Molecular Determinants Regulating the Plasticity of the MHC Class II Immunopeptidome.

In the last few years, advancement in the analysis of the MHC class II (MHC-II) ligandome in several mouse and human haplotypes has increased our understanding of the molecular components that regulate the range and selection of the MHC-II presented peptides, from MHC class II molecule polymorphisms to the recognition of different conformers, functional differences in endosomal processing along the endocytic tract, and the interplay between the MHC class II chaperones DM and DO. The sum of all these variables contributes, qualitatively and quantitatively, to the composition of the MHC II ligandome, altogether ensuring that the immunopeptidome landscape is highly sensitive to any changes in the composition of the intra- and extracellular proteome for a comprehensive survey of the microenvironment for MHC II presentation to CD4 T cells.

The Role of Angiotensin-Converting Enzyme in Immunity: Shedding Light on Experimental Findings.

Angiotensin-converting enzyme (ACE) is a zinc-dependent dicarboxypeptidase with two catalytic components, which has an important role in regulating blood pressure by converting angiotensin I to angiotensin II. ACE breaks down other peptides besides angiotensin I and has a variety of physiological effects together with renal growth and reproduction in men. ACE also acts on innate and acquired immune systems by affecting macrophage and neutrophil function, and these outcomes are exacerbated due to the overexpression of ACE. Overexpression of ACE in macrophages imposes antitumor and antimicrobial response, and it enhances the ability of neutrophils to produced super peroxide that has a bactericidal effect. ACE is also known to contribute to the expression of Major Histocompatibility Complex (MHC) class I and MHC class II peptides through enzymatic alterations of these peptides. Apprehending the expression of ACE and its effects on myeloid cell (myelogenous cells) activity can be promising in therapeutic interventions, including treatment of infection and malignancy.

Suppression of Plasmodium MIF-CD74 signaling protects against severe malaria.

The deadliest complication of infection by Plasmodium parasites, cerebral malaria, accounts for the majority of malarial fatalities. Although our understanding of the cellular and molecular mechanisms underlying the pathology remains incomplete, recent studies support the contribution of systemic and neuroinflammation as the cause of cerebral edema and blood-brain barrier (BBB) dysfunction. All Plasmodium species encode an orthologue of the innate cytokine, Macrophage Migration Inhibitory Factor (MIF), which functions in mammalian biology to regulate innate responses. Plasmodium MIF (PMIF) similarly signals through the host MIF receptor CD74, leading to an enhanced inflammatory response. We investigated the PMIF-CD74 interaction in the onset of experimental cerebral malaria (ECM) and liver stage Plasmodium development by using a combination of CD74 deficient (Cd74-/- ) hosts and PMIF deficient parasites. Cd74-/- mice were found to be protected from ECM and the protection was associated with the inability of brain microvessels to present parasite antigen to sequestered and pathogenic Plasmodium-specific CD8+ T cells. Infection of WT hosts with PMIF-deficient sporozoites or infection of Cd74-/- hosts with WT sporozoites impacted the survival of infected hepatocytes and subsequently reduced blood-stage associated inflammation, contributing to protection from ECM. We recapitulated these finding with a novel pharmacologic PMIF-selective antagonist that reduced PMIF/CD74 signaling and fully protected mice from ECM. These findings reveal a conserved mechanism for Plasmodium usurpation of host CD74 signaling and suggest a tractable approach for new pharmacologic intervention.

CD74 regulates cellularity and maturation of medullary thymic epithelial cells partially by activating the canonical NF-κB signaling pathway.

Thymic epithelial cells (TECs) are indispensable for T cell development, T cell receptor (TCR) repertoire selection, and specific lineage differentiation. Medullary thymic epithelial cells (mTECs), which account for the majority of TECs in adults, are critical for thymocyte selection and self-tolerance. CD74 is a nonpolymorphic transmembrane glycoprotein of major histocompatibility complex class II (MHCII) that is expressed in TECs. However, the exact role of CD74 in regulating the development of mTEC is poorly defined. In this research, we found that loss of CD74 resulted in a significant diminution in the medulla, a selective reduction in the cell number of mature mTECs expressing CD80 molecules, which eventually led to impaired thymic CD4+ T cell development. Moreover, RNA-sequence analysis showed that CD74 deficiency obviously downregulated the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in mTECs. Our results suggest that CD74 positively controls mTEC cellularity and maturation partially by activating the canonical NF-κB signaling pathway.

Macrophage migration inhibitory factor promotes the migration of dendritic cells through CD74 and the activation of the Src/PI3K/myosin II pathway.

Constitutively expressed by innate immune cells, the cytokine macrophage migration inhibitory factor (MIF) initiates host immune responses and drives pathogenic responses in infectious, inflammatory, and autoimmune diseases. Dendritic cells (DCs) express high levels of MIF, but the role of MIF in DC function remains poorly characterized. As migration is critical for DC immune surveillance, we investigated whether MIF promoted the migration of DCs. In classical transwell experiments, MIF-/- bone marrow-derived DCs (BMDCs) or MIF+/+ BMDCs treated with ISO-1, an inhibitor of MIF, showed markedly reduced spontaneous migration and chemotaxis. CD74-/- BMDCs that are deficient in the ligand-binding component of the cognate MIF receptor exhibited a migration defect similar to that of MIF-/- BMDCs. Adoptive transfer experiments of LPS-matured MIF+/+ and MIF-/- and of CD74+/+ and CD74-/- BMDCs injected into the hind footpads of homologous or heterologous mice showed that the autocrine and paracrine MIF activity acting via CD74 contributed to the recruitment of DCs to the draining lymph nodes. Mechanistically, MIF activated the Src/PI3K signaling pathway and myosin II complexes, which were required for the migration of BMDCs. Altogether, these data show that the cytokine MIF exerts chemokine-like activity for DC motility and trafficking.

Inhibition of BRAF Sensitizes Thyroid Carcinoma to Immunotherapy by Enhancing tsMHCII-mediated Immune Recognition.

CONTEXT: Multiple mechanisms play roles in restricting the ability of T-cells to recognize and eliminate tumor cells. OBJECTIVE: To identify immune escape mechanisms involved in papillary thyroid carcinoma (PTC) to optimize immunotherapy. SETTING AND DESIGN: iTRAQ analysis was conducted to identify proteins differentially expressed in PTC samples with or without BRAFV600E mutation. Molecular mechanisms regulating tumor cell evasion were investigated by in vitro modulations of BRAF/MAPK and related pathways. The pathological significance of identified tumor-specific major histocompatibility complex class II (tsMHCII) molecules in mediating tumor cell immune escape and targeted immune therapy was further evaluated in a transgenic mouse model of spontaneous thyroid cancer. RESULTS: Proteomic analysis showed that tsMHCII level was significantly lower in BRAFV600E-associated PTCs and negatively correlated with BRAF mutation status. Constitutive activation of BRAF decreased tsMHCII surface expression on tumor cells, which inhibited activation of CD4+ T-cells and led to immune escape. Pathway analysis indicated that the transforming growth factor (TGF)-ß1/SMAD3-mediated repression of tsMHCII, which could be reversed by BRAF inhibition (BRAFi). Targeting this pathway with a combined therapy of BRAF inhibitor PLX4032 and anti-PD-1 antibody efficiently blocked tumor growth by increasing CD4+ T-cell infiltration in a transgenic PTC mouse model. CONCLUSIONS: Our results suggest that BRAFV600E mutation in PTC impairs the expression of tsMHCII through the TGF-ß1/SMAD3 pathway to enhance immune escape. Combined treatment with PLX4032 and anti-PD-1 antibody promotes recognition and elimination of PTC by the immune system in a pre-clinical mouse model, and therefore offers an effective therapeutic strategy for patients with advanced PTC.

Lung adenocarcinoma-related TNF-α-dependent inflammation upregulates MHC-II on alveolar type II cells through CXCR-2 to contribute to Treg expansion.

MHC-II on alveolar type-II (AT-II) cells is associated with immune tolerance in an inflammatory microenvironment. Recently, we found TNF-α upregulated MHC-II in AT-II in vitro. In this study, we explored whether TNF-α-mediated inflammation upregulates MHC-II on AT-II cells to trigger Treg expansion in inflammation-driven lung adenocarcinoma (IDLA). Using urethane-induced mice IDLA model, we found that IDLA cells mainly arise from AT-II cells, which are the major source of MHC-II. Blocking urethane-induced inflammation by TNF-α neutralization inhibited tumorigenesis and reversed MHC-II upregulation on tumor cells of AT-II cellular origin in IDLA. MHC-II-dependent AT-II cells were isolated from IDLA-induced Treg expansion. In human LA samples, we found high expression of MHC-II in tumor cells of AT-II cellular origin, which was correlated with increased Foxp3+ T cells infiltration as well as CXCR-2 expression. CXCR-2 and MHC-II colocalization was observed in inflamed lung tissue and IDLA cells of AT-II cellular origin. Furthermore, at the pro-IDLA inflammatory stage, TNF-α-neutralization or CXCR-2 deficiency inhibited the upregulation of MHC-II on AT-II cells in inflamed lung tissue. Thus, tumor cells of AT-II cellular origin contribute to Treg expansion in an MHC-II-dependent manner in TNF-α-mediated IDLA. At the pro-tumor inflammatory stage, TNF-α-dependent lung inflammation plays an important role in MHC-II upregulation on AT-II cells.

HLA in Alzheimer's Disease: Genetic Association and Possible Pathogenic Roles.

Alzheimer's disease (AD) is commonly considered as the most prominent dementing disorder globally and is characterized by the deposition of misfolded amyloid-ß (Aß) peptide and the aggregation of neurofibrillary tangles. Immunological disturbances and neuroinflammation, which result from abnormal immunological reactivations, are believed to be the primary stimulating factors triggering AD-like neuropathy. It has been suggested by multiple previous studies that a bunch of AD key influencing factors might be attributed to genes encoding human leukocyte antigen (HLA), whose variety is an essential part of human adaptive immunity. A wide range of activities involved in immune responses may be determined by HLA genes, including inflammation mediated by the immune response, T-cell transendothelial migration, infection, brain development and plasticity in AD pathogenesis, and so on. The goal of this article is to review the recent epidemiological findings of HLA (mainly HLA class I and II) associated with AD and investigate to what extent the genetic variations of HLA were clinically significant as pathogenic factors for AD. Depending on the degree of contribution of HLA in AD pathogenesis, targeted research towards HLA may propel AD therapeutic strategies into a new era of development.

MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses.

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.

The Proinflammatory and Proangiogenic Macrophage Migration Inhibitory Factor Is a Potential Regulator in Proliferative Diabetic Retinopathy.

The macrophage migration inhibitory factor (MIF)/CD74 signaling pathway is strongly implicated in inflammation and angiogenesis. We investigated the expression of MIF and its receptor CD74 in proliferative diabetic retinopathy (PDR) to reveal a possible role of this pathway in the pathogenesis of PDR. Levels of MIF, soluble (s)CD74, soluble intercellular adhesion molecule-1 (sICAM-1) and vascular endothelial growth factor (VEGF) were significantly increased in the vitreous from patients with PDR compared to nondiabetic control samples. We detected significant positive correlations between the levels of MIF and the levels of sICAM-1 (r = 0.43; p = 0.001) and VEGF (r = 0.7; p < 0.001). Through immunohistochemical analysis of PDR epiretinal membranes, significant positive correlations were also found between microvessel density (CD31 expression) and the numbers of blood vessels expressing MIF (r = 0.56; p = 0.045) and stromal cells expressing MIF (r = 0.79; p = 0.001) and CD74 (r = 0.59; p = 0.045). Similar to VEGF, MIF was induced in Müller cells cultured under hypoxic conditions and MIF induced phosphorylation of ERK1/2 and VEGF production in Müller cells. Intravitreal administration of MIF in normal rats induced increased retinal vascular permeability and significant upregulation of phospho-ERK1/2, NF-κB, ICAM-1 and vascular cell adhesion molecule-1 expression in the retina. MIF induced migration and proliferation of human retinal microvascular endothelial cells. These results suggest that MIF/CD74 signaling is involved in PDR angiogenesis.

Helper CD4 T cells expressing granzyme B cause glial fibrillary acidic protein fragmentation in astrocytes in an MHCII-independent manner.

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.

Helminth Antigen-Conditioned Dendritic Cells Generate Anti-Inflammatory Cd4 T Cells Independent of Antigen Presentation via Major Histocompatibility Complex Class II.

A recently identified feature of the host response to infection with helminth parasites is suppression of concomitant disease. Dendritic cells (DCs) exposed to antigens from the tapeworm Hymenolepis diminuta significantly reduce the severity of dinitrobenzene sulfonic acid-induced colitis in mice. Here we elucidate mechanisms underlying this cellular immunotherapy. We show a requirement for Ccr7 expression on transferred H. diminuta antigen-treated (HD)-DCs, suggesting that homing to secondary lymphoid tissues is important for suppression of colitis. Furthermore, sodium metaperiodate-sensitive helminth-derived glycans are required to drive the anti-colitic response in recipient mice. Induction of Th2-type cytokines and Gata-3+Cd4+ cells in secondary lymphoid tissues is dependent on major histocompatibility complex class II (MHC II) protein expression on transferred DCs, although remarkably, transfer of MHC II-/- HD-DCs still attenuated dinitrobenzene sulfonic acid-induced colitis in recipient mice. Moreover, transfer of Cd4+ splenic T cells retrieved from mice administered MHC II-/- HD-DCs suppressed dinitrobenzene sulfonic acid-induced colitis in recipient mice. Our studies reveal that HD-DCs can suppress colitis via an alternative MHC II-independent pathway that involves, in part, mobilization of T-cell responses. These data support the utility of HD-DCs in blocking colitis, revealing a requirement for Ccr7 and providing for HD-DC autologous immunotherapy for disease in which MHC II expression and/or function is compromised.

The influence of BoLA-DRB3 alleles on incidence of clinical mastitis, cystic ovary disease and milk traits in Holstein Friesian cattle.

The major histocompatibility complex in cattle (BoLA) is regulated by genes that are closely related to the development of the immunological response to pathogens. The most polymorphic BoLA-DRB3.2 locus was analysed in 209 black-and-white Holstein-Friesian cows in Poland in order to a better explanation of influence of MHC on immunity to diseases in dairy cattle. A total of 23 alleles were identified, among which the *24, *16 and *22 alleles were observed with the highest frequency. These alleles were analysed in terms of their association with the occurrence of mastitis, ovarian cysts, retained placenta and uterine abscesses as well as their contribution to production traits (milk yield, protein and fat percentage in milk). It was determined that the BoLA-DRB3.2 *22 and *16 alleles were associated with a lower risk of clinical mastitis; however, a statistical significance was observed only for the *22 allele. Clinical mastitis was observed at a frequency lower by 8% in cows with one copy of the *22 allele compared to cows with 0 copies of the allele. The presence of the *22 allele in the genotype was also associated with higher milk yield, although this association was not statistically significant.

Contribution of the macrophage migration inhibitory factor superfamily of cytokines in the pathogenesis of preclinical and human multiple sclerosis: In silico and in vivo evidences.

Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions involved in the pathogenesis of autoimmune disorders, including Multiple Sclerosis (MS). We have first evaluated in silico the involvement of MIF, its homologue D-DT, and the receptors CD74, CD44, CXCR2 and CXCR4 in encephalitogenic T cells from a mouse model of MS, the Experimental Allergic Encephalomyelitis (EAE), as well as in circulating T helper cells from MS patients. We show an upregulation of the receptors involved in MIF signaling both in the animal model and in patients. Also, a significant increase in MIF receptors is found in the CNS lesions associated to MS. Finally, the specific inhibitor of MIF, ISO-1, improved both ex vivo and in vivo the features of EAE. Overall, our data indicate that there is a significant involvement of the MIF pathway in MS ethiopathogenesis and that interventions specifically blocking MIF receptors may represent useful therapeutic approaches in the clinical setting.

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections.IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4+ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4+ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4+ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

Class II MHC-independent suppressive adhesion of dendritic cells by regulatory T cells in vivo.

Regulatory T (T reg) cells are essential for peripheral homeostasis and known to target and suppress dendritic cells (DCs). One important mechanism is through prolonged interaction between antigen-specific T reg cells and DCs that down-regulates the co-stimulatory capacity of DCs. However, the dynamics and TCR specificities of such T reg cell-DC interaction and its relevance to the suppressive outcomes for individual DCs have not been clarified. To gain insights into the underlying cellular events in vivo, we analyzed individual T reg cell-DC interaction events in lymph nodes by intravital microscopy. Our results show that, upon exposure to interleukin-2, T reg cells formed prolonged adhesive contact with DCs, independent of antigen or MHC recognition, which significantly suppressed the contemporaneous interaction of the same DCs with antigen-specific conventional T cells and impaired T cell priming. Therefore, T reg cells may function in part as feedback regulators in inflammatory milieu, by suppressing local DCs and interrupting immune activation in a contact-dependent and class II MHC-independent manner.

Misfolded proteins complexed with MHC class II molecules are targets for autoantibodies.

Major histocompatibility complex (MHC) molecule is important for immune system through its function of presentation of peptide antigens. MHC is the gene most strongly associated with susceptibility to many autoimmune diseases. We recently found a novel function of MHC class II molecules to transport cellular misfolded proteins to the cell surface without processing to peptides. Interestingly, misfolded proteins transported to the cell surface by MHC class II molecules were found to be a specific targets for autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is strongly associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins associated with MHC class II molecules might be involved in the pathogenesis of autoimmune diseases.

Macrophage Migration Inhibitory Factor Mediates Proliferative GN via CD74.

Pathologic proliferation of mesangial and parietal epithelial cells (PECs) is a hallmark of various glomerulonephritides. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that mediates inflammation by engagement of a receptor complex involving the components CD74, CD44, CXCR2, and CXCR4. The proliferative effects of MIF may involve CD74 together with the coreceptor and PEC activation marker CD44. Herein, we analyzed the effects of local glomerular MIF/CD74/CD44 signaling in proliferative glomerulonephritides. MIF, CD74, and CD44 were upregulated in the glomeruli of patients and mice with proliferative glomerulonephritides. During disease, CD74 and CD44 were expressed de novo in PECs and colocalized in both PECs and mesangial cells. Stress stimuli induced MIF secretion from glomerular cells in vitro and in vivo, in particular from podocytes, and MIF stimulation induced proliferation of PECs and mesangial cells via CD74. In murine crescentic GN, Mif-deficient mice were almost completely protected from glomerular injury, the development of cellular crescents, and the activation and proliferation of PECs and mesangial cells, whereas wild-type mice were not. Bone marrow reconstitution studies showed that deficiency of both nonmyeloid and bone marrow-derived Mif reduced glomerular cell proliferation and injury. In contrast to wild-type mice, Cd74-deficient mice also were protected from glomerular injury and ensuing activation and proliferation of PECs and mesangial cells. Our data suggest a novel molecular mechanism and glomerular cell crosstalk by which local upregulation of MIF and its receptor complex CD74/CD44 mediate glomerular injury and pathologic proliferation in GN.