Cytotoxic and regulatory roles of mucosal-associated invariant T cells in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic ß-cells by the immune system that involves innate and adaptive immune cells. Mucosal-associated invariant T cells (MAIT cells) are innate-like T-cells that recognize derivatives of precursors of bacterial riboflavin presented by the major histocompatibility complex (MHC) class I-related molecule MR1. Since T1D is associated with modification of the gut microbiota, we investigated MAIT cells in this pathology. In patients with T1D and mice of the non-obese diabetic (NOD) strain, we detected alterations in MAIT cells, including increased production of granzyme B, which occurred before the onset of diabetes. Analysis of NOD mice that were deficient in MR1, and therefore lacked MAIT cells, revealed a loss of gut integrity and increased anti-islet responses associated with exacerbated diabetes. Together our data highlight the role of MAIT cells in the maintenance of gut integrity and the control of anti-islet autoimmune responses. Monitoring of MAIT cells might represent a new biomarker of T1D, while manipulation of these cells might open new therapeutic strategies.

Complement and MHC patterns can provide the diagnostic framework for inflammatory neuromuscular diseases.

Histopathological analysis stands as the gold standard for the identification and differentiation of inflammatory neuromuscular diseases. These disorders continue to constitute a diagnostic challenge due to their clinical heterogeneity, rarity and overlapping features. To establish standardized protocols for the diagnosis of inflammatory neuromuscular diseases, the development of cost-effective and widely applicable tools is crucial, especially in settings constrained by limited resources. The focus of this review is to emphasize the diagnostic value of major histocompatibility complex (MHC) and complement patterns in the immunohistochemical analysis of these diseases. We explore the immunological background of MHC and complement signatures that characterize inflammatory features, with a specific focus on idiopathic inflammatory myopathies. With this approach, we aim to provide a diagnostic algorithm that may improve and simplify the diagnostic workup based on a limited panel of stainings. Our approach acknowledges the current limitations in the field of inflammatory neuromuscular diseases, particularly the scarcity of large-scale, prospective studies that validate the diagnostic potential of these markers. Further efforts are needed to establish a consensus on the diagnostic protocol to effectively distinguish these diseases.

Proteogenomics Reveal the Overexpression of HLA-I in Cancer.

An accurate quantification of HLA class I gene expression is important in understanding the interplay with the tumor microenvironment of antitumor cytotoxic T cell activities. Because HLA-I sequences are highly variable, standard RNAseq and mass spectrometry-based quantification workflows using common genome and protein sequence references do not provide HLA-I allele specific quantifications. Here, we used personalized HLA-I nucleotide and protein reference sequences based on the subjects' HLA-I genotypes and surveyed tumor and adjacent normal samples from patients across nine cancer types. Mass spectrometry using data dependent acquisition data was validated to be sufficient to estimate HLA-A protein expression at the allele level. We found that HLA-I proteins were present in significantly higher levels in tumors compared to adjacent normal tissues from 41 to 63% of head and neck squamous cell carcinoma, uterine corpus endometrial carcinoma, and clear cell renal cell carcinoma patients, and this was driven by increased levels of HLA-I gene transcripts. Most immune cell types are universally enriched in HLA-I high tumors, while endothelial and neuronal cells showed divergent relationships with HLA-I. Pathway analysis revealed that tumor senescence and autophagy activity influence the level of HLA-I proteins in glioblastoma. Genes correlated to HLA-I protein expression are mostly the ones directly involved in HLA-I function in immune response and cell death, while glycosylation genes are exclusively co-expressed with HLA-I at the protein level.

Platelet EVs contain an active proteasome involved in protein processing for antigen presentation via MHC-I molecules.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

Optimized Liquid and Gas Phase Fractionation Increases HLA-Peptidome Coverage for Primary Cell and Tissue Samples.

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.

Prognostic Value of HLA Class I in Patients with Hepatocellular Carcinoma.

BACKGROUND: Downregulation of HLA class I molecules is a major tumor escape mechanism from immune attack. However, its prognostic impact for patients with hepatocellular carcinoma is still unclear. This study aimed to investigate whether HLA class I has prognostic significance in patients with hepatocellular carcinoma. METHODS: A cohort of 132 patients with hepatocellular carcinoma was enrolled. HLA class I expression was detected by immunohistochemistry. Levels of HLA class I expression were dichotomized as low and high according to staining intensity or staining percentage of positive tumor cells, respectively. Association of HLA class I expression with clinical characteristics and survival was analyzed. RESULTS: None of the clinical characteristics, including gender, age, virus infection, cirrhosis, AFP, vascular invasion, tumor size and number, was significantly associated with staining percentage of HLA class I or staining intensity (p > 0.05). Low staining percentage of HLA class I was significantly associated with a worse survival (p = 0.011), which was further confirmed by Cox regression hazards model in multivariate analysis (HR 0.416, 95% CI 0.204 - 0.849, p = 0.016). Staining intensity of HLA class I was not significantly associated with survival (p > 0.05). CONCLUSIONS: Expression of HLA class I might be a significant prognostic factor in hepatocellular carcinoma, and downregulation of HLA class I was significantly associated with a worse survival in terms of expression percentage of HLA class I.

Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?

Available evidence provides arguments both for and against a primary pathogenic role for T cells in human type 1 diabetes. Genetic susceptibility linked to HLA Class II lends strong support. Histopathology documents HLA Class I hyperexpression and islet infiltrates dominated by CD8+ T cells. While both hallmarks are near absent in autoantibody-positive donors, the variable insulitis and residual beta cells of recent-onset donors suggests the existence of a younger-onset endotype with more aggressive autoimmunity and an older-onset endotype with more vulnerable beta cells. Functional arguments from ex vivo and in vitro human studies and in vivo 'humanised' mouse models are instead neutral or against a T cell role. Clinical support is provided by the appearance of islet autoantibodies before disease onset. The faster C-peptide loss and superior benefits of immunotherapies in individuals with younger-onset type 1 diabetes reinforce the view of age-related endotypes. Clarifying the relative role of T cells will require technical advances in the identification of their target antigens, in their detection and phenotyping in the blood and pancreas, and in the study of the T cell/beta cell crosstalk. Critical steps toward this goal include the understanding of the link with environmental triggers, the description of T cell changes along the natural history of disease, and their relationship with age and the 'benign' islet autoimmunity of healthy individuals. Graphical abstract.

MHC class I loss in endometrial carcinoma: a potential resistance mechanism to immune checkpoint inhibition.

Major histocompatibility complex (MHC) class I is a membrane-bound protein complex expressed on nucleated human cells. MHC class I presents intracellular protein fragments to cytotoxic T cells and triggers an activation cascade upon neoantigen detection by these cells. MHC class I loss by tumor cells decreases tumor neoantigen presentation to the immune system and therefore represents a possible mechanism of immunotherapeutic resistance even among cancers that otherwise appear to be good candidates for checkpoint inhibition, such as mismatch repair (MMR)-deficient and PD-L1-positive malignancies. We herein assess MHC class I expression in a range of endometrial carcinomas, including MMR-deficient and PD-L1-positive cancers. Immunohistochemical staining for combined MHC class I A-, B-, and C-heavy chains was performed on 76 cases of endometrial carcinoma and was classified as present, subclonally lost, or diffusely lost. Tumoral PD-L1 expression, PD-L1 combined positive score, and CD3-positive T lymphocytes were also quantified. Forty-two percent of tumors showed loss of MHC class I expression, either in a subclonal (26%) or diffuse (16%) pattern. This included 46% of MMR-deficient and 25% of PD-L1-positive cancers. These findings suggest that tumoral MHC class I status may be an important factor to consider when selecting endometrial cancer patients for checkpoint inhibition.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

Impacts of combining anti-PD-L1 immunotherapy and radiotherapy on the tumour immune microenvironment in a murine prostate cancer model.

BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.

An effective peptide vaccine strategy circumventing clonal MHC heterogeneity of murine myeloid leukaemia.

BACKGROUND: Therapeutic cancer vaccines are an attractive approach for treating malignant tumours, and successful tumour eradication depends primarily on controlling tumour immunosuppression status as well as heterogeneity of tumour cells driven by epigenetic alterations. METHODS: Peptide-loaded dendritic cell (DC) prime and non-infectious peptide booster heterologous immunisations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides. Heterologous vaccination regimen targeting multiple shared tumour antigens simultaneously with PD-L1 blockade was assessed against murine myeloid leukaemia. RESULTS: A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumours. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukaemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumour eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukaemia. CONCLUSIONS: Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimisation of cancer vaccines for the treatment of numerous viral diseases and malignant tumours.

Tumor mutational load, CD8+ T cells, expression of PD-L1 and HLA class I to guide immunotherapy decisions in NSCLC patients.

OBJECTIVES: A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of tumor mutational load (TML), CD8+ T cell infiltration, HLA class-I and PD-L1 expression in the tumor. MATERIALS AND METHODS: Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8+ T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan-Meier methodology. RESULTS: 30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (p = 0.004) and OS (p = 0.025). Interaction analyses revealed that patients with both high TML and high total CD8+ T cell infiltrate (p = 0.023) or no loss of HLA class-I (p = 0.026), patients with high total CD8+ T cell infiltrate and no loss of HLA class-I (p = 0.041) or patients with both high PD-L1 and high TML (p = 0.003) or no loss of HLA class-I (p = 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (p = 0.007). CONCLUSION: This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8+ T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models.

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and ß2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and ß2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and ß2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic ß2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and ß2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (ß2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and ß2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.

Flow Cytometry Characterization of Pluripotent Transmembrane Glycoproteins on Resident Cervix Uteri Cells in Patients Screened for Cervical Cancer.

The aim of this study was to characterize both by flow cytometry analysis and immunohistochemistry cervix uteri cells of nulliparous women screened for cervical intraepithelial neoplasia (CIN) in comparison to a group without CIN by using mesenchymal stem cell-like and hematopoietic lineage markers. A significant expression for CD29, CD38, HLA-I, and HLA-II was correlated positively to the CIN degree and it was more relevant in patients positive for human papilloma virus (HPV). Thus, identification and detailed characterization of pluripotent resident in uteri cells could be a promising therapeutic target.

iTTCA-Hybrid: Improved and robust identification of tumor T cell antigens by utilizing hybrid feature representation.

In spite of the repertoire of existing cancer therapies, the ongoing recurrence and new cases of cancer poses a challenging health concern that prompts for novel and effective treatment. Cancer immunotherapy represents a promising venue for treatment by harnessing the body's immune system to combat cancer. Therefore, the identification of tumor T cell antigen represents an exciting area to explore. Computational tools have been instrumental in the identification of tumor T cell antigens and it is highly desirable to attain highly accurate models in a timely fashion from large volumes of peptides generated in the post-genomic era. In this study, we present a reliable, accurate, unbiased and automated sequence-based predictor named iTTCA-Hybrid for identifying tumor T cell antigens. The iTTCA-Hybrid approach proposed herein employs two robust machine learning models (e.g. support vector machine and random forest) constructed using five feature encoding strategies (i.e. amino acid composition, dipeptide composition, pseudo amino acid composition, distribution of amino acid properties in sequences and physicochemical properties derived from the AAindex). Rigorous independent test indicated that the iTTCA-Hybrid approach achieved an accuracy and area under the curve of 73.60% and 0.783, respectively, which corresponds to 4% and 7% performance increase than those of existing methods thereby indicating the superiority of the proposed model. To the best of our knowledge, the iTTCA-Hybrid is the first free web server (Available at http://camt.pythonanywhere.com/iTTCA-Hybrid) for identifying tumor T cell antigens presented by the MHC class I. The proposed web server allows robust predictions to be made without the need to develop in-house prediction models.

Global Identification of Post-Translationally Spliced Peptides with Neo-Fusion.

Post-translationally spliced peptides have recently garnered significant interest as potential targets for cancer immunotherapy and as contributors to autoimmune diseases such as type 1 diabetes, yet feasible identification methods for spliced peptides have yet to be developed. Here we present Neo-Fusion, a search program for discovering spliced peptides in tandem mass spectrometry data. Neo-Fusion utilizes two separated ion database searches to identify the two halves of each spliced peptide, and then it infers the full spliced sequence. This strategy allows for the identification of spliced peptides without peptide length constraints, providing a broadly applicable tool suitable for identification of spliced peptides in a variety of systems, such as the HLA-I and HLA-II immunopeptidomes and in vitro digested protein samples obtained from organelles, cells, or tissues of interest. Using simulated spliced peptides to benchmark Neo-Fusion, 25% of all simulated spliced peptides were identified at a measured false-discovery rate of 5% for HLA-I. Neo-Fusion provides the research community with a powerful new tool to aid in the study of the prevalence and biological significance of post-translationally spliced peptides.

Efficacy of the NCCV Cocktail-1 vaccine for refractory pediatric solid tumors: A phase I clinical trial.

Pediatric refractory solid tumors are aggressive malignant diseases, resulting in an extremely poor prognosis. KOC1, FOXM1, and KIF20A are cancer antigens that could be ideal targets for anticancer immunotherapy against pediatric refractory solid tumors with positive expression for these antigens. This nonrandomized, open-label, phase I clinical trial evaluated the safety and efficacy of the NCCV Cocktail-1 vaccine, which is a cocktail of cancer peptides derived from KOC1, FOXM1, and KIF20A, in patients with pediatric refractory solid tumors. Twelve patients with refractory pediatric solid tumors underwent NCCV Cocktail-1 vaccination weekly by intradermal injections. The primary endpoint was the safety of the NCCV Cocktail-1 vaccination, and the secondary endpoints were the immune response, as measured by interferon-r enzyme-linked immunospot assay, and the clinical outcomes including tumor response and progression-free survival. The NCCV Cocktail-1 vaccine was well tolerated. The clinical response of this trial showed that 4 patients had stable disease after 8 weeks and 2 patients maintained remission for >11 months. In 4, 8, and 5 patients, the NCCV Cocktail-1 vaccine induced the sufficient number of peptide-specific CTLs for KOC1, FOXM1, and KIF20A, respectively. Patients with high peptide-specific CTL frequencies for KOC1, FOXM1, and KIF20A had better progression-free survival than those with low frequencies. The findings of this clinical trial showed that the NCCV Cocktail-1 vaccine could be a novel therapeutic strategy, with adequate effects against pediatric refractory solid tumors. Future large-scale trials should evaluate the efficacy of the NCCV Cocktail-1 vaccination.

Multiplexed Relative Quantitation with Isobaric Tagging Mass Spectrometry Reveals Class I Major Histocompatibility Complex Ligand Dynamics in Response to Doxorubicin.

MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.

HLA class I and II alleles in susceptibility to ankylosing spondylitis.

OBJECTIVE: To examine associations of HLA class I and class II alleles with ankylosing spondylitis (AS) in three cohorts of patients of European, Asian and African ancestry. METHODS: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 and HLA-DPB1 alleles were genotyped in 1948 unrelated white and 67 African-American patients with AS from the Prospective Study of Outcomes in Ankylosing Spondylitis cohort, the North American Spondylitis Consortium and Australo-Anglo-American Spondyloarthritis Consortium, 990 white and 245 African-American Controls and HLA-B alleles in 442 Han Chinese patients with AS and 346 controls from Shanghai and Gansu, China. In addition to the case:control analyses, HLA-B*27-negative patients with AS were analysed separately, and logistic regression and 'relative predispositional effects' (RPE) analyses were carried out to control for the major effect of HLA-B*27 on disease susceptibility. RESULTS: Although numerous associations were seen between HLA alleles and AS in whites, among HLA-B*27-negative patients with AS , positive associations were seen with HLA-A*29, B*38, B*49, B*52, DRB1*11 and DPB1*03:01 and negative associations with HLA-B*07, HLA-B*57, HLA-DRB1*15:01, HLA-DQB1*02:01 and HLA-DQB1*06:02. Additional associations with HLA-B*14 and B*40 (B60) were observed via RPE analysis, which excludes the HLA-B*27 alleles. The increased frequency of HLA-B*40:01 and decreased frequency of HLA-B*07 was also seen in Han Chinese and African-Americans with AS. HLA-B*08 was decreased in whites with acute anterior uveitis. CONCLUSIONS: These data, analysing the largest number of patients with AS examined to date in three ethnic groups, confirm that other HLA class I and II alleles other than HLA-B*27 to be operative in AS predisposition.