Type 3 innate lymphoid cell-derived lymphotoxin prevents microbiota-dependent inflammation.

Splenomegaly is a well-known phenomenon typically associated with inflammation. However, the underlying cause of this phenotype has not been well characterized. Furthermore, the splenomegaly phenotype seen in lymphotoxin (LT) signaling-deficient mice is characterized by increased numbers of splenocytes and splenic neutrophils. Splenomegaly, as well as the related phenotype of increased lymphocyte counts in non-lymphoid tissues, is thought to result from the absence of secondary lymphoid tissues in LT-deficient mice. We now present evidence that mice deficient in LTα1ß2 or LTßR develop splenomegaly and increased numbers of lymphocytes in non-lymphoid tissues in a microbiota-dependent manner. Antibiotic administration to LTα1ß2- or LTßR-deficient mice reduces splenomegaly. Furthermore, re-derived germ-free Ltbr-/- mice do not exhibit splenomegaly or increased inflammation in non-lymphoid tissues compared to specific pathogen-free Ltbr-/- mice. By using various LTß- and LTßR-conditional knockout mice, we demonstrate that retinoic acid-related orphan receptor γT-positive type 3 innate lymphoid cells provide the required active LT signaling to prevent the development of splenomegaly. Thus, this study demonstrates the importance of LT-mediated immune responses for the prevention of splenomegaly and systemic inflammation induced by microbiota.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1ß2 in Newly Formed Tertiary Lymphoid Structures.

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)ßR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1ß2/LTßR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.

Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration.

Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαß rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTßR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.

Analysis of chromosomal aberrations in murine HCC.

Liver cancer-also called hepatocellular carcinoma (HCC)-is the most frequent primary liver cancer in humans. As of today, it is mainly induced by chronic virus infections such as Hepatitis B and C viruses, which induce chronic hepatitis and fibrosis, the two most important conditions predisposing towards HCC development. Besides, chronic alcohol or drug consumption contributes to chronic liver injury and HCC development. Of note, in industrialized countries virus infections have recently been outcompeted by a high-fat and high-sugar diet as the most important etiology for HCC development in humans-now representing the fastest growing cancer in the USA as of today. It is believed that soon also in Europe high-fat diet caused HCC will become the fastest growing cancer. Today more than 800,000 people die every year due to cancer; however, despite a great research effort in the last 20 years, no efficient curative therapy is available at the moment. It has turned out that various subtypes of HCC exist in humans, complicating the therapy for HCC patients in general, and leading to the need for therapies of stratified patient cohorts as the variability of HCC phenotypes (6 different subtypes exist as of today) influences the responsiveness to treatment. Thus, it is important to dissect and characterize the various HCC subtypes in humans as well as in mouse models to identify the sub-cohorts that are responsive to particular therapies. One step to do so is the characterization of HCC nodules on genetic level. Here, we describe a protocol to characterize individual HCC nodules on genomic level, enabling to stratify the respective liver carcinoma and select them for a more targeted therapy.

Innate lymphoid cells facilitate NK cell development through a lymphotoxin-mediated stromal microenvironment.

Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1ß2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORγt(+) innate lymphoid cells (ILCs), and not CD3(-)NK1.1(+) cells, express LT to drive NK development. Similar to LT(-/-) or RORγt(-/-) mice, the mice conditionally lacking LTα1ß2 on RORγt(+) ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1(+) ILCs from BM or purified RORγt(+) ILCs from lamina propria lymphocytes into LT-deficient RORγt(+) BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORγt(+) ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development.

Tumor targeting properties of antibody fusion proteins based on different members of the murine tumor necrosis superfamily.

The tumor necrosis factor superfamily (TNFSF) consists of more than 20 members that can modulate cellular and immunological functions, including cell survival and the stimulation of an inflammatory response. Many TNF superfamily members display potent anticancer activity when used as recombinant proteins in vitro and in vivo. While TNF, TRAIL and FasL have already been used as payloads in antibody-based pharmacodelivery strategies, most TNF superfamily members have not yet been investigated as antibody payloads. Here, we report the cloning, production and characterization of eight novel antibody fusion proteins based on CD40L, FasL, TRAIL, LiGHT, VEGI, lymphotoxin alpha, lymphotoxin beta and lymphotoxin alpha1/beta2. The monoclonal antibody F8 was chosen as fusion partner of proven tumor targeting performance, which recognizes the alternatively-spliced EDA domain of fibronectin, a marker of angiogenesis. A quantitative biodistribution analysis performed with radioiodinated protein preparations in tumor-bearing mice revealed that TRAIL and lymphotoxin alpha1/beta2 were able to selectively accumulate at the tumor site, while all other members of the TNF superfamily abrogated the selective tumor targeting performance of the parental antibody or accumulated also in healthy tissues. The study indicates that even cytokines, which are closely related in terms of structure and function, may have a substantially different impact on the biodistribution and functional properties of the corresponding fusions with disease-homing antibodies.

Lymphotoxin α1ß2 expression on B cells is required for follicular dendritic cell activation during the germinal center response.

CD19-deficient mice were used as a model to study follicular dendritic cell (FDC) activation because these mice have normal numbers of FDC-containing primary follicles, but lack the ability to activate FDCs or form GCs. It was hypothesized that CD19 expression is necessary for B-cell activation and upregulation of membrane lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM-1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19(+) wild-type B cells into CD19-deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19(+) donor B cells lacking mLT were unable to induce VCAM-1 expression on FDCs, furthermore FcγRII/III upregulation was impaired in FDCs stimulated with mLT-deficient B cells. VCAM-1 expression on FDCs, but not FcγRII/III, was rescued when CD19-deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19(+) , mLT-deficient B cells, suggesting that FDC activation requires the CD19-dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.

The role of core TNF/LIGHT family members in lymph node homeostasis and remodeling.

Lymph nodes (LNs) maintain active homeostasis at steady state. However, in response to changes in the local environment, such as local infection, cancer, vaccination, and autoimmune disease, dramatic remodeling of LN occurs. This remodeling includes changes in size, lymph and blood flow, immune cell trafficking and cellularity, lymphatic and blood vessel growth and activation, as well as microarchitecture. Therefore, inflammatory conditions often lead to enlarged nodes; after local inflammation resolves, LNs actively regress in size and return to steady state. Remodeling of lymphatic vessels (LVs) and blood vessels (BVs) during both the expansion and regression phases are key steps in controlling LN size as well as function. The cells, membrane-associated molecules, and soluble cytokines that are essential for LV and BV homeostasis as well as dynamic changes in the expansion and regression phases have not been well defined. Understanding the underlying cellular and molecular mechanisms behind LN remodeling would help us to better control undesired immune responses (e.g. inflammation and autoimmune diseases) or promote desired responses (e.g. antitumor immunity and vaccination). In this review, we focus on how the closely related tumor necrosis factor (TNF) members: LIGHT (TNFSF14), lymphotoxin-αß, and TNF-α contribute to the remodeling of LNs at various stages of inflammation.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway.

Adaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) beta-receptor-dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTalpha(1)beta(2)-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A-independent, LT- and B cell-dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen.

The formation of lymph nodes is a complex process crucially controlled through triggering of LTbetaR on mesenchymal cells by LTalpha(1)beta(2) expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. In this study, we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LTalpha and that these cells, expressing TNF-related activation-induced cytokine (TRANCE), are the earliest LTi cells. By paracrine signaling the first expression of LTalpha(1)beta(2) is induced. Subsequent LTbetaR triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, IL-7, as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and IL-7 will further induce the expression of LTalpha(1)beta(2) on newly arrived immature LTi cells, resulting in more LTbetaR triggering, generating a positive feedback loop. Thus, LTbetaR triggering by LTi cells during lymph node development creates a local environment to which hematopoietic precursors are attracted and where they locally differentiate into fully mature, LTalpha(1)beta(2) expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C.

The lymphotoxin LTalpha(1)beta(2) controls postnatal and adult spleen marginal sinus vascular structure and function.

The lymphotoxin LTalpha(1)beta(2) supports the development and maintenance of several aspects of spleen structure, but its significance for marginal sinus (MS) vascular organization is unclear. We showed here that, in early postnatal lymphotoxin-deficient mice, the developing Flk-1+ white pulp vessels failed to organize or upregulate MAdCAM-1, leading to altered spatial rearrangement of both the white pulp endothelial cells and the smooth muscle actin-expressing cells. In vitro, MAdCAM-1 directed the reorganization of LTbeta receptor+ endothelial cells grown on Matrigel. LTalpha(1)beta(2) also regulated the maintenance of both MAdCAM-1 expression and mature MS structure in adult mice, contributing importantly to normal trafficking of CD11b+ cells in response to bacterial antigens. Together, our studies demonstrate that LTalpha(1)beta(2) and LTbeta receptor signals control proper development and maintenance of the mature MS structure and implicate MAdCAM-1 in the structuring of the MS endothelial cells that is important for the movement of immune cells within the spleen.

Lymphotoxin-mediated crosstalk between B cells and splenic stroma promotes the initial type I interferon response to cytomegalovirus.

Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.