Modulation of TCR signalling components occurs prior to positive selection and lineage commitment in iNKT cells

AbstractiNKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their unique functional characteristics are induced by a transcriptional program initiated by positive selection mediated by CD1d expressed by CD4+CD8+ (double positive, DP) thymocytes. Here, using a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24hiCD44loNKT cells, we examined transcriptional events in four immature thymic iNKT cell subsets. A transcriptional regulatory network approach identified transcriptional changes in proximal components of the TCR signalling cascade in DP NKT cells. Subsequently, positive and negative selection, and lineage commitment, occurred at the transition from DP NKT to CD4 NKT. Thus, this study introduces previously unrecognised steps in early NKT cell development, and separates the events associated with modulation of the T cell signalling cascade prior to changes associated with positive selection and lineage commitment.

Stepwise progression of β-selection during T cell development as revealed by histone deacetylation inhibition

During T cell development, the first step in creating a unique T Cell Receptor (TCR) is the genetic recombination of the TCRβ chain. The quality of this newly recombined gene is assessed at the β-selection checkpoint, and most cells fail this checkpoint and are removed. The coordination of the complex events that combine to control fate at the β-selection checkpoint is not yet understood. We assessed the impact on T cell development of a selective inhibitor to histone deacetylase 6, ACY1215, currently in clinical use. ACY1215 led to bypass of the β-selection checkpoint such that cells in the DN4 stage often lacked expression of TCRβ, and failed to progress to the DP stage. Characterisation of the molecular basis for this bypass revealed a new, pivotal stage in β-selection, the beginning and end of which were defined by the upregulation of the TCR co-receptors, CD28 and CD2 respectively. Within this stage, termed DN3bPre, CD5 and Lef1 are upregulated to reflect pre-TCR signalling. We propose that the progressive expression of CD28, CD5 then CD2 reports and modulates the pre-TCR signal to orchestrate passage through the β-selection checkpoint. By disrupting the functional connection between CD5 and pre-TCR, ACY1215 allows cells to inappropriately bypass the β-selection checkpoint. These findings implicate a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD5 and Lef1 provides for an escalating test of TCR signalling strength, and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Gamma-Chain Receptor Cytokines & PD-1 Manipulation to Restore HCV-Specific CD8+ T Cell Response during Chronic Hepatitis C

Hepatitis C virus (HCV)-specific CD8+ T cell response is essential in natural HCV infection control, but it becomes exhausted during persistent infection. Nowadays, chronic HCV infection can be resolved by direct acting anti-viral treatment, but there are still some non-responders that could benefit from CD8+ T cell response restoration. To become fully reactive, T cell needs the complete release of T cell receptor (TCR) signalling but, during exhaustion this is blocked by the PD-1 effect on CD28 triggering. The T cell pool sensitive to PD-1 modulation is the progenitor subset but not the terminally differentiated effector population. Nevertheless, the blockade of PD-1/PD-L1 checkpoint cannot be always enough to restore this pool. This is due to the HCV ability to impair other co-stimulatory mechanisms and metabolic pathways and to induce a pro-apoptotic state besides the TCR signalling impairment. In this sense, gamma-chain receptor cytokines involved in memory generation and maintenance, such as low-level IL-2, IL-7, IL-15, and IL-21, might carry out a positive effect on metabolic reprogramming, apoptosis blockade and restoration of co-stimulatory signalling. This review sheds light on the role of combinatory immunotherapeutic strategies to restore a reactive anti-HCV T cell response based on the mixture of PD-1 blocking plus IL-2/IL-7/IL-15/IL-21 treatment.

The role of cholesterol in TCR signalling in autoimmune diseases

Cholesterol is an important structural and functional component of the plasma membrane. In this review, we focus on T cells and the role of cholesterol in T cell antigen receptor (TCR) signalling, which contributes to autoimmune diseases. Cholesterol binding to the TCR leads to an increased formation of TCR nanoclusters, increasing the avidity of T cells towards the antigen and enabling TCR cooperativity, thus increasing the sensitivity of the T cell. Further, cholesterol is important in the formation of certain lipid nanodomains, called lipid rafts that concentrate signalling molecules and thus also promote TCR signalling. T cell and lipid dysregulation play a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Typically, in SLE, hyper-responsive and exaggerated TCR signalling occurs, partially caused by cholesterol accumulation in the plasma membrane. This might lead to enhanced TCR nanoclustering and lipid raft formation. Thus, targeting lipid metabolism in T cells.

Germline genetic patterns underlying familial rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren’s syndrome highlight T cell-initiated autoimmunity

ObjectivesFamilial aggregation of primary Sjögren’s syndrome (pSS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and co-aggregation of these autoimmune diseases (ADs) (also called familial autoimmunity) is well recognised. However, the genetic predisposition variants that explain this clustering remains poorly defined.MethodsWe used whole-exome sequencing on 31 families (9 pSS, 11 SLE, 6 RA and 5 mixed autoimmunity), followed by heterozygous filtering and cosegregation analysis of a family-focused approach to document rare variants predicted to be pathogenic by in silico analysis. Potential importance in immune-related processes, gene ontology, pathway enrichment and overlap analyses were performed to prioritise gene sets.ResultsA range from 1 to 50 rare possible pathogenic variants, including 39 variants in immune-related genes across SLE, RA and pSS families, were identified. Among this gene set, regulation of T cell activation (p=4.06×10−7) and T cell receptor (TCR) signalling pathway (p=1.73×10−6) were particularly concentrated, including PTPRC (CD45), LCK, LAT–SLP76 complex genes (THEMIS, LAT, ITK, TEC, TESPA1, PLCL1), DGKD, PRKD1, PAK2 and NFAT5, shared across 14 SLE, RA and pSS families. TCR-interactive genes P2RX7, LAG3, PTPN3 and LAX1 were also detected. Overlap analysis demonstrated that the antiviral immunity gene DUS2 variant cosegregated with SLE, RA and pSS phenotypes in an extended family, that variants in the TCR-pathway genes CD45, LCK and PRKD1 occurred independently in three mixed autoimmunity families, and that variants in CD36 and VWA8 occurred in both RA-pSS and SLE-pSS families.ConclusionsOur preliminary results define common genetic characteristics linked to familial pSS, SLE and RA and highlight rare genetic variations in TCR signalling pathway genes which might provide innovative molecular targets for therapeutic interventions for those three ADs.

Differential Nr4a1 and Nr4a3 expression discriminates tonic from activated TCR signalling events in vivo

SummaryNr4a receptors are activated by T cell receptor (TCR) and B cell receptor (BCR) signalling and play key roles in T cell differentiation and promoting T cell exhaustion. How TCR signalling pathways regulate Nr4a receptors and their sensitivities to different physiological types of TCR signalling (e.g. tonic versus activating) remains unknown. Here we utilise Nr4a1/Nur77-GFP and Nr4a3-Tocky mice to elucidate the signalling pathways that govern Nr4a receptor expression in CD4+ and CD8+ T cells. Our findings reveal that Nr4a1-3 are Src family kinase-dependent. Moreover, Nr4a2 and Nr4a3 are abolished by calcineurin inhibitors and bind NFAT1, highlighting a necessary and sufficient role for NFAT in the control of Nr4a2 and Nr4a3, but redundancy for NFAT for Nr4a1. During T cell development, Nr4a1 is activated by tonic signalling during TCR-beta selection in the thymus, whilst Nr4a3 requires cognate peptide:MHC interactions for expression. Thus, due to differential sensitivity of Nr4a1 and Nr4a3 to TCR signalling pathways, T cells undergoing tonic versus activating TCR signalling events can be distinguished in vivo.

Elucidating the activation mechanisms for bifurcation of regulatory and effector T cell fates by multidimensional single cell analysis

In T cells, T cell receptor (TCR) signalling initiates downstream transcriptional mechanisms for T cell activation and differentiation. Foxp3-expressing regulatory T cells (Treg) require TCR signals for their suppressive function and maintenance in the periphery. It is, however, unclear how TCR signalling controls the transcriptional programme of Treg. Since most of studies identified the transcriptional features of Treg in comparison to naïve T cells, the relationship between Treg and non-naïve T cells including memory-phenotype T cells (Tmem) and effector T cells (Teff) is not well understood. Here we dissect the transcriptomes of various T cell subsets from independent datasets using the multidimensional analysis method Canonical Correspondence Analysis (CCA). We show that resting Treg share gene modules for activation with Tmem and Teff. Importantly, Tmem activate the distinct transcriptional modules for T cell activation, which are uniquely repressed in Treg. The activation signature of Treg is dependent on TCR signals, and is more actively operating in activated Treg. Furthermore, by analysing single cell RNA-seq data from tumour-infiltrating T cells, we revealed that FOXP3 expression occurs predominantly in activated T cells. Moreover, we identified FOXP3-driven and T follicular helper (Tfh)-like differentiation pathways in tumour microenvironments, and their bifurcation point, which is enriched with recently activated T cells. Collectively, our study reveals the activation mechanisms downstream of TCR signals for the bifurcation of Treg and Teff differentiation and their maturation processes.