CD4 Th1 Research Articles

P128 Salt-Inducible Kinase 2 is a promising therapeutic target in patients with ulcerative colitis

Abstract Background Salt-Inducible Kinases (SIKs) are a family of 3 serine/threonine kinases which represent promising therapeutic targets in a number of inflammatory diseases including ulcerative colitis (UC). Their catalytic activity is highly phosphoregulated in response to changes in cellular homeostasis. Inactivation of SIKs results in proinflammatory cytokine downregulation and the concomitant upregulation of anti-inflammatory IL-10. Of the three isozymes, SIK2 is the most active in myeloid cells and is thought to be the key driver of IL-10 production. Selective pharmacological inhibition of SIKs has thus far proved challenging: a number of SIK inhibitors have previously been developed and whilst they have demonstrated biological activity in UC, dosing has been limited due to potential side effects underlining the need for improved selectivity. We hypothesized that more selective inhibition of SIK2 represents a promising approach in ulcerative colitis. Methods We sought to examine the effects of inhibiting SIK2 with a potent small molecule in both in vitro cell cultures and in ex vivo biopsies from UC patients. CD14+ monocytes were isolated from donor PBMCs and differentiated into hMDMs (monocyte-derived macrophages) and moDCs (monocyte-derived DCs). Cultures were treated with compound followed by LPS stimulation for 4 hours (hMDMs) or maturation for 48 hours (moDCs). To recapitulate the disease microenvironment ex vivo, a colonic explant culture model was used: colonic biopsies were collected from patients with active (Mayo 1 /2) and inactive UC and cultured in the presence of compound or control for 24-48h. Supernatants were assayed for cytokine and chemokine release. Cells or biopsies were lysed for qPCR. Surface markers were assessed by flow cytometry Results Using more SIK2-selective small molecules, we show that SIK inhibition modulates inflammatory and immunometabolic pathways in human myeloid cells. Furthermore, SIK2 inhibition drives a tolerogenic phenotype in dendritic cells, as demonstrated by suppression of proinflammatory cytokines and chemokines such as TNFα and the CC/CXC chemokine family as well as downregulation of activation markers such as CD80 and 83; functionally these modulated DCs can suppress Th1 CD4 T cell IFNγ production. Modulation of cytokine and chemokine production by SIK2 inhibition was also observed in colonic explant cultures from UC patients with a reduction in levels of TNFα and the CC chemokine family. Conclusion In both myeloid cellular cultures and UC biopsies, SIK2 inhibition modulates an inflammatory environment into a pro-resolution state supporting the therapeutic potential of SIK2 inhibition in UC.

Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice

Chlamydia muridarum (Cm), an intracellular bacterium of historical importance, was recently rediscovered as moderately prevalent in research mouse colonies. Cm was first reported as a causative agent of severe pneumonia in mice about 80 y ago, and while it has been used experimentally to model Chlamydia trachomatis infection of humans, there have been no further reports of clinical disease associated with natural infection. We observed clinical disease and pathology in 2 genetically engineered mouse (GEM) strains, Il12rb2KO and STAT1KO, with impaired interferon-γ signaling and Th1 CD4+ T cell responses in a colony of various GEM strains known to be colonized with and shedding Cm. Clinical signs included poor condition, hunched posture, and poor fecundity. Histopathology revealed disseminated Cm with lesions in pulmonary, gastrointestinal, and urogenital tissues. The presence of Cm was confirmed using both immunohistochemistry for Cm major outer membrane protein-1 antigen and in situ hybridization using a target probe directed against select regions of Cm strain Nigg. Cm was also found in association with a urothelial papilloma in one mouse. These cases provide additional support for excluding Cm from research mouse colonies.

CD8 T cells are dispensable for experimental autoimmune prostatitis induction and chronic pelvic pain development.

Chronic Pelvic Pain Syndrome or Chronic Prostatitis (CPPS/CP) is the most prevalent urologic affliction among young adult men. It is a challenging condition to treat, which significantly decreases patient quality of life, mostly because of its still uncertain aetiology. In that regard, an autoimmune origin is a prominent supported theory. Indeed, studies in patients and in rodent models of Experimental Autoimmune Prostatitis (EAP) have provided compelling evidence suggesting a key role of CD4 Th1 cells in disease pathogenesis. However, the implication of other prominent effectors of the immune system, such as CD8 T cells, has yet to be studied. We herein analyzed the induction of prostatitis and the development of chronic pelvic pain in EAP using CD8 T cell-deficient animals. We found similarly elevated PA-specific immune responses, with high frequencies of specific IFNg+CD4+ and IL17+CD4+ T cells in prostate draining lymph nodes from PA-immunized either CD8 KO or wild type animals with respect to controls. Moreover, these peripheral immune responses were paralleled by the development of significant chronic pelvic pain, and accompanied by prostate histological lesions, characterized by hemorrhage, epithelial cell desquamation, marked periglandular leukocyte infiltration, and increased collagen deposition in both, PA-immunized CD8 KO and wild type animals. As expected, control animals did not develop prostate histological lesions. Our results indicate that CD8 T cells do not play a major role in EAP pathogenesis and chronic pelvic pain development. Moreover, our results corroborate the previous notion that a CD4 Th1 associated immune response drives the induction of prostate tissue inflammation and the development of chronic pelvic pain.

Unveiling the multifaceted antitumor effects of interleukin 33.

Interleukin 33 (IL-33), once predominantly recognized for its pro-tumoral activities, has emerged as a multifunctional cytokine with antitumor properties. IL-33 pleiotropic activities include activation of Th1 CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, eosinophils, as well as type 2 innate lymphoid cells. Regarding this immunomodulatory activity, IL-33 demonstrates synergistic interactions with various cancer therapies, including immune checkpoint blockade and chemotherapy. Combinatorial treatments leveraging IL-33 exhibit enhanced antitumor efficacy across different tumor models, promising novel avenues for cancer therapy. Despite its antitumor effects, the complex interplay of IL-33 within the tumor microenvironment underscores the need for further investigation. Understanding the mechanisms underlying IL-33's dual role as both a promoter and inhibitor of tumor progression is essential for refining therapeutic strategies and fully realizing its potential in cancer immunotherapy. This review delves into the intricate landscape of IL-33 effects within the tumor microenvironment, highlighting its pivotal role in orchestrating immune responses against cancer.

CD4 T cell-activating neoantigens enhance personalized cancer vaccine efficacy.

Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host’s gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3–8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8–14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB−CD8+ memory T cells and accumulation of type 2 memory T cells

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ Tcells and HLA-DR+CD4+ Tcells. In contrast to other Tcell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ Tcells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ Tcell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

EXTH-16. DEVELOPMENT OF INTRATHECAL (IT) DRUG DELIVERY PLATFORM TO CURE BREAST CANCER (BC) LEPTOMENINGEAL DISEASE (LMD) BY PEPTIDE-PULSED TYPE 1 CONVENTIONAL DENDRITIC CELL (CDC1) VACCINE IN PRECLINICAL MODEL

Abstract BACKGROUND LMD is a rare form of metastasis in the meninges. Approximately 5% of advanced stage BC will develop LMD and currently there is no cure. One of the major hurdles is drug penetration due to the blood brain barrier (BBB) and blood-cerebral spinal fluid (CSF)-barrier. While the Ommaya reservoir is used clinically to overcome this challenge, there has not been an optimized preclinical model that allows researchers to adequately test novel therapies in the CSF space. METHODS The Forsyth lab have recently developed an MRI-compatible device called the “murine Ommaya” which mimics the Ommaya reservoir and allows repeated IT administration of drugs in minute 3-7 μL directly into CSF, bypassing BBB. In a joint effort with Dr. Czerniecki, whose lab have developed a pipeline for screening immunogenic MHC class II peptides from tumor-associated oncodrivers and subsequently generating tumor-targeting cDC1s, we have created a platform to IT deliver peptide-pulsed cDC1 immunotherapy directly targeting BC-LMD. RESULTS Currently, we have tested the efficacy of treatment in HER2+ LMD and triple negative breast cancer (TNBC) LMD models and found a very favorable response; LMD mice that received IT cDC1 therapy showed tumor regression and their survival were significantly prolonged. About 70% of mice from HER2+ LMD and 30% from TNBC LMD were eventually disease-free. Cured mice also exhibited resistance against LMD recurrence. CONCLUSIONS Our preliminary data suggest IT cDC1 vaccine is effective against the incurable BC-LMD. We are currently investigating the mechanism(s) by which IT cDC1-initiated CD4 Th1 adaptive immune response against LMD. By using single-cell RNA-sequencing technique, we are analyzing the immune landscape in CSF tumor microenvironment in response to IT cDC1. FDA-approved phase 1 clinical trial has been funded by the DOD. Future approach includes employing our IT cDC1 immunotherapy platform in other cancer types, such as LMD from melanoma.

Multimodality imaging and immunophenotyping of COVID-19 related myocardial injury (the MIIC-MI Study)

Abstract Background Cardiac injury is a well-recognised complication of acute COVID-19 infection, with varied aetiologies ranging from myocarditis to endothelial dysfunction, micro-embolic phenomena, and acute coronary syndromes. While persistent cardiac symptoms in patients with post-acute COVID-19 syndrome are also common, the underlying mechanisms remain unclear. A better understanding of immune-mediated drivers of chronic cardiac injury after COVID-19 infection is urgently needed to inform the management of patients with, or at risk of, long-term sequelae. Purpose To examine immune mechanisms associated with clinically detectable imaging abnormalities across a spectrum of patients with unexplained cardiac injury and/or symptoms after COVID-19 infection. Methods Participants with a history of COVID-19 infection and suspected cardiac involvement based on: a) troponin I elevation (>99th percentile upper reference limit), b) new-onset heart failure not attributable to another cause, or c) persistence of unexplained cardiac symptoms, were enrolled at a single centre in the United Kingdom between October 2020 and February 2022. Individuals with prior myocardial infarction or heart failure and those treated with immunosuppression within the past four weeks, were excluded. Cardiac magnetic resonance imaging (MRI) was performed on a 1.5T scanner following a standard clinical protocol. In parallel to cardiac imaging, peripheral blood samples were analysed for immunophenotyping using cytometry by time of flight (Fluidigm, 32 marker panel) and proteomic analysis (Olink, Target 96 Inflammation panel). Results Twenty-one patients (mean age 47 (SD 13) years, 71% female) were enrolled who had unexplained cardiac symptoms (n=17), raised troponin I (n=2), or new-onset heart failure (n=2). In total, 9/21 (43%) patients had MRI abnormalities, including a non-ischaemic pattern of late gadolinium enhancement (Figure 1a; arrow) and/or visually overt myocardial oedema on T2-weighted imaging (Figure 1b; arrow) in 8 patients, and one with impaired ventricular function in the absence of fibrosis. Patients with MRI abnormalities had higher CCL3, CCL7 (MCP-3), FGF21, and CD4 Th2 cells, and lower CD8 T effector memory cells (Figure 2, volcano plot), compared to those without. CCL7 (MCP-3) levels were correlated with native T1 mapping (r=0.45, p<0.05) and these along with CD8 T effector memory cell counts were the strongest predictors of MRI abnormalities in a LASSO regression model incorporating all clinical, cell phenotyping and proteomic data (composite AUC 0.96, CI 0.88-1). Conclusion Here we show for the first time that COVID-19 related cardiac injury in patients with MRI abnormalities is associated with increased CCL7 (MCP-3), a chemokine known to be important in viral myocarditis1, as well as decreased CD8 T effector memory cells. These findings provide novel insights into the immune-mediated mechanisms underlying non-ischemic cardiac complications of COVID-19.Fig. 1.Post-COVID imaging abnormalitiesFig. 2.Immunophenotyping and proteomics

Gut Microbiome Links to Disease Stage and Immune Microenvironment in Patients with Chronic Lymphocytic Leukemia

Background Recent studies have shown extensive crosstalk between our immune system and the gut microbiome (GM). The host immune system plays a vital role in the maintenance of GM homeostasis by establishing a balance between eliminating invading pathogens and promoting the growth of beneficial microbes, whereas GM can modulate the immune system by cytokine production. As chronic lymphocytic leukemia (CLL) is characterized by a high rate of infectious complications and an altered immune microenvironment, it is warranted to uncover the link between microbiome composition and immune dysregulation in CLL patients. Thus, we investigated the microbiome pattern and potential connections to disease characteristics and phenotypes and distribution within the immune microenvironment in CLL patients requiring treatment. Methods Blood and feces samples were collected from 17 patients with CLL prior to targeted treatment with acalabrutinib or ibrutinib+venetoclax in clinical trials. T-cell maturation subsets, Treg/Th17 subsets, and activation- and exhaustion profiles were assessed in fresh blood samples by an 8-tube, 10 color flow cytometry panel (DuraClone), including one tube containing beads for calculating absolute concentrations. Fecal samples were subjected to shotgun sequencing using Illumina HiSeq and taxonomical profiling was done using an in-house bioinformatics pipeline. The bioinformatics analysis involved microbial species abundances comparison and co-occurrence identification across patient clusters. Electronic health records provided data on clinical baseline characteristics. Case report forms and data on use of antimicrobial drugs will be assessed at a later stage to investigate potential associations with infections or toxicity related adverse events. All patients provided written informed consent and the study was approved by the National Ethics Committee and the Data Protections Agency. Results Unsupervised clustering analyses based on GM composition data revealed two GM clusters, where we found an overlap constituting approximately 40% of the cohort between Binet stage C (7/10 patients) and GM cluster 2 (7/10 patients) (Figure 1A). GM cluster 2 was characterized by differentially abundant species such as Bacteroides fragilis and Eggerthela lenta, demonstrating the two highest fold change and elevated relative abundances (Figure 1B). Closer examination of the taxonomic composition characteristic for the 7 patients revealed a small set of indicator microbiome species, where Clostridium innocuum together with Clostridiales sp., Bacteroides fragilis and Escherichia coli, were the most significantly abundant co-occurring species for CLL patients with Binet C. Preliminary examination of T-cell subset data showed generally lower concentrations of T-cells in patients from GM cluster 2 compared to GM cluster 1, where the 7 patients overlapping with Binet C furthermore had decreased levels of Th17 CD4+ and Tc17 CD8+ cells compared to both the normal reference level and the rest of the cohort (Figure 1C). Conclusion Our preliminary results suggest a link between CLL stage at time of needing treatment, gut microbiome composition, represented by a subset of highly abundant bacterial species, and T-cell distribution with reduced Th17 and Tc17 subsets, to be further investigated. To our knowledge, we are thus among the first to present data connecting the gut microbiome to tumor microenvironmental changes in patients with CLL. Moving forward we will further explore microbiome-microenvironment interactions with focus on the production of short-chain fatty acids (bacterial metabolites which play crucial roles as immunomodulatory molecules) to investigate connections between microbiome, immune phenotypes, and clinical outcomes including infections, treatment-related toxicity, and disease progression, to light on novel avenues for therapeutic interventions in CLL.

Cytokine Profiling in Antiphospholipid Syndrome Demonstrates Persistent Immune Dysregulation and a Procoagulant Phenotype

Background: The antiphospholipid syndrome (APS) is an autoimmune thromboinflammatory disease characterized by thrombosis and the persistent detection of antiphospholipid antibodies (aPL). The rate of recurrent thrombosis in APS is between 3 and 24% despite standard of care anticoagulation with a vitamin K antagonist, suggesting a persistent prothrombotic milieu in APS which may be driven by altered cytokine signaling. Objective: To test the hypothesis that cytokine signaling is dysregulated in APS and contributes to thrombotic risk, we profiled cytokine levels in patients with thrombotic APS and healthy individuals. Methods: We measured a panel of 46 serum cytokines using the Meso Scale Discovery electrochemiluminescent platform, a validated measurement system that provides linear readouts of analytes over several logs of concentration, in 21 APS patients and 12 healthy individuals. All APS patients met the Revised Sapporo Criteria for thrombotic APS. Measurements were performed in triplicate and the log2 transformed averages were analyzed, setting significance at a FDP-adjusted p value of <0.05, while controlling for age and sex. Cytokine values were correlated with clinical history and antibody isotype profile. Results: Our cohort was 67% female and 85% identified as white. All APS patients had thrombotic disease: 57% with history of VTE, 19% with ATE, and 24% with both VTE and ATE. 14% had catastrophic APS. 67% were triple-positive for anti-cardiolipin (aCL), anti-β2 glycoprotein 1 (aβ2GP1), and lupus anticoagulant (LAC). 10% had isolated IgM isotypes of aCL or aβ2GP1. We found significantly elevated levels of TNF-α, IL-6, IL-7, IL-8, IL-10, IL-16, IL-12/IL-23p40, soluble VCAM-1, soluble ICAM-1, PIGF, SAA and CRP and significantly decreased levels of IL-17genB, IL-22 and IL-31 in APS compared with healthy controls (Figure 1). Cytokine levels were measured a median of 5 months (1-240) from the last thrombotic event, demonstrating persistence of the inflammatory response. To determine if cytokine levels were predictive of thrombotic risk, we correlated the number and pattern of positive aPL isotypes and LAC with cytokine levels. We found that TNF-α, IL-8, IL-10, sVCAM-1, sICAM-1, VEGF, IL-7, IL-12/IL-23p40, IL-4, IL-6, IL-16, PIGF, IL-23 and IL-17A each correlated with aPL positivity, with higher levels in triple-positive APS, and the highest levels observed in patients positive for all five measured antibody isotypes (aCL IgG, IgM, IgA; aβ2GP1 IgG, IgM) plus the functional LAC (Figure 2). IL-31 level inversely correlated to the number of positive aPL tests. A sub-analysis of cytokines in patients with isolated IgM aCL or aβ2GP1 antibodies with or without LAC showed significantly increased levels of TNF-α compared to healthy controls, and did not differ from APS patients with other patterns of aPL positivity. This suggests that a similar proinflammatory or procoagulant background is present in patients with isolated antiphospholipid IgM antibodies as those with IgG. Discussion: The sustained differences in cytokine levels measured in APS patients suggests a picture of ongoing immune dysregulation and compensation capable of driving vascular cell activation, and tipping the hemostasis balance into thrombosis. We find upregulation of proinflammatory cytokines, such as TNF-α and IL-6, capable of activating endothelial cells and triggering amplification loops leading to chemokine and acute phase reactant release, cell adhesion molecule expression, recruitment of leukocytes, and accumulation of complement and coagulation proteins, with the net effect of driving inflammation and thrombosis. Decreased levels of IL-17, IL-22 and IL-31 are indicative of dysregulation of the Th17 CD4+ T cell response, which has been implicated in autoimmune pathogenesis. The correlation of cytokine levels with triple or greater aPL positivity suggests a model for advancing risk stratification and has utility as a biomarker of thrombosis. Our study, although limited by size, represents one of the most extensive profiling of cytokines in thrombotic APS.

P16.03.A DEVELOPMENT OF INTRATHECAL DRUG DELIVERY PLATFORM TO CURE BREAST CANCER LEPTOMENINGEAL DISEASE BY DENDRITIC CELL VACCINE IN PRECLINICAL MODEL

Abstract BACKGROUND Leptomeningeal disease (LMD) is a rare form of metastasis in the meninges, a membrane surrounding the brain and spinal cord. Approximately 5% of advanced stage breast cancer (BC) will develop LMD and currently there is no cure. One reason for poor prognosis is the issue of drug accessibility to tumor sites due to the blood brain barrier (BBB) and blood-cerebral spinal fluid (CSF)-barrier. While the Ommaya reservoir is used clinically to overcome this challenge, there has not been an optimized preclinical model that allows researchers to adequately test novel therapies in the CSF space. MATERIAL AND METHODS The Forsyth lab have recently developed an MRI-compatible device called the “murine Ommaya” which mimics the Ommaya reservoir and allows repeated intrathecal (IT) administration of drugs in 3-7μl volume directly into CSF, bypassing BBB. In a joint effort with Dr. Czerniecki, whose lab have developed a pipeline for screening immunogenic MHC class II peptides from tumor-associated oncodrivers and subsequently generating tumor-targeting dendritic cells (DC), we have created a platform to IT deliver peptide-pulsed DCs directly targeting BC-LMD. RESULTS Currently, we have tested the efficacy of treatment in HER2+ LMD and triple negative breast cancer (TNBC) LMD models and found a very favorable response; LMD mice that received IT DC therapy exhibited reduced tumor burden and prolonged survival, and is more superior than systemic therapy. About 70% of mice from HER2+ LMD and 30% from TNBC LMD were cured of disease. Cured mice also exhibited resistance against LMD recurrence. CONCLUSION Our preliminary data suggest IT DC immunotherapy is effective against the incurable BC-LMD. We are currently investigating the mechanism(s) by which IT DC-initiated CD4 Th1 adaptive immune response against LMD. By using single-cell RNA-sequencing technique, we are analyzing the immune landscape in CSF tumor microenvironment in response to IT DC. FDA-approved phase I clinical trial has been funded by the US Department Of Defense. Future approach includes employing our IT DC vaccine platform in other cancer types, such as LMD from melanoma.

O22 Effector memory and TH17 CD4+ T cells are increased in liver transplant patients experiencing ischemia-reperfusion injury

O22 Effector memory and TH17 CD4+ T cells are increased in liver transplant patients experiencing ischemia-reperfusion injury

Immunosuppressive Effects of Annona muricata L. Leaf Extract on Cellular and Humoral Immune Responses in Male Wistar Rats.

Annona muricata</i> L. (Annonaceae) (AM)'s remarkable anti-inflammatory and anti-cancer activities make it a targeted plant to be explored for its immunomodulatory properties. Traditional practitioners have employed various components of AM to cure a variety of ailments, including cancer, diabetes, and inflammation. The present study evaluated the immunosuppressive effects of 80% ethanol extract of of AM leaves in male Wistar rats on different parameters of humoral and cellular immune responses. AM leaf extract (AMLE) was analyzed using UHPLC-MS/MS to profile its secondary metabolites. AMLE was rich in polyphenols which include (epi)catechin-(epi)catechin-(epi) catechin, caffeic acid, coumaroylquinic acid, hyperin, kaempferol, quinic acid and rutin. The rats were administered 100, 200 and 400 mg/kg bw of the extract daily for 14 days. The effects of AMLE on innate immune responses were determined by evaluating phagocytosis, neutrophils migration, reactive oxygen species (ROS) release, CD11b/CD18 integrin expression, and ceruloplasmin, lysozyme and myeloperoxidase (MPO) levels. The adaptive immune parameters were evaluated by immunizing the rats with sheep red blood cells (sRBC) on day 0 and administered orally with AMLE for 14 days. AMLE established significant immunosuppressive effects on the innate immune parameters by inhibiting the neutrophil migration, ROS production, phagocytic activity and expression of CD11b/CD18 integrin in a dose-dependent pattern. AMLE also suppressed ceruloplasmin, MPO and lysozyme expressions in the rat plasma dose-dependently. AMLE dose-dependently inhibited T and B lymphocytes proliferation, Th1 and Th2 cytokine production, CD4+ and CD8+ co-expression in splenocytes, immunoglobulins (IgM and IgG) expression and the sRBC-induced swelling rate of rat paw in delayed-type hypersensitivity (DTH). The strong inhibitory effects on the different parameters of humoral and cellular responses indicate that AMLE has potential to be an important source of effective immunosuppressive agents.

CCL21-DC in situ vaccination in murine NSCLC overcomes resistance to immunotherapy and generates systemic tumor-specific immunity

BackgroundDespite recent advances in immunotherapy, many patients with non-small cell lung cancer (NSCLC) do not respond to immune checkpoint inhibitors (ICI). Resistance to ICI may be driven by suboptimal priming...

ALCAM on human oligodendrocytes mediates CD4 T cell adhesion.

Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.

Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes.

BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

An Advax-CpG adjuvanted recombinant H5 hemagglutinin vaccine protects mice against lethal influenza infection

There is a major unmet need for strategies to improve the immunogenicity of vaccines to protect against highly pathogenic avian influenza strains with pandemic potential. This study tested the ability of adjuvants based on delta inulin (Advax™) alone or combined with a TLR9 agonist (Advax-CpG™) to enhance the immunogenicity of recombinant H5 hemagglutinin antigen expressed in insect cells (rH5HA) to protect mice against lethal influenza infection. The Advax-adjuvanted rH5HA induced high serum hemagglutination inhibition activity, as well as Th1 and Th2 cytokine secreting CD4 and CD8 T cells. Immunization protected mice against a lethal heterosubtypic H5N1 virus challenge. Mice immunized with an Advax-adjuvanted rHA2 stem antigen prepared by enzymatic cleavage of rH5HA produced serum antibodies devoid of hemagglutination inhibition activity, but these anti-HA2 antibodies were nevertheless able to transfer protection against lethal H1N1 or H3N2 infections to naïve mice. We hypothesize that the enhanced protection afforded by Advax-adjuvanted rH5HA may be mediated by the combination of neutralizing antibodies directed at the HA head, anti-HA2 stem antibodies plus memory CD4 + and CD8 + T cells. This outcome supports further development of the Advax-adjuvanted rH5 pandemic influenza vaccine platform.

Lymphocyte Expression of Intracellular Cytokines and Heat Shock Proteins in Peripheral Blood of Patients with Atopic Dermatitis.

The present study evaluates expression by activated CD4+ T helper1 (Th1) and T helper 2 (Th2) T lymphocytes of pro-inflammatory cytokines and cytoprotective heat shock proteins (HSPs) in peripheral blood of atopic dermatitis (AD) patients. This research represents preliminary work by the authors to identify correlates between critical immune parameters with the potential to serve as guidelines for the development of pharmacological strategies for altering these factors to promote the restoration of healthy immune profiles in persons afflicted with major atopic diseases. The major experimental strategy used in this research assessed immune activation by peripheral blood mononuclear cells (PBMC) from 21 AD patients and 12 age- and gender-matched healthy control subjects cultured with phorbol myristate acetate (PMA) and ionomycin (PMA/I), which are mutagenic immune activators, to induce expression of pro-inflammatory biomarkers in CD4+ T cells differentiated to express Th1 or Th2 cytokines and heme oxygenase-1 (HO-1) intracellularly (i). Evaluations were performed using an FC500 Beckman-Coulter flow cytometer. Elevated CD4+ T cell expression of cytokines, interleukin-4 (iIL-4), interleukin- 5 (iIL-5), interleukin-10 (iIL-10), interferon-gamma (iIFN-g), tumor necrosis factor-alpha (iTNF-α), were observed. Additionally, the heat shock proteins (HSP) iHO-1 and iHSP-70 were evaluated in cells from the blood of AD patients versus the control subjects. The present study demonstrated an elevated expression of both Th1 and Th2-associated cytokines in CD4+ T cells of AD patients, with a significant direct correlation between Th1 and Th2 cell populations, thus yielding insight into the immune features of the AD-associated systemic inflammatory profile. Finally, the observed increased iHO-1 and iHSP-70 expressions likely represent adaptive physiologic countermeasures to AD-associated inflammatory tissue damage, suggesting that HSP inducers are promising candidates for the management of atopic disorders.

IL-27 mediates immune response of pneumococcal vaccine SPY1 through Th17 and memory CD4+T cells

Vaccination is an effective means of preventing pneumococcal disease and SPY1 is a live attenuated pneumococcal vaccine we obtained earlier. We found IL-27 and its specific receptor (WSX-1) were increased in SPY1 vaccinated mice. Bacterial clearance and survival rates were decreased in SPY1 vaccinated IL-27Rα-/- mice. The vaccine-induced Th17 cell response and IgA secretion were also suppressed in IL-27Rα-/- mice. STAT3 and NF-κB signaling and expression of the Th17 cell polarization-related cytokines were also decreased in IL-27Rα-/- bone-marrow-derived dendritic cells(BMDC) stimulated with inactivated SPY1. The numbers of memory CD4+T cells were also decreased in SPY1 vaccinated IL-27Rα-/- mice. These results suggested that IL-27 plays a protective role in SPY1 vaccine by promoting Th17 polarization through STAT3 and NF-κB signaling pathways and memory CD4+T cells production in the SPY1 vaccine. In addition, we found that the immune protection of SPY1 vaccine was independent of aerobic glycolysis.