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B6.SJL-Ptprca Pepcb/Boy (CD45.1) mice have been used in hundreds of congenic competitive transplants, with the presumption that they differ from C57BL/6 mice only at the CD45 locus. In this study, we describe a point mutation in the natural cytotoxicity receptor 1 (Ncr1) locus fortuitously identified in the CD45.1 strain. This point mutation was mapped at the 40th nucleotide of the Ncr1 locus causing a single amino acid mutation from cysteine to arginine at position 14 from the start codon, resulting in loss of NCR1 expression. We found that these mice were more resistant to CMV due to a hyper innate IFN-γ response in the absence of NCR1. In contrast, loss of NCR1 increased susceptibility to influenza virus, a result that is consistent with the role of NCR1 in the recognition of influenza Ag, hemagglutinin. This work sheds light on potential confounding experimental interpretation when this congenic strain is used as a tool for tracking lymphocyte development.

Hematopoietic stem cell transplant therapy is limited by pulmonary infections. Mice with fully reconstituted hematopoietic compartments, including alveolar macrophages (AMs), after bone marrow transplantation (BMT) have impaired host defense against Gram-negative Pseudomonas aeruginosa. Impaired innate immunity is related to increased production of PGE2 by AMs. Cyclooxygenase (COX)-2 is the rate-limiting enzyme for synthesis of PGE2 from arachidonic acid, and COX-2 expression is elevated in AMs post-BMT. We hypothesized that epigenetic mechanisms may be responsible for upregulation of COX-2 in AMs. Using bisulfite sequencing, we observed the 5′-untranslated region and exon 1 of the COX-2 gene is hypomethylated in the AMs of BMT mice compared with control. COX-2 expression was increased in primary AMs and in the AM cell line (MHS) after treatment with 5-aza-2′-deoxycytidine (a methyltransferase inhibitor). Methylation by SssI methyltransferase of a 698-bp region of the COX-2 promoter including the beginning of exon 1 driving a luciferase reporter silenced luciferase expression. Because TGF-β1 is elevated in lungs post-BMT, we tested whether TGF-β1 could promote expression of COX-2 in a hypermethylated COX-2 vector, and observed TGF-β1–induced modest expression of COX-2, suggesting an ability to demethylate the promoter. Finally, BMTs performed with marrow from mice expressing a dominant-negative form of the TGF-βRII on CD11c-expressing cells (which includes AMs) demonstrated improved host defense and AM function. Our findings suggest impaired innate immunity and PGE2 elevation post-BMT are due to hypomethylation of the COX-2 gene, which is at least partly regulated by TGF-β1.

A large gap in our understanding of infant immunity is why natural killer (NK) cell responses are deficient, which makes infants more prone to viral infection. Here we demonstrate that transforming growth factor-β (TGF-β) was responsible for NK cell immaturity during infancy. We found more fully mature NK cells in CD11c(dnR) mice, whose NK cells lack TGF-β receptor (TGF-βR) signaling. Ontogenic maturation of NK cells progressed faster in the absence of TGF-β signaling, which results in the formation of a mature NK cell pool early in life. As a consequence, infant CD11c(dnR) mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-β in ontogeny that can explain why NK cell responses are deficient early in life.

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. Balb/c mice lacking LY49H are more susceptible to MCMV infection than B6 mice. Strikingly, CD11cdnR mice that lack TGFβR signaling in NK cells are protected from MCMV infection in both strains. It is established that unlicensed NK cells that are not inhibited by self-MHC class I can control viral infection more than licensed NK cells. We found that CD11cdnR mice have increased frequency of unlicensed (LY49G2+) NK cells and slightly decreased frequency of licensed (LY49C/I+) NK cells in B6 strain. Moreover, TGFβ-resistant NK cells showed more activated phenotypes such as increased granzyme B protein expression and more KLRG1+ expression than control NK cells in both licensed and unlicensed cells. Interestingly, in CD11cdnR mice even unlicensed NK cells have a significant capacity to produce IFN-γ when triggered by anti-NK1.1, but not in control mice. Our data suggest that inactivation of TGFβR signaling might have a critical role in the licensing of NK cell and lead to the resistance against MCMV infection.

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFβ has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFβ signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFβ signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4+ cells (that develop CR and express intragraft TGFβ) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFβ). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFβ signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4+ T cells, T cell TGFβ signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFβ-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFβ.

A small pool of NK1.1+ CD8+ T cells is harbored among the conventional CD8+ T cell compartment. Conclusions drawn from the analysis of immune responses mediated by cytotoxic CD8+ T cells are often based on the total population, which includes these contaminating NK1.1+ CD8+ T cells. An unresolved question is whether NK1.1+ CD8+ cells are conventional T cells that acquire NK1.1 expression upon activation or delineation into memory phenotype or whether they are a distinct cell population that induces immune responses in a different manner than conventional T cells. To address this question, we used the Listeria monocytogenes model of infection and followed CD8+ NK1.1+ T cells and NK1.1− CD8+ T cells during each phase of the immune response: innate, effector, and memory. Our central finding is that CD8+ NK1.1+ cells and conventional NK1.1− CD8+ T cells both contribute to the adaptive immune response to Listeria, but only CD8+ NK1.1+ cells were equipped with the ability to provide a rapid innate immune response, as demonstrated by early and Ag-independent IFN-γ production, granzyme B expression, and degranulation. More importantly, purified conventional CD8+ T cells alone, in the absence of any contaminating CD8+ NK1.1+ cells, were not sufficient to provide early protection to lethally infected mice. These results highlight the role of CD8+ NK1.1+ T cells in mounting early innate responses that are important for host defense and support the therapeutic potential of this subset to improve the effectiveness of protective immunity.

The live attenuated yellow fever vaccine 17D (YF-17D) is one of the most effective vaccines available, with a 65-yr history of use in >400 million people globally. Despite this efficacy, there is presently no information about the immunological mechanisms by which YF-17D acts. Here, we present data that suggest that YF-17D activates multiple Toll-like receptors (TLRs) on dendritic cells (DCs) to elicit a broad spectrum of innate and adaptive immune responses. Specifically, YF-17D activates multiple DC subsets via TLRs 2, 7, 8, and 9 to elicit the proinflammatory cytokines interleukin (IL)-12p40, IL-6, and interferon-α. Interestingly, the resulting adaptive immune responses are characterized by a mixed T helper cell (Th)1/Th2 cytokine profile and antigen-specific CD8+ T cells. Furthermore, distinct TLRs appear to differentially control the Th1/Th2 balance; thus, whilst MyD88-deficient mice show a profound impairment of Th1 cytokines, TLR2-deficient mice show greatly enhanced Th1 and Tc1 responses to YF-17D. Together, these data enhance our understanding of the molecular mechanism of action of YF-17D, and highlight the potential of vaccination strategies that use combinations of different TLR ligands to stimulate polyvalent immune responses.

Immunosurveillance by Natural killer (NK) cells provides an efficient effector mechanism to eliminate tumor and virally infected cells. A well-defined characteristic of NK cells is their ability to lyse target cells deficient in MHC class I expression or target cells that express non-MHC ligands as a result of infection or transformation. This observation has been the basis for identification of a number of inhibitory and activatory NK cell receptors. Although the collective effort during the last decade shed light on the function of many NK cell receptors, the role of NCR1 in NK cell biology remains highly controversial. We have recently identified a spontaneous mutation of NCR1 in our C57BL/6 mouse colony. In this mutant, NCR1 expression was absent on conventional NK cells of all lymphoid organs as well as on innate lymphocytes (ILC3) of the gut lamina propria. Of note, NCR1 expression was absent on the surface as well as in the intracellular compartment of NK cells. Likewise, lack of NCR1 expression was confirmed in mixed BM chimera indicating a cell intrinsic phenotype. Only genetic backcrossing could correct the lack of NCR1 in these mice. ORF sequencing of NCR1 gene in mutant mice revealed a point mutation (C14R) in the region of signal peptide of NCR1 protein. However, while development and homeostasis of NK cells was not affected by the lack of NCR1, infection of NCR1C14R mutant mice with murine cytomegalovirus resulted in increased resistance, an advantage that correlated with NK cell hyperresponsive production of IFN-γ, but not cytotoxic activity. Moreover, NCR1 limits NK cell IFNγ response via regulation of Dok-1 phosphorylation and p38 activation. Collectively, our data reveal the role for NCR1 in IFNγ production of NK cell.

Proliferation and differentiation of CD4 + T cells are often correlated, but it is not clear whether they are mechanistically linked. When antigen-specific T cells are present at high frequency in vivo, they all respond to antigenic peptide stimulation by expressing activation markers, but only a subset begins to proliferate. However, noncycling cells may synthesize the effector cytokine IFNγ even though their cell cycle is blocked in G1. These data show that proliferation and effector function are not rigidly linked in T cells. Instead, CD4 + T cells have the flexibility to engage in or bypass clonal expansion based on the integration of multiple signals, including the frequency of other responding T cells.

International audience; B lymphocyte generation in bone marrow (BM) compensates for cell loses. The Fas / Fas ligand (FasL) pathway has been implicated in apoptosis of various cell types. Abnormalities of the Fas receptor or of FasL expression are associated with excessive T cell proliferation and autoimmunity. To examine the role of the Fas / FasL system in B cell differentiation, we created double‐chimeric mice by transferring both C57BL / 6 (B6)‐Fas+ and lpr‐FasL+ BM cells into RAG‐2– / – hosts. Equal numbers of stem cells were co‐injected into sublethally irradiated recipients, and their progeny were studied by using antibodies directed against the B6‐Ly5.1+5.2+ and lpr‐Ly5.1–5.2+ populations. A longitudinal study lasting for up to 6 months revealed that cells of the lpr phenotype dominated the B6 phenotype in the BM, as a result of their active proliferation. Analysis of the B cell compartment showed more lpr than B6 cells among immature HSAhiB220lo populations. In contrast, the lpr and B6 phenotypes were equally represented among mature B cells. BM transfer to second hosts indicated that B6‐derived B cell progenitors were absent from the first host. These data suggest that activation of the Fas / FasL pathway disturbs the early steps of B cell development and might therefore contribute to the onset of autoimmune disorders.

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

The signals that control decisions of progenitor commitment involve the interplay of both cytokines and transcription factors. Flt3L has emerged as a potential regulator of dendritic cell (DC) development, but regulation of HSC commitment to the DC lineage remains poorly understood. Our central finding is the identification of STAT3 activation as a checkpoint of Flt3L-regulated DC development. Deletion of STAT3 caused profound deficiency in the DC compartment and abrogated Flt3L effects on DC development. DC derivation by Flt3L revealed a normal HSC pool, a 2- to 3-fold accumulation of CLP/CMP, but absence of common DC precursors as well as their DC progeny in STAT3-deficient mice. The formation of CMP and CLP represents the first decisive lineage commitment step, and in this regard we provide evidence that commitments of CLP/CMP to the DC lineage strictly depend on the interplay of both Flt3L and STAT3 activation.

International audience; Fas‐mediated apoptosis is a form of cell death that operates through a Fas‐Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas‐mutated lymphocytes. Irradiated RAG2–/– recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long‐term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas– T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL‐mediated cytotoxic effect on normal‐derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas–) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas– T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.