Search

Thymic dendritic cells (DC) play a role in central tolerance. Three thymic DC subtypes have been described: plasmacytoid DC (pDC) and two conventional DC (cDC), CD8α+ Sirpα- DC and Sirpα+ CD8α- cDC. Both pDC and Sirpα+ cDC can take up antigen in periphery and migrate into the thymus in response to chemokine signaling via CCR9 and CCR2 respectively. CCL2 is a major ligand for CCR2 and we previously showed that it was constitutively expressed in thymus, and that mice overexpressing CCL2 in thymus had reduced numbers of autoreactive T cells but elevated numbers of pDC. We have here investigated the role of CCL2-CCR2 axis in thymic pDC migration.We found that pDC expressed CCR2 at a high level and that their frequency was decreased in thymus, spleen and inguinal lymph nodes in mice lacking CCR2, but not in mice lacking CCL2. pDC migration towards the cortex or medulla within the thymus was not affected by CCL2 or CCR2 deficiency. Although some thymic progenitors expressed CCR2, this did not include those that give rise to pDC.Based on these results, we propose that CCR2 is involved in pDC homeostasis but its ligand CCL2 does not play a major role.

Objective To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn’s disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. Methods In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. Results A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [–87% to –98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84–138%] and β7+ naïve T cells [8%; 13–50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells. Conclusions Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes.

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1+Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1+Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1+Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1-TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1+Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.

Background & Aims The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. Methods Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9−/− mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. Results Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9−/− mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9+CD11b+ inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9−/− mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. Conclusions These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. Lay summary Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated fibroinflammatory condition of the biliary system which often carries a poor prognosis. Despite significant research efforts, there is no known medical therapy which can slow the progression of disease. It is linked with inflammatory bowel disease (IBD) and there are some limited data which suggest a role for gut-homing lymphocytes in the pathophysiology of the disease. I revealed via flow cytometry that patients with PSC had a reduced proportion of CCR9+ T-cells and mucosa associated innate-like T-cells (MAITs) within the peripheral blood as compared with controls. Furthermore, I was able to demonstrate an immunological difference within the ileum of patients with PSC/IBD as compared with IBD alone; namely that there is a reduced proportion of CCR9+ T-cells in the PSC ileum, lending evidence to the notion that the IBD in PSC is its own immunologically distinct disease. Utilising the technique of fine needle aspiration (FNA) of the liver, I found there was no difference in the proportion of gut-homing T-cells (i.e. CCR9+ or β7+) within the livers of patients with PSC compared with liver controls (LC). However, a subset of patients in both PSC and LC cohorts had a relative abundance of hepatic CCR9+ T-cells, though no correlation could be found with fibrosis or inflammatory measures. Patients with PSC, however, had higher hepatic proportions of MAITs and CD69+ CD8+ memory T-cells compared with LC, indicating a potential role in the pathophysiology of the disease. I was able to demonstrate that FNA liver could successfully be used for single cell RNAseq. Vedolizumab (VDZ) is a monoclonal antibody which targets α4β7 on T-cells, thereby theoretically preventing migration of lymphocytes to the gut. It is a licensed therapy in IBD but its use in PSC has not been established. I was able to demonstrate for the first time in humans that VDZ does indeed reduce the proportion of 􏰀7+ T-cells within the colon, establishing proof of concept. Furthermore, CCR9+/􏰀7+ CD4+ T-cells rise within the peripheral blood with VDZ indicating their being prevented from leaving the circulation. There was a heterogenous clinical hepatic response to VDZ among 102 patients with PSC/IBD, as evaluated by change in alkaline phosphatase (ALP). Around 20% of patients with PSC/IBD has a clinically significant drop in ALP of >20% from baseline. However, overall, the ALP rose with VDZ by a small amount. The IBD in PSC responded to a similar beneficial degree as in IBD alone, supporting its use for active IBD in PSC. A subset of patients with PSC had a significant rise in their ALP with VDZ and were investigated with liver biopsy, which revealed an increased proportion of T-cells which were CCR9+ compared with patients not on VDZ. These data suggest that in some patients with PSC, VDZ may cause a redistribution of gut-homing T-cells from the gut and peripheral blood to the liver, which has not been apparent among patients with IBD alone, though further investigation is warranted. Overall, this thesis provides a comprehensive analysis of peripheral blood, intestinal and hepatic lymphocytes in PSC compared with controls, as well as evaluating the effects of VDZ in these settings at a clinical and cellular level.

The present study aimed to determine the clinical significance and potential molecular mechanisms of C‑C motif chemokine receptor (CCR) genes in patients with early‑stage pancreatic ductal adenocarcinoma (PDAC). The transcriptomic, survival and clinical data of 112 patients with early‑stage PDAC who underwent pancreaticoduodenectomy were obtained from The Cancer Genome Atlas. The prognostic values of the CCR genes involved in early‑stage PDAC were evaluated using Kaplan‑Meier analysis and the multivariate Cox proportional risk regression model, and the potential molecular mechanisms were determined using bioinformatics tools. The identified CCRs closely interacted with each other at both the gene and protein levels. High expression levels of CCR5 [adjusted P=0.012; adjusted hazard ration (HR)=0.478, 95% confidence interval (CI)=0.269‑0.852], CCR6 (adjusted P=0.026; adjusted HR=0.527, 95% CI=0.299‑0.927) and CCR9 (adjusted P=0.001; adjusted HR=0.374, 95% CI=0.209‑0.670) were significantly associated with longer overall survival times in patients with early‑stage PDAC. The contribution of CCR5, CCR6 and CCR9 to the outcome of early‑stage PDAC was also demonstrated. Combined survival analysis of CCR5, CCR6 and CCR9 suggested that patients with high expression levels of these CCRs exhibited the most favorable outcomes. A prognostic signature was constructed in terms of the expression level of CC5, CCR6 and CCR9, and time‑dependent receiver operating characteristic curves indicated that this signature was able to effectively predict the outcome of patients with early‑stage PDAC. The potential molecular mechanisms of CCR5, CC6 and CCR9 in PDAC include its intersection of the P53, nuclear factor (NF)‑κB, generic transcription, mitogen‑activated protein kinase and STAT signaling pathways. Collectively, this highlights that CCR5, CCR6 and CCR9 are potential prognostic biomarkers for early‑stage PDAC.

There is a possible significant difficulty in differentiating between non-specific colitis (NSC) and early IBD patients with no cardinal endoscopic features. This clarifies the need to find markers with high sensitivity and specificity for distinguishing between both and other forms of specific colitis. The aim of this study to investigate the ability to use a chemokine panel (CCR9, CD146 and Foxp3) among patients with lower gastrointestinal symptoms found to have NSC (but do not have current IBD) to predict which patients progress to/develop future IBD or other diagnoses of specific colitis.Colonoscopy was done for 182 patients complaining of chronic diarrhea and or constipation, abdominal distention and pain with negative history for IBD, after Histopathological evaluation; 138 cases showing non-specific inflammation submitted for further immunohistochemical CCR9, CD146 and Foxp3 staining. On follow up patients with persistent symptoms or worsen symptoms recolonoscopy was done followed by Histopathological examination of samples and compared by the earlier results.The studied markers expressed significantly in IBD patients differentiating them from NSC patients (p 0.001) except for CCR9 expression was statistically insignificant in CD patients (p = 0.528). According to the ROC curves in prediction of progression using studied panel, the use of studied markers in combination was more statistically significant in comparison to each marker alone. Median follow up for studied patients was 12 months.This panel of markers holds a promising hope for early IBD as predictive markers, discriminating IBD from NSC and as potential therapeutic targets.

BACKGROUND: Increased frequency of CCR9(+) CD4(+) T cells in peripheral blood is linked to several gastrointestinal inflammatory diseases; however, its relationship with necrotizing enterocolitis (NEC) is not understood. We investigated whether the frequencies of CCR9(+) CD4(+) T cells and related subsets were increased in peripheral blood of both patients and mice with NEC. METHODS: CCR9(+) CD4(+) T cells and related subsets were evaluated by flow cytometry in peripheral blood collected from both patients and mice with NEC and controls. The suppressive function of CCR9(+) regulatory T (Treg) cells in NEC was assessed via in vitro suppression assay. An in vitro T cell polarization assay was performed to investigate the role of proinflammatory cytokines in Treg cell polarization. In vivo Treg cell polarization analysis was performed using NEC mice treated with anti-interleukin-6 (IL-6) receptor antibody. FINDINGS: A higher proportion of CCR9(+) CD4(+) T cells occurred in peripheral blood of both patients and mice with NEC than in controls. Elevated CCR9(+) CD4(+) T cells were primarily CCR9(+) IL-17-producing Treg cells, possessing features of conventional Treg cells, but their suppressive activity was seriously impaired and negatively correlated with the severity of intestinal tissue injury. IL-6 promoted polarization of CCR9(+) Treg cells to CCR9(+) IL-17-producing Treg cells, and blocking IL-6 signalling inhibited this conversion in vitro and ameliorated experimental NEC in vivo. INTERPRETATION: Collectively, these data suggested that CCR9(+) IL-17-producing Treg cells may be a biomarker of severity and highlight the possibility that antibodies targeting IL-6R could ameliorate NEC by modulating lymphocyte balance. FUND: This work was supported by the Science and Technology Planning Project of Guangdong Province, China (2017A020215100), the Science and Technology Foundation of Guangzhou, China (201704020086 and 201604020154), the Medical Scientific Research Foundation of Guangdong Province, China (A2017304 and A2014704), and the Social Science and Technology Development Foundation of Dongguan, China (2016108101037).

Mucosal surfaces are the primary point of entry for many infectious agents and mucosal immune responses serve as the primary defense to these pathogens. In order to mount an effective mucosal immune response, it is important to induce T cell homing to mucosal surfaces. Conventional vaccine adjuvants induce strong systemic immunity but often fail to produce mucosal immunity. We have developed an oil-in-water nanoemulsion (NE) adjuvant that provides mucosal immunity and efficient protection against mucosal pathogens when administered as part of an intranasal vaccine. In the present study, we demonstrate that intranasal immunization with NE indirectly activates the retinaldehyde dehydrogenase (RALDH) activity in dendritic cells through epithelial cell activity leading to SIgA as well as potent cellular responses and expression of α4β7 and CCR9 gut homing receptors on T cells. Confirming these findings, ex-vivo stimulation of splenocytes from NE nasally immunized animals showed increase in Th1/Th17 cytokines while suppressing Th2 responses. In examining mechanisms underlying this activation NE activated RALDH via MyD88 dependent pathways in DCs but did not activate the retinoic acid receptor directly. These results suggest that RALDH immune activities can be achieved by epithelial activation without direct RAR activation, which has significant implications for understanding mucosal immunity and the design of mucosal vaccines.

This study offers an effective strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration., CCR9+ T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9+ cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity–responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9+CD8+ T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell–dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein–1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration.

CD4+ T helper (Th) cells that express the gut homing chemokine receptor CCR9 are increased in the peripheral blood of patients with inflammatory bowel disease and Sjögren's syndrome and in the inflamed lesions of autoimmune diseases that affect the accessory organs of the digestive system. However, despite the important role of the GIT in both immunity and autoimmunity, the nature of CCR9-expressing cells in GIT lymphoid organs and their role in chronic inflammatory diseases remains unknown. In this study, we analyzed the characteristics of CCR9+ Th and T follicular helper (Tfh) cells in GIT associated lymphoid tissues in health, chronic inflammation and autoimmunity. Our findings reveal an association between the transcriptome and phenotype of CCR9+ Th in the pancreas and CCR9+ Tfh cells from GIT-associated lymphoid tissues. GIT CCR9+ Tfh cells exhibited characteristics, including a Th17-like transcriptome and production of effector cytokines, which indicated a microenvironment-specific signature. Both CCR9+ Tfh cells and CCR9+ Th cells from GIT-associated lymphoid tissues migrated to the pancreas. The expression of CCR9 was important for migration of both subsets to the pancreas, but Tfh cells that accumulated in the pancreas had downmodulated expression of CXCR5. Taken together, the findings provide evidence that CCR9+ Tfh cells and Th cells from the GIT exhibit plasticity and can accumulate in distal accessory organs of the digestive system where they may participate in autoimmunity.

Introduction Chemokine CC motif receptors 9 and 7 (CCR9 and CCR7) play a major role in the migration of T-cell precursors to the thymus to initiate T thymopoiesis. However, their role in development of T-cells in myasthenia gravis (MG) patients has not been fully elucidated. Methods Expression and distribution of CCR9+ and CCR7+ cells were detected by flow cytometry and immunofluorescence. Real-time polymerase chain reaction was used to check the adhesion molecules on CD4- CD8- double-negative (DN) thymocytes. Results CCR9 and CCR7 expression by DN thymocytes increased in the MG thymus; the levels of CCR9, CCR7, interleukin-7R mRNA increased, and CXCR4 levels decreased compared with levels in the non-MG thymus. More CCR7 and CCR9 double-positive (DP) thymocytes were gathered near the subcapsular region in MG thymus. Conclusions Enhanced expression of CCR9 and CCR7 may complicate the differentiation of DP thymocytes from the DN stage in MG thymus. Muscle Nerve, 2016 Muscle Nerve 55: 84-90, 2017.

Objective P. gingivalis is a gram-negative anaerobic bacterium and a major periodontal pathogen. LPS produced by P. gingivalis promotes osteoclast formation. TECK is a CC chemokine whose expression is increased in gingival epithelial cells exposed to P. gingivalis LPS. In this study, we investigated the effect of TECK in osteoclastogenesis induced by P. gingivalis LPS. Designs Real time reverse transcriptase polymerase chain reaction (RTPCR) analysis and western blotting were performed to confirm TECK in MG63, human osteoblast cell line and primary murine osteoblasts and CCR9 in RAW 264.7 cells and murine bone marrow macrophages (BMMs) as osteoclast precursors. P. gingivalis LPS-treated BMMs and Raw 264.7 cells were cultured with or without TECK or TECK antibody to examine the effect of TECK on osteoclast formation. Cocultures with murine osteoblasts and bone marrow cells were also treated with or without TECK or TECK antibody. Luciferase assay and western blotting were used to determine whether TECK-CCR9 induced osteoclastogenesis was mediated through NFATc1 or NF-kB signaling. Results TECK was shown to be expressed by osteoblasts, and its receptor, CCR9, by osteoclast precursors. TECK increased P. gingivalis LPS-induced osteoclast numbers in an in vitro osteoclast formation assay using osteoclast precursors. The enhanced osteoclast formation by TECK was mediated by NFATc1, but not by NF-kB signaling. Conclusion TECK may be a novel regulator of osteoclast formation induced by P. gingivalis LPS in periodontitis.

Plasmacytoid dendritic cells (pDCs) are unique bone-marrow-derived cells that produce large amounts of type I interferon in response to microbial stimulation. Furthermore, pDCs also promote T cell tolerance in sterile-inflammation conditions. However, the immunomodulatory role of aortic pDCs in atherosclerosis has been poorly understood. Here, we identified functional mouse and human pDCs in the aortic intima and showed that selective, inducible pDC depletion in mice exacerbates atherosclerosis. Aortic pDCs expressed CCR9 and indoleamine 2,3-dioxygenase 1 (IDO-1), an enzyme involved in driving the generation of regulatory T cells (Tregs). As a consequence, loss of pDCs resulted in decreased numbers of Tregs and reduced IL-10 levels in the aorta. Moreover, antigen presentation by pDCs expanded antigen-specific Tregs in the atherosclerotic aorta. Notably, Tregs ablation affected pDC homeostasis in diseased aorta. Accordingly, pDCs in human atherosclerotic aortas colocalized with Tregs. Collectively, we identified a mechanism of atheroprotection mediated by tolerogenic aortic pDCs.

As most infections by the helminth parasite elicit the recruitment of CD4(+)CD25(+)Foxp3(+) T (Treg) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated Treg cells, we compared the expression levels of Treg cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of Treg cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated Treg cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of Treg cells in the muscle tissue.

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

AIMS : To investigate whether Imiquimod (IMQ) as TLR7 ligand protects mice from colonic inflammation and the mechanisms underlying in such immunoregulatory conditions. METHODS : Murine colitis was induced to Balb/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ. Colitis was evaluated by body weight decreases and by histological score. Also colonic mRNA expression was measured by RT-PCR. To confirm the induction of Regulatory T cells (Tregs) by type-1 IFN from pDCs, we generated mouse bone marrow-derived pDCs and co-cultured these with CD4(+) T cells isolated from mouse spleen with or without IMQ stimulation. Cytokine production in the culture supernatant was measured by ELISA and the number of Tregs were analyzed by flow cytometry. Spleen and mesenteric lymph nodes (MLN) from IMQ-treated mice were collected, and mRNA expressions of cytokine were measured by RT-PCR and cytokine productions were measured by ELISA. Tregs and chemokine expressions were analyzed in colon of TNBS-induced colitis mouse by immunohistochemistry. RESULTS : Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis. In the colons of IMQ-treated mice, mRNA expression of TNF-α was decreased, and strong expressions of IL-6, IFN-β and TGF-β were detected. IL-10 and TGF-β productions were increased in the supernatant of co-cultured cells stimulated with IMQ, although we were unable to detect Treg differentiaton in IMQ-stimulated co-cultured cells. In MLN of IMQ-treated mice, strong expressions of TLR7, IFN-β, TGF-β and Foxp3 mRNA were detected. IL-10 production from MLN cells was also increased in the IMQ-treated group. Finally, Tregs in the inflamed colon and CCR9 in MLN of IMQ-treated mice were detected. CONCLUSION : These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9, which regulates accumulation of Tregs in the inflamed colon.

Airway inflammation is the most common hallmark of allergic asthma. Chemokine receptors involved in leukocyte recruitment are closely related to the pathology in asthma. CCR9 has been described as a homeostatic and inflammatory chemokine receptor, but its role and that of its ligand CCL25 during lung inflammation remain unknown. To investigate the role of CCR9 as a modulator of airway inflammation, we established an OVA-induced allergic inflammation model in CCR9-deficient mice. Here, we report the expression of CCR9 and CCL25 as early as 6 hours post-OVA challenge in eosinophils and T-lymphocytes. Moreover, in challenged CCR9-deficient mice, cell recruitment was impaired at peribronchial and perivenular levels. OVA-administration in CCR9-deficient mice leads to a less inflammatory cell recruitment, which modifies the expression of IL-10, CCL11, and CCL25 at 24 hours after OVA challenge. In contrast, the secretion of IL-4 and IL-5 was not affected in CCR9-deficient mice compared to WT mice. These results demonstrate for the first time that CCR9 and CCL25 expressions are induced in the early stages of airway inflammation and they have an important role modulating eosinophils and lymphocytes recruitment at the first stages of inflammatory process, suggesting that they might be a potential target to regulate inflammation in asthma.

Therapeutic outcome of breast cancer (BrCa) is impeded due to the intratumoral heterogeneity, as well as differences among cancer-bearing individuals. Triple-negative breast cancer (TNBC) is a highly aggressive subtype that disproportionally affects African-American (AA) women. However, the molecular basis of racial disparity in therapeutic response and clinical outcome of TNBC in AA compared to Caucasian American (CA) patients is still obscure. Our group was the first to show the involvement of CCR9 and its natural ligand, CCL25 in cancer progression and therapeutic response, including BrCa. In this study, using proteomic and bioinformatics approach we have quantified the race-specific differences in biologic pathways modulated by the CCR9/CCL25 in TNBC cells. The difference in signaling cascades following CCR9 activation by CCL25 in BrCa cell derived from AA and CA were determined using cancer signaling phospho-protein antibody microarray, which featured 269 antibodies. A heat map was generated to visualize differences in phosphorylation status among cell line derived from AA and CA patients, as well as differences between the cell lines after CCL25 treatment. Graphics of biologic functions and oncogenic signaling networks, which were altered by CCR9/CCL25 axis, were produced using GeneMANIA and Ingenuity Pathway Analysis (IPA) software. Cells derived from AA (MDA-MB-468), show activation/phosphorylation of GSK3α, Elk-1, p70S6K, BCL-2, MEK2, NFkB and STAT3 involved in cell survival and migration. However, cells derived from CA (MDA-MB-231) show activation/phosphorylation of VEGFR2, CTNNB1, FAK, and SHC, suggesting involvement of distinct pathway supporting cell survival following CCR9 activation. Hence, our data suggest that CCR9/CCL25 contributes to the race-specific difference in signaling pathways and these race-specific differences in the biology of TNBC could be the reasons for the disparity in disease and therapeutic outcome. Citation Format: Jeronay King Thomas, Neeraj Kapur, Hina Mir, Dominique N. Gales, James W. Lillard Jr., Shailesh Singh. Antibody microarray analysis of signaling networks regulated by the CCR9/CCL25 axis in African American and Caucasian American triple-negative breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C113.

Background The development of anti-TNFα immunotherapies has led to great advances in quality of life for Crohn’s and colitis patients and has allowed a reduction in steroid use for these diseases. However, a significant proportion of IBD patients is resistant to anti-TNFα agents, or becomes resistant to such treatments over time. Preclinical research and clinical trials have suggested that CCR9 antagonism can effectively reduce colon inflammation. CCR9 is expressed on a subset of T cells dedicated to monitoring the immune status of the intestine. When activated by its only known chemokine ligand, CCL25/TECK, CCR9 triggers the adhesion of gut-specific T cells to vascular endothelium and the subsequent ingress of these cells to intestinal tissues. Antagonism of this receptor inhibits the accumulation of T cells within inflamed intestinal epithelium and lamina propria. Since anti-TNFα and CCR9 antagonists act through entirely different mechanisms, we asked whether these two approaches might synergise and expand the effectiveness and scope of each therapy. Methods We used a piroxicam-accelerated version of the MDR1a−/− spontaneous colitis model in mice. In this model, 7week MDR1a−/− mice (or WT FVB controls) receive piroxicam in powdered chow for 10 days. Mice are monitored both during and after the piroxicam feeding period, for a total of 21 days. Treatment groups (eight mice each) include an antibody (a saturating dose of either anti-TNFα or its isotype-matched control) and a CCR9 antagonist (or its vehicle alone as a control). Body weight and diarrhoea score were recorded throughout the entire 21 days period, after which colons were obtained from sacrificed animals for weighing, length measurement and imaging. Results We have found that combined dosing of piroxicam-fed MDR1a−/− mice with a CCR9 antagonist and an anti-TNFα MAb significantly reverses loss of body weight, diarrhoea score and colon inflammation. The combined dosing was more effective than anti-TNFα or CCR9 antagonist alone, as measured by colon length/weight ratio, neutrophil accumulation in the colonic epithelium and reversal of the loss in body weight characteristic of this model. Conclusion These data suggest that combining a CCR9 antagonist with existing anti-TNFα treatment regimens may extend the benefits of anti-TNFα immunotherapy to a larger class of patients suffering from IBD and may prolong the effectiveness of anti-TNFα immunotherapy for those patients already undergoing such treatments.

Las células T efectoras (CD4+ y CD8+) y de memoria pueden migrar a la mayoría de los tejidos extra-linfoides en el cuerpo, pero requieren la expresión de receptores de alojamiento (homing) específicos en las células T, la integrina A4B7 y el receptor de quimiocina CCR9. Los linfocitos circulantes CD4+ o CD8+ carecen de receptores CCR9 y A4B7; sin embargo, expresan este tipo de receptores al alcanzar la mucosa intestinal. El objetivo del estudio fue determinar el efecto del ácido retinoico sobre la expresión de los receptores CCR9 y la integrina A4B7análisis in vitro de muestra de sangre circulante de alpaca. Los leucocitos obtenidos se colocaron en placas de cultivo celular en medio esencial mínimo (MEM) suplementados e inoculados con ácido retinoico a concentraciones de 10, 50 y 100 ug/ml disuelto en DMSO. Se empleó un control a base de suero fisiológico. Las placas de cultivo celular fueron incubadas por 12, 24 y 48 h a 37 ºC con CO2 al 5%. Se realizó las PCR en Tiempo Real de los ARNm del receptor CCR9 y la integrina A4B7 y la cuantificación relativa con el método 2-ÄÄCt. Las máximas expresiones para CCR9 se dieron a las 48 h para las concentraciones de 10 y 100 ug/ml de ácido retinoico y DMSO, y a las 24 h para la concentración de 50 ug/ml. El control presentó expresiones mucho más elevadas en comparación con las dosis de ácido retinoico y DMSO. No hubo expresión para la integrina A4B7., Effector T cells (CD4+ and CD8+) and memory cells can migrate to most extra lymphoid tissues in the body, but require the expression of specific homing receptors in T cells, α4β7 integrin and receptor chemokine CCR9. CD4+ or CD8+ circulating lymphocytes lack CCR9 and α4β7 receptors; however, these receptors are expressed upon reaching the intestinal mucosa. The aim of the study was to determine the effect of retinoic acid on the expression of CCR9 receptors and α4β7 integrin on alpaca lymphocytes (Vicugna pacos). An in vitro analysis of circulating alpaca blood sample was performed. The obtained leukocytes were placed in cell culture plates in minimal essential medium (MEM) supplemented with retinoic acid at concentrations of 10, 50 and 100 ìg/ml dissolved in DMSO. A control based on physiological serum was used. Cell culture plates were incubated for 12, 24 and 48 h at 37 °C with 5% CO2. Real-time PCR of the CCR9 receptor mRNA and α4β7 integrin and relative quantification with the 2-ΔΔCt method were performed. The maximum expressions for CCR9 were given at 48 h for concentrations of 10 and 100 μg/ml of retinoic acid and DMSO, and at 24 h for the concentration of 50 μg/ml. The control had much higher expressions compared to the doses of retinoic acid and DMSO. There was no expression for the α4β7 integrin.

Highlights • ATRA increased vaccine specific IgA in gut secretions to Vivotif but not Dukoral or Rotarix. • ATRA increased α4β7 and CCR9 gut marker expression in a coordinated manner only when given simultaneously with Vivotif vaccine. • In individuals with coordinated gut marker expression Vivotif specific IgA increase was much stronger., All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4β7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4β7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/β signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila . Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science , this issue p. 91 , p. 104 , p. 97 ; see also p. 32

Las células T efectoras (CD4+ y CD8+) y de memoria pueden migrar a la mayoría de los tejidos extra-linfoides en el cuerpo, pero requieren la expresión de receptores de alojamiento (homing) específicos en las células T, la integrina α4β7 y el receptor de quimiocina CCR9. Los linfocitos circulantes CD4+ o CD8+ carecen de receptores CCR9 y α4β7; sin embargo, expresan este tipo de receptores al alcanzar la mucosa intestinal. El objetivo del estudio fue determinar el efecto del ácido retinoico sobre la expresión de los receptores CCR9 y la integrina α4β7 en linfocitos sanguíneos de alpacas (Vicugna pacos). Se realizó un análisis in vitro de muestra de sangre circulante de alpaca. Los leucocitos obtenidos se colocaron en placas de cultivo celular en medio esencial mínimo (MEM) suplementados e inoculados con ácido retinoico a concentraciones de 10, 50 y 100 ìg/ml disuelto en DMSO. Se empleó un control a base de suero fisiológico. Las placas de cultivo celular fueron incubadas por 12, 24 y 48 h a 37 ºC con CO2 al 5%. Se realizó las PCR en Tiempo Real de losARNm del receptor CCR9 y la integrina α4β7 yla cuantificación relativa con el método 2-ΔΔCt. Las máximas expresiones para CCR9 se dieron a las 48 h para las concentraciones de 10 y 100 µg/ml de ácido retinoico y DMSO, y a las 24 h para la concentración de 50 µg/ml. El control presentó expresiones mucho más elevadas en comparación con las dosis de ácido retinoico y DMSO. No hubo expresión para la integrina α4β7. Effector T cells (CD4+ and CD8+) and memorycells can migrate to most extra lymphoid tissues in the body, but require the expression of specific homing receptors in T cells, α4β7 integrin and receptor chemokine CCR9. CD4+ or CD8+ circulating lymphocytes lack CCR9 and α4β7 receptors; however, these receptors are expressed upon reaching the intestinal mucosa. The aim of the study was to determine the effect of retinoic acid on the expression of CCR9 receptors and α4β7 integrin on alpaca lymphocytes (Vicugna pacos). An in vitro analysis of circulating alpaca blood sample was performed. The obtained leukocytes were placed in cell culture plates in minimal essential medium (MEM) supplemented with retinoic acid at concentrations of 10, 50 and 100 ìg/ml dissolved in DMSO. A control based on physiological serum was used. Cell culture plates were incubated for 12, 24 and 48 h at 37 °C with 5% CO2. Real-time PCR of the CCR9 receptor mRNA and α4β7 integrin and relative quantification with the 2-ΔΔCt method were performed. The maximum expressions for CCR9 were given at 48 h for concentrations of 10 and 100 µg/ml of retinoic acid and DMSO, and at 24 h for the concentration of 50 µg/ml. The control had much higher expressions compared to the doses of retinoic acid and DMSO. There was no expression for the α4β7 integrin.

Background α4β7 integrin is expressed on T-cells which migrate to gut-associated lymphoid tissues. Expression of α4β7 integrin is elevated on memory T-cells in adult hematopoietic stem cell transplant (HSCT) recipients at diagnosis of GI GVHD. No pediatric data exist regarding α4β7 integrin expression before or at onset of GVHD. We hypothesized that α4β7 integrin expression on effector memory T-cells (TEMs) would be elevated in patients before acute GI GVHD symptoms compared to patients without GVHD and may be a useful therapeutic target for the prevention of acute GI GVHD. Methods Cryopreserved T-cells were characterized in 48 children who developed acute GI GVHD (n=22), isolated skin GVHD (n=12) and no GVHD (n=14). In patients with GVHD, T-cells were characterized prior to HSCT, one week before GVHD and at GVHD diagnosis. In patients without GVHD, samples were analyzed at baseline and day+30 after HSCT. Samples were incubated with fluorochrome conjugated monoclonal antibodies directed against CD3, CD8, CCR7, α4β7 integrin and CD45RA. TEMs were defined as CD3+, CCR7-, CD45RA- lymphocytes. Co-expression of trafficking markers implicated in GI GVHD (CCR5, CCR9), and liver GVHD (CXCR6 and CCR5) were also assessed. Samples were analyzed on an LSR Fortessa flow cytometer (BD Biosciences). Data were analyzed using FCS Express (De Novo Software). Surface expression of all markers were compared using the Mann Whitney U-test. Patients with skin GVHD and no GVHD were grouped for analysis. Patients with at least 200 absolute CD8+ T-cell events were deemed evaluable. Results Twenty patients with GI GVHD, 8 with skin GVHD and 10 patients without GVHD had at least 200 absolute CD8+ events and were evaluable. Demographics of patients are shown in Figure 1. Expression of α4β7 integrin on CD8+ TEMs was upregulated in patients with GI GVHD compared to the No GI GVHD (skin GVHD + no GVHD) group one week prior to clinical symptoms (p=0.02; Figure 2A). Co-expression of CCR5 and CCR9 with α4β7 integrin was also higher on CD8+ TEM cells in the GI GVHD group compared to No GI GVHD group (p Conclusion α4β7 integrin is elevated on CD8+ TEMs prior to acute GI GVHD symptoms and co-expression of intestinal trafficking-specific markers such as CCR5 and CCR9 are also elevated prior to acute GVHD in pediatric patients. Our data suggest a rationale for the blockade of α4β7 integrin or the dual blockade of α4β7 integrin and CCR5 with clinically available therapeutics (maraviroc and vedolizumab) for acute GI GVHD prevention in pediatric allogeneic HCT patients.

Infliximab is an anti-tumor necrosis factor (TNF)-α antibody drug that suppresses TNF-α and its associated inflammatory responses. Although infliximab therapy generally results in a 75% or greater improvement in the Psoriasis Area and Severity Index from baseline in psoriasis patients, there is the heterogeneity of therapeutic efficacy in psoriasis patients among patients of a similar PASI baseline score. However, there are few published reports about the predictors of the clinical response among psoriasis patients who undergo biologic therapies. We thus evaluated the possible existence of biologic markers that would indicate poor prognosis of infliximab using skin biopsy specimens. This was because we assumed that the inhibitors for upregulated chemokine/chemokine receptors in non-responders may have the ability to reduce the occurrence of psoriatic eruptions. PCR array analyses identified that the levels of various chemokines and chemokine receptors were increased in non-responders in comparison to responders. Immunohistochemical analyses revealed that upregulation of the CCR9 protein levels was not associated with the pretherapeutic PASI score, but with poor response to infliximab. Our results indicated that the expression levels of CCR9 in lesional skin may be a useful biologic marker of the clinical efficacy of infliximab therapy in psoriasis patients.

Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, affects millions of people worldwide. CCR9 has been shown to be a key chemokine receptor mediating the local inflammatory responses in the GI tract. The CCR9 inhibitor Vercirnon advanced to phase 3 clinical trials, but carries several liabilities which we sought to improve.

Lactoferrin (LF) and retinoic acid (RA) are enriched in colostrum, milk, and mucosal tissues. We recently showed that LF-induced IgA class switching through binding to betaglycan (transforming growth factor-beta receptor III, TβRIII) and activation of canonical TGF-β signaling. We investigated the combined effect of LF and RA on the overall IgA response. An increase in IgA production by LF was further augmented by RA. This combination effect was also evident in Ig germ-line α (GLα) transcription and GLα promoter activity, indicating that LF in cooperation with RA increased IgA isotype switching. We subsequently found that RA enhanced TβRIII expression and that this increase contributed to LF-stimulated IgA production. In addition to the IgA response, LF and RA in combination also enhanced the expression of the gut-homing molecules C-C chemokine receptor 9 (CCR9) and α4β7 on B cells. Finally, peroral administration of LF and RA enhanced the frequency of CCR9+IgA+ plasma cells in the lamina propria. Taken together, these results suggest that LF in cooperation with RA can contribute to the establishment of gut IgA responses.

Current orthotopic xenograft models of human colorectal cancer (CRC) require surgery and do not robustly form metastases in the liver, the most common site clinically. CCR9 traffics lymphocytes to intestine and colorectum. We engineered use of the chemokine receptor CCR9 in CRC cell lines and patient-derived cells to create primary gastrointestinal (GI) tumors in immunodeficient mice by tail-vein injection rather than surgery. The tumors metastasize inducibly and robustly to the liver. Metastases have higher DKK4 and NOTCH signaling levels and are more chemoresistant than paired subcutaneous xenografts. Using this approach, we generated 17 chemokine-targeted mouse models (CTMMs) that recapitulate the majority of common human somatic CRC mutations. We also show that primary tumors can be modeled in immunocompetent mice by microinjecting CCR9-expressing cancer cell lines into early-stage mouse blastocysts, which induces central immune tolerance. We expect that CTMMs will facilitate investigation of the biology of CRC metastasis and drug screening.

Intestinal homeostasis plays an important role in bacteria-derived complications in cirrhosis. Intestinal lymphocytes are responsible for immune effector functions and can be modulated by certain probiotics. We evaluate the interaction between Bifidobacterium pseudocatenulatum CECT7765 and intestinal lymphocytes in mice with cirrhosis. Cirrhosis was induced by intragastrical administration of carbon tetrachloride in Balb/C mice. One week prior to laparotomy, animals received B. pseudocatenulatum CECT7765 (107, 109 or 1010 cfu/daily) or placebo. Chemokine receptor and cytokine expression were evaluated in intestinal lymphocytes. Gut permeability was studied by FITC-LPS recovery in vivo. Luminal antigens, inflammation and functional markers were evaluated in liver samples. Bifidobacterium pseudocatenulatum CECT7765 decreased the expression of pro-inflammatory chemokine receptors CCR6, CCR9, CXCR3 and CXCR6 in intestinal lymphocytes from cirrhotic mice in a concentration-dependent manner. The bifidobacterial strain induced a shift towards an anti-inflammatory cytokine profile in this cell subset. B. pseudocatenulatum CECT7765-induced inflammatory modulation was TLR2-mediated, as in vitro TLR2 blockade inhibited the reduction of TNF-alpha and its receptors and the increase of IL-10 and IL-10 receptor secretion. The recovery rate of administered fluorescence-labelled endotoxin was significantly and dose-dependently lowered with the bifidobacterial strain. The reduced intestinal permeability was associated with a decreased burden of bacterial antigens in the liver of mice treated with B. pseudocatenulatum CECT7765. Liver function and inflammation were improved with the use of the bifidobacterial strain at the highest dose tested (1010 cfu). Bifidobacterium pseudocatenulatum CECT7765 improves gut homeostasis and prevents gut-derived complications in experimental chronic liver disease.

CC chemokine receptor 9 (CCR9) serves a role in the drug resistance and metastasis of tumors. In the present study, a peptide specifically bound to CCR9 was obtained and the effect on tumor cells was observed. A Ph.D.-12 phage display peptide library was used to screen for peptides binding specifically to the second extracellular loop of CCR9. The ratios of the input and output of phage clones increased gradually following three rounds of biopanning. A total of 8 positive phage clones were identified from DNA analysis. A phage clone, C-4, was identified which exhibited higher affinity and specificity for the second extracellular loop of CCR9 in vitro compared with other clones. A peptide (P1; VHWDFRQWWQPS) was identified which may inhibit the corresponding phage, C-4, binding to the second extracellular loop of CCR9. Furthermore, P1 was able to bind specifically with MOLT4 cells which exhibit marked expression of CCR9. In addition, P1 promoted the apoptosis of MOLT4 cells induced by doxorubicin, and inhibited the migration of MOLT4 cells in the presence of chemokine (C-C motif) ligand 25. It was suggested that decreased activity in the phosphorylation of protein kinase B in MOLT4 cells may be responsible for the inhibition. In conclusion, the peptide P1 derived from a screened phage is able to specifically bind to CCR9 and inhibit the activity of CCR9. It has potential use as an antagonist in the treatment of CCR9-overexpressed carcinoma.

This review focuses on the construction and application of structural chemokine receptor models for the elucidation of molecular determinants of chemokine receptor modulation and the structure-based discovery and design of chemokine receptor ligands. A comparative analysis of ligand binding pockets in chemokine receptors is presented, including a detailed description of the CXCR4, CCR2, CCR5, CCR9, and US28 X-ray structures, and their implication for modeling molecular interactions of chemokine receptors with small-molecule ligands, peptide ligands, and large antibodies and chemokines. These studies demonstrate how the integration of new structural information on chemokine receptors with extensive structure-activity relationship and site-directed mutagenesis data facilitates the prediction of the structure of chemokine receptor-ligand complexes that have not been crystallized. Finally, a review of structure-based ligand discovery and design studies based on chemokine receptor crystal structures and homology models illustrates the possibilities and challenges to find novel ligands for chemokine receptors.

Poor clinical outcome of lung cancer (LuCa) is primarily due to lack of knowledge about specific molecules involved in its progression and metastasis. In this study, we for the first time show the clinical and biological significance of CC chemokine receptor-9 (CCR9) in non-small cell lung cancer (NSCLC). Expression of CCR9 and CCL25, the only natural ligand of CCR9, was significantly higher (p < 0.0001) in NSCLC tissues and serum respectively, compared to their respective controls. Interestingly, expression of both CCR9 and CCL25 was significantly higher in adenocarcinomas (ACs) compared to squamous cell carcinomas (SCCs) (p = 0.04, and p < 0.0001). Similar to tissues, AC and SCC cell lines were positive for CCR9 expression. Despite of marginal difference in CCR9 expression, AC cells showed higher migratory and invasive potential in response to CCL25, compared to SCC cells. This differential biological response of AC cells was primarily due to differential expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases under the influence of CCL25. Our results suggest CCR9 as a potential target for developing new treatment modality for NSCLC. Additionally, differential serum CCL25 level in ACs and SCCs, two NSCLC subtypes, suggest its potential as a non-invasive diagnostic/prognostic biomarker.