The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses.

Lymphocyte- and leukocyte-mediated lymph node (LN) lymphatic sinus growth (lymphangiogenesis) is involved in immune responses and in diseases including cancer and arthritis. We previously discovered a 10.1.1 Ab that recognizes the lymphatic endothelial cell (LEC) surface protein mCLCA1, which is an interacting partner for LFA1 and Mac-1 that mediates lymphocyte adhesion to LECs. Here, we show that 10.1.1 Ab treatment specifically induces LEC proliferation, and influences migration and adhesion in vitro. Functional testing by injection of mice with 10.1.1 Ab but not control hamster Abs identified rapid induction of LN LEC proliferation and extensive lymphangiogenesis within 23 h. BrdU pulse-chase analysis demonstrated incorporation of proliferating LYVE-1-positive LEC into the growing medullary lymphatic sinuses. The 10.1.1 Ab-induced LN remodeling involved coordinate increases in LECs and also blood endothelial cells, fibroblastic reticular cells, and double negative stroma, as is observed during the LN response to inflammation. 10.1.1 Ab-induced lymphangiogenesis was restricted to LNs, as mCLCA1-expressing lymphatic vessels of the jejunum and dermis were unaffected by 23 h 10.1.1 Ab treatment. These findings demonstrate that 10.1.1 Ab rapidly and specifically induces proliferation and growth of LN lymphatic sinuses and stroma, suggesting a key role of mCLCA1 in coordinating LN remodeling during immune responses.

Culturing purifies murine lymph node lymphatic endothelium.

BACKGROUND: Lymph node (LN) lymphatic sinuses transport lymph, cells, and antigens from the periphery through the LN. The lymphatic endothelium lining these sinuses appears to be an important contributor to the lymph node immune response. It has been challenging to obtain sufficient LN lymphatic endothelial cells for investigation of their functions, as they are minor constituents of LNs. METHODS AND RESULTS: A procedure was developed to purify lymphatic endothelial cells (LEC) from murine LNs, which yields large numbers of primary LN LEC. Two-dimensional in vitro cultures of dissociated LN stromal cells initially consist of multiple cell types, and then rapidly evolve to produce pure cultures of lymphatic endothelium within a few passages. One million LEC can be harvested after 4 weeks of culture, and much larger cell numbers can be obtained by continued culturing over long periods. The LEC cultures maintain endothelial morphology and expression of LEC markers, and preserve the same slow growth characteristics over at least 20 passages. The LEC cultures readily form tubes in Matrigel at early and at late passages, resembling those formed by LEC lines. CONCLUSIONS: A simple and economical approach to obtain purified primary murine LN LEC was developed for in vitro studies of their function. The morphology, growth characteristics, and functional behavior of these cells in tube formation assays did not change between initial and long-term passages. Large numbers of these cells can be harvested after long-term passage, so that they can be studied in biochemical and biological assays.

BATF regulates the development and function of IL-17 producing iNKT cells.

BACKGROUND: BATF plays important roles in the function of the immune system. Batf null mice are deficient in both CD4+ Th17 cells and T follicular helper cells and possess an intrinsic B cell defect that leads to the complete absence of class switched Ig. In this study, Tg mice overexpressing BATF in T cells were used together with Batf null mice to investigate how altering levels of BATF expression in T cells impacts the development and function of a recently characterized population of iNKT cells expressing IL-17 (iNKT-17). RESULTS: BATF has a direct impact on IL-17 expression by iNKT cells. However, in contrast to the Th17 lineage where BATF activates IL-17 expression and leads to the expansion of the lineage, BATF overexpression restricts overall iNKT cell numbers while skewing the compartment in vivo and in vitro toward an iNKT-17 phenotype. CONCLUSIONS: This work is the first to demonstrate that BATF joins RORγt as the molecular signature for all IL-17 producing cells in vivo and identifies BATF as a component of the nuclear protein network that could be targeted to regulate IL-17-mediated disease. Interestingly, these studies also reveal that while the Il17a gene is a common target for BATF regulation in Th17 and iNKT-17 cells, this regulation is accompanied by opposite effects on the growth and expansion of these two cell lineages.

Batf coordinates multiple aspects of B and T cell function required for normal antibody responses.

Batf belongs to the activator protein 1 superfamily of basic leucine zipper transcription factors that includes Fos, Jun, and Atf proteins. Batf is expressed in mouse T and B lymphocytes, although the importance of Batf to the function of these lineages has not been fully investigated. We generated mice (Batf(DeltaZ/DeltaZ)) in which Batf protein is not produced. Batf(DeltaZ/DeltaZ) mice contain normal numbers of B cells but show reduced numbers of peripheral CD4(+) T cells. Analysis of CD4(+) T helper (Th) cell subsets in Batf(DeltaZ/DeltaZ) mice demonstrated that Batf is required for the development of functional Th type 17 (Th17), Th2, and follicular Th (Tfh) cells. In response to antigen immunization, germinal centers were absent in Batf(DeltaZ/DeltaZ) mice and the maturation of Ig-secreting B cells was impaired. Although adoptive transfer experiments confirmed that this B cell phenotype can be driven by defects in the Batf(DeltaZ/DeltaZ) CD4(+) T cell compartment, stimulation of Batf(DeltaZ/DeltaZ) B cells in vitro, or by a T cell-independent antigen in vivo, resulted in proliferation but not class-switch recombination. We conclude that loss of Batf disrupts multiple components of the lymphocyte communication network that are required for a robust immune response.