The MHC Class II Antigen-Processing and Presentation Pathway Is Dysregulated in Type 1 Diabetes.

Peptide loading of MHC class II (MHCII) molecules is facilitated by HLA-DM (DM), which catalyzes CLIP release, stabilizes empty MHCII, and edits the MHCII-bound peptide repertoire. HLA-DO (DO) binds to DM and modulates its activity, resulting in an altered set of peptides presented at the cell surface. MHCII-peptide presentation in individuals with type 1 diabetes (T1D) is abnormal, leading to a breakdown in tolerance; however, no direct measurement of the MHCII pathway activity in T1D patients has been performed. In this study, we measured MHCII Ag-processing pathway activity in humans by determining MHCII, MHCII-CLIP, DM, and DO levels by flow cytometry for peripheral blood B cells, dendritic cells, and monocytes from 99 T1D patients and 97 controls. Results showed that MHCII levels were similar for all three APC subsets. In contrast, MHCII-CLIP levels, independent of sex, age at blood draw, disease duration, and diagnosis age, were significantly increased for all three APCs, with B cells showing the largest increase (3.4-fold). DM and DO levels, which usually directly correlate with MHCII-CLIP levels, were unexpectedly identical in T1D patients and controls. Gene expression profiling on PBMC RNA showed that DMB mRNA was significantly elevated in T1D patients with residual C-peptide. This resulted in higher levels of DM protein in B cells and dendritic cells. DO levels were also increased, suggesting that the MHCII pathway maybe differentially regulated in individuals with residual C-peptide. Collectively, these studies show a dysregulation of the MHCII Ag-processing pathway in patients with T1D.

Mouse Homologue of Human HLA-DO Does Not Preempt Autoimmunity but Controls Murine Gammaherpesvirus MHV68.

H2-O (human HLA-DO) is a relatively conserved nonclassical MHC class II (MHCII)-like molecule. H2-O interaction with human HLA-DM edits the repertoire of peptides presented to TCRs by MHCII. It was long hypothesized that human HLA-DM inhibition by H2-O provides protection from autoimmunity by preventing binding of the high-affinity self-peptides to MHCII. The available evidence supporting this hypothesis, however, was inconclusive. A possibility still remained that the effect of H2-O deficiency on autoimmunity could be better revealed by using H2-O-deficient mice that were already genetically predisposed to autoimmunity. In this study, we generated and used autoimmunity-prone mouse models for systemic lupus erythematosus and organ-specific autoimmunity (type 1 diabetes and multiple sclerosis) to definitively test whether H2-O prevents autoimmune pathology. Whereas our data failed to support any significance of H2-O in protection from autoimmunity, we found that it was critical for controlling a γ-herpesvirus, MHV68. Thus, we propose that H2-O editing of the MHCII peptide repertoire may have evolved as a safeguard against specific highly prevalent viral pathogens.

Pleiotropic consequences of metabolic stress for the major histocompatibility complex class II molecule antigen processing and presentation machinery.

Hyperglycemia and hyperlipidemia are often observed in individuals with type II diabetes (T2D) and related mouse models. One dysmetabolic biochemical consequence is the non-enzymatic reaction between sugars, lipids, and proteins, favoring protein glycation, glycoxidation, and lipoxidation. Here, we identified oxidative alterations in key components of the major histocompatibility complex (MHC) class II molecule antigen processing and presentation machinery in vivo under conditions of hyperglycemia-induced metabolic stress. These modifications were linked to epitope-specific changes in endosomal processing efficiency, MHC class II-peptide binding, and DM editing activity. Moreover, we observed some quantitative and qualitative changes in the MHC class II immunopeptidome of Ob/Ob mice on a high-fat diet compared with controls, including changes in the presentation of an apolipoprotein B100 peptide associated previously with T2D and metabolic syndrome-related clinical complications. These findings highlight a link between glycation reactions and altered MHC class II antigen presentation that may contribute to T2D complications.

Genetic Control of Neonatal Immune Tolerance to an Exogenous Retrovirus.

Viruses, including retroviruses, can be passed from mothers to their progeny during birth and breastfeeding. It is assumed that newborns may develop immune tolerance to milk-transmitted pathogens similarly to food antigens. I/LnJ mice are uniquely resistant to retroviruses acquired as newborns or as adults as they produce virus-neutralizing antibodies (Abs). A loss-of-function allele of H2-Ob (Ob), originally mapped within the virus infectivity controller 1 (vic1) locus, is responsible for production of antiretrovirus Abs in I/LnJ mice. Importantly, Ob-deficient and vic1 I/LnJ congenic mice on other genetic backgrounds produce antivirus Abs when infected as adults, but not as newborns. We report here that I/LnJ mice carry an additional genetic locus, virus infectivity controller 2 (vic2), that abrogates neonatal immune tolerance to retroviruses. Further genetic analysis mapped the vic2 locus to the telomeric end of chromosome 15. Identification of the vic2 gene and understanding of the related signaling pathways would make blocking of neonatal immune tolerance to retroviruses an achievable goal.IMPORTANCE This work describes a previously unknown genetic mechanism that allows neonates to respond to infections as efficiently as adults.

Human Hepatitis B Viral Infection Outcomes Are Linked to Naturally Occurring Variants of HLA-DOA That Have Altered Function.

HLA molecules of the MHC class II (MHCII) bind and present pathogen-derived peptides for CD4 T cell activation. Peptide loading of MHCII in the endosomes of cells is controlled by the interplay of the nonclassical MHCII molecules, HLA-DM (DM) and HLA-DO (DO). DM catalyzes peptide loading, whereas DO, an MHCII substrate mimic, prevents DM from interacting with MHCII, resulting in an altered MHCII-peptide repertoire and increased MHCII-CLIP. Although the two genes encoding DO (DOA and DOB) are considered nonpolymorphic, there are rare natural variants. Our previous work identified DOB variants that altered DO function. In this study, we show that natural variation in the DOA gene also impacts DO function. Using the 1000 Genomes Project database, we show that ∼98% of individuals express the canonical DOA*0101 allele, and the remaining individuals mostly express DOA*0102, which we found was a gain-of-function allele. Analysis of 25 natural occurring DOα variants, which included the common alleles, identified three null variants and one variant with reduced and nine with increased ability to modulate DM activity. Unexpectedly, several of the variants produced reduced DO protein levels yet efficiently inhibited DM activity. Finally, analysis of associated single-nucleotide polymorphisms genetically linked the DOA*0102 common allele, a gain-of-function variant, with human hepatitis B viral persistence. In contrast, we found that the DOα F114L null allele was linked with viral clearance. Collectively, these studies show that natural variation occurring in the human DOA gene impacts DO function and can be linked to specific outcomes of viral infections.

Deletion of Mouse Setd4 Promotes the Recovery of Hematopoietic Failure.

PURPOSE: Acquired hematopoietic failure is commonly caused by therapeutic and accidental exposure of the bone marrow (BM) to toxic agents. Efficient recovery from BM failure is dictated not only by the intrinsic sensitivity and proliferation capacity of the hematopoietic stem and progenitor cells but also by the BM environment niche. Identification of genetic factors that improve recovery from hematopoietic failure is essential. Vertebrate SETD4 is a poorly characterized and putatively nonhistone methyltransferase. This study aims to identify the roles of SETD4 in BM recovery. METHODS AND MATERIALS: An inducible SETD4 knockout mouse model (Setd4flox/flox;Rosa26-CreERT2+) was used. Adult sex-matched littermates were treated with tamoxifen to induce Setd4 deletion or oil as the control. Tamoxifen-treated Setd4wt/wt;Rosa26-CreERT2+ mice were included as another control. Those mice were irradiated to induce hematopoietic syndrome and analyzed to identify the roles and mechanisms of Setd4 in of BM recovery. RESULTS: Loss of Setd4 in adult mice improved the survival of whole-body irradiation-induced BM failure. This was associated with improved recoveries of long-term and short-term hematopoietic stem cells (HSCs) and early progenitor cells. BM transplantation analyses surprisingly showed that the improved recovery was not due to radiation resistance of the Setd4-deficient HSCs but that Setd4-deficient HSCs were actually more sensitive to radiation. However, the Setd4-deficient mice were better recipients for allogeneic HSC transplantation. Furthermore, there was enhanced splenic erythropoiesis in Setd4-deficient mice. CONCLUSION: These findings not only revealed a previously unrecognized role of Setd4 as a unique modulator of hematopoiesis but also underscored the critical role of the BM niche in recovery from hematopoietic failure. Our study also implicated Setd4 as a potential target for therapeutic inhibition to improve the conditioning of the BM niche before allogeneic transplantation.

YY1lo NKT cells are dedicated IL-10 producers.

Co-expression of Yin Yang 1 (YY1) is required for the full function of the transcription factor, PLZF, which is essential for the development of natural killer T cell (NKT cell) effector functions. Discordant expression of YY1 and PLZF, therefore, might define NKT cell subsets with distinct effector functions. A subset of NKT cells was identified that expressed low levels of YY1. YY1lo NKT cells were found in all tissues, had a mature phenotype and, distinct from other NKT cells, expressed almost no ThPOK or Tbet. When activated, YY1lo NKT cells produced little IL-4 or IFN-γ. YY1lo NKT cells were found to constitutively transcribe IL-10 mRNA and, accordingly, produced IL-10 upon primary activation. Finally, we find that tumor infiltrating NKT cells are highly enriched for the YY1lo subset. Low YY1 expression, therefore, defines a previously unrecognized NKT cell subset that is committed to producing IL-10.

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated "hot" tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can have immediate impact particularly if they repurpose drugs with additional cancer-unrelated benefits. The seasonal influenza vaccine is recommended for all persons over 6 mo without prohibitive contraindications, including most cancer patients. Here, we report that unadjuvanted seasonal influenza vaccination via intratumoral, but not intramuscular, injection converts "cold" tumors to hot, generates systemic CD8+ T cell-mediated antitumor immunity, and sensitizes resistant tumors to checkpoint blockade. Importantly, intratumoral vaccination also provides protection against subsequent active influenza virus lung infection. Surprisingly, a squalene-based adjuvanted vaccine maintains intratumoral regulatory B cells and fails to improve antitumor responses, even while protecting against active influenza virus lung infection. Adjuvant removal, B cell depletion, or IL-10 blockade recovers its antitumor effectiveness. Our findings propose that antipathogen vaccines may be utilized for both infection prevention and repurposing as a cancer immunotherapy.

Loss of Setd4 delays radiation-induced thymic lymphoma in mice.

Radiation-induced lymphomagenesis results from a clonogenic lymphoid cell proliferation due to genetic alterations and immunological dysregulation. Mouse models had been successfully used to identify risk and protective factors for radiation-induced DNA damage and carcinogenesis. The mammalian SETD4 is a poorly understood putative methyl-transferase. Here, we report that conditional Setd4 deletion in adult mice significantly extended the survival of radiation-induced T-lymphoma. However, in Tp53 deficient mice, Setd4 deletion did not delay the radiation-induced lymphomagenesis although it accelerated the spontaneous T-lymphomagenesis in non-irradiated mice. The T-lymphomas were largely clonogenic in both Setd4flox/flox and Setd4Δ/Δ mice based on sequencing analysis of the T-cell antigen ß receptors. However, the Setd4Δ/Δ T-lymphomas were CD4+/CD8+ double positive, while the littermate Setd4flox/floxtumor were largely CD8+ single positive. A genomic sequencing analysis on chromosome deletion, inversion, duplication, and translocation, revealed a larger contribution of inversion but a less contribution of deletion to the overall chromosome rearrangements in the in Setd4Δ/Δ tumors than the Setd4flox/flox tumors. In addition, the Setd4flox/flox mice died more often from the large sizes of primary thymus lymphoma at earlier time, but there was a slight increase of lymphoma dissemination among peripheral organs in Setd4Δ/Δ at later times. These results suggest that Setd4 has a critical role in modulating lymphomagenesis and may be targeted to suppress radiation-induced carcinogenesis.

The Transcription Factor PLZF Is Necessary for the Development and Function of Mouse Basophils.

Basophils are innate immune cells associated with type 2 immunity, allergic reactions, and host defense against parasite infections. In this study, we show that the transcription factor PLZF, which is known for its essential role in the function and development of several innate lymphocyte subsets, is also important for the myeloid-derived basophil lineage. PLZF-deficient mice had decreased numbers of basophil progenitors in the bone marrow and mature basophils in multiple peripheral tissues. Functionally, PLZF-deficient basophils were less responsive to IgE activation and produced reduced amounts of IL-4. The altered function of basophils resulted in a blunted Th2 T cell response to a protein allergen. Additionally, PLZF-deficient basophils had reduced expression of the IL-18 receptor, which impacted migration to lungs. PLZF, therefore, is a major player in controlling type 2 immune responses mediated not only by innate lymphocytes but also by myeloid-derived cells.

Coexpression of YY1 Is Required to Elaborate the Effector Functions Controlled by PLZF in NKT Cells.

The promyelocytic leukemia zinc-finger transcription factor (PLZF) is essential for nearly all of the unique, innate-like functions and characteristics of NKT cells. It is not known, however, if the activity of PLZF is regulated by other factors. In this article, we show that the function of PLZF is completely dependent on the transcription factor Yin Yang 1 (YY1). Mouse NKT cells expressing wild-type levels of PLZF, but deficient for YY1, had developmental defects, lost their characteristic "preformed" mRNA for cytokines, and failed to produce cytokine protein upon primary activation. Immunoprecipitation experiments showed that YY1 and PLZF were coassociated. Taken together, these biochemical and genetic data show that the broadly expressed transcription factor, YY1, is required for the cell-specific "master regulator" functions of PLZF.

Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob.

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling.

The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge.

The Paf oncogene is essential for hematopoietic stem cell function and development.

Hematopoietic stem cells (HSCs) self-renew to maintain the lifelong production of all blood populations. Here, we show that the proliferating cell nuclear antigen-associated factor (Paf) is highly expressed in cycling bone marrow HSCs and plays a critical role in hematopoiesis. Mice lacking Paf exhibited reduced bone marrow cellularity; reduced numbers of HSCs and committed progenitors; and leukopenia. These phenotypes are caused by a cell-intrinsic blockage in the development of long-term (LT)-HSCs into multipotent progenitors and preferential loss of lymphoid progenitors caused by markedly increased p53-mediated apoptosis. In addition, LT-HSCs from Paf(-/-) mice had increased levels of reactive oxygen species (ROS), failed to maintain quiescence, and were unable to support LT hematopoiesis. The loss of lymphoid progenitors was likely due the increased levels of ROS in LT-HSCs caused by treatment of Paf(-/-) mice with the anti-oxidant N-acetylcysteine restored lymphoid progenitor numbers to that of Paf(+/+) mice. Collectively, our studies identify Paf as a novel and essential regulator of early hematopoiesis.

CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain.

MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

Right place, right time, right peptide: DO keeps DM focused.

Peptide loading of major histocompatibility class II molecules is catalyzed in late endosomal and lysosomal compartments of cells by the catalytic action of human leukocyte antigen (HLA)-DM (H-2M in mice). In B cells, dendritic cells and thymic epithelial cells, the peptide loading of class II molecules is modified by the expression of the non-classical class II molecule, HLA-DO (H-2O in mice). Collectively, studies to date support that DO/H-2O expression inhibits the presentation of antigens acquired by cells via fluid phase endocytosis. However, in B cells, the expression of H-2O promotes the presentation of antigens internalized by the B-cell receptor. In this review, we summarize the literature pertaining to DO assembly, transport, and function, with an emphasis on the function of DO/H-2O.

Cathepsin V is involved in the degradation of invariant chain in human thymus and is overexpressed in myasthenia gravis.

Stepwise degradation of the invariant chain (Ii) is required for the binding of antigenic peptides to MHC class II molecules. Cathepsin (Cat) L in the murine thymus and Cat S in peripheral APCs have both been implicated in the last step of Ii degradation that gives rise to the class II-associated invariant chain peptides (CLIP). Cat V has been recently described as highly homologous to Cat L and exclusively expressed in human thymus and testis, but with no mouse orthologue. We report that Cat V is the dominant cysteine protease in cortical human thymic epithelial cells, while Cat L and Cat S seem to be restricted to dendritic and macrophage-like cells. Active Cat V in thymic lysosomal preparations was demonstrated by active-site labeling. Recombinant Cat V was capable of converting Ii into CLIP efficiently, suggesting that Cat V is the protease that controls the generation of alphabeta-CLIP complexes in the human thymus, in analogy to Cat L in mouse. Comparison of Cat V expression between thymi from patients with myasthenia gravis and healthy controls revealed a significantly higher expression level in the pathological samples, suggesting a potential involvement of this protease in the immunopathogenesis of myasthenia gravis, an autoimmune disease almost invariably associated with thymic pathology.

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor.

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.

Germinal center B cells regulate their capability to present antigen by modulation of HLA-DO.

Peptide acquisition by MHC class II molecules is catalyzed by HLA-DM (DM). In B cells, HLA-DO (DO) inhibits or modifies the peptide exchange activity of DM. We show here that DO protein levels are modulated during B cell differentiation. Remarkably, germinal center (GC) B cells, which have low levels of DO relative to naive and memory B cells, are shown to have enhanced antigen presentation capabilities. DM protein levels also were somewhat reduced in GC B cells; however, the ratio of DM to DO in GC B cells was substantially increased, resulting in more free DM in GC B cells. We conclude that modulation of DM and DO in distinct stages of B cell differentiation represents a mechanism by which B cells regulate their capacity to function as antigen-presenting cells. Efficient antigen presentation in GC B cells would promote GC B cell-T cell interactions that are essential for B cells to survive positive selection in the GC.