Current developments in the use of biomarkers for juvenile idiopathic arthritis.

Use of biomarkers in clinical practice has proved extremely valuable and is a rapidly expanding field. However, despite the huge potential of biomarkers, for juvenile idiopathic arthritis (JIA) there are currently no validated paediatric biomarkers available to help with setting up a more tailored approach on which drug choice could be based, to achieve remission early in the course of disease. Early remission reduces burden of disease, limits side effects from toxic and unnecessary medication, and, most importantly, enhances quality of life. Several studies have suggested promising biomarkers: these may be a protein, cellular component, mRNA, or genetic component, for example a single nucleotide polymorphism (SNP). Here we describe recent developments in the use of biomarkers for JIA and their potential to assist in management of disease by predicting disease phenotype, severity, progression, and response to treatment, and determining when patients have reached stable remission and can safely discontinue treatment.

Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells.

During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

CD161 expression and regulation defines rapidly responding effector CD4+ T cells associated with improved survival in HPV16-associated tumors.

BACKGROUND: Expression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown. METHODS: We examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies. RESULTS: Central and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)ß1. CONCLUSION: High levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells.

CD163+ cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking. METHODS: We analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16+ patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses. RESULTS: We show that the tumor microenvironment of HPV16+ OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163+ cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163+ cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFNγ and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163+ cDC2 positively correlated with the infiltration by Tbet+ and tumor-specific T cells, and with prolonged survival. CONCLUSIONS: These data suggest an important role for intratumoral CD163+ cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects.

Innate Lymphoid Cells and T Cells Contribute to the Interleukin-17A Signature Detected in the Synovial Fluid of Patients With Juvenile Idiopathic Arthritis.

OBJECTIVE: Evidence suggests that aberrant function of innate lymphoid cells (ILCs), whose functional and transcriptional profiles overlap with those of Th cell subsets, contributes to immune-mediated pathologies. To date, analysis of juvenile idiopathic arthritis (JIA) immune pathology has concentrated on the contribution of CD4+ T cells; we have previously identified an expansion of Th17 cells within the synovial fluid (SF) of JIA patients. We undertook this study to extend this analysis to further investigate the role of ILCs and other interleukin-17 (IL-17)-producing T cell subsets in JIA. METHODS: ILCs and CD3+ T cell subsets were defined in peripheral blood mononuclear cells (PBMCs) from healthy adults, healthy children, and JIA patients and in SF mononuclear cells (SFMCs) from JIA patients using flow cytometry. Defined subsets in SFMCs were correlated with clinical measures including physician's global assessment of disease activity on a visual analog scale, number of joints with active disease, and erythrocyte sedimentation rate. Transcription factor and cytokine profiles of sorted ILCs were assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Group 1 ILCs (ILC1s), NKp44- group 3 ILCs (natural cytotoxicity receptor-negative [NCR-] ILC3s), and NKp44+ ILC3s (NCR+ ILC3s) were enriched in JIA SFMCs compared to PBMCs, which corresponded to an increase in transcripts for TBX21, IFNG, and IL17A. Of the ILC subsets, the frequency of NCR- ILC3s in JIA SFMCs displayed the strongest positive association with clinical measures, which was mirrored by an expansion in IL-17A+CD4+, IL-17A+CD8+, and IL-17A+ γδ T cells. CONCLUSION: We demonstrate that the strength of the IL-17A signature in JIA SFMCs is determined by multiple lymphoid cell types, including NCR- ILC3s and IL-17A+CD4+, IL-17A+CD8+, and IL-17A+ γδ T cells. These observations may have important implications for the development of stratified therapeutics.

The Anatomical Location Shapes the Immune Infiltrate in Tumors of Same Etiology and Affects Survival.

PURPOSE: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. EXPERIMENTAL DESIGN: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)-induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). RESULTS: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. CONCLUSIONS: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

CD19+CD24hiCD38hi B Cells Are Expanded in Juvenile Dermatomyositis and Exhibit a Pro-Inflammatory Phenotype After Activation Through Toll-Like Receptor 7 and Interferon-α.

Juvenile dermatomyositis (JDM) is a rare form of childhood autoimmune myositis that presents with proximal muscle weakness and skin rash. B cells are strongly implicated in the pathogenesis of the disease, but the underlying mechanisms are unknown. Therefore, the main objective of our study was to investigate mechanisms driving B cell lymphocytosis and define pathological features of B cells in JDM patients. Patients were recruited through the UK JDM Cohort and Biomarker study. Peripheral blood B cell subpopulations were immunophenotyped by flow cytometry. The results identified that immature transitional B cells were significantly expanded in active JDM, actively dividing, and correlated positively with disease activity. Protein and RNAseq analysis revealed high interferon alpha (IFNα) and TLR7-pathway signatures pre-treatment. Stimulation of B cells through TLR7/8 promoted both IL-10 and IL-6 production in controls but failed to induce IL-10 in JDM patient cells. Interrogation of the CD40-CD40L pathway (known to induce B cell IL-10 and IL-6) revealed similar expression of IL-10 and IL-6 in B cells cultured with CD40L from both JDM patients and controls. In conclusion, JDM patients with active disease have a significantly expanded immature transitional B cell population which correlated with the type I IFN signature. Activation through TLR7 and IFNα may drive the expansion of immature transitional B cells in JDM and skew the cells toward a pro-inflammatory phenotype.

CD161+ Tconv and CD161+ Treg Share a Transcriptional and Functional Phenotype despite Limited Overlap in TCRß Repertoire.

Human regulatory T cells (Treg) are important in immune regulation, but can also show plasticity in specific settings. CD161 is a lectin-like receptor and its expression identifies an effector-like Treg population. Here, we determined how CD161+ Treg relate to CD161+ conventional T cells (Tconv). Transcriptional profiling identified a shared transcriptional signature between CD161+ Tconv and CD161+ Treg, which is associated with T helper (Th)1 and Th17 cells, and tissue homing, including high expression of gut-homing receptors. Upon retinoic acid (RA) exposure, CD161+ T cells were more enriched for CCR9+ and integrin α4+ß7+ cells than CD161- T cells. In addition, CD161+ Tconv and CD161+ Treg were enriched at the inflamed site in autoimmune arthritis, and both CD161+ and CD161- Treg from the inflamed site were suppressive in vitro. CD161+ T cells from the site of autoimmune arthritis showed a diminished gut-homing phenotype and blunted response to RA suggesting prior imprinting by RA in the gut or at peripheral sites rather than during synovial inflammation. TCRß repertoires of CD161+ and CD161- Tconv and Treg from blood showed limited overlap whereas there was clear overlap between CD161+ and CD161- Tconv, and CD161+ and CD161- Treg from the inflamed site suggesting that the inflamed environment may alter CD161 levels, potentially contributing to disease pathogenesis.