Marc-André Wurbel

Selective Generation of Gut Tropic T Cells in Gut-associated Lymphoid Tissue (GALT) Requirement for GALT Dendritic Cells and Adjuvant

In the current study, we address the underlying mechanism for the selective generation of gut-homing T cells in the gut-associated lymphoid tissues (GALT). We demonstrate that DCs in the GALT are unique in their capacity to establish T cell gut tropism but in vivo only confer this property to T cells in the presence of DC maturational stimuli, including toll-like receptor-dependent and -independent adjuvants. Thus, DCs from mesenteric LNs (MLNs), but not from spleen, supported expression of the chemokine receptor CCR9 and integrin α4β7 by activated CD8+ T cells. While DCs were also required for an efficient down-regulation of CD62L, this function was not restricted to MLN DCs. In an adoptive CD8+ T cell transfer model, antigen-specific T cells entering the small intestinal epithelium were homogeneously CCR9+α4β7 +CD62Llow, and this phenotype was only generated in GALT and in the presence of adjuvant. Consistent with the CCR9+ phenotype of the gut-homing T cells, CCR9 was found to play a critical role in the localization of T cells to the small intestinal epithelium. Together, these results demonstrate that GALT DCs and T cell expression of CCR9 play critical and integrated roles during T cell homing to the gut.

Chemokine Receptor CCR9 Contributes to the Localization of Plasma Cells to the Small Intestine

Humoral immunity in the gut-associated lymphoid tissue is characterized by the production of immunoglobulin A (IgA) by antibody-secreting plasma cells (PCs) in the lamina propria. The chemokine CCL25 is expressed by intestinal epithelial cells and is capable of inducing chemotaxis of IgA+ PCs in vitro. Using a newly generated monoclonal antibody against murine CCR9, we show that IgA+ PCs express high levels of CCR9 in the mesenteric lymph node (MLN) and Peyers patches (PPs), but down-regulate CCR9 once they are located in the small intestine. In CCR9-deficient mice, IgA+ PCs are substantially reduced in number in the lamina propria of the small intestine. In adoptive transfer experiments, CCR9-deficient IgA+ PCs show reduced migration into the small intestine compared with wild-type controls. Furthermore, CCR9 mutants fail to mount a regular IgA response to an orally administered antigen, although the architecture and cell type composition of PPs and MLN are unaffected and are functional for the generation of IgA PCs. These findings provide profound in vivo evidence that CCL25/CCR9 guides PCs into the small intestine.

Cutting Edge: Chemokine Receptor CCR4 Is Necessary for Antigen-Driven Cutaneous Accumulation of CD4 T Cells under Physiological Conditions

Dual expression of chemokine receptor CCR4 and E-selectin ligand is characteristic of skin-tropic CD4 T cells from blood, lymphoid organs, and the skin itself. A strong and specific correlation exists among CCR4, its ligand CCL17/TARC, and the cutaneous lymphocyte-homing process. Nevertheless, whether CCR4 function is required for skin-specific trafficking remains an open question, which we address in this study. We developed an Ag-specific, TCR-transgenic, murine CD4 T cell adoptive transfer model that induces a mixed Th1 and Th17 cutaneous response. Within the hosts, both CCR4+/+ and CCR4−/− donor CD4 T cells contribute equally well to the circulating E-selectin ligand+ pool in response to Ag. However, only CCR4+/+ donor cells accumulate efficiently within the skin. CCR4−/− cells home normally to the peritoneum, showing that they do not have a general defect in lymphocyte trafficking. We conclude that under physiological conditions, CCR4 is a nonredundant, necessary component of skin-specific lymphocyte trafficking.

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis.

Background & Aims CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine. Methods Acute inflammation and recovery in wild-type (WT) and CCR9−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level. Results CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production. Conclusions Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.

Impaired Accumulation of Antigen-Specific CD8 Lymphocytes in Chemokine CCL25-Deficient Intestinal Epithelium and Lamina Propria

CCL25 and CCR9 constitute a chemokine/receptor pair involved in T cell development and in gut-associated immune responses. In this study, we generated CCL25−/− mice to answer questions that could not be addressed with existing CCR9−/− mice. Similar phenotypes were observed for both CCL25−/− and CCR9−/− mice, consistent with the notion that CCL25 and CCR9 interact with each other exclusively. We assessed the requirement for CCL25 in generating CCR9high CD8 intestinal memory-phenotype T cells and the subsequent accumulation of these cells within effector sites. TCR-transgenic naive CD8 T cells were transferred into wild-type or CCL25-deficient hosts. Oral sensitization with Ag allowed these naive donor cells to efficiently differentiate into CCR9high memory-phenotype cells within the mesenteric lymph nodes of wild-type hosts. This differentiation event occurred with equal efficiency in the MLN of CCL25-deficient hosts, demonstrating that CCL25 is not required to induce the CCR9high memory phenotype in vivo. However, we found that CCL25 deficiency severely impaired the Ag-dependent accumulation of donor-derived CD8 T cells within both lamina propria and epithelium of the small intestine. Thus, although CCL25 is not necessary for generating memory-phenotype CD8 T cells with “gut-homing” properties, this chemokine is indispensable for their trafficking to the small intestine.

A role for CCR4 in development of mature circulating cutaneous T helper memory cell populations.

Expression of the chemokine receptor CCR4 is strongly associated with trafficking of specialized cutaneous memory T helper (Th) lymphocytes to the skin. However, it is unknown whether CCR4 itself participates in the development of cutaneous Th populations. We have addressed this issue via competitive bone marrow (BM) reconstitution assays; equal numbers of BM cells from CCR4+/+ and CCR4−/− donors were allowed to develop side-by-side within RAG-1−/− hosts. Cells from both donor types developed equally well into B cells, naive CD8 T cells, naive CD4 T cells, interferon-γ+ Th1 cells, and interleukin-4+ Th2 cells. In marked contrast, circulating cutaneous memory Th cells (i.e., E-selectin ligand+ [E-lig+]) were more than fourfold more likely to be derived from CCR4+/+ donors than from CCR4−/− donors. Most of this effect resides within the CD103+ subset of the E-lig+ Th population, in which donor CCR4+/+ cells can outnumber CCR4−/− cells by >12-fold. No similar effect was observed for α4β7+ intestinal memory Th cells or CD103+/E-lig− Th cells. We conclude that CCR4 expression provides a competitive advantage to cutaneous Th cells, either by participating in their development from naive Th cells, or by preferentially maintaining them within the memory population over time.

Complex regulation of CCR9 at multiple discrete stages of T cell development

We have conducted a comprehensive assessment of CCR9 expression and function at the important milestone stages of murine thymocyte development. We reveal an unusually complex regulatory pattern, in which CCR9 influences T cell development at several widely dispersed stages. We find that CCR9 is not expressed within the thymus until the double‐negative (DN)3 stage, although it appears to contribute to T cell precursor development prior to residence in the thymus. CCR9 expression is influenced by pre‐T cell receptor signals, and is dramatically up‐regulated in a population that appears to be transitional between the DN4 and double‐positive stages. In the periphery, functional CCR9 is expressed by all naive CD8 T cells, but not by naive CD4 T cells. To our knowledge, this latter finding is the first difference observed in homing receptor expression between naive lymphocyte populations. This suggests that naive CD8 T cells might have access to lymphoid microenvironments from which naive CD4 T cells are excluded.

Epicutaneous challenge of orally immunized mice redirects antigen-specific gut-homing T cells to the skin

Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4-/- mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4-/- mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.

iNKT Cells Require CCR4 to Localize to the Airways and to Induce Airway Hyperreactivity

iNKT cells are required for the induction of airway hyperreactivity (AHR), a cardinal feature of asthma, but how iNKT cells traffic to the lungs to induce AHR has not been previously studied. Using several models of asthma, we demonstrated that iNKT cells required the chemokine receptor CCR4 for pulmonary localization and for the induction of AHR. In both allergen-induced and glycolipid-induced models of AHR, wild-type but not CCR4−/− mice developed AHR. Furthermore, adoptive transfer of wild-type but not CCR4−/− iNKT cells reconstituted AHR in iNKT cell-deficient mice. Moreover, we specifically tracked CCR4−/− vs wild-type iNKT cells in CCR4−/−:wild-type mixed BM chimeric mice in the resting state, and when AHR was induced by protein allergen or glycolipid. Using this unique model, we showed that both iNKT cells and conventional T cells required CCR4 for competitive localization into the bronchoalveolar lavage/airways compartment. These results establish for the first time that the pulmonary localization of iNKT cells critical for the induction of AHR requires CCR4 expression by iNKT cells.

Maintenance of Peripheral Tolerance through Controlled Tissue Homing of Antigen-Specific T Cells in K14-mOVA Mice

Immunological tolerance is crucial to avoid autoimmune and inflammatory diseases; however, the mechanisms involved are incompletely understood. To study peripheral tolerance to skin-associated Ags, we generated new transgenic mice expressing a membrane-bound form of OVA in skin under the human keratin 14 (K14) promoter (K14-mOVA mice). In contrast to other transgenic mice expressing similar self-Ags in skin, adoptive transfer of Ag-specific T cells does not induce inflammatory skin disease in our K14-mOVA mice. OVA-specific T cells transferred into K14-mOVA mice are activated in lymphoid tissues, undergo clonal expansion, and eventually acquire effector function. Importantly, these Ag-specific T cells selectively up-regulate expression of E-selectin ligand in cutaneous lymph nodes but not in mesenteric lymph nodes and spleen, demonstrating that expression of endogenous self-Ags in skin dictates imprinting of skin tissue homing in vivo. However, an additional inflammatory signal, here induced by tape stripping, is required in K14-mOVA mice to induce T cell migration to skin and development of inflammatory skin disease. Depletion of regulatory CD4+CD25+ T cells did not provoke homing of transferred T cells to skin under steady-state conditions, indicating that these cells are not the key regulators for inhibiting T cell homing in K14-mOVA mice. Both skin-derived and lymph node-resident CD8α+ dendritic cells are responsible for Ag presentation in vivo and induce tolerance to skin Ags, as we show by selective depletion of langerin+ and CD11c+ dendritic cells. Taken together, controlled skin homing of T cells is critical for the maintenance of peripheral immune tolerance to epidermal self-Ags.