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Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

Lymphocytes recirculate continuously between the blood and lymphoid organs, a process that is of fundamental importance for proper functioning of the immune system. The molecular mechanisms underlying lymphocyte trafficking to the spleen remain an enigma. Here, we show that lymphocytes enter the spleen preferentially from vessels in the red pulp rather than the marginal sinus or the vasculature in the white pulp. Ex vivo adhesion assays in mice and humans, together with genetic ablation of Clever-1 in mice, indicate that CD8+T cell and B220+B cell homing to the spleen via the red pulp is Clever-1 dependent. Moreover, absence of Clever-1 leads to down-regulation of the B cell attractant chemokine, CXCL13, on spleen endothelium. CXCL13 is known to guide B cell trafficking to lymphoid organs, and its lack may contribute to the observed decrease in B cell trafficking into the spleen as well. In summary, this study identifies Clever-1 as an important molecule controlling lymphocyte entry into the spleen, along with a critical role for the splenic red pulp in this regulated trafficking. Furthermore, the results demonstrate that location-specific homing-associated molecules guide lymphocyte entry into the spleen.

BackgroundElevated levels of IL-17A were detected in the airways of patients with cystic fibrosis (CF), but its cellular sources and role in the pathogenesis of CF lung disease remain poorly understood. The aim of this study was to determine the sources of IL-17A and its role in airway inflammation and lung damage in CF.MethodsWe performed flow cytometry to identify IL-17A-producing cells in lungs and peripheral blood from CF patients and β-epithelial Na+ channel transgenic (Scnn1b-Tg) mice with CF-like lung disease and determined effects of genetic deletion of Il17a and Rag1 on the pulmonary phenotype of Scnn1b-Tg mice.ResultsT helper (Th)17 cells, CD3+CD8+, γδ T cells, invariant natural killer T cells (iNKT) and innate lymphoid cells (ILCs) contribute to IL-17A secretion in lung tissue, lymph nodes and peripheral blood of patients with CF. Scnn1b-Tg mice displayed increased pulmonary expression of Il17a and elevated IL-17A-producing innate and adaptive lymphocytes with a major contribution by γδ T cells. Lack of IL-17A, but not RAG1 reduced neutrophilic airway inflammation in Scnn1b-Tg mice. Genetic deletion of Il17a or Rag1 had no effect on mucus obstruction, but reduced structural lung damage and revealed an IL-17A-dependent macrophage activation in Scnn1b-Tg mice.ConclusionWe identify innate and adaptive sources of IL-17A in CF lung disease. Our data demonstrate that IL-17A contributes to airway neutrophilia, macrophage activation and structural lung damage in CF-like lung disease in mice. These results suggest IL-17A as a novel target for anti-inflammatory therapy of CF lung disease.

Mendelian susceptibility to mycobacterial disease is a rare primary immunodeficiency characterized by severe infections caused by weakly virulent mycobacteria. Biallelic null mutations in genes encoding interferon gamma receptor 1 or 2 ( or ) result in a life-threatening disease phenotype in early childhood. Recombinant interferon γ (IFN-γ) therapy is inefficient, and hematopoietic stem cell transplantation has a poor prognosis. Thus, we developed a hematopoietic stem cell (HSC) gene therapy approach using lentiviral vectors that express either constitutively or myeloid specifically. Transduction of mouse HSCs led to stable IFNγR1 expression on macrophages, which rescued their cellular responses to IFN-γ. As a consequence, genetically corrected HSC-derived macrophages were able to suppress T-cell activation and showed restored antimycobacterial activity against and Bacille Calmette-Guérin (BCG) in vitro. Transplantation of genetically corrected HSCs into mice before BCG infection prevented manifestations of severe BCG disease and maintained lung and spleen organ integrity, which was accompanied by a reduced mycobacterial burden in lung and spleen and a prolonged overall survival in animals that received a transplant. In summary, we demonstrate an HSC-based gene therapy approach for IFNγR1 deficiency, which protects mice from severe mycobacterial infections, thereby laying the foundation for a new therapeutic intervention in corresponding human patients.

Objectives The contribution of adaptive vs. innate lymphocytes to IL‐17A and IL‐22 secretion at the end stage of chronic lung diseases remains largely unexplored. In order to uncover tissue‐ and disease‐specific secretion patterns, we compared production patterns of IL‐17A and IL‐22 in three different human end‐stage lung disease entities. Methods Production of IL‐17A, IL‐22 and associated cytokines was assessed in supernatants of re‐stimulated lymphocytes by multiplex assays and multicolour flow cytometry of conventional T cells, iNKT cells, γδ T cells and innate lymphoid cells in bronchial lymph node and lung tissue from patients with emphysema ( n = 19), idiopathic pulmonary fibrosis ( n = 14) and cystic fibrosis ( n = 23), as well as lung donors ( n = 17). Results We detected secretion of IL‐17A and IL‐22 by CD4 + T cells, CD8 + T cells, innate lymphoid cells, γδ T cells and iNKT cells in all end‐stage lung disease entities. Our analyses revealed disease‐specific contributions of individual lymphocyte subpopulations to cytokine secretion patterns. We furthermore found the high levels of microbial detection in CF samples to associate with a more pronounced IL‐17A signature upon antigen‐specific and unspecific re‐stimulation compared to other disease entities and lung donors. Conclusion Our results show that both adaptive and innate lymphocyte populations contribute to IL‐17A‐dependent pathologies in different end‐stage lung disease entities, where they establish an IL‐17A‐rich microenvironment. Microbial colonisation patterns and cytokine secretion upon microbial re‐stimulation suggest that pathogens drive IL‐17A secretion patterns in end‐stage lung disease.

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