αvß8 integrin-expression by BATF3-dependent dendritic cells facilitates early IgA responses to Rotavirus.

Secretory intestinal IgA can protect from re-infection with rotavirus (RV), but very little is known about the mechanisms that induce IgA production during intestinal virus infections. Classical dendritic cells (cDCs) in the intestine can facilitate both T cell-dependent and -independent secretory IgA. Here, we show that BATF3-dependent cDC1, but not cDC2, are critical for the optimal induction of RV-specific IgA responses in the mesenteric lymph nodes. This depends on the selective expression of the TGFß-activating integrin αvß8 by cDC1. In contrast, αvß8 on cDC1 is dispensible for steady state immune homeostasis. Given that cDC2 are crucial in driving IgA during steady state but are dispensable for RV-specific IgA responses, we propose that the capacity of DC subsets to induce intestinal IgA responses reflects the context, as opposed to an intrinsic property of individual DC subsets.

TGFßR signalling controls CD103+CD11b+ dendritic cell development in the intestine.

CD103+CD11b+ dendritic cells (DCs) are unique to the intestine, but the factors governing their differentiation are unclear. Here we show that transforming growth factor receptor 1 (TGFßR1) has an indispensable, cell intrinsic role in the development of these cells. Deletion of Tgfbr1 results in markedly fewer intestinal CD103+CD11b+ DCs and a reciprocal increase in the CD103-CD11b+ dendritic cell subset. Transcriptional profiling identifies markers that define the CD103+CD11b+ DC lineage, including CD101, TREM1 and Siglec-F, and shows that the absence of CD103+CD11b+ DCs in CD11c-Cre.Tgfbr1 fl/fl mice reflects defective differentiation from CD103-CD11b+ intermediaries, rather than an isolated loss of CD103 expression. The defect in CD103+CD11b+ DCs is accompanied by reduced generation of antigen-specific, inducible FoxP3+ regulatory T cells in vitro and in vivo, and by reduced numbers of endogenous Th17 cells in the intestinal mucosa. Thus, TGFßR1-mediated signalling may explain the tissue-specific development of these unique DCs.Developmental cues for the different dendritic cell (DC) subsets in the intestine are yet to be defined. Here the authors show that TGFßR1 signalling is needed for development of CD103+CD11b+ intestinal DCs from CD103-CD11b+ cells and that they contribute to the generation of Th17 and regulatory T cells.

Hydrodynamic gene delivery in human skin using a hollow microneedle device.

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVß and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50µL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVß reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery.

Tissue-specific differentiation of colonic macrophages requires TGFß receptor-mediated signaling.

Intestinal macrophages (mφ) form one of the largest populations of mφ in the body and are vital for the maintenance of gut homeostasis. They have several unique properties and are derived from local differentiation of classical Ly6Chi monocytes, but the factors driving this tissue-specific process are not understood. Here we have used global transcriptomic analysis to identify a unique homeostatic signature of mature colonic mφ that is acquired as they differentiate in the mucosa. By comparing the analogous monocyte differentiation process found in the dermis, we identify TGFß as an indispensable part of monocyte differentiation in the intestine and show that it enables mφ to adapt precisely to the requirements of their environment. Importantly, TGFßR signaling on mφ has a crucial role in regulating the accumulation of monocytes in the mucosa, via mechanisms that are distinct from those used by IL10.

Allergen-loaded strontium-doped hydroxyapatite spheres improve allergen-specific immunotherapy in mice.

BACKGROUND: Immunomodulatory interventions play a key role in the treatment of infections and cancer as well as allergic diseases. Adjuvants such as micro- and nanoparticles are often added to immunomodulatory therapies to enhance the triggered immune response. Here, we report the immunological assessment of novel and economically manufactured microparticle adjuvants, namely strontium-doped hydroxyapatite porous spheres (SHAS), which we suggest for the use as adjuvant and carrier in allergen-specific immunotherapy (ASIT). METHODS AND RESULTS: Scanning electron microscopy revealed that the synthesis procedure developed for the production of SHAS results in a highly homogeneous population of spheres. Strontium-doped hydroxyapatite porous spheres bound and released proteins such as ovalbumin (OVA) or the major cat allergen Fel d 1. SHAS-OVA were taken up by human monocyte-derived dendritic cells (mdDCs) and murine DCs and did not have any necrotic or apoptotic effects even at high densities. In a murine model of ASIT for allergic asthmatic inflammation, we found that OVA released from subcutaneously injected SHAS-OVA led to a sustained stimulation of both CD4+ and CD8+ T cells. Allergen-specific immunotherapy with SHAS-OVA as compared to soluble OVA resulted in similar humoral responses but in a higher efficacy as assessed by symptom scoring. CONCLUSION: We conclude that SHAS may constitute a suitable carrier and adjuvant for ASIT with great potential due to its unique protein-binding properties.

Exploitation of Langerhans cells for in vivo DNA vaccine delivery into the lymph nodes.

There is no clinically available cancer immunotherapy that exploits Langerhans cells (LCs), the epidermal precursors of dendritic cells (DCs) that are the natural agent of antigen delivery. We developed a DNA formulation with a polymer and obtained synthetic 'pathogen-like' nanoparticles that preferentially targeted LCs in epidermal cultures. These nanoparticles applied topically under a patch-elicited robust immune responses in human subjects. To demonstrate the mechanism of action of this novel vaccination strategy in live animals, we assembled a high-resolution two-photon laser scanning-microscope. Nanoparticles applied on the native skin poorly penetrated and poorly induced LC motility. The combination of nanoparticle administration and skin treatment was essential both for efficient loading the vaccine into the epidermis and for potent activation of the LCs to migrate into the lymph nodes. LCs in the epidermis picked up nanoparticles and accumulated them in the nuclear region demonstrating an effective nuclear DNA delivery in vivo. Tissue distribution studies revealed that the majority of the DNA was targeted to the lymph nodes. Preclinical toxicity of the LC-targeting DNA vaccine was limited to mild and transient local erythema caused by the skin treatment. This novel, clinically proven LC-targeting DNA vaccine platform technology broadens the options on DC-targeting vaccines to generate therapeutic immunity against cancer.

Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors.

Macrophages (mφ) are essential for intestinal homeostasis and the pathology of inflammatory bowel disease (IBD), but it is unclear whether discrete mφ populations carry out these distinct functions or if resident mφ change during inflammation. We show here that most resident mφ in resting mouse colon express very high levels of CX3CR1, are avidly phagocytic and MHCII(hi), but are resistant to Toll-like receptor (TLR) stimulation, produce interleukin 10 constitutively, and express CD163 and CD206. A smaller population of CX3CR1(int) cells is present in resting colon and it expands during experimental colitis. Ly6C(hi)CCR2(+) monocytes can give rise to all mφ subsets in both healthy and inflamed colon and we show that the CX3CR1(int) pool represents a continuum in which newly arrived, recently divided monocytes develop into resident CX3CR1(hi) mφ. This process is arrested during experimental colitis, resulting in the accumulation of TLR-responsive pro-inflammatory mφ. Phenotypic analysis of human intestinal mφ indicates that analogous processes occur in the normal and Crohn's disease ileum. These studies show for the first time that resident and inflammatory mφ in the intestine represent alternative differentiation outcomes of the same precursor and targeting these events could offer routes for therapeutic intervention in IBD.

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of alphaIIb/beta3.

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor gammadelta(+) gut intraepithelial lymphocytes.

CC chemokine receptor (CCR) 9, the receptor for the CC-chemokine CCL25/thymus-expressed chemokine (TECK), is mainly expressed by thymocytes and by intraepithelial (IEL) and lamina propria lymphocytes of the small intestine. To study the biologic role of CCR9, a mouse strain was generated in which the CCR9 gene was deleted. In spite of the high level of CCR9 found in double- and single-positive thymocytes and of the expression of its corresponding ligand on thymic stromal cells, CCR9 deletion had no major effect on intrathymic T-cell development. It was noted that there was only a one-day lag in the appearance of double-positive cells during fetal ontogeny in CCR9(-/-) thymi. When tested in chemotaxis assay, thymocytes isolated from CCR9(-/-) mice failed to respond to TECK/CCL25. Taken together, these results suggest that in thymocytes, CCR9 is the only physiologic receptor for TECK/CCL25, and that it is dispensable for proper T-cell development. Bone marrow pre-pro-B cells migrate in response to TECK/CCL25, but more mature B cells do not. Consistent with this observation, it was shown that there are fewer pre-pro-B cells in CCR9(-/-) mice than in wild-type mice. However, this diminution does not appear to have a detectable effect on the generation of a normal complement of mature B cells. Finally, it was shown that in the small intestine of CCR9-deficient mice, the intraepithelial T-cell-to-epithelial cell ratio is decreased, an observation that can be accounted for by a marked diminution of the T-cell receptor gammadelta(+) compartment.

The tight interallelic positional coincidence that distinguishes T-cell receptor Jalpha usage does not result from homologous chromosomal pairing during ValphaJalpha rearrangement.

The T-cell receptor (TCR) alpha locus is thought to undergo multiple cycles of secondary rearrangements that maximize the generation of alphabeta T cells. Taking advantage of the nucleotide sequence of the human Valpha and Jalpha segments, we undertook a locus-wide analysis of TCRalpha gene rearrangements in human alphabeta T-cell clones. In most clones, ValphaJalpha rearrangements occurred on both homologous chromosomes and, remarkably, resulted in the use of two neighboring Jalpha segments. No such interallelic coincidence was found for the position of the two rearranged Valpha segments, and there was only a loose correlation between the 5' or 3' chromosomal position of the Valpha and Jalpha segments used in a given rearrangement. These observations question the occurrence of extensive rounds of secondary Valpha-->Jalpha rearrangements and of a coordinated and polarized usage of the Valpha and Jalpha libraries. Fluorescence in situ hybridization analysis of developing T cells in which TCRalpha rearrangements are taking place showed that the interallelic positional coincidence in Jalpha usage cannot be explained by the stable juxtaposition of homologous Jalpha clusters.

T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase.

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.

The mouse and human IGSF6 (DORA) genes map to the inflammatory bowel disease 1 locus and are embedded in an intron of a gene of unknown function.

We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Crystal structure of a T cell receptor bound to an allogeneic MHC molecule.

Many T cell receptors (TCRs) that are selected to respond to foreign peptide antigens bound to self major histocompatibility complex (MHC) molecules are also reactive with allelic variants of self-MHC molecules. This property, termed alloreactivity, causes graft rejection and graft-versus-host disease. The structural features of alloreactivity have yet to be defined. We now present a basis for this cross-reactivity, elucidated by the crystal structure of a complex involving the BM3.3 TCR and a naturally processed octapeptide bound to the H-2Kb allogeneic MHC class I molecule. A distinguishing feature of this complex is that the eleven-residue-long complementarity-determining region 3 (CDR3) found in the BM3.3 TCR alpha chain folds away from the peptide binding groove and makes no contact with the bound peptide, the latter being exclusively contacted by the BM3.3 CDR3 beta. Our results formally establish that peptide-specific, alloreactive TCRs interact with allo-MHC in a register similar to the one they use to contact self-MHC molecules.

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9.

Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.

Relationships between natural T cells, atopy, IgE levels, and IL-4 production.

BACKGROUND: Th2 cells govern allergic disorders. Mechanisms leading to the Th2 commitment are dominated by the requirement of IL-4. A potential source of this triggering IL-4 could be the CD4 + subset of a small population of T cells, natural T (NT) cells. Indeed, this subset is involved in IgE responses in mice and produces promptly high amounts of IL-4 in both mice and man. METHODS: NT cells were identified in peripheral blood by flow cytometry with antibodies against Valpha24 and Vbeta11, recognizing the T-cell receptor specific for NT cells. Simultaneous staining with anti-CD3, anti-CD4, or anti-CD8 antibodies was performed. The frequency of NT cells in man was studied according to the presence of atopy defined by the positivity of skin tests, according to total IgE levels in serum, and according to IL-4 concentration of whole-blood culture supernatants determined by a flow cytometer microsphere-based assay. RESULTS: Seventy subjects were included, of whom 30 were atopic. The number of CD4+ NT cells was higher in atopics than in nonatopics (P=0.009). This number was correlated to the total IgE levels (r = 0.34, P = 0.03). In addition, the number of CD4 + NT cells, but also of CD8 + NT cells, was correlated to the levels of IL-4 (r=0.71, P=0.01, and r=0.6, P=0.03, respectively). CONCLUSIONS: These results show that the number of NT cells, particularly the CD4+ subset, is related to atopy, IL-4 production, and IgE levels. Therefore, this population of T cells is likely to play a role in the Th2 commitment initiating atopic diseases.