Although it is reversible upon T reg cell disengagement, this sequestration of essential cytoskeletal components causes a lethargic state of DCs, leading to reduced T cell priming. Our results reveal a dynamic cytoskeletal component underlying T reg cell-mediated DC suppression in a contact-dependent manner.

PMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion.

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment.

Antigen presentation kinetics control T cell/dendritic cell interactions and follicular helper T cell generation in vivo.

The production of high affinity, class switched antibodies produced by B cells hinges on the effective differentiation of T follicular helper (Tfh) cells. Here we define conditions specifically enhancing Tfh differentiation and providing protection in a model of influenza infection. Tfh responses were associated with prolonged antigen presentation by dendritic cells (DCs), which maintained T cell/DC interactions into stage 3 (>72 hr) of activation. Blocking stage 3 interactions ablated Tfh generation, demonstrating a causal link between T cell-DC behaviour and functional outcomes. The current data therefore explain how duration of antigen presentation affects the dynamics of T cell-DC interactions and consequently determine Tfh cell differentiation in the developing immune response.

CD28-CD80 interactions control regulatory T cell motility and immunological synapse formation.

Regulatory T cells (Tregs) are essential for tolerance to self and environmental Ags, acting in part by downmodulating costimulatory molecules on the surface of dendritic cells (DCs) and altering naive CD4 T cell-DC interactions. In this study, we show that Tregs form stable conjugates with DCs before, but not after, they decrease surface expression of the costimulatory molecule CD80 on the DCs. We use supported planar bilayers to show that Tregs dramatically slow down but maintain a highly polarized and motile phenotype after recognizing Ag in the absence of costimulation. These motile cells are characterized by distinct accumulations of LFA-1-ICAM-1 in the lamella and TCR-MHC in the uropod, consistent with a motile immunological synapse or "kinapse." However, in the presence of high, but not low, concentrations of CD80, Tregs form stationary, symmetrical synapses. Using blocking Abs, we show that, whereas CTLA-4 is required for CD80 downmodulation, CD28-CD80 interactions are critical for modulating Treg motility in the presence of Ag. Taken together, these results support the hypothesis that Tregs are tuned to alter their motility depending on costimulatory signals.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions.

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

Ex Vivo Assessment of Transplantation Tolerance Using Time Lapse Microscopy.

Background: Identifying patients in whom immunosuppression might be decreased remains challenging. Ex vivo visualization of T cell-dendritic cell (T-DC) interactions has been informative in autoimmune models suggesting that a similar approach might provide a functional measure of alloreactivity. Hypothesis and methods: CBA (H-2k) CD4+ T cells were incubated with syngeneic (CBA) or allogeneic (B6, H-2b) DCs. Individual T-DC contact times were obtained by time lapse microscopy over 40 minutes. We hypothesized that contact times would differ between autologous, allogeneic and tolerised T cell populations. Results: Allogeneic interactions ranged from short (<100s) to prolonged (>2000s, Fig 1A). To distinguish simple collisions from TCR-MHC-mediated interactions, a CD4 antibody was added at a concentration known to block allogeneic T cell responses. The antibody had little impact on short-term contacts but reduced those lasting ≥500s (Fig 1B) suggesting 500s as a threshold for defining synapse-dependent interactions. In keeping with these findings, 12.3% of allogeneic interactions were ≥500s compared with 4.2% of syngeneic ones (Fig 2).Figure: No Caption available.Figure: No Caption available.To test this system in a transplant setting, CD4+ T cells were isolated from naïve CBA mice and CBA mice tolerised to B6 alloantigens using a tolerance induction protocol. While 10.7% of naïve T cell-DC interactions exceeded 500s, T cells from tolerised mice showed a 2-fold reduction in long-term contacts (5.4%, Fig 3).Figure: No Caption available.These observations indicate that assessment of T cell-DC interaction times in vitro may be an accessible and sensitive measure of alloreactivity which could potentially be used to guide minimisation of immunosuppression.

Mechanisms of effector T cell suppression by CD4+Foxp3+ Treg in inflamed peripheral tissue (IRC4P.482)

Abstract In secondary lymphoid organs (SLO), CD4+Foxp3+ regulatory T cells (Treg) control immunity by preventing prolonged T cell-DC interactions. Whether Treg suppressor function in inflamed non-lymphoid tissues differs from SLO remains unclear. Thus, we used 2-photon intravital microscopy to dissect how Treg prevent allograft rejection by effector T cells (Teff) in transplanted islets. To restrain the immune reaction to the transplant site, we adoptively transferred Teff, Treg, or both (2-3x106 each) to B6 mice lacking SLO (splenectomized LTβR-/- mice) 3-4 days after transplantation of BALB/c islets. Teff alone induced acute allograft rejection. However, the co-transfer of Teff + Treg led to long-term allograft survival in ~50% of recipients. Analysis of adoptively transferred CFSE-labeled Teff recovered from the graft on day 3 and 5 revealed that Treg did not affect either Teff migration or proliferation at that site (compared to Teff alone), despite the presence of Treg in significant numbers. Intravital microscopy of transplanted islets on day 2 showed that Treg were found in the peri-islet region, where they made brief (&amp;lt;1 min) contacts with DCs. Interestingly, Treg restrained the motility parameters of Teff in the peri-islet region, and significantly reduced Teff intra-islet infiltration, compared to Teff alone. These results indicate that Treg can function directly in inflamed non-lymphoid tissues, but that their mechanisms of action may differ from those used in SLO.

CXCR3 Chemokine Receptor-Ligand Interactions in the Lymph Node Optimize CD4+ T Helper 1 Cell Differentiation

Differentiation of naive CD4(+) Tcells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, but it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon-γ (IFN-γ)-secreting Th1 cells invivo. By using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 whereas hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated Tcell-DC interactions in LNs during Tcell priming while both chemokines guided intranodal positioning of CD4(+) Tcells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and both functions contribute to Th1 cell differentiation.

Memory CD4 T cells induce selective expression of IL-27 in CD8+ dendritic cells and regulate homeostatic naive T cell proliferation.

Naive T cells undergo robust proliferation in lymphopenic conditions, whereas they remain quiescent in steady-state conditions. However, a mechanism by which naive T cells are kept from proliferating under steady-state conditions remains unclear. In this study, we report that memory CD4 T cells are able to limit naive T cell proliferation within lymphopenic hosts by modulating stimulatory functions of dendritic cells (DC). The inhibition was mediated by IL-27, which was primarily expressed in CD8(+) DC subsets as the result of memory CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition, as naive T cells deficient in IL-27R were resistant to memory CD4 T cell-mediated inhibition. Finally, IL-27-mediated regulation of T cell proliferation was also observed in steady-state conditions as well as during Ag-mediated immune responses. We propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8(+) DC-derived IL-27 in vivo.