Ronald N. Germain

House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells

Barrier epithelial cells and airway dendritic cells (DCs) make up the first line of defense against inhaled substances such as house dust mite (HDM) allergen and endotoxin (lipopolysaccharide, LPS). We hypothesized that these cells need to communicate with each other to cause allergic disease. We show in irradiated chimeric mice that Toll-like receptor 4 (TLR4) expression on radioresistant lung structural cells, but not on DCs, is necessary and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony–stimulating factor, interleukin-25 and interleukin-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM-driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma, including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.

Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody.

CD8+ T lymphocytes recognize antigens as short peptides bound to MHC class I molecules. Available methods cannot determine the number and distribution of these ligands on individual cells or detect antigen-presenting cells in tissues. Here we describe a method for eliciting and identifying monoclonal antibodies specific for a particular peptide-MHC class I combination. One such antibody can identify antigen complexes with a limit of detection approaching that of T cells. We used this antibody to determine the number of peptide-class I complexes generated upon viral infection, to identify antigen-presenting cells in cell mixtures, to determine the site of peptide-MHC class I interaction inside cells, and to visualize cells bearing specific peptide-MHC class I complexes after in vivo infection. Similar antibodies may prove useful for diagnostic or therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement

Cells lining the gastrointestinal tract serve as both a barrier to and a pathway for infectious agent entry. Dendritic cells (DCs) present in the lamina propria under the columnar villus epithelium of the small bowel extend processes across this epithelium and capture bacteria, but previous studies provided limited information on the nature of the stimuli, receptors, and signaling events involved in promoting this phenomenon. Here, we use immunohistochemical as well as dynamic explant and intravital two-photon imaging to investigate this issue. Analysis of CD11c–enhanced green fluorescent protein (EGFP) or major histocompatibility complex CII-EGFP mice revealed that the number of trans-epithelial DC extensions, many with an unusual “balloon” shape, varies along the length of the small bowel. High numbers of such extensions were found in the proximal jejunum, but only a few were present in the terminal ileum. The extensions in the terminal ileum markedly increased upon the introduction of invasive or noninvasive Salmonella organisms, and chimeric mouse studies revealed the key role of MyD88-dependent Toll-like receptor (TLR) signaling by nonhematopoietic (epithelial) elements in the DC extension response. Collectively, these findings support a model in which epithelial cell TLR signaling upon exposure to microbial stimuli induces active DC sampling of the gut lumen at sites distant from organized lymphoid tissues.

Chemokines enhance immunity by guiding naive CD8 + T cells to sites of CD4 + T cell–dendritic cell interaction

CD8+ T cells have a crucial role in resistance to pathogens and can kill malignant cells; however, some critical functions of these lymphocytes depend on helper activity provided by a distinct population of CD4+ T cells. Cooperation between these lymphocyte subsets involves recognition of antigens co-presented by the same dendritic cell, but the frequencies of such antigen-bearing cells early in an infection and of the relevant naive T cells are both low. This suggests that an active mechanism facilitates the necessary cell–cell associations. Here we demonstrate that after immunization but before antigen recognition, naive CD8+ T cells in immunogen-draining lymph nodes upregulate the chemokine receptor CCR5, permitting these cells to be attracted to sites of antigen-specific dendritic cell–CD4+ T cell interaction where the cognate chemokines CCL3 and CCL4 (also known as MIP-1α and MIP-1β) are produced. Interference with this actively guided recruitment markedly reduces the ability of CD4+ T cells to promote memory CD8+ T-cell generation, indicating that an orchestrated series of differentiation events drives nonrandom cell–cell interactions within lymph nodes, optimizing CD8+ T-cell immune responses involving the few antigen-specific precursors present in the naive repertoire.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists

Small changes in the peptide-major histocompatibility complex (MHC) molecule ligands recognized by antigen-specific T cell receptors (TCRs) can convert fully activating complexes into partially activating or even inhibitory ones. This study examined early TCR-dependent signals induced by such partial agonists or antagonists. In contrast to typical agonist ligands, both an antagonist and several partial agonists stimulated a distinct pattern of zeta chain phosphorylation and failed to activate associated ZAP-70 kinase. These results identify a specific step in the early tyrosine phosphorylation cascade that is altered after TCR engagement with modified peptide-MHC molecule complexes. This finding may explain the different biological responses to TCR occupancy by these variant ligands.

SAP-controlled T-B cell interactions underlie germinal centre formation

Generation of long-term antibody-mediated immunity depends on the germinal centre reaction, which requires cooperation between antigen-specific T and B lymphocytes. In human X-linked lymphoproliferative disease and its gene-targeted mouse model, loss-of-function mutations in signalling lymphocyte activation molecule-associated protein (SAP, encoded by SH2D1a) cause a profound defect in germinal centre formation by an as yet unknown mechanism. Here, using two-photon intravital imaging, we show that SAP deficiency selectively impairs the ability of CD4+ T cells to stably interact with cognate B cells but not antigen-presenting dendritic cells. This selective defect results in a failure of antigen-specific B cells to receive adequate levels of contact-dependent T-cell help to expand normally, despite Sap-/- T cells exhibiting the known characteristics of otherwise competent helper T cells. Furthermore, the lack of stable interactions with B cells renders Sap-/- T cells unable to be efficiently recruited to and retained in a nascent germinal centre to sustain the germinal centre reaction. These results offer an explanation for the germinal centre defect due to SAP deficiency and provide new insights into the bi-directional communication between cognate T and B cells in vivo.

Extrafollicular Activation of Lymph Node B Cells by Antigen-Bearing Dendritic Cells

In contrast to naïve T cells that recognize short antigen-derived peptides displayed by specialized antigen-presenting cells, immunoglobulin receptors of B lymphocytes primarily recognize intact proteins. How and where within a lymph node such unprocessed antigens become available for naïve B cell recognition is not clear. We used two-photon intravital imaging to show that, after exiting high-endothelial venules and before entry into lymph node follicles, B cells survey locally concentrated dendritic cells. Engagement of the B cell receptor by the dendritic cell (DC)–associated antigen leads to lymphocyte calcium signaling, migration arrest, antigen acquisition, and extrafollicular accumulation. These findings suggest a possible role for antigen-specific B-DC interactions in promoting T cell–dependent antibody responses in vivo.

Constitutive Presentation of a Natural Tissue Autoantigen Exclusively by Dendritic Cells in the Draining Lymph Node

The major histocompatibility complex (MHC)-dependent presentation of processed tissue-specific self-antigens can contribute to either peripheral (extrathymic) tolerance or the differentiation of autoreactive T cells. Here, we have studied the MHC class II molecule presentation of gastric parietal cell (PC)-specific H+/K+-ATPase, which induces a destructive autoimmune gastritis in BALB/c mice lacking CD4+ CD25+ regulatory T cells. Immunofluorescence microscopy showed physical association of CD11c+ dendritic cells (DCs) with PCs in the gastric mucosa. H+/K+-ATPase protein was found within vesicular compartments of a few CD11c+ DCs only in the draining gastric lymph node (LN) and these antigen-containing DCs increased markedly in number with the onset of tissue destruction in autoimmune animals. Both CD8αhi and CD8αlo gastric DCs, but not peripheral or mesenteric DCs, showed evidence of constitutive in vivo processing and presentation of H+/K+-ATPase. These data provide direct support for a widely held model of local tissue antigen uptake and trafficking by DCs in normal animals and demonstrate that DCs in the draining LN can present a tissue-specific self-antigen under noninflammatory conditions without fully deleting autoreactive T cells or inducing active autoimmunity.

TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways

Functional discrimination between structurally similar self and foreign antigens is a main attribute of adaptive immunity. Here we describe two feedback mechanisms in T lymphocytes that together sharpen and amplify initial signaling differences related to the quality of T cell receptor (TCR) engagement. Weakly binding ligands predominantly trigger a negative feedback loop leading to rapid recruitment of the tyrosine phosphatase SHP-1, followed by receptor desensitization through inactivation of Lck kinase. In contrast, strongly binding ligands efficiently activate a positive feedback circuit involving Lck modification by ERK, preventing SHP-1 recruitment and allowing the long-lasting signaling necessary for gene activation. The characteristics of these pathways suggest that they constitute an important part of the mechanism allowing T cells to discriminate between self and foreign ligands.