Tissue-resident Antigen-presenting Cells Research Articles

Neonatal monocytes demonstrate impaired homeostatic extravasation into a microphysiological human vascular model

Infections are most frequent at the extremes of life, especially among newborns, reflecting age-specific differences in immunity. Monocytes maintain tissue-homeostasis and defence-readiness by escaping circulation in the absence of inflammation to become tissue-resident antigen presenting cells in vivo. Despite equivalent circulating levels, neonates demonstrate lower presence of monocytes inside peripheral tissues as compared to adults. To study the ability of monocytes to undergo autonomous transendothelial extravasation under biologically accurate circumstances we engineered a three-dimensional human vascular-interstitial model including collagen, fibronectin, primary endothelial cells and autologous untreated plasma. This microphysiological tissue construct enabled age-specific autonomous extravasation of monocytes through a confluent human endothelium in the absence of exogenous chemokines and activation. Both CD16− and CD16+ newborn monocytes demonstrated lower adherence and extravasation as compared to adults. In contrast, pre-activated tissue constructs were colonized by newborn monocytes at the same frequency than adult monocytes, suggesting that neonatal monocytes are capable of colonizing inflamed tissues. The presence of autologous plasma neither improved newborn homeostatic extravasation nor shaped age-specific differences in endothelial cytokines that could account for this impairment. Newborn monocytes demonstrated significantly lower surface expression of CD31 and CD11b, and mechanistic experiments using blocking antibodies confirmed a functional role for CD31 and CD54 in neonatal homeostatic extravasation. Our data suggests that newborn monocytes are intrinsically impaired in extravasation through quiescent endothelia, a phenomenon that could contribute to the divergent immune responsiveness to vaccines and susceptibility to infection observed during early life.

In situ neutrophil efferocytosis shapes T cell immunity to influenza infection.

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.

Role of M Cells in Human Mucosal Immunity to Mycobacterium tuberculosis

BackgroundMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a bacterial pathogen that infects roughly one-third of the worldÕs population and causes 1–2 million deaths per year. The current paradigm is that phagocytosis of Mtb by patrolling alveolar macrophages initiates Mtb infection. While this model can account for pulmonary TB, it does not adequately explain the occurrence of extrapulmonary forms of TB that manifest in the absence of obvious lung involvement, such as tuberculous cervical lymphadenitis, also known as scrofula. We hypothesized that specialized epithelial cells called microfold cells (M cells) may be an alternate portal of entry for Mtb. Previously we demonstrated that Mtb is able to transcytose across an epithelial barrier in an M cell dependent manner and that M cell mediated transcytosis is vital for Mtb pathogenesis in a mouse model of tuberculosis.MethodsWe used an in vitro M-cell mediated translocation assay and a Mtb mutant lacking a key virulence factor, ESAT6. We used biochemistry and genetics to identify a novel receptor for ESAT6. We also developed a novel explanted human adenoid Mtb infection model to study mucosal immunity.ResultsWe now demonstrate that the Mtb virulence factor ESAT6 is necessary and sufficient to mediate binding and transcytosis by M cells in vitro and in vivo, and that uptake of Mtb by M cells requires a unique cell surface ESAT6 receptor. We developed a novel explanted human adenoid model of M cell biology and demonstrate rapid Mtb transcytosis by primary human tissue within 60–120 minutes. Using flow cytometry we find that Mtb is first ingested by M cells and then after transcytosis, by tissue resident antigen-presenting cells. Explanted adenoids from 10 independent donors display a wide range of Mtb uptake.ConclusionWe conclude that Mtb ESAT6 is necessary for Mtb uptake by M-cells and that binding and transcytosis require a host receptor. Because explanted adenoids display a wide range of Mtb uptake, M cell mediated transcytosis may confer differential susceptibility to scrofula and disseminated disease. These findings are significant as M cells could potentially serve as the basis for novel therapeutic targets against primary Mtb infection.Disclosures All authors: No reported disclosures.

Papers of note in Science 357 (6355)

This week’s articles describe an alternative mechanism of secretion that is deployed by intestinal cells in the presence of pathogens; how the immune system protects the lungs from inhaled fungal spores; and metabolic reprogramming of tissue-resident antigen-presenting cells in the lung.

T Cells Motility in the Colonic Gvhd Is Influenced By Both Cognate Interaction and Microenvironment

Graft-versus-host disease (GVHD) accounts for substantial morbidity and mortality after allogeneic bone marrow transplantation. Allograft T cells are stimulated by both host and donor-derived antigen presenting cells (APCs). After differentiating into T effector cells, donor T cells migrate to GVHD target organs where they mediate damage directly and indirectly. However, very little is known about the dynamics of T cells in GVHD target tissues and how their behaviors are affected by the local environments, including by tissue-resident APCs. We utilized two-photon intravital microscopy (2PIM) to analyze T cell locations, motility and interactions with APCs. 129S1/SVLmJ (129) (H-2b) hosts were irradiated and reconstituted with C57Bl/6 (B6) (H-2b) CD11-YFP transgenic mice bone marrow together with B6 dsRed (RFP) CD8 cells and cyan fluorescent protein (CFP) CD4 cells. Mice were imaged from days 25-42 post-transplant. We focused on the colon which is strongly targeted in this model and in humans and which previously has been difficult to image by 2PIM. In syngeneic B6 into B6 recipients, donor CD11c+, CD8+ and CD4+ cells populate the colonic mucosa. However, a greater number of cells accumulated in the allogenic as compared to syngeneic recipients. Based on the analysis of 7-8 mice per group, T cells in allogenic mice were less motile. The mean CD8 speeds in syngeneic and allogeneic mice were 3.38 ± 0.07 µm/min and 2.21 ± 0.03 µm/min, respectively. Mean CD4 speeds in syngeneic and allogeneic mice were 3.11 ± 0.08 µm/min and 2.38 ± 0.04 µm/min, respectively. The majority of T cells were stationary, with few entering or leaving the imaged volume. Perfusion was confirmed by i.v. rhodamine-dextran; therefore, stationary behaviors was not due to tissue viability. Using optical clearing we imaged whole-mount colonic tissue from mucosa to serosa. T cells were aggregated in the sub-mucosa and infiltrated crypts and surprisingly also the muscular layer. Ki67+ T cells were found throughout, especially in the submucosa. Given the 2PIM data which show few new T cells entering the imaged volumes, these cells were likely stimulated to divide locally. Preliminary data with EdU pulsing suggest this to be the case. To determine whether T cell stability is microenvironment- or antigen-driven, we injected OT-1 TCR transgenic effectors which do not recognize any antigen in the recipient and imaged the next day. Most colonic OT-1 cells showed the same stationary behavior as nearby donor-derived CD4 and CD8 cells, suggesting that factors in the GVHD colon microenvironment drive T cell stability. Nonetheless there was also an antigen-driven component as injection of an anti-MHCII antibody (but not isotype control Ab) increased CD4+ T cell motility presumably by disrupting TCR:MHCII interaction. These results show the motility of T cells in the GVHD colon is influence by both TCR:MHC cognate interactions and by the microenvironment. That T cells are dividing and may be activated in situ, suggesting GVHD may be maintained locally. Current studies are focusing on what antigen-independent factors affect T cell motility and on defining the roles played by tissue APCs. DisclosuresNo relevant conflicts of interest to declare.

Comparison of microglia and infiltrating CD11c+ cells as antigen presenting cells for T cell proliferation and cytokine response

BackgroundTissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages.MethodsIn this work we compared the antigen presenting capacity of CD11c+ and CD11c− microglia subsets with infiltrating CD11c+ APC, which include DC. The microglial subpopulations (CD11c− CD45dim CD11b+ and CD11c+ CD45dim CD11b+) as well as infiltrating CD11c+ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR for cytokine expression. They were co-cultured with primed T cells to measure induction of T cell proliferation and cytokine response.ResultsThe number of CD11c+ microglia cells increased dramatically in EAE. They expressed equivalent levels of major histocompatibility complex and co-stimulatory ligands CD80 and CD86 as those expressed by CD11c+ cells infiltrating from blood. CD11c+ microglia differed significantly from blood-derived CD11c+ cells in their cytokine profile, expressing no detectable IL-6, IL-12 or IL-23, and low levels of IL-1β. By contrast, CD11c− microglia expressed low but detectable levels of all these cytokines. Transforming growth factor β expression was similar in all three populations. Although CNS-resident and blood-derived CD11c+ cells showed equivalent ability to induce proliferation of myelin oligodendrocyte glycoprotein-immunised CD4+ T cells, CD11c+ microglia induced lower levels of T helper (Th)1 and Th17 cytokines, and did not induce Th2 cytokines.ConclusionsOur findings show distinct subtypes of APC in the inflamed CNS, with a hierarchy of functional competence for induction of CD4+ T cell responses.

Myelin antigen load influences antigen presentation and severity of central nervous system autoimmunity

This study was designed to understand the impact of self-antigen load on manifestation of organ specific autoimmunity. Using a transgenic mouse model characterized by CNS hypermyelination, we show that larger myelin content results in greater severity of experimental autoimmune encephalomyelitis attributable to an increased number of microglia within the hypermyelinated brain. We conclude that a larger self-antigen load affects an increase in number of tissue resident antigen presenting cells (APCs) most likely due to compensatory antigen clearance mechanisms thereby enhancing the probability of productive T cell–APC interactions in an antigen abundant environment and results in enhanced severity of autoimmune disease.

Induction of an Immune Response for Imaging Antigen-Presenting Cell/T–Cell Interactions

INTRODUCTIONActivated dendritic cells (DCs) are the most efficient antigen-presenting cells (APCs) in the immune system. This protocol describes three different methods to activate antigen-specific CD4+ T cells using (i) bone-marrow-derived DCs, (ii) endogenous dermal DCs that drain to the local lymph node (LN), and (iii) tissue-resident APCs in a delayed-type hypersensitivity (DTH) response.

T-cell receptor– and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

AbstractLocalization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)– and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into nonlymphoid antigen-rich tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signaling pathways that regulate T-cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1−/− T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1−/− T cells in vivo. In contrast, Vav1−/− T-cell retention into antigen-rich tissue was severely impaired, reflecting T cells’ inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, and TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T cell–mediated tissue damage.

Chimerism Analysis of Langerhans Cells Early after T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation.

Skin is the most frequently affected organ in acute graft versus host disease (GVHD). Data from murine studies support the hypothesis that the interaction of residing host Langerhans cells of the epidermis (LC) and donor T cells is crucial for the initiation of acute GVHD. Donor T cells are also necessary to induce the switch of LC from host to donor origin after allogeneic stem cell transplantation (SCT). In an ongoing clinical protocol applying alemtuzumab-based T cell depleted (TCD) allogeneic SCT (Meyer, Blood 2007; 109:374), we observed acute skin GVHD occurring early after transplantation. We therefore intended to analyse the LC chimerism in patients undergoing this protocol. So far, LC-chimerism analysis in humans has been performed by the detection of the Y-chromosome restricting it to sex-mismatched donor/recipient pairs. Here we introduce a new method to isolate LC from small skin samples at high purity for a sensitive STR-based chimerism analysis of general applicability. Epidermal skin layers were obtained from 6 mm punch biopsies by dispase I digestion. A small slice of epidermis was used for immunofluorescent staining. The remaining sample was digested by trypsin, and CD1a/MHC-class II-positive LC were sorted by flow cytometry. This approach resulted in a mean purity of > 96% with skin of healthy individuals. However, the density of LC early after SCT following non-TCD myeloablative regimens had previously been shown to be much lower compared to healthy individuals. By CD1a-staining, we were able to show that this is also the case after TCD reduced intensity SCT. Nevertheless, LC could be purified in all of 8 analyzed patients. The isolated LC numbers ranged from 10 to more than 1000. In 4 patients we performed a re-analysis of the isolated cells by flow cytometry and confirmed a purity exceeding 97%. We obtained reliable results for LC chimerism in 6 of 8 patients. After the RNA-isolation protocol was further improved, we were able to detect signals even with 35 isolated LC in patient MZ-47. In two patients, the majority of isolated LC were of donor origin whereas the other 4 patients had predominantly host LC (patients' characteristics and chimerism results are summarized in Table 1). None of the patients developed spontaneous acute GVHD so far. For patients MZ-37 and MZ-43 LC-chimerism was also performed after day +50 post HSCT and showed a switch to >97% donor chimerism at that time. In summary, we have established a sensitive method that enables the chimerism analysis on highly purified LC independent of sex-mismatched donor/recipient pairs. Our results on a few patients' samples can not yet be related to clinical events. This assay, however, allows the comprehensive investigation of the chimerism of LC and potentially of other tissue-resident antigen presenting cells to study their impact on GVHD in humans.Table 1patientdonortypesex (patient/donor)LC (n)purity (%)donor chimerism (%)37MSDF/M112797,7<339MMUDF/M75098,6>9740MMUDM/M180098,31343MMUDM/M10n.a.n.a.44MSDM/M157n.a.n.a.45MSDM/F325> 983646MMUDM/M355n.a.4847MSDM/M35n.a.>90MSD, matched sibling donor; MMUD, mismatched unrelated donor; n.a., not applicable; M, male; F, female

A PCR-Based Assay for the Detection of Langerhans-Cell Chimerism after Allogeneic Stem Cell Transplantation.

Early acute GVHD of the skin frequently occurs in patients after allogeneic hematopoietic stem cell transplantation. Although T cell depletion reduces the incidence and severity, it does not completely prevent skin GVHD. This leads to a prolonged need for immunosuppressive medication in a significant number of patients. For the induction of acute GVHD, the stimulation of donor T cells by residing host antigen presenting cells such as Langerhans cells of the skin (LCs) plays a central role. The absence of donor T cells after depletion, however, seems to hamper an early switch of LCs from host to donor origin. Therefore, the monitoring of LC chimerism is of great interest. We and others have provided evidence for a delayed switch in LC chimerism after T cell depleted reduced intensity stem cell transplantation. However, the assays used so far either are imprecise when applying low numbers of isolated cells or they depend on the detection of the Y-chromosome in skin sections of sex-mismatched transplants. In an attempt to set up a more sensitive assay of general applicability, we combined the detection of donor chimerism and tissue specific markers in a single multiplex PCR. We established PCRs for 10 different cDNA regions of constitutively expressed genes containing single nucleotide polymorphisms (SNPs). The SNP-containing products of the multiplex PCR were subsequently analyzed by the primer extension method (minisequencing) and subsequently analyzed by capillary electrophoresis. All SNP-containing cDNAs were expressed in peripheral blood mononuclear cells (PBMCs) as well as in isolated CD4- and CD8-positive T-lymphocytes, in myeloid dendritic cells, LCs, and keratinocytes. When we tested this approach on PBMCs of 10 patients and their HLA-matched sibling donors, the assay distinguished all pairs in 1 to 6 out of 10 systems. In a subsequent step, the 10plex PCR was combined with the tissue specific markers langerin for LCs and cytokeratin 10 to distinguish LCs from keratinocytes. Their expression was detected using gene-specific probes in the same minisequencing reaction used for the detection of SNPS. The resulting 12plex assay distinguished sibling donors from the patients with the same specificity and, in the same reaction, detected Langerin as well as cytokeratin 10 in purified LCs and keratinocytes, respectively. In summary, we established a sensitive assay allowing the simultaneous detection of donor chimerism together with the tissue specificity of isolated LCs that is independent of sex-mismatched donors. The addition of further tissue specific markers might allow performing chimerism studies on other tissue resident antigen presenting cells.

Evolution of the T‐Cell Repertoire during the Course of Experimental Immune‐Mediated Demyelinating Diseases

Fig. 6 depicts a model for epitope spreading in T cell-mediated demyelination. The acute phase of disease is due to T cells specific for the initiating epitope, which can be either a determinant on the CNS target organ of the autoimmune response or a determinant on a persisting, CNS-tropic virus. The primary T cell response is responsible for the initial tissue damage by the production of proinflammatory Th1 cytokines which can affect myelination directly (Selmaj et al. 1991) and indirectly by their ability to recruit and activate macrophages to phagocytize myelin (Cammer et al. 1978). As a result of myelin damage and opening of the blood-brain-barrier during acute disease, T cells specific for endogenous epitopes on the same and/or different myelin proteins are primed and expand either in the periphery or locally in the CNS. These secondary T cells initiate an additional round of myelin destruction, leading to a clinical relapse by production of additional pro-inflammatory cytokines, similar to the bystander demyelination operative during acute disease. It will be of great interest to determine the relative contributions of local and systemic immune responses to these endogenous neuroepitopes. It is possible that local CNS presentation of endogenous neuroepitopes following acute CNS damage could be mediated by infiltrating inflammatory macrophages, activated microglial cells, endothelial cells and/or astrocytes. These tissue resident antigen presenting cells have been shown to upregulate expression of MHC class II (Sakai et al. 1986, Traugott & Lebon 1988), certain adhesion molecules (Cannella et al. 1990), and B7 costimulatory molecules (K. M. Nikcevich, J. A. Bluestone, and S. D. Miller, in preparation) in response to pro-inflammatory cytokines. The data on epitope spreading provided by the murine demyelinating disease models clearly illustrate the dynamic nature of the T cell repertoire during chronic inflammation in a specific target organ. The contribution of epitope spreading to chronic CNS demyelination could be considered to be a special case since tolerance to myelin epitopes would be expected to be inefficient due to their sequestration behind the blood-brain-barrier. However, the recent description of epitope spreading in response to pancreatic antigens in spontaneous diabetes in the NOD mouse may indicate that this phenomenon is operative in a variety of organ-specific experimental and spontaneous autoimmune diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

IL-1α-induced tumor immunogenicity: Endogenous cytokine expressed by tumor cells confers reduced tumorigenicity

A direct correlation between the constitutive expression of IL-1α and reduced tumorigenicity of fibrosarcomas was observed. This was established in fibrosarcoma cell lines which produce IL-1α ‘spontaneously’, possibly as an aberration of oncogene-mediated transformation or upon IL-1α gene transfer. In fibroblasts intracellular or membrane-associated IL-1α is expressed, whereas the secreted form of the cytokine (IL-1β) is absent. Studies on the mechanisms of tumor regression of the IL-1α-positive fibroblastoid cell lines indicated that IL-1α potentiates the development of tumor cell-specific CTLs, which are of importance for tumor eradication. Thus, IL-1α induces enhanced helper T cell activity which provides auxiliary signals for the growth/development of CTLs. Non-adaptive effector cells, activated locally by IL-1α-expressing fibrosarcoma cells, also contribute to the eradication of IL-1α-expressing fibrosarcomas. Local IL-1α expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, thus further potentiating anti-tumor immune responses. Mice, in which IL-1α-producing tumors were regressed, developed an immune memory and rejected a challenge with an IL-1 non-producing violent tumor cell line. Endogenous IL-1α activates a cytokine cascade (i.e.; IL-6, CSF), produced by the malignant cells and possibly also by stromal cells. However, IL-1α expression is essential for fibrosarcoma eradication, while other cytokines possibly amplify and sustain its action. IL-1 non-producing fibrosarcomas can be induced to express IL-1α in a transient manner, by treatment in culture with cytokine/LPS, and subsequently they shift from progressor to regressor tumors. Positive immunotherapeutical effects were obtained when treating mice bearing IL-1 non-producing fibrosarcomas with cells from the same line, induced to express IL-1α in a constitutive or transient manner. Thus, IL-1α expression by malignant cells bears the potential to be applied in immunotherapy, as it is assumed that most naturally occurring tumors do not express IL-1α activity. Further studies are aimed at the elucidation of the mechanisms of IL-1α-mediated tumor regression and on the development of immunotherapy protocols based on its expression by malignant cells.

Cytokine-induced tumor immunogenicity: endogenous interleukin-1α expressed by fibrosarcoma cells confers reduced tumorigenicity

A direct correlation between the constitutive expression of IL-1α and reduced tumorigenicity of fibrosarcomas was observed. This was established in fibrosarcoma cell lines which produce IL-1α ‘spontaneously’, possibly as an aberration of oncogene-mediated transformation or upon IL-1α gene transfer. In fibroblasts intracellular or membrane-associated IL-1α is expressed, whereas the secreted form of the cytokine (IL-1β) is absent. Studies on the mechanisms of tumor regression of the IL-1α-positive fibroblastoid cell lines indicated that IL-1α potentiates the development of tumor cell-specific CTLs, which are of importance for tumor eradication. Thus, IL-1α induces enhanced helper T cell activity which provides auxiliary signals for the growth/development of CTLs. Non-adaptive effector cells, activated locally by IL-1α-expressing fibrosarcoma cells, also contribute to the eradication of IL-1α-expressing fibrosarcomas. Local IL-1α expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, thus further potentiating anti-tumor immune responses. Mice, in which IL-1α-producing tumors were regressed, developed an immune memory and rejected a challenge with an IL-1 non-producing violent tumor cell line. Endogenous IL-1α activates a cytokine cascade (i.e.; IL-6, CSF), produced by the malignant cells and possibly also by stromal cells. However, IL-1α expression is essential for fibrosarcoma eradication, while other cytokines possibly amplify and sustain its action. IL-1 non-producing fibrosarcomas can be induced to express IL-1α in a transient manner, by treatment in culture with cytokine/LPS, and subsequently they shift from progressor to regressor tumors. Positive immunotherapeutical effects were obtained when treating mice bearing IL-1 non-producing fibrosarcomas with cells from the same line, induced to express IL-1α in a constitutive or transient manner. Thus, IL-1α expression by malignant cells bears the potential to be applied in immunotherapy, as it is assumed that most naturally occurring tumors do not express IL-1α activity. Further studies are aimed at the elucidation of the mechanisms of IL-1α-mediated tumor regression and on the development of immunotherapy protocols based on its expression by malignant cells.