Cutting Edge: CCR8 Signaling Regulates IL-25- and IL-33-Responsive Skin Group 2 Innate Lymphoid Cell Migration and Function.

Group 2 innate lymphoid cells (ILC2s) are sentinels of barrier immunity, and their activation by the epithelial alarmins IL-25 and IL-33 is a defining trait. In this study, we identified a role for the chemokine receptor CCR8 in modulating skin ILC2 abundance and activation. CCR8 signaling facilitated IL-25-induced increases in skin and lung ILC2s, ILC2 activation and systemic IL-13 production, and ligand-directed ILC2 entry into skin and lung. CCR8 controlled ILC2 tissue entry in IL-25-treated naive mice, but only transferred bone marrow ILC2 progenitors were equipped to enter the skin, whereas multiple tissue-sourced ILC2s entered the lung. CCR8 selectively regulated IL-33-induced increases in skin ILC2s, their proliferation, and production of IL-13/IL-5, as well as IL-33-responsive transferred ILC2 trafficking only to the skin. Collectively, we illuminate (to our knowledge) novel aspects of CCR8 signaling-regulated ILC2 motility and function, especially in the skin, in response to two hallmark ILC2-activating alarmins.

Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells.

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.

The transcriptional repressor BLIMP1 curbs host defenses by suppressing expression of the chemokine CCL8.

The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.

T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation.

Limb transplantation is a life-changing procedure for amputees. However, limb recipients have a 6-fold greater rejection rate than solid organ transplant recipients, related in part to greater immunogenicity of the skin. Here, we report a detailed immunological and molecular characterization of individuals who underwent bilateral limb transplantation at our institution. Circulating Th17 cells are increased in limb transplant recipients over time. Molecular characterization of 770 genes in skin biopsies reveals upregulation of T cell effector immune molecules and chemokines, particularly CCL18. Skin antigen-presenting cells primarily express the chemokine CCL18, which binds to the CCR8 receptor. CCL18 treatment recruits more allo-T cells to the skin xenograft in a humanized skin transplantation model, leading to signs of accelerated graft rejection. Blockade of CCR8 remarkedly decreases CCL18-induced allo-T cell infiltration. Our results suggest that targeting the CCL18:CCR8 pathway could be a promising immunosuppressive approach in transplantation.

BLT1-mediated T cell trafficking is critical for rejection and obliterative bronchiolitis after lung transplantation.

Leukotriene B4 is a lipid mediator that recently has been shown to have potent chemotactic activity for effector T lymphocytes mediated through its receptor, BLT1. Here, we developed a novel murine model of acute lung rejection to demonstrate that BLT1 controls effector CD8+ T cell trafficking into the lung and that disruption of BLT1 signaling in CD8+ T cells reduces lung inflammation and mortality in the model. In addition, we used BLT1-deficient mice and a BLT1 antagonist in two tracheal transplant models of lung transplantation to demonstrate the importance of BLT1 for the recruitment of T cells into tracheal allografts. We also show that BLT1-mediated CD8+ T cell recruitment plays an important role in the development of airway fibroproliferation and obliteration. Finally, in human studies of lung transplant recipients, we found that BLT1 is up-regulated on T lymphocytes isolated from the airways of patients with obliterative bronchiolitis. These data demonstrate that BLT1 contributes to the development of lung rejection and obliterative bronchiolitis by mediating effector T lymphocyte trafficking into the lung. This is the first report that describes a pathologic role for BLT1-mediated T lymphocyte recruitment in disease and identifies BLT1 as a potential therapeutic target after lung transplantation.

CCR5 is essential for NK cell trafficking and host survival following Toxoplasma gondii infection.

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5-/-) mice were less able to generate an inflammatory response, had decreased chemokine and interferon gamma production, and had higher parasite burden. As a result, CCR5-/- mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5+/+ NK cells into CCR5-/- mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense.

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation.

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation.

Identification of human CCR8 as a CCL18 receptor.

The CC chemokine ligand 18 (CCL18) is one of the most highly expressed chemokines in human chronic inflammatory diseases. An appreciation of the role of CCL18 in these diseases has been hampered by the lack of an identified chemokine receptor. We report that the human chemokine receptor CCR8 is a CCL18 receptor. CCL18 induced chemotaxis and calcium flux of human CCR8-transfected cells. CCL18 bound with high affinity to CCR8 and induced its internalization. Human CCL1, the known endogenous CCR8 ligand, and CCL18 competed for binding to CCR8-transfected cells. Further, CCL1 and CCL18 induced heterologous cross-desensitization of CCR8-transfected cells and human Th2 cells. CCL18 induced chemotaxis and calcium flux of human activated highly polarized Th2 cells through CCR8. Wild-type but not Ccr8-deficient activated mouse Th2 cells migrated in response to CCL18. CCL18 and CCR8 were coexpressed in esophageal biopsy tissue from individuals with active eosinophilic esophagitis (EoE) and were present at markedly higher levels compared with esophageal tissue isolated from EoE patients whose disease was in remission or in normal controls. Identifying CCR8 as a chemokine receptor for CCL18 will help clarify the biological role of this highly expressed chemokine in human disease.

T cell homing to epithelial barriers in allergic disease.

Allergic inflammation develops in tissues that have large epithelial surface areas that are exposed to the environment, such as the lung, skin and gut. In the steady state, antigen-experienced memory T cells patrol these peripheral tissues to facilitate swift immune responses against invading pathogens. In at least two allergy-prone organs, the skin and the gut, memory T cells are programmed during the initial antigen priming to express trafficking receptors that enable them to preferentially home to these organs. In this review we propose that tissue-specific memory and inflammation-specific T cell trafficking facilitates the development of allergic disease in these organs. We thus review recent advances in our understanding of tissue-specific T cell trafficking and how regulation of T cell trafficking by the chemokine system contributes to allergic inflammation in mouse models and in human allergic diseases of the skin, lung and gut. Inflammation- and tissue-specific T lymphocyte trafficking pathways are currently being targeted as new treatments for non-allergic inflammatory diseases and may yield effective new therapeutics for allergic diseases.

The leukotriene B4 lipid chemoattractant receptor BLT1 defines antigen-primed T cells in humans.

We have recently shown that the leukotriene B(4) (LTB(4))-BLT1 pathway is important in early effector T-cell recruitment in mouse models of inflammation. Here we characterize the phenotype and function of human peripheral blood BLT1(+) T cells in health and illustrate their involvement in asthma and acute infection. In healthy individuals, BLT1(+) T cells are a rare peripheral blood T-cell population enriched for the activation markers CD38 and HLA-DR. Compared with BLT1(-) T cells, a larger proportion of peripheral blood BLT1(+) T cells express the effector cytokines IFNgamma and IL-4 and inflammatory chemokine receptors, CCR1, CCR2, CCR6, and CXCR1. Consequently, in healthy individuals peripheral blood BLT1(+) T cells are a rare antigen-primed T-cell subset with unique phenotypic, migratory, and functional properties. BLT1 expression on T cells is tightly regulated by inflammation and only transiently expressed after naive T-cell activation by dendritic cells. Although rare in the peripheral blood of healthy individuals, BLT1(+) T cells are markedly increased in frequency in the peripheral blood in response to acute Epstein-Barr virus (EBV) infection and moderately increased in the airways of asymptomatic allergic asthmatics. Our studies provide novel insights into the LTB(4)-BLT1 lipid chemoattractant pathway in human T-cell responses, and how it may link innate and adaptive immunity.

Examination of the highly diverse CD4(+) T-cell repertoire directed against an influenza peptide: a step towards TCR proteomics.

We combined several recent technological advances in immunology and molecular biology to identify and sequence a large number of T-cell receptor (TCR) genes specific for a particular antigen. We utilized class II MHC tetramers and interferon-gamma surface capture to isolate from samples of peripheral blood the population of CD4(+) T cells responding to a peptide derived from influenza hemagglutinin and restricted by HLA-DR1. Detailed analysis of hundreds of clones from three different patients revealed an extremely diverse repertoire, with little overlap between patients. We observed no dominant usage of particular Vbeta segments nor any clear CDR3 sequence motif in the responding T cells, but most of the clones appear to utilize acidic residues in the CDR1 and CDR3 regions, presumably to interact with the exposed basic residues in the MHC-peptide complex. This methodology could be expanded to a large scale to identify the generalized rules governing TCR-MHC engagement and factors which shape the T-cell repertoire after vaccination and in autoimmune pathologies.

Clonotype tracking of TCR repertoires during chronic virus infections.

Human viral infections such as HIV and EBV typically evoke a strong and diverse CD8(+) T cell response. Relatively little is known about the extent to which TCR repertoire evolution occurs during viral infection or how repertoire evolution affects the efficacy of the CD8(+) T cell response. In this study we describe a general approach for tracking TCR repertoire evolution during viral infection. IFNgamma surface capture and MHC class I tetramer staining were independently used to isolate EBV-specific CD8(+) T cells from peripheral blood. Anchored RT-PCR and clonotype TCR repertoire analysis were performed immediately after isolating the cells. We find that the TCR repertoires of the IFNgamma-secreting and MHC class I tetramer staining populations were similar. In one subject a detailed analysis of the TCR repertoire during the first year of EBV infection was performed and over 600 TCR sequences targeting an EBV-immunodominant epitope were analyzed. Although some repertoire evolution occurred during the year, in general, the degree of repertoire drift was small. TCR repertoire analysis for an HIV-immunodominant epitope revealed a highly conserved amino acid motif in the Dbeta region of TCR that recognizes the epitope and suggested that T cell precursor frequency influences which epitopes are targeted early in HIV infection. This methodology, which allows one to sort antigen-specific T cells based on different functional assays and to obtain a snapshot of their TCR repertoire with relative ease, should lead to a richer understanding of the rules underlying antigen recognition and T cell evolution during viral infection.

Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment.

Leukotriene B4 (LTB4) was originally described as a potent lipid myeloid cell chemoattractant, rapidly generated from innate immune cells, that activates leukocytes through the G protein-coupled receptor BLT1. We report here that BLT1 is expressed on effector CD4+ T cells generated in vitro as well as in vivo when effector T cells migrate out of the lymphoid compartment and are recruited into peripheral tissues. BLT1 mediated LTB4-induced T helper type 1 (T(H)1) and T(H)2 cell chemotaxis and firm adhesion to endothelial cells under flow, as well as early CD4+ and CD8+ T cell recruitment into the airway in an asthma model. Our findings show that the LTB4-BLT1 pathway is involved in linking early immune system activation and early effector T cell recruitment.