cell-DC Interactions Research Articles

In Vivo Assessment of T Effector, T Regulatory Cell, Dendritic Cell Interaction in Lymphoid Tissue Using Intravital Imaging During Acute GvHD

Abstract 818Graft-versus-host disease (GvHD) is a complication that results from minor and/or major MHC incompatibilities between the donor and the recipient after hematopoietic stem cell transplantation (HSCT). Although the importance of donor T effector cells (Teffs) in inducing GvHD is well-established, the early events by which host antigen presenting cells (APCs) activate allogeneic T cells are not well described. In pathogen specific immunity, after T cells migrate to the lymph node, they initially form brief contacts with dendritic cells (DCs). This phase lasts for 6–8 hours, which is termed random-walk. Following this phase, T cells establish long-lasting arrest on DCs. At least 6 hours of stable T cell-DC interaction are required for na•ve T cells to undergo clonal expansion. After the phase of long-lasting contact, T cells start to proliferate and expand. At the same time (20∼24 hrs), their interactions with DCs become very short again. It is not clear if allogeneic T cells follow a similar tri-phasic activation process. In addition, studies on autoimmunity have shown that Tregs can prevent T cell activation by prolonged interaction with APCs. However, the mechanisms by which donor regulatory T cells (Tregs) regulate T effector responses in GvHD in lymphoid tissue has not described previously.In this study, we have used intravital microscopy to monitor the movement of donor na•ve T cells and Tregs within lymph nodes after bone marrow transplantation (BMT) in a C57BL/6 to BALB/c murine model (Figure 1A, DCs are green, T cells are red). We have found that donor na•ve T cells do not follow the triphasic activation process demonstrated by von Andrian’s group for pathogen-specific transgenic T cells. Instead, a substantial minority of MHC mismatched allogeneic T cells demonstrate a diminished velocity within 4 hours of adoptive transfer. These data indicate that prolonged interactions with host DCs develop rapidly post transplant. The velocity of allogeneic T cells increased at 18 hours post BMT. Peak velocity of Teff cells occurred 24 hours post BMT, consistent with the movement pattern of activated syngeneic T cells. [Display omitted] We have also observed that although the speed of donor Treg movement is very similar to na•ve T cell 4∼6 hours after BMT, the velocity of donor Tregs is much slower than the velocity of Teffs 18 hours post BMT. This slow speed persists even at 24 hours post BMT, indicating that Tregs form very stable contacts with host DCs. Interestingly, donor na•ve T cells move significantly faster when transplanted with Tregs at a 2:1 (T:Treg) ratio (Figure 1B), suggesting that Tregs interfere with interactions between na•ve T cells and DCs.We conclude that allogeneic na•ve T cells do not go through a random-walk phase in order to interact with host DCs early post HSCT. The transplantation of Tregs with donor T cells and bone marrow cells significantly increased the velocity of donor na•ve T cells, probably diminishing the interaction of Teff cells with DCs after BMT. We believe that the large number of APCs able to present antigen in the allogeneic setting allows for rapid activation of donor Teff cells and precludes the necessity of the random-walk phase characteristic of pathogen-specific T cells. Disclosures:No relevant conflicts of interest to declare.

Memory CD4 T cells induce selective expression of IL-27 in CD8+ dendritic cells and maintain homeostatic control of naïve T cell proliferation (104.8)

Abstract Naïve CD4 T cells undergo robust proliferation in lymphopenic conditions, while they remain quiescent in steady-state conditions. Mechanisms involved in the induction of the proliferation have been extensively examined. However, a mechanism of how naïve T cells are kept from proliferating under steady-state conditions remains unclear. Here we demonstrate that memory CD4 T cells directly inhibit naïve T cell proliferation within lymphopenic hosts by modulating stimulatory functions of DCs. The inhibition occurred only when both memory and naïve CD4 T cells interact with the same DCs. Thus, when naïve CD4 T cells were introduced into a recipient where a portion of DCs do not interact with memory CD4 T cells, the inhibition was lost. Moreover, the inhibition was mediated by DC-derived IL-27, which was directly induced in CD8+ DC subsets following CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition as naïve T cells deficient in IL-27R were resistant to memory CD4 T cell mediated inhibition. Finally, IL-27-mediated regulation of memory T cell homeostasis was also observed in steady-state conditions. Taken together, we propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8+ DC-derived IL-27 in vivo.

Visualizing the Functional Diversification of CD8+ T Cell Responses in Lymph Nodes

CD8(+) Tcell responses generate effector cells endowed with distinct functional potentials but the contribution of early events in this process is unclear. Here, we have imaged Tcells expressing a fluorescent reporter for the activation of the interferon-γ (IFN-γ) locus during priming in lymph nodes. We have demonstrated marked differences in the efficiency of gene activation during stable Tcell-dentritic cell (DC) contacts, influenced in part by signal strength. Imaging the first cell division, we have demonstrated that heterogeneity in Tcell functional potential was largely apparent as Tcells initiated clonal expansion. Moreover, by analyzing the fate of single activated Tcells exvivo, we have provided evidence that these early differences resulted in clonal progenies with distinct functional properties. Thus, the early set of Tcell-DC interactions in lymph nodes largely contribute tothe heterogeneity of Tcell responses through the generation of functionally divergent clonal progenies.

Dendritic cells control T cell tonic signaling required for responsiveness to foreign antigen.

Dendritic cells (DCs) are key components of the adaptive immune system contributing to initiation and regulation of T cell responses. T cells continuously scan DCs in lymphoid organs for the presence of foreign antigen. However, little is known about the functional consequences of these frequent T cell-DC interactions without cognate antigen. Here we demonstrate that these contacts in the absence of foreign antigen serve an important function, namely, induction of a basal activation level in T cells required for responsiveness to subsequent encounters with foreign antigens. This basal activation is provided by self-recognition of MHC molecules on DCs. Following DC depletion in mice, T cells became impaired in TCR signaling and immune synapse formation, and consequently were hyporesponsive to antigen. This process was reversible, as T cells quickly recovered when the number of DCs returned to a normal level. The extent of T cell reactivity correlated with the degree of DC depletion in lymphoid organs, suggesting that a full DC compartment guarantees optimal T cell responsiveness. These findings indicate that DCs are specialized cells that not only present foreign antigen, but also promote a "tonic" state in T cells for antigen responsiveness.

Selective Expression of the Chemokine Receptor XCR1 on Cross-presenting Dendritic Cells Determines Cooperation with CD8+ T Cells

The expression of the chemokine receptor XCR1 and the function of its ligand XCL1 (otherwise referred to as ATAC, lymphotactin, or SCM-1) remained elusive to date. In the present report we demonstrated that XCR1 is exclusively expressed on murine CD8(+) dendritic cells (DCs) and showed that XCL1 is a potent and highly specific chemoattractant for this DC subset. CD8(+) T cells abundantly secreted XCL1 8-36 hr after antigen recognition on CD8(+) DCs in vivo, in a period in which stable T cell-DC interactions are known to occur. Functionally, XCL1 increased the pool of antigen-specific CD8(+) T cells and their capacity to secrete IFN-gamma. Absence of XCL1 impaired the development of cytotoxicity to antigens cross-presented by CD8(+) DCs. The XCL1-XCR1 axis thus emerges as an integral component in the development of efficient cytotoxic immunity in vivo.

Live imaging of peripheral T cell-DC interactions in the islets: An emerging axis of autoimmune infiltration (48.15)

Abstract Defining processes of islet infiltration and destruction in Type 1 diabetes (T1D) is important for its prevention and preventing destruction of transplanted islets in diabetic individuals. We hypothesize that T1D onset and persistence is maintained by T cell-dendritic cell (DC) interactions in both the draining pancreatic lymph node (PLN) and the islets. In the steady-state, we found that intra-islet DCs are a uniform semi-mature population that constitutively take up β cell antigens in preparation for antigen presentation. Islet-antigen specific CD8 T cells activated in the PLN trafficked to the islets where they induced maturation of islet DCs. Leukocytic infiltration of the islets and islet DC maturation were dependent on IFN-γ production by T cells. Blockade of antigen processing following T cell activation in the PLN also blocked islet DC maturation but not islet infiltration. This suggests that the DCs require productive antigen-presenting interactions with T cells in the islets for maturation to occur. Through 2-photon imaging of the islets, we visualized sustained interactions of islet-antigen specific CD8 T cells with islet DCs and characterized features of cytotoxicity and dwell-time in this setting. Taken together, these data suggest that directed and synaptic delivery of IFN-γ to intra-islet DCs induces their maturation. This work was funded by the Larry L. Hillblom Foundation and the Juvenile Diabetes Research Foundation.

CXCR6/CXCL16 is required for optimal NKT cell and dendritic cell interactions (33.2)

Abstract Natural killer T (NKT) cells are a conserved T cell sublineage which rapidly produces cytokines upon activation by glycolipid antigens (i.e. α-GalCer) presented by dendritic cells (DC). Cross-talk between NKT cells and DCs is important in the initiation of anti-tumour responses. The objective of this study was to determine the role of CXCR6/CXCL16 in NKT cell-DC interactions. The chemokine receptor, CXCR6, is highly expressed on NKT cells, while its ligand, CXCL16, is expressed on DCs. When liver NKT cells were cultured with recombinant CXCL16 in vitro they were not activated directly. However, CXCL16 did enhance IFN-γ production in vitro when NKT cells were stimulated with suboptimal doses of plate bound anti-CD3. The presence of CXCL16 on the surface of DC is also important for NKT cell production of IFN-γ. NKT cells cultured with α-GalCer loaded CXCL16+ DC produced more IFN-γ than those cultured with CXCL16- DC. CXCL16+ DC stimulated more NKT cell IFN-γ production than CXCL16- DC in vivo as well. CD8+ DC constitutively express CXCL16 while all other DC subsets upregulate CXCL16 expression following α-GalCer stimulation. This is dependent on NKT cells since CXCL16 upregulation did not occur in NKT cell deficient (Jα18-/-) mice. DC maturation is also attenuated in the absence of CXCL16 signaling. These results suggest that CXCR6/CXCL16 is required for full NKT cell and dendritic cell activation/maturation.

Decoding the dynamics of T cell–dendritic cell interactions in vivo

T lymphocytes receive activation signals during their encounters with antigen-bearing dendritic cells (DCs) in secondary lymphoid organs. With the recent application of two-photon imaging to visualize immune responses as they happen, the dynamics of T cell-DC interactions have been dissected in several mouse models. As we are integrating the results of these new studies, we are learning that the dynamics of T cell-DC interactions are regulated by multiple immunological parameters and, most importantly, that the spatiotemporal characteristics of these cell-cell contacts encode part of the T-cell fate.

T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation

After homing to lymph nodes, CD8+ T cells are primed by dendritic cells (DCs) in three phases. During phase one, T cells undergo brief serial contacts with DCs for several hours, whereas phase two is characterized by stable T cell-DC interactions. We show here that the duration of phase one and T cell activation kinetics correlated inversely with the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC and with the density of antigen-presenting DCs per lymph node. Very few pMHC complexes were necessary for the induction of full-fledged T cell activation and effector differentiation. However, neither T cell activation nor transition to phase two occurred below a threshold antigen dose determined in part by pMHC stability. Thus, phase one permits T cells to make integrated 'measurements' of antigen dose that determine subsequent T cell participation in immune responses.

Real-Time Manipulation of T Cell-Dendritic Cell Interactions In Vivo Reveals the Importance of Prolonged Contacts for CD4 + T Cell Activation

T cells interact with dendritic cells (DCs) for periods lasting from minutes to hours. However, a causal link between the duration of this interaction and the efficiency of T cell activation has not been established in vivo. Employing intravital two-photon imaging, we manipulated T cell-DC interactions in real time and found that the first T cell-DC encounter often resulted in a long-lived interaction. Moreover, the cessation of T cell receptor-major histocompatibility complex signals promoted cellular dissociation, suggesting that antigen availability on DCs regulates contact duration. Finally, at least 6 hr of in vivo T cell-DC interaction were required for naive CD4(+) T cells to undergo clonal expansion. These results establish the importance of prolonged T cell-DC interactions for efficient CD4(+) T cell activation in vivo.

In situ visualization of CD8 T cell-DC interactions reveals distinct features of the early and late response to antigen after influenza virus infection (87.27)

Abstract The anatomical characteristics of T cell activation following infection are poorly described. Using transferred CD8 T cells and confocal microscopy we show that small clusters of virus-specific T cells formed transiently in the lung-draining lymph nodes (DLN) early in the response to influenza virus infection. Several weeks after infection naïve nucleoprotein (NP)-specific CD8 T cells still responded to antigen in the DLN, but remained predominantly in large stable clusters that included CD11c+ APCs. Many activated cells upregulated CD69 and PD-1, but did not lose CD62L-expression and remained clustered with CD11c+ APCs in the paracortex of the DLN until at least 7 days after transfer. Some central/memory cells also entered the DLN after transfer, but did not colocalize with the clustered naïve cells or show any signs of a response to the antigen. These data indicated a change in the response to antigen stimulation in the DLN as the time interval after infection increased and suggested that naïve and memory F5 CD8 T cells used different mechanisms to locate antigen-bearing APCs within the pLN. Some endogenous NP366-374/Db-specific CD8 T cells were also retained in the MLN and showed signs of a continuing response to antigen stimulation, while PA324-332/Db-specific cells were predominantly found in non-lymphoid tissues. Overall all the data revealed a dichotomy in the response to late antigen presentation which could influence epitope selection during reinfection by favoring preferential expansion of the NP366-374/Db-specific cells in the DLN. This work was supported by the American Lung Association RG1119 (L.S.C.), the Damon Runyon foundation (K.M.K.) and National Institutes of Health grants; R21-AI065895 (L.S.C.) and DK45260, AI41576 and AI56172 (LL).

A new view on cutaneous dendritic cell subsets in experimental leishmaniasis.

Because of their anatomical distribution epidermal Langerhans cells (LCs) are discussed to be crucial for antigen uptake and subsequent presentation to naïve T cells in skin-draining lymph nodes. The use of LC-specific markers like Langerin or knock-in mice expressing green fluorescent protein under the control of the Langerin promotor now facilitates the dissection of LCs from other dendritic cell (DC) subsets. Surprisingly, current data indicate that LCs are not generally involved in the induction of cellular immune responses. Moreover, the widely accepted paradigm postulating that LCs in principle act as T cell activators is contested by recent publications. Consequently, the biological role of LCs, in particular in cutaneous immune responses, needs to be revisited. The experimental model of leishmaniasis represents a suitable model to study the origin of an antigen-specific T cell response in mice. With this model the transport and presentation of skin derived Leishmania (L.) major antigens can be monitored in vivo. Furthermore, the quality of T cell-DC interactions can be determined. Considering recent progress in LC research we propose a novel concept of LCs in T cell meditated immunity against L. major parasites.

Diesel Exhaust Particle-Exposed Human Bronchial Epithelial Cells Induce Dendritic Cell Maturation

Increased exposure to air pollutants such as diesel exhaust particles (DEP) has been proposed as one mechanism to explain the rise in allergic disorders. However, the immunologic mechanisms by which DEP enhance allergic sensitization and asthma remain unclear. We hypothesized that DEP act as an adjuvant for immature dendritic cell (DC) maturation via its effect on airway epithelial cell-derived microenvironment for DC. Immature monocyte-derived DC (iMDDC) failed to undergo phenotypic (CD80, CD83, CD86) or functional (T cell activation) maturation in response to exposure to DEP (0.001-100 mug/ml). In contrast, primary cultures of human bronchial epithelial cells (HBEC) treated with DEP induced iMDDC phenotypic maturation (2.6 +/- 0.1-fold increase in CD83 expression, n = 4, p < 0.05) and functional maturation (2.6 +/- 0.2-fold increase in T cell activation, n = 4, p < 0.05). Functional maturation of iMDDC was induced by conditioned medium derived from DEP-treated HBEC, and was inhibited in cultures with DEP-treated HBEC and blocking Abs against GM-CSF, or GM-CSF-targeted small interfering RNA. These data suggest that DEP induce Ag-independent DC maturation via epithelial cell-DC interactions mediated by HBEC-derived GM-CSF. Although additional signals may be required for polarization of DC, these data suggest a novel mechanism by which environmental pollutants alter airway immune responses.

Functional comparison of mouse CIRE/mouse DC-SIGN and human DC-SIGN

CIRE/mDC-SIGN is a C-type lectin we originally identified as a molecule differentially expressed by mouse dendritic cell (DC) populations. Immunostaining with a CIRE/mDC-SIGN-specific mAb revealed that CIRE/mDC-SIGN is indeed on the surface of some CD4+, CD4−8− DCs and plasmacytoid pre-DCs, but not on CD8+ DCs. It has been proposed that CIRE/mDC-SIGN is the functional orthologue of human DC-SIGN (hDC-SIGN), a molecule that both enhances T cell responses and facilitates antigen uptake. We assessed if CIRE/mDC-SIGN and hDC-SIGN exhibit functional similarities. CIRE/mDC-SIGN is down-regulated upon activation, but unlike hDC-SIGN, incubation with IL-4 and IL-13 did not enhance CIRE/mDC-SIGN expression, indicating differences in gene regulation. Like hDC-SIGN, CIRE/mDC-SIGN bound mannosylated residues. However, we could detect no role for CIRE/mDC-SIGN in T cell–DC interactions and the protein did not bind to pathogens known to interact with hDC-SIGN, including Leishmania mexicana, cytomegalovirus, HIV and lentiviral particles bearing the Ebolavirus glycoprotein. The binding of CIRE/mDC-SIGN to hDC-SIGN ligands was not rescued when CIRE/mDC-SIGN was engineered to express the stalk region of hDC-SIGN. We conclude that there are significant differences in the fine specificity of the C-type lectin domains of hDC-SIGN and CIRE/mDC-SIGN and that these two molecules may not be functional orthologues.

Regulated Recruitment of MHC Class II and Costimulatory Molecules to Lipid Rafts in Dendritic Cells

T cell activation has long been associated with the partitioning of Ag receptors and associated molecules to lipid microdomains. We now show that dendritic cells (DCs) also accomplish the selective recruitment to lipid rafts of molecules critical for Ag presentation. Using mouse bone marrow-derived DCs, we demonstrate that MHC class II molecules become substantially localized to rafts upon DC maturation. Even more striking is the fact that CD86 is recruited to rafts upon T cell-DC interaction. Recruitment is Ag dependent and requires CD28 on T cells. Despite the regulated recruitment of MHC class II and CD86 to rafts, unlike the counter-receptors in T cells, DCs do not polarize these molecules to sites of DC-T cell contact. This difference may reflect the necessity for DCs to interact with multiple T cells simultaneously and emphasizes that the biochemical and morphological correlates of lipid rafts are not necessarily equivalent.

Malassezia enhances natural killer cell-induced dendritic cell maturation.

Human natural killer (NK) cells can induce cell death in autologous dendritic cells (DCs), though an interaction between these two cell types can also lead to a reciprocal activation. We have recently shown cell contact between NK cells and DCs in vivo, in Malassezia-induced lesional skin of patients with atopic eczema, where the yeast acts as an allergen although it is part of the normal skin microflora. Here we characterize the interaction of human NK cells and monocyte-derived DCs (MDDCs) by using an in vitro system where short-term activated polyclonal NK cells are cocultured with autologous, immature, Malassezia-stimulated or lipopolysaccharide-matured MDDCs. We found that the number of CD83(+) MDDCs increased in the immature and Malassezia-stimulated MDDCs upon coculture with NK cells, while an increased number of CD86(+) cells was detected in the Malassezia-stimulated MDDCs. NK cell-MDDC interaction induced the production of interleukin-8 (IL-8). In conclusion, our results imply that NK cells provide maturation signals and may play a role in inducing IL-8 production in DCs. Furthermore, the increased expression of CD86 on Malassezia-stimulated MDDCs might have a function in subsequent T-cell activation by DCs, and indicate a role for NK cell-DC interaction in modulating the immune responses to microbial stimuli.

CIITA-regulated plexin-A1 affects T-cell-dendritic cell interactions.

The major histocompatibility complex (MHC) class II transactivator (CIITA) is the 'master coactivator' of MHC class II genes. To identify new targets of CIITA, we analyzed cDNA microarrays of dendritic cells (DCs) from CIITA-deficient, MHC class II-deficient and control mice. We found the semaphorin receptor plexin-A1 was expressed in DCs, but not in other immune cells, and was strongly induced by CIITA. RNA interference by short hairpin RNA specific for plexin-A1, but not a single-nucleotide mutant, greatly reduced plexin-A1 expression and T cell stimulation by protein- or peptide-antigen-pulsed DCs.Plexin-A1 is not required for peptide binding to MHC. These data indicate involvement of plexin-A1 in T cell-DC interactions but not antigen processing or binding.

Peripheral blood-derived bovine dendritic cells promote IgG1-restricted B cell responses in vitro.

Regulation of humoral responses involves multiple cell types including the requirements for cognate interactions between T and B cells to drive CD40-dependent responses to T-dependent antigens. A third cell type has also been shown to play an essential role, the dendritic cell (DC). We demonstrate that bovine peripheral blood-derived (PB)-DC are similar in function to features described for human interstitial DC including the production of signature type 2 cytokines [interleukin (IL)-13, IL-10]. PB-DC express moderate-to-high costimulatory molecule expression, and major histocompatibility complex class II is negative for CD14 expression and has low or no expression of CD11c. Consistent with the interstitial phenotype is the ability of PB-DC to influence B cell activation and differentiation via direct expression of CD40L and type 2 cytokines. Collectively, these results suggest that direct B cell-DC interactions may promote an immunoglobulin-isotype expression pattern consistent with type 2 responses, independent of direct T cell involvement.

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.

Effective antigen presentation by dendritic cells is NF-kappaB dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines.

Antigen presentation is a key rate-limiting step in the immune response. Dendritic cells (DC) are the most potent antigen-presenting cells for naive T cells, due to their high expression of MHC and co-stimulatory molecules, but little is known about the biochemical pathways that regulate this function. We here demonstrate that monocyte-derived mature DC can be infected with adenovirus at high efficiency (>95%) and that this procedure can be used to dissect out which pathways are essential for inducing DC antigen presentation to naive T cells. Using adenoviral transfer of the endogenous inhibitor of NF-kappaB, IkappaBalpha, we show that DC antigen presentation is NF-kappaB dependent. The mechanism for this is that NF-kappaB is essential for three aspects of antigen-presenting function: blocking NF-kappaB coordinately down-regulates HLA class II, co-stimulatory molecules like CD80, CD86 and CD40, and immuno-stimulatory cytokines like IL-12 and tumor necrosis factor-alpha. In contrast adhesion molecules are up-regulated after infection with the adenovirus transferring IkappaBalpha, indicating that NF-kappaB also regulates the duration of T cell-DC interaction. These results establish NF-kappaB as an effective target for blocking DC antigen presentation and inhibiting T cell-dependent immune responses, and this finding has potential implications for the development of therapeutic agents for use in allergy, autoimmunity and transplantation.