Depletion Of Immune Cells Research Articles

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model

BackgroundCD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model.MethodsThe contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments.ResultsWe showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells.ConclusionsThe study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

Restriction of viral replication, rather than T cell immunopathology, drives lethality in MNV CR6-infected STAT1-deficient mice

Abstract Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. Here, we used colonization of C57BL6/J mice with the model commensal murine norovirus (MNV CR6) to interrogate host-directed mechanisms of viral regulation, and show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In STAT1-sufficient mice MNV CR6 is asymptomatic and restricted to the colon, despite the persistence of high viral loads. However, in the absence of STAT1 viral replication is disseminated and observed in key innate and adaptive immune populations. STAT1-deficient mice develop dysregulated inflammatory CD8+ and CD4+ T cell responses, which coincide with weight loss and severe necrosis of the spleen and liver. Despite these altered T cell responses which resemble those that mediate lethal immunopathology following other model viral infections of STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. In contrast, therapeutic administration of an antiviral compound limited viral replication, preventing viral-induced tissue damage and death despite ongoing inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa. Unlike other model viral infections, CR6 induces disease in STAT1-deficient mice via uncontrolled viral replication rather than the concomitant development of dysregulated antiviral T cell responses. Research in the Osborne lab is supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, the Canada Research Chair program and scholarships from the University of British Columbia.

Abstract 379: Smooth Muscle-specific Lncrna Carmn Plays A Potential Role In Aortic Aneurysm

Background: Aortic aneurysm is a life-threatening vascular disease characterized by vascular smooth muscle cell (VSMC) depletion, ECM degradation, and infiltration of immune cells. Previous studies have shown that Carmn , a SMC-enriched long non-coding RNA, is critical for maintaining VSMC contractile phenotype. However, its functional role in aortic aneurysm remains completely unknown. Methods and Results: We re-analyzed the publicly available aortic aneurysm bulk RNA-seq and scRNA-seq datasets in mouse and human. These unbiased analyses revealed that Carmn is downregulated in the SMCs of aortic aneurysm samples. To examine the potential role of Carmn in aortic aneurysm, we generated Carmn global knockout (gKO) mice or inducible SMC-specific KO (iKO) mice driven by Myh11 -Cre ER T2, respectively. Unexpectedly, both Carmn gKO and iKO mice exhibited premature lethality, due to a severe colonic pseudo-obstruction resulting from deletion of Carmn in visceral SMCs. Despite the dominant gastrointestinal phenotype observed in Carmn KO mice, morphological analysis of the Carmn KO thoracic aorta revealed thinner vascular wall compared to that of controls. Results from TEM showed that the VSMC in the Carmn KO thoracic aorta are disorganized and degradative with aneurysmal dissection. Data from bulk RNA-seq of Carmn -deficient aorta revealed that the down-regulated genes are associated with degradation of ECM while the up-regulated genes are associated with cell death. Moreover, scRNA-seq of Carmn -deficient aorta revealed that the percentage of the SMCs is decreased while the percentage of infiltrated immune cells is increased. These data indicated that Carmn deficiency may play a critical role in promoting the development of aortic aneurysm. To circumvent the lethal visceral SMC phenotype, we are in the progress of crossing Carmn flox mice with the novel vascular SMC-specific inducible Itga8 -Cre ER T2 mouse to generate VSMC-specific Carmn KO mice. Conclusions: Our data suggest that Carmn is indispensable for maintaining gastrointestinal contractile function, and implicate that Carmn deficiency plays a potential role in contributing the development of aortic aneurysm.

Protein Kinase Inhibitor-Mediated Immunoprophylactic and Immunotherapeutic Control of Colon Cancer.

Immunotherapy has allowed major advances in oncology in the past years, in particular with the development of immune checkpoint inhibitors, but the clinical benefits are still limited, particularly in colorectal cancer (CRC). Our scientific approach is based on the search for innovative immunotherapy with a final goal that aims to induce an effective antitumor immune response in CRC. Here, we focused on a multikinase inhibitor, H89. We carried out in vivo experiments based on syngeneic mouse models of colon cancer in BALB/c mice and chemically colon tumorigenesis. Flow cytometry, RNAseq, RT-qPCR, antibody-specific immune cell depletion, and Western blot were used to identify the immune cell type involved in the preventive and antitumor activity of H89. We demonstrated that H89 delays colon oncogenesis and prevents tumor growth. This latter effect seems to involve NK cells. H89 also inhibits colon tumor growth in a T-cell-dependent manner. Analysis of the immune landscape in the tumor microenvironment showed an increase of CD4+ Th1 cells and CD8+ cytotoxic T cells but a decrease of CD4+ Treg cell infiltration. Mechanistically, we showed that H89 could promote naïve CD4+ T-cell differentiation into Th1, a decrease in Treg differentiation, and an increase in CD8+ T-cell activation and cytotoxicity ex vivo. Furthermore, H89 induced overexpression of genes involved in antitumor immune response, such as IL-15RA, which depletion counteracts the antitumor effect of H89. We also found that H89 regulated Akt/PP2A pathway axis, involved in TCR and IL-15 signaling transduction. Our findings identify the H89 as a potential strategy for immune system activation leading to the prevention and treatment of CRC.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

The interaction between end-metabolites and immune escape

Abstract Emerging data from metabolites-relating trails in cancers demonstrate that a common mechanism of resistance to many novel classes of immune therapeutics is the emergence of immune escape due to the reprogramming of cellular metabolism. Among them, current work about end-metabolites mostly focuses on the intersection between lactate acid, adenosine, reactive oxygen species (ROS), and tumour immune escape. In this article, we aim to review the evidence to date for the dynamic interplay between the three end-metabolites and tumour immune escape for potential approaches to overcome obstacles in the efficacy and durability of immune cancer therapies. We have organized known end-metabolites-associated immune escape mechanisms into three hallmarks: (1) decreased immunogenicity of cancer cells which constitutes defective antigen presentation and the attenuated expression of costimulatory molecules on tumour cells, (2) immunosuppressive microenvironment with aberrant angiogenesis inhibits the differentiation, maturation, and immune deviation of immune cells while drives the activation of immunosuppressive cells by immune-suppressive mediators (cytokines and other factors), (3) immune tolerance retained by inhibitory molecules and depletion of immune cells.

Short-Time Effect of Alemtuzumab Induction Therapy on B- and T-cell Subsets After Lung Transplantation

<h3>Purpose</h3> Alemtuzumab is a monoclonal antibody targeting CD52, one of the most predominant antigens on the surface of lymphocytes. It induces cell lysis and leads to a profound immune cell depletion. However, little is known how T- and B-cell repopulate and if there is predominance towards certain subtypes. <h3>Methods</h3> B- and T cell subsets were analyzed from whole blood samples of 25 patients, prospectively collected at time of listing (=baseline) and 6 months post-transplant. Cell subsets were measured by FACS. Patients received alemtuzumab induction followed by low-dose tacrolimus and steroids. <h3>Results</h3> Median age of the cohort was 59 years (51-64) and 36% of patients were female. Underlying diagnoses were COPD (36%), fibrosis (20%), CF (12%), PPH (4%), EAA (12%) and others (16%). B- and T cell counts significantly decreased compared to baseline (p=0.009, p<0.001). Within B cell population, relative frequency of IgM⁺CD27⁻CD38^high, IgM⁺CD27⁺CD38^high and regulatory B cells increased 6 months after Tx (p=0.017, p=0.016, p=0.028, respectively). Within T cell population, CD4⁺ T cells decreased 6 months after Tx (p<0.001), while relative frequency of CD8⁺ T cells was significantly higher (p<0.001). A detailed analysis revealed a shift towards CD4⁺CD57⁺(p=0,002), PD1⁺CD4⁺(p<0.001), CD45RA+CD197-CD4⁺ (p=0.0013), CD45RA⁻CD197⁻CD4⁺ (p<0.001), CD27⁻CD28⁺ (p<0.001), CD27⁺CD28⁺ (p<0.001), PD1⁺CD8⁺ (p<0.001), CD45RA⁺CD197⁻CD8⁺ (p=0.039) and regulatory FoxP3⁺-T cells (p=0.005) at 6 months after Tx. <h3>Conclusion</h3> After alemtuzumab induction therapy, the composition of B and T cell subsets is altered compared to baseline. Expansion of subsets such as PD1 T cells and regulatory B- and T cells suggests an immunomodulatory effect of alemtuzumab on cell repopulation.

Aggregates Associated with Instability of Antibodies during Aerosolization Induce Adverse Immunological Effects.

Background: Immunogenicity refers to the inherent ability of a molecule to stimulate an immune response. Aggregates are one of the major risk factors for the undesired immunogenicity of therapeutic antibodies (Ab) and may ultimately result in immune-mediated adverse effects. For Ab delivered by inhalation, it is necessary to consider the interaction between aggregates resulting from the instability of the Ab during aerosolization and the lung mucosa. The aim of this study was to determine the impact of aggregates produced during aerosolization of therapeutic Ab on the immune system. Methods: Human and murine immunoglobulin G (IgG) were aerosolized using a clinically-relevant nebulizer and their immunogenic potency was assessed, both in vitro using a standard human monocyte-derived dendritic cell (MoDC) reporter assay and in vivo in immune cells in the airway compartment, lung parenchyma and spleen of healthy C57BL/6 mice after pulmonary administration. Results: IgG aggregates, produced during nebulization, induced a dose-dependent activation of MoDC characterized by the enhanced production of cytokines and expression of co-stimulatory markers. Interestingly, in vivo administration of high amounts of nebulization-mediated IgG aggregates resulted in a profound and sustained local and systemic depletion of immune cells, which was attributable to cell death. This cytotoxic effect was observed when nebulized IgG was administered locally in the airways as compared to a systemic administration but was mitigated by improving IgG stability during nebulization, through the addition of polysorbates to the formulation. Conclusion: Although inhalation delivery represents an attractive alternative route for delivering Ab to treat respiratory infections, our findings indicate that it is critical to prevent IgG aggregation during the nebulization process to avoid pro-inflammatory and cytotoxic effects. The optimization of Ab formulation can mitigate adverse effects induced by nebulization.

Abstract P3-11-01: Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse

Abstract Syngeneic mouse tumor models are critical to investigating new immunotherapeutic modalities and providing a rationale for clinical translation. Oncolytic herpes simplex virus (oHSV) is an emerging treatment strategy for breast cancer. Genetically modified oHSV selectively replicates in cancer cells (sparring healthy cells), induces immunogenic cancer cell death, and stimulates innate/adaptive anti-tumor immune responses. The therapeutic success of oHSV depends on a dynamic interaction between oHSV, the tumor, and the host. Here, we evaluated the anti-tumor effects of G47Δ-IL12, a genetically engineered oHSV expressing interleukin-12 (IL-12), in two orthotopic immunocompetent mouse models of breast cancer, E0771 (some classified this model as luminal B subtype and some as triple-negative) and EMT6 (triple-negative subtype), which are syngeneic to C57BL/6 and BALB/c mice, respectively. Intratumoral G47Δ-IL12 treatment led to complete eradication of orthotopic E0771 and EMT6 mammary tumors and prevention of lung metastases, resulting in 100% long-term survivors, which remained tumor-free during the observation period (&amp;gt;100 days following the last G47Δ-IL12 treatment). In a contralateral orthotopic mammary tumor model, G47Δ-IL12 treatment of primary tumors led to the eradication of both treated and untreated distal tumors, confirming the abscopal and anti-metastatic effect of G47Δ-IL12 virotherapy. All cured mice from G47Δ-IL12-treated groups rejected lethal tumor re-challenge in the contralateral naïve mammary fat pad, indicating successful generation of a protective memory response. In both E0771 and EMT6 models, cell-specific IFN-γ responses and the presence of effector memory T cells (CD4+CD44highCD62Llow phenotype) in spleens of long-term survivors, possibly contributing to tumor relapse prevention. G47Δ-IL12-mediated anti-tumor efficacy was associated with significant increases in CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) (vs. mock group) in both E0771 and EMT6 tumor lesions. G47Δ-IL12 monotherapy also led to a significantly increased CD8+/FoxP3+ ratio (an important parameter for immunotherapy success) in both tumor models. Interestingly, immune cell depletion studies demonstrated that efficacy requires both CD4+ and CD8+ T cells in the E0771 model while only CD8+ T cells were required in the EMT6 model. Conclusions: G47Δ-IL12 monotherapy was effective in eradicating both orthotopic and metastatic tumors and generating long-term protective memory responses in two syngeneic breast cancer models. Treatment efficacy of G47Δ-IL12 was associated with significant immunologic alterations of the TME. G47Δ-IL12 utilized distinct immunologic mechanisms of action to eradicate tumors: CD8+ T cell-dependent action in the EMT6 model, whereas CD4+ and CD8+ T cell-dependent efficacy in the E0771 model. Altogether, our data suggest that G47Δ-IL12 is an efficient inflammatory modulator and beneficially exploits the immune system to eradicate breast tumors. These studies provide the necessary supporting evidence for the clinical translation of this agent into breast cancer patients. Citation Format: Hong-My Nguyen, Othon Almanza, Dipongkor Saha. Oncolytic herpes simplex virus immunovirotherapy eradicates breast cancer and prevents tumor relapse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-11-01.

Restriction of Viral Replication, Rather than T Cell Immunopathology, Drives Lethality in Murine Norovirus CR6-Infected STAT1-Deficient Mice.

Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. In this study, we used colonization with the model commensal murine norovirus (MNV; strain CR6) to interrogate host-directed mechanisms of viral regulation, and we show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In addition to restricting CR6 replication to the intestinal tract, we show that STAT1 regulates antiviral CD4+ and CD8+ T cell responses and prevents systemic viral-induced tissue damage and disease. Despite altered T cell responses that resemble those that mediate lethal immunopathology in systemic viral infections in STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. However, therapeutic administration of an antiviral compound limited viral replication, preventing virus-induced tissue damage and death without impacting the generation of inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa and that uncontrolled viral replication mediates disease rather than the concomitant development of dysregulated antiviral T cell responses in STAT1-deficient mice. IMPORTANCE The intestinal microbiota is a collection of bacteria, archaea, fungi, and viruses that colonize the mammalian gut. Coevolution of the host and microbiota has required development of immunological tolerance to prevent ongoing inflammatory responses against intestinal microbes. Breakdown of tolerance to bacterial components of the microbiota can contribute to immune activation and inflammatory disease. However, the mechanisms that are necessary to maintain tolerance to viral components of the microbiome, and the consequences of loss of tolerance, are less well understood. Here, we show that STAT1 is integral for preventing escape of a commensal-like virus, murine norovirus CR6 (MNV CR6), from the gut and that in the absence of STAT1, mice succumb to infection-induced disease. In contrast to the case with other systemic viral infections, mortality of STAT1-deficient mice is not driven by immune-mediated pathology. Our data demonstrate the importance of host-mediated geographical restriction of commensal-like viruses.

Immune Landscape Refines the Classification of Colorectal Cancer With Heterogeneous Prognosis, Tumor Microenvironment and Distinct Sensitivity to Frontline Therapies.

The immune microenvironment has profound impacts on the initiation and progression of colorectal cancer (CRC). Therefore, the goal of this article is to identify two robust immune subtypes in CRC, further provide novel insights for the underlying mechanisms and clinical management. In this study, two CRC immune subtypes were identified using the consensus clustering of immune-related gene expression profiles in the meta-GEO dataset (n = 1,198), and their reproducibility was further verified in the TCGA-CRC dataset (n = 638). Subsequently, we characterized the immune escape mechanisms, gene alterations, and clinical features of two immune subtypes. Cluster 1 (C1) was defined as the “immune cold subtype” with immune cell depletion and deficiency, while cluster 2 (C2) was designed as the “immune hot subtype”, with abundant immune cell infiltration and matrix activation. We also underlined the potential immune escape mechanisms: lack of MHC molecules and defective tumor antigen presentation capacity in C1, increased immunosuppressive molecules in C2. The prognosis and sensitivity to 5-FU, Cisplatin and immunotherapy differed between two subtypes. According to the two immune subtypes, we developed a prognosis associated risk score (PARS) with the accurate performance for predicting the prognosis. Additionally, two nomograms for overall survival (OS) and disease-free survival (DFS) were further constructed to facilitate clinical management. Overall, our research provides new references and insights for understanding and refining the CRC.

Neurological update: treatment escalation in multiple sclerosis patients refractory to fingolimod\u2014potentials and risks of subsequent highly active agents

A critical issue in the management of relapsing MS (RMS) is the discontinuation of disease-modifying treatments (DMT) due to lack of efficacy, intolerability or impending risks. With new therapeutic agents introduced into the treatment of RMS, immediate- and long-term consequences of sequential drug use, as well as the effect of the sequence in which the drugs are given, are unclear but may affect efficacy, adverse events, and long-term immunocompetence. In the absence of clinical studies specifically addressing these concerns, observations from clinical practice are of particular value in guiding current management algorithms. Prompted by a study published by Ferraro et al. in this journal, we set out to provide an overview of the published real-world evidence on the effectiveness and safety of switching from fingolimod to another DMT in patients with active RMS. Seventeen publications reporting relevant information were identified. The literature suggests that immune cell depletion induced by alemtuzumab or ocrelizumab is associated with an increased risk of relapse and worsening disability in patients switching from fingolimod compared to patients switching from other therapeutic agents. However, the evidence reported for natalizumab and cladribine is inconclusive. While shortening of the washout period may limit early disease reactivation after fingolimod discontinuation, there is no strong evidence that the duration of the washout period or the absolute lymphocyte count at baseline are predictors of attenuated long-term efficacy. Further real-world studies are required to better understand outcomes among patients who are under-represented in controlled trials.

Induction Therapy and Therapeutic Antibodies.

Prevention of allograft rejection is one of the crucial goals in solid organ transplantation to ensure durability of the graft and is chiefly mediated by cellular and humoral pathways targeting cell surface alloantigens. The risk of rejection is highest in the first post-transplant year and wanes with time albeit the risk always exists and varies with the type of organ transplanted. Induction therapies refer to the use of high-intensity immunosuppression in the immediate post-operative period to mitigate the highest risk of rejection. This term encompasses chiefly the use of antibody therapies directed against one of the key pathways in T-cell activation or abrogating effects of circulating alloantibodies. These antibodies carry more potent immunomodulatory effect than maintenance immunosuppressive therapy alone and many of them lead to durable immune cell depletion. A variety of monoclonal and polyclonal antibodies have been utilized for use not only for induction therapy, but also for treatment of allograft rejection when it occurs and as components of desensitization therapy before and after transplantation to modulate circulating alloantibodies.

Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

Abstract PO-096: The synaptic protein Netrin G1 ligand (NGL-1) modulates tumorigenesis and immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer, with a 5-year survival of 10%. A major feature of PDAC is the presence of a dense fibrous stroma, due to the expansion of cancer associated fibroblasts (CAFs) and their extracellular matrix. This unique environment represents a challenge for therapies as it promotes immunosuppression, limits access to nutrients, and excludes or inactivates antitumor immune cells. Recently, we identified the ectopic expression of the neuronal protein Netrin G1 Ligand (NGL-1) in PDAC tissue, including its novel expression in immune cells and CAFs. However, the roles of NGL-1 in the tumor microenvironment (TME) of PDAC and in immune cell function are unknown and warranted further investigation. The contribution of NGL-1 to PDAC tumorigenesis was assessed by measuring the expression of NGL-1 in different models of PDAC and by orthotopically injecting PDAC cells in wild type (WT) or NGL-1 full body knockout mice (KO). Using our in vitro 3D system we evaluated if NGL-1+ CAFs, compared to NGL-1 knockdown (KD) CAFs, produced less immunosuppressive factors and were able to rescue PDAC cell survival under nutrient deprivation. For NGL-1 dependent immune cell functions we isolated naïve immune cells from WT and KO mice and performed ex-vivo functional assays. NGL-1 expression in fibroblasts correlated with disease development in different models of PDAC, and myeloid, T and NK cells from tumor bearing mice tended to overexpress NGL-1 when compared with cells from naïve mice. Accordingly, NGL-1 KO mice orthotopically injected with PDAC cells developed smaller tumors with decreased secretion of immunosuppressive factors, increased presence of CD8+ T cells and CD4+ T cells expressing less pro-tumor markers. Single cell RNA sequencing data from tumors from KO mice showed downregulation of pro-tumor genes in different cell populations, with the fibroblastic populations differing between WT and KO mice. In order to evaluate the contribution of the immune system for tumorigenesis in WT and KO mice, we performed bone marrow chimeras and depletion of specific immune cells. Functionally, CD8+ and CD4+ T cells from KO mice proliferated more when stimulated in vitro, suggesting that NGL-1 could represent a functional brake for T cells, inhibiting their anti-tumor capacity. The lack of NGL-1 in stimulated bone marrow-derived macrophages decreased pro-inflammatory cytokine secretion, further suggesting a functional role for NGL-1 in myeloid cells. Of note, NGL-1 KD CAFs did not support PDAC cell survival in vitro and produced less immunosuppressive cytokines, which was phenocopied by the treatment with a peptide targeting NGL-1. Translationally, we assessed the overall survival of 140 PDAC patients according to NGL-1 expression in the TME, where low expression of NGL-1 in CAFs and immune cells correlated with better survival of PDAC patients. Overall, this suggests NGL-1 as potential new target in PDAC, that could be manipulated in different compartments in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Nina Steele, Benjamin Allen, Marina Pasca di Magliano, Charline Ogier, Igor Astsaturov, Kathy Q. Cai, Andres J. Klein-Szanto, Huamin Wang, Kerry Campbell, Edna Cukierman. The synaptic protein Netrin G1 ligand (NGL-1) modulates tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-096.

HYPERTENSION IN RELATION TO IMMUNE SYSTEM AND WAY OF LIFE ALONG WITH TREATMENT

The objective of the review is to explain the pathophysiology, different causes and various treatments involved in hypertension. This article discusses the disease's pathogenesis, etiology, diagnosis, and immunity. This review looks at the main significant epidemiological and clinical studies on the role of several lifestyle factors in hypertension development. This review examines the numerous mechanisms that cause hypertension in order to discover new treatments. In addition, it covers the many types of hypertension therapy. According to different studies, lifestyle habits may have an impact on blood pressure levels.&#x0D; Moreover, the importance of chronic inflammation in hypertension and its repercussions has been confirmed in genetically engineered mice lacking components of innate and/or adaptive immunity. Immune cell depletion enhances endothelial function, lowers oxidative stress, lowers the vascular tone, and protects against renal interstitial infiltrates, salt retention, and kidney injury. Based on existing literature, there is strong evidence that lifestyle variables can affect blood pressure levels. Then, in hypertensive people, lifestyle changes can help by lowering overall cardiovascular risk and death from any cause. The involvement of immunity as a common thread in the hypertension processes of many organ systems.

Cladribine Alters Immune Cell Surface Molecules for Adhesion and Costimulation: Further Insights to the Mode of Action in Multiple Sclerosis.

Cladribine (CLAD) is a deoxyadenosine analogue prodrug which is given in multiple sclerosis (MS) as two short oral treatment courses 12 months apart. Reconstitution of adaptive immune function following selective immune cell depletion is the presumed mode of action. In this exploratory study, we investigated the impact of CLAD tablets on immune cell surface molecules for adhesion (CAMs) and costimulation (CoSs) in people with MS (pwMS). We studied 18 pwMS who started treatment with CLAD and 10 healthy controls (HCs). Peripheral blood mononuclear cells were collected at baseline and every 3 months throughout a 24-month period. We analysed ICAM-1, LFA-1, CD28, HLADR, CD154, CD44, VLA-4 (CD49d/CD29), PSGL-1 and PD-1 with regard to their expression on B and T cells (T helper (Th) and cytotoxic T cells (cT)) and surface density (mean fluorescence intensity, MFI) by flow cytometry. The targeted analysis of CAM and CoS on the surface of immune cells in pwMS revealed a higher percentage of ICAM-1 (B cells, Th, cT), LFA-1 (B cells, cT), HLADR (B cells, cT), CD28 (cT) and CD154 (Th). In pwMS, we found lower frequencies of Th and cT cells expressing PSGL-1 and B cells for the inhibitory signal PD-1, whereas the surface expression of LFA-1 on cT and of HLADR on B cells was denser. Twenty-four months after the first CLAD cycle, the frequencies of B cells expressing CD44, CD29 and CD49d were lower compared with the baseline, together with decreased densities of ICAM-1, CD44 and HLADR. The rate of CD154 expressing Th cells dropped at 12 months. For cT, no changes were seen for frequency or density. Immune reconstitution by oral CLAD was associated with modification of the pro-migratory and -inflammatory surface patterns of CAMs and CoSs in immune cell subsets. This observation pertains primarily to B cells, which are key cells underlying MS pathogenesis.

DPP inhibition alters the CXCR3 axis and enhances NK and CD8+ T cell infiltration to improve anti-PD1 efficacy in murine models of pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer death in the USA by 2030. Immune checkpoint inhibitors fail to control most PDAC tumors because...

Imaging Tolerance Induction in Neonatal Mice: Hierarchical Interplay Between Allogeneic Adult and Neonatal Immune Cells.

In Medawar's murine neonatal tolerance model, injection of adult semiallogeneic lymphohematopoietic cells (spleen cells [SC] and bone marrow cells [BMC]) tolerizes the neonatal immune system. An eventual clinical application would require fully allogeneic (allo) cells, yet little is known about the complex in vivo/in situ interplay between those cells and the nonconditioned neonatal immune system. To this end, labeled adult SC and BMC were injected into allogeneic neonates; interactions between donor and host cells were analyzed and modulated by systematic depletion/inactivation of specific donor and host immune effector cell types. Consistent with effector cell compositions, allo-SC and allo-SC/BMC each induced lethal acute graft-versus-host disease, whereas allo-BMC alone did so infrequently. CD8 T cells from SC inoculum appeared naïve, while those of BMC were more memory-like. Age-dependent, cell-type dominance defined the interplay between adult donor cells and the neonatal host immune system such that if the dominant adult effector type was removed, then the equivalent neonatal one became dominant. Depletion of donor/host peripheral T cells protected against acute graft-versus-host disease and prolonged heart allograft survival; peripheral CD8 T-cell depletion together with CD4 T cell-costimulation blockade induced more robust tolerance. This comprehensive study provides direct observation of the cellular interplay between allogeneic donor and host immune systems, adds to our previous work with semiallogeneic donor cells, and provides important insights for robust tolerance induction. Induction of transplant tolerance in neonates will likely require "crowd sourcing" of multiple tolerizing cell types and involve depletion of immune effector cells with costimulation blockade.