Single-cell atlas of the human brain vasculature across development, adulthood and disease.

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

Distribution of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 allele frequencies in patients with COVID-19 bilateral pneumonia in Russians, living in the Chelyabinsk region (Russia).

In this population-based case-control study conducted in the Chelyabinsk region of Russia, we examined the distribution of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, in a group of 100 patients with confirmed COVID-19 bilateral pneumonia. Typing was performed by NGS and statistical calculations were carried out with the Arlequin program. HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were compared between patients with COVID-19 and 99 healthy controls. We identified that COVID-19 susceptibility is associated with alleles and genotypes rs9277534A (disequilibrium with HLA-DPB1*02:01, -02:02, -04:01, -04:02, -17:01 alleles) with low expression of protein products HLA-DPB1 (pc < 0.028) and homozygosity at HLA-C*04 (p = 0.024, pc = 0.312). Allele HLA-A*01:01 was decreased in a group of patients with severe forms of bilateral pneumonia, and therefore it may be considered as a protective factor for the development of severe symptoms of COVID-19 (p = 0.009, pc = 0.225). Our studies provide further evidence for the functional association between HLA genes and COVID-19.

Marginal zone B cells acquire dendritic cell functions by trogocytosis.

Marginal zone (MZ) B cells produce broad-spectrum antibodies that protect against infection early in life. In some instances, antibody production requires MZ B cells to display pathogen antigens bound to major histocompatibility complex class II (MHC II) molecules to T cells. We describe the trogocytic acquisition of these molecules from conventional dendritic cells (cDCs). Complement component 3 (C3) binds to murine and human MHC II on cDCs. MZ B cells recognize C3 with complement receptor 2 (CR2) and trogocytose the MHC II-C3 complexes, which become exposed on their cell surface. The ubiquitin ligase MARCH1 limits the number of MHC II-C3 complexes displayed on cDCs to prevent their elimination through excessive trogocytosis. Capture of C3 by MHC II thus enables the transfer of cDC-like properties to MZ B cells.

HLA Class II Polymorphisms Modulate Gut Microbiota and Experimental Autoimmune Encephalomyelitis Phenotype.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91-110)-induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8-specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

Analysis of B Cell Receptor-Mediated Antigen Extraction by B Lymphocytes from Plasma Membrane Sheets Using Confocal Microscopy.

High-resolution confocal imaging has provided new insights in the process of receptor-mediated endocytosis in variety of cell types. We describe here the protocol for investigating B cell receptor (BCR)-mediated internalization of membrane bound antigens using confocal microscopy. We describe the method to prepare plasma membrane sheets (PMS) in a small area, bind fluorescently tagged antigens to the PMS and activate B cells on the PMS. We also describe the method for analyzing antigen internalization using confocal microscopy and computational image analysis. This protocol is useful for the study of antigen internalization by B cells and can be applied for studying receptor-mediated endocytosis in other cells as well. The setup we describe here is especially useful for studying rare cell types when the number of cells available is limiting.

The MHC-II antigen presentation machinery and B7 checkpoint ligands display distinctive patterns correlated with acute myeloid leukaemias blast cells HLA-DR expression.

Acute Myeloid Leukaemia (AML) is a neoplasia characterised by rapid proliferation and an increased rate of relapses. The AML blasts display features of antigen-presenting cells (APC), and thus can directly modulate the anti-tumour T cell responses. The bone marrow of a group consisting of 30 newly diagnosed patients and four healthy donors (HD) was investigated for the expression of HLA-DR, several molecules involved in MHC-II antigen-presentation and MHC-II groove editing, like HLA-DM, CD74 and CLIP, as well as a set of immune checkpoint ligands, like ICOS-L, B7.2, PD-L2 and B7-H3. The patients were further characterised for their genetic anomalies and distributed to favourable, intermediate and adverse ELN risk categories. We were able to show that while 23% of our patients displayed a low level of HLA-DR surface expression, all patients displayed higher HLA-DM and CD74 expression compared to HD. However, a higher CLIP expression was noticed only in the HLA-DR low patients. The co-inhibitory PD-L2 and B7-H3 molecules were increased in the cases with normal HLA-DR expression; oppositely, the co-stimulatory ICOS-L and the dual function B7.2 were significantly increased in the cases with HLA-DR low expression. Furthermore, no favourable ELN risk cases were found within the HLA-DR low group. All in all, these data show that the AML with low versus normal HLA-DR expression display different profiles of MHC class II machinery molecules and B7 ligands, which are correlated with distinct ELN stratification. Furthermore, as our study included healthy individuals, it offers valuable information about the expression levels that should be considered as normal for these markers known to cause differences in peptide repertoires, reflected further in distinct T-cells polarisation pathways.

Risk factors and associated outcomes of ventilator-associated events developed in 28 days among sepsis patients admitted to intensive care unit.

We hypothesized that Ventilator-Associated Event (VAE) within 28 days upon admission to medical intensive care units (ICUs) can be a predictor for poor outcomes in sepsis patients. We aimed to determine the risk factors and associated outcomes of VAE. A total of 453 consecutive mechanically ventilated (MV) sepsis patients were enrolled. Of them, 136 patients had immune profile study. Early VAE (< 7-day MV, n = 33) was associated with a higher mortality (90 days: 81.8% vs. 23.0% [non-VAE], P < 0.01), while late VAE (developed between 7 and 28 days, n = 85) was associated with longer MV day (43.8 days vs. 23.3 days [non-VAE], P < 0.05). The 90-day Kaplan-Meier survival curves showed three lines that separate the groups (non-VAE, early VAE, and late VAE). Cox regression models with time-varying coefficient covariates (adjusted for the number of days from intubation to VAE development) confirmed that VAE which occurred within 28 days upon admission to the medical ICUs can be associated with higher 90-day mortality. The risk factors for VAE development include impaired immune response (lower human leukocyte antigen D-related expression, higher interleukin-10 expression) and sepsis progression with elevated SOFA score (especially in coagulation sub-score).

HLA-DM catalytically enhances peptide dissociation by sensing peptide-MHC class II interactions throughout the peptide-binding cleft.

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Differences in Expression of Human Leukocyte Antigen Class II Subtypes and T Cell Subsets in Behçet's Disease with Arthritis.

It has been reported Human Leukocyte Antigen (HLA) gene polymorphism is a risk factor for the development of Behçet's disease (BD). In this study, the association of HLA class II subtypes HLA-DP, DQ, DR, and T cell subsets in BD patients with arthritis was evaluated. Frequencies of HLA-DP, DQ, DR positive cells, and T cell subsets in peripheral blood leukocytes (PBL) were measured by flow cytometric analysis in BD, and compared to rheumatoid arthritis as disease controls and healthy controls. Frequencies of HLA-DQ were significantly decreased in whole PBL and granulocytes of BD active patients as compared to healthy controls. In monocytes populations, proportions of HLA-DR positive cells were significantly increased in BD active patients as compared to healthy controls. Proportions of CD4+CCR7+ and CD8+CCR7+ cells were significantly higher in BD active patients than in BD inactive in whole PBL. Frequencies of CD4+CD62L- and CD8+CD62L- cells in lymphocytes were significantly decreased in active BD than those in inactive BD. There were also correlations between disease activity markers and T cell subsets. Our results revealed HLA-DP, DQ, and DR expressing cell frequencies and several T cell subsets were significantly correlated with BD arthritis symptoms.

Defining HLA-II Ligand Processing and Binding Rules with Mass Spectrometry Enhances Cancer Epitope Prediction.

Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.

E-Selectin-Binding Peptide-Modified Bovine Serum Albumin Nanoparticles for the Treatment of Acute Lung Injury.

Currently, there is no specific treatment for acute lung injury (ALI). E-selectin-binding peptide (Esbp), a high-affinity peptide that delivers drugs targeting inflammatory vascular endothelial cells, can bind to E-selectin and act as a targeting ligand for selective drug delivery. In this study, we coupled the thiol groups of Esbp to the amino groups on the surface of bovine serum albumin (BSA) using succinimidyl iodoacetic acid to make Esbp-modified BSA nanoparticles (BSANPs) at the average ratio of 19.3 µg Esbp to 1 mg BSA. The Esbp-modified BSANPs were spherical in shape and had a particle size of 266.7 ± 2.7 nm, polydispersity index of 0.165 ± 0.02, zeta potential of - 33.64 ± 1.23 mV, encapsulation efficiency of 84.3 ± 2.3%, and drug loading of 6.7 ± 0.32%. The cumulative release rate of dexamethasone-loaded Esbp-modified BSANPs was 51.2% within 12 h, significantly lower than that of 88.2% of free drugs. Moreover, Esbp-modified BSANPs could be uptaken in vitro by activated human umbilical vein endothelial cells and in vivo by the lungs of the established ALI mouse model. These results indicated that our Esbp-modified BSANPs delivery system has characteristics of good targeting ability and biocompatibility and is able to inhibit inflammation. Overall, our Esbp-modified BSANPs delivery system has therapeutic potentials as a new targeting drug system for the treatment of ALI in the future.

HLA-DM Stabilizes the Empty MHCII Binding Groove: A Model Using Customized Natural Move Monte Carlo.

MHC class II molecules bind peptides derived from extracellular proteins that have been ingested by antigen-presenting cells and display them to the immune system. Peptide loading occurs within the antigen-presenting cell and is facilitated by HLA-DM. HLA-DM stabilizes the open conformation of the MHCII binding groove when no peptide is bound. While a structure of the MHCII/HLA-DM complex exists, the mechanism of stabilization is still largely unknown. Here, we applied customized Natural Move Monte Carlo to investigate this interaction. We found a possible long-range mechanism that implicates the configuration of the membrane-proximal globular domains in stabilizing the open state of the empty MHCII binding groove.

What to do with HLA-DO/H-2O two decades later?

The main objective of antigen processing is to orchestrate the selection of immunodominant epitopes for recognition by CD4 T cells. To achieve this, MHC class II molecules have evolved with a flexible peptide-binding groove in need of a bound peptide. Newly synthesized MHC-II molecules bind a class II invariant chain (Ii) upon synthesis and are shuttled to a specialized compartment, where they encounter exogenous antigens. Ii serves multiple functions, one of which is to maintain the shape of the MHC-II groove so that it can readily bind exogenous antigens upon dissociation of the Ii peptide in MHC- II compartment. MIIC contains processing enzymes, one or both accessory molecules, HLA-DM/H2-M (DM) and HLA-DO/H2-O (DO), and optimal denaturing conditions. In a process known as "editing," DM facilitates the dissociation of the invariant chain peptide, CLIP, for exchange with exogenous antigens. Despite the availability of mechanistic insights into DM functions, understanding how DO contributes to epitope selection has proven to be more challenging. The current dogma assumes that DO inhibits DM, whereas an opposing model suggests that DO fine-tunes the epitope selection process. Understanding which of these, or potentially other models of DO function is important, as DO variants have been linked to autoimmunity, cancer, and the generation of broadly neutralizing antibodies to viruses. This review therefore attempts to evaluate experimental evidence in support of these hypotheses, with an emphasis on the less discussed model, and to explore intriguing questions about the importance of DO in biology.

Low expression of a Ddm7/Ldm7-hybrid mutant (D/Ldm7) in the novel haplotype H-2nc identified in atopic dermatitis model NC/Nga mice.

Environmental factors and the major histocompatibility complex (MHC) are involved in the pathogenesis of atopic dermatitis (AD). However, MHC type (H2 haplotype) of AD model mice NC/Nga is poorly understood. Alloreactive CD8+ or CD4+ T cells in NC/Nga strongly responded to each antigen-presenting cells (A/J: H-2a, C57BL/6: H-2b, BALB/c: H-2d, or C3H/HeJ: H-2k), suggesting that NC/Nga has other H2 haplotype. Polymorphic microsatellite (CA)n repeats in TNF-α gene differ based on the H2 haplotype at present. NC/Nga's (CA)n repeats (n = 19) were different from other examined strains, A/J (n = 14), BALB/c (n = 14), C3H/HeJ (n = 16), and C57BL/6 (n = 20). Using flow cytometry and genotyping, we demonstrated the NC/Nga H2 haplotype had a unique phenotype (Kd, I-Ak, and I-Ek) in which Dd and Ld lacked as protein despite sensitive mRNA detection. The loss of Dd and Ld was caused by forming a unique Ddm7/Ldm7-hybrid mutant (D/Ldm7). We propose to call this novel H2 haplotype the "H-2nc," and provide the important information regarding the AD research using NC/Nga mice.

HLA class II expression on tumor cells and low numbers of tumor-associated macrophages predict clinical outcome in oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a highly immunogenic tumor and differences in tumor microenvironment might contribute to the improved survival of HPV-positive OPSCC patient. METHODS: A comprehensive multivariate analysis with clinical and immune variables (human leukocyte antigen [HLA] I/II, programmed death ligand 1 (PD-L1), programmed death receptor 1 (PD1), T cells, and macrophages) was performed in 142 OPSCC patients. RESULTS: We found an inverse correlation between the expression of HLA class II molecules on tumor cells and CD68+ CD163+ tumor-associated macrophages (TAMs). High HLA-DP/DQ/DR expression and low number of TAMs were associated with longer disease-specific survival and disease-free survival (DFS). Furthermore, a new population of CD8+ FoxP3+ T cells was correlated with shorter DFS in multivariate analysis. CONCLUSIONS: \We identified new prognostic markers for patients with oropharyngeal cancer, which can be used for selecting patients that can benefit from immunotherapy.

APP21 transgenic rats develop age-dependent cognitive impairment and microglia accumulation within white matter tracts.

BACKGROUND: Most of the animal models commonly used for preclinical research into Alzheimer's disease (AD) largely fail to address the pathophysiology, including the impact of known risk factors, of the widely diagnosed sporadic form of the disease. Here, we use a transgenic rat (APP21) that does not develop AD-like pathology spontaneously with age, but does develop pathology following vascular stress. To further the potential of this novel rat model as a much-needed pre-clinical animal model of sporadic AD, we characterize APP21 transgenic rats behaviorally and histologically up to 19 months of age. METHODS: The open field test was used as a measure of activity; and the Morris water maze was used to assess learning, memory, and strategy shift. Neuronal loss and microglia activation were also assessed throughout the brain. RESULTS: APP21 transgenic rats showed deficits in working memory from an early age, yet memory recall performance after 24 and 72 h was equal to that of wildtype rats and did not deteriorate with age. A deficit in strategy shift was observed at 19 months of age in APP21 transgenic rats compared to Fischer wildtype rats. Histologically, APP21 transgenic rats demonstrated accelerated white matter inflammation compared to wildtype rats, but interestingly no differences in neuron loss were observed. CONCLUSIONS: The combined presence of white matter pathology and executive function deficits mirrored what is often found in patients with mild cognitive impairment or early dementia, and suggests that this rat model will be useful for translationally meaningful studies into the development and prevention of sporadic AD. The presence of widespread white matter inflammation as the only observed pathological correlate for cognitive deficits raises new questions as to the role of neuroinflammation in cognitive decline.

Mechanisms underlying the lack of endogenous processing and CLIP-mediated binding of the invariant chain by HLA-DP84Gly.

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPß84Gly (DP84Gly) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.

Analysis of the regulatory function of natural killer cells from patients with systemic lupus erythematosus.

Natural killer (NK) cells participate in the regulation of the immune response. However, the immunomodulatory function of NK cells in systemic lupus erythematosus (SLE) is not well understood. The aim of this study was to evaluate the regulatory function of NK cells in SLE patients and to identify the NK cells involved in the pathogenesis of this complex disease. We analysed the expression of NK receptors and co-stimulatory molecules in peripheral NK cells (CD3- CD56+ ) from SLE patients, as well as the numbers of human leucocyte antigen D-related (HLA-DR)/CD11c+ NK cells. In addition, NK cell regulatory function was assessed by the detection of NK cell-mediated dendritic cell (DC) lysis. We found that SLE patients showed increased numbers of immunoglobulin-like transcript 2 (ILT2)+ , CD86+ and CD134+ NK cells. Furthermore, NK cells from SLE patients induced higher levels of DC lysis. We were able to identify a new subset of NK cells co-expressing CD11c and HLA-DR. These atypical NK cells were increased in SLE patients when compared with controls. We have identified an expanded new subset of NK cells in SLE patients. This is the first study, to our knowledge, which demonstrates that NK cells in SLE patients have an altered phenotype with a high expression of receptors characteristic of dendritic cells. Our results suggest that the impairment in the regulatory function of NK cells, together with the increased number of DC-like NK cells, could play an important role in the development of SLE and highlight the importance of NK cells as a future therapeutic target.