Gene of the month: lymphocyte-activation gene 3 (LAG-3)

Lymphocyte-activation gene 3 (LAG-3) is a coreceptor found on activated T-lymphocytes activated B-lymphocytes and natural killer (NK) cells. It is closely related to CD4 where it shares multiple common and divergent features. It contains specific binding sites with high affinity to major histocompatibility complex (MHC) Class II and functions as an inhibitor of T-cell signalling. Tumour-infiltrating lymphocytes with high LAG-3 expression have been found in many solid tumours including ovarian cancer, melanoma, colorectal cancer and haematological malignancies including Hodgkin and diffuse large B-cell lymphoma. LAG-3 antagonism has been demonstrated to restore the anti-tumourigenic function of T-cells in vivo, however, mechanistic knowledge remains relatively poorly defined. As other immune checkpoint inhibitors have transformed the management of difficult to treat cancers, such as melanoma, it is hoped that LAG-3 might have the same potential. This review will explore LAG-3 modulation as an anticancer therapy, highlighting recent clinical developments.

B7-H7 Is Inducible on T Cells to Regulate Their Immune Response and Serves as a Marker for Exhaustion

The discovery of immune checkpoints highlights the complexity of T cell signalling during an immune response. Upon activation, T cells express several molecules to regulate their function and to prevent overactivation. B7 homolog 7 (B7-H7) is expressed in tumours and associated with a worse prognosis. However, conflicting data regarding its function suggest that it can be both stimulatory and inhibitory. In this study we report that B7-H7 is also expressed on T cells upon cross-linking of CD3 and CD28 and that additional stimulation via CD137 further enhances the expression of B7-H7. B7-H7 is preferentially expressed on exhausted Th1 and Tc1 cells with an impaired secretion of TNF-α and IFN-γ. Blockade of B7-H7 with its natural receptor, recombinant CD28H, enhances T cell proliferation and activation. Thus, B7-H7 represents another target for immunotherapy and a biomarker to select for active effector T cells with relevance for adoptive cell transfer therapy.

THU0047 THE SYNOVIUM REWIRES AN IMMUNOLOGICAL RHEOSTAT THAT DEFINES TWO FUNCTIONALLY DISPARATE PATHOGENIC CD4+HLA-DR+ SUBSETS IN HUMAN ARTHRITIS

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

P084 Cannabis use promotes lymphocyte responsiveness and modifies T-cell signalling in adolescent IBD patients

Background The prevalence of inflammatory bowel disease (IBD) has increased dramatically in recent years, particularly in paediatric populations. The efficacy of available therapies is limited and often transient, leading patients to seek alternative therapies for symptom relief, including the use of medical marijuana (Cannabis sativa). There is some evidence to suggest that cannabinoids exert anti-inflammatory effects in preclinical colitis models. In addition, a number of small studies have reported improvements in non-empirical disease measures and reductions in visceral pain in response to cannabis use. However, associations between cannabis use in IBD and increased need for surgical interventions have also been reported. Methods Therefore, determining the direct impact of cannabis use on immune modulation in IBD patients is of critical importance. Blood samples collected from paediatric IBD patients who reported cannabis use for symptom control were analysed for cytokine expression and T-cell signalling pathway activation compared with non-users. Concentrations of serum phytocannabinoids were determined by HPLC and correlated with subsequent ex vivo assay parameters. Results Results demonstrated elevated levels of a myriad of pro-inflammatory cytokines in users vs. non-users upon ex vivo restimulation. This coincided with an expansion of pathogenic Th17 cells in the periphery. Differences in signalling cascades of activated T cells between users and non-users were also observed. Conclusion These results suggest that cannabis exposure, which can desensitise cannabinoid receptors, may prime pro-inflammatory pathways in paediatric IBD patients. Future studies should address the limitations of observational studies through the use of randomised controlled trials of cannabis use in paediatric IBD populations. This research was funded by the Colorado Department of Public Health and Environment.

Abstract 455: HDL-Small RNA Gene Regulatory Networks Alter T Cell Signalling in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a debilitating disease primarily in women involving complex T and B cell dysregulation. SLE presents with dysfunctional HDL and we have previously found that HDL-microRNAs (miRNA) are significantly altered in SLE; however, miRNAs are just one of many types of small non-coding RNAs (sRNA). As such, we hypothesized that HDL-sRNA cargo and cell-to-cell communication in SLE extend beyond miRNAs. Using high-throughput sRNA sequencing (sRNA-seq), we found that tRNA-derived sRNAs (tDRs) were highly abundant on HDL and were significantly altered in SLE subjects (n=9) compared to controls (n=8, P <0.05). In addition, circulating levels of angiogenin, an RNaseIII enzyme responsible for tDR cleavage from parent tRNAs, was also found to be significantly increased in plasma ( P <0.05) from SLE subjects compared to controls. To determine if tDRs are altered in CD4+ T cells in SLE subjects, real-time PCR was used to quantify candidate tDRs, and we found that tDR-GlyGCC levels were significantly increased 4.2-fold in SLE ( P <0.01) and readily exported to HDL. Strikingly, total RNAseq, in silico analysis, and mRNA sequencing suggested that ROCK2, a critical regulator of CD4+ T cell differentiation, is a direct tDR-GlyGCC target gene which was confirmed with gene reported (luciferase) assays. Moreover, activated human CD4+ T cells transfected with tDR-GlyGCC mimetics, demonstrated reduced ROCK2 protein levels and STAT3 phosphorylation, and consequently reduced inflammatory cytokine secretion (IL-17 and IL-21; P <0.05). To determine if T cell exported tDR-GlyGCC is transferred between cells by HDL, ex vivo studies were completed using Trans-PhotoActivatable-Ribonucleoside-CrossLinking-ImmunoPrecipitation high-throughput Sequencing (Trans-PAR-CLIPseq). Using this approach, we found a cassette of CD4+ T cell-originating sRNAs, including tDR-GlyGCC, that were transferred by HDL to recipient immune cells. Here, we demonstrate that HDL facilitates intercellular transfer of tDRs between immune cells and a critical role for tDR-GlyGCC in regulating T cell signalling.