Fate mapping and scRNA sequencing reveal origin and diversity of lymph node stromal precursors.

Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells.

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues.

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-ß receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.

Engineered Treg cells as putative therapeutics against inflammatory diseases and beyond.

Regulatory T (Treg) cells ensure tolerance against self-antigens, limit excessive inflammation, and support tissue repair processes. Therefore, Treg cells are currently attractive candidates for the treatment of certain inflammatory diseases, autoimmune disorders, or transplant rejection. Early clinical trials have proved the safety and efficacy of certain Treg cell therapies in inflammatory diseases. We summarize recent advances in engineering Treg cells, including the concept of biosensors for inflammation. We assess Treg cell engineering possibilities for novel functional units, including Treg cell modifications influencing stability, migration, and tissue adaptation. Finally, we outline perspectives of engineered Treg cells going beyond inflammatory diseases by using custom-designed receptors and read-out systems, aiming to use Treg cells as in vivo diagnostic tools and drug delivery vehicles.

Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis.

Lymph nodes (LNs) are strategically situated throughout the body at junctures of the blood vascular and lymphatic systems to direct immune responses against antigens draining from peripheral tissues. The current paradigm describes LN development as a programmed process that is governed through the interaction between mesenchymal lymphoid tissue organizer (LTo) cells and hematopoietic lymphoid tissue inducer (LTi) cells. Using cell-type-specific ablation of key molecules involved in lymphoid organogenesis, we found that initiation of LN development is dependent on LTi-cell-mediated activation of lymphatic endothelial cells (LECs) and that engagement of mesenchymal stromal cells is a succeeding event. LEC activation was mediated mainly by signaling through receptor activator of NF-κB (RANK) and the non-canonical NF-κB pathway and was steered by sphingosine-1-phosphate-receptor-dependent retention of LTi cells in the LN anlage. Finally, the finding that pharmacologically enforced interaction between LTi cells and LECs promotes ectopic LN formation underscores the central LTo function of LECs.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

Biosensors for inflammation as a strategy to engineer regulatory T cells for cell therapy.

Engineered regulatory T cell (Treg cell) therapy is a promising strategy to treat patients suffering from inflammatory diseases, autoimmunity, and transplant rejection. However, in many cases, disease-related antigens that can be targeted by Treg cells are not available. In this study, we introduce a class of synthetic biosensors, named artificial immune receptors (AIRs), for murine and human Treg cells. AIRs consist of three domains: (a) extracellular binding domain of a tumor necrosis factor (TNF)-receptor superfamily member, (b) intracellular costimulatory signaling domain of CD28, and (c) T cell receptor signaling domain of CD3-ζ chain. These AIR receptors equip Treg cells with an inflammation-sensing machinery and translate this environmental information into a CD3-ζ chain-dependent TCR-activation program. Different AIRs were generated, recognizing the inflammatory ligands of the TNF-receptor superfamily, including LIGHT, TNFα, and TNF-like ligand 1A (TL1A), leading to activation, differentiation, and proliferation of AIR-Treg cells. In a graft-versus-host disease model, Treg cells expressing lymphotoxin ß receptor-AIR, which can be activated by the ligand LIGHT, protect significantly better than control Treg cells. Expression and signaling of the corresponding human AIR in human Treg cells prove that this concept can be translated. Engineering Treg cells that target inflammatory ligands leading to TCR signaling and activation might be used as a Treg cell-based therapy approach for a broad range of inflammation-driven diseases.

Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-ß receptor (LTßR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTßR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTßR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Lymphotoxin-ß-receptor (LTßR) signaling on hepatocytes is required for liver regeneration after partial hepatectomy.

Lymphotoxin-ß-receptor deficient (LTßR-/-) and Tumor Necrosis Factor Receptor p55 deficient (TNFRp55-/-) mice show defects in liver regeneration (LR) after partial hepatectomy (PHx) with significantly increased mortality. LTßR and TNFRp55 belong to the core members of the TNF/TNFR superfamily. Interestingly, combined failure of LTßR and TNFRp55 signaling after PHx leads to a complete defect in LR. Here, we first addressed the question which liver cell population crucially requires LTßR signaling for efficient LR. To this end, mice with a conditionally targeted LTßR allele (LTßRfl/fl) were crossed to AlbuminCre and LysozymeMCre mouse lines to unravel the function of the LTßR on hepatocytes and monocytes/macrophages/Kupffer cells, respectively. Analysis of these mouse lines clearly reveals that LTßR is required on hepatocytes for efficient LR while no deficit in LR was found in LTßRfl/fl × LysMCre mice. Second, the molecular basis for the cooperating role of LTßR and TNFRp55 signaling pathways in LR was investigated by transcriptome analysis of etanercept treated LTßR-/- (LTßR-/-/ET) mice. Bioinformatic analysis and subsequent verification by qRT-PCR identified novel target genes (Cyclin-L2, Fas-Binding factor 1, interferon-related developmental regulator 1, Leucyl-tRNA Synthetase 2, and galectin-4) that are upregulated by LTßR/TNFRp55 signaling after PHx and fail to be upregulated after PHx in LTßR-/-/ET mice.

RelA and RelB transcription factors in distinct thymocyte populations control lymphotoxin-dependent interleukin-17 production in γδ T cells.

The NF-κB transcription factor regulates numerous immune responses but its contribution to interleukin-17 (IL-17) production by T cells is largely unknown. Here, we report that IL-17, but not interferon-γ (IFN-γ), production by γδ T cells required the NF-κB family members RelA and RelB as well as the lymphotoxin-ß-receptor (LTßR). In contrast, LTßR-NF-κB signaling was not involved in the differentiation of conventional αß Th17 cells. Impaired IL-17 production in RelA- or RelB-deficient T cells resulted in a diminished innate immune response to Escherichia coli infection. RelA controlled the expression of LT ligands in accessory thymocytes whereas RelB, acting downstream of LTßR, was required for the expression of RORγt and RORα4 transcription factors and the differentiation of thymic precursors into γδT17 cells. Thus, RelA and RelB within different thymocyte subpopulations cooperate in the regulation of IL-17 production by γδ T cells and contribute to the host's ability to fight bacterial infections.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 µmol/L to 11.8 ± 2.8 µmol/L in all post-transplant samples and to 3.5 ± 3 µmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

Lymphotoxin ß receptor activation on macrophages induces cross-tolerance to TLR4 and TLR9 ligands.

Our previous studies indicated that lymphotoxin ß receptor (LTßR) activation controls and downregulates inflammatory reactions. In this study, we report that LTßR activation on primary mouse macrophages results in induction of tripartite motif containing (TRIM) 30α, which negatively regulates NF-κB activation induced by TLR signaling. LTßR activation results in a downregulation of proinflammatory cytokine and mediator expression upon TLR restimulation, demonstrating that LTßR signaling is involved in the induction of TLR cross-tolerance. Specific knockdown experiments using TRIM30α-specific small interfering RNA abolished the LTßR-dependent induction of TRIM30α and LTßR-mediated TLR cross-tolerance. Concordantly, LTßR activation on bone marrow-derived macrophages induced cross-tolerance to TLR4 and TLR9 ligands in vitro. Furthermore, we have generated cell type-specific LTßR-deficient mice with ablation of LTßR expression on macrophages/neutrophils (LTßR(flox/flox) × LysM-Cre). In bone marrow-derived macrophages derived from these mice LTßR-induced cross-tolerance to TLR4 and TLR9 ligands was impaired. Additionally, mice with a conditional ablation of LTßR expression on macrophages (LTßR(flox/flox) × LysM-Cre) are resistant to LTßR-induced TLR4 tolerance in vivo. Collectively, our data indicate that LTßR activation on macrophages by T cell-derived lymphotoxin α(1)ß(2) controls proinflammatory responses by activation of a TRIM30α-controlled, counterregulatory signaling pathway to protect against exacerbating inflammatory reactions.

Mouse ß-defensin 14 (Defb14) promotes tumor growth by inducing angiogenesis in a CCR6-dependent manner.

ß-Defensins are known for their antimicrobial activity and belong to the molecular barrier of the innate immune system against invading pathogens. In addition, it has been shown that some members of the ß-defensin superfamily have the capacity to promote local innate inflammatory and systemic adaptive immune responses, mediated in part by the interaction with CCR6. We found that mouse ß-defensin 14 (mBD14, Defb14), a newly identified member of the mouse ß-defensin superfamily, is expressed in mouse fibrosarcoma tumor tissue. Tumor cells overexpressing mBD14 demonstrated enhanced solid tumor growth in syngeneic C57BL/6 mice concomitant with increased vascularization of these tumors. Furthermore, mBD14-overexpressing tumors demonstrated increased expression of proangiogenic MIP-2 (CXCL2) ex vivo. In contrast, vascular endothelial growth factor expression was not affected. Cellular analysis of tumor-infiltrating leukocytes revealed a significant increase of CCR6(+) B220(+) lymphocytes in solid tumors derived from mBD14-overexpressing tumor cells. Enhanced tumor growth of mBD14-overexpressing fibrosarcomas was abolished in CCR6-deficient mice, which was paralleled by decreased infiltration of CCR6(+) B220(+) lymphocytes, indicating the requirement of CCR6 expression on host cells. Previously, the interaction of activated, LTαß(+), lymphocytes with lymphotoxin ß-receptor-expressing fibrosarcoma tumor cells has been identified as a new CXCL2-dependent proangiogenic pathway. Coexpression of a soluble lymphotoxin ß-receptor:Ig fusion protein, an inhibitor of CXCL2-dependent angiogenesis, in mBD14-overexpressing fibrosarcoma tumor cells abolished enhanced solid tumor growth. Thus, we conclude that mBD14 expression by tumor-infiltrating host cells results in the chemoattraction of CCR6(+) B220(+) lymphocytes, which in turn initiates a proangiogenic pathway leading to enhanced angiogenesis and organized tumor tissue development.

The effector program of human CD8 T cells supports tissue remodeling.

CD8 T lymphocytes are classically viewed as cytotoxic T cells. Whether human CD8 T cells can, in parallel, induce a tissue regeneration program is poorly understood. Here, antigen-specific assay systems revealed that human CD8 T cells not only mediated cytotoxicity but also promoted tissue remodeling. Activated CD8 T cells could produce the epidermal growth factor receptor (EGFR)-ligand amphiregulin (AREG) and sensitize epithelial cells for enhanced regeneration potential. Blocking the EGFR or the effector cytokines IFN-γ and TNF could inhibit tissue remodeling. This regenerative program enhanced tumor spheroid and stem cell-mediated organoid growth. Using single-cell gene expression analysis, we identified an AREG+, tissue-resident CD8 T cell population in skin and adipose tissue from patients undergoing abdominal wall or abdominoplasty surgery. These tissue-resident CD8 T cells showed a strong TCR clonal relation to blood PD1+TIGIT+ CD8 T cells with tissue remodeling abilities. These findings may help to understand the complex CD8 biology in tumors and could become relevant for the design of therapeutic T cell products.

Human beta-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2.

Beta-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain beta-defensin family members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However, because beta-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human beta-defensin (hBD)2 and 3 and their mouse orthologs, beta-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages, and neutrophils. These beta-defensins, fused to the Fc region of human IgG1, showed binding to CCR2-transfected HEK293 cells, as revealed by flow cytometry. The beta-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by beta-defensins. Conversely, preincubation with hBD2:Ig or hBD3:Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice failed to migrate toward these fusion proteins. In conclusion, the beta-defensins used in this study contribute to the innate and adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse orthologs (beta-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner.

Propionate induces cross-tolerance to TLR1/2 and TLR4 agonists in an IFIT-dependent manner.

In this study, we have identified Interferon-stimulated genes (ISGs), especially IFIT1, 2 and 3, as target genes of propionate-induced signalling in the human epithelial cell line A549, the monocytic cell line THP-1 as well as in primary, human peripheral blood-derived macrophages (PBMs). Induction of the IFIT gene family by propionate negatively regulates TLR-induced signalling. Propionate stimulation results in downregulation of pro-inflammatory cytokine and chemokine expression as well as MHC class II expression upon TLR1/2 and TLR4 re-stimulation in A549 and THP-1 cells as well as in PBMs, demonstrating that propionate-induced signalling is involved in the induction of TLR cross-tolerance. Signalling pathway analysis clearly demonstrates that propionate-induced IFIT expression is mediated by FFAR2 in a Gαq/11 signalling pathway-dependent manner. Furthermore, propionate-induced IFIT expression is dependent on IFN type I and/or type III-mediated signalling since pre-treatment of A549 cells with Ruxolitinib, a specific JAK1/2 tyrosine kinase inhibitor, prior to stimulation with propionate, inhibited the upregulation of IFIT1 expression. The hypo-responsiveness towards TLR1/2 and TLR4 agonists seems to be mediated by different members of the IFIT gene family in a cell type-specific manner. Collectively, our data indicate that propionate-induced signalling controls pro-inflammatory responses by activation of IFN type I and/or type III-induced and IFIT-mediated counter-regulatory mechanisms in order to protect against exacerbating inflammatory reactions.

Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function.

Innate lymphoid cells (ILCs) govern immune cell homeostasis in the intestine and protect the host against microbial pathogens. Various cell-intrinsic pathways have been identified that determine ILC development and differentiation. However, the cellular components that regulate ILC sustenance and function in the intestinal lamina propria are less known. Using single-cell transcriptomic analysis of lamina propria fibroblasts, we identify fibroblastic reticular cells (FRCs) that underpin cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Genetic ablation of lymphotoxin-ß receptor expression in Ccl19-expressing FRCs blocks the maturation of CPs into mature ILFs. Interactome analysis shows the major niche factors and processes underlying FRC-ILC crosstalk. In vivo validation confirms that a sustained lymphotoxin-driven feedforward loop of FRC activation including IL-7 generation is critical for the maintenance of functional ILC populations. In sum, our study indicates critical fibroblastic niches within the intestinal lamina propria that control ILC homeostasis and functionality and thereby secure protective gut immunity.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTßR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Specific binding and chemotactic activity of mBD4 and its functional orthologue hBD2 to CCR6-expressing cells.

Beta-defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG(1) to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these beta-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the beta-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both beta-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the beta-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and beta-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the beta-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific beta-defensin receptor interactions.

Lymphotoxin-ß receptor activation by lymphotoxin-α(1)ß(2) and LIGHT promotes tumor growth in an NFκB-dependent manner.

Lymphotoxin beta receptor (LTßR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTßR, namely LTα(1) ß(2) and LIGHT, are involved in the activation of LTßR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTßR in these fibrosarcoma tumors, we used conditional LTß-deficient mice that specifically lack LTß expression either on T cells (T-LTß(-/-)) or on B cells (B-LTß(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTßR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTßR in BFS-1 fibrosarcomas. LTßR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTßR activation. Our data support the idea that the ablation of LTßR signaling should be considered for cancer treatment.