Immune Cell Trafficking Research Articles

The ebb and flow of cardiac lymphatics: a tidal wave of new discoveries.

The heart is imbued with a vast lymphatic network that is responsible for fluid homeostasis and immune cell trafficking. Disturbances in the forces that regulate microvascular fluid movement can result in myocardial edema, which has profibrotic and proinflammatory consequences and contributes to cardiovascular dysfunction. This review explores the complex relationship between cardiac lymphatics, myocardial edema, and cardiac disease. It covers the revised paradigm of microvascular forces and fluid movement around the capillary as well as the arsenal of preclinical tools and animal models used to model myocardial edema and cardiac disease. Clinical studies of myocardial edema and their prognostic significance are examined in parallel to the recent elegant animal studies discerning the pathophysiological role and therapeutic potential of cardiac lymphatics in different cardiovascular disease models. This review highlights the outstanding questions of interest to both basic scientists and clinicians regarding the roles of cardiac lymphatics in health and disease.

Gut immune cell trafficking: inter-organ communication and immune-mediated inflammation.

Immune cell trafficking is a complex and tightly regulated process that is indispensable for the body's fight against pathogens. However, it is also increasingly acknowledged that dysregulation of cell trafficking contributes to the pathogenesis of immune-mediated inflammatory diseases (IMIDs) in gastroenterology and hepatology, such as inflammatory bowel disease and primary sclerosing cholangitis. Moreover, altered cell trafficking has also been implicated as a crucial step in the immunopathogenesis of other IMIDs, such as rheumatoid arthritis and multiple sclerosis. Over the past few years, a central role of the gut in mediating these disorders has progressively emerged, and the partly microbiota-driven imprinting of particular cell trafficking phenotypes in the intestine seems to be crucially involved. Therefore, this Review highlights achievements in understanding immune cell trafficking to, within and from the intestine and delineates its consequences for immune-mediated pathology along the gut-liver, gut-joint and gut-brain axes. We also discuss implications for current and future therapeutic approaches that specifically interfere with homing, retention, egress and recirculation of immune cells.

Abstract P3066: Macrophage-derived Ccl24 Worsens Cardiac Dysfunction Following Pressure-overload

Heart failure (HF) is the leading cause of death worldwide. Systemic inflammation is associated with HF and is considered an independent predictor of adverse cardiac outcomes. Macrophages are the most abundant immune cells in the heart and have integral roles in cardiac homeostasis and remodeling. Despite their unique identity and homeostatic functions, the precise role of cardiac macrophages during cardiac remodeling is poorly understood. We have previously reported that cardiac resident macrophages expand early in response to pressure overload injury and provide cardioprotection. Using single-cell mRNA sequencing, we revealed that these macrophages consist of distinct subsets including a population that highly expresses C-C Motif Chemokine Ligand 24 (CCL24). CCL24 is a chemokine that promotes immune cell trafficking and activation through its receptor expressed on immune cells, fibroblasts, and cardiomyocytes. Here, we tested whether CCL24 regulates cardiac adaptation to pressure overload by performing transverse aortic constriction (TAC) in whole-body CCL24 knockout (CCL24KO) mice. Aortic constriction during TAC increases the pressure overload in ventricular chambers leading to an early compensated hypertrophy and diastolic dysfunction followed by systolic dysfunction and decompensated heart failure. Despite no differences at steady state, CCL24KO mice showed an improved cardiac function after TAC-induced pressure overload. Compared with WT controls, CCL24KO mice had a higher ejection fraction and fractional shortening. Furthermore, CCL24KO mice had increased hypertrophy but improved diastolic function. These results suggest that CCL24 worsens cardiac dysfunction leading to decompensated HF in response to pressure overload. Interestingly, CCL24KO mice have higher proportions of neutrophils and eosinophils at steady state, suggesting that the presence of these cells may facilitate cardiac repair. Overall, our findings indicate that macrophage-derived CCL24 is detrimental to cardiac function in response to pressure overload injury. The results of this study uncover CCL24 as a central mechanism by which cardiac resident macrophages promote cardiac inflammation and HF.

Vascular and lymphatic regulation of gastrointestinal function and disease risk

The vascular and lymphatic systems in the gut regulate lipid transport while restricting transfer of commensal gut microbiota and directing immune cell trafficking. Increased permeability of the endothelial systems in the intestine associates with passage of antigens and microbiota from the gut into the bloodstream leading to tissue inflammation, the release of pro-inflammatory mediators and ultimately to abnormalities of systemic metabolism. Recent studies show that lipid metabolism maintains homeostasis and function of intestinal blood and lymphatic endothelial cells, BECs and LECs, respectively. This review highlights recent progress in this area, and information related to the contribution of the lipid transporter CD36, abundant in BECs and LECs, to gastrointestinal barrier integrity, inflammation, and to gut regulation of whole body metabolism. The potential role of endothelial lipid delivery in epithelial tissue renewal after injury and consequently in the risk of gastric and intestinal diseases is also discussed.

Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues.

Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus. It affects many women during their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, which limits early diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared them to matched eutopic endometrium, unaffected endometrium and organoids derived from these tissues, generating data on over 122,000 cells across 14 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell specific to the peritoneal lesions, with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesion microenvironments and an unreported progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment that represents a comprehensive cell atlas of the disease in individuals undergoing hormonal treatment, providing essential information for future therapeutics and diagnostics.

767 A biopsy-sized 3D model of skin vascular plexus and appendages enables monitoring T cell trafficking

The human skin vasculature has distinct anatomy with two vascular plexuses and specialized capillary loops near skin appendages and rete ridges, and has diverse functions including thermal regulation and immune cell trafficking. There is currently no engineered 3D skin model to capture this level of complexity. The goal of this study is to generate an anatomically-relevant 3D skin model with five different skin cell types, patterned hair follicles, and a perfusable microvasculature mimicking the superficial vascular plexus of the skin.

Full spectrum of vitamin D immunomodulation in multiple sclerosis: mechanisms and therapeutic implications.

Vitamin D deficiency has been associated with the risk of multiple sclerosis, disease activity and progression. Results from in vitro experiments, animal models and analysis of human samples from randomized controlled trials provide comprehensive data illustrating the pleiotropic actions of Vitamin D on the immune system. They globally result in immunomodulation by decreasing differentiation of effector T and B cells while promoting regulatory subsets. Vitamin D also modulates innate immune cells such as macrophages, monocytes and dendritic cells, and acts at the level of the blood–brain barrier reducing immune cell trafficking. Vitamin D exerts additional activity within the central nervous system reducing microglial and astrocytic activation. The immunomodulatory role of Vitamin D detected in animal models of multiple sclerosis has suggested its potential therapeutic use for treating multiple sclerosis. In this review, we focus on recent published data describing the biological effects of Vitamin D in animal models of multiple sclerosis on immune cells, blood–brain barrier function, activation of glial cells and its potential neuroprotective effects. Based on the current knowledge, we also discuss optimization of therapeutic interventions with Vitamin D in patients with multiple sclerosis, as well as new technologies allowing in-depth analysis of immune cell regulations by vitamin D.

P793: SCREENING OF NATURALLY OCCURRING CXCR4 VARIANTS FOR IDENTIFICATION OF NOVEL PATHOGENIC MUTATIONS FOR WHIM SYNDROME

Background: WHIM (Warts, Hypogammaglobulinemia, Infections, Myelokathexis) syndrome is a rare, autosomal-dominant primary immunodeficiency marked by neutropenia and lymphopenia. Classically, WHIM syndrome pathogenesis is causally linked to a variety of heterozygous gain-of-function mutations in the C-terminus of chemokine receptor CXCR4, a master regulator of immune cell trafficking and homeostasis. As of February 2022, 18 variants in CXCR4 have been implicated in WHIM syndrome, while a substantial number remain unexplored for their association with the disease. Using in vitro functional assays, we previously found that impaired receptor internalization and enhanced chemotaxis are conserved in all CXCR4WHIM variants and that defective internalization correlates with neutropenia, the most penetrant phenotype of patients with WHIM syndrome. We used this approach to confirm functional defects in primary cells isolated from patients harboring a novel variant, suggesting that the in vitro profiling approach can potentially drive identification of pathogenic variants. Aims: We aimed to functionally characterize multiple previously uncharacterized variants of CXCR4 using in vitro assays. We also examined allele frequencies of these novel variants to estimate the potential number of individuals harboring these variants with a long-term goal of determining the actual prevalence of WHIM syndrome. Methods: The CXCR4-negative K562 cell line was used as a model system to express 53 novel CXCR4 variants identified in patient and population databases (ClinVar, Ensembl, CentoMD, GnomAD) and genetic screening initiatives (Invitae PATH4WARD) found in the C-terminus (hotspot of CXCR4WHIM mutations) as well as throughout the protein. The effects of these mutations on CXCR4 internalization and chemotaxis were studied in cells stimulated with the C-X-C chemokine ligand 12 (CXCL12). The in vitro functional parameters were compared with known pathogenic CXCR4 variants and were used to assign a cumulative score (impaired internalization + enhanced chemotaxis) of functional defect severity. Results: Out of 53 selected variants, 42 were missense (ms), 6 were frameshift (fs), 3 were nonsense (ns), and 2 were transcript variants (tv). Eighteen of the screened variants led to a decreased internalization of CXCR4 in comparison to the wild-type (WT) receptor upon stimulation with CXCL12 while 20 variants demonstrated enhanced chemotaxis of cells toward CXCL12. Overall, 34 variants showed defects in at least one CXCR4-dependent marker. Correlation analysis and scoring of the functional parameters in cells expressing mutated or CXCR4WT revealed 17 variants (9 ms, 5 fs, 2 ns, 1 tv) that co-segregated with the known pathogenic WHIM variants. Finally, white blood cell counts in 3 patients harboring novel variants showed that CXCR4 internalization defects in vitro correlated with the severity of neutropenia. Summary/Conclusion: This is the first study characterizing functional impairments in naturally occurring CXCR4 variants via a pipeline of assays intended to predict pathogenicity. Seventeen CXCR4 variants were identified (several outside the C-terminus of CXCR4) that caused in vitro functional defects resembling those exhibited by known WHIM variants. Many (10/17) of these newly identified variants are present at high frequencies (4.4 × 10-4–8.0 × 10-6) in genomic databases. Potential association of these variants with WHIM syndrome is under investigation and is likely to expand current estimates of WHIM syndrome prevalence.

Abstract 3874: Engineered microphysiological systems for testing effectiveness of cell-based cancer immunotherapies in small cell lung cancer

Abstract Background: Immune cell trafficking into solid tumors has proven challenging due to immune cell exclusion by vascular barriers. Additionally, testing next-generation immune therapies remains difficult in animal models and requires sophisticated ex vivo systems to study human tumor biology and predict treatment response in real time. SCLC comprises approximately 15% of all lung cancer cases and despite recent incorporation of immune checkpoint blockade (ICB) into first line treatment, patients with SCLC continue to have a poor prognosis and limited treatment options. Notably, recent transcriptomic profiling of SCLC has indicated significant inter-tumoral and intra-tumoral heterogeneity. We have previously demonstrated that neuroendocrine SCLC subpopulations downregulate MHC I, whereas non-neuroendocrine SCLC subpopulations exhibit increased innate immune signaling and robust upregulation of MHC I antigen presentation. Here we interrogate the vulnerability of SCLC populations to NK cells and investigate their trafficking using a novel microphysiological ex vivo model. Methods: We developed a 3-dimensional (3D) microphysiological TME model of vascularized SCLC, comprising tumor spheroids embedded in a microvascular network self-assembled by endothelial cells and lung fibroblasts. Real time fluorescence imaging, cell-titer Glo and flow cytometry were used to quantify tumor cell viability in 2D and 3D microfluidic co-culture cytotoxicity assays, using primary NK cells and the NK92 cell line. Results: We demonstrated robust targeting of MHC-I low SCLC cells by primary human NK cells and NK92, and found that MHC-I high SCLC subpopulations show resistance to NK cell-mediated killing. To dissect the influence of vascular barriers on immune cell trafficking, we developed a 3D microphysiological TME model of vascularized SCLC, which successfully recapitulated tumor-vascular-immune interactions in a human-relevant platform as evidenced by vascular perfusion by NK cells. Using this model, we were able to demonstrate that the activation of cGAS-STING signaling in endothelial cells results in the secretion of CXCL10 and upregulation of adhesion molecules (ICAM-1, VCAM-1, E-selectin), promoting immune cell recruitment. Conclusions: We successfully developed a 3D microphysiological model of vascularized SCLC TME and show proof of principle that this model can be used for preclinical studies of cell therapies. Using this novel microphysiological system, we revealed that co-opting STING signaling can promote NK cell recruitment to the SCLC TME. Taken together, we show that microphysiological systems can be used to identify and test therapeutic vulnerabilities while studying immune infiltration and killing in the TME, offering an accurate and adaptable platform for biologic discovery and therapeutic development. Citation Format: Marco Campisi, Minyue Chen, Nathaniel Spicer, Patrick Lizotte, Erik H. Knelson, Cloud P. Paweletz, David A. Barbie, Navin R. Mahadevan. Engineered microphysiological systems for testing effectiveness of cell-based cancer immunotherapies in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3874.

Abstract 1718: Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing

Abstract Background: Radiation therapy is delivered to approximately 50% of all cancer patients. It has long been known that radiation causes not only DNA strand breaks, apoptosis, and necrosis, but also immunogenic modulation. Lately, there has been renewed interest in the potential of irradiation in colorectal cancer, not only as an adjuvant and palliative treatment, but also as an immune stimulant. Materials and Methods: CT26 murine colorectal carcinoma cells were subcutaneously implanted on the flank of BALB/c mice. Tumors were exposed to sham, single dose or fractionated radiotherapy regimens via a 300kV, 10mA X-ray with additional filtration to give a radiation quality of 2.3mm Cu half-value layer (HVL). Mice were anaesthetized, restrained and delivered a single uniform dose of radiation at a rate of 0.696Gy/min with the whole mouse being lead shielded except for a 1cm hole in the lead above the tumor site. Tumors were harvested at different timepoints post irradiation and dissociated into single cell suspensions. The tumor microenvironment and immune response within the tumors were analyzed with flow cytometry comparing the different treatment regimens. The individual CD45+ cell profile was investigated using 10x gene expression. 10x sequencing was demultiplexed using CellRanger and data analyzed using Seurat and ACTINN. Results: Following the initial cytotoxicity, flow cytometry showed a clear immune stimulatory effect of radiotherapy with immune cells infiltrating the irradiated tumors. The level of infiltration was dependent on the treatment scheduling. For single cell assessments, we observed approximately 8000 reads/cell and T-SNE cluster analysis using Seurat identified 16 clusters in control and treated samples. Identification of clusters by ACTINN demonstrated that CD8+ T-cells were increased 8-fold in irradiated samples whereas monocytes numbers decreased 3-fold. In irradiated tumors, the main population of CD8+ T-cells with increased numbers also expressed NKG7. Conclusion: These data provide evidence that radiotherapy can enhance immune cell trafficking to locally treated CT26 tumors. Furthermore, 10x genomics single cell sequencing revealed increased levels of cytotoxic T-cells expressing NKG7, a protein implicated in enhanced killing of extracellular targets. This response could prime the tumors such that they may be exploited by immune modulating drugs. Citation Format: Frida Ponthan, Valentina Ubertini, Gary Beale. Characterization of infiltrating immune cells after irradiation using 10x genomics single cell sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1718.

The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cell homing to bone.

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.

Role of hair follicles in the pathogenesis of arsenical-induced cutaneous damage.

Arsenical vesicants cause skin inflammation, blistering, and pain. The lack of appropriate animal models causes difficulty in defining their molecular pathogenesis. Here, Ptch1+/- /C57BL/6 mice were employed to investigate the pathobiology of the arsenicals lewisite and phenylarsine oxide (PAO). Following lewisite or PAO challenge (24h), the skin of animals becomes grayish-white, thick, leathery, and wrinkled with increased bi-fold thickness, Draize score, and necrotic patches. In histopathology, infiltrating leukocytes (macrophages and neutrophils), epidermal-dermal separation, edema, apoptotic cells, and disruption of tight and adherens junction proteins can be visualized. PCR arrays and nanoString analyses showed significant increases in cytokines/chemokines and other proinflammatory mediators. As hair follicles (HFs), which provide an immune-privileged environment, may affect immune cell trafficking and consequent inflammatory responses, we compared the pathogenesis of these chemicals in this model to that in Ptch1+/- /SKH-1 hairless mice. Ptch1+/- /SKH-1 mice have rudimentary, whereas Ptch1+/- /C57BL/6 mice have well-developed HFs. Although no significant differences were observed in qualitative inflammatory responses between the two strains, levels of cytokines/chemokines differed. Importantly, the mechanism of inflammation was identical; both reactive oxygen species induction and consequent activation of unfolded protein response signaling were similar. These data reveal that the acute molecular pathogenesis of arsenicals in these two murine models is similar.

Lymphatic Clearance and Pump Function.

Lymphatic vessels have an active role in draining excess interstitial fluid from organs and serving as conduits for immune cell trafficking to lymph nodes. In the central circulation, the force needed to propel blood forward is generated by the heart. In contrast, lymphatic vessels rely on intrinsic vessel contractions in combination with extrinsic forces for lymph propulsion. The intrinsic pumping features phasic contractions generated by lymphatic smooth muscle. Periodic, bicuspid valves composed of endothelial cells prevent backflow of lymph. This work provides a brief overview of lymph transport, including initial lymph formation along with cellular and molecular mechanisms controlling lymphatic vessel pumping.

CXCR4 overexpression: An indicator of poor survival and predictor of response to immunotherapy in patients with metastatic colorectal cancer.

3546 Background: CXC-chemokine receptor 4 (CXCR4) is a ubiquitous chemokine receptor activated by the CXCL12 ligand and is implicated in tumor invasion, metastasis, and immune cell (IC) trafficking. High CXCR4 expression is associated with poor prognosis in colorectal cancer (CRC). < 10% of metastatic CRC cases harbor microsatellite instability (MSI-H) and demonstrate lower tumor mutation burden (TMB), decreased IC infiltration, and lack of response to current immunotherapy regimens. This study aims to interrogate the role of CXCR4 mRNA expression on the the tumor microenvironment (TME) and its prognostic and predictive value to tailor immunotherapeutic treatment strategies in CRC. Methods: A total of 15,026 CRC samples were analyzed using whole-exome sequencing, whole-transcriptome sequencing, and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). Study cohort was stratified by CXCR4 mRNA expression levels in quartiles (Q1 (low) vs Q4 (high)). IC fraction was calculated by QuantiSeq, and real-world overall survival information was obtained from insurance claims data and calculated from tissue collection time to last day of contact. Statistical significance was determined using chi-square/Fisher-Exact and adjusted for multiple comparisons (q < 0.05). Results: Samples obtained from metastatic sites showed higher CXCR4 mRNA expression than those from primary tumors (22.7 vs 18.6 median transcripts per million (TPM), p < 0.001). CXCR4 mRNA expression was significantly lower in liver metastases than in non-liver metastases (21.2 vs 24.8 TPM, p < 0.001). Median CXCR4 mRNA expression was highest in the consensus molecular subtypes 4 (33.3 TPM) and lowest in 3 (13.0 TPM, p < 0.05). CXCR4 mRNA expression was positively associated with TMB-H, MSI-H/dMMR, and positive PD-L1 IHC status. In the TME, high CXCR4 mRNA expression was observed in tumors with a higher IC infiltration including B cells, M1/M2 macrophages, NK cells, CD8+ T cells and T-regs, regardless of MSI status. High CXCR4 mRNA expression in the primary tumor was associated with poor prognosis (HR 0.77, 95% CI 0.70-0.85; p < 0.001), regardless of MSI-status. In metastatic tumors, low mRNA expression was correlated with improved survival (HR 0.89, 95% CI 0.80-0.99; p = 0.34); however, this did not reach statistical significance in the MSS cohort (HR 0.90, 95% CI 0.80-1.0; p = 0.06). Of note, high CXCR4 mRNA expression was associated with improved survival in all patients with CRC who received pembrolizumab (HR 2.12, 95% CI 1.16-3.91; p = 0.013). Conclusions: This is the largest clinical dataset to date demonstrating high CXCR4 expression as a predictor for poor survival in CRC. Furthermore, high CXCR4 expression was associated with improved outcome after checkpoint inhibition immunotherapy, indicating its strong potential as a predictive biomarker that could inform immunotherapeutic strategies in CRC.

Gut microbiome-mediated regulation of neuroinflammation.

The intestinal microbiome influences neuroinflammatory disease in animal models, and recent studies have identified multiple pathways of communication between the gut and brain. Microbes are able to produce metabolites that enter circulation, can alter inflammatory tone in the intestines, periphery, and central nervous system (CNS), and affect trafficking of immune cells into the brain. Additionally, the vagus nerve that connects the enteric nervous system to the CNS is implicated in modulation of brain immune responses. As preclinical research findings and concepts are applied to humans, the potential impacts of the gut microbiome-brain axis on neuroinflammation represent exciting frontiers for further investigation.

Photodynamique therapy relieves tumor vascular anergy and promotes immune cell trafficking in an orthotopic mouse model of malignant pleural mesothelioma

Abstract Objective Malignant pleural mesothelioma (MPM) is a deadly disease with limited treatment options. Recently, dual immune checkpoint inhibition therapy (ICI) showed improved patient survival. However, only a fraction of patients were responsive to immunotherapy. One potential mechanism of MPM resistance to ICIs could be their endothelial anergy that hampers leukocyte trafficking to the tumor bulk. Here, we hypothesized that vascular-targeted low dose photodynamic therapy (L-PDT), treatment of MPM could relieve tumor endothelial anergy and improve immunotherapy efficacy. Methods Using an orthotopic syngeneic MPM murine model (AB12 cells injected in the pleura of BALB/c mice), we determined the impact of L-PDT on the endothelial expression of E-Selectin, a key molecule involved in leukocyte diapedesis by immunohistochemistry. Furthermore, to confirm the role of E-selectin, we determined the extravasation of effector T cells (CD8+/CD4+) by immunostaining in L-PDT treated tumors in the presence or absence of an E-selectin blocking antibody. Finally, we assessed tumor growth/survival of our MPM murine model treated with L-PDT alone or combined to ICIs. Results L-PDT pre-treatment enhanced MPM endothelial E-Selectin expression in vivo. The latter was associated with increased CD4+ and CD8+ lymphocyte infiltration of MPM following L-PDT which did not occur after E-Selectin blockade. Also, L-PDT pre-treatment of MPM influenced favorably tumor control, mouse survival and the impact of ICIs compared to controls. Conclusion L-PDT pre-treatment relieves endothelial anergy in MPM which improves antitumor immunity and response to ICI. This approach could constitute a promising pre-treatment option, in combination with ICIs, for the management of this deadly disease.

Abstract No. 354 Effect of variable radiofrequency ablation (RFA) thermal doses on intratumoral antigen presenting cell (APC) trafficking

Intratumoral APCs such as dendritic cells (DCs) and tumor-associated macrophages (TAMs) play a key role in initiation of anti-tumor antigen specific immunity especially when polarized to mature CD80 positive (DCs) vs immature phenotypes and pro-inflammatory phenotypes (M1) vs tissue healing phenotypes (M2). Thermal tumor ablation (with RFA) can induce increased and altered intratumoral immune cell trafficking. However, variable thermal doses have been shown to induce different intratumoral cellular infiltration patterns.

High-Fat Nutritional Challenge Reshapes Circadian Signatures in Murine Extraorbital Lacrimal Glands.

PurposeA high-fat diet (HFD) increases the risk of developing many systemic diseases; however, the effects of high fat intake on lacrimal gland functions and the molecular mechanisms underlying these effects are unknown. We explored the effects of an HFD on the circadian rhythms of the extraorbital lacrimal glands (ELGs).MethodsMale C57BL/6J mice maintained on a 12/12-hour light/dark cycle were fed an ad libitum HFD or normal chow (NC) for 2 weeks. The ELGs were collected from euthanized animals every 3 hours throughout the circadian cycle (24 hours). Using high-throughput RNA-sequencing (RNA-Seq), we studied the circadian transcriptomic profile of the ELGs. Circadian oscillations in cell size, secretion response, lipid deposition, and immune cell trafficking of the ELGs were also analyzed.ResultsAn HFD modulated the circadian transcriptomic profile of the ELGs, including the composition, phase, and amplitude of cyclical transcript oscillations, and affected the associated signaling pathways at spatiotemporal levels. HFD feeding significantly altered the normal rhythmic oscillations of ELG cell size, immune cell trafficking, secretion response, and lipid deposition. After dietary reversal in HFD-fed animals, the activity, core temperature, and lipid accumulation in lacrimal glands recovered partially to the level of NC-fed animals. However, the average cell size of the ELGs, the recruitment of immune cells, and the rhythm of lacrimal secretion did not return to the levels of the NC-fed group.ConclusionsHFD perturbation interferes with the cyclical transcriptomic profile, cell size, immune cell trafficking, and secretion function of the ELGs with a strikingly high sensitivity.

Immune-vascular mural cell interactions: consequences for immune cell trafficking, cerebral blood flow, and the blood-brain barrier.

.Brain barriers are crucial sites for cerebral energy supply, waste removal, immune cell migration, and solute exchange, all of which maintain an appropriate environment for neuronal activity. At the capillary level, where the largest area of brain–vascular interface occurs, pericytes adjust cerebral blood flow (CBF) by regulating capillary diameter and maintain the blood–brain barrier (BBB) by suppressing endothelial cell (EC) transcytosis and inducing tight junction expression between ECs. Pericytes also limit the infiltration of circulating leukocytes into the brain where resident microglia confine brain injury and provide the first line of defence against invading pathogens. Brain “waste” is cleared across the BBB into the blood, phagocytosed by microglia and astrocytes, or removed by the flow of cerebrospinal fluid (CSF) through perivascular routes—a process driven by respiratory motion and the pulsation of the heart, arteriolar smooth muscle, and possibly pericytes. “Dirty” CSF exits the brain and is probably drained around olfactory nerve rootlets and via the dural meningeal lymphatic vessels and possibly the skull bone marrow. The brain is widely regarded as an immune-privileged organ because it is accessible to few antigen-primed leukocytes. Leukocytes enter the brain via the meninges, the BBB, and the blood-CSF barrier. Advances in genetic and imaging tools have revealed that neurological diseases significantly alter immune–brain barrier interactions in at least three ways: (1) the brain’s immune-privileged status is compromised when pericytes are lost or lymphatic vessels are dysregulated; (2) immune cells release vasoactive molecules to regulate CBF, modulate arteriole stiffness, and can plug and eliminate capillaries which impairs CBF and possibly waste clearance; and (3) immune–vascular interactions can make the BBB leaky via multiple mechanisms, thus aggravating the influx of undesirable substances and cells. Here, we review developments in these three areas and briefly discuss potential therapeutic avenues for restoring brain barrier functions.

Multiparameter Longitudinal Imaging of Immune Cell Activity in Chimeric Antigen Receptor T Cell and Checkpoint Blockade Therapies.

Longitudinal multimodal imaging presents unique opportunities for noninvasive surveillance and prediction of treatment response to cancer immunotherapy. In this work we first designed a novel granzyme B activated self-assembly small molecule, G-SNAT, for the assessment of cytotoxic T lymphocyte mediated cancer cell killing. G-SNAT was found to specifically detect the activity of granzyme B within the cytotoxic granules of activated T cells and engaged cancer cells in vitro. In lymphoma tumor-bearing mice, the retention of cyanine 5 labeled G-SNAT-Cy5 correlated to CAR T cell mediated granzyme B exocytosis and tumor eradication. In colorectal tumor-bearing transgenic mice with hematopoietic cells expressing firefly luciferase, longitudinal bioluminescence and fluorescence imaging revealed that after combination treatment of anti-PD-1 and anti-CTLA-4, the dynamics of immune cell trafficking, tumor infiltration, and cytotoxic activity predicted the therapeutic outcome before tumor shrinkage was evident. These results support further development of G-SNAT for imaging early immune response to checkpoint blockade and CAR T-cell therapy in patients and highlight the utility of multimodality imaging for improved mechanistic insights into cancer immunotherapy.