Blockade of CCR5 and CXCR3 attenuates murine acute graft-versus-host disease through modulating donor-derived T-cell distribution and function.

Lymphocyte trafficking via chemokine receptors such as C-C chemokine receptor 5 (CCR5) and CXCR3 plays a critical role in the pathogenesis of acute graft-versus-host disease (aGVHD). Our previous studies showed that the addition of CCR5 or CXCR3 antagonists could only slightly alleviate the development of aGVHD. Given the specificity of T lymphocytes bearing CXCR3 and CCR5, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD. A mouse model of aGVHD was established to assess the efficacy of CCR5 and/or CXCR3 blockade on the development of aGVHD. The distribution of lymphocytes was calculated by quantification of immunostaining cells. The immunomodulatory effect on T cells was assessed by evaluating T-cell proliferation, viability, and differentiation. Using the murine allogeneic hematopoietic stem cell transplantation model, we demonstrated that blockade of both CCR5 and CXCR3 could efficiently alleviate the development of aGVHD. Further investigation on the immune mechanisms for this prophylactic effect showed that more T cells were detained into secondary lymphoid organs (SLOs), which may lead to reduced infiltration of T cells into GVHD target organs. Our study also showed that T cells detained in SLOs dampened the activation, suppressed the polarization toward T helper type 1 (Th1) and T cytotoxic type 1 (Tc1) cells, and induced the production of Treg cells. These data suggest that concurrent blockade of CCR5 and CXCR3 attenuates murine aGVHD through modulating donor-derived T-cell distribution and function, and this might be applicable for aGVHD prophylaxis in clinical settings.

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models.

For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.

Solid Tumor Microenvironment Can Harbor and Support Functional Properties of Memory T Cells.

Robust T cell responses are crucial for effective anti-tumor responses and often dictate patient survival. However, in the context of solid tumors, both endogenous T cell responses and current adoptive T cell therapies are impeded by the immunosuppressive tumor microenvironment (TME). A multitude of inhibitory signals, suppressive immune cells, metabolites, hypoxic conditions and limiting nutrients are believed to render the TME non-conducive to sustaining productive T cell responses. In this study we conducted an in-depth phenotypic and functional comparison of tumor-specific T cells and tumor-nonspecific bystander memory T cells within the same TME. Using two distinct TCR transgenic and solid-tumor models, our data demonstrate that despite exposure to the same cell-extrinsic factors of the TME, the tumor-nonspecific bystander CD8 T cells retain the complete panoply of memory markers, and do not share the same exhaustive phenotype as tumor-reactive T cells. Compared to tumor-specific T cells, bystander memory CD8 T cells in the TME also retain functional effector cytokine production capabilities in response to ex vivo cognate antigenic stimulation. Consistent with these results, bystander memory T cells isolated from tumors showed enhanced recall responses to secondary bacterial challenge in a T cell transplant model. Importantly, the tumor-resident bystander memory cells could also efficiently utilize the available resources within the TME to elaborate in situ recall effector functions following intra-tumoral peptide antigen injection. Additionally, CRISPR-Cas9 gene deletion studies showed that CXCR3 was critical for the trafficking of both tumor antigen-specific and bystander memory T cells to solid tumors. Collectively, these findings that T cells can persist and retain their functionality in distinct solid tumor environments in the absence of cognate antigenic stimulation, support the notion that persistent antigenic signaling is the central driver of T cell exhaustion within the TME. These studies bear implications for programming more efficacious TCR- and CAR-T cells with augmented therapeutic efficacy and longevity through regulation of antigen and chemokine receptors.

Pro-inflammatory microenvironment and systemic accumulation of CXCR3+ cell exacerbate lung pathology of old rhesus macaques infected with SARS-CoV-2.

Understanding the pathological features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in an animal model is crucial for the treatment of coronavirus disease 2019 (COVID-19). Here, we compared immunopathological changes in young and old rhesus macaques (RMs) before and after SARS-CoV-2 infection at the tissue level. Quantitative analysis of multiplex immunofluorescence staining images of formalin-fixed paraffin-embedded (FFPE) sections showed that SARS-CoV-2 infection specifically induced elevated levels of apoptosis, autophagy, and nuclear factor kappa-B (NF-κB) activation of angiotensin-converting enzyme 2 (ACE2)+ cells, and increased interferon α (IFN-α)- and interleukin 6 (IL-6)-secreting cells and C-X-C motif chemokine receptor 3 (CXCR3)+ cells in lung tissue of old RMs. This pathological pattern, which may be related to the age-related pro-inflammatory microenvironment in both lungs and spleens, was significantly correlated with the systemic accumulation of CXCR3+ cells in lungs, spleens, and peripheral blood. Furthermore, the ratio of CXCR3+ to T-box protein expression in T cell (T-bet)+ (CXCR3+/T-bet+ ratio) in CD8+ cells may be used as a predictor of severe COVID-19. These findings uncovered the impact of aging on the immunopathology of early SARS-CoV-2 infection and demonstrated the potential application of CXCR3+ cells in predicting severe COVID-19.

CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions.

The development of pancreatic cancer requires recruitment and activation of different macrophage populations. However, little is known about how macrophages are attracted to the pancreas after injury or an oncogenic event, and how they crosstalk with lesion cells or other cells of the lesion microenvironment. Here, we delineate the importance of CXCL10/CXCR3 signaling during the early phase of murine pancreatic cancer. We show that CXCL10 is produced by pancreatic precancerous lesion cells in response to IFNγ signaling and that inflammatory macrophages are recipients for this chemokine. CXCL10/CXCR3 signaling in macrophages mediates their chemoattraction to the pancreas, enhances their proliferation, and maintains their inflammatory identity. Blocking of CXCL10/CXCR3 signaling in vivo shifts macrophage populations to a tumor-promoting (Ym1+, Fizz+, Arg1+) phenotype, increases fibrosis, and mediates progression of lesions, highlighting the importance of this pathway in PDA development. This is reversed when CXCL10 is overexpressed in PanIN cells.

CXCR3 and CXCR5 are highly expressed in HIV-1-specific CD8 central memory T cells from infected patients.

New ways of characterizing CD8+ memory T cell responses in chronic infections are based on the measurement of chemokine receptor expression (CXCR3, CXCR5, and CX3CR1). We applied these novel phenotyping strategies to chronic HIV infection by comparing healthy donors (HDs), HIV-infected patients receiving antiretroviral therapy (ART), and spontaneous HIV controllers (HICs). In all groups, the memory cells exhibited high proportion of CXCR3+ cells. Proportions of CXCR5+ and CX3CR1+ cells were preferentially observed among central memory cells (Tcm) and effector memory cells (Tem) respectively. Chronic controlled HIV infection impacted the chemokine receptor profile of both HIV-specific and nonspecific CD8+ T cells. In total CD8+ T cells, the proportions of CXCR3- CXCR5- CX3CR1- Tcm and Tem were lower in HIV-infected patients than in HDs with subtle differences between ART and HICs. Such phenotyping strategy also revealed differences in exhaustion and senescence phenotypes, the CXCR3+ CXCR5+ CX3CR1- being more exhausted and senescent than the CXCR3+ CXCR5- CX3CR1- Tcm fraction. Among HIV-specific CD8+ T cells, the vast majority of Tcm cells were CXCR3+ and CXCR5+ cells in contrast with their nonspecific counterparts. In conclusion, the addition of migration markers contributes to better characterize Tcm/Tem compartment.

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Long-lived T-memory stem cells (TSCM ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA+ CCR7+ CD95+ phenotype. Significant heterogeneity within the TSCM population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 TSCM subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The TSCM CD122hi -expressing subset (versus CD122lo ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The TSCM CXCR3lo subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the TSCM CD122hi population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the TSCM CD122hi phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.

Ingredients such as trehalose and hesperidin taken as supplements or foods reverse alterations in human T cells, reducing asbestos exposure-induced antitumor immunity.

Exposure of human immune cells to asbestos causes a reduction in antitumor immunity. The present study aimed to investigate the recovery of reduced antitumor immunity by several ingredients taken as supplements or foods, including trehalose (Treh) and glycosylated hesperidin (gHesp). Peripheral blood CD4+ cells were stimulated with IL­2, anti­CD3 and anti­CD28 antibodies for 3 days, followed by further stimulation with IL­2 for 7 days. Subsequently, cells were stimulated with IL­2 for an additional 28 days. During the 28 days, cells were cultured in the absence or presence of 50 µg/ml chrysotile asbestos fibers. In addition, cells were treated with 10 mM Treh or 10 µM gHesp. Following culture for 28 days, reverse transcription­quantitative PCR was performed to assess the expression levels of transcription factors, cytokines and specific genes, including matrix metalloproteinase­7 (MMP­7), nicotinamide nucleotide transhydrogenase (NNT) and C­X­C motif chemokine receptor 3, in unstimulated cells (fresh) and cells stimulated with PMA and ionomycin (stimuli). The results demonstrated that compared with the control group, chrysotile­exposure induced alterations in MMP­7, NNT and IL­17A expression levels were not observed in the 'Treh' and 'gHesp' groups in stimulated cells. The results suggested that Treh and gHesp may reverse asbestos exposure­induced reduced antitumor immunity in T helper cells. However, further investigation is required to confirm the efficacy of future trials involving the use of these compounds with high­risk human populations exposed to asbestos, such as workers involved in asbestos­handling activities.

Ovariectomy induces bone loss via microbial-dependent trafficking of intestinal TNF+ T cells and Th17 cells.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.

The association of Epstein-Barr virus infection with CXCR3+ B-cell development in multiple sclerosis: impact of immunotherapies.

Epstein-Barr virus (EBV) infection of B cells is associated with increased multiple sclerosis (MS) susceptibility. Recently, we found that CXCR3-expressing B cells preferentially infiltrate the CNS of MS patients. In chronic virus-infected mice, these types of B cells are sustained and show increased antiviral responsiveness. How EBV persistence in B cells influences their development remains unclear. First, we analyzed ex vivo B-cell subsets from MS patients who received autologous bone marrow transplantation (n = 9), which is often accompanied by EBV reactivation. The frequencies of nonclass-switched and class-switched memory B cells were reduced at 3-7 months, while only class-switched B cells returned back to baseline at 24-36 months posttransplantation. At these time points, EBV DNA load positively correlated to the frequency of CXCR3+ , and not CXCR4+ or CXCR5+ , class-switched B cells. Second, for CXCR3+ memory B cells trapped within the blood of MS patients treated with natalizumab (anti-VLA-4 antibody n = 15), latent EBV infection corresponded to enhanced in vitro formation of anti-EBNA1 IgG-secreting plasma cells under GC-like conditions. These findings imply that EBV persistence in B cells potentiates brain-homing and antibody-producing CXCR3+ subsets in MS.

Wrinkle in the plan: miR-34a-5p impacts chemokine signaling by modulating CXCL10/CXCL11/CXCR3-axis in CD4+, CD8+ T cells, and M1 macrophages.

BACKGROUND: In 2016 the first-in-human phase I study of a miRNA-based cancer therapy with a liposomal mimic of microRNA-34a-5p (miR-34a-5p) was closed due to five immune related serious adverse events (SAEs) resulting in four patient deaths. For future applications of miRNA mimics in cancer therapy it is mandatory to unravel the miRNA effects both on the tumor tissue and on immune cells. Here, we set out to analyze the impact of miR-34a-5p over-expression on the CXCL10/CXCL11/CXCR3 axis, which is central for the development of an effective cancer control. METHODS: We performed a whole genome expression analysis of miR-34a-5p transfected M1 macrophages followed by an over-representation and a protein-protein network analysis. In-silico miRNA target prediction and dual luciferase assays were used for target identification and verification. Target genes involved in chemokine signaling were functionally analyzed in M1 macrophages, CD4+ and CD8+ T cells. RESULTS: A whole genome expression analysis of M1 macrophages with induced miR-34a-5p over-expression revealed an interaction network of downregulated target mRNAs including CXCL10 and CXCL11. In-silico target prediction in combination with dual luciferase assays identified direct binding of miR-34a-5p to the 3'UTRs of CXCL10 and CXCL11. Decreased CXCL10 and CXCL11 secretion was shown on the endogenous protein level and in the supernatant of miR-34a-5p transfected and activated M1 macrophages. To complete the analysis of the CXCL10/CXCL11/CXCR3 axis, we activated miR-34a-5p transfected CD4+ and CD8+ T cells by PMA/Ionomycin and found reduced levels of endogenous CXCR3 and CXCR3 on the cell surface. CONCLUSIONS: MiR-34a-5p mimic administered by intravenous administration will likely not only be up-taken by the tumor cells but also by the immune cells. Our results indicate that miR-34a-5p over-expression leads in M1 macrophages to a reduced secretion of CXCL10 and CXCL11 chemokines and in CD4+ and CD8+ T cells to a reduced expression of CXCR3. As a result, less immune cells will be attracted to the tumor site. Furthermore, high levels of miR-34a-5p in naive CD4+ T cells can in turn hinder Th1 cell polarization through the downregulation of CXCR3 leading to a less pronounced activation of cytotoxic T lymphocytes, natural killer, and natural killer T cells and possibly contributing to lymphocytopenia.

Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

In Vitro Immunological Effects of CXCR3 Inhibitor AMG487 on Dendritic Cells.

AMG 487 is the targeted blocker of chemokine receptor CXCR3 and improves inflammatory symptoms by blocking the inflammatory cycle. Here we investigated whether AMG 487 affects dendritic cell (DC) biology and function. The expression of co-stimulatory markers on DCs was reduced, indicating the semi-mature state of DC when AMG 487 was added throughout the in vitro differentiation period. Additionally, when added solely during the final lipopolysaccharide-induced activation step, AMG 487 inhibited DC activation, as demonstrated by a decreased expression of activation markers. AMG487 also promoted the expression of PD-L2 and impaired the ability to induce antigen-specific T cell responses. Our results demonstrated that AMG 487 significantly affects DC maturity in vitro and function leading to impaired T cell activation, inducing DCs to have characteristics similar to tolerogenic DCs. AMG 487 may directly play an immunomodulatory role during DC development and functional shaping.

B cell-Derived IL35 Drives STAT3-Dependent CD8+ T-cell Exclusion in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell- but not regulatory T cell-specific production of IL35. We also demonstrated that B cell-specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti-PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3-CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell-mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

Human pDCs Are Superior to cDC2s in Attracting Cytolytic Lymphocytes in Melanoma Patients Receiving DC Vaccination.

Plasmacytoid dendritic cells (pDCs) and type 2 conventional dendritic cells (cDC2s) are currently under evaluation for use in cancer vaccines. Although both DC subsets can activate adaptive and innate lymphocytes, their capacity to recruit such cells is rarely considered. Here, we show that pDCs and cDC2s display a striking difference in chemokine secretion, which correlates with the recruitment of distinct types of immune effector cells. Activated pDCs express high levels of CXCR3 ligands and attract more CD8+ T cells, CD56+ T cells, and γδ T cells in vitro, compared to cDC2s. Skin from melanoma patients shows an influx of immune effector cells following intradermal vaccination with pDCs or cDC2s, with pDCs inducing the strongest influx of lymphocytes known to possess cytolytic activity. These findings suggest that combining both DC subsets could unite the preferred chemoattractive properties of pDCs with the superior T cell priming properties of cDC2s to ultimately enhance vaccine efficacy.

Inactivation of the PD-1-Dependent Immunoregulation in Mice Exacerbates Contact Hypersensitivity Resembling Immune-Related Adverse Events.

Blockade of PD-1, an indispensable physiological immunoregulatory mechanism, enhances immune activities and is widely used in the immunotherapy of cancer. This treatment often accompanies inflammatory complication called immune-related adverse events (irAE), most frequently in the skin. To analyze how skin inflammation develops by the blockade of PD-1-dependent immunoregulation, we studied the exacerbation of oxazolone-induced contact hypersensitivity by PD-L1 blockade. The inactivation of PD-1 signaling enhanced swelling of the skin with massive CD8+ T cell infiltration. Among PD-1-expressing cells, T cells were the predominant targets of anti-PD-L1 mAb treatment since PD-L1 blockade did not affect skin inflammation in RAG2-/- mice. PD-L1 blockade during immunization with oxazolone significantly promoted the development of hapten-reactive T cells in the draining lymph nodes. The enhancement of local CD8+ T cell-dominant immune responses by PD-L1 blockade was correlated with the upregulation of CXCL9 and CXCL10. Challenges with a low dose of oxazolone did not demonstrate any significant dermatitis; however, the influence of PD-L1 blockade on T cell immunity was strong enough to cause the emergence of notable dermatitis in this suboptimal dosing, suggesting its relevance to dermal irAE development. In the low-dose setting, the blockade of CXCR3, receptor of CXCL9/10, prevented the induction of T cell-dominant inflammation by anti-PD-L1 mAb. This experimental approach reproduced CD8+ T cell-dominant form of cutaneous inflammation by the blockade of PD-L1 that has been observed in dermal irAE in human patients.

CXCR3 from chemokine receptor family correlates with immune infiltration and predicts poor survival in osteosarcoma.

BACKGROUND: Chemokine receptors have a crucial role in regulating tumor mediating immunity and are also implicated in the prognosis of some cancers. Here, the association between CXC chemokine receptors (CXCR2-5) and prognosis in osteosarcoma was studied. METHODS: Differences between CXCR2, CXCR3, CXCR4, and CXCR5 expression and overall survival (OS) and event-free survival (EFS) were compared using Kaplan-Meier analyses. The associations of CXCR3 expression with clinical features and the prognosis were also analyzed. The signaling pathways modulated by CXCR3 were investigated. The correlations between CXCR3 and immune infiltrates were investigated. RESULTS: The expression of CXCR2, CXCR4, and CXCR5 was not associated with the prognosis, but CXCR3 low expression was correlated with worse OS and EFS of osteosarcoma, especially for female, patients aged less than 15.1 years, or patients without metastasis. Low CXCR3 expression was related to tumor site and histologic response (P<0.05), but not associated with other clinical characteristics. Multivariate Cox analysis revealed that CXCR3 remained independently associated with the prognosis, especially for OS (hazard ratio (HR) = 3.26, 95% CI = 1.15-9.24, P=0.026). The cell adhesion, apoptosis, metabolism, KRAS, P53, NOTCH, reactive oxygen species (ROS), PI3K/Akt/mTOR, vascular endothelial growth factor (VEGF), inflammation, and immune-related pathways such as IL-6/JAK/STAT3, TNF-α via NF-κB, Toll/NOD-like receptor, and complement were modulated by CXCR3. CXCR3 expression showed an especially positive correlation with immune infiltration of T cells CD8, macrophages M1, plasma cells, and NK cells activated. CONCLUSIONS: CXCR3 may be an independent risk factor for the prognosis and is most likely to benefit from immunotherapy in osteosarcoma.

Infection and RNA-seq analysis of a zebrafish tlr2 mutant shows a broad function of this toll-like receptor in transcriptional and metabolic control and defense to Mycobacterium marinum infection.

BACKGROUND: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.