CXCR3 Blockade Research Articles

Adverse effects of CXCR2 deficiency in mice reared under non-gnotobiotic conditions

The family of pro-inflammatory and pro-angiogenic chemokines including Interleukin-8 (IL-8, aka CXCL8) and its homologues (CXCL1,2,3,5,6, and 7) exhibit promiscuous binding and activation of several G-protein-coupled receptors (i.e., CXCR2, CXCR1, and the atypical chemokine receptor (ACKR1)). A high proportion of their biological activity is attributed to CXCR2 activation, thus many CXCR2 inhibitors are in clinical trials for several chronic diseases. However, CXCR2 inhibition is often only investigated acutely in these trials or in Cxcr2−/− mice grown in gnotobiotic conditions. Since humans do not live in germ-free environments, our first goal is to highlight novel retinal and systemic observations in Cxcr2−/− mice grown in non-gnotobiotic conditions that suggest potential harmful consequences of long-term CXCR2 deficiency or blockade. Beyond confirmation of circulating blood/immune cell-related phenotypes, we report novel findings in Cxcr2−/− mice including: (1) delayed dye transit to the retinal vasculature, (2) alterations in the density and distribution of retinal vessels, astrocytes and microglia, (3) decreased electroretinogram a- and b-wave amplitudes, (4) reduced visual acuity, and (5) increased polymorphonuclear cell accumulation in vascular lumina abutting venular walls in the retina and in vital non-ocular tissues (lung and liver). Furthermore, PheWAS of CXCR2 CXCR1, and ACKR1 gene variants using data from UK Biobank participants suggest clinical associations with both retinal and vascular disease phenotypes. We conclude that chronic CXCR2 deficiency in mice contributes to functional damage to the retina and that the long-term safety of CXCR1/2 inhibitors designed for chronic use in humans should be explored before clinical adoption to safeguard sight and overall vascular health.

Non-homogenous intratumor ionizing radiation doses synergize with PD1 and CXCR2 blockade

The efficacy and side effects of radiotherapy (RT) depend on parameters like dose and the volume of irradiated tissue. RT induces modulations of the tumor immune microenvironment (TIME) that are dependent on the dose. Low dose RT (LDRT, i.e., single doses of 0.5–2 Gy) has been shown to promote immune infiltration into the tumor. Here we hypothesize that partial tumor irradiation combining the immunostimulatory/non-lethal properties of LDRT with cell killing/shrinkage properties of high dose RT (HDRT) within the same tumor mass could enhance anti-tumor responses when combined with immunomodulators. In models of colorectal and breast cancer in immunocompetent female mice, partial irradiation (PI) with millimetric precision to deliver LDRT (2 Gy) and HDRT (16 Gy) within the same tumor induces substantial tumor control when combined with anti-PD1. Using flow cytometry, cytokine profiling and single-cell RNA sequencing, we identify a crosstalk between the TIME of the differentially irradiated tumor volumes. PI reshapes tumor-infiltrating CD8+ T cells into more cytotoxic and interferon-activated phenotypes but also increases the infiltration of pro-tumor neutrophils driven by CXCR2. The combination of the CXCR2 antagonist SB225002 with PD1 blockade and PI improves tumor control and mouse survival. Our results suggest a strategy to reduce RT toxicity and improve the therapeutic index of RT and immune checkpoint combinations.

Single-cell multiomic analysis identifies macrophage subpopulations in promoting cardiac repair.

Cardiac mononuclear phagocytic cells (Cardiac MPCs) participate in maintaining homeostasis and orchestrating cardiac responses upon injury. However, the function of specific MPC subtypes and the related cell fate commitment mechanisms remain elusive in regenerative and nonregenerative hearts due to their cellular heterogeneities. Using spatiotemporal single-cell epigenomic analysis of cardiac MPCs in regenerative (P1) and nonregenerative (P10) mouse hearts after injury, we found that P1 hearts accumulate reparative Arg1+ macrophages, while proinflammatory S100a9+Ly6c+ monocytes are uniquely abundant during nonregenerative remodeling. Moreover, blocking chemokine CXCR2 to inhibit the specification of the S100a9+Ly6c+-biased inflammatory fate in P10 hearts resulted in elevated wound repair responses and marked improvements in cardiac function after injury. Single-cell RNA-Seq further confirmed an increased Arg1+ macrophage subpopulation after CXCR2 blockade, which was accomplished by increased expression of wound repair-related genes and reduced expression of proinflammatory genes. Collectively, our findings provide instructive insights into the molecular mechanisms underlying the function and fate specification of heterogeneous MPCs during cardiac repair and identify potential therapeutic targets for myocardial infarction.

Abstract Mo017: CXCR3: A Tumor-Conscious Target to Treat Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs, such as anti-CTLA-4 or anti-PD-1) have especially been effective in cancer therapies. However, ICIs treatment can result in myocarditis, the most serious complication of ICIs, which has a case fatality rate as high as 40%. Here, we investigate CXCR3 blockade for targeted treatment of myocardial inflammation in ICI myocarditis while considering concurrent tumor treatment. We hypothesize that CXCR3 blockade will have a direct effect on reducing CD8+ T-cell mediated cardiomyocyte cardiotoxicity without affecting tumor treatment. Methods: To study the impact of CXCR3 blockade on myocardial inflammation, we established an in vitro cardiomyocyte apoptosis model by co-culturing cardiomyocytes and MRL-WT mice CD8+ T-cells with/without ICI stimulation or MRL- Pdcd1-/- mice CD8+ T-cells. We then blocked CXCR3 to assess the effect on CD8+ T cell-mediated cytotoxicity against cardiomyocytes. To assess CXCR3 blockade in an in vivo model of ICI myocarditis accounting for tumor, we treated MRL mice inoculated with B16F1 melanoma cells with six doses of 400 µ g each of anti-PD1 and anti-CTLA4 and investigated the effects of CXCR3-CXCL9/10 blockade on hearts and tumors. Results: In vitro CD8+ T-cell and cardiomyocyte co-culture demonstrated mitigation of cardiomyocytes apoptosis with CXCR3 blockade ( Fig. 1 A - C) , suggesting decreased CD8+ T-cell cytotoxicity with CXCR3 blockade. In vivo treatment with dual immunotherapy (anti-PD1/anti-CTLA4 monoclonal antibodies) in the tumor MRL mouse model demonstrated significant tumor volume reduction, which was not impacted by concurrent treatment with anti-CXCR3 or anti-CXCL9/10 (Fig. 1 D - F) . Concurrently, myocarditis occurred in the group treated with immunotherapy alone, not in anti-CXCL9/10 or anti-CXCR3 (Fig. 1 G - H). Conclusions: CXCR3 blockade reduces CD8+ T-cell cytotoxicity against cardiomyocytes and mitigates myocarditis in an in vivo tumor ICI myocarditis mouse model without affecting anti-tumor ICI efficacy, positioning CXCR3 as a promising target for therapeutic intervention.

BMAL1 plays a crucial role in immune homeostasis during sepsis-induced acute lung injury

Sepsis is a widespread and life-threatening disease characterised by infection-triggered immune hyperactivation and cytokine storms, culminating in tissue damage and multiple organ dysfunction syndrome. BMAL1 is a pivotal transcription factor in the circadian clock that plays a crucial role in maintaining immune homeostasis. BMAL1 dysregulation has been implicated in inflammatory diseases and immunodeficiency. However, the mechanisms underlying BMAL1 disruption in sepsis-induced acute lung injury (ALI) remain poorly understood. In vitro, we used THP1 and mouse peritoneal macrophages to elucidate the potential mechanism of BMAL1 function in sepsis. In vivo, an endotoxemia model was used to investigate the effect of BMAL1 on sepsis and the therapeutic role of targeting CXCR2. We showed that BMAL1 significantly affected the regulation of innate immunity in sepsis-induced ALI. BMAL1 deficiency in the macrophages exacerbated systemic inflammation and sepsis-induced ALI. Mechanistically, BMAL1 acted as a transcriptional suppressor and regulated the expression of CXCL2. BMAL1 deficiency in macrophages upregulated CXCL2 expression, increasing the recruitment of polymorphonuclear neutrophils and the formation of neutrophil extracellular traps (NETs) by binding to the chemokine receptor CXCR2, thereby intensifying lung injury in a sepsis model. Furthermore, a selective inhibitor of CXCR2, SB225002, exerted promising therapeutic effects by markedly reducing neutrophil infiltration and NETs formation and alleviating lung injury. Importantly, CXCR2 blockade mitigated multiple organ dysfunction. Collectively, these findings suggest that BMAL1 controls the CXCL2/CXCR2 pathway, and the therapeutic efficacy of targeting CXCR2 in sepsis has been validated, presenting BMAL1 as a potential therapeutic target for lethal infections.

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.

Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment

BackgroundChemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked.MethodsWe employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1−/−, Cxcr2−/− or Ccr1−/−Cxcr2−/− mice.ResultsMyeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1−/−Cxcr2−/− BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2−/− BM significantly suppressed tumor growth and liver metastasis.ConclusionDual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.

Abstract 727: FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis

Abstract Cancer-associated fibroblasts (CAFs) contribute to cancer progression via crosstalk with cancer and immune cells in the tumor microenvironment (TME). Despite the significant role of CAF, the understanding of the trigger mechanisms of cancer-mediated CAF activation remains unclear. We previously reported the mechanism by which FGFR4 in colon cancer secretes amphiregulin (AREG), resulting in anti-EGFR chemoresistance. It motivated us to investigate whether FGFR4 overexpressing tumors may change the TME, so we explored the mechanism of CAF activation under the FGFR4 signaling pathway. CAF was isolated from BALB/C mice after injecting 1 × 106 CT-26/EV or FGFR4 cells using microbeads and conditioned media (CM) was prepared by filtering after 48h cancer cell culture with serum-free medium In mice model, CT-26/FGFR4 showed more significant tumor progression and CAF abundance (α-SMA, FAP, PDGFR, and vimentin) than CT-26/EV. Additionally, CT26/FGFR4-derived CAFs showed significantly increased invasiveness and contractility, indicating the highly activated status of CAFs. In order to verify the effect of cancer secretome from FGFR4 in cancer cells, normal fibroblast (NIH/3T3) were co-cultured with either stable cancer cells or CM. As a result, CT-26/FGFR4 cells robustly induced the expression of CAF markers in NIH/3T3 cells, indicating the promotion of CAF differentiation from resting fibroblasts. Also, CT-26/FGFR4-derived CM significantly promoted further activation of CAFs compared to CT-26/EV-derived CM. Taken together, FGFR4 contributes not only to CAF differentiation but also to CAF activation via secretome. Next, we performed RNA seq of CT-26/EV and CT-26/FGFR4 cells and identified the upregulated genes related with the interferon (IFN) signaling. Among these genes, we identified IFNβ and CXCL10 as targetable molecules via the activation of TLR3-TBK-IRF and IFN-STAT-CXCL10 signaling pathways. These results were confirmed by RT-PCR and western blot assay. Secreted CXCL10 induced gene expression of CAF markers in fibroblast and increased CAF functions such as migration, invasion and contractibility, reflecting CAF activation. Blocking antibodies and knocking down of CXCL10 inhibited the cancer secretome-mediated CAF markers. The blockade of CXCR3, which is the CXCL10 cognate receptor, also showed an inhibitory effect on the FGFR4-overexpressing cancer-educated CAF. In human colon cancer specimens, the expression of FGFR4, CXCL10, and CAF markers showed a positive correlation with each other. Finally, dual inhibition of FGFR4 and CXCR3 in tumor mouse model suppressed the tumor growth, accompanied by CAF suppression. Our findings reveal the novel mechanism of FGFR4 leading CAF differentiation/activation in TME via the CXCL10-CXCR3 axis. This suggests that FGFR4/CXCR3 inhibitor could be used as a potential therapeutic strategy to attenuate CAF in stromal dominant tumors. Citation Format: Sang-Hee Cho, Hyunjin Bang, Eun-Gene Sun, Ji-Na Choi, Mi-Ra Park, Hyun-Jeong Shim, Jun-Eul Hwang, Woo-Kyun Bae, Ik Joo Chung. FGFR4 promotes colon cancer progression through CAF activation via the CXCL10-CXCR3 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 727.

Abstract 1188: Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast cancer

Abstract Metastatic triple-negative breast cancer (mTNBC) currently has the poorest prognosis among subtypes, with limited treatment options due to molecular characteristics that render conventional targeted therapies ineffective. Immunotherapy, specifically anti-PD-(L)1 (ICIs), has revolutionized certain cancer treatments, but its efficacy in mTNBC has been disappointing. To enhance tumor responses to ICIs, combining them with immunogenic chemotherapy has been explored. Studies like IMpassion130 and KEYNOTE 522 indicate benefits of chemo-immunotherapy based on taxanes, especially in PD-L1+ tumors. PD-L1 expression often correlates with TILs infiltration, particularly cytotoxic T lymphocytes (CTLs), indicating an activated immune response in the tumor. However, tumors with poor CTL infiltration, known as "Cold" phenotype, are often associated with a lack of induction of CXCR3-associated chemokines (CXCL9, 10, and 11), crucial for CTLs recruitment in the tumor microenvironment. The limited efficacy of chemo-immunotherapy in PD-L1-negative tumors may stem from chemotherapy's inability to induce immunogenic cancer cell death, CXCR3-associated chemokines, and CTL recruitment. Internal data from a 4T1 TNBC murine model demonstrated that the taxane paclitaxel failed to induce CXCL10, CTL recruitment, and sensitization to PD-L1 blockade. The preclinical research project aimed to identify a combination therapy, inspired by current clinical practices, capable of inducing CXCL10 expression and restoring CTL recruitment in the 4T1 "cold" mTNBC model. We initially screened standard TNBC chemotherapies and selected carboplatin for its in vitro potential to induce CXCL10 production and its clinical use in association with ICIs in the TNBC. Combining carboplatin with a library of over 400 targeted therapies, we identified histone deacetylase (HDAC) inhibitors as significantly enhancing CXCL10 secretion. In vivo, this combination demonstrated therapeutic additivity, increasing immune recruitment, particularly CD8+ T lymphocytes, with the therapeutic effect reduced by CXCR3 blockade. Mechanistic exploration in vitro revealed that the combination activated the type I interferon pathway, inducing CXCL10 secretion. In vivo confirmation validated the role of the type I interferon pathway and CXCL10 in the combination's efficacy. This data outlines a chemotherapy and HDAC inhibitor combination capable of inducing CTL recruitment via increased CXCL10 production. Future project phases will focus on understanding HDACi's mechanism of action on CXCL10 production and the combination's potential to sensitize tumors to ICIs. Overall, this project underscores the role of epigenetic modifications in amplifying carboplatin-induced CXCL10 secretion, potentially converting "cold" ICI-resistant tumors into a "hot" phenotype. Citation Format: Laura Kalfeist, Stacy Petit, Loick Galland, Cyriane Poirrier, Romain Aucagne, François Ghiringhelli, Sylvain Ladoire, Emeric Limagne. Identification of CXCL10-inducing chemotherapy/targeted therapy combinations for PD-1 blockade sensitization in “cold” triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1188.

Targeting ESE3/EHF With Nifurtimox Inhibits CXCR2+ Neutrophil Infiltration and Overcomes Pancreatic Cancer Resistance to Chemotherapy and Immunotherapy

Background & aimsSince pancreatic cancer responds poorly to chemotherapy and immunotherapy, it is necessary to identify novel targets and compounds to overcome resistance to treatment. MethodsThis study analyzed genomic SNP sequencing, single-cell RNA sequencing, and spatial transcriptomics. Ehf-knockout mice, KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+ and Pdx1-Cre) mice, CD45.1+BALB/C nude mice, and CD34+humanized mice were also used as subjects. Multiplexed immunohistochemistry and flow cytometry were performed to investigate the proportion of tumor-infiltrated CXCR2+neutrophils. In addition, multiplexed cytokines assays and chromatin immunoprecipitation assays were employed to examine the mechanism. ResultsThe TP53 mutation-mediated loss of tumoral EHF increased the recruitment of CXCR2+neutrophils, modulated their spatial distribution, and further induced chemo- and immuno-therapy resistance in clinical cohorts and pre-clinical syngeneic mice models. Mechanistically, EHF deficiency induced CXCL1 transcription to enhance in vitro and in vivo CXCR2+neutrophils migration. Moreover, CXCL1 or CXCR2 blockade completely abolished the effect, indicating that EHF regulated CXCR2+neutrophils migration in a CXCL1-CXCR2-dependent manner. The depletion of CXCR2+neutrophils also blocked the in vivo effects of EHF deficiency on chemotherapy and immunotherapy resistance. The scRNA-seq results of PDAC treated with Nifurtimox highlighted the therapeutic significance of Nifurtimox by elevating the expression of tumoral EHF and decreasing the weightage of CXCL1-CXCR2 pathway within the microenvironment. Importantly, by simultaneously inhibiting the JAK1/STAT1 pathway, it could significantly suppress the recruitment and function of CXCR2+neutrophils, further sensitizing PDAC to chemotherapy and immunotherapies. ConclusionsThe study demonstrated the role of EHF in the recruitment of CXCR2+neutrophils and the promising role of Nifurtimox in sensitizing pancreatic cancer to chemotherapy and immunotherapy.

The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis.

The dual role of CD8+ T cells in influenza control and lung pathology is increasingly appreciated. To explore whether protective and pathological functions can be linked to specific subsets, we dissected CD8+ T responses in influenza-infected murine lungs. Our single-cell RNA-sequencing (scRNA-seq) analysis revealed notable diversity in CD8+ T subpopulations during peak viral load and infection-resolved state. While enrichment of a Cxcr3hi CD8+ T effector subset was associated with a more robust cytotoxic response, both CD8+ T effector and central memory exhibited equally potent effector potential. The scRNA-seq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The late-stage CD8+ T blockade in influenza-cleared lungs or continuous CXCR3 blockade mitigated lung injury without affecting viral clearance. Furthermore, adoptive transfer of wild-type CD8+ T cells exacerbated influenza lung pathology in Cxcr3-/- mice. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.

Radiation-Induced Innate Neutrophil Response in Tumor Is Mediated by the CXCLs/CXCR2 Axis

The early events that lead to the inflammatory and immune-modulatory effects of radiation therapy (RT) in the tumor microenvironment (TME) after its DNA damage response activating the innate DNA-sensing pathways are largely unknown. Neutrophilic infiltration into the TME in response to RT is an early innate inflammatory response that occurs within 24–48 h. Using two different syngeneic murine tumor models (RM-9 and MC-38), we demonstrated that CXCR2 blockade significantly reduced RT-induced neutrophilic infiltration. CXCR2 blockade showed the same effects on RT-induced tumor inhibition and host survival as direct neutrophil depletion. Neutrophils highly and preferentially expressed CXCR2 compared to other immune cells. Importantly, RT induced both gene and protein expression of CXCLs in the TME within 24 h, attracting neutrophils into the tumor. Expectedly, RT also upregulated the gene expression of both cGAS and AIM2 DNA-sensing pathways in cGAS-positive MC-38 tumors but not in cGAS-negative RM-9 tumors. Activation of these pathways resulted in increased IL-1β, which is known to activate the CXCLs/CXCR2 axis. Gene ontology analysis of mRNA-Seq supported these findings. Taken together, the findings suggest that the CXCLs/CXCR2 axis mediates the RT-induced innate inflammatory response in the TME, likely translating the effects of innate DNA-sensing pathways that are activated in response to RT-induced DNA damage.

Abstract 16577: Unraveling T-cell and Macrophage Chemokine Crosstalk in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that activate immunity against tumor cells but can cause immune related adverse events such as myocarditis, a rare but serious side effect with up to 40% mortality. Hypothesis: Macrophages contribute to recruitment of cytotoxic T-cell subsets to the heart in the setting of ICI, causing myocarditis. Methods: To study the role of macrophage depletion on ICI myocarditis, we treated MRL/Pdcd1-/- mice with liposomal clodronate. To identify timing of immune infiltration, we performed serial single cell T-cell receptor and RNA sequencing of CD45+ cells from the hearts/blood of MRL/Pdcd1-/- myocarditis mice at 1, 2, and 4 weeks of age. We treated MRL mice with anti-PD1/CTLA4 to induce myocarditis and assessed if anti-CXCR3 could decrease effector CD8+ T-cell cardiac infiltration and mitigate myocarditis. We studied the effects of in vitro CXCR3 blockade on CXCR3+ T-cell migration in a transwell assay. Results: Serial single-cell TCR sequencing/scRNA-seq of immune cells from MRL/ Pdcd1-/- vs. MRL control hearts showed infiltration of CXCL9/10+ macrophages ( Fig. 1, A ) along with progressive clonal expansion of CXCR3+ effector CD8+ T-cells in the heart starting at 2 weeks of age, prior to clinical onset of myocarditis. Macrophage depletion with liposomal clodronate reduced myocarditis and improved survival in MRL/ Pdcd1-/- mice ( Fig. 1, B-C) . CXCR3 blockade with anti-CXCR3 antibody reduced myocarditis, decreased CXCR3+ CD8+ T-cell infiltration, and improved survival in mice treated with anti-CTLA4/anti-PD1 antibodies (Fig. 1, D-E) . CXCR3 blockade in transwell assay also reduced migration of CD8+CXCR3+ T-cells towards the macrophage compartment (Fig. 1, F) . Conclusions: CXCL9/10+ macrophages likely play a critical role in development of ICI myocarditis through the recruitment of CXCR3+ effector T-cells into the heart, making this pathway an attractive therapeutic target.

Targeting Members of the Chemokine Family as a Novel Approach to Treating Neuropathic Pain.

Neuropathic pain is a debilitating condition that affects millions of people worldwide. Numerous studies indicate that this type of pain is a chronic condition with a complex mechanism that tends to worsen over time, leading to a significant deterioration in patients' quality of life and issues like depression, disability, and disturbed sleep. Presently used analgesics are not effective enough in neuropathy treatment and may cause many side effects due to the high doses needed. In recent years, many researchers have pointed to the important role of chemokines not only in the development and maintenance of neuropathy but also in the effectiveness of analgesic drugs. Currently, approximately 50 chemokines are known to act through 20 different seven-transmembrane G-protein-coupled receptors located on the surface of neuronal, glial, and immune cells. Data from recent years clearly indicate that more chemokines than initially thought (CCL1/2/3/5/7/8/9/11, CXCL3/9/10/12/13/14/17; XCL1, CX3CL1) have pronociceptive properties; therefore, blocking their action by using neutralizing antibodies, inhibiting their synthesis, or blocking their receptors brings neuropathic pain relief. Several of them (CCL1/2/3/7/9/XCL1) have been shown to be able to reduce opioid drug effectiveness in neuropathy, and neutralizing antibodies against them can restore morphine and/or buprenorphine analgesia. The latest research provides irrefutable evidence that chemokine receptors are promising targets for pharmacotherapy; chemokine receptor antagonists can relieve pain of different etiologies, and most of them are able to enhance opioid analgesia, for example, the blockade of CCR1 (J113863), CCR2 (RS504393), CCR3 (SB328437), CCR4 (C021), CCR5 (maraviroc/AZD5672/TAK-220), CXCR2 (NVPCXCR220/SB225002), CXCR3 (NBI-74330/AMG487), CXCR4 (AMD3100/AMD3465), and XCR1 (vMIP-II). Recent research has shown that multitarget antagonists of chemokine receptors, such as CCR2/5 (cenicriviroc), CXCR1/2 (reparixin), and CCR2/CCR5/CCR8 (RAP-103), are also very effective painkillers. A multidirectional strategy based on the modulation of neuronal-glial-immune interactions by changing the activity of the chemokine family can significantly improve the quality of life of patients suffering from neuropathic pain. However, members of the chemokine family are still underestimated pharmacological targets for pain treatment. In this article, we review the literature and provide new insights into the role of chemokines and their receptors in neuropathic pain.

Dual IL-6 and CTLA-4 blockade regresses pancreatic tumors in a T cell- and CXCR3-dependent manner.

This study aimed to enhance antitumor immune responses to pancreatic cancer via Ab-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing s.c. or orthotopic pancreatic tumors were treated with blocking Abs to IL‑6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3-specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for antitumor efficacy. Both CD4+ and CD8+ T cells were required for the antitumor activity of this combination therapy, as their in vivo depletion via Abs impaired outcomes. These data represent the first report to our knowledge of IL-6 and CTLA‑4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

Optimized CAR-T therapy based on spatiotemporal changes and chemotactic mechanisms of MDSCs induced by hypofractionated radiotherapy

Poor intratumoral infiltration is the major challenge for chimeric antigen receptor (CAR)-T cell therapy in solid tumors. Hypofractionated radiotherapy (HFRT) has been reported to induce immune cell infiltration and reshape the tumor immune microenvironment. Here, we showed that HFRT (5×5 Gy)-mediated an early accumulation of intratumoral myeloid-derived suppressor cells (MDSCs) and decreased infiltration of T cells in the tumor microenvironment (TME) of immunocompetent mice bearing triple-negative breast cancer (TNBC) or colon cancer, which was further confirmed in tumors from patients. RNA sequencing (RNA-seq) and cytokine profiling analysis revealed that HFRT-induced the activation and proliferation of tumor-infiltrated MDSCs, and which was mediated by the interactions of multiple chemokines and chemokine receptors. Further investigation showed that when combined with HFRT, CXCR2 blockade significantly inhibited MDSCs trafficking to tumors, and effectively enhanced the intratumoral infiltration and treatment efficacy of CAR-T cells. Our study demonstrates that MDSCs blockade combined with HFRT is promising for CAR-T cell therapy optimization in solid tumors.

IMMU-43. CXCR3 SIGNALING ENGAGES GLIOMA INFILTRATING T CELLS AND IS REQUIRED FOR THE ANTITUMOR EFFICACY OF INNATE STIMULATING IMMUNOTHERAPY

Abstract BACKGROUND T cell dysfunction is well documented in patients with gliomas, yet mechanisms explaining T cell dysfunction in gliomas are lacking. Recent evidence links CXCR3 signaling and functional immune surveillance in both preclinical models and human data sets. METHODS Bulk and single cell RNA-seq data sets were used to compare CXCR3 ligand expression in gliomas to that of other solid tumor types. The role of CXCR3 signaling in mediating the recruitment and phenotype of tumor infiltrating lymphocytes (TILs) was tested using CXCR3 blockade and genetic deletion of CXCR3 in murine tumor models. The capacity of relevant innate stimulating immunotherapies to induce CXCR3 ligands in human glioma tissue was assessed using an ex vivo glioma slice culture assay, and the contribution of CXCR3 signaling to polio virotherapy (PVSRIPO) and STING agonist therapy was tested in murine models. RESULTS Relative to other solid tumors, gliomas express significantly lower levels of CXCR3 ligands (CXCL9, 10, and 11), which are largely expressed by tumor associated myeloid cells. Blocking CXCR3 signaling profoundly restricted TIL recruitment in murine gliomas, and resulted in decreased TIL stemness features coinciding with exacerbated exhaustion phenotypes. Polio virotherapy and STING agonism efficiently induced CXCR3 ligand production in fresh human glioma tissue in a manner dependent upon myeloid cells, and at levels similar to that induced by the same agents in melanoma and breast cancer tissue. CXCR3+ TILs expressed higher levels of memory and activation markers, proliferated more, and were required for the antitumor efficacy of both polio virotherapy and a STING agonist in mice. CONCLUSION This work indicates that CXCR3 signaling, a key contributor to cancer immune surveillance, is disrupted in human gliomas relative to other solid tumor types. Intratumor virotherapy and STING agonists hold potential to rectify diminished CXCR3 ligand expression in gliomas.

Anomalous diffusion and asymmetric tempering memory in neutrophil chemotaxis.

The motility of neutrophils and their ability to sense and to react to chemoattractants in their environment are of central importance for the innate immunity. Neutrophils are guided towards sites of inflammation following the activation of G-protein coupled chemoattractant receptors such as CXCR2 whose signaling strongly depends on the activity of Ca2+ permeable TRPC6 channels. It is the aim of this study to analyze data sets obtained in vitro (murine neutrophils) and in vivo (zebrafish neutrophils) with a stochastic mathematical model to gain deeper insight into the underlying mechanisms. The model is based on the analysis of trajectories of individual neutrophils. Bayesian data analysis, including the covariances of positions for fractional Brownian motion as well as for exponentially and power-law tempered model variants, allows the estimation of parameters and model selection. Our model-based analysis reveals that wildtype neutrophils show pure superdiffusive fractional Brownian motion. This so-called anomalous dynamics is characterized by temporal long-range correlations for the movement into the direction of the chemotactic CXCL1 gradient. Pure superdiffusion is absent vertically to this gradient. This points to an asymmetric ‘memory’ of the migratory machinery, which is found both in vitro and in vivo. CXCR2 blockade and TRPC6-knockout cause tempering of temporal correlations in the chemotactic gradient. This can be interpreted as a progressive loss of memory, which leads to a marked reduction of chemotaxis and search efficiency of neutrophils. In summary, our findings indicate that spatially differential regulation of anomalous dynamics appears to play a central role in guiding efficient chemotactic behavior.

Local delivery of a CXCR3 antagonist decreases the progression of bone resorption induced by LPS injection in a murine model.

This experimental study was carried out to investigate the effects of locally delivered nanoparticles (AMG-487 NP) containing a CXCR3 antagonist in inhibiting the progression of LPS-induced inflammation, osteoclastic activity, and bone resorption on a murine model. Thirty, 7-week-old C57BL/6J male mice were used. Inflammatory bone loss was induced by Porphyromonas gingivalis-lipopolysaccharide (P.g.-LPS) injections between the first and second maxillary molars, bilaterally, twice a week for 6weeks (n = 20). AMG-487 NP were incorporated into a liposome carrier and locally delivered on sites where P.g.-LPS was injected. Control mice (n = 10) were injected with vehicle only. Experimental groups included (1) control, (2) LPS, and (3) LPS + NP. At the end of 1 and 6weeks, mice were euthanized, maxillae harvested, fixed, and stored for further analysis. Volumetric bone loss analysis revealed, at 1week, an increase in bone loss in the LPS group (47.9%) compared to control (27.4%) and LPS + NP (27.8%) groups. H&E staining demonstrated reduced inflammatory infiltrate in the LPS + NP group compared to LPS group. At 6weeks, volumetric bone loss increased in all groups; however, treatment with the CXCR3 antagonist (LPS + NP) significantly reduced bone loss compared to the LPS group. CXCR3 antagonist treatment significantly reduced osteoclast numbers when compared to LPS group at 1 and 6weeks. This study showed that local delivery of a CXCR antagonist, via nanoparticles, in a bone resorption model, induced by LPS injection, was effective in reducing inflammation, osteoclast numbers, and bone loss. CXCR3 blockade can be regarded as a novel target for therapeutic intervention of bone loss. It can be a safe and convenient method for periodontitis treatment or prevention applicable in clinical practice.