Cross-presenting CD103+ dendritic cells are protected from influenza virus infection

CD8⁺ cytotoxic T cells are critical for viral clearance from the lungs upon influenza virus infection. The contribution of antigen cross-presentation by DCs to the induction of anti-viral cytotoxic T cells remains controversial. Here, we used a recombinant influenza virus expressing a nonstructural 1–GFP (NS1-GFP) reporter gene to visualize the route of antigen presentation by lung DCs upon viral infection in mice. We found that lung CD103⁺ DCs were the only subset of cells that carried intact GFP protein to the draining LNs. Strikingly, lung migratory CD103⁺ DCs were not productively infected by influenza virus and thus were able to induce virus-specific CD8⁺ T cells through the cross-presentation of antigens from virally infected cells. We also observed that CD103⁺ DC resistance to infection correlates with an increased anti-viral state in these cells that is dependent on the expression of type I IFN receptor. These results show that efficient cross-priming by migratory lung DCs is coupled to the acquisition of an anti-viral status, which is dependent on the type I IFN signaling pathway.     

Major Histocompatibility Complex Class I–restricted Cross-presentation Is Biased towards High Dose Antigens and Those Released during Cellular Destruction

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I–restricted presentation to CD8⁺ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8⁺ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8⁺ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8⁺ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.     

PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Viral vector–based vaccines that induce protective CD8⁺ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non–DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8⁺ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1–based vaccination potentiated CD8⁺ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12–producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8⁺ T cell immunity induced by soluble PD1–based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.     

Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells

In recent years, human dendritic cells (DCs) could be subdivided into CD304⁺ plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter encompassing the CD1c⁺, CD16⁺, and CD141⁺ DC subsets. To date, the low frequency of these DCs in human blood has essentially prevented functional studies defining their specific contribution to antigen presentation. We have established a protocol for an effective isolation of pDC and cDC subsets to high purity. Using this approach, we show that CD141⁺ DCs are the only cells in human blood that express the chemokine receptor XCR1 and respond to the specific ligand XCL1 by Ca²⁺ mobilization and potent chemotaxis. More importantly, we demonstrate that CD141⁺ DCs excel in cross-presentation of soluble or cell-associated antigen to CD8⁺ T cells when directly compared with CD1c⁺ DCs, CD16⁺ DCs, and pDCs from the same donors. Both in their functional XCR1 expression and their effective processing and presentation of exogenous antigen in the context of major histocompatibility complex class I, human CD141⁺ DCs correspond to mouse CD8⁺ DCs, a subset known for superior antigen cross-presentation in vivo. These data define CD141⁺ DCs as professional antigen cross-presenting DCs in the human.The adaptive immune response is initiated through presentation of antigen to T cells by DCs. In the mouse, DCs can be broadly grouped into plasmacytoid DCs (pDCs) and conventional DCs (cDCs; earlier termed myeloid DCs). Mouse cDCs can be further subdivided into several DC types, which are apparently specialized for optimal antigen uptake, processing, and presentation to T cells in different body compartments (Steinman and Banchereau, 2007; Heath and Carbone, 2009; Segura and Villadangos, 2009). One particular type of antigen presentation is cross-presentation: in this case, extracellular antigen is not classically presented in the context of MHC-II but is instead shunted into the MHC-I presentation pathway (Bevan, 2006; Shen and Rock, 2006; Villadangos et al., 2007). CD8⁺ T cells can thus be activated by antigens taken up from the extracellular space and then differentiate into cytotoxic T cells. This mechanism is thought to be of major importance for the recognition of viral or bacterial antigens when DCs are not directly infected. In these instances, debris of cells that were infected and have subsequently undergone apoptosis as part of a cellular stress reaction is taken up and cross-presented by specialized DCs. Through this type of processing, the antigenic composition of the pathogen can become visible to the CD8⁺ T cell immune system. In the mouse, extensive experimentation has demonstrated that within cDCs, CD8⁺ DCs are the most effective in antigen cross-presentation (den Haan et al., 2000; Iyoda et al., 2002; Schulz and Reis e Sousa, 2002; Heath et al., 2004). Whether mouse pDCs play a significant role in antigen presentation and more so in antigen cross-presentation is controversial (Colonna et al., 2004; Liu, 2005; Villadangos and Young, 2008).We have recently shown in the mouse system that splenic CD8⁺ DCs (and their counterparts in other organs) are the only cells in the body expressing XCR1, a chemokine receptor with a unique ligand, XCL1 (Dorner et al., 2009). In vitro, XCL1 induces potent chemotaxis of XCR1⁺ CD8⁺ DCs. In vivo, XCL1 secreted by activated CD8⁺ T cells augments their expansion and differentiation into cytotoxic T cells when the antigen is cross-presented by CD8⁺ DCs in the context of MHC-I (Dorner et al., 2009). Collectively, these observations indicate that the XCL1–XCR1 communication axis optimizes the cooperation of antigen-specific CD8⁺ T cells with XCR1⁺ DCs, which cross-present antigen to them.Based on our studies in the mouse, we were interested to determine whether human DCs express XCR1. Human DCs have been extensively phenotyped in the past and subdivided again into pDC and into CD1c⁺ (BDCA-1⁺), CD16⁺, and CD141⁺ (BDCA-3⁺) cDC subsets (Dzionek et al., 2000; MacDonald et al., 2002; Piccioli et al., 2007; for review see Ju et al., 2010). Meticulous gene expression analyses of all human and mouse DCs have recently revealed a large gene expression program shared by human and mouse pDCs, and also led to the suggestion that human CD141⁺ DCs correspond to mouse CD8⁺ DCs (Robbins et al., 2008). In spite of this groundbreaking work on the subdivision of human DCs into subsets, information on the function of human primary DCs remained very scarce, apparently because of the limitations imposed by the very low frequencies of DCs in human blood (CD1c⁺ DCs, 0.31 ± 0.14% SD; CD16⁺ DCs, 0.75 ± 0.41%; CD141⁺ DCs, 0.04 ± 0.03%; pDCs, 0.29 ± 0.08%; n = 8; not depicted). Instead, antigen cross-presentation in the human system was essentially analyzed with DCs derived from monocytes in culture (Fonteneau et al., 2003), a system that may not reflect all of the functional properties of primary DCs.In the present study, we demonstrate that CD141⁺ DCs are the only population in human blood that expresses the chemokine receptor XCR1. Human CD141⁺ DCs react to the chemokine XCL1 by mobilization of intracellular Ca²⁺ ([Ca²⁺]_i) and by strong chemotaxis in vitro. More importantly, our experiments demonstrate that primary CD141⁺ DCs excel in cross-presentation of antigen when directly compared with CD1c⁺ DCs, CD16⁺ DCs, and pDCs from the same donors. Collectively, these functional data strongly indicate that human CD141⁺ DCs are the homologue of mouse CD8⁺ DCs. At the same time, the professional capacity of human CD141⁺ DCs to cross-present antigen is of major interest in the ongoing quest to develop vaccines capable of inducing antiviral or antitumor cytotoxicity in the human.     

CD4+ T Cell Help Impairs CD8+ T Cell Deletion Induced by Cross-presentation of Self-Antigens and Favors Autoimmunity

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I–restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8⁺ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4⁺ T cell help on this process. Small numbers of autoreactive OVA-specific CD8⁺ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic β cells. Coinjection of OVA-specific CD4⁺ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8⁺ T cells indicated that CD4⁺ T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8⁺ T cell tolerance induced by cross-presentation.There is now considerable evidence that CD8⁺ T cell responses can be induced in vivo by professional APCs capable of MHC class I–restricted presentation of exogenous antigens (1–3). This mechanism is known as cross-presentation and was suggested to be instrumental in the immune response to pathogens that avoid professional APCs (2–4). However, if this pathway was only directed towards induction of immunity, cross-presentation of self-antigens to autoreactive CD8⁺ T cells would result in autoimmunity. Recently, in studies using transgenic mice that express a membrane-bound form of OVA under the control of the rat insulin promoter (RIP-mOVA), we have shown that this is not the case. RIP-mOVA mice express membrane-bound OVA in pancreatic islets, kidney proximal tubular cells, thymus and testis. In these mice, OVA was found to enter the class I presentation pathway of a bone marrow– derived cell population and then activate transgenic OVA-specific CD8⁺ T cells (OT-I cells) (3) in LNs draining the sites of antigen expression. Although this form of activation initially led to proliferation of OT-I cells, it ultimately caused their deletion (5). Thus, cross-presentation can remove autoreactive CD8⁺ T cells, and may tolerize the CD8⁺ T cell compartment to self-antigens. These studies, however, did not explain why cross-presentation of a self-antigen induced CD8⁺ T cell tolerance, whereas foreign antigens induced immunity (1–4, 6).In numerous models, CD4⁺ T cell help has been shown to be important for the induction or maintenance of immune responses by CD8⁺ T cells (7–11), but such help is not always essential (12–14). CD4⁺ T cell help has also been shown to be important for avoiding CTL tolerance induction (15–17). In these reports, however, it was not known whether CD8⁺ T cells were activated by cross-presentation or by direct recognition of antigen. Thus, whether CD4⁺ T cell help can affect tolerance induced by cross-presentation has not been addressed. Recently, we demonstrated that cross-priming by foreign antigens requires CD4⁺ T cell help for induction of CTL immunity (6). In this study, we have investigated the influence of such help on the deletion of CD8⁺ T cells induced by cross-presentation of self-antigens.     

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4⁺ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8⁺ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8⁺ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell–mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8⁺ CTL-mediated antitumor immunity and indicate that Th9 cell–based cancer immunotherapy may be a promising therapeutic approach.     

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8⁺ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1–specific human CD8⁺ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.     

The route of priming influences the ability of respiratory virus–specific memory CD8+ T cells to be activated by residual antigen

After respiratory virus infections, memory CD8⁺ T cells are maintained in the lung airways by a process of continual recruitment. Previous studies have suggested that this process is controlled, at least in the initial weeks after virus clearance, by residual antigen in the lung-draining mediastinal lymph nodes (MLNs). We used mouse models of influenza and parainfluenza virus infection to show that intranasally (i.n.) primed memory CD8⁺ T cells possess a unique ability to be reactivated by residual antigen in the MLN compared with intraperitoneally (i.p.) primed CD8⁺ T cells, resulting in the preferential recruitment of i.n.-primed memory CD8⁺ T cells to the lung airways. Furthermore, we demonstrate that the inability of i.p.-primed memory CD8⁺ T cells to access residual antigen can be corrected by a subsequent i.n. virus infection. Thus, two independent factors, initial CD8⁺ T cell priming in the MLN and prolonged presentation of residual antigen in the MLN, are required to maintain large numbers of antigen-specific memory CD8⁺ T cells in the lung airways.In recent years, there has been considerable progress in understanding the mechanisms regulating the tissue-specific migration of lymphocytes to peripheral sites. An evolving concept is that environmental factors at the site of initial priming induce the expression of tissue-selective homing molecules on activated lymphocytes. In support of this, numerous studies have demonstrated a pivotal role for antigen-presenting cells in the programming of lymphocyte trafficking patterns during priming (Mora et al., 2003; Iwata et al., 2004; Sigmundsdottir et al., 2007). In contrast, several recent studies suggest a pliable property of memory T cells in terms of their tissue tropism. Adoptive transfer and parabiosis studies have shown that the location of initial priming has little impact on the ability of circulating effector memory T cells (T_EMs) to migrate to different nonlymphoid sites (Klonowski et al., 2004; Masopust et al., 2004). One explanation for this pleotropic homing ability is that activated CD8⁺ T cells disseminate from LNs draining the site of infection to distant LNs, where they acquire additional tissue-homing molecules associated with the local microenvironment (Liu et al., 2006). Moreover, the migration of circulating central memory T cells (T_CMs) to nonlymphoid tissues also results in functional and phenotypic conversion to tissue-resident T_EM phenotype (Laouar et al., 2005, 2007; Kohlmeier et al., 2007; Marzo et al., 2007). Together, these studies demonstrate that the site of initial priming, the continued maturation of activated T cells in nondraining lymphoid tissues, and the local environment within nonlymphoid tissues all contribute the migratory properties of memory CD8⁺ T cells.Studies in both humans and mice have shown that substantial numbers of T_EM persist in the lung airways after the resolution of respiratory virus infections. The numbers of T_EM in the lung airways gradually decline over the first 6 mo after infection and then stabilize as a relatively small population of memory T cells that is maintained in the lung airways indefinitely (Ostler et al., 2001; Hogan et al., 2001a; Wiley et al., 2001; de Bree et al., 2005; van Panhuys et al., 2005). This decline and stabilization in the number of memory T cells in the lung airways correlates with a progressive decline in cell-mediated protection from a secondary challenge (Liang et al., 1994; Kündig et al., 1996; Hogan et al., 2001b; Ray et al., 2004; Bachmann et al., 2005a,b). Unlike memory T cell populations that reside in other anatomical locations, lung airway memory T cells are not directly maintained through cytokine-driven homeostatic proliferation within the lung airways. Rather, antigen-specific memory T cells present in the lung airways represent a dynamic population that is maintained by continual recruitment from the systemic memory T cell pool under steady-state conditions (Ely et al., 2006). The accumulation of memory T cells in the airways under steady-state conditions is determined by migration from the circulation and cell death within the airways, a process which we refer to as continual recruitment. A recent study has demonstrated that residual antigen is maintained in the local draining LNs for several months after respiratory virus infection, and it has suggested a model in which recent stimulation by residual antigen is required for continual recruitment of memory CD8⁺ T cells to the airways (Zammit et al., 2006). In addition, we previously demonstrated that systemic memory CD8⁺ T cells generated after a respiratory virus infection could migrate to the airways in the absence of cognate antigen, albeit at low levels (Kohlmeier et al., 2007). However, it is not known how the route of priming impacts the ability of these antigen-dependent and -independent mechanisms to promote the recruitment of memory CD8⁺ T cells to the lung airways.To better understand the mechanisms regulating the continual recruitment of memory CD8⁺ T cells to the lung airways, we investigated the localization of memory CD8⁺ T cells that had been elicited by intranasal (i.n.) versus i.p. infection. The data show that i.n.-primed memory CD8⁺ T cells were preferentially recruited and maintained in the lung airways compared with i.p.-primed CD8⁺ T cells, and the defective recruitment of i.p.-primed memory CD8⁺ T cells to the lung airways was not corrected by the presence of cognate residual antigen in the mediastinal LN (MLN). Importantly, the ability of virus-specific memory CD8⁺ T cells to be activated by residual antigen in the MLN was restricted to i.n.-primed cells, and this activation resulted in multiple phenotypic changes which are associated with lung airway-resident cells. Collectively, the data suggest that not only the prolonged presentation of cognate antigen in the MLN but also T cell priming in the LNs that drain the respiratory tract during the primary response are required for the continual recruitment of memory CD8⁺ T cells to the lung airways.     

Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions

Chemokine receptor CX3CR1⁺ dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103⁺ DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103⁺ DC, CX3CR1⁺ cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103⁺ DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1⁺ cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103⁺ DC. These findings indicate that selectively CD103⁺ DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1⁺ populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.The intestinal mucosa contains large numbers of mononuclear cells, including macrophages and DCs, which together are believed to play a central role in regulating mucosal innate and adaptive immune responses in both the steady-state and inflammatory setting (Kelsall, 2008). Expression of CD11c and CD11b has been used to distinguish DC subsets from macrophages in the intestine. However, separating subsets of mononuclear cells based on CD11c is complicated because this marker is also expressed on tissue macrophages (Hume, 2008). Furthermore, there is currently little evidence to suggest that subdividing mononuclear lamina propria (LP) cells (LPCs) based on CD11c and CD11b defines populations with distinct turnover rates, origins, and functional properties.Based on the observation that DC can penetrate epithelial monolayers in vitro and a close association of CD11c-expressing cells with the gut epithelium in vivo, Rescigno et al. (2001) put forward the idea of epithelial-associated DC that is capable of capturing luminal bacteria and transporting them into the intestinal LP. Further studies using transgenic mice expressing GFP under the control of the CX3CR1-promoter (CX3CR1^+/GFP mice) confirmed that a major subset of mononuclear cells in the intestinal LP are capable of extending processes across the epithelial layer into the intestinal lumen (Niess et al., 2005). In vivo invasion-defective Salmonella enterica were associated with CX3CR1-expressing cells in the intestinal LP of transepithelial-proficient, but not transepithelial dendrite-deficient, CX3CR1^GFP/GFP mice, suggesting that transepithelial dendrites play an important role in sampling and uptake of luminal microbes (Niess et al., 2005; Hapfelmeier et al., 2008). Such transepithelial dendrites have also been observed in CD11c and MHCII reporter mice (Niess et al., 2005; Chieppa et al., 2006) even though their frequency and distribution remains controversial (Chieppa et al., 2006; Vallon-Eberhard et al., 2006). Together, these studies have led to the prevailing assumption that epithelial-associated CX3CR1⁺ mononuclear LPCs are DCs that play an important role in the initiation of intestinal adaptive immune response (Rescigno, 2003; Chieppa et al., 2006; Niess and Reinecker, 2006). Nevertheless, definitive evidence that CX3CR1⁺ LPCs are tissue DCs that are capable of migrating to draining mesenteric LN (MLN) and priming T cell responses has not been provided.We, and others, have identified a subset of DC in the intestinal LP and MLN which expresses the integrin α chain CD103. These cells display an intrinsic ability to induce the gut-homing receptors CCR9 and α4β7-integrin on responding T cells (Annacker et al., 2005; Johansson-Lindbom et al., 2005) and FoxP3 Treg differentiation in vitro (Coombes et al., 2007; Sun et al., 2007). Both of these functions appear to be caused by an enhanced ability of intestinal CD103⁺ DC to induce retinoic acid (RA) receptor signaling in responding T cells (Jaensson et al., 2008; Svensson et al., 2008) and are associated with higher expression of the gene encoding RALDH2 (retinaldehyde dehydrogenase 2), aldh1a2 (Coombes et al., 2007), a key enzyme involved in retinal metabolism. Importantly, CD103⁺ DCs are also present in human MLN and selectively induce RA receptor–dependent CCR9 expression on allogenic T cells (Jaensson et al., 2008). Thus, expression of CD103 identifies a DC subset that, under the influence of environmental conditioning in the gut and in the MLN (Agace, 2008; Hammerschmidt et al., 2008), confers typical properties of intestinal immune responses. CD103⁺ DCs are dramatically reduced in the MLN, but not LP, of CCR7^−/− mice (Johansson-Lindbom et al., 2005; Worbs et al., 2006) and, in BrdU pulse-chase experiments, have been shown to turn over rapidly in the LP in the steady state and with delayed kinetics in the MLN, suggesting that CD103⁺ MLN DCs represent LP-derived migratory DCs (Jaensson et al., 2008). Finally, CD103⁺, but not CD103⁻, MLN DCs are capable of priming OT-I and OT-II cells ex vivo after oral administration of OVA, indicating that these cells play a central role in initiating T cell responses to soluble luminal antigen (Coombes et al., 2007; Jaensson et al., 2008). Despite these findings, it remains unclear whether intestinal CD103⁺ DC and CX3CR1⁺ cells represent phenotypically and functionally overlapping or distinct populations of cells in the intestinal LP.In this paper, we demonstrate that CD103 and CX3CR1 expression defines distinct LPC populations. Although CD103⁺ DCs are short lived, travel via intestinal lymph to the gut draining MLN, and efficiently present antigens to naive T cells, CX3CR1⁺ cells are comparably longer lived LP-resident cells, cannot be observed in intestinal lymph, and are poor at presenting antigen to naive T cells. Together, these results suggest a division of labor between gut-resident CX3CR1⁺ LPC and migratory antigen-presenting CD103⁺ DC in the intestinal immune system.     

Laminins affect T cell trafficking and allograft fate

Lymph nodes (LNs) are integral sites for the generation of immune tolerance, migration of CD4⁺ T cells, and induction of Tregs. Despite the importance of LNs in regulation of inflammatory responses, the LN-specific factors that regulate T cell migration and the precise LN structural domains in which differentiation occurs remain undefined. Using intravital and fluorescent microscopy, we found that alloreactive T cells traffic distinctly into the tolerant LN and colocalize in exclusive regions with alloantigen-presenting cells, a process required for Treg induction. Extracellular matrix proteins, including those of the laminin family, formed regions within the LN that were permissive for colocalization of alloantigen-presenting cells, alloreactive T cells, and Tregs. We identified unique expression patterns of laminin proteins in high endothelial venule basement membranes and the cortical ridge that correlated with alloantigen-specific immunity or immune tolerance. The ratio of laminin α4 to laminin α5 was greater in domains within tolerant LNs, compared with immune LNs, and blocking laminin α4 function or inducing laminin α5 overexpression disrupted T cell and DC localization and transmigration through tolerant LNs. Furthermore, reducing α4 laminin circumvented tolerance induction and induced cardiac allograft inflammation and rejection in murine models. This work identifies laminins as potential targets for immune modulation.     

Lymph node stromal cells acquire peptide–MHCII complexes from dendritic cells and induce antigen-specific CD4+ T cell tolerance

Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8⁺ T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4⁺ T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide–MHCII complexes from DCs and induce CD4⁺ T cell dysfunction by presenting transferred complexes to naive CD4⁺ T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4⁺ T cells through the presentation of self-antigens via transferred peptide–MHCII complexes of DC origin.Self-reactive T cells that escape thymic negative selection are kept in check by peripheral tolerance mechanisms that include T cell anergy and deletion. Research into how self-reactive T cells are tolerized in LNs has focused largely on DCs. Depending on their functional status, antigen presentation by DCs can indeed lead to different forms of T cell tolerance (Steinman et al., 2003; Helft et al., 2010). Recently, however, LN-resident radio-resistant cells, the LN stromal cells (LNSCs), have been suggested to contribute to peripheral T cell tolerance. These cells can be discriminated based on their lack of CD45 expression and the differential expression of podoplanin (gp38) and PECAM (CD31). Fibroblastic reticular cells (FRCs, gp38⁺CD31⁻) produce chemokines such as CCL19 and CCL21, thereby providing a scaffold on which the CC-chemokine receptor 7 (CCR7)⁺ T cells and DCs can migrate and establish contact (Turley et al., 2010). In LNs, blood endothelial cells (BECs, gp38⁻CD31⁺) lining the high endothelial venules are crucial for lymphocyte entry (Mueller and Germain, 2009). Afferent lymphatic endothelial cells (LECs, gp38⁺CD31⁺) promote DC entry (Johnson et al., 2006; Acton et al., 2012), as well as antigen delivery (Sixt et al., 2005; Roozendaal et al., 2009), into LNs, whereas efferent LECs regulate T cell egress from LNs (Cyster and Schwab, 2012). The function of so-called double-negative (DN) stromal cells (gp38⁻CD31⁻) remains unknown. For many years, LNSCs were thought to only play an architectural role in LN construction and homeostasis. More recently, however, studies have identified LNSCs as active players in modulating adaptive immune responses (Swartz and Lund, 2012). In vitro, DC adhesion to LECs leads to decreased levels of co-stimulatory molecules by DCs (Podgrabinska et al., 2009). Furthermore, FRCs inhibit the proliferation of newly activated T cells through a NOS2-dependent mechanism, but also indirectly affect T cell proliferation by suppressing DC functions (Khan et al., 2011; Lukacs-Kornek et al., 2011; Siegert et al., 2011). In addition, FRCs can suppress acute T cell proliferation both in vitro and in vivo (Siegert et al., 2011). Other studies have convincingly demonstrated a role for LNSCs in maintaining peripheral CD8⁺ T cell tolerance via direct presentation of self-antigens to self-reactive CD8⁺ T cells. Unlike DCs, which acquire antigens and subsequently cross-present self-peptides to CD8⁺ T cells in the draining LNs, LNSCs ectopically express and present PTAs (peripheral tissue antigens) to CD8⁺ T cells, and consequently induce clonal deletion of self-reactive CD8⁺ T cells (Lee et al., 2007; Nichols et al., 2007; Gardner et al., 2008; Magnusson et al., 2008; Yip et al., 2009; Cohen et al., 2010; Fletcher et al., 2010). In addition, we have recently shown that tumor-associated LECs can scavenge tumor antigens and cross-present them to cognate CD8⁺ T cells, driving their dysfunctional activation (Lund et al., 2012). The lack of expression of co-stimulatory molecules such as CD80/86, and high PD-L1 expression levels at the surface of LECs (Fletcher et al., 2010; Tewalt et al., 2012), were proposed as the major mechanisms by which these cells induce deletional CD8⁺ T cell tolerance.While accumulating evidence suggests that direct antigen presentation by LNSCs promotes CD8⁺ T cell deletion, it is unknown whether LNSCs can similarly contribute to CD4⁺ T cell tolerance. As previously described, FRCs, BECs, and LECs express MHCII under virally induced inflammatory conditions or IFN-γ treatment (Malhotra et al., 2012; Ng et al., 2012). However, little is known about the regulation of MHCII expression by LNSCs.Here, we show that endogenous MHCII expression by LNSCs is controlled by the IFN-γ–inducible promoter IV (pIV) of class II transactivator (CIITA). Due to basal pIV activity, LNSCs express low levels of MHCII upon steady state and up-regulate these molecules when exposed to IFN-γ. Unexpectedly, in addition to low endogenous basal expression, the majority of MHCII molecules detected at LEC, BEC, and FRC surface were acquired from DCs. Furthermore, antigen-presenting DCs transfer antigenic peptide–MHCII (pMHCII) complexes to LNSCs, in a process dependent on both cell–cell contact and DC-derived exosomes. Importantly, acquired pMHCII complexes were presented by LECs, BECs, and FRCs to CD4⁺ T cells and promoted cognate CD4⁺ T cell dysfunction by impairing their survival and response to further restimulation. These data suggest that LNSCs serve more diverse roles than previously thought in regulating CD4⁺ T cell immunity.     

An AXL/LRP-1/RANBP9 complex mediates DC efferocytosis and antigen cross-presentation in vivo

The phagocytosis of apoptotic cells (ACs), or efferocytosis, by DCs is critical for self-tolerance and host defense. Although many efferocytosis-associated receptors have been described in vitro, the functionality of these receptors in vivo has not been explored in depth. Using a spleen efferocytosis assay and targeted genetic deletion in mice, we identified a multiprotein complex — composed of the receptor tyrosine kinase AXL, LDL receptor–related protein–1 (LRP-1), and RAN-binding protein 9 (RANBP9) — that mediates DC efferocytosis and antigen cross-presentation. We found that AXL bound ACs, but required LRP-1 to trigger internalization, in murine CD8α⁺ DCs and human-derived DCs. AXL and LRP-1 did not interact directly, but relied on RANBP9, which bound both AXL and LRP-1, to form the complex. In a coculture model of antigen presentation, the AXL/LRP-1/RANBP9 complex was used by DCs to cross-present AC-associated antigens to T cells. Furthermore, in a murine model of herpes simplex virus–1 infection, mice lacking DC-specific LRP-1, AXL, or RANBP9 had increased AC accumulation, defective viral antigen-specific CD8⁺ T cell activation, enhanced viral load, and decreased survival. The discovery of this multiprotein complex that mediates functionally important DC efferocytosis in vivo may have implications for future studies related to host defense and DC-based vaccines.     

The Peripheral Deletion of Autoreactive CD8+ T Cells Induced by Cross-presentation of Self-antigens Involves Signaling through CD95 (Fas,Apo-1)

Recently, we demonstrated that major histocompatibility complex class I–restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8⁺ T cells. In these studies, naive ovalbumin (OVA)-specific CD8⁺ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8⁺ T cells in the whole animal.     

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-γ production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c⁺ DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 µm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-γ production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.     

Annexin1 regulates DC efferocytosis and cross-presentation during Mycobacterium tuberculosis infection

The phagocytosis of apoptotic cells and associated vesicles (efferocytosis) by DCs is an important mechanism for both self tolerance and host defense. Although some of the engulfment ligands involved in efferocytosis have been identified and studied in vitro, the contributions of these ligands in vivo remain ill defined. Here, we determined that during Mycobacterium tuberculosis (Mtb) infection, the engulfment ligand annexin1 is an important mediator in DC cross-presentation that increases efferocytosis in DCs and intrinsically enhances the capacity of the DC antigen–presenting machinery. Annexin1-deficient mice were highly susceptible to Mtb infection and showed an impaired Mtb antigen–specific CD8⁺ T cell response. Importantly, annexin1 expression was greatly downregulated in Mtb-infected human blood monocyte–derived DCs, indicating that reduction of annexin1 is a critical mechanism for immune evasion by Mtb. Collectively, these data indicate that annexin1 is essential in immunity to Mtb infection and mediates the power of DC efferocytosis and cross-presentation.     

Pivotal role of dendritic cell-derived CXCL10 in the retention of T helper cell 1 lymphocytes in secondary lymph nodes

Various immune diseases are considered to be regulated by the balance of T helper (Th)1 and Th2 subsets. Although Th lymphocytes are believed to be generated in draining lymph nodes (LNs), in vivo Th cell behaviors during Th1/Th2 polarization are largely unexplored. Using a murine granulomatous liver disease model induced by Propionibacterium acnes, we show that retention of Th1 cells in the LNs is controlled by a chemokine, CXCL10/interferon (IFN) inducible protein 10 produced by mature dendritic cells (DCs). Hepatic LN DCs preferentially produced CXCL10 to attract 5'-bromo-2'-deoxyuridine (BrdU)+CD4+ T cells and form clusters with IFN-gamma-producing CD4+ T cells by day 7 after antigen challenge. Blockade of CXCL10 dramatically altered the distribution of cluster-forming BrdU+CD4+ T cells. BrdU+CD4+ T cells in the hepatic LNs were selectively diminished while those in the circulation were significantly increased by treatment with anti-CXCL10 monoclonal antibody. This was accompanied by accelerated infiltration of memory T cells into the periphery of hepatic granuloma sites, most of them were in cell cycle and further produced higher amount of IFN-gamma leading to exacerbation of liver injury. Thus, mature DC-derived CXCL10 is pivotal to retain Th1 lymphocytes within T cell areas of draining LNs and optimize the Th1-mediated immune responses.     

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

Human BDCA3⁺ dendritic cells (DCs) were suggested to be homologous to mouse CD8α⁺ DCs. We demonstrate that human BDCA3⁺ DCs are more efficient than their BDCA1⁺ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8⁺ T cells, which is similar to mouse CD8α⁺ DCs as compared with CD11b⁺ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α⁺, human BDCA3⁺, and sheep CD26⁺ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8⁺ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8⁺ T lymphocytes. Finally, XCR1^−/− mice have decreased early CD8⁺ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α⁺ DCs in higher vertebrates and promotes their ability to activate early CD8⁺ T cell defenses against an intracellular pathogenic bacteria.DCs are central to immune defenses in mammals. In mice, three subsets of DCs are resident of lymphoid organs (Crozat et al., 2010). Plasmacytoid DCs (pDCs) are professional producers of IFN-α and -β, contributing to immune defenses against viruses (Baranek et al., 2009). CD11b⁺ DCs preferentially prime CD4⁺ T cells and promote humoral immunity (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are endowed with a unique efficiency in priming CD8⁺ T cells and in cross-presenting exogenous antigens (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are required for the natural induction of strong CD8⁺ T cell responses against tumors (Hildner et al., 2008; Sancho et al., 2008) or West Nile virus (Hildner et al., 2008). Specific delivery of vaccine antigens to CD8α⁺ DCs is especially efficient for vaccination against intracellular pathogens or tumors (Bonifaz et al., 2004; Nchinda et al., 2008). Therefore, identification of human DC subsets functionally homologous to mouse CD8α⁺ DCs (CD8α⁺-type DCs) should be a major step forward for the design of innovative vaccination or immunotherapeutic strategies against cancer or infections (Crozat et al., 2010). So far, no conserved marker has been identified to specifically and unambiguously define CD8α⁺-type DCs in several mammalian species. Although human BDCA3⁺ and mouse CD8α⁺ DCs express the C-type lectin CLEC9A, which is known to be involved in cross-presentation in mice, this marker is also found on some human CD14⁺ monocytes and on mouse pDCs (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). It is of note that no orthologue of CLEC9A has been identified yet in non mammalian vertebrate species.We have recently performed comparative genomics studies of human, mouse, and sheep DC subsets to help identify potential homologies between these cell types across mammalian species (Robbins et al., 2008; unpublished data). We found that human blood BDCA3⁺ DCs share a specific gene signature with mouse CD8α⁺ DCs and proposed that they could be human professional cross-presenting DCs (Robbins et al., 2008; Crozat et al., 2010). In this paper, we demonstrate that human BDCA3⁺ DCs are more potent than their BDCA1⁺ counterparts or than pDCs for CD8⁺ T cell activation through antigen cross-presentation. We established elsewhere that sheep lymph CD26⁺ DCs (Epardaud et al., 2004) are also equivalents to mouse CD8α⁺ DCs based on gene expression and functions such as superior efficacy for presentation of soluble antigen to CD8⁺ T cells (unpublished data). In this paper, we identify the XC chemokine receptor 1 (XCR1) as the first universal marker specifically expressed by the CD8α⁺-type DCs from three different mammalian species: ovine CD26⁺ DCs, mouse CD8α⁺ DCs, and human BDCA3⁺ DCs. We show that the Xcr1 gene is present and well conserved in all higher vertebrates from reptiles to human. The ligand of XCR1, chemokine (C motif) ligand 1 (XCL1), is specifically expressed by activated NK and CD8⁺ T cells in mouse and human. We show that Xcl1 mRNA is stored selectively in memory CD8⁺ T cells, allowing them to rapidly produce high levels of this chemokine upon stimulation. Finally, we show that XCR1^−/− mice have decreased CD8⁺ T cell responses to Listeria monocytogenes (Lm) associated with higher bacterial loads early after infection. Overall, our study strongly suggests an important and conserved role in mammals for XCR1 in the cross talk between NK or CD8⁺ T cells and CD8α⁺-type DCs, identifying this molecule as a novel tool to survey and target the DCs endowed with the best cross-presentation capacity across species.     

In Vivo Imaging of Lymph Node Migration of MNP- and 111In-Labeled Dendritic Cells in a Transgenic Mouse Model of Breast Cancer (MMTV-Ras)

Purpose

The authors present a protocol for the in vivo evaluation, using different imaging techniques, of lymph node (LN) homing of tumor-specific dendritic cells (DCs) in a murine breast cancer model.

Procedures

Bone marrow DCs were labeled with paramagnetic nanoparticles (MNPs) or ¹¹¹In-oxine. Antigen loading was performed using tumor lysate. Mature DCs were injected into the footpads of transgenic tumor-bearing mice (MMTV-Ras) and DC migration was tracked by magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). Ex vivo analyses were performed to validate the imaging data.

Results

DC labeling, both with MNPs and with ¹¹¹In-oxine, did not affect DC phenotype or functionality. MRI and SPECT allowed the detection of iron and ¹¹¹In in both axillary and popliteal LNs. Immunohistochemistry and ??-counting revealed the presence of DCs in LNs.

Conclusions

MRI and SPECT imaging, by allowing in vivo dynamic monitoring of DC migration, could further the development and optimization of efficient anti-cancer vaccines.