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 (BatfΔZ/ΔZ) in which Batf protein is not produced. BatfΔZ/ΔZ 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 BatfΔZ/ΔZ 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 BatfΔZ/ΔZ 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 BatfΔZ/ΔZ CD4+ T cell compartment, stimulation of BatfΔZ/ΔZ 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.

BackgroundBATF 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).ResultsBATF 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.ConclusionsThis 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.

Activator protein-1 (AP-1) is a dimeric transcription factor composed of the Jun, Fos and Atf families of proteins. Batf is expressed in the immune system and participates in AP-1 dimers that modulate gene expression in response to a variety of stimuli. Transgenic (Tg) mice overexpressing human BATF in T cells were generated using the human CD2 promoter (CD2-HA (hemagglutinin antigen) - BATF). By 1 year of age, over 90% of the mice developed a lymphoproliferative disorder (LPD). The enlarged lymph nodes characteristic of this LPD contain a polyclonal accumulation of T cells with a CD4+ bias, yet efforts to propagate these tumor cells in vitro demonstrate that they do not proliferate as well as wild-type CD4+ T cells. Instead, the accumulation of these cells is likely due to an apoptotic defect as CD2-HA-BATF Tg T cells challenged by trophic factor withdrawal in vitro resist apoptosis and display a pro-survival pattern of Bcl-2 family protein expression. As elevated levels of Batf expression are a feature of lymphoid tumors in both humans and mice, these observations support the use of CD2-HA-BATF mice as a model for investigating the molecular details of apoptotic dysregulation in LPD.

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.

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.

The lymphatic system delivers fluid and molecules from tissues to the lymph node (LN) to mediate immune system surveillance of self and foreign antigens. LN lymphatic endothelial cells (LECs) function in orchestration of the immune response, and in reinforcement of peripheral tolerance. Extensive LN lymphatic sinus growth (lymphangiogenesis) is involved in multiple diseases including cancer, arthritis, and inflammation. Lymphocytes and leukocytes regulate this lymphangiogenesis; however, the mechanism is not well defined. We identified a monoclonal antibody (10.1.1) that recognizes the LEC protein mCLCA1. Our studies identified mCLCA1 as an interacting partner for LFA-1 and Mac-1 that mediates lymphocyte and leukocyte adhesion. The 10.1.1 Ab blocks adhesion of lymphocytes to LECs in vitro to a greater extent than ICAM-1 neutralizing Ab, suggesting that while ICAM-1 interactions predominate in vascular endothelium, mCLCA1 interaction is more important for adhesion to LECs. In addition to mediating adhesion, the 10.1.1 Ab (but not control Ab) induces rapid LN lymphangiogenesis within 23 hours after injection into mice, which involves proliferation of LN LECs. This lymphangiogenesis is accompanied by LN remodeling involving multiple stromal cell types, resembling the LN response to inflammation. These findings suggest that 10.1.1 Ab mimics lymphocyte or leukocyte B2-integrin binding to LEC mCLCA1, to drive LN remodeling and lymphangiogenesis during immune responses.

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