Molecular basis for antibody recognition of multiple drug-peptide/MHC complexes.

The HapImmuneTM platform exploits covalent inhibitors as haptens for creating major histocompatibility complex (MHC)-presented tumor-specific neoantigens by design, combining targeted therapies with immunotherapy for the treatment of drug-resistant cancers. A HapImmune antibody, R023, recognizes multiple sotorasib-conjugated KRAS(G12C) peptides presented by different human leukocyte antigens (HLAs). This high specificity to sotorasib, coupled with broad HLA-binding capability, enables such antibodies, when reformatted as T cell engagers, to potently and selectively kill sotorasib-resistant KRAS(G12C) cancer cells expressing different HLAs upon sotorasib treatment. The loosening of HLA restriction could increase the patient population that can benefit from this therapeutic approach. To understand the molecular basis for its unconventional binding capability, we used single-particle cryogenic electron microscopy to determine the structures of R023 bound to multiple sotorasib-peptide conjugates presented by different HLAs. R023 forms a pocket for sotorasib between the VH and VL domains, binds HLAs in an unconventional, angled way, with VL making most contacts with them, and makes few contacts with the peptide moieties. This binding mode enables the antibody to accommodate different hapten-peptide conjugates and to adjust its conformation to different HLAs presenting hapten-peptides. Deep mutational scanning validated the structures and revealed distinct levels of mutation tolerance by sotorasib- and HLA-binding residues. Together, our structural information and sequence landscape analysis reveal key features for achieving MHC-restricted recognition of multiple hapten-peptide antigens, which will inform the development of next-generation therapeutic antibodies.

Selection, engineering, and in vivo testing of a human leukocyte antigen-independent T-cell receptor recognizing human mesothelin.

BACKGROUND: Canonical α/ß T-cell receptors (TCRs) bind to human leukocyte antigen (HLA) displaying antigenic peptides to elicit T cell-mediated cytotoxicity. TCR-engineered T-cell immunotherapies targeting cancer-specific peptide-HLA complexes (pHLA) are generating exciting clinical responses, but owing to HLA restriction they are only able to target a subset of antigen-positive patients. More recently, evidence has been published indicating that naturally occurring α/ß TCRs can target cell surface proteins other than pHLA, which would address the challenges of HLA restriction. In this proof-of-concept study, we sought to identify and engineer so-called HLA-independent TCRs (HiTs) against the tumor-associated antigen mesothelin. METHODS: Using phage display, we identified a HiT that bound well to mesothelin, which when expressed in primary T cells, caused activation and cytotoxicity. We subsequently engineered this HiT to modulate the T-cell response to varying levels of mesothelin on the cell surface. RESULTS: The isolated HiT shows cytotoxic activity and demonstrates killing of both mesothelin-expressing cell lines and patient-derived xenograft models. Additionally, we demonstrated that HiT-transduced T cells do not require CD4 or CD8 co-receptors and, unlike a TCR fusion construct, are not inhibited by soluble mesothelin. Finally, we showed that HiT-transduced T cells are highly efficacious in vivo, completely eradicating xenografted human solid tumors. CONCLUSION: HiTs can be isolated from fully human TCR-displaying phage libraries against cell surface-expressed antigens. HiTs are able to fully activate primary T cells both in vivo and in vitro. HiTs may enable the efficacy seen with pHLA-targeting TCRs in solid tumors to be translated to cell surface antigens.

Differences in F pocket impact on HLA I genetic associations with autoimmune diabetes.

Introduction: Human leukocyte antigen (HLA) I molecules present antigenic peptides to activate CD8+ T cells. Type 1 Diabetes (T1D) is an auto-immune disease caused by aberrant activation of the CD8+ T cells that destroy insulin-producing pancreatic ß cells. Some HLA I alleles were shown to increase the risk of T1D (T1D-predisposing alleles), while some reduce this risk (T1D-protective alleles). Methods: Here, we compared the T1D-predisposing and T1D-protective allotypes concerning peptide binding, maturation, localization and surface expression and correlated it with their sequences and energetic profiles using experimental and computational methods. Results: T1D-predisposing allotypes had more peptide-bound forms and higher plasma membrane levels than T1D-protective allotypes. This was related to the fact that position 116 within the F pocket was more conserved and made more optimal contacts with the neighboring residues in T1D-predisposing allotypes than in protective allotypes. Conclusion: Our work uncovers that specific polymorphisms in HLA I molecules potentially influence their susceptibility to T1D.

Generation of Highly Functional Hepatocyte-like Organoids from Human Adipose-Derived Mesenchymal Stem Cells Cultured with Endothelial Cells.

Previously, we successfully established a highly functional, three-dimensional hepatocyte-like cell (3D-HLC) model from adipose-derived mesenchymal stem cells (ADSCs) via a three-step differentiation protocol. The aim of the present study was to investigate whether generating hepatocyte-like organoids (H-organoids) by adding endothelial cells further improved the liver-like functionality of 3D-HLCs and to assess H-organoids' immunogenicity properties. Genes representing liver maturation and function were detected by quantitative reverse transcription-PCR analysis. The expression of hepatic maturation proteins was measured using immunofluorescence staining. Cytochrome P (CYP)450 metabolism activity and ammonia metabolism tests were used to assess liver function. H-organoids were successfully established by adding human umbilical vein endothelial cells at the beginning of the definitive endoderm stage in our 3D differentiation protocol. The gene expression of alpha-1 antitrypsin, carbamoyl-phosphate synthase 1, and apolipoprotein E, which represent liver maturation state and function, was higher in H-organoids than non-organoid 3D-HLCs. H-organoids possessed higher CYP3A4 metabolism activity and comparable ammonia metabolism capacity than 3D-HLCs. Moreover, although H-organoids expressed human leukocyte antigen class I, they expressed little human leukocyte antigen class II, cluster of differentiation (CD)40, CD80, CD86, and programmed cell death ligand 1, suggesting their immunogenicity properties were not significantly upregulated during differentiation from ADSCs. In conclusion, we successfully established an H-organoid model with higher liver-like functionality than previously established 3D-HLCs and comparable immunogenicity to ADSCs.

The electrostatic landscape of MHC-peptide binding revealed using inception networks.

Predictive modeling of macromolecular recognition and protein-protein complementarity represents one of the cornerstones of biophysical sciences. However, such models are often hindered by the combinatorial complexity of interactions at the molecular interfaces. Exemplary of this problem is peptide presentation by the highly polymorphic major histocompatibility complex class I (MHC-I) molecule, a principal component of immune recognition. We developed human leukocyte antigen (HLA)-Inception, a deep biophysical convolutional neural network, which integrates molecular electrostatics to capture non-bonded interactions for predicting peptide binding motifs across 5,821 MHC-I alleles. These predictions of generated motifs correlate strongly with experimental peptide binding and presentation data. Beyond molecular interactions, the study demonstrates the application of predicted motifs in analyzing MHC-I allele associations with HIV disease progression and patient response to immune checkpoint inhibitors. A record of this paper's transparent peer review process is included in the supplemental information.

Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

T-Cell Mediated Immune Rejection of Beta-2-Microglobulin Knockout Induced Pluripotent Stem Cell-Derived Kidney Organoids.

Immune evasive induced pluripotent stem cell (iPSC)-derived kidney organoids, known as "stealth" organoids, hold promise for clinical transplantation. To address immune rejection, we investigated the impact of genetically modifying human leukocyte antigen (HLA) class I in kidney organoids prior to transplantation. By using CRISPR-Cas9, we successfully knocked out beta-2-microglobulin (B2M), resulting in iPSCs devoid of HLA class I surface expression. In vitro, the B2M knockout protected kidney organoids derived from these iPSCs against T-cell rejection. To assess in vivo protection, unmodified (control) and B2M-/- kidney organoids were transplanted into humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs). Successful engraftment of human PBMCs was confirmed, and after 4 weeks, we observed no discernible difference in the infiltration rate, proliferation, or cytotoxicity of CD4+ and CD8+ T cells between control and B2M-/- organoids. Both groups of organoids showed compromised tissue integrity, displaying tubulitis and loss of tubule integrity. Notably, while B2M-/- organoids failed to express HLA class I on their cell surface, there was preexisting expression of HLA class II in both control and B2M-/- organoids transplanted into mice with human PBMCs. HLA class II expression was not limited to antigen-presenting cells but also evident in epithelial cells of the kidney organoid, posing an additional immunological challenge to its transplantation. Consequently, we conclude that B2M knockout alone is insufficient to protect iPSC-derived kidney organoids from T-cell-mediated immune rejection. Additionally, our findings suggest that modulating HLA class II signaling will be necessary to prevent rejection following transplantation.

Structure of the murine CD94-NKG2A receptor in complex with Qa-1b presenting an MHC-I leader peptide.

The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell surface expression levels through binding to MHC class Ia-derived leader sequence peptides presented by HLA class I histocompatibility antigen, alpha chain E (HLA-E; in humans) or H-2 class I histocompatibility antigen, D-37 (Qa-1b; in mice). Although the molecular basis underpinning human CD94-NKG2A recognition of HLA-E is known, the equivalent interaction in the murine setting is not. By determining the high-resolution crystal structure of murine CD94-NKG2A in complex with Qa-1b presenting the Qa-1 determinant modifier peptide (QDM), we resolved the mode of binding. Compared to the human homologue, the murine CD94-NKG2A-Qa-1b-QDM displayed alterations in the distribution of interactions across CD94 and NKG2A subunits that coincide with differences in electrostatic complementarity of the ternary complex and the lack of cross-species reactivity. Nevertheless, we show that Qa-1b could be modified through W65R + N73I mutations to mimic HLA-E, facilitating binding with both human and murine CD94-NKG2A. These data underscore human and murine CD94-NKG2A cross-species heterogeneity and provide a foundation for humanising Qa-1b in immune system models.

Generation of a bank of clinical-grade, HLA-homozygous iPSC lines with high coverage of the Spanish population.

BACKGROUND: Induced pluripotent stem cell (iPSC)-derived cell therapies are an interesting new area in the field of regenerative medicine. One of the approaches to decrease the costs of iPSC-derived therapies is the use of allogenic homozygous human leukocyte antigen (HLA)-matched donors to generate iPSC lines and to build a clinical-grade iPSC bank covering a high percentage of the Spanish population. METHODS: The Spanish Stem Cell Transplantation Registry was screened for cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes. Seven donors were selected with haplotypes covering 21.37% of the haplotypes of the Spanish population. CD34-positive hematopoietic progenitors were isolated from the mononuclear cell fraction of frozen cord blood units from each donor by density gradient centrifugation and further by immune magnetic labeling and separation using purification columns. Purified CD34 + cells were reprogrammed to iPSCs by transduction with the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit. RESULTS: The iPSCs generated from the 7 donors were expanded, characterized, banked and registered. Master cell banks (MCBs) and working cell banks (WCBs) from the iPSCs of each donor were produced under GMP conditions in qualified clean rooms. CONCLUSIONS: Here, we present the first clinical-grade, iPSC haplobank in Spain made from CD34 + cells from seven cord blood units homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes within the Spanish population. We describe their generation by transduction with Sendai viral vectors and their GMP-compliant expansion and banking. These haplolines will constitute starting materials for advanced therapy medicinal product development (ATMP).

TScan-II: A genome-scale platform for the de novo identification of CD4+ T cell epitopes.

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.

Induction of broad multifunctional CD8+ and CD4+ T cells by hepatitis B virus antigen-based synthetic long peptides ex vivo.

Introduction: Therapeutic vaccination based on synthetic long peptides (SLP®) containing both CD4+ and CD8+ T cell epitopes is a promising treatment strategy for chronic hepatitis B infection (cHBV). Methods: We designed SLPs for three HBV proteins, HBcAg and the non-secreted proteins polymerase and X, and investigated their ability to induce T cell responses ex vivo. A set of 17 SLPs was constructed based on viral protein conservation, functionality, predicted and validated binders for prevalent human leukocyte antigen (HLA) supertypes, validated HLA I epitopes, and chemical producibility. Results: All 17 SLPs were capable of inducing interferon gamma (IFNÉ£) production in samples from four or more donors that had resolved an HBV infection in the past (resolver). Further analysis of the best performing SLPs demonstrated activation of both CD8+ and CD4+ multi-functional T cells in one or more resolver and patient sample(s). When investigating which SLP could activate HBV-specific T cells, the responses could be traced back to different peptides for each patient or resolver. Discussion: This indicates that a large population of subjects with different HLA types can be covered by selecting a suitable mix of SLPs for therapeutic vaccine design. In conclusion, we designed a set of SLPs capable of inducing multifunctional CD8+ and CD4+ T cells ex vivo that create important components for a novel therapeutic vaccine to cure cHBV.

Creating an HLA-homozygous iPS cell bank for the Brazilian population: Challenges and opportunities.

Identifying human leukocyte antigen (HLA) haplotype-homozygous donors for the generation of induced pluripotent stem (iPS) cell lines permits the construction of biobanks immunologically compatible with significant numbers of individuals for use in therapy. However, two questions must be addressed to create such a bank: how many cell lines are necessary to match most of the recipient population and how many people should be tested to find these donors? In Japan and the UK, 50 and 100 distinct HLA-A, -B, and -DRB1 triple-homozygous haplotypes would cover 90% of those populations, respectively. Using data from the Brazilian National Registry of Bone Marrow Donors (REDOME), encompassing 4,017,239 individuals, we identified 1,906 distinct triple-homozygous HLA haplotypes. In Brazil, 559 triple-homozygous cell lines cover 95% of the population, and 3.8 million people would have to be screened. Finally, we show the contribution of the 30 most frequent triple-homozygous HLA haplotypes in Brazil to populations of different countries.

A retrospective observational study to estimate the risk of HLA alloimmunization with blood transfusion: Can the risk be reduced by leucodepletion?

BACKGROUND: In this retrospective study, our aim was to find the effect of leucodepleted (LD) blood transfusions on the formation of anti-HLA-antibodies when compared to non-leucodepleted (non-LD) transfusions using Luminex-based method. METHODS: In this study, Luminex single antigen bead assay (L-SAB) and HLA typing were performed on 310 patients. Test positivity rates (as MFI - Mean florescence intensity) were analyzed according to the different sensitization events and gender. RESULTS: Of the 310 patients included in the study, 58.06% (180) patients were male and 41.93% (130) were female. The average age of the patients was 42.86 (±12.37) years. In this study, test positivity rates were significantly lower in the patients who received LD RBC units than in those who received non-LD RBC units (28.43% = 29 of 102 Vs 55.22% = 74 of 134, p < 0.05). In our study, transfusion combined with a history of pregnancy had higher number of significant HLA antibodies compared to cases where transfusion was the only sensitization event (81.81% = 18/22 Vs 39.71% = 85/214, p < 0.05). In addition, anti-HLA-antibodies-MFI were significantly (p < 0.01) higher in non-LD patients compared to LD patients. CONCLUSION: Patients who received LD RBC units had a significantly lower rate of transfusion-associated alloimmunization compared to those who received non-LD RBC units. Multiparous women had a high risk for transfusion-related alloimmunization compared to both nulliparous women and male patient. Furthermore, class I-anti-HLA-antibodies (HLA-B and HLA-A + B) were significantly associated with pregnancy sensitization and/or blood transfusion as a single sensitization.

What do cancer-specific CD8+ T cells see? The contribution of immunopeptidomics.

Immunopeptidomics is the survey of all peptides displayed on a cell or tissue when bound to human leukocyte antigen (HLA) molecules using tandem mass spectrometry. When attempting to determine the targets of tumour-specific CD8+ T cells, a survey of the potential ligands in tumour tissues is invaluable, and, in comparison with in-silico predictions, provides greater certainty of the existence of individual epitopes, as immunopeptidomics-confirmed CD8+ T-cell epitopes are known to be immunogenic, and direct observation should avoid the risk of autoreactivity which could arise following immunisation with structural homologues. The canonical sources of CD8+ T-cell tumour specific epitopes, such as tumour associated antigens, may be well conserved between patients and tumour types, but are often only weakly immunogenic. Direct observation of tumour-specific neoantigens by immunopeptidomics is rare, although valuable. Thus, there has been increasing interest in the non-canonical origins of tumour-reactive CD8+ T-cell epitopes, such as those arising from proteasomal splicing events, translational/turnover defects and alternative open reading frame reads. Such epitopes can be identified in silico, although validation is more challenging. Non-self CD8+ T-cell epitopes such as viral epitopes may be useful in certain cancer types with known viral origins, however these have been relatively unexplored with immunopeptidomics to date, possibly due to the paucity of source viral proteins in tumour tissues. This review examines the latest evidence for canonical, non-canonical and non-human CD8+ T-cell epitopes identified by immunopeptidomics, and concludes that the relative contribution for each of these sources to anti-tumour CD8+ T-cell reactivity is currently uncertain.

Defining the role of natural killer cells in COVID-19.

Natural killer (NK) cells are critical effectors of antiviral immunity. Researchers have therefore sought to characterize the NK cell response to coronavirus disease 2019 (COVID-19) and the virus that causes it, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The NK cells of patients with severe COVID-19 undergo extensive phenotypic and functional changes. For example, the NK cells from critically ill patients with COVID-19 are highly activated and exhausted, with poor cytotoxic function and cytokine production upon stimulation. The NK cell response to SARS-CoV-2 is also modulated by changes induced in virally infected cells, including the ability of a viral peptide to bind HLA-E, preventing NK cells from receiving inhibitory signals, and the downregulation of major histocompatibility complex class I and ligands for the activating receptor NKG2D. These changes have important implications for the ability of infected cells to escape NK cell killing. The implications of these findings for antibody-dependent NK cell activity in COVID-19 are also reviewed. Despite these advances in the understanding of the NK cell response to SARS-CoV-2, there remain critical gaps in our current understanding and a wealth of avenues for future research on this topic.

HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I.

Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation.

[The expression of long non-coding RNA human leukocyte antigen complex P5(lncRNA HCP5) in synovial tissue of patients with rheumatoid arthritis is up-regulated and correlated with immune cell infiltration].

Objective To identify the potential long non-coding RNA (lncRNA) expressed in rheumatoid arthritis (RA) synovium key to RA onset and investigate its association with immune cell infiltration. Methods RA synovium data were downloaded from the GEO database and normalized. The lncRNAs key to RA onset were identified using multiple machine learning methods. Infiltration of 22 immune cell populations in RA synovium was measured by cell-type identification by estimating relative subsets of RNA transcripts (CIBER-SORT). The relationship between the key lncRNA and infiltrating immune cells was analyzed. Finally, real-time quantitative PCR was applied to validate the expression of the key lncRNA in RA synovial cells. Results lncRNA human leukocyte antigen complex P5(HCP5) was identified as the key lncRNA associated with RA onset. Infiltration analysis revealed increased abundance of CD8+ T cells, γδ T cells, and M1 macrophages while decreased abundance of M2 macrophages in RA synovial tissue. Correlation analysis demonstrated that the lncRNA HCP5 expression was positively associated with the infiltration abundance of CD8+ T cells, γδ T cells, and M1 macrophages in RA synovial tissue. Furthermore,the expression of lncRNA HCP5 in RA synovial cells was up-regulated. Conclusion lncRNA HCP5 expression is up-regulated in RA synovial tissue and potentially associated with immune cells infiltration.

Major histocompatibility complex class II in the tumor microenvironment: functions of nonprofessional antigen-presenting cells.

Major histocompatibility complex class-II-restricted presentation by nonprofessional antigen-presenting cells in the tumor microenvironment can regulate antitumor T-cell responses. In murine models, tumor cell-specific MHC class II expression decreases in vivo tumor growth, dependent on T cells. Tumor cell-specific MHC class II expression is associated with improved survival and response to immune checkpoint inhibitors in human cancers. Antigen-presenting cancer-associated fibroblasts (apCAF) present MHC class-II-restricted antigens and activate CD4 T cells. The role of MHC class II on apCAFs depends on the cell of origin. MHC class II on tumoral lymphatic endothelial cells leads to expansion of regulatory T cells and increased in vivo tumor growth.

Human leukocyte antigen F-associated transcript 10 regulates the IKs potassium channel by competing for Kv7.1 ubiquitination.

The protein expression and function changes from the slow-delayed rectifying K+ current, IKs, are tightly associated with ventricular cardiac arrhythmias. Human leukocyte antigen F-associated transcript 10 (FAT10), a member of the ubiquitin-like-modifier family, exerts a protective effect against myocardial ischaemia. However, whether or how FAT10 influences the function of IKs remains unclear. Here, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Fat10 knockout HEK293 (Fat10-/-) cells through CRISPR-Cas9 technology were used to evaluate the novel modulation of FAT10 in IKs function. Patch-clamp studies showed that the overexpression of FAT10 significantly enhanced the current density of IKs both in hiPSC-CMs and HEK293-Fat10-/- cells. In addition, a shortened action potential duration (APD) was seen from hiPSC-CMs transfected with the ad-Fat10 virus. Then, a series of molecular approaches from neonatal rat cardiomyocytes, H9C2 cells and HEK293 cells were used to determine the regulatory mechanism of FAT10 in IKs. First, western blot assays indicated that the expression of Kv7.1, the alpha-subunit of IKs, was increased when FAT10 was overexpressed. Furthermore, immunofluorescence and co-immunoprecipitation assays demonstrated that FAT10 could interact with Kv7.1. Notably, FAT10 impedes Kv7.1 ubiquitination and degradation, thereby stabilizing its expression. Finally, a hypoxia model of hiPSC-CMs was established, and the overexpression of FAT10 showed a protective effect against hypoxia-induced decreases in the current density of IKs. Taken together, these findings revealed a novel role of FAT10 in the regulation of the IKs potassium channel by competing for Kv7.1 ubiquitination, which provides a new electrophysiological insight that FAT10 could modulate Kv7.1. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.