HLA Molecules Research Articles

Increased soluble HLA in COVID-19 present a disease-related, diverse immunopeptidome associated with Tcell immunity.

HLA-presented antigenic peptides are central components of Tcell-based immunity in infectious disease. Beside HLA molecules on cell surfaces, soluble HLA molecules (sHLA) are released in the blood suggested to impact cellular immune responses. We demonstrated that sHLA levels were significantly increased in COVID-19 patients and convalescent individuals compared to a control cohort and positively correlated with SARS-CoV-2-directed cellular immunity. Of note, patients with severe courses of COVID-19 showed reduced sHLA levels. Mass spectrometry-based characterization of sHLA-bound antigenic peptides, the so-called soluble immunopeptidome, revealed a COVID-19-associated increased diversity of HLA-presented peptides and identified a naturally presented SARS-CoV-2-derived peptide from the viral nucleoprotein in the plasma of COVID-19 patients. Of interest, sHLA serum levels directly correlated with the diversity of the soluble immunopeptidome. Together, these findings suggest an inflammation-driven release of sHLA in COVID-19, directly influencing the diversity of the soluble immunopeptidome with implications for SARS-CoV-2-directed Tcell-based immunity and disease outcome.

Differences in Presentation of SARS-CoV-2 Omicron Strain Variant BA.1-BA.5 Peptides by HLA Molecules.

In this work, we analyzed the binding affinities of mutated peptides of Omicron strain variants BA.1-BA.5 and the worldwide prevalent HLA alleles. Bioinformatics analysis was conducted with the use of T-CoV web portal. We showed that, for all five viral variants, mutations cause a significant reduction in the number of tightly binding peptides for HLA-B*07:02 and HLA-C*01:02 molecules. At the same time, there were novel potential mutant epitopes (binding affinity less than 50 nM) in case of HLA-A*32:01 allele. Interestingly, mutations caused multidirectional effect on the binding affinities of the viral peptides and HLA-DRB1*03:01. Specifically, Spike protein mutations in the BA.1 variant caused more than 100-fold decrease in PINLVRDLPQGFSAL binding affinity, 10-fold decrease in affinity in the case of BA.2, BA.4, and BA.5 variants, and 30% increase in affinity for the BA.3 variant.

Abstract B003: Cross-reacting antigens between the intestinal microbiome and tumor neoantigen are associated with immune checkpoint inhibitor response in a gastrointestinal tumor cohort

Abstract Background: Immune checkpoint inhibitor (ICI) is a revolutionary therapy that promotes the activation of effector T cells against tumors. Despite the potential for long-term benefit, a large proportion of patients does not respond to ICI therapy. One reason for this heterogenic response is due to the high variability in the intestinal microbiota among different patients. The abundance of certain bacteria has been associated with different responses and fecal meta transplant from responding donors was shown to rescue a beneficial phenotype in refractory patients. The presence of cross-reactive antigens (CRAgs) between the gut microbiome and tumor neoantigens has been proposed as a potential mechanism that could link the intestinal microbiota with ICI response. However, empirical evidence of CRAg’s role in ICI therapy is limited to a few studies. Methods: We investigated ICI response in a cohort of 22 stage IV gastrointestinal (GI) tumor patients in which whole-exome and gut metagenomic sequencing data were available. This cohort included patients with gastric cancer (n=11), esophageal carcinoma (n=6), and colon cancer (n=5). For each patient, we characterized the tumor neoantigen load based on epitopes that show high affinity and presentability in HLA molecules. Neoantigens were matched to the intestinal microbiome to identify corresponding CRAgs. CRAg quality, which ranks CRAgs according to their immunogenic potential, was defined based on matches in immunogenic protein or epitope databases. Results: We found the total number of CRAgs varied per tumor type, with a higher abundance in colon cancer (median µ=619) followed by esophageal (µ =287.5) and gastric (µ = 243). Interestingly, colon cancer patients showed a better progression-free survival (PFS) curve followed by esophageal and gastric cancer patients, respectively. We also observed that CRAg load was better associated with longer PFS (p=.03) than neoantigen load (p=0.07). Moreover, CRAg’s quality showed to be an important predictor of PFS. Patients with a high proportion of CRAgs matching at least one of the quality databases showed better PFS (p=0.02). Further, a taxa-specific analysis indicated that commensal CRAgs load was a better predictor of PFS (p<0.008) than relative abundance. Altogether, our work supports a mechanistic link between the microbiome, neoantigens, and immuno-therapy. Citation Format: Ravin Poudel, Die Dai, Fang Li, Xiaochen Yin, Yan Kou, Antonio L. C. Gomes. Cross-reacting antigens between the intestinal microbiome and tumor neoantigen are associated with immune checkpoint inhibitor response in a gastrointestinal tumor cohort [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B003.

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

An Approach to Identify HLA Class II Immunogenic Epitopes in the Greek Population through Machine Learning Algorithms

Current pre-transplantation routine matching involves serum anti-HLA antibodies quantification but cannot always preclude unfavorable graft outcomes. Epitope-based matching is proposed as a more precise approach, but to date no epitope-matching algorithm provides a satisfactory predictive tool for transplantation outcomes. In this study, anti-HLA-II loci responses from 1748 patients were analyzed with unsupervised machine learning algorithms, namely principal component analysis (PCA) and antigenic distances, projected as dendrograms. PCA for anti-HLA-DR anti-bodies revealed three main clusters of responses: anti-HLA-DR51 combined with anti-HLA-DRB1*01, anti-HLA-DR52 combined with anti-HLA-DRB1*08 and anti-HLA-DR53 combined with anti-HLA-DRB1*10. The dendrogram for anti-HLA-DR confirmed the pattern and showed further bisection of each cluster. Common epitopes present exclusively in all HLA molecules of each cluster were determined following the HLA epitope registry. Thus, we propose that 19 out of 123 HLA-DR epitopes are those that mainly lead anti-HLA-DR responses in the studied population. Likewise, we identified 22 out of 83 epitopes responsible for anti-HLA-DQ and 13 out of 62 responsible for anti-HLA-DP responses. Interpretation of these results may elucidate mechanisms of interlocus cross-reactivity, providing an alternative way of estimating the significance of each epitope in a population and thus suggesting a novel strategy towards optimal donor selection.

Site-directed mutagenesis of HLA molecules reveals the functional epitope of a human HLA-A1/A36-specific monoclonal antibody.

Eplet 44KM is currently listed in the HLA Epitope Registry but does not adhere to the eplet definition of an amino acid configuration within a 3.5Å radius. Eplet 44KM has been previously redefined to the antibody-verified reactivity pattern 44K/150V/158V, based on reactivity analysis of monoclonal antibody VDK1D12. Since the three residues are always simultaneously present on common HLA alleles, methods to define which residue is crucial for antibody-induction and binding are limited. In this proof-of-concept study, we performed site-directed mutagenesis to narrow down the antibody-verified reactivity pattern 44K/150V/158V to a single amino acid and defined 44K as the eplet or functional epitope of mAb VDK1D12.

From the Wright Brothers to the A380: Developing Transplantation Takes Teams of Teams.

From the Wright Brothers to the A380: Developing Transplantation Takes Teams of Teams.

Ex Vivo Generation and Infusion of Anti-Minor Histocompatibility Antigen Expanded T Cells for Patients Who Relapse after Allogeneic HLA-Matched Stem Cell Transplantation

Rationale Long-term clinical remissions of hematologic malignancies after allogeneic hematopoietic stem cell transplantation (HSCT) largely rely on the graft-versus-leukemia effect (GVL). In a fully HLA-matched setting, the GVL effect is mediated primarily by T cell immune responses against host minor histocompatibility antigens (MiHAs) found on the surface of malignant cells. Upon relapse of leukemia following HSCT, donor lymphocyte infusions (DLI) have unfortunately failed to exert efficient GVL activity. This is likely due to the insufficient number of anti-MiHA T cells within DLIs and to the risk of graft-vs-host disease (GVHD) that prevents the administration of high numbers of T cells. To generate potent and selective anti-leukemia activity, we have developed a strategy for the ex vivo selection and expansion of T cells targeting MiHAs preferentially expressed on hematopoietic cells (Perreault, Leukemia 2016). Thus, these cells should display augmented GVL immune reactivity while harboring a low risk of causing GVHD. To our knowledge, this is the first clinical study targeting several different molecularly defined MiHAs and on different HLA molecules. Patients and Methods In an open-label, multi-center phase 1 clinical trial (CR-MIHA-001; NCT 03091933), patients with hematologic malignancies who relapsed after an allogeneic HLA-matched HSCT were treated with sibling donor-derived, ex vivo- expanded T cells selected and expanded for reactivity toward tissue-restricted recipient MiHAs (GLIDE: Guided Lymphocyte Immunopeptide Derived Expansion). After donor cell collection, patients received fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 3 days followed by infusion of GLIDE, consisting of donor T-lymphocytes expanded ex vivo following exposure to dendritic cells coated with immunogenic peptides (MiHAs) present on the surface of host hematopoietic cells, at a single target dose of 4x107 viable T-cells/m2. A second dose of GLIDE, aiming at 3-5 times the original dose, could be administered to patients at a minimum of 6 weeks after the first infusion. GLIDE cell characteristics were evaluated using multi-parameter flow cytometry and TCR sequencing. Patients were followed up for one year post-GLIDE infusion for toxicity and clinical outcome measures. Results Ten patients, median age of 58 (range 33-69), 4 females/6 males with AML (n=7), NHL (1), CLL (1), and myelofibrosis (1) were treated with GLIDE. These patients had relapsed at a median of 12 months (range 5-126 mo) after allogeneic HSCT. GLIDE cells were personalized to target single MiHAs in 9 patients and two MiHAs in 1 patient. Overall, GLIDE cell products were directed at 9 different MiHAs on 6 different HLA-class 1 molecules (HLA-A02:01, A24:02, B07:02, B18:01, B44:02, and B44:03). A first dose of GLIDE was administered at 4.0x107 CD3+ cells/m2 to 8 patients, and 2 patients received slightly lower doses (2.6 and 3.7 x 107 CD3+ cells/m2). Final GLIDE products consisted mainly of T-cells (median: 98,7%), with CD8>CD4>NKT>NK cells. Most CD8+ T cells were effector memory and central memory T cells (Figure 1). At the end of the expansion period, CD8+ T cells expressed activation markers (CD25+ = 67%) with low levels of PD1+, PDL1+, CD57+ and KLRG1+ as markers of exhaustion. Not surprisingly, GLIDE cells had a more restricted TCR β chain profile than initial lymphopheresis cells. Six patients received a second dose of GLIDE (mean: 12 x 107 CD3+ cells/m2) at 53 days after the first infusion (median, range: 48-118 days). There were no infusional toxicities, and cells were well-tolerated by patients. Only one patient developed thrombocytopenia and subsequent chronic GVHD. There were no other adverse events potentially related to GLIDE. According to TCR β sequencing data, T cells present in the adoptively transferred anti-MiHA GLIDE product persisted in peripheral blood up to one year after infusion. Survival at one year was observed in 4 patients with 3 patients in sustained remission (Figure 2). Conclusion This pilot study demonstrates the feasibility of anti-MiHA T cell ex vivo generation (GLIDE) and safety of its administration to patients relapsing after allogeneic HSCT, as well as the clinical potential of these cells. The persistent remissions and low occurrence of GVHD support further development of GLIDE to target acute myeloid leukemia and other hematologic malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune Checkpoint Molecules Regulate Paroxysmal Nocturnal Hemoglobinuria Clonal Evolution

In paroxysmal nocturnal hemoglobinuria (PNH), the acquisition of somatic PIGA mutations provides a selection advantage to hematopoietic stem cells from aplastic anemia (AA) immune attack. In addition to this immune evasion mechanism, HLA mutations, deletions, and somatic UPD involving 6p21 locus have been also described in AA, all of which implicated in such adaptive clonal escape. It is well-known that blood cells derived from a PIGA-mutant clone lack membrane-bound complement regulators (e.g., CD55, CD59) and exhibit an increased vulnerability to complement attack. Nevertheless, PIGA-mutant cells may still be susceptible targets for AA T cell-mediated immune attack. In conjunction with mutational analysis, HLA protein expression may be investigated by flow cytometry to identify a haploinsufficient (clonal) vs. possible adaptive down-modulation. Indeed, the absence of these otherwise constitutionally expressed proteins represents an excellent marker of clonality. Additionally, in an attempt to further hinder the creation of a successful immunological synapsis, other mechanisms, including modulation of immune checkpoints expression, may be also involved in such AA/PNH intertwine. Whether these immune escape mechanisms are convergent or instead represent alternative pathways is a subject of intense research. Here, we comprehensively explore the dynamics governing PNH immune ontogenesis by combining HLA/PIGA genomics and flow cytometric measurements of HLA (A, B, C, DQ, DR, DP) and the immune checkpoint B7/CD28H family (PD1, PDL1, PDL2, VISTA, B7-H6, HHLA2, CD28H), stimulatory (GITR, CD30L) and inhibitory immune checkpoints (CD47) in CD45+CD15+CD59+ and CD59- fractions. A deep targeted sequencing panel covering HLA classical loci along with an in-house developed pipeline was also applied to assess fine HLA aberrations (mutational and copy number events), whereas multi-amplicon deep sequencing was deployed to identify PIGA mutational status. A total of 27 PNH patients classified as fully blown hemolytic PNH (n=15) or PNH/AA overlap (n=12) were included in this study with additional 4 healthy controls (HC). Overall, 41% of our patients harboured concomitant myeloid clones (most common hits were in DNMT3A, TET2, ASXL1, BCOR), while 58% of cases carried somatic HLA alterations, involving allelic losses or mutations. The latter were nonsense (33%), missense (11%), or 5’ or 3’ UTR (56%) hits with a median VAF of 11% (3-95%). Somatic lesions (including losses) were found in HLA-A (10%), B (30%), DQA1 (10%), or DQB1 (50%) loci. Since HLA aberrations may constitute the extreme pole of immune evasion and a marker of enhanced immune selection, we performed sorting/sequencing experiments on CD59+vs CD59- cells. A similar distribution and clonal burden of HLA-mutant subclones was found between the 2 fractions, indicating that PNH cells do not provide absolute immune privilege.1 Next, we compared the expression of HLA and other immunomodulatory molecules across HC, CD59+ cells from PNH patients and their corresponding CD59- cells (GPI+ and GPI-, respectively). Using MFI as a readout, GPI- cells showed similar expression levels of HLA-DP, -DR, PDL2, HHLA2 and GITR as compared to HC cells, while these molecules were significantly 1.5 to 5-fold upregulated on corresponding GPI+ cells consistent with their trigger/target function. This conclusion was also supported by the higher levels of CD28H found on GPI+ cells vs. GPI-(median MFI, 13.3 vs. 6.0; p<0.0001). Of note is that GPI- cells exhibited lower levels of HLA-DQ (0 vs. 7.0, p=0.01), B7H6 (0 vs. 39, p<0.0001) and CD30L (0 vs. 41, p <0.0001) vs. HC cells and upregulation of VISTA (460 vs. 227, p=0.006), suggesting that PNH clones may exhibit additional (to PNH phenotype) immune deterrence pathways. HLA alterations resulted in downregulation of HLA-DQ (0 vs. 29, p=.03), -DR (20 vs. 136, p=.03), B7H6 (0 vs. 45, p=.02), and CD30L (7.5 vs. 52, p=.02), as well as upregulation of HLA-B (2671 vs. 1909, p=.03) and VISTA (561 vs. 291, p=.02) in GPI- vs. GPI+, while no difference was noted in HLAWT cases. In conclusion, differential expression of immunomodulatory molecules on PNH vs. residual phenotypically normal cells in PNH patients and HC highlights various stimulatory and immune escape mechanisms operating in this disease. Understanding these processes may enable the identification of therapeutically relevant immune pathways.

A Phase 1/1b Multicenter Ascending Dose Study to Evaluate the Safety of HA-1 Minor Histocompatibility Antigen-Reactive TCR-Modified T Cells (BSB-1001) in Patients Undergoing HLA-Matched Allogenic Hematopoietic Stem Cell Transplant (alloSCT) for MRD+ AML or ALL or Poor/Very Poor Risk MDS

Background: AlloSCT can cure patients with acute leukemia and high-risk myelodysplastic syndromes (MDS). However, post-transplant relapse remains the greatest single cause of post-transplant death, occurring in up to 80% of high-risk patients. In HLA-matched alloSCT, alloreactive donor T cells target minor histocompatibility antigens (miHAs), which are peptide products of coding polymorphisms that distinguish recipients from donors. These alloreactive T cells can target leukemic cells, mediating the graft-vs-leukemia (GVL) effect; unfortunately, they also can cause graft-vs-host disease (GVHD). Pre-clinical mouse models (Blood, 2011) demonstrate that CD8+ T cells that target miHAs with expression limited to hematopoietic cells can mediate GVL without causing GVHD. To translate this concept for how to safely augment GVL to the clinic, BlueSphere Bio (BSB) used its high throughput T cell receptor (TCR) discovery and characterization platforms to clone a panel of TCRs reactive against the HLA-A*02-restricted miHA, HA-1, which is expressed mostly in hematopoietic cells. These receptors were cloned from a multiparous A*02:01 HA-1- woman who was immunized to HA-1 through pregnancy. A high affinity TCR specific for HA-1 and not reactive against allogeneic HLA molecules was chosen to create a cellular therapeutic designed to mediate GVL and enhance donor engraftment with a lower risk for GVHD, relative to polyclonal donor T cells. The anti-HA-1 T cell product (BSB-1001) is composed of donor CD8+ T cells transduced with a lentivirus vector encoding the anti-HA-1 TCR and the RQR8 tag, which expresses an epitope from CD20, thereby enabling the in vivo killing of BSB-1001 cells with rituximab. CRISPR-cas9 editing of the CD8+ T cells deletes the endogenous TCRα and β chains with >95% efficiency. This increases expression of the TCR and reduces risk of GVHD caused by native TCRs, as well as possible off-target reactivities generated by pairing of endogenous and introduced TCR chains. BSB-1001 cells specifically kill HLA-A*02:01, HA-1+ targets, with no activity against cells that are HLA-A*02:01+ but HA-1-, or cells that are HA-1+ but are HLA-A*02:01-. BSB-1001 is also not reactive against a panel of B lymphoblastic cell lines (LCL) expressing allogeneic HLA molecules. Importantly, efficient target killing is seen at effector:target ratios as low as 0.1, and maintained after several rounds of ex-vivo expansion following target cell challenge, a likely predictor of potency and persistence in vivo. Study Design: The first in human Phase 1/1b study is a multicenter, open-label, single dose study with a 3+3 ascending design with the option to expand accrual at a promising dose. Subjects must have ALL, AML or high-risk MDS with evidence of disease after the last therapy, be HLA-A*02:01 HA-1+ and have a 10/10 HLA-matched HA-1- related or unrelated donor. GCSF-mobilized donors will undergo leukapheresis. From this product, a small fraction of cells will be removed for BSB-1001 manufacturing; the remainder will undergo CD34-selection and cryopreservation until SCT. After BSB-1001 manufacturing is complete, patients will undergo ablative conditioning (choice of three regimens), followed by infusion of the CD34-selected cells and then BSB-1001. Patients will not receive anti-T cell antibodies and will receive no prophylactic pharmacologic immunosuppression. Subjects must otherwise be suitable for ablative conditioning using standard criteria. The primary objective is to establish a recommended dose of BSB-1001 based on its dose-limiting toxicities and the magnitude of post-infusion BSB-1001 expansion. The primary endpoint is determining the incidence of treatment-emergent adverse events, including dose-limiting toxicities. Additional endpoints include rates of engraftment, incidences of cytokine release syndrome (CRS) and acute and chronic GVHD, quantitation of malignant hematopoiesis and lineage-specific donor chimerism. CRS and GVHD, should they occur, will be treated per institutional standards. Rituximab can be administered to kill BSB-1001 cells in case of refractory CRS or GVHD. Enrollment is anticipated to begin in 1Q2023. BSB is also developing panels of TCRs targeting additional relatively hematopoietically-restricted miHAs with prioritization based on the frequencies of the restricting HLAs and the immunogenic/nonimmunogenic miHA alleles.

Limited T-Cell-Stimulating Effect of Cytochalasin-B-Induced Membrane Vesicles Isolated from Artificial Antigen-Presenting Cells.

Artificial antigen-presenting cells (aAPCs) that stably express particular HLA and co-stimulatory molecules by gene transfer have been developed to effectively stimulate T cells. To investigate whether cytochalsin-B-induced membrane vesicles derived from aAPCs (AP-CIMVs) have similar antigen-presenting functions as a cell-free system, T cell responses to different types of antigen presentation were measured using Jurkat reporter cells. First, the aggregation of AP-CIMV, which affects the measurement of function, was inhibited by nuclease treatment to produce uniform AP-CIMVs. The Green fluorescent protein (GFP) expression in Jurkat reporter cells was induced in a dose-dependent manner in groups stimulated with anti-CD3 antibody-coated AP-CIMVs and aAPCs, and anti-CD3/CD28 Dynabead. When Jurkat reporter cells expressing specific T cell receptors were stimulated by AP-CIMVs and aAPCs loaded with CMV pp65 peptide, AP-CIMVs showed similar stimulatory effects to that by aAPC. However, when these Jurkat reporter cells were stimulated by aAPCs endogenously expressing CMV pp65 antigen and their AP-CIMVs, the GFP expression rate by AP-CIMVs was 8.4%, which was significantly lower than 53.2% by aAPCs. Although this study showed a limited T-cell-stimulating effect of AP-CIMVs on endogenously processed antigen presentation, these results provide useful information for the development of improved cell-free systems for T cell stimulation in the future.

A simple approach for detecting HLA-A*02 alleles in archival formalin-fixed paraffin-embedded tissue samples and an application example for studying cancer immunoediting.

The HLA system represents a central component of the antigen presentation machinery. As every patient possesses a defined set of HLA molecules, only certain antigens can be presented on the cell surface. Thus, studying HLA type-dependent antigen presentation can improve the understanding of variation in susceptibility to various diseases, including infectious diseases and cancer. In archival formalin-fixed paraffin-embedded (FFPE) tissue, the HLA type is difficult to analyze because of fragmentation of DNA, hindering the application of commonly used assays that rely on long DNA stretches. Addressing these difficulties, we present a refined approach for characterizing presence or absence of HLA-A*02, the most common HLA-A allele in the Caucasian population, in archival samples. We validated our genotyping strategy in a cohort of 90 samples with HLA status obtained by an NGS-based method. 90% (n=81) of the samples could be analyzed with the approach. For all of them, the presence or absence of HLA-A*02 alleles was correctly determined with the method, demonstrating 100% sensitivity and specificity (95% CI: 91.40%-100% and 91.19%-100%). Furthermore, we provide an example of application in an independent cohort of 73 FFPE microsatellite-unstable (MSI) colorectal cancer samples. As MSI cancer cells encompass a high number of mutations in coding microsatellites, leading to the generation of highly immunogenic frameshift peptide antigens, they are ideally suited for studying relations between the mutational landscape of tumor cells and interindividual differences in the immune system, including the HLA genotype. Overall, our method can help to promote studying HLA type-dependency during the pathogenesis of a wide range of diseases, making archival and historic tissue samples accessible for identifying HLA-A*02 alleles.

Priming Mesenchymal Stem/Stromal Cells with a Combination of a Low Dose of IFN-γ and Bortezomib Results in Potent Suppression of Pathogenic Th17 Immunity Through the IDO1-AHR Axis.

Preconditioning of mesenchymal stem/stromal cells (MSCs) with the inflammatory cytokine IFN-γ enhances not only their immunosuppressive activity but also their expression of HLA and proinflammatory genes. We hypothesized that prevention of the upregulation of inflammatory cytokines and HLA molecules in IFN-γ-primed MSCs would render these cells more immunosuppressive and less immunogenic. In this study, we discovered the following findings supporting this hypothesis: (1) activated human T cells induced the expression of IDO1 in MSCs via IFN-γ secretion and those MSCs in turn inhibited T-cell proliferation in an AHR-dependent fashion; (2) there was no difference in the expression of IDO1 and HLA-DR in MSCs after priming with a low dose (25 IU/mL) versus a high dose (100 IU/mL) of IFN-γ; (3) the transient addition of bortezomib, a proteasome inhibitor, to culture MSCs after IFN-γ priming decreased the expression of HLA-DR, inflammatory cytokine genes and Vcam1 while increasing the expression of IDO1 and the production of L-kynurenine; finally, MSCs primed with a combination of a low dose of IFN-γ and bortezomib were more effective in inhibiting Th17-mediated idiopathic pneumonia syndrome (IPS) and chronic colitis than unprimed MSCs. Our results suggest that bortezomib significantly eliminates the unfavorable effects of IFN-γ priming of MSCs (increased expression of MHC molecules and inflammatory cytokines and cell aggregation genes) and simultaneously increases their immunosuppressive activity by upregulating IDO1. Taken together, our newly established MSC priming method may contribute to MSC-based cell therapy for inflammatory diseases.

HLA-G expression in Merkel cell carcinoma and the correlation with Merkel cell polyomavirus infection.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine cutaneous carcinoma with a high mortality rate. The MCC etiology is not fully understood. Merkel cell-associated polyomavirus (MCPyV) was found in MCC patients, indicating a risk factor for the tumor. Caucasian, elderly, and immunocompromised individuals are more likely to develop this tumor. HLA-G consists of a non-classical class I (Ib) HLA molecule with an immunoregulatory function and was associated with tumor escape in different types of tumors, nonetheless, never been studied in MCC. The purpose of this study was to evaluate the HLA-G expression and also to detect the MCPyV in MCC patients and correlate it with the clinical course of the disease. Forty-five MCC patients were included in a retrospective study. Formalin-fixed paraffin-embedded cutaneous skin biopsies were used by immunohistochemistry and RT-PCR to verify the HLA-G expression and MCPyV infection. HLA-G expression was found in 7 (15.6%), while the presence of MCPyV was detected in 28 (62.2%) of the studied patients. No significant association was found between HLA-G expression and MCPyV infection (p = 0.250). The presence of MCPyV was associated with areas of low sunlight exposure (p = 0.042) and the HLA-G expression with progression to death (p = 0.038). HLA-G expression was detected in MCC patients, as well as the MCPyV presence was confirmed. These markers could represent factors with a possible impact on patient survival; however, further studies with a greater number of patients are needed, to better elucidate the possible role in disease progression.

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

Cross-Talk between HLA Class I and TLR4 Mediates P-Selectin Surface Expression and Monocyte Capture to Human Endothelial Cells.

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin β4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.

Association between the HLA-DQB1 Gene Polymorphism and Chronic Progression of Hepatitis B in Indonesia

BACKGROUND: The progression of hepatitis B is affected by the activity of T lymphocytes. Activation of T lymphocytes requires a primary signal originating from the presentation of antigen by HLA molecules to T cell receptors. HLA-DQB1 gene polymorphisms can affect the ability of HLA to bind and present viral antigens to T cells, thus affecting T cell activation and potentially associated with the progression of chronic hepatitis B. AIM: We aimed to investigate the polymorphisms of the HLA-DQB1 gene and its influence on chronic hepatitis B progression of chronic hepatitis B patients in Indonesia. METHODS: This cross-sectional research studied chronic hepatitis B patients at the Internal Medicine Department, Arifin Ahmad Hospital, Pekanbaru, from January 2018 to December 2018. Subjects were grouped into three categories: (1) Inactive chronic hepatitis B, (2) active chronic hepatitis B, and (3) end stage liver disease (ESLD) which consisted of patients with liver cirrhosis and hepatocellular carcinoma. Examination of the HLA-DQB1 gene polymorphism was performed with the SSP-PCR method and sequenced to verify the PCR results. Analysis results of p &lt; 0.05 were considered statistically significant. RESULTS: The most common allele in patients with chronic hepatitis B was the HLA-DQB1 0301. The HLA-DQB1 0301 allele found primarily in the inactive chronic hepatitis B group. The DQB1 0501 allele found to be more abundant in patients with active chronic hepatitis B. The HLA DQB1 0502 allele only found in patients with chronic hepatitis B with ESLD. CONCLUSIONS: The HLA-DQB1 gene polymorphism is associated with the progression of chronic hepatitis B in chronic hepatitis B patients in Indonesia.

Membrane attack complexes, endothelial cell activation, and direct allorecognition.

Endothelial cells (ECs) form a critical immune interface regulating both the activation and trafficking of alloreactive T cells. In the setting of solid organ transplantation, donor-derived ECs represent sites where alloreactive T cells encounter major and minor tissue-derived alloantigens. During this initial encounter, ECs may formatively modulate effector responses of these T cells through expression of inflammatory mediators. Direct allorecognition is a process whereby recipient T cells recognize alloantigen in the context of donor EC-derived HLA molecules. Direct alloresponses are strongly modulated by human ECs and are galvanized by EC-derived inflammatory mediators.Complement are immune proteins that mark damaged or foreign surfaces for immune cell activation. Following labeling by natural IgM during ischemia reperfusion injury (IRI) or IgG during antibody-mediated rejection (ABMR), the complement cascade is terminally activated in the vicinity of donor-derived ECs to locally generate the solid-phase inflammatory mediator, the membrane attack complex (MAC). Via upregulation of leukocyte adhesion molecules, costimulatory molecules, and cytokine trans-presentation, MAC strengthen EC:T cell direct alloresponses and qualitatively shape the alloimmune T cell response. These processes together promote T cell-mediated inflammation during solid organ transplant rejection.In this review we describe molecular pathways downstream of IgM- and IgG-mediated MAC assembly on ECs in the setting of IRI and ABMR of tissue allografts, respectively. We describe work demonstrating that MAC deposition on ECs generates ‘signaling endosomes’ that sequester and post-translationally enhance the stability of inflammatory signaling molecules to promote EC activation, a process potentiating EC-mediated direct allorecognition. Additionally, with consideration to first-in-human xenotransplantation procedures, we describe clinical therapeutics based on inhibition of the complement pathway. The complement cascade critically mediates EC activation and improved understanding of relevant effector pathways will uncover druggable targets to obviate dysregulated alloimmune T cell infiltration into tissue allografts.

Redundancy and complementarity between ERAP1 and ERAP2 revealed by their effects on the Behçet’s disease-associated HLA-B*51 peptidome

The endoplasmic reticulum aminopeptidases (ERAP)1 and ERAP2 trim peptides to be loaded onto the HLA class I molecules, including the main risk factor for Behçet’s disease HLA-B*51. The low activity ERAP1 variant is also a risk factor for this disease among HLA-B*51-positive individuals, whereas no association is known with ERAP2. This study aimed at defining the mutual relationships between both enzymes in the processing of an HLA-bound peptidome and at gaining insight into their differential association with Behçet’s disease. CRISPR/Cas9 was used to generate knock outs of ERAP1, ERAP2 or both enzymes from transfectant 721.221-HLA-B*51:01 cells. The surface expression of HLA-B*51 was reduced in all cases. The effects of depleting each or both enzymes on the B*51:01 peptidome was analyzed by quantitative label-free mass spectrometry. Substantial quantitative alterations of peptide length, subpeptidome balance, N-terminal residue usage, affinity and presentation of non-canonical li-gands were observed. These effects were often different in the presence or absence of the other enzyme, revealing their mutual dependency. In the absence of ERAP1, ERAP2 showed significant processing of B*51:01 ligands, indicating functional redundancy. A high overlap between the peptidomes of wildtype and double KO cells was observed, indicating that a large majority of B*51:01 ligands are present in the ER without the need of N-terminal processing, while ERAP1 and ERAP2 shape the peptidomes by altering the quantities of available peptides to be presented by the HLA molecule. These results indicate that both enzymes have distinct, but complementary and partially redundant effects on the B*51:01 pep-tidome, leading to its optimization and maximal surface expression. The distinct effects of both enzymes on the HLA-B*51 peptidome provide a basis for their differential association with Behçet’s disease and suggest a pathogenetic role of the B*51:01 peptidome.

Enzymatic processing and MHC loading in cancer immunotherapyAdvanced immunopeptidomics based discovery engine for the development of personalized cancer immunotherapy

The remarkable clinical efficacy of the immune checkpoint blockade therapies has motivated researchers to discover immunogenic epitopes and exploit them for personalized vaccines. Mutated human leukocyte antigen binding peptides (HLAp) are currently the leading targets. Most studies attempt to identify neoantigens based on predicted affinity to HLA molecules. We have shown that the direct identification of tissue-derived neoantigens by mass spectrometry is becoming feasible and we have recently designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides that is suitable for both cell lines and tissues. The massive amount of HLAp data we acquire while hunting down the neo-antigens is highly valuable. We have compiled a large immunopeptidomics database across dozens of cell types and HLA allotypes. First, we have shown that by taking advantage of co-occurring HLA-I alleles across dozens of immunopeptidomics datasets we can rapidly and accurately identify HLA-I binding motifs. Consequently, training HLA-I ligand predictors on refined motifs significantly improves the identification of neoantigens. Recently, we have acquired the largest reported high-quality HLA-II peptidomics dataset. We introduced completely novel algorithmic tools to analyze such data and developed for the first time HLA-II epitope prediction tool trained on peptidomics data that results in major improvements in prediction accuracy. Second, our database captures the global nature of the in vivo peptidome averaged over many HLA alleles, and therefore, reflects the propensity of peptides to be presented on HLA complexes, which is complementary to the existing neoantigen prediction features. We have shown as a proof of concept that our immunopeptidomics MS-based features improved neoantigen prioritization by up to 50%. Overall, immunopeptidomics facilitates direct identification of neoantigens and it can also improve the prediction of clinically relevant neoantigens, and we develop a novel clinical pipeline for target selection for personalized anti-cancer vaccines.