CD4 Th1 Research Articles

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.

A tumor mutational burden-derived immune computational framework selects sensitive immunotherapy/chemotherapy for lung adenocarcinoma populations with different prognoses.

Lung adenocarcinoma (LUAD) kills millions of people every year. Recently, FDA and researchers proved the significance of high tumor mutational burden (TMB) in treating solid tumors. But no scholar has constructed a TMB-derived computing framework to select sensitive immunotherapy/chemotherapy for the LUAD population with different prognoses. The datasets were collected from TCGA, GTEx, and GEO. We constructed the TMB-derived immune lncRNA prognostic index (TILPI) computing framework based on TMB-related genes identified by weighted gene co-expression network analysis (WGCNA), oncogenes, and immune-related genes. Furthermore, we mapped the immune landscape based on eight algorithms. We explored the immunotherapy sensitivity of different prognostic populations based on immunotherapy response, tumor immune dysfunction and exclusion (TIDE), and tumor inflammation signature (TIS) model. Furthermore, the molecular docking models were constructed for sensitive drugs identified by the pRRophetic package, oncopredict package, and connectivity map (CMap). The TILPI computing framework was based on the expression of TMB-derived immune lncRNA signature (TILncSig), which consisted of AC091057.1, AC112721.1, AC114763.1, AC129492.1, LINC00592, and TARID. TILPI divided all LUAD patients into two populations with different prognoses. The random grouping verification, survival analysis, 3D PCA, and ROC curve (AUC=0.74) firmly proved the reliability of TILPI. TILPI was associated with clinical characteristics, including smoking and pathological stage. Furthermore, we estimated three types of immune cells threatening the survival of patients based on multiple algorithms. They were macrophage M0, T cell CD4 Th2, and T cell CD4 memory activated. Nevertheless, five immune cells, including B cell, endothelial cell, eosinophil, mast cell, and T cell CD4 memory resting, prolonged the survival. In addition, the immunotherapy response and TIDE model proved the sensitivity of the low-TILPI population to immunotherapy. We also identified seven intersected drugs for the LUAD population with poor prognosis, which included docetaxel, gemcitabine, paclitaxel, palbociclib, pyrimethamine, thapsigargin, and vinorelbine. Their molecular docking models and best binding energy were also constructed and calculated. We divided all LUAD patients into two populations with different prognoses. The good prognosis population was sensitive to immunotherapy, while the people with poor prognosis benefitted from 7 drugs.

Exploring the Regulatory Role of XIST-microRNAs/mRNA Network in Circulating CD4+ T Cells of Hepatocellular Carcinoma Patients.

Hepatocellular carcinoma (HCC) is one of the most common cancers and the main cause of cancer-related death globally. Immune dysregulation of CD4+ T cells has been identified to play a role in the development of HCC. Nevertheless, the underlying molecular pathways of CD4+ T cells in HCC are not completely known. Thus, a better understanding of the dysregulation of the lncRNA-miRNA/mRNA network may yield novel insights into the etiology or progression of HCC. In this study, circulating CD4+ T cells were isolated from the whole blood of 10 healthy controls and 10 HCC patients for the next-generation sequencing of the expression of lncRNAs, miRNAs, and mRNAs. Our data showed that there were different expressions of 34 transcripts (2 lncRNAs, XISTs, and MIR222HGs; 29 mRNAs; and 3 other types of RNA) and 13 miRNAs in the circulating CD4+ T cells of HCC patients. The expression of lncRNA-XIST-related miRNAs and their target mRNAs was confirmed using real-time quantitative polymerase chain reaction (qPCR) on samples from 100 healthy controls and 60 HCC patients. The lncRNA-miRNA/mRNA regulation network was created using interaction data generated from ENCORI and revealed there are positive correlations in the infiltration of total CD4+ T cells, particularly resting memory CD4+ T cells, and negative correlations in the infiltration of Th1 CD4+ T cells.

Glycan dependent phenotype differences of HIV-1 generated from macrophage versus CD4+ T helper cell populations.

Human immunodeficiency virus type 1 (HIV-1) is able to infect a variety of cell types with differences in entry efficiency and replication kinetics determined by the host cell type or the viral phenotype. The phenotype of the virus produced from these various cell types, including infectivity, co-receptor usage and neutralisation sensitivity, may also be affected by the characteristics of the producing cell. This can be due to incorporation of variant cell-specific molecules or differences in post-translational modifications of the gp41/120 envelope. In this study we produced genetically identical virus strains from macrophages, CD4-enriched lymphocytes as well as Th1 and Th2 CD4+ cell lines and compared each different virus stock for their infectivity in various cell types and sensitivity to neutralisation. In order to study the effect of the producer host cell on the virus phenotype, virus stocks were normalised on infectivity and were sequenced to confirm env gene homogeneity. Virus production by Th1 or Th2 cells did not compromise infectivity of the variant cell types tested. We observed no difference in sensitivity to co-receptor blocking agents upon viral passage through Th1 and Th2 CD4+ cell lineages nor did this affect DC-SIGN-mediated viral capture as measured in a transfer assay to CD4+ lymphocytes. Virus produced by macrophages was comparably sensitive to CC-chemokine inhibition as was virus generated from the array of CD4+ lymphocytes. We identified that virus produced from macrophages was fourteen times more resistant to 2G12 neutralisation than virus produced from CD4+ lymphocytes. Macrophage-produced dual-tropic (R5/X4) virus was six times more efficiently transmitted to CD4+ cells than lymphocyte-derived HIV-1 (p<0.0001) after DCSIGN capture. These results provide further insights to what extent the host cell influences viral phenotype and thereby various aspects of HIV-1 pathogenesis but suggest that viruses generated from Th1 versus Th2 cells are consistent in phenotype.

A phase II study of concurrent WOKVAC vaccination with neoadjuvant chemotherapy and HER2-targeted monoclonal antibody therapy.

TPS636 Background: HER2+ breast cancer (BCa) is an aggressive breast cancer subtype. The treatment of Stage I-III HER2+ BCa with neoadjuvant chemotherapy and HER2 monoclonal antibodies (trastuzumab and pertuzumab) induces a pathologic complete response in 43-67% of patients. Residual HER2+ disease (i.e. residual cancer burden) after neoadjuvant therapy is associated with a significantly higher risk of recurrence. Evidence supports that HER2 immunity is progressively lost in HER2 oncogenesis and that restoration of HER2 specific CD4+ Th1 immunity is associated with a lower risk of recurrence. Trastuzumab can partially restore HER2 specific immunity, but only in a minority of patients. We have demonstrated that HER2 specific vaccination can augment HER2 specific immunity. While taxane chemotherapy can deplete immunosuppressive cells (MDSCs) 13 – 17 days after treatment creating an optimal time for vaccination. The WOKVAC vaccine is a plasmid vaccine encoding Th1 selective epitopes from HER2 and two other high-risk cancer antigens IGFBP-2 and IGF-1R. The Phase I trial of this vaccine reported that it is safe and all doses induced antigen specific Type I immune responses. We hypothesize that addition of WOKVAC to neoadjuvant chemo/HER2 targeted therapy will induce a significant increase in the number of patients with Th1 CD4+ and CD8+ T cells after neoadjuvant vaccine/chemo/HER2 therapy. Methods: Trial Design: Phase II single arm trial of WOKVAC vaccination with concurrent neoadjuvant taxane based chemotherapy, trastuzumab, and pertuzumab in patients with Stage I-III HER2+ (HR+/-) breast cancer. Enrolled patients will receive WOKVAC vaccine 150mcg on day 13 of cycles 1-3. WOKVAC vaccination may be given with either TCHP or THP. A maximum of 16 patients will be enrolled in this study. Objectives: The primary objective is to evaluate whether concurrent WOKVAC vaccine with chemotherapy and HER2 antibody therapy can significantly increase T-bet+ CD4+ and CD8+ TIL. Secondary objectives are to determine: (1) whether concurrent WOKVAC with neoadjuvant chemo/HER2 therapy is safe, (2) if the magnitude of vaccine induced antigen specific Th1 immunity after neoadjuvant therapy is associated with pathologic response. Statistical Methods: (1) In order to detect a 30% increase in the number of patients with Tbet+ CD4+ and CD8+ TIL in the enrolled patients with 80% power and a one-side alpha of 0.05, our enrollment goal for this study will be 16 patients. (2) Safety will be assessed per CTCAE v5.0, Benchmarks for safety needed to advance this trial concept to a Phase III study will be a grade 3 toxicity rate of ≤13% and a grade 4 toxicity rate of ≤ 7%. (3) The induction of Th1 immunity will be compared against the presence or absence of a complete pathologic response to determine if there is a significant correlation using Pearson r correlation analysis. Targeted Accrual: The study will accrue up to 16 patients. Clinical trial information: NCT04329065 .

Neoadjuvant therapy of HER2 directed conventional dendritic cell (DC1) intratumoral (IT) therapy plus weekly paclitaxel, trastuzumab, and pertuzumab in patients with HER-2 positive breast cancer: NATASHA trial.

596 Background: Early stage HER2 positive breast cancer (BC) patients (pts) are commonly treated with neoadjuvant HER2-targeted therapy. Optimizing neoadjuvant therapy by improving efficacy while reducing toxicity is a critical unmet need. IT DC 1 therapy combined with an IgG1 antibody mediating ADCC drives strong anti-HER-2 CD4 Th1 responses and complete tumor regression in preclinical models. We initiated a phase I/II neoadjuvant study to explore the efficacy and immune stimulation effects of an initial 6 weeks of immunotherapy phase (IP) using IT DC1 vaccine plus trastuzumab (H) and pertuzumab (P) (as source of IgG1) followed by 12 weeks of paclitaxel (T) and HP. Methods: Early stage HER2+ BC pts with tumor ≥ 1cm were eligible. Treatment included IT DC1 weekly x6 followed by paclitaxel 80 mg/m2 IV weekly x 12. Trastuzumab IV every (q) 3 weeks (8 mg/kg loading dose, then 6 mg/m2) and pertuzumab IV q 3 weeks (840 mg loading dose, then 420 mg) x 6 cycles starting from day 1. Two dose levels (DL) of IT DC1 (DL1 = 50 million and DL2 = 100 million cells) were evaluated including 6 pts in each DL. Once the optimal DL is determined based on toxicities, additional 22 patients will be enrolled in expansion phase 2 trial. The primary end point of the phase 1 is the safety, immune responses (measured by ELISPOT). MRI breast was performed at the baseline, post-immunotherapy and post-chemotherapy. Here we are reporting the safety and immune correlates in phase 1 study. Results: Twelve pts were enrolled between 10/2021 and 10/2022. Median age was 57 (range 34-74). Nine pts had hormone receptor positive disease with clinical stage I/II/III (5/5/2) and 4 pts had node positive disease. All pts completed IP and 10 patients completed THP chemotherapy as of 2/1/2023. Radiologic response per MRI breast on pre and post-IP showed 6 partial response, 3 complete response and 3 stable disease. Nine pts underwent surgery and 6 out of 9 pts had pCR (RCB 0). The most frequently observed toxicities were chills (50%), diarrhea (42%), nausea (42%), fatigue (42%), and headache (42%). ELISPOT demonstrated a significant decrease of HER2- specific T cell response following 6 weeks of DC1 IT injections in DL2 compared to DL1. Conclusions: The addition of IT DC1/HP to neoadjuvant chemotherapy was well tolerated with manageable toxicities. The 100 million DC1 led to a significant decrease in peripherally circulating anti-HER2 T cell response at week 6 following IP and improved radiologic tumor responses which correlates with a higher likelihood of pCR. Data from another IT DC1 trial suggests decreased circulating HER2 responsive T cells are due to increased trafficking of reactive T cells out of circulation into the tumor possibly leading to an enhanced anti-tumor effect. Updated results including correlative biomarkers will be presented at the meeting. Clinical trial information: NCT05325632 .