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ObjectiveDendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC.DesignHuman DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction.ResultsA lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1β) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4+FoxP3+IL-10+ (regulatory) T cells. There were enhanced proportions of CD103+Sirpα− DC in the colon, with increased proportions of CD103+Sirpα+ DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103+Sirpα+ DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103+ DC, in particular CD103+Sirpα+ DC. However, expression of ILT3 was associated with CD103− DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells.ConclusionsThe regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

Dendritic cells (DCs) are antigen-presenting cells specialized for activating T cells to elicit effector T-cell functions. Cross-presenting DCs are a DC subset capable of presenting antigens to CD8⁺ T cells and play critical roles in cytotoxic T-cell–mediated immune responses to microorganisms and cancer. Although their importance is known, the spatiotemporal dynamics of cross-presenting DCs in vivo are incompletely understood. Here, we study the T-cell zone in skin-draining lymph nodes (SDLNs) and find it is compartmentalized into regions for CD8⁺ T-cell activation by cross-presenting DCs that express the chemokine (C motif) receptor 1 gene, Xcr1 and for CD4⁺ T-cell activation by CD11b⁺ DCs. Xcr1-expressing DCs in the SDLNs are composed of two different populations: migratory (CD103hi) DCs, which immigrate from the skin, and resident (CD8αhi) DCs, which develop in the nodes. To characterize the dynamic interactions of these distinct DC populations with CD8⁺ T cells during their activation in vivo, we developed a photoconvertible reporter mouse strain, which permits us to distinctively visualize the migratory and resident subsets of Xcr1-expressing DCs. After leaving the skin, migratory DCs infiltrated to the deep T-cell zone of the SDLNs over 3 d, which corresponded to their half-life in the SDLNs. Intravital two-photon imaging showed that after soluble antigen immunization, the newly arriving migratory DCs more efficiently form sustained conjugates with antigen-specific CD8⁺ T cells than other Xcr1-expressing DCs in the SDLNs. These results offer in vivo evidence for differential contributions of migratory and resident cross-presenting DCs to CD8⁺ T-cell activation.

Background and Objectives: Parkinson’s disease (PD) entails the progressive degeneration of dopaminergic neurons in the substantia nigra (SN), accompanied by α-synuclein (α-syn)-enriched Lewy bodies. ITGA7 mediates cell–extracellular matrix adhesion and modulates apoptosis, though its involvement in PD pathogenesis warrants further investigation. Although acupuncture demonstrates neuroprotective effects in PD models, its precise molecular mechanisms remain incompletely understood; therefore, in this study, we explored the relationship between ITGA7 and α-synuclein expression in an MPTP-induced PD mouse model to determine the association between LR3/GB34 acupuncture-induced changes in α-synuclein levels and ITGA7 modulation. Materials and Methods: In the in vivo model, MPTP-induced PD mice underwent immunohistochemistry, immunofluorescence, and Western blotting to assess ITGA7, α-synuclein, and TH levels in the SN and striatal tissues following LR3/GB34 acupuncture. In parallel, for the in vitro mechanistic study, SH-SY5Y neuroblastoma cells treated with MPP+ and transfected with ITGA7-siRNA were utilized to examine the involvement of apoptosis-related signaling pathways. Results: In the in vivo model, MPTP administration downregulated ITGA7 and upregulated α-synuclein in SN tissues. Similarly, in vitro exposure of SH-SY5Y cells to MPP+ yielded comparable results, revealing an inverse correlation between ITGA7 and α-synuclein. LR3/GB34 acupuncture treatment in the mouse model significantly increased ITGA7 and TH expression while reducing α-synuclein accumulation. To further understand the specific role of ITGA7 observed in these animal findings, we silenced ITGA7 in the MPP+-treated cellular model. ITGA7 silencing exacerbated the neurotoxic effects, leading to further TH downregulation, α-synuclein upregulation, Bcl-2 reduction, and Bax/Bcl-2 ratio elevation. Conclusions: Collectively, the histological preservation of dopaminergic neurons following LR3/GB34 acupuncture in the PD mouse model appears to be linked to ITGA7 upregulation. Furthermore, our in vitro findings implicate ITGA7 in the regulation of apoptosis-related signaling cascades, supporting its potential role in mitigating α-synuclein pathology.

The human lung T cell compartment contains many CD8⁺ T cells specific for respiratory viruses, suggesting that the lung is protected from recurring respiratory infections by a resident T cell pool. The entry site for respiratory viruses is the epithelium, in which a subset of lung CD8⁺ T cells expressing CD103 (αE integrin) resides. Here, we determined the specificity and function of CD103⁺CD8⁺ T cells in protecting human lung against viral infection. Mononuclear cells were isolated from human blood and lung resection samples. Variable numbers of CD103⁺CD8⁺ T cells were retrieved from the lung tissue. Interestingly, expression of CD103 was seen only in lung CD8⁺ T cells specific for influenza but not in those specific for EBV or CMV. CD103⁺ and influenza-reactive cells preferentially expressed NKG2A, an inhibitor of CD8⁺ T cell cytotoxic function. In contrast to CD103⁻CD8⁺ T cells, most CD103⁺CD8⁺ cells did not contain perforin or granzyme B. However, they could quickly upregulate these cytotoxic mediators when exposed to a type I IFN milieu or via contact with their specific antigen. This mechanism may provide a rapid and efficient response to influenza infection, without inducing cytotoxic damage to the delicate epithelial barrier.

Nivolumab, an immune checkpoint blocker, has been approved for advanced gastric cancer (GC), but predictive factors of nivolumab’s efficacy in patients with GC, especially immune cells such as tissue-resident memory T cells or those forming tertiary lymphoid structures (TLS), remain unclear. Tissue samples were obtained from surgically resected specimens of patients with GC who were treated with nivolumab as third-line or later treatment. Immunohistochemical staining was performed to detect the presence of TLS and CD103 + T cells and assess the association between TLSs and response to nivolumab treatment. A total of 19 patients were analyzed. In patients with partial response (PR) to nivolumab, numerous TLS were observed, and CD103 + T cells were found in and around TLS. Patients with many TLS experienced immune-related adverse events more often than those with few TLS ( p = 0.018). The prognosis of patients with TLS high was better than those with TLS low. Patients with a combination of TLS high and CD103 high tended to have a better prognosis than other groups. Our results suggested that TLS status might be a predictor of nivolumab effectiveness.

ObjectiveCD103+ gut dendritic cells (DCs) have been shown to be required for de novo conversion of adaptive T regulatory (Treg) cells. Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in tryptophan catabolism that is expressed by DCs isolated from tumour-draining lymph nodes. IDO-expressing DCs sustain and differentiate Tregs. The aim of this study was to investigate the expression and the possible physiological role of IDO in the tolerogenic properties of intestinal DCs.DesignThe expression level of IDO in CD103+ and CD103− DCs was analysed by qRT-PCR, western blot and immunofluorescence. CD103+ and CD103− DCs were sorted from mesenteric lymph nodes (MLNs) and the small intestinal lamina propria, and the role of IDO in the conversion of Tregs and Th effector cell development was evaluated via specific inhibition or gene deletion. Oral tolerance, experimental colitis and T cell differentiation in vivo were assessed upon IDO inactivation.ResultsWe show that, primarily, CD103+ but not CD103− gut DCs express IDO whose inhibition results in reduced CD4+Foxp3+ T regulatory cell conversion and enhanced T cell proliferation. When IDO was inhibited or genetically deleted there was an increase in Th1 and Th17 differentiation both in vitro and in vivo. Finally, in vivo IDO blockade affected the development of Tregs specific for orally administered antigens, impaired oral tolerance induction and exacerbated colitis.ConclusionsWe identified a new IDO-dependent pathway leading to acquisition of tolerogenic functions in mucosal CD103-expressing DCs, indicating IDO as a possible therapeutic target for gut disorders.

Bronchoalveolar lavage (BAL) is a useful procedure for differential diagnosis of interstitial lung diseases (ILDs) and for identification of granulomatous lung diseases. We investigated a panel of biomarkers from BAL fluid of ILD patients to evaluate their utility in differentiating ILDs. Bronchoscopy with BAL was performed in 100 consecutive patients with suspected ILD (41 sarcoidosis, 11 cHP and 24 other ILDs); the 24 patients negative for ILD diagnosis were included as control group. BAL phenotypes and cell profiles (CD4+/CD8+ ratio, NK and CD103+ cell counts, chitotriosidase and KL-6 levels in BAL) were determined by flow cytometry. A decision-tree statistical algorithm was applied. Sarcoidosis was discriminated by a higher BAL CD4+/CD8+ ratio (p = 5.8E−05), a lower BAL CD103+CD4+ count (p = 5.0E−02) and lower BAL NK percentages (p = 8.8E−03) than the other groups. BAL KL-6 concentrations were higher in sarcoidosis than in other ILDs (p = 1.5E−02) and were directly correlated with CD4+/CD8+ ratio. We used decision-tree statistical analysis to combine our biomarkers into two diagnostic algorithms for differential diagnosis of ILDs. A panel of BAL biomarkers for diagnosis of ILDs is proposed; CD4+/CD8+ ratio, KL-6 concentrations, and NK and CD103+CD4+ cell percentages in BAL could improve the identification and differential diagnosis of sarcoidosis.

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8 + and CD4 + tumor-specific reactive TILs in vitro , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8 + TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4 + and CD8 + tumor-specific reactive TILs. In situ , the combined detection of TNFRSF9 , TNF , and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro , which can be rapidly adopted in most cancer immunology laboratories.

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC--exhibiting the CD103(+) and CD11b(hi) phenotype, respectively--are the primary DC presenting antigen to naïve CD4(+) and CD8(+) T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103(+) RDC subset preferentially drives efficient proliferation and differentiation of naive CD8(+) T cells responding to infection into effector cells, and only the CD103(+) RDC subset can present to naïve CD8(+) T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103(+) and CD11b(hi) RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.

Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal that GM-CSF-dependent cross-talk between IV-infected AECs and CD103+ DCs is crucial for effective viral clearance and recovery from injury, which has potential implications for GM-CSF treatment in severe IV pneumonia.