Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

IL-4 acts on skin-derived dendritic cells to promote the TH2 response to cutaneous sensitization and the development of allergic skin inflammation.

BACKGROUND: Atopic dermatitis is characterized by scratching and a TH2-dominated local and systemic response to cutaneously encountered antigens. Dendritic cells (DCs) capture antigens in the skin and rapidly migrate to draining lymph nodes (dLNs) where they drive the differentiation of antigen-specific naive T cells. OBJECTIVE: We sought to determine whether non-T-cell-derived IL-4 acts on skin-derived DCs to promote the TH2 response to cutaneously encountered antigen and allergic skin inflammation. METHODS: DCs from dLNs of ovalbumin (OVA)-exposed skin were analyzed by flow cytometry and for their ability to polarize OVA-specific naive CD4+ T cells. Skin inflammation following epicutaneous sensitization of tape-stripped skin was assessed by flow cytometry of skin cells and real-time quantitative PCR of cytokines. Cytokine secretion and antibody levels were evaluated by ELISA. RESULTS: Scratching upregulated IL4 expression in human skin. Similarly, tape stripping caused rapid basophil-dependent upregulation of cutaneous Il4 expression in mouse skin. In vitro treatment of DCs from skin dLNs with IL-4 promoted their capacity to drive TH2 differentiation. DCs from dLNs of OVA-sensitized skin of Il4-/- mice and CD11c-CreIl4rflox/- mice, which lack IL-4Rα expression in DCs (DCΔ/Δll4ra mice), were impaired in their capacity to drive TH2 polarization compared with DCs from controls. Importantly, OVA-sensitized DCΔ/Δll4ra mice demonstrated impaired allergic skin inflammation and OVA-specific systemic TH2 response evidenced by reduced TH2 cytokine secretion by OVA-stimulated splenocytes and lower levels of OVA-specific IgE and IgG1 antibodies, compared with controls. CONCLUSIONS: Mechanical skin injury causes basophil-dependent upregulation of cutaneous IL-4. IL-4 acts on skin DCs that capture antigen and migrate to dLNs to promote their capacity for TH2 polarization and drive allergic skin inflammation.

T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Ubiquitin-Specific Protease 2 in the Ventromedial Hypothalamus Modifies Blood Glucose Levels by Controlling Sympathetic Nervous Activation.

Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.

Cutting Edge: Recruitment, Retention, and Migration Underpin Functional Phenotypic Heterogeneity of Regulatory T Cells in Tumors.

Tumor-infiltrating regulatory T cells (Tregs) have been extensively studied as therapeutic targets. However, not all infiltrating T cells exert their functions equally, presumably because of their heterogeneity and substantial turnover in tissues. In this study, we hypothesized that intertissue migration underlies the functional heterogeneity of Tregs. To test this, we applied in vivo photolabeling to examine single-cell diversity of immunosuppressive molecules in mouse Tregs migrating to, remaining in, and emigrating from MC38 tumors. Neuropilin-1 (Nrp1) expression was inversely correlated with that of six other molecules associated with Treg function. Unsupervised clustering analyses revealed that clusters containing Tregs that were retained in tumors expressed high levels of the six functional molecules but not of Nrp1. However, these clusters represented only half of the Tregs migrating to the tumor, suggesting evolving heterogeneity of tumor-infiltrating Tregs. Thus, we propose progressive pathways of Treg activation and migration between tumors and draining lymph nodes.

Circulation of gut-preactivated naïve CD8+ T cells enhances antitumor immunity in B cell-defective mice.

The gut microbiome has garnered attention as an effective target to boost immunity and improve cancer immunotherapy. We found that B cell-defective (BCD) mice, such as µ-membrane targeted deletion (µMT) and activation-induced cytidine deaminase (AID) knockouts (KOs), have elevated antitumor immunity under specific pathogen-free but not germ-free conditions. Microbial dysbiosis in these BCD mice enriched the type I IFN (IFN) signature in mucosal CD8+ T cells, resulting in up-regulation of the type I IFN-inducible protein stem cell antigen-1 (Sca-1). Among CD8+ T cells, naïve cells predominantly circulate from the gut to the periphery, and those that had migrated from the mesenteric lymph nodes (mLNs) to the periphery had significantly higher expression of Sca-1. The gut-educated Sca-1+ naïve subset is endowed with enhanced mitochondrial activity and antitumor effector potential. The heterogeneity and functional versatility of the systemic naïve CD8+ T cell compartment was revealed by single-cell analysis and functional assays of CD8+ T cell subpopulations. These results indicate one of the potential mechanisms through which microbial dysbiosis regulates antitumor immunity.

Near-infrared photoimmunotherapy induced tumor cell death enhances tumor dendritic cell migration.

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326+ DCs to dLNs. This effect was abolished by blocking adenosine triphosphate (ATP), one of the DAMPs molecules, as well as by inhibition of Gαi signaling by pertussis toxin. Thus, ICD induction by NIR-PIT stimulates Ti-DC migration to dLNs via ATP-P2X7 receptor and Gαi protein-coupled receptor signaling pathways and may augment tumor antigen presentation to induce anti-tumor T cells in dLNs.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens.

The generation of tumor-directed cytotoxic T lymphocytes is considered crucial for the induction of antitumor immunity. To activate these CD8(+) T cells, antigen-presenting cells (APCs) must initially acquire tumor cell-associated antigens. The major source of tumor antigens is dead tumor cells, but little is known about how APCs in draining lymph nodes acquire and crosspresent these antigens. Here we show that CD169(+) macrophages phagocytose dead tumor cells transported via lymphatic flow and subsequently crosspresent tumor antigens to CD8(+) T cells. Subcutaneous immunization with irradiated tumor cells protects mice from syngenic tumor. However, tumor antigen-specific CD8(+) T cell activation and subsequent antitumor immunity are severely impaired in mice depleted with CD169(+) macrophages. Neither migratory dendritic cells (DCs) nor lymph node-resident conventional DCs are essential for the crosspresentation of tumor antigens. Thus, we have identified CD169(+) macrophages as lymph node-resident APCs dominating early activation of tumor antigen-specific CD8(+) T cells.

In vivo photolabeling of tumor-infiltrating cells reveals highly regulated egress of T-cell subsets from tumors.

Immune therapy is rapidly gaining prominence in the clinic as a major weapon against cancer. Whereas much attention has been focused on the infiltration of tumors by immune cells, the subsequent fate of these infiltrates remains largely unexplored. We therefore established a photoconversion-based model that allowed us to label tumor-infiltrating immune cells and follow their migration. Using this system, we identified a population of tumor-experienced cells that emigrate from primary tumors to draining lymph nodes via afferent lymphatic vessels. Although the majority of tumor-infiltrating cells were myeloid, T cells made up the largest population of tumor-egressing leukocytes. Strikingly, the subset composition of tumor-egressing T cells was greatly skewed compared with those that had infiltrated the tumor and those resident in the draining lymph node. Some T-cell subsets such as CD8+ T cells emigrated more readily; others including CD4-CD8- T cells were preferentially retained, suggesting that specific mechanisms guide immune cell egress from tumors. Furthermore, tumor-egressing T cells were more activated and displayed enhanced effector function in comparison with their lymph node counterparts. Finally, we demonstrated that tumor-infiltrating T cells migrate to distant secondary tumors and draining lymph nodes, highlighting a mechanism whereby tumor-experienced effector T cells may mediate antitumor immunity at metastatic sites. Thus, our results provide insights into migration and function of tumor-infiltrating immune cells and the role of these cells in tumor immunity outside of primary tumor deposits.

Occludin Expression in Epidermal γδ T Cells in Response to Epidermal Stress Causes Them To Migrate into Draining Lymph Nodes.

Epidermal γδ T cells that reside in the front line of the skin play a pivotal role in stress immune surveillance. However, it is not clear whether these cells are involved in further induction of immune responses after they are activated in dysregulated epidermis. In this study, we found that activated γδ T cells expressed occludin and migrated into draining lymph nodes in an occludin-dependent manner. Epidermal γδ T cells in occludin-deficient mice exhibited impairments in morphology changes and motility, although they expressed activation markers at levels comparable to those in wild-type cells. Occludin deficiency weakened the induction of allergen-induced contact hypersensitivity, primarily as the result of the impaired migration of epidermal γδ T cells. Thus, occludin expression by epidermal γδ T cells upon activation in response to epidermal stress allows them to move, which could be important for augmentation of immune responses via collaboration with other cells.

Retention of Ag-specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations.

Several different memory T-cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4+ T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non-photoconverted Ag-specific CD4+ T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4+ T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non-lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4+ T-cell populations are generated in peripheral lymph nodes following immunisation.

Epithelial membrane protein 3 (Emp3) downregulates induction and function of cytotoxic T lymphocytes by macrophages via TNF-α production.

Tetraspanin membrane protein, epithelial membrane protein 3 (Emp3), is expressed in lymphoid tissues. Herein, we have examined the Emp3 in antigen presenting cell (APC) function in the CD8+ cytotoxic T lymphocytes (CTLs) induction. Emp3-overexpressing RAW264.7 macrophage cell line derived from BALB/c mice reduced anti-C57BL/6 alloreactive CTL induction, while Emp3-knockdown RAW264.7 enhanced it compared with parent RAW267.4. Emp3-overexpressing RAW264.7 inhibited, but Emp3-knockdown RAW264.7 augmented, CD8+ T cell proliferation, interferon-γ secretion, IL-2 consumption, and IL-2Rα expression on CD8+ T cells. The supernatant from co-culture with Emp3-overexpressing RAW264.7 contained higher amount of TNF-α, and TNF- α neutralization significantly restored all these inhibitions and the alloreactive CTL induction. These results suggest that Emp3 in allogeneic APCs possesses the inhibitory function of alloreactive CTL induction by downregulation of IL-2Rα expression CD8+ T cells via an increase in TNF-α production. This demonstrates a novel mechanism for regulating CTL induction by Emp3 in APCs through TNF-α production.

Involvement of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-induced Incomplete Cytokinesis in the Polyploidization of Osteoclasts.

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.

Tracking of intertissue migration reveals the origins of tumor-infiltrating monocytes.

Myeloid cells such as monocytes and monocyte-derived macrophages promote tumor progression. Recent reports suggest that extramedullary hematopoiesis sustains a sizable reservoir of tumor-infiltrating monocytes in the spleen. However, the influence of the spleen on tumor development and the extent to which spleen monocytes populate the tumor relative to bone marrow (BM) monocytes remain controversial. Here, we used mice expressing the photoconvertible protein Kikume Green-Red to track the redistribution of monocytes from the BM and spleen, and mice expressing fluorescent ubiquitination-based cell-cycle indicator proteins to monitor active hematopoiesis in these tissues. In mice bearing late-stage tumors, the BM, besides being the major site of monocyte production, supplied the expansion of the spleen reservoir, replacing 9% of spleen monocytes every hour. Deployment of monocytes was equally rapid from the BM and the spleen. However, BM monocytes were younger than those in the spleen and were 2.7 times more likely to migrate into the tumor from the circulation. Partly as a result of this intrinsic difference in migration potential, spleen monocytes made only a minor contribution to the tumor-infiltrating monocyte population. At least 27% of tumor monocytes had traveled from the BM in the last 24 h, compared with only 2% from the spleen. These observations highlight the importance of the BM as the primary hematopoietic tissue and monocyte reservoir in tumor-bearing mice, despite the changes that occur in the spleen monocyte reservoir during tumor development.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses.

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use.

Recirculating memory T cells are a unique subset of CD4+ T cells with a distinct phenotype and migratory pattern.

Several populations of memory T cells have been described that differ in their migration and function. In this study, we have identified a unique subset of memory T cells, which we have named recirculating memory T cells (T(RCM)). By exposing Kaede transgenic mouse skin to violet light, we tracked the fate of cutaneous T cells. One population of memory CD4(+) T cells remained in the skin. A second population migrated from the skin into draining lymph nodes (LNs) in a CCR7-dependent manner. These migrating CD4(+) T cells expressed a novel cell surface phenotype (CCR7(int/+)CD62L(int)CD69(-)CD103(+/-) E-selectin ligands(+)) that is distinct from memory T cell subsets described to date. Unlike memory T cell subsets that remain resident within tissues long-term, or that migrate either exclusively between lymphoid tissues or into peripheral nonlymphoid sites, CD4(+) T(RCM) migrate from the skin into draining LNs. From the draining LNs, CD4(+) T(RCM) reenter into the circulation, distal LNs, and sites of non-specific cutaneous inflammation. In addition, CD4(+) T(RCM) upregulated CD40L and secreted IL-2 following polyclonal stimulation. Our results identify a novel subset of recirculating memory CD4(+) T cells equipped to deliver help to both distal lymphoid and cutaneous tissues.

Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques.

Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue-resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation "parabiosis" with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX(3)CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP(+) osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP(+) osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP(+) osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR(+) monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted "red" KikGR(+) osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.