CXCR4 Ligand CXCL12 Research Articles

Cytotoxic T cells drive doxorubicin-induced cardiac fibrosis and systolic dysfunction.

Doxorubicin, the most prescribed chemotherapeutic drug, causes dose-dependent cardiotoxicity and heart failure. However, our understanding of the immune response elicited by doxorubicin is limited. Here we show that an aberrant CD8+ T cell immune response following doxorubicin-induced cardiac injury drives adverse remodeling and cardiomyopathy. Doxorubicin treatment in non-tumor-bearing mice increased circulating and cardiac IFNγ+CD8+ T cells and activated effector CD8+ T cells in lymphoid tissues. Moreover, doxorubicin promoted cardiac CD8+ T cell infiltration and depletion of CD8+ T cells in doxorubicin-treated mice decreased cardiac fibrosis and improved systolic function. Doxorubicin treatment induced ICAM-1 expression by cardiac fibroblasts resulting in enhanced CD8+ T cell adhesion and transformation, contact-dependent CD8+ degranulation and release of granzyme B. Canine lymphoma patients and human patients with hematopoietic malignancies showed increased circulating CD8+ T cells after doxorubicin treatment. In human cancer patients, T cells expressed IFNγ and CXCR3, and plasma levels of the CXCR3 ligands CXCL9 and CXCL10 correlated with decreased systolic function.

Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans.

Chemokine-driven leukocyte recruitment is a key component of the immune response and of various diseases. Therapeutically targeting the chemokine system in inflammatory disease has been unsuccessful, which has been attributed to redundancy. We investigated why chemokines instead have specific, specialized functions, as demonstrated by multiple studies. We analyzed the expression of genes encoding chemokines and their receptors across species, tissues, and diseases. This analysis revealed complex expression patterns such that genes encoding multiple chemokines that mediated recruitment of the same leukocyte type were expressed in the same context, such as the genes encoding the CXCR3 ligands CXCL9, CXCL10, and CXCL11. Through biophysical approaches, we showed that these chemokines differentially interacted with extracellular matrix glycosaminoglycans (ECM GAGs), which was enhanced by sulfation of specific GAGs. Last, in vivo approaches demonstrated that GAG binding was critical for the CXCL9-dependent recruitment of specific T cell subsets but not of others, irrespective of CXCR3 expression. Our data demonstrate that interactions with ECM GAGs regulated whether chemokines were presented on cell surfaces or remained more soluble, thereby affecting chemokine availability and ensuring specificity of chemokine action. Our findings provide a mechanistic understanding of chemokine-mediated immune cell recruitment and identify strategies to target specific chemokines during inflammatory disease.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses invitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

XCR1+ conventional dendritic cell-induced CD4+ T helper 1 cell activation exacerbates cardiac remodeling after ischemic myocardial injury

Cardiac remodeling has no established therapies targeting inflammation. CD4+ T-cell subsets have been reported to play significant roles in healing process after ischemic myocardial injury, but their detailed mechanisms of activation remain unknown. To explore immune reactions during cardiac remodeling, we applied a non-surgical model of coronary heart disease (CHD) induced by a high-fat diet (HFD-CHD) in SR-BI−/−/ApoeR61h/h mice. Flow cytometry analyses throughout the period of progressive cardiac dysfunction revealed that CD4+ T Helper 1 (Th1) cells were predominantly activated in T-cell subsets. Probucol was reported to attenuate cardiac dysfunction after coronary artery ligation model (ligation-MI) in rats. To determine whether probucol suppress cardiac remodeling after HFD-CHD, we treated SR-BI−/−/ApoeR61h/h mice with probucol. We found treatment with probucol in HFD-CHD mice reduced cardiac dysfunction, with attenuated activation of Th1 cells. RNA-seq analyses revealed that probucol suppressed the expression of CXCR3, a Th1-related chemokine receptor, in the heart. XCR1+ cDC1 cells, which highly expresses the CXCR3 ligands CXCL9 and CXCL10, were predominantly activated after HFD-CHD. XCR1+ cDC1 lineage skewing of pre-DC progenitors was observed in bone marrow, with subsequent systemic expansion of XCR1+ cDC1 cells after HFD-CHD. Activation of CXCR3+ Th1 cell and XCR1+ cDC1 cells was also observed in ligation-MI. Notably, post-MI depletion of XCR1+ cDC1 cells suppressed CXCR3+ Th1 cell activation and prevented cardiac dysfunction. In patient autopsy samples, CXCR3+ Th1 and XCR1+ cDC1 cells infiltrated the infarcted area. In this study, we identified a critical role of XCR1+ cDC1-activated CXCR3+ Th1 cells in ischemic cardiac remodeling.

CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.

Abstract B034: CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

Characterization of a Novel Missense CXCR4 Mutation in a Patient with WHIM-like Syndrome

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare primary immunodeficiency with a heterogeneous presentation of symptoms defining its acronym as well as panleukopenia. The majority of cases are inherited in an autosomal dominant manner, with gain-of-function mutations in the C-terminus of the C-X-C chemokine receptor 4 (CXCR4) (McDermott D, et al. Immunol Rev. 2019;287:91-102). To our knowledge, there are no literature reports implicating CXCR4WHIM mutations outside the CXCR4 C-terminus. Here we report the clinical presentation of a patient with the novel mutation CXCR4D84H and characterize the functional effects of the mutation on CXCR4 trafficking and chemotaxis in in vitro and ex vivo assays. Further, CXCR4D84H is reported in 3 population genetic databases (average allele frequency 3.8 × 10 -5) and is also found in 5 unrelated patients in another rare disease database (CentoMD).C ase Report: A 40-year-old female presented with a history of recurrent vulvovaginal and anal dysplasia and carcinoma in situ requiring multiple surgeries starting at age 20 years. Cytopenia was first documented at age 15 years in the context of mononucleosis, which resolved, although cytopenias persisted through adulthood. To date, the patient has decreased absolute neutrophil counts (600-1100/mm 3 range), WBC counts ~2000/mm 3, and normal immunoglobulin levels. Bone marrow biopsy revealed bilobed neutrophils and granulocyte precursors. The patient has no history of recurrent infections besides HPV/EBV and no family history of warts, immunodeficiency, or squamous cell carcinoma. Genetic testing revealed a heterozygous mutation in CXCR4 (c.250G>C, D84H) that has not been previously reported. This missense mutation occurs in the transmembrane domain of CXCR4 in proximity of the residues involved in signal initiation, and hence, may alter signaling responses.Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from this patient and 2 healthy controls and analyzed for functionality ex vivo including CXCR4 internalization assays and chemotaxis to exogenously added CXCR4-ligand CXCL12. The CXCR4-negative K562 cell line was transiently transfected to express CXCR4D84H, CXCR4WT, and known CXCR4WHIM mutations for further comparison of their functional responses to CXCL12.Results: Flow cytometry data shows abnormal peripheral leukocyte frequency and count. Lymphocyte subpopulations showed 33%-57% reduced CXCR4 internalization in response to CXCL12 compared to controls. Chemotactic response to CXCL12 was increased in PBMC subsets including natural killer T (NKT) cells compared to healthy donor cells. These observations are comparable to previously published responses to pathogenic CXCR4WHIM variants, but, chemotactic responses of NKT cells is a novel finding. In K562-transfected cells, CXCR4D84H demonstrated impaired CXCL12-induced internalization compared to CXCR4WT at a level comparable to the CXCR4E343K variant, the only CXCR4 missense variant known to date to cause WHIM syndrome. Chemotaxis of cells expressing CXCR4D84H in response to CXCL12 is increased by 1.25- to 2.4-fold compared to CXCR4WTdepending on CXCL12 concentration, in line with the observation of increased chemotaxis of cells expressing known CXCR4WHIM variants. Overall, the CXCR4D84H mutation recapitulates the phenotypes exhibited by known CXCR4WHIM variants in both internalization and chemotaxis assays.Conclusions: We show the functional effects of a novel missense mutation CXCR4D84H found in a patient with clinical WHIM syndrome recapitulates the internalization and chemotaxis findings of other CXCR4WHIM mutations. This is the first report of a missense mutation in CXCR4 outside of the C-terminus causing a WHIM phenotype, and additionally highlights defective chemotaxis in NKT cells, which may be relevant in this patient's HPV-associated carcinoma in situ phenotype. Based on an analysis of population databases, and assuming a conservative 5-10% penetrance, there are potentially ~1250-2500 individuals in the United States with disease due to the p.D84H variant alone, highlighting the under-recognition of WHIM syndrome. Patients who have similar clinical presentation as described in this case study should be evaluated for WHIM syndrome and other primary immunodeficiencies by using genetic screening for CXCR4 and diagnostic assays described. DisclosuresPawar: X4 Pharmaceuticals: Current Employment. Zmajkovicova: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Wiest: X4 Pharmaceuticals: Current Employment. Nguyen: X4 Pharmaceuticals: Current Employment. Monticelli: X4 Pharmaceuticals: Current Employment. Maier-Munsa: X4 Pharmaceuticals: Current Employment. Sondheimer: X4 Pharmaceuticals: Consultancy. Walter: Octapharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmig: Consultancy, Membership on an entity's Board of Directors or advisory committees. Taveras: X4 Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Badarau: X4 Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Tarrant: ThermoFisher Scientific: Consultancy; X4 Pharmaceuticals: Consultancy, Research Funding; Department of Justice: Consultancy; Abbvie: Research Funding; Viela Bio: Research Funding.

Imperfect storm: is interleukin-33 the Achilles heel of COVID-19?

The unique cytokine signature of COVID-19 might provide clues to disease mechanisms and possible future therapies. Here, we propose a pathogenic model in which the alarmin cytokine, interleukin (IL)-33, is a key player in driving all stages of COVID-19 disease (ie, asymptomatic, mild–moderate, severe–critical, and chronic–fibrotic). In susceptible individuals, IL-33 release by damaged lower respiratory cells might induce dysregulated GATA-binding factor 3-expressing regulatory T cells, thereby breaking immune tolerance and eliciting severe acute respiratory syndrome coronavirus 2-induced autoinflammatory lung disease. Such disease might be initially sustained by IL-33-differentiated type-2 innate lymphoid cells and locally expanded γδ T cells. In severe COVID-19 cases, the IL-33–ST2 axis might act to expand the number of pathogenic granulocyte–macrophage colony-stimulating factor-expressing T cells, dampen antiviral interferon responses, elicit hyperinflammation, and favour thromboses. In patients who survive severe COVID-19, IL-33 might drive pulmonary fibrosis by inducing myofibroblasts and epithelial–mesenchymal transition. We discuss the therapeutic implications of these hypothetical pathways, including use of therapies that target IL-33 (eg, anti-ST2), T helper 17-like γδ T cells, immune cell homing, and cytokine balance.

CXCR4 Signaling Has a CXCL12-Independent Essential Role in Murine MLL-AF9-Driven Acute Myeloid Leukemia

SUMMARYAcute myeloid leukemia (AML) is defined by an accumulation of immature myeloid blasts in the bone marrow. To identify key dependencies of AML stem cells in vivo, here we use a CRISPR-Cas9 screen targeting cell surface genes in a syngeneic MLL-AF9 AML mouse model and show that CXCR4 is a top cell surface regulator of AML cell growth and survival. Deletion of Cxcr4 in AML cells eradicates leukemia cells in vivo without impairing their homing to the bone marrow. In contrast, the CXCR4 ligand CXCL12 is dispensable for leukemia development in recipient mice. Moreover, expression of mutated Cxcr4 variants reveals that CXCR4 signaling is essential for leukemia cells. Notably, loss of CXCR4 signaling in leukemia cells leads to oxidative stress and differentiation in vivo. Taken together, our results identify CXCR4 signaling as essential for AML stem cells by protecting them from differentiation independent of CXCL12 stimulation.

Analysis of associations of CXCR3 ligands with immune microenvironment and aggressiveness in murine and human pancreatic ductal adenocarcinoma.

762 Background: The complex milieu of cytokines within pancreatic ductal adenocarcinoma (PDAC) promotes tumor progression and immune suppression thereby contributing to the dismal prognosis of patients with PDAC. However, the roles of many cytokines, including CXCR3 ligands, in PDAC have not been thoroughly investigated. Methods: Bioinformatics analyses of PDAC microarray and TCGA datasets were used to identify cytokines overexpressed in PDAC, their association with patient survival as well as the expression of cognate cytokine receptors. Comparative analysis of cytokine expression in KrasLSL-G12D-P53LSL-R172H-Pdx1-Cre (KPC) and KrasLSL-G12D-Pdx1-Cre (KC) murine PDAC models were used to validate these findings. Pathway and CIBERSORT analyses were employed to determine mechanistic basis of altered survival associated with cytokines of interest. Results: Of the 149 cytokines analyzed, CXCR3 ligands CXCL9 and CXCL10 were highly and consistently overexpressed in PDAC datasets. Concurrently, CXCL9, CXCL10 and PF4 were overexpressed in the aggressive KPC murine model compared to the indolent KC model. CXCR3 showed robust expression in PDAC in microarray, TCGA and IHC analyses. Interestingly, high expression of CXCR3 ligands was associated with shorter overall survival (p = 0.04 for CXCL9, 10 and 11 and p = 0.02 for PF4) while high expression of CXCR3 was associated with increased overall survival (p = 0.03). Pathway analysis of genes correlated with CXCR3 and/or its ligands showed that CXCR3 ligands may promote T-cell exhaustion (p < 0.001). Finally, CIBERSORT analysis of TCGA data demonstrated that high CXCR3 expression was associated with increased CD8 T-cell and naïve B-cell signatures and loss of plasma cells signatures. High CXCR3 ligand expression was associated with increased CD8 T-cell, and M1 macrophage, and loss of NK-cell signatures(p < 0.05). Conclusions: CXCR3 ligands are overexpressed in PDAC and are associated with poor survival, likely related to alterations in tumor immune infiltrate/activity and may represent targets to augment anti-tumor immunity.

BATF3-dependent dendritic cells drive both effector and regulatory T-cell responses in bacterially infected tissues

The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion.

Plasmodium chabaudi AS Infection Induces CD4+ Th1 Cells and Foxp3+T-bet+ Regulatory T Cells That Express CXCR3 and Migrate to CXCR3 Ligands.

Control and elimination of blood-stage Plasmodium chabaudi AS infection requires CD4+ Th1 cells that secrete IFN-γ and T follicular help (Tfh) cells together with B cell production of antibody. Foxp3+ regulatory T cells (Tregs) are also crucial to protect the host from immunopathology and severe disease, but these cells can suppress protective immune responses to malaria. The chemokine receptor CXCR3 expressed by activated T cells is important for trafficking of CD4+ Th1 cells to sites of inflammation and infection. Previous studies demonstrated CXCR3 is expressed on CD4+ T cells in the spleen during malaria, but the phenotype was not defined. We identified the phenotype of CD4+ T cells that expressed CXCR3 in C57BL/6 (B6) mice during acute P. chabaudi AS infection by analyzing expression of the transcription factors T-bet and Foxp3. We also investigated if CXCR3 contributes to control of parasite replication and survival. The frequency and number of CD4+CXCR3+ T cells increased dramatically in the spleen of infected B6 mice coincident with increased CD4+IFN-γ+ T cells. CXCR3 was up-regulated on effector CD4+Foxp3− T cells as well as Foxp3+ Tregs. Consistent with our previous observations, CD4+T-bet+Foxp3− T cells increased in B6 mice during acute infection. T-bet+Foxp3+ Tregs also increased significantly and a high frequency of these cells expressed CXCR3 supporting the notion that these cells may be Th1-like Tregs. Despite this, the percentage of CD4+Foxp3+ Tregs from infected B6 mice that migrated in vitro to the CXCR3 ligands CXCL9 and CXCL10 was significantly less than naïve mice. To investigate the in vivo contribution of CXCR3 to control of acute blood-stage malaria, we compared the course and outcome of P. chabaudi AS infection in wild-type (WT) B6 and CXCR3-deficient mice. Parasitemia levels were significantly higher around the time of peak parasitemia in CXCR3−/− compared to WT mice but survival was similar suggesting a role for CXCR3 in controlling parasite replication during acute P. chabaudi AS infection. Together, our findings indicate Th1-like CD4+T-bet+Foxp3+ Tregs that express CXCR3 are induced during acute blood-stage malaria and suggest CXCR3 expression on CD4+ Th1 cells may contribute to their migration to the spleen.

CCL21-expression and accumulation of CCR7+ NK cells in livers of patients with primary sclerosing cholangitis.

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7-ligand CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7+ NK cells in the inflamed liver tissue.

Herpes Simplex Virus Type 2 Infection-Induced Expression of CXCR3 Ligands Promotes CD4+ T Cell Migration and Is Regulated by the Viral Immediate-Early Protein ICP4.

HSV-2 infection-induced CXCR3 ligands are important for the recruitment of virus-specific CD8+ T cells, but their impact on CD4+ T cell trafficking remains to be further determined. Given that recruitment of CD4+ T cells to infection areas may be one of the mechanisms that account for HSV-2 infection-mediated enhancement of HIV-1 sexual transmission, here we investigated the functionality of HSV-2 infection-induced CXCR3 ligands CXCL9, CXCL10, and CXCL11 in vivo and in vitro, and determined the viral components responsive for such induction and the underlying mechanisms. We first found that the expression of CXCR3 ligands CXCL9, CXCL10, and CXCL11 was increased in mice following vaginal challenge with HSV-2, while CXCL9 played a predominant role in the recruitment of CD4+ T cells to the vaginal foci of infected mice. HSV-2 infection also induced the production of CXCL9, CXCL10, and CXCL11 in human cervical epithelial cells. Of note, although HSV-2 induced the expression of all the three CXCR3 ligands, the induced CXCL9 appeared to play a predominant role in promoting CD4+ T cell migration, reflecting that the concentrations of CXCL10 and CXCL11 required for CD4+ T cell migration are higher than that of CXCL9. We further revealed that, ICP4, an immediate-early protein of HSV-2, is crucial in promoting CXCR3 ligand expression through the activation of p38 MAPK pathway. Mechanistically, ICP4 binds to corresponding promoters of CXCR3 ligands via interacting with the TATA binding protein (TBP), resulting in the transcriptional activation of the corresponding promoters. Taken together, our study highlights HSV-2 ICP4 as a vital viral protein in promoting CXCR3 ligand expression and CXCL9 as the key induced chemokine in mediating CD4+ T cell migration. Findings in this study have shed light on HSV-2 induced leukocyte recruitment which may be important for understanding HSV-2 infection-enhanced HIV-1 sexual transmission and the development of intervention strategies.

Regulatory T cells control endothelial chemokine production and migration of T cells into intestinal tumors of APCmin/+ mice

Tumor-infiltrating lymphocytes are crucial for anti-tumor immunity. We have previously shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo. Treg depletion also resulted in increased levels of the chemokines CXCL9 and CXCL10 specifically in the tumors. In this study, we investigated the mechanisms for Treg mediated suppression of T-cell migration into intestinal tumors in the APCmin/+ mouse model. By breeding APCmin/+ mice with DEREG mice, which harbour a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, we were able to deplete Treg in tumor-bearing mice. Using adoptive transfer experiments, we could document a markedly increased migration of T cells specifically into Treg depleted tumors, and that Treg depletion results in increased production of the CXCR3 ligand CXCL10 from endothelial cells in the tumors. Furthermore, we were able to demonstrate that T cells use CXCR3 to migrate into intestinal tumors. In addition, human colon adenocarcinomas express high levels of mRNA CXCR3 ligands and tumor endothelial cells produce CXCL9 and CXCL10 ex vivo. In conclusion, this study demonstrates that Treg reduce endothelial CXCL10 production, inhibit T-cell migration into tumors and that CXCR3 mediated signalling is crucial for lymphocyte accumulation in intestinal tumors. Thus, immunotherapy aimed at Treg depletion may be effective by increasing not only T effector cell activity, but also their accumulation in tumors.

Bi‐directional Macrophage‐Fibroblast Crosstalk Directs Wound Resolution Factors

IntroductionDuring the wound repair process, fibroblasts secrete abundant extracellular matrix (ECM) proteins and participate in remodeling of the provisional ECM. Macrophages and other immune cells also play an important role in all stages of wound healing. Classically activated macrophages (CAMs/M1) contribute to inflammation early on, while alternatively activated macrophages (AAMs/M2) secrete ECM‐degrading enzymes including matrix metallopeptidases (MMPs) during the resolution phase. It is also understood that macrophages instruct fibroblast activities and that crosstalk is largely coordinated by secreted factors. However, the differential effect of CAMs and AAMs on fibroblasts remains elusive. CXCR3 is a chemokine receptor known to modulate both fibroblast and macrophage activity during wound healing and its activation acts as a critical stop signal to halt continuous ECM remodeling by fibroblasts. Yet, the importance of CXCR3 in macrophage‐fibroblast crosstalk during tissue repair remains explored. Therefore, the goal of this study was to investigate the interplay between macrophages and fibroblasts and elucidate the role of CXCR3 signaling in this interaction.MethodsWe differentiated naïve bone marrow‐derived macrophages (M0) into either CAMs/M1 using IFN‐gamma and LPS, or AAMs/M2 using IL‐4. To study the effects of secreted and cell‐contact dependent factors, we used both indirect and direct co‐culture systems. The role of CXCR3 in macrophages and fibroblasts was examined using cells from wild‐type (WT) and CXCR3−/− knockout mice.ResultsCytokine profiling revealed that fibroblasts treated with media conditioned (CM) by CAMs secreted higher levels of CXCR3 ligands CXCL10 and CXCL11 compared to untreated fibroblasts. Because both fibroblasts and macrophages express CXCR3, it was unclear which cell type might respond to these ligands. Investigation using qRT‐PCR, showed that treatment with WT CAM CM resulted in increased fibroblast expression of MMP8 and MMP9 compared to no treatment. Fibroblast induction of MMPs by WT CAM CM persisted when CXCR3−/− fibroblasts were treated with WT CAM CM, but was greatly diminished when fibroblasts were cultured with CXCR3−/− CAM CM, suggesting that this crosstalk requires CXCR3 expression on CAMs but not on fibroblasts. The influence of macrophage subtypes on MMP8, MMP9, and TGF‐β‐induced α‐SMA and ECM protein production was observed by IF staining.ConclusionsOur data indicate that CAMs induce fibroblast expression of CXCR3 ligands and key matrix degrading enzymes MMP8 and MMP9, with MMP expression requiring CXCR3 expression on CAMs. Thus, our study suggests for the first time that paracrine interaction between fibroblasts and macrophages dictate the effector functions of fibroblasts and the wound healing process.Support or Funding InformationSupported by grants from NIAMS and NIAD (AR68317 CCY and R56A1097679 NI), and support in kind from the Pittsburgh VA Medical Center (AW), the School of Nursing, and McGowan Institute of Regenerative Medicine (BNB, CCY).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4770: Novel CXCR3 allosteric modulators inhibit breast cancer stem cells and metastasis

Abstract There is an urgent need to identify molecular targets that are relevant to metastatic and/or stem-like cancer cells (CSC). Theoretically, surface-expressed G-protein coupled receptors on CSC are attractive therapeutic targets. There is considerable evidence that the inflammatory milieu of the tumor microenvironment drives CSC and we now show that two isoforms of the chemokine receptor CXCR3 play important but distinct roles in cancer behavior. In the bulk tumor cell population, CXCR3, isoform A is highly expressed relative to CXCR3B. CXCR3A is critical to metastatic success. CXCR3, isoform B, is highly upregulated in breast cancer CSC. When CXCR3B is gene-silenced in a model of basal-type, metastatic breast cancer, tumorsphere-forming capacity is reduced and the aldehyde dehydrogenase-positive (ALDH1+) population is correspondingly decreased. Conversely, overexpression of CXCR3B enhances these CSC properties. Thus, CXCR3B is critical to maintenance of the phenotype and function of breast CSC. CXCR3 ligands CXCL9, CXCL10 and CXCL11 all bind with high affinity to CXCR3, however, each ligand is coupled to different intracellular signaling pathways. For example, others have reported that, in HEK cells, CXCL10 strongly induces ERK activation, but CXCL11 does not. In our hands, CXCL11 more potently induces an ALDH1+ population in MDA-MB-231 cells compared to CXCL10. We have developed novel allosteric modulators of CXCR3 that demonstrate probe (ligand)-dependence. BD64 preferentially inhibits CXCL11-mediated β-arrestin 2 recruitment relative to inhibition of GPCR signaling; BD103 is significantly more effective at blocking CXCL11 versus CXCL10-mediated GPCR signaling. Neither BD64 nor BD103 affects the proliferation of non-CSC, however both compounds inhibit the ability of CSC to form tumorspheres. Furthermore, both CXCR3 antagonists reverse ligand-mediated induction of the ALDH1+ fraction. Most interesting is that both BD64 and BD103 effectively inhibit tumor cell colonization of the lung. These data suggest that optimal tumor control will be achieved when both metastatic, CXCR3Ahigh as well as CXCR3Bhigh CSC populations are inhibited by highly specific CXCR3 modulators. Note: This abstract was not presented at the meeting. Citation Format: Namita Kundu, Regine Brox, Xinrong Ma, Jocelyn Reader, Nuska Tschammer, Amy M. Fulton. Novel CXCR3 allosteric modulators inhibit breast cancer stem cells and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4770. doi:10.1158/1538-7445.AM2017-4770

Identification of an immunogenic neo-epitope encoded by mouse sarcoma using CXCR3 ligand mRNAs as sensors

ABSTRACTThe CXCR3 ligands CXCL9, 10, and 11 play critical roles in the amplification of immune responses by recruiting CXCR3+ immune effector cells to the tumor site. Taking advantage of this property of CXCR3 ligands, we aimed to establish a novel approach to identify immunogenic mutated-antigens. We examined the feasibility of using CXCR3 ligand mRNAs as sensors for detection of specific immune responses in human and murine systems. We further investigated whether this approach is applicable for the identification of immunogenic mutated-antigens by using murine sarcoma lines. Rapid synthesis of CXCR3 ligand mRNAs occurred shortly after specific immune responses in both human and murine immune systems. Particularly, in CMS5 tumor-bearing mice, we detected specific immune responses to mutated mitogen-activated protein kinase 2 (ERK2), which has previously been identified as an immunogenic mutated-antigen. Furthermore, by combining this approach with whole-exome and transcriptome sequencing analyses, we identified an immunogenic neo-epitope derived from mutated staphylococcal nuclease domain-containing protein 1 (Snd1) in CMS7 tumor-bearing mice. Most importantly, we successfully detected the specific immune response to this neo-epitope even without co-administration of anti-cytotoxic T-lymphocyte protein-4 (CTLA-4), anti-programmed cell death-1 (PD-1) and anti-glucocorticoid-induced TNFR-related protein (GITR) antibodies, which vigorously augmented the immune response and consequently enabled us to detect the specific immune response to this neo-epitope by conventional IFNγ intracellular staining method.Our data indicate the potential usefulness of this strategy for the identification of immunogenic mutated-antigens. We propose that this approach would be of great help for the development of personalized cancer vaccine therapies in future.

Equine herpesvirus type 1 modulates inflammatory host immune response genes in equine endothelial cells

Equine herpesvirus myeloencephalopathy (EHM), a disease caused by equine herpesvirus type 1 (EHV-1), is characterized by severe inflammation, thrombosis, and hypoxia in central nervous system (CNS) endothelial cells, which can result in a spectrum of clinical signs including urinary incontinence, ataxia, and paralysis. Strains of EHV-1 that contain a single point mutation within the viral DNA polymerase (nucleotide A2254>G2254: amino acid N752→D752) are isolated from EHM afflicted horses at higher frequencies than EHV-1 strains that do not harbor this mutation. Due to the correlation between the DNA Pol mutation and EHM disease, EHV-1 strains that contain the mutation have been designated as neurologic. In this study, we measured virus replication, cell to cell spread efficacy, and host inflammatory responses in equine endothelial cells infected with 12 different strains of EHV-1. Two strains, T953 (Ohio 2003) (neurologic) and Kentucky A (KyA) (non-neurologic), have well described disease phenotypes while the remaining strains used in this study are classified as neurologic or non-neurologic based solely on the presence or absence of the DNA pol mutation, respectively. Results show that the neurologic strains do not replicate better or spread more efficiently in endothelial cells. Also, the majority of the host inflammatory genes were modulated similarly regardless of EHV-1 genotype. Analyses of host gene expression showed that a subset of pro-inflammatory cytokines, including the CXCR3 ligands CXCL9, CXCL10, and CXCL11, as well as CCL5, IL-6 and TNF-α were consistently up-regulated in endothelial cells infected with each EHV-1 strain. The identification of specific pro-inflammatory cytokines in endothelial cells that are modulated by EHV-1 provides further insight into the factors that contribute to the immunopathology observed after infection and may also reveal new targets for disease intervention.

The pathogenic effect of TNF-α on development of Sjögren’s syndrome in non-obese diabetic mice

Abstract Tumor necrosis factor-alpha (TNF-α), is a proinflammatory cytokine involved in the pathogenesis of various autoimmune diseases including rheumatoid arthristis, inflammatory bowel disease and psoriasis. Previous studies showed that patients with Sjögren’s syndrome(SS), a prevalent systemic autoimmune disease primarily affecting tear and saliva secretion, exhibit enhanced expression of TNF-α in saliva and salivary gland. However, the exact role of TNF-α in SS disease development is not yet fully determined. Our current study employed non-obese diabetic (NOD) mice, a model of SS-like diseases, to investigate the function of endogenous TNF-α in pathogenesis of SS. We observed that administration of a neutralizing anti-TNF-α antibody significantly improved salivary secretion in female NOD mice, indicating a remission of clinical symptom of SS. TNF-α neutralization also decreased the number of leukocytic foci and reduced the number of total and activated CD4 T cells and B cells in the submandibular gland of NOD mice. Moreover, neutralization of TNF-α reduced the number of CXCR3-expressing CD4 T cells and T-bet protein level in the submandibular gland, suggesting a decrease in the infiltrating T helper 1 and T cytotoxic 1 cells. In addition, the decrease in CXCR3+ CD4 T cells was associated with reduced expression of CXCR3 ligand CXCL9. Overall, the results presented here indicate that endogenous TNF-α plays a pathogenic role in the initiation and development of SS disease and neutralization of TNF-α may be greatly beneficial for the prevention and treatment of this prevalent autoimmune disease.