Stefanie Willenzon

Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

CC chemokine receptor 7 and 9 double-deficient hematopoietic progenitors are severely impaired in seeding the adult thymus

T-cell development depends on recruitment of bone marrow-derived precursor cells to the thymus via a multistep adhesion cascade involving the chemokine receptor CCR9. However, CCR9 deficiency does not result in complete abrogation of progenitor entry into the adult thymus. Therefore, we tested the hypothesis that additional chemokine/chemokine receptor systems might play a role in this process. To this end, we generated mice deficient in both CCR9 and CCR7. Deficiency in both chemokine receptors resulted in severely reduced numbers of early T-cell progenitors and in near-complete abrogation of thymus reconstitution. Progenitors in bone marrow and peripheral blood remained largely unaffected in CCR7(-/-)CCR9(-/-) mice, and direct intrathymic transfer of precursors from CCR7(-/-)CCR9(-/-) mice as well as single-mutant mice showed that intrathymic differentiation of these precursors remained functional. Thus, our data reveal a previously unrecognized role of CCR7 in progenitor seeding of the adult thymus, which is largely masked by compensatory effects of CCR9 signals. In turn, CCR7 signals can partially compensate for CCR9 signals, thus explaining the rather mild phenotype of CCR9(-/-) mice with respect to progenitor seeding.

Generalized multi‐organ autoimmunity in CCR7‐deficient mice

Development of autoimmunity is a multi‐factorial process involving genetic predisposition as well as environmental and stochastic factors. Although the mechanisms responsible for the initiation of autoimmunity remain only partially understood, several studies have demonstrated that genetic predisposition plays a major role in this process. In the present study, we analyzed the influence of CCR7 signaling in the development of autoimmunity, because this chemokine receptor is essentially involved in the functional organization of thymus architecture. We demonstrate that CCR7‐deficient mice are prone to develop generalized multi‐organ autoimmunity. The autoimmune phenotype of CCR7–/– mice encompasses the presence of lymphocyte infiltrates in several peripheral organs, circulating autoantibodies against a multitude of tissue‐specific antigens and IgG deposition on renal glomeruli. Additionally, CCR7‐deficient mice show increased susceptibility to streptozotocin‐induced diabetes and spontaneously display signs of chronic autoimmune renal disease. Thus, this study identifies CCR7 as a genetic factor involved in the regulation of autoimmunity.

IL-17–induced CXCL12 recruits B cells and induces follicle formation in BALT in the absence of differentiated FDCs

The requirements for BALT formation are pathogen-dependent and, in the absence of FDC maturation, IL-17 can drive BALT formation via CXCL12 B cell recruitment.

Lymph Node T Cell Homeostasis Relies on Steady State Homing of Dendritic Cells

Little is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

Induction of BALT in the absence of IL-17.

1 and Il17a–/–Il17f –/– mice in follicle formation, as described above. In summary, our data show that BALT can be efficiently induced in the complete absence of IL-17A and its homolog IL-17F. Therefore, we believe that the general conclusion reached by Rangel-Moreno et al. that iBALT formation depends on IL-17 (ref. 1) is inappropriate. The function of IL-17 in the induction of BALT needs clarification for understanding of the role of this cytokine in the biology of ectopic lymphoid structures.

Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin.

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of β2 and β7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either β2 or β7 integrin but not of β2-deficient lymphocytes, which in addition were blocked by anti-β7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Gαi signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.

Application of light sheet microscopy for qualitative and quantitative analysis of bronchus-associated lymphoid tissue in mice.

Bronchus-associated lymphoid tissue (BALT) develops at unpredictable locations around lung bronchi following pulmonary inflammation. The formation and composition of BALT have primarily been investigated by immunohistology that, due to the size of the invested organ, is usually restricted to a limited number of histological sections. To assess the entire BALT of the lung, other approaches are urgently needed. Here, we introduce a novel light sheet microscopy-based approach for assessing lymphoid tissue in the lung. Using antibody staining of whole lung lobes and optical clearing by organic solvents, we present a method that allows in-depth visualization of the entire bronchial tree, the lymphatic vasculature and the immune cell composition of the induced BALT. Furthermore, three-dimensional analysis of the entire lung allows the qualitative and quantitative enumeration of the induced BALT. Using this approach, we show that a single intranasal application of the replication-deficient poxvirus MVA induces BALT that constitutes up to 8% of the entire lung volume in mice deficient in CCR7, in contrast to wild type mice (WT). Furthermore, BALT induced by heat-inactivated E. coli is dominated by a pronounced T cell infiltration in Cxcr5-deficient mice, in contrast to WT mice.

Cardiomyocytes display low mitochondrial priming and are highly resistant toward cytotoxic T‐cell killing

Following heart transplantation, alloimmune responses can cause graft rejection by damaging donor vascular and parenchymal cells. However, it remains unclear whether cardiomyocytes are also directly killed by immune cells. Here, we used two‐photon microscopy to investigate how graft‐specific effector CD8+ T cells interact with cardiomyocytes in a mouse heart transplantation model. Surprisingly, we observed that CD8+ T cells are completely impaired in killing cardiomyocytes. Even after virus‐mediated preactivation, antigen‐specific CD8+ T cells largely fail to lyse these cells although both cell types engage in dynamic interactions. Furthermore, we established a two‐photon microscopy‐based assay using intact myocardium to determine the susceptibility of cardiomyocytes to undergo apoptosis. This feature, also known as mitochondrial priming reveals an unexpected weak predisposition of cardiomyocytes to undergo apoptosis in situ. These observations together with the early exhaustion phenotype of graft‐infiltrating specific T cells provide an explanation why cardiomyocytes are largely protected from direct CD8+ T‐cell‐mediated killing.