Potentiality of multiple modalities for single-cell analyses to evaluate the tumor microenvironment in clinical specimens.

Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.

Multi-dimensional analysis identified rheumatoid arthritis-driving pathway in human T cell.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease accompanied by lymphocyte infiltration into joint synovium. While T cells are considered to be important for its pathogenesis, the features that are the most relevant to disease and how they change after treatment remain unclear. The aim of this study was to clarify the characteristics of T cells in RA, comprehensively. METHODS: We enrolled a total of 311 patients with RA and 73 healthy participants, and carefully classified them by disease state, constructed multiple cohorts and analysed clinical samples from them in a stepwise manner. We performed immunophenotyping with multiple evaluation axes, and two independent transcriptome analyses complementary to each other. RESULTS: We identified that 'effector memory-Tfh' subset was specifically expanded in the peripheral blood (PB) of patients with RA in correlation with disease activity, and reverted after treatment. Besides, we revealed distinct features of T cells in synovial fluid (SF) that the expression of Tfh/Tph-related genes and pro-inflammatory cytokines and chemokines, including CXCL13, were significantly enriched, whereas these phenotype were Th1-like. Finally, we identified specific pathways, such as mTORC1, IL-2-stat5, E2F, cell cycle and interferon-related genes, that were significantly enriched in SF, in particular, as well as PB of untreated patients with RA, and notably, these features reverted after treatment. CONCLUSION: Our multi-dimensional investigation identified disease relevant T-cell subsets and gene signatures deeply involved in pathogenesis of RA. These findings could aid in our understanding of essential roles of T cells in RA and will facilitate to development better diagnostic and therapeutic interventions.

Gene Signature-Based Approach Identified MEK1/2 as a Potential Target Associated With Relapse After Anti-TNFα Treatment for Crohn's Disease.

Background: Anti-tumor necrosis factor alpha (anti-TNFα) therapy has become the mainstay of therapy for Crohn's disease (CD). However, post-therapy, the recurrence rate is still high. The aim of this study was to dissect the molecular mechanism for recurrence of CD treated with anti-TNFα therapy and investigate novel therapeutic options that could induce complete remission. Methods: We re-analyzed publicly available mucosal gene expression data from CD patients pre- and post-infliximab therapy to extract the transcriptional differences between responders and healthy controls. We used a systematic computational approach based on identified differences to discover novel therapies and validated this prediction through in vitro and in vivo experimentation. Results: We identified a set of 3545 anti-TNFα therapy-untreatable genes (TUGs) that are significantly regulated in intestinal epithelial cells, which remain altered during remission. Pathway enrichment analysis of these genes clearly showed excessive growth state and suppressed terminal differentiation, whereas immune components were clearly resolved. Through in silico screening strategy, we observed that MEK inhibitors were predicted to revert expression of genes dysregulated in infliximab responders. In vitro transcriptome analysis demonstrated that selective MEK1/2 inhibitor significantly normalized reference genes from TUGs. In addition, in vitro functional study proved that MEK1/2 inhibitor facilitated intestinal epithelial differentiation. Finally, using murine colitis model, administration of MEK1/2 inhibitor significantly improved diarrhea and histological score. Conclusions: Our data revealed the abnormalities in anti-TNFα responders' CD colons that would be cause of recurrence of CD. Also, we provided evidence regarding MEK1/2 inhibitor as a potential treatment against CD to achieve sustainable remission.

Rap1 controls lymphocyte adhesion cascade and interstitial migration within lymph nodes in RAPL-dependent and -independent manners.

The small GTPase Rap1 and its effector RAPL regulate lymphocyte adhesion and motility. However, their precise regulatory roles in the adhesion cascade preceding entry into lymph nodes and during interstitial migration are unclear. Here, we show that Rap1 is indispensably required for the chemokine-triggered initial arrest step of rolling lymphocytes through LFA-1, whereas RAPL is not involved in rapid arrest. RAPL and talin play a critical role in stabilizing lymphocyte arrest to the endothelium of blood vessels under flow or to the high endothelial venules of peripheral lymph nodes in vivo. Further, mutagenesis and peptide studies suggest that release of a trans-acting restraint from the beta2 cytoplasmic region of LFA-1 is critical for Rap1-dependent initial arrest. Rap1 or RAPL deficiency severely impaired lymphocyte motility over lymph node stromal cells in vitro, and RAPL deficiency impaired high-velocity directional movement within lymph nodes. These findings reveal the several critical steps of Rap1, which are RAPL-dependent and -independent, in lymphocyte trafficking.

Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes.

The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20-like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1(-/-) lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1(-/-) lymphocytes on endothelium was markedly reduced, whereas their L-selectin-dependent rolling/tethering and transition to LFA-1-mediated arrest were not affected. Mst1(-/-) lymphocytes were also defective in the stabilization of adhesion through alpha4 integrins. Consequently, Mst1(-/-) mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1(-/-) lymphocytes had impaired motility over lymph node-derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration.

Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs.

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.

Regulation of leukocyte migration across endothelial barriers by ECTO-5'-nucleotidase-generated adenosine.

CD73-deficient mice are valuable for evaluating the ability of CD73-generated adenosine to modulate adenosine receptor-mediated responses. Here we report the role of CD73 in regulating lymphocyte migration across two distinct barriers. In the first case, CD73-generated adenosine restricts the migration of lymphocytes across high endothelial venules (HEV) into draining lymph nodes after an inflammatory stimulus, apparently by triggering A(2B) receptors on HEV. Secondly, CD73 promotes the migration of pathogenic T cells into the central nervous system during experimental autoimmune encephalomyelitis. Experiments are in progress to determine whether this effect is also adenosine receptor-mediated and to identify the relevant adenosine receptor.

CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

Dynamic expression of chemokines and the infiltration of inflammatory cells in the HSV-infected cornea and its associated tissues.

BACKGROUND: The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown. OBJECTIVES: To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues. STUDY DESIGN: Eight-week-old female BALB/c mice were infected with 10 mu l HSV-1 (CHR3 strain: 5 x 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry. RESULT: After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated. CONCLUSION: Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

Nepmucin, a novel HEV sialomucin, mediates L-selectin-dependent lymphocyte rolling and promotes lymphocyte adhesion under flow.

Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function-associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains.

CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes.

Although dendritic cells (DCs) located in the small intestinal lamina propria (LP-DCs) migrate to mesenteric lymph nodes (MLNs) constitutively, it is unclear which chemokines regulate their trafficking to MLNs. In this study we report that LP-DCs in unperturbed mice require CCR7 to migrate to MLNs. In vitro, LP-DCs expressing CCR7 migrated toward CCL21, although the LP-DCs appeared morphologically and phenotypically immature. In MLNs, DCs bearing the unique LP-DC phenotype (CD11chighCD8alphaintCD11blowalphaLlowbeta7high and CD11chighCD8alpha-CD11bhighalphaLlowbeta7high) were abundant in wild-type mice, but were markedly fewer in CCL19-, CCL21-Ser-deficient plt/plt mice and were almost absent in CCR7-deficient mice, indicating the critical importance of CCR7 in LP-DC trafficking to MLNs. Interestingly, CCR7+ DCs in MLNs with the unique LP-DC phenotype had numerous vacuoles containing cellular debris in the cytoplasm, although MLN-DCs themselves were poorly phagocytic, suggesting that the debris was derived from the LP, where the LP-DCs ingested apoptotic intestinal epithelial cells (IECs). Consistent with this, LP-DCs ingested IECs vigorously in vitro. By presenting IEC-associated Ag, the LP-DCs also induce T cells to produce IL-4 and IL-10. Collectively, these results strongly suggest that LP-DCs with unique immunomodulatory activities migrate to MLNs in a CCR7-dependent manner to engage in the presentation of IEC-associated Ags acquired in the LP.

CXCL13 is an arrest chemokine for B cells in high endothelial venules.

Chemokine receptor signaling is critical for lymphocyte trafficking across high endothelial venules (HEVs), but the exact mode of action of individual chemokines expressed in the HEVs is unclear. Here we report that CXCL13, expressed in a substantial proportion of HEVs in both lymph nodes (LNs) and Peyer patches (PPs), serves as an arrest chemokine for B cells. Whole-mount analysis of mesenteric LNs (MLNs) showed that, unlike T cells, B cellsa dhere poorly to the HEVs of CXCL13-/- mice and that B-cell adhesion is substantially restored in CXCL13-/- HEVs when CXCL13 is added to the MLNs by superfusion, as we have previously observed in PP HEVs by intravital microscopy. In vitro, CXCL13 activated the small guanosine triphosphatase (GTPase) Rap1 in B cells, and corroborating this observation, a deficiency of RAPL, the Rap1 effector molecule, caused a significant reduction in shear-resistant B-cell adhesion to intercellular adhesion molecule 1 (ICAM-1). In addition, CXCL13 induced B-cell adhesion to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) by activating alpha4 integrin. These data identify CXCL13 as an arrest chemokine for B cells in HEVs and show that CXCL13 plays an important role in B-cell entry into not only PPs but also MLNs.

Endomucin, a sialomucin expressed in high endothelial venules, supports L-selectin-mediated rolling.

Lymphocyte homing to lymph nodes is regulated by transient but specific interactions between lymphocytes and high endothelial venules (HEVs), the initial phase of which is mainly governed by the leukocyte adhesion molecule L-selectin, which recognizes sulfated and sialylated O-linked oligosaccharides displayed on sialomucin core proteins. One of the sialomucin proteins, endomucin, is predominantly expressed in vascular endothelial cells of a variety of tissues including the HEVs of lymph nodes; however, whether it functions as a ligand for L-selectin remains to be formally proven. Here we show that the endomucin splice isoform a is predominantly expressed in PNAd+ HEVs and MAdCAM-1+ HEVs, as seen in non-HEV-type vascular endothelial cells. Using affinity purification with soluble L-selectin, we found that HEV endomucin is specifically modified with L-selectin-reactive oligosaccharides and can bind L-selectin as well as an HEV-specific mAb, MECA-79. Our results also indicated that a 90-100 kDa endomucin species is preferentially decorated with L-selectin-reactive sugar chains, whereas an 80 kDa species represents conventional forms expressed in non-HEV-type vascular endothelial cells in lymph nodes. Furthermore, a CHO cell line expressing endomucin together with a specific combination of carbohydrate-modifying enzymes [core-2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT), alpha-1,3-fucosyltransferase VII (FucTVII) and L-selectin ligand sulfotransferase (LSST)] showed L-selectin-dependent rolling under flow conditions in vitro. These results suggest that when endomucin is appropriately modified by a specific set of glycosyltransferases and a sulfotransferase, it can function as a ligand for L-selectin, and that the endomucin expressed in HEVs may represent another sialomucin ligand for L-selectin.

Molecular determinants controlling homeostatic recirculation and tissue-specific trafficking of lymphocytes.

The homeostasis of the immune system is maintained by the recirculation of naïve lymphocytes through the secondary lymphoid tissues, such as the lymph nodes, Peyer's patches, and spleen. Upon insult by pathogens or antigens, lymphocytes become activated, and the regulated trafficking of these cells results in the integration of systemic and regional immune responses. The exquisite specificity of such lymphocyte trafficking is determined by tissue-specific guidance signals expressed by the endothelial cells of postcapillary venules, combined with counterreceptors expressed by the circulating lymphocytes. The high endothelial venules can selectively guide naïve lymphocytes into the lymph nodes and Peyer's patches by expressing a unique combination of vascular addressins, lymphocyte-specific chemokines, and chemokine-binding molecules. The inflamed postcapillary venules in extralymphoid tissues, such as the skin and intestinal lamina propria, also use a distinct array of endothelial adhesion molecules and tissue- selective chemokines, and support the recruitment of effector and memory lymphocytes that express the appropriate receptors for tissue-specific trafficking. In this review, we summarize the present understanding of the homeostatic recirculation of naïve lymphocytes through the secondary lymphoid tissues and the specific targeting of antigen-experienced lymphocytes into the effector sites. We also revisit some previous studies that reported apparently conflicting observations.

A high endothelial venule-expressing promiscuous chemokine receptor DARC can bind inflammatory, but not lymphoid, chemokines and is dispensable for lymphocyte homing under physiological conditions.

Chemokines displayed on the luminal surface of blood vessels play pivotal roles in inflammatory and homeostatic leukocyte trafficking in vivo. However, the mechanisms underlying the functional regulation of chemokines on the endothelial cell surface remain ill-defined. A promiscuous chemokine receptor, the Duffy antigen receptor for chemokines (DARC), has been implicated in the regulation of chemokine functions. Here we show that DARC is selectively expressed at the mRNA and protein levels in the high endothelial venules (HEV) of unstimulated lymph nodes (LN). To examine the biological significance of DARC expression in HEV, we performed competitive binding experiments with 20 different chemokines. The results showed that DARC selectively bound distinct members of the pro-inflammatory chemokines such as CXCL1, CXCL5, CCL2, CCL5 and CCL7, but not lymphoid chemokines such as CCL21, CCL19, CXCL12 and CXCL13 that are normally expressed in HEV. CCL2 bound to DARC failed to induce a significant cytosolic [Ca(2+)] elevation in CCR2B-expressing cells, whereas the free form of CCL2 induced a distinct [Ca(2+)] elevation, suggesting that DARC down-regulates activities of pro-inflammatory chemokines upon binding. Targeted disruption of the gene encoding DARC did not induce any obvious changes in the cell number or leukocyte subsets in the peripheral and mesenteric LN. Neither did DARC deficiency significantly affect lymphocyte migration into LN. These results suggest that DARC may be a scavenger for pro-inflammatory chemokines, but not a presenting molecule for lymphoid chemokines at HEV and that it is probably functionally dispensable for lymphocyte trafficking to HEV-bearing lymphoid tissues under physiological conditions.

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.

Gene expression profiling of mucosal addressin cell adhesion molecule-1+ high endothelial venule cells (HEV) and identification of a leucine-rich HEV glycoprotein as a HEV marker.

High endothelial venule (HEV) cells support lymphocyte migration from the peripheral blood into secondary lymphoid tissues. Using gene expression profiling of mucosal addressin cell adhesion molecule-1(+) mesenteric lymph node HEV cells by quantitative 3'-cDNA collection, we have identified a leucine-rich protein, named leucine-rich HEV glycoprotein (LRHG) that is selectively expressed in these cells. Northern blot analysis revealed that LRHG mRNA is approximately 1.3 kb and is expressed in lymph nodes, liver, and heart. In situ hybridization analysis demonstrated that the mRNA expression in lymph nodes is strictly restricted to the HEV cells, and immunofluorescence analysis with polyclonal Abs against LRHG indicated that the LRHG protein is localized mainly to HEV cells and possibly to some lymphoid cells surrounding the HEVs. LRHG cDNA encodes a 342-aa protein containing 8 tandem leucine-rich repeats of 24 aa each and has high homology to human leucine-rich alpha(2)-glycoprotein. Similar to some other leucine-rich repeat protein family members, LRHG can bind extracellular matrix proteins that are expressed on the basal lamina of HEVs, such as fibronectin, collagen IV, and laminin. In addition, LRHG binds TGF-beta. These results suggest that LRHG is likely to be multifunctional in that it may capture TGF-beta and/or other related humoral factors to modulate cell adhesion locally and may also be involved in the adhesion of HEV cells to the surrounding basal lamina.