Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry. This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor. While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents. Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection. Mononuclear cells are purified from peripheral blood by Ficoll underlay. CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads. Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2. The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25. After labeling, the cells can be directly analyzed by flow cytometry. The tetramer positive cells typically form a distinct population among the expanded CD4+ cells. Tetramer positive cells are usually CD25+ and often CD4 high. Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer. Multiple variations of this basic assay are possible. Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays. Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies. Open in a separate windowClick here to view.^{(85M, flv)}     

Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.     

Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo

Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors.     

Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis

Background

During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite.

Methods and Findings

We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-α expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression.

Conclusion

These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host''s response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.     

Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.     

Metabolic Responses of Primary and Transformed Cells to Intracellular Listeria monocytogenes

The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using ¹³C-labelled glucose or glutamine as carbon tracers. The ¹³C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.     

Short-Lived and Phosphorylated Proteins Contribute to Carrier-Mediated Efflux, but Not to Influx, of Auxin in Suspension-Cultured Tobacco Cells

Auxin is transported across the plasma membrane of plant cells by diffusion and by two carriers operating in opposite directions, the influx and efflux carriers. Both carriers most likely play an important role in controlling auxin concentration and distribution in plants but little is known regarding their regulation. We describe the influence of modifications of the transmembrane pH gradient and the effect of agents interfering with protein synthesis, protein traffic, and protein phosphorylation on the activity of the auxin carriers in suspension-cultured tobacco (Nicotiana tabacum L.) cells. Carrier-mediated influx and efflux were monitored independently by measuring the accumulation of [¹⁴C]2,4-dichlorophenoxyacetic acid and [³H]naphthylacetic acid, respectively. The activity of the influx carrier decreased on increasing external pH and on decreasing internal pH, whereas that of the efflux carrier was only impaired on internal acidification. The efflux carrier activity was inhibited by cycloheximide, brefeldin A, and the protein kinase inhibitors staurosporine and K252a, as shown by the increased capability of treated cells to accumulate [³H]naphthylacetic acid. Kinetics and reversibility of the effect of brefeldin A were consistent with one or several components of the efflux system being turned over at the plasma membrane with a half-time of less than 10 min. Inhibition of efflux by protein kinase inhibitors suggested that protein phosphorylation was essential to sustain the activity of the efflux carrier. On the contrary, the pharmacological agents used in this study failed to inhibit [¹⁴C]2,4-dichlorophenoxyacetic acid accumulation, suggesting that rapidly turned-over proteins or proteins activated by phosphorylation are not essential to carrier-mediated auxin influx. Our data support the idea that the efflux carrier in plants constitutes a complex system regulated at multiple levels, in marked contrast with the influx carrier. Physiological implications of the kinetic features of this regulation are discussed.     

Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific,Autoreactive CD4 T Cells In Situ

We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139–151; the sections were costained with IA^s/PLP 139–151 (specific) or Theiler''s murine encephalomyelitis virus (TMEV) 70–86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139–151 but not for TMEV 70–86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139–151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer⁺ CD4⁺ T cells in the ‘Z’ serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334–352. Heart sections prepared from immunized mice were costained with Myhc 334–352 (specific) or bovine ribonuclease 43–56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.     

MHC II in Dendritic Cells is Targeted to Lysosomes or T Cell-Induced Exosomes Via Distinct Multivesicular Body Pathways

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.     

ZBTB32 Is an Early Repressor of the CIITA and MHC Class II Gene Expression during B Cell Differentiation to Plasma Cells

CIITA and MHC class II expression is silenced during the differentiation of B cells to plasma cells. When B cell differentiation is carried out ex vivo, CIITA silencing occurs rapidly, but the factors contributing to this event are not known. ZBTB32, also known as repressor of GATA3, was identified as an early repressor of CIITA in an ex vivo plasma cell differentiation model. ZBTB32 activity occurred at a time when B lymphocyte-induced maturation protein-1 (Blimp-1), the regulator of plasma cell fate and suppressor of CIITA, was minimally induced. Ectopic expression of ZBTB32 suppressed CIITA and I-A gene expression in B cells. Short hairpin RNA depletion of ZBTB32 in a plasma cell line resulted in re-expression of CIITA and I-A. Compared with conditional Blimp-1 knockout and wild-type B cells, B cells from ZBTB32/ROG-knockout mice displayed delayed kinetics in silencing CIITA during ex vivo plasma cell differentiation. ZBTB32 was found to bind to the CIITA gene, suggesting that ZBTB32 directly regulates CIITA. Lastly, ZBTB32 and Blimp-1 coimmunoprecipitated, suggesting that the two repressors may ultimately function together to silence CIITA expression. These results introduce ZBTB32 as a novel regulator of MHC-II gene expression and a potential regulatory partner of Blimp-1 in repressing gene expression.     

Focus: Allergic Diseases and Type II Immunity: Mast Cells as Regulators of Adaptive Immune Responses in Food Allergy

Mast cells are a critical first line of defense against endogenous and environmental threats. Their participation in innate immunity is well characterized; activation of toll like receptors as well as receptors for complement, adenosine, and a host of other ligands leads to mast cell release of preformed mediators contained within granules along with newly synthesized arachidonic acid metabolites, cytokines, and chemokines. These confer protective effects including the induction of mucus secretion, smooth muscle contraction, and activation of common itch and pain sensations, all of which act to promote expulsion of noxious agents. While their innate immune role as sentinel cells is well established, recent research has brought into focus their separate but also critical function in adaptive immunity particularly in the setting of IgE mediated food allergies. Crosslinking of FcεR1, the high affinity receptor for IgE, when bound to IgE and antigen, triggers the release of the same factors and elicits the same physiologic responses that occur after activation by innate stimuli. Though IgE-activated mast cells are best known for their role in acute allergic reactions, including the most severe manifestation, anaphylaxis, accumulating evidence has suggested an immunoregulatory effect in T cell-mediated immunity, modulating the balance between type 2 immunity and tolerance. In this review, we outline how mast cells act as adjuvants for food antigen driven Th2 cell responses, while curtailing Treg function.     

Modulation of Cytokine Secretion Allows CD4 T Cells Secreting IL-10 and IL-17 to Simultaneously Participate in Maintaining Tolerance and Immunity

CD4 T cells secreting IL-10 or IL-17 are frequent at mucosal sites, where their equilibrium is important for simultaneously maintaining tolerance and immunity to the resident microbiota. The mode of action of these cells, however, is as yet incompletely understood. In this study, we have combined ex vivo analysis of CD4 T cells producing IL-10 or/and IL-17 with assessment of clonal populations isolated ex vivo using a cytokine catch assay. We found that circulating CD4 T cells secreting IL-10 or/and IL-17 ex vivo include both conventional FOXP3^- CD4 T cells and FOXP3⁺ Helios^- Treg. Upon assessment of clonal populations derived from single ex vivo isolated cytokine secreting cells, we found that IL-10 or/and IL-17 secreting cells prevalently secrete one or the other cytokine depending on the type of stimulation, the time after stimulation and the presence of microbial products. Namely, IL-10 secretion by clonal cells was prevalent at early time points after TCR mediated stimulation, was independent of co-stimulation and was increased in the presence of the microbial fermentation product butyrate. In contrast, IL-17 secretion was higher at later time points after TCR mediated stimulation and in the presence of co-stimulatory signals. Taken together, these results provide insights into the mechanisms that, through modulation of cytokine secretion depending on conditions, allow IL-10 and IL-17 producing CD4 T cells to contribute to maintain tolerance to microbes locally, while retaining the ability to participate in protective immune responses at distant sites.     

All β Cells Contribute Equally to Islet Growth and Maintenance

In healthy adult mice, the β cell population is not maintained by stem cells but instead by the replication of differentiated β cells. It is not known, however, whether all β cells contribute equally to growth and maintenance, as it may be that some cells replicate while others do not. Understanding precisely which cells are responsible for β cell replication will inform attempts to expand β cells in vitro, a potential source for cell replacement therapy to treat diabetes. Two experiments were performed to address this issue. First, the level of fluorescence generated by a pulse of histone 2B–green fluorescent protein (H2BGFP) expression was followed over time to determine how this marker is diluted with cell division; a uniform loss of label across the entire β cell population was observed. Second, clonal analysis of dividing β cells was completed; all clones were of comparable size. These results support the conclusion that the β cell pool is homogeneous with respect to replicative capacity and suggest that all β cells are candidates for in vitro expansion. Given similar observations in the hepatocyte population, we speculate that for tissues lacking an adult stem cell, they are replenished equally by replication of all differentiated cells.     

IL-17A and IL-2-Expanded Regulatory T Cells Cooperate to Inhibit Th1-Mediated Rejection of MHC II Disparate Skin Grafts

Several evidences suggest that regulatory T cells (Treg) promote Th17 differentiation. Based on this hypothesis, we tested the effect of IL-17A neutralization in a model of skin transplantation in which long-term graft survival depends on a strong in vivo Treg expansion induced by transient exogenous IL-2 administration. As expected, IL-2 supplementation prevented rejection of MHC class II disparate skin allografts but, surprisingly, not in IL-17A-deficient recipients. We attested that IL-17A was not required for IL-2-mediated Treg expansion, intragraft recruitment or suppressive capacities. Instead, IL-17A prevented allograft rejection by inhibiting Th1 alloreactivity independently of Tregs. Indeed, T-bet expression of naive alloreactive CD4+ T cells and the subsequent Th1 immune response was significantly enhanced in IL-17A deficient mice. Our results illustrate for the first time a protective role of IL-17A in CD4+-mediated allograft rejection process.     

Age-Associated Differences in MiRNA Signatures Are Restricted to CD45RO Negative T Cells and Are Associated with Changes in the Cellular Composition,Activation and Cellular Ageing

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- T_EMRA compared to CD45RO-CCR7+ T_NAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic T_NAIVE cells as compared to thymic CD45RO-CD31+ T_NAIVE cells. Upon activation of CD45RO- T_NAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ T_EMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.     

Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells.

Phagocytic processing of heat-killed Listeria monocytogenes by peritoneal macrophages resulted in degradation of these bacteria in phagolysosomal compartments and processing of bacterial antigens for presentation to T cells by class II MHC molecules. Within 20 min of uptake by macrophages, Listeria peptide antigens were expressed on surface class II MHC molecules, capable of stimulating Listeria-specific T cells. Within this period, degradation of labeled bacteria to acid-soluble low molecular weight catabolites also commenced. Immunoelectron microscopy was used to evaluate the compartments involved in this processing. Upon uptake of the bacteria, phagosomes containing Listeria fused rapidly with both lysosomes and endosomes. Class II MHC molecules were present in a tubulo-vesicular lysosome compartment, which appeared to fuse with phagosomes, as well as in the resulting phagolysosomes containing internalized Listeria; these compartments were all positive for Lamp 1 and cathepsin D and lacked 46-kD mannose-6-phosphate receptors. In addition, class II MHC and Lamp 1 were co-localized in vesicles of the trans Golgi reticulum, where they were segregated from 46-kD mannose-6-phosphate receptors. Vesicles containing both Listeria-derived components and class II MHC molecules were also observed; some of these may represent vesicles recycling from phagolysosomes, potentially bearing processed immunogenic peptides complexed with class II MHC. These results support a central role for lysosomes and phagolysosomes in the processing of bacterial antigens for presentation to T cells. Tubulo-vesicular lysosomes appear to represent an important convergence of endocytic, phagocytic and biosynthetic pathways, where antigens may be processed to allow binding to class II MHC molecules and recycling to the cell surface.