CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

BACKGROUND/AIM: The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. MATERIALS AND METHODS: We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. RESULTS: CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. CONCLUSION: This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

The CCL27-CCR10 axis contributes to promoting proliferation, migration, and invasion of lung squamous cell carcinoma.

Lung cancer is characterized by its high mortality and morbidity. A deep understanding of the molecular mechanisms of lung cancer tumorigenesis helps to develop novel lung cancer diagnostic and therapeutic strategies. However, the picture of the associated molecular landscape is not yet complete. As understood, chemokine-receptor interactions contribute much to lung cancer tumorigenesis, in which CCR10 also plays an important role. This study aimed to expand the knowledge of CCR10 in lung squamous cell carcinoma (LUSC) in the manner of molecular mechanism and biological functions. Using GEPIA database, the survival analysis between LUSC patients with high and low CCR10 expressions was performed, showing that CCR10 could be regarded as a risk factor for LUSC patients. Subsequently, CCR10 protein and mRNA expressions in LUSC were examined by qRT-PCR and western blot respectively. The results indicated that CCR10 was highly expressed in LUSC cells. The results of CCK-8, colony formation, and Transwell assays presented that CCL27, the ligand of CCR10, promoted proliferative, migratory, and invasive abilities of LUSC cells by activating CCR10. Also, the PI3K/AKT signaling pathway was verified as the involved pathway by western blot. Overall, it could be concluded that the CCL27-CCR10 regulatory axis can activate the PI3K/AKT pathway fostering the malignant features of LUSC cells.

CCL28 promotes locomotor recovery after spinal cord injury via recruiting regulatory T cells.

BACKGROUND: Chemokines play a key role in post-traumatic inflammation and secondary injury after spinal cord injury (SCI). CCL28, the chemokine CC-chemokine ligand 28, is involved in the epithelial and mucosal immunity. However, whether CCL28 participates in the physiopathologic processes after SCI remains unclear. RESULTS: CCL28 is upregulated in the spinal cord after SCI. In addition, neutralizing antibodies against IL-1ß or TNF-α, or treatment of ML120B, a selective inhibitor of IKK-ß, remarkably decrease CCL28 upregulation, suggesting that CCL28 upregulation relies on NF-κB pathway activated by IL-1ß and TNF-α after SCI. Moreover, CD4+CD25+FOXP3+ regulatory T (Treg) cells that express CCR10, a receptor of CCL28, are enriched in the spinal cord after SCI. We further demonstrate that the spinal cord recruits Treg cells through CCL28-CCR10 axis, which in turn function to suppress immune response and promote locomotor recovery after SCI. In contrast, neutralizing CCL28 or CCR10 reduces Treg cell recruitment and delays locomotor recovery. METHODS: The neutralizing antibodies and recombinant CCL28 were injected intraspinally into the mice prior to SCI, which was established via hemitransection. RT-qPCR analysis was performed to determine transcript level, and Western blot analysis and ELISA assay were used to detect protein expression. Immune cells were analyzed by flow cytometry and visualized by immunofluorescence. The chemotaxis was assessed by in vitro transwell migration assay. The mouse locomotor activity was assessed via the Basso Mouse Scale (BMS) system. CONCLUSIONS: These results indicate that NF-κB pathway-regulated CCL28 production plays a protective role after SCI through recruiting CCR10-expressing and immunosuppressive Treg cells, and suggest that interfering CCL28-CCR10 axis might be of potential clinical benefit in improving SCI recovery.

The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation.

G-protein-coupled receptor (GPCR)-related proteins are dysregulated and the GPCR CC-chemokine receptor 10 (CCR10) is significantly upregulated in inflammation-driven HCC. However, CCR10's role in inflammation-driven hepatocarcinogenesis remains unknown. The aim of this study was to evaluate the role of CCR10 in inflammation-driven hepatocarcinogenesis. Via a targeted gene expression microarray screening alterations in GPCR family gene expression, we found CCR10 to be significantly upregulated in hepatocytes isolated from inflammation-driven human HCC tumors and matching paracancerous tissues. Tetrachloromethane (CCl4)-induced and diethylnitrosamine (DEN)-induced murine models of inflammatory hepatocarcinogenesis displayed significant hepatocellular TNF and CCR10 upregulation. Exogenous TNF applied to HepG2 and LO2 cell lines as well as wild-type (WT) mice significantly upregulated hepatocellular CCR10 expression, Akt phosphorylation, PCNA expression, and hepatocellular proliferation. Additionally, exogenous TNF significantly upregulated secretion of the natural CCR10 ligand-agonist CCL28 from both cell lines. Transgenic CCR10-knockout (CCR10 KO) in DEN-treated mice significantly increased hepatocellular apoptosis levels and significantly lowered compensatory hepatocellular proliferation but did not affect upstream TNF expression. In addition, DEN-treated CCR10 KO mice showed a significantly lower liver weight/body weight ratio, significantly lower liver tumor incidence, and significantly smaller tumors. Moreover, exogenous CCR10 expression significantly raised xenograft tumor growth in Balb/c nude mice. In vitro, CCR10 transfection or CCL28 treatment in HepG2 and LO2 cell lines significantly increased Akt phosphorylation, PCNA expression, and cell proliferation, while CCR10 silencing or Akt inhibition produced the opposite effects. In vivo, hepatocytes isolated from HCC tumor tissue and matching paracancerous tissue in DEN-treated CCR10 KO mice showed significantly lower Akt phosphorylation and PCNA expression relative to WT hepatocytes. In conclusion, inflammation-induced TNF promotes hepatocellular CCR10 expression and downstream PI3K/Akt-mediated hepatocarcinogenesis. CCR10 appears to function as a linkage between TNF stimulation and downstream PI3K/Akt pathway activation and shows promise as a potential therapeutic target for inflammation-driven HCC.

CCR10 activation stimulates the invasion and migration of breast cancer cells through the ERK1/2/MMP-7 signaling pathway.

CCR10, a member of the chemokine receptor subfamily, is overexpressed in several tumors and play a crucial role in cancer development and progression. However, the functions of CCR10 in breast cancer are unknown. Here, we detected the protein levels of CCR10 in breast cancer cells by western blotting, and examined CCR10 expression in breast cancer tissues via immunohistochemical assay. The results showed that CCR10 expression was elevated in breast cancer MCF7, BT-474 and MDA-MB-231 cells. Further, 63 of 89 cases (70.8%) had positive CCR10 staining, and the CCR10 level was closely related to capsular invasion, lymph node metastasis and tumor stage. Moreover, CCL27, the ligand of CCR10, dose-dependently stimulated the invasion and migration of breast cancer cells, and promoted MMP-7 expression and ERK1/2 activation. CCR10 knockdown in breast cancer cells through siRNA transfection attenuated CCL27-induced cell invasiveness, and suppressed MMP-7 expression and ERK1/2 activation. Additionally, blocking the ERK1/2 pathway inhibited the CCL27/CCR10-promoted cell invasion of breast cancer cells. Together, these data suggest that CCL27/CCR10 interaction induces the ERK1/2 pathway, which then increases MMP-7 expresion and subsequently promotes breast cancer cell invasion and migration. Thus, CCR10 may be a key regulator in breast cancer cell invasion and migration.

Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells.

Herpes simplex virus (HSV) infection is restricted to epithelial cells and neurons and is controlled by CD8 T cells. These cells both traffic to epithelial sites of recurrent lytic infection and to ganglia and persist at the dermal-epidermal junction for up to 12 weeks after lesion resolution. We previously showed that cutaneous lymphocyte-associated antigen (CLA), a functional E-selectin ligand (ESL), is selectively expressed on circulating HSV-2-specific CD8 T cells. CLA/ESL mediates adhesion of T cells to inflamed vascular endothelium. Later stages in T-cell homing involve chemokines (Ch) and lymphocyte chemokine receptors (ChR) for vascular wall arrest and diapedesis. Several candidate ChR have been implicated in skin homing. We measured cell surface ChR on HSV-specific human peripheral blood CD8 T cells and extended our studies to HSV-1. We observed preferential cell surface expression of CCR10 and CXCR3 by HSV-specific CD8 T cells compared to CD8 T cells specific for control viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and compared to bulk memory CD8 T cells. CXCR3 ligand mRNA levels were selectively increased in skin biopsy specimens from persons with recurrent HSV-2, while the mRNA levels of the CCR10 ligand CCL27 were equivalent in lesion and control skin. Our data are consistent with a model in which CCL27 drives baseline recruitment of HSV-specific CD8 T cells expressing CCR10, while interferon-responsive CXCR3 ligands recruit additional cells in response to virus-driven inflammation.IMPORTANCE HSV-2 causes very localized recurrent infections in the skin and genital mucosa. Virus-specific CD8 T cells home to the site of recurrent infection and participate in viral clearance. The exit of T cells from the blood involves the use of chemokine receptors on the T-cell surface and chemokines that are present in infected tissue. In this study, circulating HSV-2-specific CD8 T cells were identified using specific fluorescent tetramer reagents, and their expression of several candidate skin-homing-associated chemokine receptors was measured using flow cytometry. We found that two chemokine receptors, CXCR3 and CCR10, are upregulated on HSV-specific CD8 T cells in blood. The chemokines corresponding to these receptors are also expressed in infected tissues. Vaccine strategies to prime CD8 T cells to home to HSV lesions should elicit these chemokine receptors if possible to increase the homing of vaccine-primed cells to sites of infection.

CCL5 Promotes Resolution-Phase Macrophage Reprogramming in Concert with the Atypical Chemokine Receptor D6 and Apoptotic Polymorphonuclear Cells.

The engulfment of apoptotic polymorphonuclear cells (PMN) during the resolution of inflammation leads to macrophage reprogramming culminating in reduced proinflammatory and increased anti-inflammatory mediator secretion. The atypical chemokine receptor D6/ACKR2 is expressed on apoptotic PMN and plays an important role in regulating macrophage properties during and after engulfment. In this study, we found that the inflammatory chemokine CCL5 is mostly retained (75%) during the resolution of zymosan A peritonitis in mice. Moreover, this chemokine is secreted by resolution-phase macrophages (2.5 ng/ml) and promotes their reprogramming in vivo in D6+/+ mice (2-fold increase in IL-10/IL-12 ratio) but not their D6-/- counterparts. In addition, CCL5 enhanced macrophage reprogramming ex vivo exclusively when bound to D6+/+ apoptotic PMN. Signaling through p38MAPK and JNK in reprogrammed macrophages was enhanced by CCL5-bound apoptotic PMN (3.6-4 fold) in a D6-dependent manner, and was essential for reprogramming. Thus, CCL5 exerts a novel proresolving role on macrophages when acting in concert with apoptotic PMN-expressed D6.

CCR10/CCL27 crosstalk contributes to failure of proteasome-inhibitors in multiple myeloma.

The bone marrow microenvironment plays a decisive role in multiple myeloma progression and drug resistance. Chemokines are soluble mediators of cell migration, proliferation and survival and essentially modulate tumor progression and drug resistance. Here we investigated bone marrow-derived chemokines of naive and therapy-refractory myeloma patients and discovered that high levels of the chemokine CCL27, known so far for its role in skin inflammatory processes, correlated with worse overall survival of the patients. In addition, chemokine levels were significantly higher in samples from patients who became refractory to bortezomib at first line treatment compared to resistance at later treatment lines.In vitro as well as in an in vivo model we could show that CCL27 triggers bortezomib-resistance of myeloma cells. This effect was strictly dependent on the expression of the respective receptor, CCR10, on stroma cells and involved the modulation of IL-10 expression, activation of myeloma survival pathways, and modulation of proteasomal activity. Drug resistance could be totally reversed by blocking CCR10 by siRNA as well as blocking IL-10 and its receptor.From our data we suggest that blocking the CCR10/CCL27/IL-10 myeloma-stroma crosstalk is a novel therapeutic target that could be especially relevant in early refractory myeloma patients.

Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse.

PURPOSE: Chemokines may play vital roles in breast cancer progression and metastasis. The primary members of chemokine decoy receptors (CDR), DARC and D6, are expressed in breast tumors and lymphatic/hematogenous vessels. CDRs sequestrate the pro-malignant chemokines. We hypothesized that breast cancer patients carrying different levels of CDR expression in tumor and/or in host might have differing clinical outcomes. METHODS: This prospective observational study measured both expression and germline genotype of DARC and D6 in 463 primary breast cancer patients enrolled between 2004 and 2006. The endpoint was breast cancer relapse-free survival (RFS). RESULTS: There was a significant association between the co-expression of CDR (immunohistochemical expression of both DARC and D6) with RFS (hazard ratio [HR] of 0.32, 95% confidence interval [CI] 0.19 to 0.54). Furthermore, the co-genotype of two non-synonymous polymorphisms (with two major alleles of DARC-rs12075 and D6-rs2228468 versus the others) significantly related to relapse. Mechanistically, the variant-alleles of these two polymorphisms significantly decreased by 20-30% of CCL2/CCL5 (CDR ligands) levels relative to their major counterparts. Multivariate analysis highlighted that the co-expression and co-genotype of CDR were independent predictors of RFS, with HR of 0.46 (95% CI 0.27 to 0.80) and 0.56 (95% CI 0.37 to 0.85), respectively. The addition of host CDR genetic information to tumor-based factors (including co-expression of CDR) improved the relapse prediction ability (P = 0.02 of AUC comparison). CONCLUSION: The host genotype and tumor phenotype of CDR integrally affect breast cancer relapse. Host-related factors should be considered for individualized prediction of prognosis.

Characterising the expression and function of CCL28 and its corresponding receptor, CCR10, in RA pathogenesis.

OBJECTIVE: This study was conducted to determine the expression pattern, regulation and function of CCL28 and CCR10 in rheumatoid arthritis (RA) pathogenesis. METHODS: Expression of CCL28 and CCR10 was assessed in RA compared with other arthritis synovial tissues (STs) or fluids (SFs) by histology or ELISA. The factors modulating CCL28 and CCR10 expression were identified in RA myeloid and endothelial cells by ELISA, FACS and Western blotting. The mechanism by which CCL28 ligation promotes RA angiogenesis was examined in control and CCR10-knockdown endothelial cell chemotaxis and capillary formation. RESULTS: CCL28 and/or CCR10 expression levels were accentuated in STs and SFs of patients with joint disease compared with normal controls and they were predominately coexpressed in RA myeloid and endothelial cells. We show that protein expression of CCL28 and CCR10 was modulated by tumour necrosis factor (TNF)-α and toll-like receptor 4 ligation in RA monocytes and endothelial cells and by interleukin (IL)-6 stimulation in RA macrophages. Neutralisation of CCL28 in RA SF or blockade of CCR10 on human endothelial progenitor cells (EPCs) significantly reduced SF-induced endothelial migration and capillary formation, demonstrating that ligation of joint CCL28 to endothelial CCR10+ cells is involved in RA angiogenesis. We discovered that angiogenesis driven by ligation of CCL28 to CCR10 is linked to the extracellular signal regulated kinase (ERK) cascade, as CCR10-knockdown cells exhibit dysfunctional CCL28-induced ERK signalling, chemotaxis and capillary formation. CONCLUSIONS: The overexpression of CCL28 and CCR10 in RA ST and their contribution to EPC migration into RA joints support the CCL28/CCR10 cascade as a potential therapeutic target for RA.

Upregulation of chemokine receptor CCR10 is essential for glioma proliferation, invasion and patient survival.

Human gliomas are characterized by their invasion of normal brain structures irrespective of their grade of malignancy. Tumor cell invasion share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptor CCR10 is highly expressed in human glioblastoma compared with control brain tissue. In vitro, signaling through CCL27-CCR10 mediates activation of p-Akt, and subsequently induces proliferation and invasive responses. Cell proliferation and invasion promoted by CCL27 were blocked by inhibition of p-Akt or CCR10. In vivo, down-regulation of CCR10 significantly impairs growth of glioma. Clinically, High CCR10 expression in GBM correlated with p-Akt, shorter overall survival and progression-free survival (P < 0.05). Together, these findings suggest that elevated CCR10 is a critical molecular event associated with gliomagenesis.

Microarray analysis of gene expression by microdissected epidermis and dermis in mycosis fungoides and adult T-cell leukemia/lymphoma.

The characteristic histopathological feature of mycosis fungoides (MF) and adult T-cell leukemia/lymphoma (ATLL) is epidermotropism. To identify the mechanism for epidermotropism of lymphoma cells, total RNAs were obtained from skin biopsies of epidermis and dermis of MF and ATLL patients by means of laser capture microdissection, and used for subsequent complementary DNA (cDNA) microarray experiments. This procedure has made it possible for us to observe and evaluate the regional environment of MF and ATLL. Hierarchical cluster analysis revealed that the cDNAs could be clearly differentiated into MF and ATLL. CCL27 was expressed in the dermis generated from keratinocytes, CCR4/CCR6/CCR7/CCR10/cutaneous lymphocyte-associated antigen (CLA) lymphoma cells in the dermis, and CCL21 in the extracellular matrix (stroma). Lymphotoxin (LT) ß and CCL21 expression was significantly higher and that of CCR10 relatively for MF, while CCR4 and CLA expression was relatively higher for ATLL. In the epithelium, keratinocytes expressed CCL20/CCL27, and lymphoma cells CCR4/CCR6/CCR10, while CCR4, CCR6, CCL20 and CCL27 expression was relatively higher for ATLL than MF. The dermis of MF, but not that of ATLL, showed correlation between CCR7 and CCL21. These findings support the suggestion that chemokines and chemokine receptors are involved in the pathogenesis of MF and ATLL, indicate that cutaneous homing seems to be different for MF and ATLL, and point to the possibility that cutaneous T-cell lymphomas originate in regulatory T cells, especially in the case of ATLL.

Expression of the chemokine decoy receptor D6 is decreased in colon adenocarcinomas.

Recruitment of immune cells to tumors is a complex process crucial for both inflammation-driven tumor progression and specific anti-tumor cytotoxicity. Chemokines control the directed migration of immune cells, and their actions are partly controlled by nonsignaling chemokine decoy receptors. The role of the receptors such as D6, Duffy antigen receptor for chemokines and ChemoCentryx chemokine receptor in immunity to tumors is still unclear. Using real-time PCR, we detected significantly decreased expression of D6 mRNA in colon tumors compared to unaffected mucosa. D6 protein was expressed by lymphatic endothelium and mononuclear cells in the colon lamina propria and detected by immunohistochemistry in two out of six tissue samples containing high D6 mRNA levels, whereas no staining was observed in any tissue samples expressing low mRNA levels. When examining the density of lymphatic vessels in colon tumors, we detected a marked increase in vessels identified by the lymphatic endothelial marker Lyve-1, excluding passive regulation of D6 due to decreased lymphatic vessel density. In parallel, the Treg-recruiting chemokine CCL22, which is sequestered by D6, was threefold increased in tumor tissue. Furthermore, we could show that low D6 expression correlated to more invasive tumors and that tumor location influences D6 expression, which is lower in the more distal parts of the colon. The data support that regulation of D6 by colon tumors results in altered levels of proinflammatory CC chemokines, thereby shaping the local chemokine network to favor tumor survival. This may have implications for the design of future immunotherapy for colon cancer.

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1ß, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

An analysis of the function and expression of D6 on lymphatic endothelial cells.

The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.

Mucosal plasma cell barrier disruption during intestine transplant rejection.

BACKGROUND: Intestinal allograft mucosa undergoes repopulation with host immunocytes. However, critical changes within key immunocyte subsets are not known. METHODS: To explain acute cellular rejection after intestine transplantation (ITx) on the basis of altered mucosal immunocytes, rejecting and rejection-free ITx allografts (n=17) were compared with genome-wide expression arrays. Cells identified by cell/lineage-specific genes were evaluated by immunohistochemistry. The corresponding phenotype and donor-specific alloreactivity were characterized in peripheral blood. Time-dependent changes in candidate cell(s) were evaluated in biopsies from an independent cohort of 12 children with ITx. RESULTS: Among 107 differentially expressed genes, three B-cell lineage-specific genes, CCR10, STAP1, and IGLL1, were down-regulated during ITx rejection and were selected for and achieved technical quantitative reverse transcription polymerase chain reaction replication. Down-regulation of the immunoglobulin (Ig)A+ plasma cell-specific CCR10 gene correlated with decreased mature mucosal CD138+ plasma cell numbers in corresponding biopsy specimens (r=0.761, P=0.006) and inversely correlated with enhanced alloreactivity of CD154+ T-cytotoxic memory cells (r=-0.56, P=0.031), which predict acute cellular rejection with high sensitivity. An independent cohort of serial biopsy specimens from 12 ITx recipients (1) confirmed relative CD138+ plasma cell depletion during rejection (P=0.042) and (2) showed increased IgG+-to-IgA+ cell ratios within 4 hr of reperfusion in rejection-prone allografts (P=0.037) and during ITx rejection (P=0.025), compared with rejection-free allografts. No differences existed late after ITx. Increased peripheral IgG+ CD27+ CD19+ memory B cells (P=0.004) were seen during ITx rejection in archived peripheral blood lymphocyte from test and replication cohorts. CONCLUSIONS: Protracted depletion of the mucosal CD138+ plasma cell barrier and early mucosal infiltration with memory IgG+ cells characterize the rejection-prone intestine allograft. Mucosal IgA+ plasma cell barrier reconstitution may augur resolution of ITx rejection.

CCR10 and its ligands in regulation of epithelial immunity and diseases.

Epithelial tissues covering the external and internal surface of a body are constantly under physical, chemical or biological assaults. To protect the epithelial tissues and maintain their homeostasis, multiple layers of immune defense mechanisms are required. Besides the epithelial tissue-resident immune cells that provide the first line of defense, circulating immune cells are also recruited into the local tissues in response to challenges. Chemokines and chemokine receptors regulate tissue-specific migration, maintenance and functions of immune cells. Among them, chemokine receptor CCR10 and its ligands chemokines CCL27 and CCL28 are uniquely involved in the epithelial immunity. CCL27 is expressed predominantly in the skin by keratinocytes while CCL28 is expressed by epithelial cells of various mucosal tissues. CCR10 is expressed by various subsets of innate-like T cells that are programmed to localize to the skin during their developmental processes in the thymus. Circulating T cells might be imprinted by skin-associated antigen- presenting cells to express CCR10 for their recruitment to the skin during the local immune response. On the other hand, IgA antibody-producing B cells generated in mucosa-associated lymphoid tissues express CCR10 for their migration and maintenance at mucosal sites. Increasing evidence also found that CCR10/ligands are involved in regulation of other immune cells in epithelial immunity and are frequently exploited by epithelium-localizing or -originated cancer cells for their survival, proliferation and evasion from immune surveillance. Herein, we review current knowledge on roles of CCR10/ligands in regulation of epithelial immunity and diseases and speculate on related important questions worth further investigation.

The atypical chemokine receptor D6 controls macrophage efferocytosis and cytokine secretion during the resolution of inflammation.

The resolution of acute inflammation is hallmarked by the apoptotic death of inflammatory polymorphonuclear (PMN) cells, followed by their clearance by macrophages. In turn, resolution-phase macrophages exert reduced proinflammatory cytokine production, termed immune silencing. In this study, we found that the atypical chemokine receptor D6 plays an important and chemokine scavenging-independent role in promoting macrophage-mediated resolution. D6(-/-) mice displayed increased numbers of macrophages (2.2-fold increase), but not neutrophils, in their peritonea during the resolution of murine zymosan A-initiated peritonitis, in comparison to D6(+/+) animals. Moreover, D6-deficient macrophages engulfed higher numbers of apoptotic PMN cells in vivo (1.6-fold increase), and secreted higher amounts of TNF-α, CCL3, and CCL5 ex vivo than their wild-type (WT) counterparts. In addition, D6 was found to be expressed on apoptotic neutrophils from healthy humans and rodents. Moreover, the immune silencing of LPS-stimulated macrophages following their incubation with senescent PMN cells ex vivo (in terms of TNF-α, IL-1ß, and CCL5 secretion) was diminished (50-65% decrease) when D6(-/-) PMN cells were applied. Accordingly, the adhesive responses induced by macrophage interactions with senescent PMN cells were reduced with D6-deficient PMN cells. Thus, our results indicate a novel mode of action for D6 during the resolution of inflammation that is instrumental to the shaping of resolving macrophage phenotypes and the completion of resolution.

Toll-like receptor 2 induces mucosal homing receptor expression and IgA production by human B cells.

There is a need for developing vaccines that elicit mucosal immunity. Although oral or nasal vaccination methods would be ideal, current strategies have yielded mixed success. Toll-like receptor 2 (TLR2) ligands are effective adjuvants and are currently used in the Haemophilus influenzae type B vaccine. Induction of humoral immunity in the mucosa is critical for effective vaccination; thus, we sought to determine the effects of TLR2 ligands on human mucosal B cell differentiation. We demonstrate that TLR2 ligands induce CCR9 and CCR10 expression by circulating B cells and increased chemotaxis to cognate chemokines CCL25 and CCL28 suggesting that TLR2 induces B cell homing to the gastrointestinal tract. TLR2 stimulation of B cells also induced J chain and IgA production demonstrating the induction of mucosal-like antibody secreting cells. These observations suggest that vaccines containing TLR2-ligands as adjuvants could induce mucosal B cell immunity even when delivered in a non-mucosal manner.