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Background/Purpose: The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in significant mortality, social disorder and economic hardships. We set out to understand why certain individuals could resolve the disease successfully without serious complications by immune profiling of PBMCs from convalescent COVID-19 patients (n = 14) with mild disease trajectory. Methods: We designed 7 peptides targeting the receptor binding region (RBD) of the spike protein of SARS-CoV-2, that encompass broad HLA class I / II alleles, with the aid of the Immune Epitope Database and Analysis Resource (IEDB) website. The RBD site was chosen as it was shown to be protective when neutralising antibodies against this region could negate binding of viral spike protein to host cell ACE-2 receptor. Convalescent COVID-19 patients enrolled in this study were screened and selected for positive antibody titre against the RBD region. PBMCs from convalescent COVID-19 patients were stimulated with/without the peptides for 72hrs and immune profiled (n = 70 markers across two panels) with the high dimensional single cell mass cytometry platform (CyToF). Results: Convalescent COVID-19 patients elicited a robust recall memory T follicular helper response (CD3 + CD4 + CD45RO + CXCR5 + Tigit + ;∗∗∗∗ p < 0.0001) demonstrating peptide efficacy. Unsupervised clustering (FlowSOM) of the CD4 T cell immune landscape reaffirmed increase in memory T follicular subsets and additionally CD4 + CD45RO + CXCR5 -subsets. Further gating of antigen specific memory cells (CD45RO + CD69 + ) revealed an increase in Tbet + CXCR3 + T effectors in both CD4 and CD8 compartments. Strikingly we detected a parallel increase in CD4 + Treg (CD25 + FoxP3 + ) CXCR3 + Tbet + CD45RO + CD152 + Tigit + expression. Conclusions: COVID-19 patients that successfully resolve the viral infection not only mount a robust T effector and follicular response but also in tandem a similar T regulatory profile.

IL-17-producing CD8 (Tc17) T cells have been shown to play an important role in infection and chronic inflammation, however their implications in hepatocellular carcinoma (HCC) remain elusive. In this study, we performed cytometry by time-of-flight (CyTOF) and revealed the distinctive immunological phenotypes of two IFNγ

Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated.We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRWe uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

Background & Aims: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. Methods: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. Results: Single-cell analyses identified CXCR3+ CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+ CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. Conclusions: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. National Medical Research Council (NMRC) Published version This work was supported by the National Medical Research Council (NMRC), Singapore (ref numbers: NMRC/OFLCG/003/2018, NMRC/TCR/015-NCC/2016, NMRC/CSA-SI/0013/2017, NMRC/CSASI/0018/2017 and NMRC/STaR/020/2013).

ObjectivesBiologics treatment with antitumour necrosis factor alpha (TNFα) is efficacious in patients with juvenile idiopathic arthritis (JIA). Despite displaying clinical inactivity during treatment, many patients will flare on cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown.MethodsWe interrogated the circulatory reservoir of CD4+ immune subsets at the single-cell resolution with mass cytometry (cytometry by time of flight) of patients with JIA (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16) the dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with NanoString.ResultsAn inflammatory memory subset of CD3+CD4+CD45RA−TNFα+ T cells deficient in immune checkpoints (PD1−CD152−) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease-centric pathways involving (1) T-cell receptor activation, (2) apoptosis, (3) TNFα, (4) nuclear factor-kappa B and (5) mitogen-activated protein kinase signalling.ConclusionsA unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.

Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4+ T cells, CD8+ T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8+PD-1+ T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8+PD-1- T cells and non-Treg CD4+FOXP3- T cells; but increased exhausted CD8+PD-1+ T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1+CD8+ T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive fibrosis of skin and internal organs, and vascular dysfunction. Association of T and B cell subsets has been reported in SSc; however, there is lack of systematic studies of functional relations between immune cell subsets in this disease. This lack of mechanistic knowledge hampers targeted intervention. In the current study we sought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients. Mononuclear cells from blood of SSc patients (n = 20) and healthy controls (n = 10) were analyzed by mass cytometry using a 36-marker (cell surface and intracellular) panel. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and mucosal associated invariant T (MAIT) cells in patients, accompanied by increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Overall, this study provides an in-depth analysis of the systemic immunome in SSc, highlighting the potential pathogenic role of inflammation and chronic stimulation-mediated “functional adaptation” of immune cells.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV., A Zika virus (ZIKV) vaccine should provide long-lasting immunity, which may be achieved with a live-attenuated vaccine. Here, Kwek et al. select an interferon-restricted, attenuated ZIKV variant and evaluate replication and immunogenicity in mouse and mosquito models.

ObjectivesYttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response.DesignTime-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE.ResultsTILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8+ T cells, CD56+ NK cells and CD8+ CD56+ NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB+CD8+ T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8+ and CD4+ T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1+/Tim-3+CD8+ T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs.ConclusionHigh-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterised by excessive fibrosis of skin and internal organs, and vascular dysfunction (1). Association of T and B cell subsets have been reported in SSc, however there is lack of systematic studies of functional relations between immune cell subsets in this disease (2,3,4). This lack of mechanistic knowledge hampers targeted intervention. Objectives: In the current study we ought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients and its impact on disease severity and progression. Methods: Mononuclear cells from blood of SSc patients (n=20) and healthy controls (n=10) were analysed by mass cytometry using a 36 marker (cell-surface and intracellular) panel to aid in identification of major PBMC lineages including T cells, B cells, monocytes and NK cells and their subsets. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering of mass cytometry data was performed using in-house developed analysis software MARVIS. This software combines dimension reduction and clustering steps to identify all possible cellular subsets. Further, custom R scripts helped in identifying nodes that were differentially expressed between the study groups and also phenotype of these nodes. Results: Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and MAIT cells in patients, accompanied with increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Conclusion: Overall this study provides an in depth analysis of systemic immunome in SSc, highlighting role of inflammation and chronic stimulation mediated “premature senescence” of immune cells, with implications to delineate mechanism of pathogenesis and identify diagnostic/therapeutic targets. References: [1] Denton, C. P. and D. Khanna (2017). “Systemic sclerosis.” Lancet 390(10103): 1685-1699. [2] Fuschiotti, P. (2017). “Current perspectives on the role of CD8+ T cells in systemic sclerosis.” Immunol Lett. In Press [3] Liu, M., et al. (2016). “New insights into CD4(+) T cell abnormalities in systemic sclerosis.” Cytokine Growth Factor Rev 28: 31-36. [4] Sanges, S., et al. (2017). “Role of B cells in the pathogenesis of systemic sclerosis.” Rev Med Interne 38(2): 113-124. Disclosure of Interests: None declared

ObjectivesBiologics treatment with anti-TNFα is efficacious in juvenile idiopathic arthritic (JIA) patients. Despite displaying clinical inactivity during treatment, many patients will flare upon cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown.MethodsWe interrogated the circulatory reservoir of CD4+ immune subsets at the single cell resolution with mass cytometry (CyToF) of JIA patients (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα, and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16), the continued dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with Nanostring.ResultsAn inflammatory memory subset of CD3+CD4+CD45RA−TNFα+ T cells deficient in immune checkpoints (PD1−CD152−) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease centric pathways involving (a) TCR activation, (b) apoptosis, (c) TNFα, (d) NF-kB and (e) MAPK signalling.ConclusionsA unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.

Systemic sclerosis (SSc) is an autoimmune disease characterised by excessive fibrosis of skin and internal organs, and vascular dysfunction. Association of T and B cell subsets have been reported in SSc, however there is lack of systematic studies of functional relations between immune cell subsets in this disease. This lack of mechanistic knowledge hampers targeted intervention. In the current study we sought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients. Mononuclear cells from blood of SSc patients and healthy controls (HC) were analysed by mass cytometry using a 36 marker (cell-surface and intracellular) panel. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and MAIT cells in patients, accompanied with increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Overall this study provides an in-depth analysis of the systemic immunome in SSc, highlighting the potential pathogenic role of inflammation and chronic stimulation mediated “premature senescence” of immune cells.ONE SENTENCE SUMMARYThe immune architecture in Systemic sclerosis is altered and reminiscent of chronic stimulatory environment led premature senescence of immune cells.

ObjectivesThe imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation.MethodsWe compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions.ResultsWe found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells.ConclusionsUsing an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity.

Background:Despite advances in understanding how the adaptive T cell landscape is affected in human arthritis, specific T cell subset knowledge has yet to be utilised in clinical settings. We have previously discovered within active arthritic patients, a circulating pathogenic-like lymphocyte (CPLs; CD4+HLA-DR+) within the T-effector compartment, that is phenotypically similar to their synovial counterparts. CPLs are inflammatory, correlate with disease activity and overlap in synovial TCR repertoire. A similar inflammation-associated T-regulatory (iaTreg; CD4+HLA-DR+) subset, that is activated, poised to migrate to inflamed site and sharing synovial TCR overlap, suggest a common disease ontogeny that may exist between CPLs and iaTregs.Objectives:Here we seek to determine whether and how the synovial microenvironment plays a role in modulating these two functionally divergent (Teff/Treg compartments) yet pathogenically homologous subsets. This modulation, akin to an immunological rheostat, may be a feature of the disease process.Methods:We examined CD45+ immune cells from synovial and PBMCs (active JIA, inactive JIA, paediatric healthy) through mass cytometry (CyToF). CD4 T cells were sorted into CPLs, iaTregs, Teff and Treg through FACS Aria II, from active JIA PBMCs, paired JIA SFMCs and healthy paediatric PBMCs and examined through ngRNASEQ.Results:Mass cytometric analysis reveal a significant enrichment of synovium signatures in both circulatory CPLs and iaTregs subsets from active arthritic PBMCs, as compared with the conventional pool of Teff/Tregs. This immunological relationship between CPLs/iaTregs is reaffirmed by comparative differential gene expression (DEG) and phylogenetic tree analysis, which indicated transcriptomic convergence between circulatory pathogenic CPLs/iaTreg subsets and divergence from their respective conventional Teff/Treg pools. Circulatory CPLs/iaTregs exhibit (a) common pathway dysregulation in T cell signalling, (b) restriction in TCR oligoclonality and (c) common transcription factor drivers within the gene regulatory network, suggesting a common pathogenic mechanism acting on these two disparate compartments.To understand how the microenvironment plays a role in modulating these two subsets, we compared the transcriptome of CPLs/iaTreg and conventional Teff/Treg subsets from (a) healthy PBMCs, (b) JIA PBMCs and (c) paired JIA SFMCs. The convergence between CPLs/iaTreg increases across the spatial/disease continuum, culminating in 7 key common dysregulated pathways within synovium CPLs/iaTregs. Importantly we detected higher clonotypic sharing of TCRs in CPLs/iaTregs across the spatial and disease continuum, suggesting a common precursor driven by antigenic selection.Conclusion:Our data suggest that CPLs/iaTregs are dichotomic components of a systemic immune rheostat, shape through the synovium environment, modulating autoimmunity in human arthritis. As iaTreg and CPL most likely have the capacity to morph into each other, the molecular crossroads which control this plasticity represent novel therapeutic targets.Disclosure of Interests:None declared

Background Systemic sclerosis (SSc) is an autoimmune disease characterised by excessive fibrosis of skin and internal organs, and vascular dysfunction1. Association of T and B cell subsets have been reported in SSc, however there is lack of systematic studies of functional relations between immune cell subsets in this disease2,3,4. This lack of mechanistic knowledge hampers targeted intervention. Objectives In the current study we ought to determine differential immune cell composition and heterogeneity in peripheral blood of SSc patients and its impact on disease severity and progression. Methods Mononuclear cells from blood of SSc patients with interstitial lung disease (ILD, n=10) or No ILD (n=10) and healthy controls (n=10) were analysed by mass cytometry using a 36 marker (cell-surface and intracellular) panel to aid in identification of major PBMC lineages including T cells, B cells, monocytes and NK cells and their subsets. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering of mass cytometry data was performed using in-house developed analysis software MARVIS. This software combines dimension reduction and clustering steps to identify all possible cellular subsets. Further, custom R scripts helped in identifying nodes that were differentially expressed between the study groups and also phenotype of these nodes. Results Unsupervised clustering performed revealed significant differences in the frequencies of T cell and B cell subsets. Most strikingly we identify a 3 fold decrease in frequencies of Va7.2+CD161+mucosal associated invariant T cells (MAIT) in SSc patients and 2 fold increase in total B cells, particularly CD19 +CD27 naive cells. A subset of memory CD8 +T cell, expressing CXCR3 was found to be increased in SSc patients as compared to healthy controls. Transcriptome analysis of sorted B cell and T cell subsets showed decrease in genes related to survival and increased expression of apoptotic genes in CD4,CD8 T and MAIT cells from SSc patients. Genes related to exhaustion and leukocyte migration were highly expressed in T cells from patients. Conclusions This study provides an in depth analysis of systemic immune composition in SSc with the potential to delineate mechanisms of pathogenesis and identify diagnostic and/or therapeutic targets. This is the first demonstration of dysfunction of MAIT cells in SSc and further characterisation of their function in this context is required. References [1] Denton CP, Khanna D. ‘Systemic sclerosis.’Lancet2017;390(10103):1685–1699. [2] Fuschiotti P. Current perspectives on the role of CD8+ T cells in systemic sclerosis. Immunol Lett2017. [3] Liu M, et al. New insights into CD4(+) T cell abnormalities in systemic sclerosis.’Cytokine Growth Factor Rev2016;28:31–36. [4] Sanges S, et al. ‘Role of B cells in the pathogenesis of systemic sclerosis.’Rev Med Interne2017;38(2):113–124. Disclosure of Interest None declared

Background JIA patients respond well to anti-TNFA biologics, with up to 80% of patients achieving clinical remission. In spite of this success, 50%–80% will relapse upon therapy withdrawal, indicating a large proportion of patients had yet to fully resolve their disease. Compounded by concerns with drug toxicities and financial burden, there is a genuine interest to find predictors for successful drug cessation and devise new avenues of therapy. Objectives To determine how subclinical persistence of disease occurs despite therapy, we compare JIA individuals destined to flare or remain inactive, prior to (To) and after therapy withdrawal (Tend). Previous publications have reveal that CD4 T cells play a vital role in disease pathogenesis in JIA patients. We aim to dissect the CD4 landscape (a) through CyToF, to decipher the CD4 subsets responsible for disease persistence, (b) to unravel the pathways involved through mRNA analysis with Nanostring. Methods Patients treated with anti-TNF-alpha biologics were recruited with clinically inactive disease on treatment and initiated with therapy discontinuation. The patients designated as flare (n=24) and inactive (n=24) based on 6 JIA core set parameters. Healthy paediatric controls (n=17) with no inflammatory disease were recruited pre-operatively during day surgeries. A separate study with active JIA patients recruited pre/post treatment (n=4 paired) with anti-TNFA biologics and achieving recent clinical remission. Results Interrogation of PBMCs with CyToF reveal the persistence of a subset of CD3 +CD4+inflammatory memory CD45RA- TNFA +PD1- CTLA4- T cells (p Conclusions These results highlight a strong immunological memory dysregulation in a subset of CD4 T cells in JIA patients that is predictive of clinical fate and providing new therapeutic insights. Disclosure of Interest None declared

Background One of the key elements of immune pathogenesis of human autoimmune arthritis is the resilience of pathogenic T cells. We have previously described that CD4 +T cells in patients with arthritis have an increased level of autophagy than their healthy equivalents. Here, we sought to explore at epigenetic and transcriptional levels the concept of persisting increased autophagy as the consequence of “autophagic memory”, as one of the mechanisms conferring resilience to pathogenic T cells, in particular to a subset of CD4 +T cells (CPL: Circulating Pathogenic-like Lymphocytes), which are significantly more represented in patients with active arthritis and resistant to therapy with biologics Objectives To understand molecular mechanism of resilience and persistence in pathogenic T cells in rheumatoid arthritis. Methods Autophagy in T cells were analysed using CytoID autophagy detection kit. Jurkat cells pre-starved and control were harvested at various time points, and RNA was extracted for RNA-sequencing and DNA methylation analysis. Illumina paired end sequencing was performed and data was analysed using open source tools in R statistical programming software. CD4 +memory and naive T cells were sorted using flow cytometer for qPCR analysis. The CD4 +memory and naive cells were sorted using Flow cytometer. RNA extracted and converted to cDNA for qPCR analysis of key genes Results First, we demonstrated elevated autophagic levels in CD4 +memory T cells when compared to naive CD4 +T cells. Second, we showed that autophagic levels are increased in naive and CD4 +T cells from RA patients compared to healthy controls. Using next generation RNA-sequencing, transcription factor gene regulatory network (TF-GRN) and methylation analyses, we identified MYC as key regulator of autophagic memory in a human T cell line. Transcriptome and network analysis of RNA-seq data from patients’ CPLs confirmed MYC as key modulator of autophagy. Importantly, inhibitor of MYC increases autophagy Conclusions The present study suggests that autophagic memory is retained both at the transcriptional and epigenetic levels as an integral part of mechanisms of efficient activation and survival of memory T cells. This mechanism is particularly relevant for cells subsets, such as CPLs, which are relevant to the immunopathogenesis of autoimmune diseases, such as arthritis. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target. Disclosure of Interest None declared

Background Psoriasis (Ps) currently inflicts 2%–3% of the population globally and one-third of patients have psoriatic arthritis (PsA). Up to 30% of PsA patients with active psoriasis (Ps) do not respond adequately to any treatment. Understanding the immune mechanisms contributing to the initiation and disease progression of PsA and Ps is crucial for devising novel therapeutic strategies. Objectives To address the current unmet clinical need and bridge the knowledge gap in the pathogenesis underlying PsA/Ps, we perform transcriptomic analyses of the skin microenvironment and deep immunophenotyping of immune cells from PsA patients with active disease. We hypothesise that the interaction between the tissue microenvironment and the peripheral immune system dictates the immune response that impacts upon the development and progression of PsA/Ps. This multi-dimensional strategy will also enable the distillation of immune cell subsets in the periphery that can potentiate pathogenic responses in the microenvironment. Methods Total RNA was extracted from skin punch biopsies of lesional and morphologically normal sites from 7 patients with active disease. RNASeq was performed to decipher the transcriptomes of the skin punch biopsies. Peripheral Blood Mononuclear Cells (PBMCs) from 17 PsA patients and 12 healthy donors were stimulated with PMA-Ionomycin, stained with 37 phenotypic T cells markers and interrogated with the CyTOF platform. Dimensional reduction and unsupervised clustering analyses were performed with Multi-dimensional Automated Reduction and Visualisation (MARVis). Results Transcriptomic analysis of skin punch biopsies of psoriatic and morphologically normal sites revealed a gene signature in lesional skin that promotes the infiltration of multiple immune cell subsets into the microenvironment. The expression of chemokine genes such as CXCL8, CCL4, and CCL20 suggests a role for the accumulation of neutrophils, monocytes, natural killer (NK) cells and lymphocytes in the establishment of a pro-inflammatory microenvironment that is perpetuated by the presence of TNF and IFNγ. Examination of the immune landscapes of PsA patients highlights multiple perturbations in various immune cell subsets. Specifically, we observed declines in CD8 +CD161+TCRVα7.2+Mucosal Associated Invariant T (MAIT), CD4 +CD45RO+CXCR5+TFH as well as CD45RO+Tbet+IFNγ+TNFα+IL17A+memory TH1 cells in PsA patients. This decline is potentially attributed to the trafficking of these immune cell subsets into the microenvironment in response to chemokine signals. Conversely, we observed enrichments of CD56 +Tbet+GranB +IFNγ+NK and activated CD4 +CD127+CCR7+CD69+effector T cells in PsA patients that can contribute to the pathogenic immune response. Conclusions Our multi-dimensional approach resolves the complex interactions between the tissue microenvironment and the peripheral immunome that shapes the immune response and dictates the cellular composition in lesional skin. These findings possess translational value and will facilitate the identification of novel immune therapeutic targets. Disclosure of Interest None declared

Background and aimsChronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics.MethodsWe interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays.ResultsIn-depth interrogation of the immune landscapes showed that regulatory T cells (TREG) and CD8+ resident memory T cells (TRM) were enriched in HBV-related HCC, whereas Tim-3+CD8+ T cells and CD244+ natural killer cells were enriched in non-viral-related HCC. NGS of isolated TREG and TRM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. TREG and TRM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1+ TREG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that TREG and TRM contributed to overall patient survival: TREG were associated with a poor prognosis and TRM were associated with a good prognosis in HCC.ConclusionWe have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.

The innate immune response shapes the development of adaptive immunity following infections and vaccination. However, it can also induce symptoms such as fever and myalgia, leading to the possibility that the molecular basis of immunogenicity and reactogenicity of vaccination are inseparably linked. To test this possibility, we used the yellow fever live-attenuated vaccine (YFLAV) as a model to study the molecular correlates of reactogenicity or adverse events (AEs). We analyzed the outcome of 68 adults who completed a YFLAV clinical trial, of which 43 (63.2%) reported systemic AEs. Through whole-genome profiling of blood collected before and after YFLAV dosing, we observed that activation of innate immune genes at day 1, but not day 3 after vaccination, was directly correlated with AEs. These findings contrast with the gene expression profile at day 3 that we and others have previously shown to be correlated with immunogenicity. We conclude that although the innate immune response is a double-edged sword, its expression that induces AEs is temporally distinct from that which engenders robust immunity. The use of genomic profiling thus provides molecular insights into the biology of AEs that potentially forms a basis for the development of safer vaccines.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8+ T cells (TRMs), resident natural killer cells (NKRs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3+CD4+ and PD-1+CD8+ T cells. Interestingly, Tregs and TRMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and TRMs isolated from the NTME. We found PD-1+ TRMs were the predominant T-cell subset responsive to anti–PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8+ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NKRs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed.