Faculty Bibliography

Interleukin-15 receptor alpha (IL-15R alpha) is a pleiotropically expressed molecule that chaperones and trans-presents IL-15 to NK and T cells. To investigate whether IL-15R alpha presented by different cells perform distinct physiological functions, we have generated four lines of mice lacking IL-15R alpha in various cell types. We find that IL-15R alpha expression on macrophages but not dendritic cells (DCs) supports the early transition of antigen specific effector CD8(+) T cells to memory cells. After memory CD8(+) T cell differentiation, IL-15R alpha expression on DCs selectively supports central memory CD8(+) T cells, whereas IL-15R alpha expression on macrophages supports both central and effector memory CD8(+) T cells. By contrast, mice lacking IL-15R alpha on macrophages, DCs, or both, exhibit equivalent defects in NK cell homeostasis and activation. These studies define unique roles for macrophage expression of IL-15R alpha and show that NK cells rely upon distinct IL-15R alpha dependent IL-15 signals than memory CD8(+) T cells. Moreover, they demonstrate the diversity, specification, and geographic restriction of cytokine signals.

T follicular helper (Tfh) cells represent a recently defined CD4(+) T cell subset characterized by the expression of the chemokine receptor CXCR5 and an enhanced ability to support B cells to mount antibody responses. Here, we demonstrate that lymph-node-resident CXCR5(+) Tfh cells and gut-homing integrin alpha(4)beta(7)-expressing T helper cells are generated as separate subsets in the gut-draining mesenteric lymph nodes. Type I interferon signaling in dendritic cells and in nonhematopoietic cells selectively stimulates Tfh cell development in response to antigen in conjunction with Toll-like receptor (TLR)3 or TLR4 agonists. Consistent with this, the ability of dendritic cells to produce the cytokine IL-6, required for in vivo Tfh differentiation, and antibody affinity maturation are both reduced in absence of type I interferon signaling. Thus, our results identify type I interferon as a natural adjuvant that selectively supports the generation of lymph node resident Tfh cells.

The development, homeostasis, and function of B lymphocytes involve multiple rounds of B-cell receptor (BCR)-controlled proliferation and prolonged maintenance. We analyzed the role of transcription factor Zfx, a recently identified regulator of hematopoietic stem cell maintenance, in B-cell development and homeostasis. Panhematopoietic or B cell-specific deletion of Zfx in the bone marrow blocked B-cell development at the pre-BCR selection checkpoint. Zfx deficiency in peripheral B cells caused accelerated B-cell turnover, depletion of mature recirculating B cells, and delayed T-dependent antibody responses. In addition, the numbers and function of B-1 cell lineage were reduced. Zfx-deficient B cells showed normal proximal BCR signaling, but impaired BCR-induced proliferation and survival in vitro. This was accompanied by aberrantly enhanced and prolonged integrated stress response and by delayed induction of cyclin D2 and Bcl-xL proteins. Thus, Zfx restrains the stress response and couples antigen receptor signaling to cell expansion and maintenance during B-cell development and peripheral homeostasis. These results identify a novel transcriptional regulator of the B-cell lineage and highlight the common genetic control of stem cell maintenance and lymphocyte homeostasis.

IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.

The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses. Plasmacytoid dendritic cell-derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus (MHV). The aim of this study was to determine the major target cell populations of this first wave of type I IFNs. Generation of bone marrow-chimeric mice expressing the type I IFN receptor (IFNAR) on either hemopoietic or non-bone marrow-derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells. To establish which cell population responds most efficiently to type I IFNs, mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV. This genetic analysis revealed that IFNAR expression on LysM+ macrophages and CD11c+ dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease. This study identifies type I IFN-mediated cross-talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other, as an essential cellular pathway for the control of fatal cytopathic virus infection.

Dendritic cells are critically involved in the promotion and regulation of T cell responses. Here, we report a mouse strain that lacks conventional CD11c(hi) dendritic cells (cDCs) because of constitutive cell-type specific expression of a suicide gene. As expected, cDC-less mice failed to mount effective T cell responses resulting in impaired viral clearance. In contrast, neither thymic negative selection nor T regulatory cell generation or T cell homeostasis were markedly affected. Unexpectedly, cDC-less mice developed a progressive myeloproliferative disorder characterized by prominent extramedullary hematopoiesis and increased serum amounts of the cytokine Flt3 ligand. Our data identify a critical role of cDCs in the control of steady-state hematopoiesis, revealing a feedback loop that links peripheral cDCs to myelogenesis through soluble growth factors, such as Flt3 ligand.

An early granulomatous response, characterized by collections of white blood cells at foci surrounding pathogens, occurs after infection by many intracellular organisms, including Listeria, but how these clusters become organized and for what purpose remain poorly understood. Here, we showed that dendritic cell (DC) activation by Listeria nucleated rapid clustering of innate cells, including granulocytes, natural killer (NK) cells, and monocytes, to sites of bacteria propagation where interleukin-12 was expressed in the spleen. Clustered NK cells expressed interferon-gamma (IFN-gamma), which was necessary for the activation and maturation of colocalized monocytes to tumor necrosis factor- and inducible nitric oxide synthase-producing DCs (TipDCs). NK cell clustering was necessary for IFN-gamma production and required pertussis-toxin-sensitive recruitment, in part mediated by the chemokine receptor CCR5, and MyD88 adaptor-mediated signaling. Thus, spatial organization of the immune response by DCs between 6 and 24 hr ensures functional activation of innate cells, which restricts pathogens before adaptive immunity is fully activated.

Plasmacytoid dendritic cells (PDCs) represent a unique immune cell type specialized in type I interferon (IFN) secretion in response to viral nucleic acids. The molecular control of PDC lineage specification has been poorly understood. We report that basic helix-loop-helix transcription factor (E protein) E2-2/Tcf4 is preferentially expressed in murine and human PDCs. Constitutive or inducible deletion of murine E2-2 blocked the development of PDCs but not of other lineages and abolished IFN response to unmethylated DNA. Moreover, E2-2 haploinsufficiency in mice and in human Pitt-Hopkins syndrome patients was associated with aberrant expression profile and impaired IFN response of the PDC. E2-2 directly activated multiple PDC-enriched genes, including transcription factors involved in PDC development (SpiB, Irf8) and function (Irf7). These results identify E2-2 as a specific transcriptional regulator of the PDC lineage in mice and humans and reveal a key function of E proteins in the innate immune system.

Toll-like receptors (TLRs) play prominent roles in initiating immune responses to infection, but their roles in particular cell types in vivo are not established. Here we report the generation of mice selectively lacking the crucial TLR-signaling adaptor MyD88 in dendritic cells (DCs). In these mice, the early production of inflammatory cytokines, especially IL-12, was substantially reduced after TLR stimulation. Whereas the innate interferon-gamma response of natural killer cells and of natural killer T cells and the Th1 polarization of antigen-specific CD4(+) T cells were severely compromised after treatment with a soluble TLR9 ligand, they were largely intact after administration of an aggregated TLR9 ligand. These results demonstrate that the physical form of a TLR ligand affects which cells can respond to it and that DCs and other innate immune cells can respond via TLRs and collaborate in promoting Th1 adaptive immune responses to an aggregated stimulus.

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.