Low Major Histocompatibility Complex Research Articles

Different MHC class I cell surface expression levels in diverse chicken lines, associations with B blood group, and proposed relationship to antigen-binding repertoire

The Major Histocompatibility Complex (MHC) is a cluster of genes with primarily immune-related functions. The MHC class I genes are responsible for self- versus non-self-recognition and viral antigen presentation to T lymphocytes. The chicken MHC class I protein binds its cognate antigen(s) over a repertoire spectrum ranging from promiscuous (generalist) to fastidious (specialist). The MHC class I protein expression level at the cell surface is inversely related to the promiscuity of its peptide-binding repertoire. In our study, erythrocytes from 6 diverse and highly inbred lines of chickens, a closed broiler line, and a highly advanced intercross line were evaluated for MHC class I antigen expression level by flow cytometry using monoclonal antibodies to chicken MHC class I molecules. In chickens, the B blood group antigens include the MHC class I antigen expressed from the MHC. Thus, the B blood group has historically been used as a genetic marker for Marek's Disease virus response. Erythrocytes of the inbred lines were blood typed by serology. The B21 blood type is widely recognized as relatively resistant to Marek's disease and regarded as an MHC class I generalist with low MHC class I expression. The Spanish line, which types serologically as B21.1 (similar to B21), was the lowest MHC class I expressing line. The two sublines (B5.1 and B15.2) of the Fayoumi breed, which significantly differed in their MHC class I expression, also differ in response to multiple pathogens. These defined genetic lines of chickens, with distinct MHC class I expression levels, provide an excellent platform to further interrogate the hypothesis of high or low MHC class I expression (antigenic specialists or generalists, respectively) determining diverse responses to pathogens.

Mechanisms of Low MHC I Expression and Strategies for Targeting MHC I with Small Molecules in Cancer Immunotherapy.

Major histocompatibility complex (MHC) class I load antigens and present them on the cell surface, which transduces the tumor-associated antigens to CD8+ T cells, activating the acquired immune system. However, many tumors downregulate MHC I expression to evade immune surveillance. The low expression of MHC I not only reduce recognition by- and cytotoxicity of CD8+ T cells, but also seriously weakens the anti-tumor effect of immunotherapy by restoring CD8+ T cells, such as immune checkpoint inhibitors (ICIs). Accumulated evidence suggested that restoring MHC I expression is an effective strategy for enhancing tumor immunotherapy. This review focuses on mechanisms underlying MHC I downregulation include gene deletion and mutation, transcriptional inhibition, reduced mRNA stability, increased protein degradation, and disruption of endocytic trafficking. We also provide a comprehensive review of small molecules that restore or upregulate MHC I expression, as well as clinical trials involving the combination of ICIs and these small molecule drugs.

DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Comprehensive single-cell analysis reveals novel anergic antigen-presenting cell subtypes in human sepsis.

Sepsis is a life-threatening condition with high mortality. A few studies have emerged utilizing single-cell RNA sequencing (scRNA-seq) to analyze gene expression at the single-cell resolution in sepsis, but a comprehensive high-resolution analysis of blood antigen-presenting cells has not been conducted. All published human scRNA-seq data were downloaded from the single cell portal database. After manually curating the dataset, we extracted all antigen-presenting cells, including dendritic cells (DCs) and monocytes, for identification of cell subpopulations and their gene profiling and intercellular interactions between septic patients and healthy controls. Finally, we further validated the findings by performing deconvolution analysis on bulk RNA sequencing (RNA-seq) data and flow cytometry. Within the traditional DC populations, we discovered novel anergic DC subtypes characterized by low major histocompatibility complex class II expression. Notably, these anergic DC subtypes showed a significant increase in septic patients. Additionally, we found that a previously reported immunosuppressive monocyte subtype, Mono1, exhibited a similar gene expression profile to these anergic DCs. The consistency of our findings was confirmed through validation using bulk RNA-seq and flow cytometry, ensuring accurate identification of cell subtypes and gene expression patterns. This study represents the first comprehensive single-cell analysis of antigen-presenting cells in human sepsis, revealing novel disease-associated anergic DC subtypes. These findings provide new insights into the cellular mechanisms of immune dysregulation in bacterial sepsis.

Functional immune diversity in reindeer reveals a high Arctic population at risk

Climate changes the geographic range of both species as well as pathogens, causing a potential increase in the vulnerability of populations or species with limited genetic diversity. With advances in high throughput sequencing (HTS) technologies, we can now define functional expressed genetic diversity of wild species at a larger scale and identify populations at risk. Previous studies have used genomic DNA to define major histocompatibility complex (MHC) class II diversity in reindeer. Varying numbers of expressed genes found in many ungulates strongly argues for using cDNA in MHC typing strategies to ensure that diversity estimates relate to functional genes. We have used available reindeer genomes to identify candidate genes and established an HTS approach to define expressed MHC class I and class II diversity. To capture a broad diversity we included samples from wild reindeer from Southern Norway, semi-domesticated reindeer from Northern Norway and reindeer from the high Artic archipelago Svalbard. Our data show a medium MHC diversity in semi-domesticated and wild Norwegian mainland reindeer, and low MHC diversity reindeer in Svalbard reindeer. The low immune diversity in Svalbard reindeer provides a potential risk if the pathogenic pressure changes in response to altered environmental conditions due to climate change, or increased human-related activity.

Connection between MHC class II binding and aggregation propensity: The antigenic peptide 10 of Paracoccidioides brasiliensis as a benchmark study

The aggregation of epitopes that are also able to bind major histocompatibility complex (MHC) alleles raises questions around the potential connection between the formation of epitope aggregates and their affinities to MHC receptors. We first performed a general bioinformatic assessment over a public dataset of MHC class II epitopes, finding that higher experimental binding correlates with higher aggregation-propensity predictors. We then focused on the case of P10, an epitope used as a vaccine candidate against Paracoccidioides brasiliensis that aggregates into amyloid fibrils. We used a computational protocol to design variants of the P10 epitope to study the connection between the binding stabilities towards human MHC class II alleles and their aggregation propensities. The binding of the designed variants was tested experimentally, as well as their aggregation capacity. High-affinity MHC class II binders in vitro were more disposed to aggregate forming amyloid fibrils capable of binding Thioflavin T and congo red, while low affinity MHC class II binders remained soluble or formed rare amorphous aggregates. This study shows a possible connection between the aggregation propensity of an epitope and its affinity for the MHC class II cleft.

Unravelling the sex-specific diversity and functions of adrenal gland macrophages.

SummaryDespite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.

Expression of the Nonclassical MHC Class I, Saha-UD in the Transmissible Cancer Devil Facial Tumour Disease (DFTD).

Devil facial tumour disease (DFTD) is a transmissible cancer that has circulated in the Tasmanian devil population for >25 years. Like other contagious cancers in dogs and devils, the way DFTD escapes the immune response of its host is a central question to understanding this disease. DFTD has a low major histocompatibility complex class I (MHC-I) expression due to epigenetic modifications, preventing host immune recognition of mismatched MHC-I molecules by T cells. However, the total MHC-I loss should result in natural killer (NK) cell activation due to the ‘missing self’. Here, we have investigated the expression of the nonclassical MHC-I, Saha-UD as a potential regulatory or suppressive mechanism for DFTD. A monoclonal antibody was generated against the devil Saha-UD that binds recombinant Saha-UD by Western blot, with limited crossreactivity to the classical MHC-I, Saha-UC and nonclassical Saha-UK. Using this antibody, we confirmed the expression of Saha-UD in 13 DFTD tumours by immunohistochemistry (n = 15) and demonstrated that Saha-UD expression is heterogeneous, with 12 tumours showing intratumour heterogeneity. Immunohistochemical staining for the Saha-UD showed distinct patterns of expression when compared with classical MHC-I molecules. The nonclassical Saha-UD expression by DFTD tumours in vivo may be a mechanism for immunosuppression, and further work is ongoing to characterise its ligand on immune cells.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Updated Overall Survival Data and Predictive Biomarkers of Sintilimab Plus Pemetrexed and Platinum as First-Line Treatment for Locally Advanced or Metastatic Nonsquamous NSCLC in the Phase 3 ORIENT-11 Study

IntroductionSintilimab plus chemotherapy significantly prolonged progression-free survival (PFS) compared with chemotherapy alone in nonsquamous NSCLC in the ORIENT-11 study. Updated overall survival (OS) and PFS data and corresponding biomarker analyses are reported here. MethodsIn this study, a total of 397 patients with previously untreated, locally advanced or metastatic nonsquamous NSCLC were assigned to sintilimab plus chemotherapy combination treatment (combo) group or placebo plus chemotherapy treatment group. The patients were stratified by programmed death-ligand 1 (PD-L1) expression levels. Immune signature profiles from tumor RNA sequencing and PD-L1 immunohistochemistry were correlated with clinical outcome to identify predictive biomarkers. ResultsAs of January 2021, with median follow-up of 22.9 months, median OS was significantly improved in the combo group compared with the placebo plus chemotherapy treatment group (not reached versus 16.8 mo; hazard ratio [HR] = 0.60, 95% confidence interval [CI]: 0.45–0.79, p = 0.0003). High or medium immune cell infiltration was strongly associated with improved PFS in the combo group, in contrast to absent or low immune cell infiltration, which suggests that chemotherapy could not prime “immune deserts” to obtain benefit from programmed cell death protein-1 inhibition. In particular, high major histocompatibility complex (MHC) class II presentation pathway expression was significantly correlated with prolonged PFS (HR = 0.32, 95% CI: 0.19–0.54, p < 0.0001) and OS (HR = 0.36, 95% CI: 0.20–0.64, p = 0.0005) in the combo group. Importantly, patients with low or absent PD-L1 but high MHC class II expression could still benefit from the combo treatment. In contrast, MHC class I antigen presentation pathway was less relevant in this combination setting. ConclusionsThe addition of sintilimab to chemotherapy resulted to significantly longer OS in nonsquamous NSCLC. Expression of MHC class II antigen presentation pathway could identify patients benefiting most from this combination.

Parasitic fauna of Eurasian beavers (Castor fiber) in Sweden (1997\u20131998)

BackgroundThe parasitic fauna of beavers (Castor fiber and C. canadensis) has been well studied in many parts of their respective areas of distribution. In Scandinavia there have, however, been limited investigations conducted on the parasites of beavers in recent times. The present study is the first quantitative survey of parasites on beavers living in Sweden and elsewhere in Scandinavia. We investigated the parasitic fauna of the Eurasian beaver (C. fiber) in a North–South gradient in Sweden. The aim of the study was to investigate parasite distribution and prevalence in particular, related to average yearly air temperature and different age groups of beavers. A total of 30 beavers were sampled at eight localities, spanning a 720 km North–South gradient during the springs of 1997 and 1998.ResultsFive parasite taxa were identified. Four of these were present in all of the examined beavers, Stichorchis subtriquetrus (trematode), Travassosius rufus (nematode), Platypsyllus castoris (coleopteran), and Schizocarpus spp. (arachnid). A higher number of new infections of S. subtriquetrus, and more adults of T. rufus, were seen in beavers in southern Sweden where temperatures are higher. One-year old beavers had a higher infestation of S. subtriquetrus, but not of T. rufus, than older individuals.ConclusionsThe parasite fauna of Swedish beavers mirrored the impoverished parasite fauna of the original Norwegian population, and the high prevalence of parasites could be due to low major histocompatibility complex (MHC) polymorphism. Young beavers had a higher load of trematodes, probably depending on behavioural and ecological factors. Warmer temperatures in southern localities likely contributed to increased endoparasite loads.

The HDAC Inhibitor Domatinostat Promotes Cell-Cycle Arrest, Induces Apoptosis, and Increases Immunogenicity of Merkel Cell Carcinoma Cells

Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer for which immune modulation by immune checkpoint inhibitors shows remarkable response rates. However, primary or secondary resistance to immunotherapy prevents benefits in a significant proportion of patients. For MCC, one immune escape mechanism is insufficient for recognition by T cells owing to the downregulation of major histocompatibility complex I surface expression. Histone deacetylase inhibitors have been demonstrated to epigenetically reverse the low major histocompatibility complex I expression caused by the downregulation of the antigen-processing machinery. Domatinostat, an orally available small-molecule inhibitor targeting histone deacetylase class I, is currently in clinical evaluation to overcome resistance to immunotherapy. In this study, we present preclinical data on domatinostat's efficacy and mode of action in MCC. Single-cell RNA sequencing revealed a distinct gene expression signature of antigen processing and presentation, cell-cycle arrest, and execution phase of apoptosis on treatment. Accordingly, functional assays showed that domatinostat induced G2M arrest and apoptosis. In the surviving cells, antigen-processing machinery component gene transcription and translation were upregulated, consequently resulting in increased major histocompatibility complex I surface expression. Altogether, domatinostat not only exerts direct antitumoral effects but also restores HLA class I surface expression on MCC cells, therefore, restoring surviving MCC cells' susceptibility to recognition and elimination by cognate cytotoxic T cells.

Depauperate major histocompatibility complex variation in the endangered reticulated flatwoods salamander (Ambystoma bishopi).

Reticulated flatwoods salamander (Ambystoma bishopi) populations began decreasing dramatically in the1900s. Contemporary populations are small, isolated, and may be susceptible to inbreeding and reduced adaptive potential because of low genetic variation. Genetic variation at immune genes is especially important as it influences disease susceptibility and adaptation to emerging infectious pathogens, a central conservation concern for declining amphibians. We collected samples from across the extant range of this salamander to examine genetic variation at major histocompatibility complex (MHC) class Iα and IIβ exons as well as the mitochondrial control region. We screened tail or toe tissue for ranavirus, a pathogen associated with amphibian declines worldwide. Overall, we found low MHC variation when compared to other amphibian species and did not detect ranavirus at any site. MHC class Iα sequencing revealed only three alleles with a nucleotide diversity of 0.001, while MHC class IIβ had five alleles with a with nucleotide diversity of 0.004. However, unique variation still exists across this species' range with private alleles at three sites. Unlike MHC diversity, mitochondrial variation was comparable to levels estimated for other amphibians with nine haplotypes observed, including one haplotype shared across all sites. We hypothesize that a combination of a historic disease outbreak and a population bottleneck may have contributed to low MHC diversity while maintaining higher levels of mitochondrial DNA variation. Ultimately, MHC data indicated that the reticulated flatwoods salamander may be at an elevated risk from infectious diseases due to low levels of immunogenetic variation necessary to combat novel pathogens.

Development and functional analysis of an anticancer T-cell medicine with immune checkpoint inhibitory ability.

Adoptive cell therapy using patients' own T-cells is expected to be an ideal cancer treatment strategy with excellent antitumor effects and low side effects. However, this therapy targeting solid tumors is unlikely to be effective because tumor tissues have an environment that suppresses T-cell function. In particular, interaction between programmed death-1 (PD-1) and its ligand (PD-L1) inhibits T-cell activation by which T-cells eliminate tumor cells. Here, we attempted to develop T-cells that can exert potent antitumor activity even in tumor tissues by genetically modifying them to express the anti-PD-L1 membrane-anchoring type single chain variable fragment (M-scFv) that can inhibit PD-L1/PD-1 interaction. Anti-PD-L1 M-scFv could be expressed on T-cells while maintaining PD-L1-binding ability. Although T-cell proliferation induced by CD3 stimulation was decreased depending on the PD-L1 stimulation intensity, M-scFv-expressing T-cells showed high proliferative activity even in the presence of PD-L1 by avoiding the PD-L1/PD-1-mediated suppression. Furthermore, M-scFv-expressing T-cells showed higher cytotoxic activity against PD-L1high tumor cells than that of mock T-cells. The effect of PD-L1/PD-1 blockade was more pronounced when the therapeutic target was low-antigenic tumor cells with low major histocompatibility complex expression, presenting only the shared antigen. These results indicated that anti-PD-L1 M-scFv expression was functional in avoiding T-cell dysfunction by PD-L1/PD-1 interaction. Our concept of anti-PD-L1 M-scFv-expressing T-cells is thus expected to improve the efficacy of T-cell therapy and contribute to simplify the treatment system and reduce treatment costs compared with the combination therapy of T-cells and antibodies.

Efficient Cardiac Differentiation of Human Amniotic Fluid-Derived Stem Cells into Induced Pluripotent Stem Cells and Their Potential Immune Privilege.

Mature mammalian hearts possess very limited regenerative potential. The irreversible cardiomyocyte loss after heart injury can lead to heart failure and death. Pluripotent stem cells (PSCs) can differentiate into cardiomyocytes for cardiac repair, but there are obstacles to their clinical application. Among these obstacles is their potential for post-transplant rejection. Although human amniotic fluid-derived stem cells (hAFSCs) are immune privileged, they cannot induce cardiac differentiation. Thus, we generated hAFSC-derived induced PSCs (hAFSC-iPSCs) and used a Wnt-modulating differentiation protocol for the cardiac differentiation of hAFSC-iPSCs. In vitro studies using flow cytometry, immunofluorescence staining, and patch-clamp electrophysiological study, were performed to identify the characteristics of hAFSC-iPSC-derived cardiomyocytes (hAFSC-iPSC-CMs). We injected hAFSC-iPSC-CMs intramuscularly into rat infarcted hearts to evaluate the therapeutic potential of hAFSC-iPSC-CM transplantation. At day 21 of differentiation, the hAFSC-iPSC-CMs expressed cardiac-specific marker (cardiac troponin T), presented cardiomyocyte-specific electrophysiological properties, and contracted spontaneously. Importantly, these hAFSC-iPSC-CMs demonstrated low major histocompatibility complex (MHC) class I antigen expression and the absence of MHC class II antigens, indicating their low immunogenicity. The intramyocardial transplantation of hAFSC-iPSC-CMs restored cardiac function, partially remuscularized the injured region, and reduced fibrosis in the rat infarcted hearts. Therefore, hAFSC-iPSCs are potential candidates for the repair of infarcted myocardium.

Improved Anti-Tumour Adaptive Immunity Can Overcome the Melanoma Immunosuppressive Tumour Microenvironment.

Clinical benefits obtained from checkpoint blockade regimens demonstrate the importance of overcoming the immunosuppressive tumour microenvironment (TME) in cancer immunotherapy. Intravenous (i.v.) injection of B16 melanoma cells (H-2Kb) leads to lethal disseminated pulmonary metastasis in Balb/c recipients (H-2Kd). This lack of immune control is related to low major histocompatibility complex (MHC) expression on B16 cells which is associated with delayed and decreased anti-tumour adaptive immune responses (e.g., alloantibody formation) as: (i) other tumour types with normal H-2Kb expression are rejected with concomitant antibody production; (ii) preincubation of B16 with IFN-gamma to upregulate H-2Kb expression resulted in improved antibody production and anti-tumour activity. The delayed/decreased anti-tumour adaptive immune responses induced by B16 inoculation is not able to interrupt progression of primary metastases, while it is able to effectively eliminate secondary inoculated subcutaneously (s.c.) B16 cells from progression. This is due to the presence of an immunosuppressive TME within the primary metastases characterized by increased regulatory T cells (Tregs) and an increased T helper cells (Th) 2/1 profile. These tumour-induced immunosuppressive T cell populations are counteracted by improved adaptive immunity via active and passive immunization, resulting in effective elimination of the TME, destruction of the metastatic tumour and a reversal of Th2/1 profile in a time-sensitive manner. Thus, we here demonstrate that the TME is not irreversible and adaptive immunity is able to eradicate established solid tumour and its immunosuppressive TME. This study will help design treatments to overcome the immunosuppressive effect of the TME and improve efficacy of cancer immunotherapy.

Tumor venéreo transmissível canino com resistência quimioterápica e metástase esplênica - Relato de caso

O Tumor Venéreo Transmissível Canino (TVTC) é uma neoplasia de células redondas que tem a particularidade de se implantar em mucosas que tenham perdido a sua integridade. Nesse local o tumor prolifera e ocasionalmente origina metástase. Em geral, o tumor responde ao tratamento com sulfato de vincristina, porém a resistência quimioterápica associada ao fenótipo tumoral tem sido documentada. Objetivou-se relatar um caso de TVTC genital de fenótipo citológico misto com metástase esplênica e o insucesso da quimioterapia com sulfato de vincristina, em uma fêmea canina, da raça Australian Cattle Dog, de cinco anos de idade. Após diagnóstico citológico e histológico, o tumor primário foi ainda caracterizado em fase de progressão e mostrou baixa expressão de moléculas do complexo principal de histocompatibilidade MHC (4,4 ± 2% classe I e 11 ± 4,1% classe II). A cadela foi submetida à ovariohisterectomia e esplenectomia terapêutica e não apresentou recidiva do tumor após 12 meses de acompanhamento clínico.

Natural Spleen Cell Ligandome in Transporter Antigen Processing-Deficient Mice.

Peptides generated by proteases in the cytosol must be translocated to endoplasmic reticulum lumen by the transporter associated with antigen processing (TAP) prior to their assembly with major histocompatibility complex (MHC) class I molecules. Nonfunctional TAP complexes produce a drastic decrease of the MHC class I/peptide complexes presented on the cell surface. Previously, the cellular MHC class I ligandome from TAP-deficient cell lines was determined, but similar analysis from normal tissues remains incomplete. Using high-throughput mass spectrometry to analyze the MHC-bound peptide pools isolated from ex vivo spleen cells of TAP-deficient mice, we identified 210 TAP-independent ligands naturally presented by murine MHC class I molecules. This ligandome showed increased peptide lengths, presence of multiple nested set peptides, and low theoretical MHC binding affinity. The gene ontology enrichment analysis of parental proteins of this TAP-independent subligandome showed almost exclusively enrichment in tissue-specific biological processes related to the immune system as would be expected. Also, cellular components of the extracellular space (namely proteins outside the cell but still within the organism excluding the extracellular matrix) were specifically associated with TAP-independent antigen processing from these ex vivo mice cells. In addition, functional protein association network analysis revealed low protein-protein interactions between parental proteins from the TAP-independent ligandome. Finally, predominant endoproteolytic peptidase specificity for Leu/Phe residues in the P1 position of the scissile bond at both ligand termini was found for the ex vivo TAP-independent ligands. These data indicate that the TAP-independent ligandome from ex vivo cells derives from a more diverse collection of both endoprotease activities and parental proteins and where the cell origin and contribution of the extracellular environment are more relevant than in its equivalent cell lines.

Immunological MHC supertypes and allelic expression: how low is the functional MHC diversity in free-ranging Namibian cheetahs?

Cheetahs (Acinonyx jubatus) are a textbook example of how habitat loss, human-wildlife conflicts and historic bottlenecks depleted genetic variability, both genome-wide and at the major histocompatibility complex (MHC), which plays an integral role in the adaptive immune response. However, free-ranging Namibian cheetahs show no signs of impaired immunocompetence or health. This contradicts theoretical expectations and poses the question whether the manner by which MHC diversity is judged needs to be revised. Here, we show that free-ranging Namibian cheetahs still harbour MHC alleles that are divergent enough to cover several functionally distinct MHC supertypes and thus are probably capable of binding and presenting a relatively broad range of antigens to T-cells. We detected a similar pattern in three other free-ranging, strongly bottlenecked cat species, supporting the hypothesis that species with a low MHC allelic diversity might be able to retain functional diversity not within but across loci. Moreover, the allelic composition influences the level of MHC class I and class II expression which also might play a significant role in pathogen defence. Thus, our study indicates that the evolutionary role of MHC diversity goes beyond counting the remaining number of MHC alleles and offers an explanation as to how cat species might have avoided impaired immuno-competence, despite showing low MHC allelic diversity. Although the low MHC diversity currently seems to be sufficient to ensure the health of free-ranging cheetahs, it is currently unknown whether it can provide sufficient protection from future threats through emerging new pathogens.

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients

ABSTRACTPurpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma.Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line).Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells’ cytotoxicity.Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.