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Antibody-mediated rejection (AMR) is a major barrier to long-term graft survival following solid organ transplantation (SOT). Major histocompatibility antigens mismatched between donor and recipient are well-recognized targets of humoral alloimmunity in SOT and thought to drive most cases of AMR. In contrast, the implication of minor histocompatibility antigens (mHAs) in AMR has not been fully investigated, and their clinical relevance remains controversial. Recent technological advances, allowing for genome-wide comparisons between donors and recipients, have uncovered novel, polymorphic mHA targets with potential influence on the graft outcome following SOT. Here, we review these latest studies relating to mHAs and discuss their clinical significance.

Except for two reports on Lebanese immigrants, there have been no studies on the major histocompatibility [MHC] antigens in the Lebanese population. We describe the frequency and distribution of MHC class I antigens present in the A, B and C loci based on data obtained from 200 healthy unrelated individuals from different parts of Lebanon. The highest gene frequencies were as follows:A2 [24.8%], B35 [17.9%] and Cw4 [18.6%], making this haplotype the commonest. Comparison of genetic distances revealed a pattern closer to the Caucasoid population than to the Mongoloid, Oriental or Black populations

The immune system’s ability to recognize peptides on major histocompatibility molecules (pMHCs) contributes to eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. We employ synTacs, dimeric pMHC scaffolds of defined composition, which enable clonal-selective delivery of a variety of signaling, recruitment, and imaging modalities. We show that synTacs, when labeled with positron-emitting isotopes, can non-invasively image antigen-specific CD8 T cells in vivo. We imaged human papillomavirus (HPV16) E7-specific CD8 T cells by positron emission tomography with an HPV16 E7 peptide-loaded synTac in HPV16-positive tumors, following administration of a therapeutic vaccine. We also imaged influenza A virus (IAV) nucleoprotein-specific CD8 T cells in the lungs of IAV-infected mice, using an isotopically labeled flu-specific synTac. It is thus possible to visualize antigen-specific CD8 T cell populations in vivo, which may serve prognostic and diagnostic roles.

Malaria remains a large-scale public health problem, killing more than 400,000 people and infecting up to 230 million worldwide, every year. Unfortunately, despite numerous efforts and research concerning vaccine development, results to date have been low and/or strain-specific. This work describes a strategy involving Plasmodium falciparum Duffy binding-like (DBL) and reticulocyte-binding protein homologue (RH) family-derived minimum functional peptides, netMHCIIpan3.2 parental and modified peptides’ in silico binding prediction and modeling some Aotus major histocompatibility class II (MHCII) molecules based on known human molecules’ structure to understand their differences. These are used to explain peptides’ immunological behaviour when used as vaccine components in the Aotus model. Despite the great similarity between human and Aotus immune system molecules, around 50% of Aotus allele molecules lack a counterpart in the human immune system which could lead to an Aotus-specific vaccine. It was also confirmed that functional Plasmodium falciparum’ conserved proteins are immunologically silent (in both the animal model and in-silico prediction); they must therefore be modified to elicit an appropriate immune response. Some peptides studied here had the desired behaviour and can thus be considered components of a fully-protective antimalarial vaccine.

Mesenchymal stem cells have been applied to treat graft versus host disease as they have immunosuppressive ability and can overcome the major histocompatibility complex–histocompatibility barrier. The potential of allogeneic mesenchymal stem cells in treating systemic lupus erythematosus (SLE) was investigated in this study. MRL/lpr mice which can develop acquired SLE-like phenotypes were selected as an animal model. Mesenchymal stem cells obtained from green fluorescent protein-transgenic ICR mice were infused into MRL/lpr mice at either the early or late stage of disease. The dosage was 1 × 106/mice per infusion. Mice were stratified into six groups including negative controls and those receiving one, two, three, four or five doses at 2-weekly intervals. The phenotypes were monitored regularly. After treatment, the spleen CD3+CD4–CD8– T and CD19+ B cells of two-dose mesenchymal stem cell-treated mice were significantly lower than those of the phosphate-buffered saline control. In terms of reducing the severity of SLE such as hair loss, skin ulcers, proteinuria and anti-dsDNA level, mesenchymal stem cells given at the early stage responded better and mice receiving two doses of mesenchymal stem cells performed better than those receiving either a lower dose (one dose) or higher doses (three, four or five doses). In conclusion, early treatment and an optimal dose of mesenchymal stem cells can effectively suppress the murine SLE model.

Science is a tedious and painstaking business. Many discoveries are considered incremental, individually not necessarily earth shattering, but collectively providing the critical broad framework on which pivotal insights can emerge. Transformational discoveries spring from this knowledge legacy of others and spur a fervent discovery process, often driven by technological developments. The seminal discovery of major histocompatibility class restriction I (MHCI) and its role in antiviral infections by Doherty and Zinkernagel in 1974 was one such discovery-the key that unlocked the treasure chest to the rich tapestry of the diversity of the immune system. An army of researchers have teased apart the different elements of the immune response, which now brings us to a deeper understanding of immune memory and protective immunity. In this process, it has uncovered a multitude of cell types that bridge the innate and adaptive arms of the immune system-blurring the line between these two branches-and ultimately fortifying the development of long-term immune protection.

Immunopeptidomics aims to identify immunopeptides, which are presented on Major Histocompatibility Complexes (MHC) on every cell and can be used to develop vaccines against pathogens and cancer. However, existing immunopeptidomics data analysis pipelines have some major hurdles to overcome, mostly due to the non-tryptic nature of immunopeptides, which complicates their identification. Previously, the machine and deep learning tools MS2PIP and DeepLC have shown to improve tryptic peptide identifications by accurately predicting tandem mass spectrometry (MS2) peak intensities and retention times, respectively, and by using these predictions to rescore peptide-spectrum matches (PSMs) with post-processing tool Percolator. However, MS2PIP was still tailored towards tryptic peptides and fragmentation patterns of immunopeptides are drastically different. To enable MS2PIP-based rescoring of immunopeptide PSMs, we have retrained MS2PIP to include non-tryptic peptides. These newly trained MS2PIP models greatly improve the predictions for immunopeptides and, surprisingly, also for tryptic peptides. Next, the new MS2PIP models, DeepLC, and Percolator were integrated into one software package, called MS2Rescore. Using MS2Rescore, 46% more spectra and 36% more unique peptides were identified at 1% false discovery rate (FDR), with even more extreme differences at 0.1% FDR, in comparison with standard Percolator rescoring. Due to the innovative extraction of MS2PIP-, DeepLC and search engine-based features, MS2Rescore even outperforms current state-of-the-art immunopeptide rescoring efforts. Thus, the integration of the new immunopeptide MS2PIP models, DeepLC, and Percolator into MS2Rescore shows great promise to substantially improve the identification of novel epitopes from immunopeptidomics workflows.

BACKGROUND: As dendritic cells (DCs) are the major antigen-presenting cells of the immune system, understanding their role in esophageal cancer is essential for the development of preventative and treatment strategies. This study investigated the expression level and clinical value of tumor infiltrating dendritic cells (TIDCs) in tumor tissues of patients with esophageal cancer. METHODS: From January 2019 to January 2021, 184 patients with esophageal cancer treated were prospectively enrolled as the observation group and 184 patients with benign esophageal tumors were selected as the control group. Tumor tissue samples were obtained and the expression level and phenotypes of the TIDCs were analyzed. The correlation between TIDC expression and clinical characteristics of patients with esophageal cancer was investigated. RESULTS: The density of the TIDCs in the observation group was lower than that in the control group (8.76±2.25 vs. 9.97±2.19; P=0.000). Furthermore, the percentage of major histocompatibility complex-II (MHC-II) positive DCs and the percentage of CD54 positive DCs were relatively lower in the observation group compared to the control group (6.60%±2.12% vs. 9.34%±2.41%; P=0.000 and 7.41%±2.36% vs. 9.98%±2.47%; P=0.000, respectively). Esophageal cancer patients with lymph node metastasis had lower TIDC density, lower percentage of MHC-II positive DCs, and lower percentage of CD54 positive DCs compared to patients without node metastasis (P

T cells are key effectors of anticancer immunity. They are capable of distinguishing tumor cells from normal ones by recognizing major histocompatibility complex–bound cancer-specific peptides. Accumulating evidence suggests that peptides associated with T cell–mediated tumor rejection arise predominantly from somatically mutated proteins and are unique to every patient's tumor. Knowledge of an individual's cancer mutanome (the entirety of cancer mutations) allows harnessing this enormous tumor cell–specific repertoire of highly immunogenic antigens for individualized cancer vaccines. This review outlines the preclinical and clinical state of individualized cancer vaccine development and the challenges ahead.

Machine learning with multi-layered artificial neural networks, also known as “deep learning,” is effective for making biological predictions. However, model interpretation is challenging, especially for sequential input data used with recurrent neural network architectures. Here, we introduce a framework called “Positional SHAP” (PoSHAP) to interpret models trained from biological sequences by utilizing SHapely Additive exPlanations (SHAP) to generate positional model interpretations. We demonstrate this using three long short-term memory (LSTM) regression models that predict peptide properties, including binding affinity to major histocompatibility complexes (MHC), and collisional cross section (CCS) measured by ion mobility spectrometry. Interpretation of these models with PoSHAP reproduced MHC class I (rhesus macaque Mamu-A1*001 and human A*11:01) peptide binding motifs, reflected known properties of peptide CCS, and provided new insights into interpositional dependencies of amino acid interactions. PoSHAP should have widespread utility for interpreting a variety of models trained from biological sequences.

Summary Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides., Graphical abstract, Highlights • In-house production and Sepharose conjugation of anti-MHC class I antibodies • Affinity purification of MHC-I complex from cultured cancer cell lysates • LC-MS/MS analysis of purified MHC-I bound peptides, Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.

The interactions between peptide(p)-bound major histocompatibility complexes (MHCs) and T cell receptors (TCRs) play an important role during the immune response. Here, we report on a molecular dynamics (MD) simulation study of free and TCR-bound pMHC-I proteins of the LC13-HLA-B* 44:05-pEEYLQAFTY complex. We performed long-term MD simulations of 4 μs each for both free and bound pMHC-TCR systems, employing 10 independent 400 ns replicas for each state. We observed a reduced dynamic flexibility in the central residues of the peptide and the MHC α1-helix upon TCR ligation, indicating a restricted conformational space in the MHC binding groove. Moreover, our data suggest a minimum of 50 to 100 ns simulation time of individual runs and 5 to 10 independent replicas to permit reliable conclusions for biomolecules of comparable complexity and similar descriptors.

Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle. Fil: Bellido, Demian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Baztarrica, Josefina. No especifíca; Fil: Rocha, Lucía. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Pecora, Andrea. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Acosta, Mario. No especifíca; Fil: Escribano, José M.. No especifíca; Fil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina Fil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Virología E Innovaciones Tecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Instituto de Virología E Innovaciones Tecnológicas; Argentina

Major histocompatibility complex-II (MHC-II)-Associated Peptide Proteomics (MAPPs) is a mass spectrometry-based approach to identify and relatively quantitate naturally processed and presented MHC-II-associated peptides that can potentially activate T cells and contribute to the immunogenicity of a drug. Acceptance of the MAPPs technology as an appropriate preclinical (and potentially clinical) immunogenicity risk assessment tool depends not only on its technical stability and robustness but also on the ability to compare results across experiments and donors. To this end, we developed a specialized MAPPs data processing pipeline, dataMAPPs, which presents complex mass spectrometric data sets in the form of heat maps (heatMAPPs), enabling rapid and convenient comparison between conditions and donors. A customized normalization procedure based on identified endogenous peptides standardizes signal intensities within and between donors and enables cross-experimental comparison. We evaluated the technical reproducibility of the MAPPs platform using tool compounds with respect to the most prominent experimental factors and found that the systematic biological differences across donors by far outweighed any technical source of variation. We illustrate the capability of the MAPPs platform to generate data that may be used for preclinical risk assessment of drug-induced immunogenicity and discuss its applicability in the clinics.

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

25 p.-5 fig.-5 fig. supl.-2 tab. supl., MHCII in antigen-presenting cells (APCs) is a key regulator of adaptive immune responses. Expression of MHCII genes is controlled by the transcription coactivator CIITA, itself regulated through cell type-specific promoters. Here we show that the transcription factor NFAT5 is needed for expression of Ciita and MHCII in macrophages, but not in dendritic cells and other APCs. NFAT5-deficient macrophages showed defective activation of MHCII-dependent responses in CD4(+) T lymphocytes and attenuated capacity to elicit graft rejection in vivo. Ultrasequencing analysis of NFAT5-immunoprecipitated chromatin uncovered an NFAT5-regulated region distally upstream of Ciita. This region was required for CIITA and hence MHCII expression, exhibited NFAT5-dependent characteristics of active enhancers such as H3K27 acetylation marks, and required NFAT5 to interact with Ciita myeloid promoter I. Our results uncover an NFAT5-regulated mechanism that maintains CIITA and MHCII expression in macrophages and thus modulates their T lymphocyte priming capacity., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), Agencia Estatal de Investigación, and European Regional Development Fund(SAF2012-36535, SAF2015-71363-R, and BFU2016-77961-P), and Fundació la Marató de TV3 (1225-30 and 201619-30). We also acknowledge funding support from Generalitat de Catalunya(2014SGR1153, 2017SGR888, and 2017SGR702) and MINECO through the “Unidad de Excelencia María de Maeztu” funded by MINECO (MDM-2014-0370). H. Huerga Encabo was supported by a predoctoral fellowship of the Spanish Ministerio de Educación, Cultura y Deporte (FPU13/01798). M. Tellechea was supported by fellowships from Fundació Catalunya-La Pedrera(2011) and Generalitat de Catalunya (FI-DGR program 2013). C. López-Rodríguez is a recipient of an ICREA Acadèmia award from Institució Catalana de Recerca i Estudis Avançats (ICREA,Generalitat de Catalunya)

In recent vaccine studies, DNA immunization was found to effectively stimulate both innate and adaptive immunities to elicit high levels of antigen-specific antibody responses. The DNA molecule itself can activate multiple pathways of innate immunity. The in vivo production of antigens allows for presentation by major histocompatibility complexes, so that T-cell responses are generated to help in the development of antigen-specific B cells, leading to high-affinity antibody response. By using the same process, DNA immunization should also be effective at producing functionally potent monoclonal antibodies (mAbs). Furthermore, the in vivo expressed proteins can maximally maintain the native structures and go through appropriate post-transcriptional modifications. By combining such advantages, DNA immunization can be expected to play more important roles in the future to elicit mAbs against difficult targets from a wide range of host systems. The current report shares our group's experience in using DNA immunization to elicit mAbs in the mouse, rabbit, and human models.

T cells initiate and regulate adaptive immune responses that can clear infections. To do this, they use their T cell receptors (TCRs) to continually scan the surfaces of other cells for cognate peptide antigens presented on major histocompatibility complexes (pMHCs). Experimental work has established that as few 1-10 pMHCs are sufficient to activate T cells. This sensitivity is remarkable in light of a number of factors, including the observation that the TCR and pMHC are short molecules relative to highly abundant long surface molecules, such as CD45, that can hinder initial binding, and moreover, the TCR/pMHC interaction is of weak affinity with solution lifetimes of approximately 1 second. Here, we review experimental and mathematical work that has contributed to uncovering molecular mechanisms of T cell sensitivity. We organize the mechanisms by where they act in the pathway to activate T cells, namely mechanisms that (a) promote TCR/pMHC binding, (b) induce rapid TCR signaling, and (c) amplify TCR signaling. We discuss work showing that high sensitivity reduces antigen specificity unless molecular feedbacks are invoked. We conclude by summarizing a number of open questions.

Chaperones TAPBPR and tapasin associate with class-I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR, and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product, and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, where the lifetime and structural features of transiently bound peptides controls the regulation of a conformational switch, located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex., Graphical abstract

Little is known about the relationship of Gammaherpesviruses with their bat hosts. Gammaherpesviruses are of interest because of their long–term infection of lymphoid cells and their potential to cause cancer. Here, we report the characterization of a novel bat herpesvirus isolated from a big brown bat (Eptesicus fuscus) in Canada. The genome of the virus, tentatively named Eptesicus fuscus herpesvirus (EfHV), is 166,748 base pairs. Phylogenetically EfHV is a member of Gammaherpesvirinae, in which it belongs to the Genus Rhadinovirus and is closely related to other bat Gammaherpesviruses. In contrast to other known Gammaherpesviruses, the EfHV genome contains coding sequences similar to those of class I and II host major histocompatibility antigens. The virus is capable of infecting and replicating in human, monkey, cat and pig cell lines. Although we detected EfHV in 20 of 28 big brown bats tested, these bats lacked neutralizing antibodies against the virus.

Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4+ T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-Ab by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model ( Apoe−/− mice on Western diet). Immunization with each of the three peptides (1 time in complete Freund’s adjuvant subcuntaneously and 4 time in incomplete Freund’s adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4+ T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3+ regulatory CD4+ T cells and more than tripled the number of CCR5+FoxP3+ cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection. NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4+ T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.

The clinical use of frozen, human allogeneic skin grafts is considered a suitable alternative to freshly harvested allogeneic skin grafts when the latter are not available. However, limited functional and histological information exists regarding the effects of cryopreservation on allogeneic skin grafts, especially those across mismatched histocompatibility barriers. Thus, we performed a side-by-side comparative study of fresh vs frozen skin grafts, across both minor and major histocompatibility barriers, in a miniature swine model. Since porcine skin shares many physical and immunological properties with human skin, our findings have relevance to current clinical practices involving allogeneic grafting and may support future, temporary wound therapies involving frozen xenografts, comprised genetically modified porcine skin. Four miniature swine underwent harvest and grafting of split-thickness skin, with and without cryopreservation, in order to observe autologous grafts and grafts across minor and major histocompatibility barriers. A biopsy of the grafts was done at regular intervals for study of architecture, vascularization, and outcomes. All grafts vascularized without technical complications. Differences were noted in the early appearance of some fresh vs frozen grafts, but no significant difference was observed in overall survival times in any of the experimental groups. These results demonstrate that despite early observable differences in the healing process, cryopreservation and thawing does not significantly affect long-term graft survival or time to rejection, thus supporting the clinical and experimental use of fresh and frozen split-thickness skin grafts as comparable and interchangeable.

Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-mannosidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new therapeutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the pathophysiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent transient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytometry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock transfected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune disease, making ERAD an interesting therapeutic target.

The binding of peptides to major histocompatibility complex class I (MHCI) molecules is a key step for regulating the immune response. Hundreds of crystal structures of the MHCI in complex with the peptide, which sometimes include the T-cell receptor (TCR), have been produced to help understand this key interaction. To further understand the conformations of peptides bound to MHCI, we examined all peptide structures in peptide-MHCI dimers or TCR-peptide-MHCI trimers. We observed that peptides with the same sequence in different peptide-MHCI/TCR-peptide-MHCI complexes can either take similar conformations in both backbone and side chain, similar conformations in backbone and distinct conformations in side chains, and also distinct conformations in both backbone and side chains. These conformational varieties can be used to support the modeling of peptide-MHC complexes and the structure-based prediction of the binding affinity of the MHC-peptide complex.

There is growing clinical interest in the utilization of mesenchymal stem cells (MSCs) in the management of acute graft-versus-host disease (aGvHD), yet the effect of major histocompatibility complexes (MHCs) on B lymphocytes in this process has been less well documented. Working in an MHC fully mismatched murine aGvHD model, we found that MSC co-transfer significantly prolonged the survival time of the recipients. More interestingly, analysis on immunophenotypic profiles of posttransplant splenocytes showed that surface expression of CD69 (an early activation marker) and CD86 (a costimulatory molecule) was suppressed predominantly on donor derived B lymphocytes by MSC infusion. Additionally, mRNA level of interleukin-4, a potent B lymphocyte stimulator, was strikingly reduced from MSC-treated mice, while interleukin-10, the regulatory B lymphocytes inductor, was increased; these may underlie the lesser activation of B lymphocytes. In consistence, depletion of B lymphocytes in the transfusion inoculum further prolonged the survival time of aGvHD mice regardless of MSC administration. Therefore, B lymphocytes played an important role in the development of aGvHD, and they are targets in MSC-regulated immune response cascade in vivo. This study may provide a mechanistic clue for the treatment of human clinical aGvHD.

For several malignant and nonmalignant disorders such as leukemias, lymphomas, or inborn errors of hematopoiesis, stem cell transplantation is the only curative option. Depending on the underlying cause of the disease, the conditioning regimens, source of the stem cells, and graft composition may vary. Possible stem cell donors are selected from databases considering existing major histocompatibility genes of the donor and the recipient. This is currently performed by matching human leukocyte antigen (HLA)-A, -B, and -C for class I, as well as HLA-DRB1 and -DQB1 for class II. Stem cell transplantation for nonmalignant disorders is a specialty of pediatrics. While algorithms for donor selection in these cases are generally similar, the objective of optimizing a possible graft-versus-leukemia effect is less important. In this article, we aim to provide an overview on the current methods for HLA typing and the algorithms for HLA matching. We also address ethical aspects regarding children and minors as stem cell donors.

There is a sharp difference in how one views TCR structure-function-behaviour dependent on whether its recognition of major histocompatibility complex-encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αβ TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele-specific. The establishing of allele-specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele-specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.