Application of the immunoregulatory receptor LILRB1 as a crystallisation chaperone for human class I MHC complexes

X-ray crystallographic studies of class I peptide-MHC molecules (pMHC) continue to provide important insights into immune recognition, however their success depends on generation of diffraction-quality crystals, which remains a significant challenge. While protein engineering techniques such as surface-entropy reduction and lysine methylation have proven utility in facilitating and/or improving protein crystallisation, they risk affecting the conformation and biochemistry of the class I MHC antigen binding groove. An attractive alternative is the use of noncovalent crystallisation chaperones, however these have not been developed for pMHC. Here we describe a method for promoting class I pMHC crystallisation, by exploiting its natural ligand interaction with the immunoregulatory receptor LILRB1 as a novel crystallisation chaperone. First, focussing on a model HIV-1-derived HLA-A2-restricted peptide, we determined a 2.4 Å HLA-A2/LILRB1 structure, which validated that co crystallisation with LILRB1 does not alter conformation of the antigenic peptide. We then demonstrated that addition of LILRB1 enhanced the crystallisation of multiple peptide-HLA-A2 complexes, and identified a generic condition for initial co-crystallisation. LILRB1 chaperone-based crystallisation enabled structure determination for HLA-A2 complexes previously intransigent to crystallisation, including both conventional and post-translationally-modified peptides, of diverse lengths. Since both the LILRB1 recognition interface on the HLA-A2 α3 domain molecule and HLA-A2 mediated crystal contacts are predominantly conserved across class I MHC molecules, the approach we outline could prove applicable to a diverse range of class I pMHC. LILRB1 chaperone-mediated crystallisation should expedite molecular insights into the immunobiology of diverse immune-related diseases and immunotherapeutic strategies, particularly involving class I pMHC complexes that are challenging to crystallise

The Biological Influence and Clinical Relevance of Polymorphism Within the NKG2D Ligands

NKG2D is a major regulator of the activity of cytotoxic cells and interacts with eight different ligands (NKG2DL) from two families of MIC and ULBP proteins. The selective forces that drove evolution of NKG2DL are uncertain, but are likely to have been dominated by infectious disease and cancer. Of interest, NKG2DL are some of the most polymorphic genes outside the MHC locus and the study of these is uncovering a range of novel observations regarding the structure and function of NKG2DL. Polymorphism is present within all NKG2DL members and varies markedly within different populations. Allelic variation influences functional responses through three major mechanisms. First, it may drive differential levels of protein expression, modulate subcellular trafficking, or regulate release of soluble isoforms. In addition, it may alter the affinity of interaction with NKG2D or modulate cytotoxic activity from the target cell. In particular, ligands with high affinity for NKG2D are associated with down regulation of this protein on the effector cell, effectively limiting cytotoxic activity in a negative-feedback circuit. Given these observations, it is not surprising that NKG2DL alleles are associated with relative risk for development of several clinical disorders and the critical role of the NKG2D:NKG2DL interaction is demonstrated in many murine models. Increased understanding of the biophysical and functional consequences of this polymorphism is likely to provide insights into novel immunotherapeutic approaches

Modifications outside CDR1, 2 and 3 of the TCR variable β domain increase TCR expression and antigen-specific function

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable β domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing

Structural and Functional Insights into Alpha-actinin Isoforms and their Implications in Cardiovascular Disease

Alpha-actinin (ACTN) is a pivotal member of the actin-binding protein family, crucial for the anchoring and organisation of actin filaments within the cytoskeleton. Four isoforms of alpha-actinin exist: two non-muscle isoforms (ACTN1 and ACTN4) primarily associated with actin stress fibres and focal adhesions, and two muscle-specific isoforms (ACTN2 and ACTN3) localised to the Z-disk of the striated muscle. Although these isoforms share structural similarities, they exhibit distinct functional characteristics that reflect their specialised roles in various tissues. Genetic variants in alpha-actinin isoforms have been implicated in a range of pathologies, including cardiomyopathies, thrombocytopenia, and non-cardiovascular diseases, such as nephropathy. However, the precise impact of these genetic variants on the alpha-actinin structure and their contribution to disease pathogenesis remain poorly understood. This review provides a comprehensive overview of the structural and functional attributes of the four alpha-actinin isoforms, emphasising their roles in actin crosslinking and sarcomere stabilisation. Furthermore, we present detailed structural modelling of select ACTN1 and ACTN2 variants to elucidate mechanisms underlying disease pathogenesis, with a particular focus on macrothrombocytopenia and hypertrophic cardiomyopathy. By advancing our understanding of alpha-actinin’s role in both normal cellular function and disease states, this review lays the groundwork for future research and the development of targeted therapeutic interventions

Superficial back line release versus trigger points release in chronic nonspecific low back pain patients

The aim of this study was to investigate the effect of Superficial back line (SBL) release on pain, Pressure Pain Threshold (PPT), lumbar function, lumbar ROM, and balance in chronic nonspecific low back pain (CNSLBP) patients. Additionally, it aims to compare the effect of SBL release versus myofascial trigger points (MTrPs) release on the same variables. This was a Randomized Controlled Trial (RCT) involving forty-eight male and female patients with CNSLBP. They were randomized via an online randomization web service into 3 groups: group A (n=16) received SBL release and exercises, group B (n=16) received MTrPs release and exercises, while group C (n=16) received exercises only. There were statistically significant improvements in all outcomes post-treatment in comparison to pre-treatment within the three groups (p<0.05). After treatment, significant differences emerged between the groups in pain, PPT, lumbar extension ROM, and left anterior balance (p < 0.05). Group A showed the most favorable results in pain reduction, lumbar extension ROM, and left anterior balance compared to groups B and C, while Group B demonstrated the most favorable results in PPT compared to groups A and C. In conclusion, SBL release and MTrPs release are effective in improving pain, lumbar function, MTrPs PPT, lumbar ROM, and balance in patients with CNSLBP. SBL release is particularly effective for pain, lumbar extension ROM, and left anterior balance, while MTrPs release is most effective for improving PPT

Modifications outside CDR1, 2 and 3 of the TCR variable β domain increase TCR expression and antigen-specific function

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable β domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing

Butyrophilin-like 3 Directly Binds a Human Vγ4⁺ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4+ TCR, “LES” with an affinity (∼15–25 μM) comparable to many αβ TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined. Butyrophilin (BTN) and butyrophilin-like (BTNL) molecules powerfully influence selection and activation of specific γδ lymphocyte subsets, but whether they directly bind the γδ TCR has remained contentious. Willcox et al. show that BTNL3 directly binds to human Vγ4+ TCRs via a superantigen-like binding mode that is focused on germline-encoded TCR regions.</p