MHC-I Proteins Research Articles

Adaptive humoral immune responses to type III capsular polysaccharide of Streptococcus pneumoniae.

Abstract Capsular polysaccharides (CPSs) are important vaccine candidates given that they are located on the outermost surface of bacteria and have distinct structures. To induce CPS specific adaptive immune responses (i.e., T cell-mediated B cell responses), CPSs are conjugated with carrier proteins, to make glycoconjugate vaccines. The production of the current generation of glycoconjugate vaccines does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective IgG antibodies. Our previous discovery demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells (i.e., Tcarbs) that recognize carbohydrate epitopes. Here, we investigate the interactions of carbohydrate epitopes with the key molecules of the adaptive immune system, MHCII proteins and T cell receptors (TCRs). This knowledge will have fundamental biological implications in our comprehension of how the adaptive immune system functions, and in future knowledge-based vaccine design. We employed a model bacterial polysaccharide antigen (capsular polysaccharide of type 3 Streptococcus pneumoniae, Pn3P) and its protein or peptide conjugates. We have collected evidence for the presence of a Tcarb repertoire that recognizes MHCII-presented Pn3P epitopes and drives Pn3P-specific IgG production by B cells. We have also generated Pn3 oligosaccharides through controlled depolymerization of Pn3P to be used in interation studies. Building on our expertise in these systems, we will define the MHCII and TCR interactions of processed Pn3P epitopes, and characterize immune responses that result from these interactions.

The length of the N-terminal region of a photocleavable peptide bound to DR1 determines the kinetics of photocleavable peptide fragment release

Abstract MHC class II molecules (MHCII) bind peptides and present them to CD4+ T cells. As prepared as a recombinant protein, DR1 (a MHCII) is in equilibrium between peptide-receptive and peptide-averse conformations. In order to dissect which step of the peptide binding reaction is being affected in DR1 mutants with altered peptide affinity, we wanted to generate DR1 fully in the peptide-receptive conformation. To generate this form, we used a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII proteins. We used variants of a well-characterized peptide derived from influenza hemagglutinin (HA), with different truncations at the N-terminal end, expecting to generate peptides with different affinity for DR1. This was confirmed by higher IC50 values obtained for the shorter peptides compared to the longer ones. All of the studied peptides were cleaved as expected after UV treatment as shown by mass spectrometry. However, when DR1-photocleavable peptide complexes were exposed to UV light, they generated different amounts of peptide-receptive DR1 measured by their ability to bind fluorescently-labeled HA peptide. The shorter the peptide, the more peptide-receptive DR1 was generated. This suggests that minimizing the interaction between photocleavable peptide and DR1 allows faster dissociation of the photo-generated fragments, and thus faster association of an incoming peptide. These results showed that the kinetics of photocleavable peptide fragment release can interfere with the kinetics of subsequent reactions.

Requirement of EHD family of endocytic recycling regulators for T-cell function.

Abstract Regulatory mechanisms that ensure effective antigen-specific responses by T-cells without evoking autoimmune disease are of great interest in basic and clinical immunology. While signaling through T-cell receptor (TCR) and positive and negative co-receptors have received great attention, mechanisms that control the dynamic traffic of such receptors between the cell surface and intracellular compartments have received less attention. Based on the well-known ability of the TCR to undergo constitutive internalization and recycling to the cell surface, we hypothesized C-terminal Eps-15 Homology Domain-containing (EHD) proteins may play important roles in TCR-mediated T-cell functions by regulating the recycling of TCR and other receptors on T-cells. EHD proteins (EHD1-4) are key regulators of endocytic recycling of surface receptors, including the MHC-I and MHC-II proteins, but their role in TCR traffic or T-cell function is unknown. We found EHD1, 3 and 4 to be expressed in CD4+T cells. Accordingly, we generated a conditional knockout model in which floxed EHD1, 3 and 4 are deleted in CD4+T cells (CD4-Cre) of mice bearing a myelin oligodendrocyte glycoprotein (MOG)-specific TCR transgene. In vitro, CD4+T-cells from these mice exhibit reduced antigen-driven cell proliferation and IL-2 secretion. In vivo, these mice exhibit reduced severity of experimental autoimmune encephalomyelitis (EAE) upon immunization with antigen. Initial analyses suggest that the recycling of TCR (CD3ɛ) and the surface levels of TCR, CD28, LFA-1 and CD25 on the surface of CD4+ T cells are reduced by EHD1/3/4 knockout. Our studies reveal a novel role of the endocytic recycling proteins of the EHD family in TCR-mediated T-cell activation.

MHC-class-II are expressed in a subpopulation of human neural stem cells in vitro in an IFNγ-independent fashion and during development.

Expression of major histocompatibility antigens class-2 (MHC-II) under non-inflammatory conditions is not usually associated with the nervous system. Comparative analysis of immunogenicity of human embryonic/fetal brain-derived neural stem cells (hNSCs) and human mesenchymal stem cells with neurogenic potential from umbilical cord (UC-MSCs) and paediatric adipose tissue (ADSCs), while highlighting differences in their immunogenicity, led us to discover subsets of neural cells co-expressing the neural marker SOX2 and MHC-II antigen in vivo during human CNS development. MHC-II proteins in hNSCs are functional, and differently regulated upon differentiation along different lineages. Mimicking an inflammatory response using the inflammatory cytokine IFNγ induced MHC-II up-regulation in both astrocytes and hNSCs, but not in UC-MSCs and ADSCs, either undifferentiated or differentiated, though IFNγ receptor expression was comparable. Together, hypoimmunogenicity of both UC-MSCs and ADSCs supports their suitability for allogeneic therapy, while significant immunogenicity of hNSCs and their progeny may at least in part underlie negative effects reported in some patients following embryonic neural cell grafts. Crucially, we show for the first time that MHC-II expression in developing human brains is not restricted to microglia as previously suggested, but is present in discrete subsets of neural progenitors and appears to be regulated independently of inflammatory stimuli.

Zinc Methionine Supplementation Impacts Gene and Protein Expression in Calf-Fed Holstein Steers with Minimal Impact on Feedlot Performance.

Providing cattle a more bioavailable zinc (Zn) source prior to administering a beta adrenergic agonist (βAA) may enhance the metabolic pool of primary nutrients that will influence the magnitude of the βAA response. Calf-fed Holstein steers were supplemented with a Zn methionine supplement (ZnMet; ZINPRO®; Zinpro Corporation, Eden Prairie, MN) for 115 ± 5 days prior to harvest along with zilpaterol hydrochloride (ZH; Zilmax®; Merck Animal Health, Summit, NJ) for the last 20 days with a 3-day withdrawal to evaluate the effects on growth and carcass performance together with gene and protein expression of skeletal muscle, adipose tissue, and fatty acid composition of polar and neutral lipid depots. Steers (n = 1296; initial weight = 468.5 ± 0.5 kg) were sorted by weight, blocked by harvest date, and randomly assigned to pens (n = 12) and treatments: control (90 ppm Zn from ZnSO4) and ZnMet (Control plus 720 mg Zn from ZnMet/hd/d). There were no differences (P > 0.05) in growth performance or carcass characteristics. The ZnMet-fed cattle had reduced (P < 0.05) abundance of myosin heavy chain (MHC)-IIX, β1-adrenergic receptor (βAR), peroxisome proliferator-activated receptor gamma, and stearoyl-CoA desaturase mRNA in skeletal muscle tissue. The ZnMet cattle had greater (P < 0.05) abundance of MHC-II protein, increased MHC-IIA and IIX cross-sectional areas (P < 0.05), an increased percentage of MHC-I fibers (P < 0.05), and a decreased percentage of MHC-IIX fibers (P < 0.05). The combination of ZnMet and ZH had positive biological effects on musculoskeletal tissue; however, these molecular effects were not significant enough to impact overall feedlot and carcass performance.

Captopril inhibits maturation of dendritic cells and maintains their tolerogenic property in atherosclerotic rats

Atherosclerosis (AS) is a systemic disease of the immune system featuring hyperactive dendritic cells (DCs) in atherosclerotic plaques and organs. Captopril, a representative medicine of angiotensin-converting enzyme inhibitors, has been demonstrated to be effective in treating AS. However, captopril's anti-atherosclerotic mechanism is still poorly understood. Therefore, this study was primarily performed to investigate the effects of captopril on the function of DCs in vivo. AS in rats was induced by feeding them with atherogenic diets, and it was evaluated by the levels of plasma lipids and aortic cholesterol. DCs' activity was appraised by endocytic activity, mixed lymphocyte reactions and cytokine secretion. The markers of DCs (CD103, CD80, CD86 and MHC-II) and Treg (CD4+, CD25+ and Foxp3+) were assayed by western blotting analysis and flow cytometry. Cytokine level was measured by an enzyme-linked immunosorbent assay. The results showed that captopril treatment (10, 20mg/kg/d) obviously improved dyslipidemia and reduced the levels of aortic cholesterol. Captopril significantly reduced CD103, CD80, CD86 and MHC-II protein expression while increasing that of Foxp3 in aortic tissue. Further study indicated oral administration of captopril up-regulated endocytic activity and reduced the immunostimulatory function of splenic DCs. Captopril treatment also promoted IL-10 & TGF-β production while decreasing that of IL-6 & IL-12 in splenic DCs. Finally, the results of flow cytometry indicated that captopril obviously inhibited DC maturation and promoted Treg polarization. Captopril treatment was able to inhibit DC maturation and maintain their tolerogenic property, which is closely associated with DC anti-atherosclerosis activity.

Visualizing Interactions Between HIV-1 Nef and Host Cellular Proteins Using Ground-State Depletion Microscopy.

The HIV-1 accessory protein Nef is essential for HIV disease progression and pathogenesis. A major Nef function is the downregulation of cell surface MHC-I, which contributes to the ability of HIV-1 to evade immune surveillance by cytotoxic T cells.1 This function is mediated by multiple Nef binding interfaces that interact with the cytosolic tail of MHC-I and multiple membrane trafficking regulator proteins, including the heterotetrameric adaptor protein 1 (AP-1) and phosphofurin acidic cluster sorting proteins 1 and 2 (PACS-1 and 2).1 Both AP-1 and PACS-1 have been demonstrated to mediate the trafficking of cargo proteins between early endocytic vesicles and the trans-Golgi network. However, the specific cellular compartments implicated in Nef-mediated trafficking of MHC-I away from the cell surface are largely undercharacterized. Our research program utilizes bimolecular fluorescence complementation (BiFc) to study the interaction between Nef and cellular binding partners. BiFc is a powerful tool for assessing the interaction of two protein-binding partners fused to nonfluorescent fragments of a split fluorescent protein, with interactions between the two proteins driving reconstitution of a functioning fluorophore.2 The BiFc fluorophore reconstitution occurs only when both proteins of interest are within 10 angstroms. Therefore, this technique permits the visualization of protein–protein interactions and the identification of distinct subcellular compartments where the interaction occurs. Indeed, we have previously used BiFc to demonstrate that Nef interacts with PACS-1 and PACS-2 at both early and late endosomes, and that this interaction is critical for MHC-I downregulation.3 In Fig. 1 we have combined BiFc with ground-state depletion superresolution microscopy (GSDM) to study the interaction between Nef and MHC-I. GSDM images are acquired by repeatedly imaging a sample while stochastically switching fluorophores in and out of a nonfluorescent dark state. By mapping the position of the active fluorophores at high precision in each acquisition, images with a resolution of up to 20 nm can be formed at 10 to 20 times better resolution than confocal microscopy.4 When combined with our recently developed algorithms, we can begin to spatially segregate protein interactions within the cell. Figure 1 demonstrates the interaction between Nef and MHC-I via BiFc, with GSDM determining that this interaction occurs partially within Rab5-positive early endosomes. This result is consistent with previous findings that Nef reroutes MHC-I from the cell surface through the endocytic network.3 This image illustrates for the first time that Nef and MHC-I interact along early endosomes and contributes to our understanding of the trafficking itinerary undertaken by MHC-I away from the cell surface. FIG. 1. Visualizing the Nef/MHC-I interaction along Rab5-positive endosomes. Here we imaged a HeLa cell utilizing ground-state depletion microscopy. Cells were cotransfected with plasmids expressing the HIV-1 accessory protein Nef and MHC-I fused to bimolecular ... In summary, by coupling the powerful techniques of BiFc and GSD microscopy, our research demonstrates a novel method to study HIV-1 Nef interactions at a per cell level. The analysis of such images will enable us to understand the location of specific protein complexes utilized by Nef to mediate MHC-I downregulation.

Regulation of the immunological synapse and T cell function by the c-terminal eps-15 homology domain-containing protein-dependent endocytic recycling. (IRM7P.711)

Abstract The immunological synapse (IS) is a specialized membrane domain with localized display of T-cell receptor (TCR) in contact with a similar display of peptide-loaded MHC on antigen presenting cells (APCs). Aberrant IS function is linked to ineffective tumor killing and autoimmunity. Accumulating evidence indicates a key role of endocytic traffic in the generation and maintenance of the IS. The C-terminal Eps-15 Homology Domain-containing proteins (EHD1-4) are essential regulators of endocytic recycling of surface receptors. Yet, nothing is known about their role in immune cell function. EHD-regulated receptors include the MHC-I and MHC-II proteins, and EHDs regulate vesicle budding at neural synapse. Our hypothesis is that EHD-dependent recycling of TCR and accessory receptors on T cells and MHC-II on APCs is required for the formation and stability of the IS. Indeed, using antigen-specific TCR transgenic CD4+ T-cells, we observe an accumulation of EHD proteins at the interface of T cells and APCs. CD4+ T-cells from floxed EHD1,3,4 mice bearing CD4-Cre and an antigen-specific TCR transgene demonstrate reduced antigen-driven proliferation. Further in vitro and in vivo analyses are being carried out to test the idea that endocytic recycling orchestrated by EHD proteins plays an important functional role in the formation and/or maintenance of IS and in T-cell function.

Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Inhibits Major Histocompatibility Complex Class II Expression by Disrupting Enhanceosome Assembly through Binding with the Regulatory Factor X Complex.

Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4(+) T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify a novel mechanism used by KSHV to downregulate the expressions of MHC-II genes. Kaposi's sarcoma-associated herpesvirus is the causative agent of multiple human malignancies. It establishes a lifelong latent infection and persists in infected cells without being detected by the host's immune surveillance system. Only a limited number of viral proteins are expressed during latency, and these proteins play a significant role in suppressing both the innate and adaptive immunities of the host. Latency-associated nuclear antigen (LANA) is one of the major proteins expressed during latent infection. Here, we show that LANA blocks MHC-II gene expression to subvert the host immune system by disrupting the MHC-II enhanceosome through binding with RFX transcription factors. Therefore, this study identifies a novel mechanism utilized by KSHV LANA to deregulate MHC-II gene expression, which is critical for CD4(+) T cell responses in order to escape host immune surveillance.

Human B Cell-Derived Lymphoblastoid Cell Lines Constitutively Produce Fas Ligand and Secrete MHCII(+)FasL(+) Killer Exosomes.

Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for immunotherapy in a variety of inflammatory contexts, including allogeneic transplantation. Exosomes containing the apoptosis-inducing molecule Fas ligand (FasL) have demonstrated efficacy in inhibiting antigen-specific immune responses upon adoptive transfer in animal models. We report here that a very high frequency of human B cell-derived lymphoblastoid cell lines (LCL) constitutively produce MHCII+FasL+ exosomes that can induce apoptosis in CD4+ T cells. All LCL tested for this study (>20 independent cell lines) showed robust expression of FasL, but had no detectable FasL on the cell surface. Given this intracellular sequestration, we hypothesized that FasL in LCL was retained in the secretory lysosome and secreted via exosomes. Indeed, we found both MHCII and FasL proteins present in LCL-derived exosomes, and using a bead-based exosome capture assay demonstrated the presence of MHCII+FasL+ exosomes among those secreted by LCL. Using two independent experimental approaches, we demonstrated that LCL-derived exosomes were capable of inducing antigen-specific apoptosis in autologous CD4+ T cells. These results suggest that LCL-derived exosomes may present a realistic source of immunosuppressive exosomes that could reduce or eliminate T cell-mediated responses against donor-derived antigens in transplant recipients.

Early changes in costameric and mitochondrial protein expression with unloading are muscle specific.

We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (−23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

Carrying Yourself: Self Antigen Composition of the Lymphatic Fluid

Advances in proteomics methodology and instrumentation have allowed detailed characterization of the composition of lymph. Far from being a simple ultrafiltrate of blood plasma, lymph has been shown to carry a rich repertoire of proteins and peptides reflecting the tissue of origin and its physiological state. Peptides derived from lymph can be loaded on the MHCII proteins, particularly those present on immature and/or inactivated antigen presenting cells, and may play an important role in maintenance of peripheral tolerance.

How does HLA-DM catalyze peptide exchange? Insights into the molecular mechanism of HLA-DM action from structural and biochemical studies (P5012)

Abstract Efficient antigen presentation in the class II MHC pathway requires the non-classical MHC-II protein HLA-DM to catalyze peptide exchange on classical MHC-II molecules. Recent crystal structures of HLA-DM bound to the peptide-loading inhibitor HLA-DO and to a tethered MHC-peptide complex have clarified the nature of the HLA-DM / MHC-II interaction and revealed substantial MHC-II conformational lability around the N-terminal side of the peptide binding site. However, the molecular mechanism by which HLA-DM binding to a MHC-peptide complex induces peptide release still is not clear, and several conflicting models have been proposed. We evaluated the roles of various MHC-peptide and MHC-DM interactions in equilibrium binding and peptide exchange kinetics studies. The results suggest a mechanism in which DM binding induces MHC conformational changes that result in loss of key peptide main-chain hydrogen bonding interactions in the vicinity of the P1 residue, and alteration of peptide side-chain interactions in the P1 pocket, leading to release of peptide and formation of a meta-stable DM-MHC complex. Peptide binding driven by interactions all along the binding site can reverse these conformational changes. Thus resistance to HLA-DM mediated peptide exchange and ultimately peptide immunogenicity is related to the strength of MHC-peptide interactions throughout binding site and not simply occupancy of the P1 pocket.

The relationship between structural/MHC changes in upper airway palatopharyngeal muscle morphology and obstructive sleep apnea/hypopnea syndrome

The aim of this study is to explore the relationship between structural/MHC changes in upper airway palatopharyngeal muscle morphology and obstructive sleep apnea/hypopnea syndrome. Palatopharyngeal muscle specimens were taken from 51 patients with obstructive sleep apnea hypopnea syndrome (OSAHS) who underwent uvulopalatopharyngoplasty (UPPP) resection. Patients were divided into light, medium and severe in terms of the severity of their OSAHS. There were 17 patients in each severity group. Palatopharyngeal muscle specimens were also taken from 17 patients suffering from chronic tonsillitis for comparison as the control group. All specimens were stained using Masson and observed for structural changes, especially in muscle fiber morphology, density and arrangement, as well as intermuscular connective tissues, under light microscopy. All specimens were also analyzed for MHC-I, MHC-IIa and MHC-IIb phenotype and protein expression differences using mRNA quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. The results from each group were then statistically analyzed using semi-quantitative analysis. Light microscopy with Masson staining revealed that in the control group, the muscle fibers are closely connected and arranged neatly. In specimens from patients suffering from OSAHS, the palatopharyngeal muscle fibers are larger with obvious hypertrophy and there was an increase in elastic fibers. The mucosal lamina propria was thickened, and the density of muscle fibers was reduced. Muscle fibers are not neatly arranged and degeneration was observed. The amount of muscular pathology and fibrosis corresponds to the severity of disease in the patients. In patients with severe OSAHS, the proportion of collagen to muscle fibers was increased significantly. Immunofluorescence results reveal that there were significantly more fast muscle fibers and less slow muscle fibers in the study group than the control group. mRNA quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed similar results, i.e., the proportion of MHC-II palatopharyngeal muscle fibers is higher in the study group than the control group, and increases with the severity of OSAHS. Pathological change occurs in both the collagen and muscle of OSAHS patients and corresponds to the degree of severity of OSAHS. Pathological change in palatopharyngeal muscle tissues is therefore, likely to be related to the occurrence and development of OSAHS. The increase in the proportion of the MHC-1I type fibers in OSAHS patients is likely to have an effect on the amount of airway support conferred by the muscle. This is likely the reason behind the lack of clinical improvement in some patients with severe OSAHS despite surgical treatment.

Immunohistochemical Detection of Dendritic Cell Markers in Cattle

Dendritic cells (DCs) are "professional" antigen-presenting cells with a critical role in the regulation of innate and adaptive immune responses and thus have been considered of great interest in the study of a variety of infectious diseases. The objective of this investigation was to characterize the in vivo distribution of DCs in bovine tissues by using potential DC markers to establish a basis for the study of DCs in diseased tissues. Markers evaluated included MHCII, CD208, CD1b, CD205, CNA.42, and S100 protein, the latter 2 being expressed by follicular dendritic cells whose origin and role are different from the rest of hematopoietic DCs. Paraffin wax-embedded tissues from 6 healthy Friesian calves were subjected to the avidin-biotin-peroxidase method, and the most appropriate fixatives, dilutions, and antigen retrieval pretreatments were studied for each of the primary antibodies. The most significant results included the localization of CD208-positive cells not only in the T zone of lymphoid organs but also within lymphoid follicles; CD1b-positive cells were mainly found in thymus and interfollicular areas of some lymph nodes; cells stained with anti-CD205 antibody were scarce, and their location was mainly in nonlymphoid tissues; and CNA.42- and S100 protein-positive cells localized in primary lymphoid follicles and light zones of germinal centers, although showing differences in the staining pattern. Furthermore, MHCII was established as one of the most sensitive markers for any DC of hematopoietic origin. These results increase our understanding of DC immunolabeling and will help in future DC studies of both healthy and diseased tissues.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb

The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.

Vav1 regulates MHCII expression in murine resting and activated B cells

Vav1 is a guanine nucleotide exchange factor (GEF) for Rho GTPases, which is exclusively expressed in cells of the hematopoietic system. In addition to its well-documented GEF activity, it was suggested to have other functions due to the presence of multiple domains and nuclear localization signals in its protein structure. Although GEF-dependent and GEF-independent functions of vav have been implicated in T-cell development and T-cell receptor signaling, the role of vav1 in antigen-presenting cells is poorly understood. We found that vav1 is an important regulator of MHCII expression and transport. Microarray analysis of unstimulated bone marrow-derived macrophages revealed a novel role of vav1 in transcriptional regulation of the MHCII locus, possibly by indirect means. Primary immune cells from vav1-deficient mice had a significantly lower constitutive surface expression of MHCII with the strongest impact observed on splenic and peritoneal B cells. Impaired MHCII expression resulted in a diminished capacity for T-cell activation. Using 6-thio-GTP, a specific inhibitor of the GEF function of vav1, we were able to show that the GEF activity is required for MHCII upregulation in B cells after stimulation with LPS. Furthermore, our data show that vav1 not only affects transcription of the MHCII locus but also is an important regulator of MHCII protein transport to the cell surface.

Modulation of the host Th1 immune response in pigeon protozoal encephalitis caused by Sarcocystis calchasi

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon (IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic phase of disease. Concomitantly, no cellular immune response was observed in histopathology while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor (TNF) α-related cytokines were significantly up-modulated, which correlated with a prominent MHC-II protein expression in areas of mononuclear cell infiltration and necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune cell response appears unrelated to the infectious dose, although the severity and onset of the central nervous signs clearly was dose-dependent. We identified no or only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the extensive cerebral lesions during the late neurological phase of disease.

Natural killer (NK)–dendritic cell interactions generate MHC class II-dressed NK cells that regulate CD4+T cells

Natural killer (NK) cells contribute to not only innate but also to adaptive immunity by interacting with dendritic cells (DCs) and T cells. All activated human NK cells express HLA-DR and can initiate MHCII-dependent CD4(+) T-cell proliferation; however, the expression of MHCII by mouse NK cells and its functional significance are controversial. In this study, we show that NK-DC interactions result in the emergence of MHCII-positive NK cells. Upon in vitro or in vivo activation, mouse conventional NK cells did not induce MHCII transcripts, but rapidly acquired MHCII protein from DCs. MHCII H2-Ab1-deficient NK cells turned I-A(b)-positive when adoptively transferred into wild-type mice or when cultured with WT splenic DCs. NK acquisition of MHCII was mediated by intercellular membrane transfer called "trogocytosis," but not upon DAP10/12- and MHCI-binding NK cell receptor signaling. MHCII-dressed NK cells concurrently acquired costimulatory molecules such as CD80 and CD86 from DCs; however, their expression did not reach functional levels. Therefore, MHCII-dressed NK cells inhibited DC-induced CD4(+) T-cell responses rather than activated CD4(+) T cells by competitive antigen presentation. In a mouse model for delayed-type hypersensitivity, adoptive transfer of MHCII-dressed NK cells attenuated footpad swelling. These results suggest that MHCII-dressed NK cells generated through NK-DC interactions regulate T cell-mediated immune responses.

A Single T Cell Receptor Bound to Major Histocompatibility Complex Class I and Class II Glycoproteins Reveals Switchable TCR Conformers

Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how Tcell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα β strand, switching Vα's position relative to Vβ. In several other structures, either Vα or Vβ undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.