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).

HLA-DRB1*04:05 is involved in the development of Vogt-Koyanagi-Harada disease-like immune-related adverse events in patients receiving immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) activate anti-tumor activity by inhibiting immune checkpoint molecules that suppress inflammatory T-cell activity. However, ICIs can initiate excessive immune responses, thereby causing immune-related adverse events (irAEs). ICI-associated uveitis (ICIU) is an irAE that affects the eyes. Although Vogt-Koyanagi-Harada disease (VKH)-like uveitis is a common form of ICIU, it is unclear which factors determine the ICIU form. We retrospectively reviewed the medical records of nine ICIU cases treated with ICIs for malignancies. We also performed HLA typing in seven cases to investigate the association between HLA and disease type. Fisher's exact test was used for the statistical analysis. Five of the ICIU cases were VKH-like ICIUs, and four were non-VKH-like ICIUs. No association was found between mean age, sex, primary disease, ICI, time to onset, and disease type. Four patients with VKH-like uveitis underwent HLA genotyping and were all positive for HLA-DRB1*04:05. All 3 patients with non-VKH-like uveitis were negative for HLA-DRB1*04:05. Statistical analysis showed that HLA-DRB1*04:05 was significantly associated with developing VKH-like ICIU (P = 0.029). In ICIU, HLA-DRB1*04:05 was associated with the pathogenesis of VKH-like uveitis, suggesting that ICI-associated VKH-like uveitis has a similar pathogenesis to VKH.

Cysteine carboxyethylation generates neoantigens to induce HLA-restricted autoimmunity.

Autoimmune diseases such as ankylosing spondylitis (AS) can be driven by emerging neoantigens that disrupt immune tolerance. Here, we developed a workflow to profile posttranslational modifications involved in neoantigen formation. Using mass spectrometry, we identified a panel of cysteine residues differentially modified by carboxyethylation that required 3-hydroxypropionic acid to generate neoantigens in patients with AS. The lysosomal degradation of integrin αIIb [ITGA2B (CD41)] carboxyethylated at Cys96 (ITGA2B-ceC96) generated carboxyethylated peptides that were presented by HLA-DRB1*04 to stimulate CD4+ T cell responses and induce autoantibody production. Immunization of HLA-DR4 transgenic mice with the ITGA2B-ceC96 peptide promoted colitis and vertebral bone erosion. Thus, metabolite-induced cysteine carboxyethylation can give rise to pathogenic neoantigens that lead to autoreactive CD4+ T cell responses and autoantibody production in autoimmune diseases.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

West Nile virus-associated HLA-DRB1 alleles in the Greek population: A structural perspective.

The HLA system plays a significant role via the regulation of the immune system and contributes to the progression and protection of many diseases. In our previous study, several HLA-DRB1 alleles were found to have a susceptible or protective role toward infection and neuroinvasion of West Nile Virus (WNV) in the Greek population. As expected, the majority of polymorphic positions are located in the peptide-binding region of the molecule. In the present work, the structure of these alleles was studied in silico, to examine the effect of polymorphism on the conformation of DRB1 proteins, with the aspect of WNV association. More specifically, molecular dynamics simulations were used for structural prediction of 23 available alleles. These modeled alleles were evaluated using root-mean-square deviation (RMSD) and root-mean-square fluctuation analysis. Low RMSD values indicate that different alleles have similar structures. Furthermore, low fluctuation was observed in the peptide-binding region between alleles with the higher and the lowest RMSD values. These findings indicate that probably variable residues do not affect the behavior of DRB1 alleles in WNV disease, by causing structural differences between them.

MHC2AffyPred: A machine-learning approach to estimate affinity of MHC class II peptides based on structural interaction fingerprints.

Understanding how MHC class II (MHC-II) binding peptides with differing lengths exhibit specific interaction at the core and extended sites within the large MHC-II pocket is a very important aspect of immunological research for designing peptides. Certain efforts were made to generate peptide conformations amenable for MHC-II binding and calculate the binding energy of such complex formation but not directed toward developing a relationship between the peptide conformation in MHC-II structures and the binding affinity (BA) (IC50 ). We present here a machine-learning approach to calculate the BA of the peptides within the MHC-II pocket for HLA-DRA1, HLA-DRB1, HLA-DP, and HLA-DQ allotypes. Instead of generating ensembles of peptide conformations conventionally, the biased mode of conformations was created by considering the peptides in the crystal structures of pMHC-II complexes as the templates, followed by site-directed peptide docking. The structural interaction fingerprints generated from such docked pMHC-II structures along with the Moran autocorrelation descriptors were trained using a random forest regressor specific to each MHC-II peptide lengths (9-19). The entire workflow is automated using Linux shell and Perl scripts to promote the utilization of MHC2AffyPred program to any characterized MHC-II allotypes and is made for free access at https://github.com/SiddhiJani/MHC2AffyPred. The MHC2AffyPred attained better performance (correlation coefficient [CC] of .612-.898) than MHCII3D (.03-.594) and NetMHCIIpan-3.2 (.289-.692) programs in the HLA-DRA1, HLA-DRB1 types. Similarly, the MHC2AffyPred program achieved CC between .91 and .98 for HLA-DP and HLA-DQ peptides (13-mer to 17-mer). Further, a case study on MHC-II binding 15-mer peptides of severe acute respiratory syndrome coronavirus-2 showed very close competency in computing the IC50 values compared to the sequence-based NetMHCIIpan v3.2 and v4.0 programs with a correlation of .998 and .570, respectively.

Metabolic heterogeneity caused by HLA-DRB1*04:05 and protective effect of inosine on autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune disease caused by disruption of liver immune homeostasis. Genetic studies have revealed the predisposition of AIH with the human leukocyte antigen (HLA) region. Recently, metabolomics integrated with genomics has identified many genetic loci of biomedical interest. However, there is no related report in AIH. In the present study, we found that HLA-DRB1*04:05 was linked to the clinical features and prognosis of AIH in Chinese patients. Furthermore, our patients were divided into DRB1*04:05 positive and DRB1*04:05 negative groups and the metabolic profiling was done by HPLC/MS. We chose inosine, one of the highly altered metabolites, to explore the effect on an acute severe hepatitis murine model. The results showed that inosine treatment attenuated hepatocyte apoptosis, enhanced antioxidant ability and inhibited the activation and glycolysis of CD4+ T cell. We propose that inosine participates in the regulation of AIH through its protective effect on hepatocytes and inhibition of overactivated immune cells, which might provide a potential novel approach in treating acute form of AIH.

Molecular Mimicry of the Rheumatoid Arthritis-Related Immunodominant T-Cell Epitope within Type II Collagen (CII260-270) by the Bacterial L-Asparaginase.

The etiology of most autoimmune diseases, including rheumatoid arthritis (RA), remains unclear. Both genetic and environmental factors are believed to be involved in pathogenesis. Molecular mimicry is considered one of the mechanisms for the occurrence of autoimmune diseases. The aim of the study was to determine whether the bacterial peptide L-ASNase67-81, which mimics the immunodominant T-cell epitope CII259-273, can induce T-cell reactivity in blood samples from RA patients and healthy subjects through molecular mimicry. Using bioinformatic molecular modeling methods, we first determined whether the L-ASNase67-81 peptide binds to the HLA-DRB1*04:01 molecule and whether the formed MHCII-peptide complex interacts with the corresponding T-cell receptor. To validate the obtained results, leukocytes isolated from early RA patients and healthy individuals were stimulated in vitro with L-ASNase67-81 and CII259-273 peptides as well as with bacterial L-asparaginase or human type II collagen (huCII). The activated T cells (CD4+CD154+) were analyzed by flow cytometry (FACS), and the levels of cytokines produced (IL-2, IL-17A/F, and IFN-γ) were measured by ELISA. Our in silico analyses showed that the bacterial peptide L-ASNase67-81 binds better to HLA-DRB1*04:01 compared to the immunodominant T-cell epitope CII259-273, mimicking its structure and localization in the binding groove of MHCII. Six contact points were involved in the molecular interaction of the peptide with the TCR. FACS data showed that after in vitro stimulation with the L-ASNase67-81 peptide, the percentage of activated T cells (CD154+CD4+) was significantly increased in both cell cultures isolated from ERA patients and those isolated from healthy individuals, as higher values were observed for the ERA group (9.92 ± 0.23 vs. 4.82 ± 0.22). Furthermore, the ELISA assays revealed that after stimulation with L-ASNase67-81, a significant increase in the production of the cytokines IL-2, IL-17A/F, and IFN-γ was detected in the group of ERA patients. Our data showed that the bacterial L-ASNase67-81 peptide can mimic the immunodominant T-cell epitope CII259-273 and activate HLA-DRB1*04:01-restricted T cells as well as induce cytokine production in cells isolated from ERA patients. These results are the first to demonstrate that a specific bacterial antigen could play a role in the pathogenesis of RA, mimicking the immunodominant T-cell epitope from type II collagen.

Epigenetic/genetic variations in CG-rich elements of immune-related genes contribute to food allergy development during childhood.

BACKGROUND: Genetic areas of FOXP3 TSDR, human leukocyte antigen-G (HLA-G) upstream of CpG island 96, CpG41 and CpG73 islands of the HLA-DRB1 and HLA-DQB1 genes respectively, previously documented to display immune-modulatory properties, were subjected to epigenetic/genetic analysis to assess their influence in IgE-mediated food allergy (FA) development in children. METHODS: Sixty-four orally challenged and IgE-tested food allergic subjects together with 44 controls were recruited. Targeted pyrosequencing analysis to detect DNA methylation status and genetic variations was utilized and experimental results obtained were analyzed by a statistical software platform and correlated to clinical data. Also, transcription factor (TF) binding sites in study areas were unmasked by the JASPAR prediction database. RESULTS: Parents' smoking was significantly correlated with aberrant methylation patterns, regardless of food allergic or control status. HLA-G promoter region showed a trend for hypomethylation in food allergic subjects, with one of the CG sites displaying significantly decreased methylation values. Rs1233333, residing within the HLA-G promoter region preserved a protective role toward DNA methylation. Variable methylation patterns were recorded for CpG41 of the HLA-DRB1 gene and hypermethylation of the region was significantly correlated with the presence of single nucleotide polymorphisms (SNPs). TFs' recognition sites, located in studied genetic areas and exerting pivotal regulatory biological roles, are potentially affected by divergent DNA methylation status. CONCLUSIONS: We propose that HLA-G expression is triggered by food-derived allergens, providing a TregFoxP3-/HLA-G+ subpopulation generation to promote direct immune tolerance. Furthermore, clear evidence is provided for the underlying co-operation of genetic polymorphisms with epigenetic events, mainly at the CpG41 island of the HLA-DRB1 gene, which needs an extended investigation and elucidation.

PAD2 immunization induces ACPA in wild-type and HLA-DR4 humanized mice.

Rheumatoid arthritis (RA) is associated with HLA-DRB1 alleles expressing the "shared epitope." RA is usually preceded by the emergence of anti-citrullinated protein autoantibodies (ACPAs). ACPAs recognize citrulline residues on numerous proteins. Conversion of arginine into citrulline is performed by enzymes called peptidyl arginine deiminases (PADs). We have previously demonstrated that C3H mice immunized with PADs can produce ACPAs by a hapten-carrier mechanism. Here, we address the influence of HLA-DR alleles in this model in mice expressing RA-associated HLA-DRB1*04:01 (KO/KI*04:01), HLA-DRB1*04:04 (KO/KI*04:04), or non-RA-associated HLA-DRB1*04:02 (KO/KI*04:02) after murine PAD2 immunization. Immunization with mPAD2 triggers production of ACPAs in wild-type (WT) and HLA-DR4 C57BL/6 mice. Both I-Ab and HLA-DR are involved in the activation of mPAD2-specific T lymphocytes. Among HLA-DR4 mice, mice expressing RA-associated HLA-DRB1*04:01 are the best responders to mPAD2 and the best anti-citrullinated peptide antibody producers.

High resolution HLA-DRB1 analysis and shared molecular amino acid signature of DRß1 molecules in Occult hepatitis B infection.

AIM: To investigate the association of human leukocyte antigen (HLA)-DRB1 alleles and the variations of polymorphic amino acid changes in DRß1 chain in Shaanxi Han population with Occult hepatitis B infection (OBI). METHODS: High-resolution HLA-DRB1 genotyping was performed in 107 OBI carriers and 280 normal controls. Sequence information was used to assign which amino acids were encoded at all polymorphic positions. Three-dimensional modeling was performed to explore the effect of the key residues on the HLA-DRB1 molecule. RESULTS: Strong susceptible association for allele DRB1*07:01 was observed in OBI carriers. The amino acid variation at HLA-DRß1 molecule revealed susceptible associations for residues Gln4ß, Val57ß(P9), Ser60ß(P9) and Val78ß(P4), the amino acids Arg4ß, Asp57ß(P9), Tyr60ß(P9) and Tyr78ß(P4) showed protective associations. CONCLUSION: Alleles DRB1*07:01 showed strong susceptible associations in OBI carriers. The amino acid variations in DRß molecules revealed significant molecular markers for susceptibility and protection from OBI in Shaanxi Han population.

HLA autoimmune risk alleles restrict the hypervariable region of T cell receptors.

Polymorphisms in the human leukocyte antigen (HLA) genes strongly influence autoimmune disease risk. HLA risk alleles may influence thymic selection to increase the frequency of T cell receptors (TCRs) reactive to autoantigens (central hypothesis). However, research in human autoimmunity has provided little evidence supporting the central hypothesis. Here we investigated the influence of HLA alleles on TCR composition at the highly diverse complementarity determining region 3 (CDR3), which confers antigen recognition. We observed unexpectedly strong HLA-CDR3 associations. The strongest association was found at HLA-DRB1 amino acid position 13, the position that mediates genetic risk for multiple autoimmune diseases. We identified multiple CDR3 amino acid features enriched by HLA risk alleles. Moreover, the CDR3 features promoted by the HLA risk alleles are more enriched in candidate pathogenic TCRs than control TCRs (for example, citrullinated epitope-specific TCRs in patients with rheumatoid arthritis). Together, these results provide genetic evidence supporting the central hypothesis.

The influence of HLA-DRB1*15 on the relationship between microglia and neurons in multiple sclerosis normal appearing cortical grey matter.

Cortical tissue injury is common in multiple sclerosis (MS) and associates with disability progression. We have previously shown that HLA-DRB1*15 genotype status associates with the extent of cortical inflammatory pathology. In the current study, we sought to examine the influence of HLA-DRB1*15 on relationships between inflammation and neurodegeneration in MS. Human post-mortem MS cases (n = 47) and controls (n = 10) were used. Adjacent sections of motor cortex were stained for microglia (Iba1+, CD68+, TMEM119+), lymphocytes (CD3+, CD8+), GFAP+ astrocytes, and neurons (NeuN+). A subset of MS cases (n = 20) and controls (n = 7) were double-labeled for neurofilament and glutamic acid decarboxylase 65/67 (GAD+) to assess the extent of the inhibitory synaptic loss. In MS cases, microglial protein expression positively correlated with neuron density (Iba1+: r = 0.548, p < 0.001, CD68+: r = 0.498, p = 0.001, TMEM119+ r = 0.437, p = 0.003). This finding was restricted to MS cases not carrying HLA-DRB1*15. Evidence of a 14% reduction in inhibitory synapses in MS was detected (MS: 0.299 ± 0.006 synapses/µm2 neuronal membrane versus control: 0.348 ± 0.009 synapses/µm2 neuronal membrane, p = 0.005). Neurons expressing inhibitory synapses were 24% smaller in MS cases compared to the control (MS: 403 ± 15 µm2 versus control: 531 ± 29 µm2 , p = 0.001), a finding driven by HLA-DRB1*15+ cases (15+: 376 ± 21 µm2 vs. 15-: 432 ± 22 µm2 , p = 0.018). Taken together, our results demonstrate that HLA-DRB1*15 modulates the relationship between microglial inflammation, inhibitory synapses, and neuronal density in the MS cortex.

Computational identification and characterization of antigenic properties of Rv3899c of Mycobacterium tuberculosis and its interaction with human leukocyte antigen (HLA).

A rise in drug-resistant tuberculosis (TB) cases demands continued efforts towards the discovery and development of drugs and vaccines. Secretory proteins of Mycobacterium tuberculosis (H37Rv) are frequently studied for their antigenicity and their scope as protein subunit vaccines requires further analysis. In this study, Rv3899c of H37Rv emerges as a potential vaccine candidate on its evaluation by several bioinformatics tools. It is a non-toxic, secretory protein with an 'immunoglobulin-like' fold which does not show similarity with a human protein. Through BlastP and MEME suite analysis, we found Rv3899c homologs in several mycobacterial species and its antigenic score (0.54) to compare well with the known immunogens such as ESAT-6 (0.56) and Rv1860 (0.52). Structural examination of Rv3899c predicted ten antigenic peptides, an accessibility profile of the antigenic determinants constituting B cell epitope-rich regions and a low abundance of antigenic regions (AAR) value. Significantly, STRING analysis showed ESX-2 secretion system proteins and antigenic PE/PPE proteins of H37Rv as the interacting partners of Rv3899c. Further, molecular docking predicted Rv3899c to interact with human leukocyte antigen HLA-DRB1*04:01 through its antigenically conserved motif (RAAEQQRLQRIVDAVARQEPRISWAAGLRDDGTT). Interestingly, the binding affinity was observed to increase on citrullination of its Arg1 residue. Taken together, the computational characterization and predictive information suggest Rv3899c to be a promising TB vaccine candidate, which should be validated experimentally.

HLA-DRB1*15:01 is a co-receptor for Epstein-Barr virus, linking genetic and environmental risk factors for multiple sclerosis.

The strongest genetic and environmental risk factors for MS, an inflammatory CNS disease, are HLA-DRB1*15:01 and EBV. This work shows that HLA-DRB1*15:01 acts as a co-receptor for EBV infection of a B cell line, suggesting a mechanistic link between both risk factors for MS.

The shared susceptibility epitope of HLA-DR4 binds citrullinated self-antigens and the TCR.

Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell-mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibß-74cit69-81 peptide led to a population of HLA-DR4Fibß-74cit69-81 tetramer+ T cells that exhibited biased T cell receptor (TCR) ß chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibß-72,74cit69-81) altered the responding HLA-DR4 tetramer+ T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR ß chain usage toward the Fibß-74cit69-81 peptide was observed in healthy HLA-DR4+ individuals and patients with HLA-DR4+ RA, thereby suggesting a link to human RA.

Rituximab and Corticosteroid Effect on Desmoglein-Specific B Cells and Desmoglein-Specific T Follicular Helper Cells in Pemphigus.

Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.

Terminally Differentiated CD4+ T Cells Promote Myocardial Inflammaging.

The cardiovascular and immune systems undergo profound and intertwined alterations with aging. Recent studies have reported that an accumulation of memory and terminally differentiated T cells in elderly subjects can fuel myocardial aging and boost the progression of heart diseases. Nevertheless, it remains unclear whether the immunological senescence profile is sufficient to cause age-related cardiac deterioration or merely acts as an amplifier of previous tissue-intrinsic damage. Herein, we sought to decompose the causality in this cardio-immune crosstalk by studying young mice harboring a senescent-like expanded CD4+ T cell compartment. Thus, immunodeficient NSG-DR1 mice expressing HLA-DRB1*01:01 were transplanted with human CD4+ T cells purified from matching donors that rapidly engrafted and expanded in the recipients without causing xenograft reactions. In the donor subjects, the CD4+ T cell compartment was primarily composed of naïve cells defined as CCR7+CD45RO-. However, when transplanted into young lymphocyte-deficient mice, CD4+ T cells underwent homeostatic expansion, upregulated expression of PD-1 receptor and strongly shifted towards effector/memory (CCR7- CD45RO+) and terminally-differentiated phenotypes (CCR7-CD45RO-), as typically seen in elderly. Differentiated CD4+ T cells also infiltrated the myocardium of recipient mice at comparable levels to what is observed during physiological aging. In addition, young mice harboring an expanded CD4+ T cell compartment showed increased numbers of infiltrating monocytes, macrophages and dendritic cells in the heart. Bulk mRNA sequencing analyses further confirmed that expanding T-cells promote myocardial inflammaging, marked by a distinct age-related transcriptomic signature. Altogether, these data indicate that exaggerated CD4+ T-cell expansion and differentiation, a hallmark of the aging immune system, is sufficient to promote myocardial alterations compatible with inflammaging in juvenile healthy mice.

Peptides Derived From Mismatched Paternal Human Leukocyte Antigen Predicted to Be Presented by HLA-DRB1, -DRB3/4/5, -DQ, and -DP Induce Child-Specific Antibodies in Pregnant Women.

Predicted Indirectly ReCognizable Human Leukocyte Antigen (HLA) Epitopes (PIRCHE) are known to be a significant risk factor for the development of donor HLA-specific antibodies after organ transplantation. Most previous studies on PIRCHE limited their analyses on the presentation of the HLA-DRB1 locus, although HLA-DRB3/4/5, -DQ, and -DP are also known for presenting allopeptides to CD4+ T cells. In this study, we analyzed the impact of predicted allopeptides presented by these additional loci on the incidence of HLA-specific antibodies after an immunization event. We considered pregnancy as a model system of an HLA immunization and observed child-specific HLA antibody (CSA) development of 231 mothers during pregnancy by samples being taken at delivery. Our data confirm that PIRCHE presented by HLA-DRB1 along with HLA-DRB3/4/5, -DQ, and -DP are significant predictors for the development of CSA. Although there was limited peptidome overlap observed within the mothers' presenting HLA proteins, combining multiple presenting loci in a single predictor improved the model only marginally. Prediction performance of PIRCHE further improved when normalizing scores by the respective presenters' binding promiscuity. Immunogenicity analysis of specific allopeptides could not identify significant drivers of an immune response in this small cohort, suggesting confirmatory studies.

Do RA associated HLA-DR molecules bind citrullinated peptides or peptides from PAD4 to help the development of RA specific antibodies to citrullinated proteins?

PURPOSE: Rheumatoid arthritis (RA) is associated with HLA-DRB1 genes encoding a five amino acid basic motive, the shared epitope SE). Each HLA-DRB1 genotype defines a genotype specific risk of developing RA. RA is preceded by the emergence of anti citrullinated protein antibodies (ACPAs). Citrullin is a neutral version of arginin, a basic amino acid, formed after post translational modification by Peptidyl Arginyl Deiminases (PADs). HLA-DRB1 genes associated with RA are also associated with ACPAs. Two models might explain this association. Here we tested both models for prediction of HLA-DRB1 genotypic risks of developing RA. METHODS: We calculated the likelihoods for the 2 HLA-DR molecules encoded by 12 common HLA-DRB1 genotypes to bind at least one randomly chosen peptide from PAD4 or fibrinogen(native or citrullinatd) and compared them with the 12 respective HLA-DRB1genotypic risks of developing RA. RESULTS: HLA-DRB1 Genotypic risks of developing RA correlate with likelihoods of binding PAD4 peptides, not citrullinated Fibrinogen peptides. Thus, the molecular basis for the association of HLA-DR and ACPA positive RA is most likely the capability for RA associated HLA-DR molecules to bind peptides(s) from PAD4.