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Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication

B-cell functions in antitumor immunity are not well understood. In this study,we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag),D5 mouse melanoma,or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments,spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors),IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells. View Publication

In herpesvirus infections,the virus persists for life but is contained through T-cell-mediated immune surveillance. How this immune surveillance operates is poorly understood. Recent studies of other persistent infections have indicated that virus persistence is associated with functional deficits in the CD8(+) T-cell response. To test whether this is the case in a herpesvirus infection,we used a mutant murine gammaherpesvirus that is defective in its ability to persist in the host. By comparing the immune response to this virus with a revertant virus that can persist,we were able to dissect the changes in the antiviral CD8(+) T-cell response that are induced by virus persistence. Surprisingly,persistently infected mice controlled a secondary challenge infection more rapidly than nonpersistently infected mice,indicating enhanced rather than diminished effector functions. Consistent with this,virus-specific CD8 T cells from these mice exhibited faster upregulation of the cytotoxic mediator granzyme B. Another unexpected finding was that CD8(+) T cells from neither infection responded efficiently to homeostatic cytokines. The unresponsiveness of the memory cells from the nonpersistently infected mice appears to be linked to the prolonged replication of virus within the lungs. Other changes seen in different chronic infection models were also observed,such as changes in Bcl-2 levels,interleukin-2 production,and the immunodominance hierarchy. These data show persistence of gammaherpesvirus type 68 alters the properties of CD8(+) T cells and illustrates that immune surveillance does not require CD8 T cells with the same attributes as classical" memory CD8(+) T cells." View Publication

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition,vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy. View Publication

Interferon regulatory factor-1 (IRF-1) is an important transcription factor that mediates interferon-gamma (IFN-gamma)-induced cell-signaling events. In this study,we examined whether 17beta-estradiol alters IRF-1 in splenic lymphocytes,in view of the immunomodulatory effects of this natural female sex hormone including its ability to alter IFN-gamma levels. We find that IRF-1 expression is markedly downregulated in splenocytes or purified T-cells from estrogen-treated mice at all time points studied when compared with their placebo counterparts. This decrease in IRF-1 in splenocytes from estrogen-treated mice is neither due to upregulation of IRF-1-interfering proteins (nucleophosmin or signal transducer and activator of transcription (STAT)-5) nor due to alternatively spliced IRF-1 mRNA. Given that IFN-gamma is a potent inducer of IRF-1,direct addition of recombinant IFN-gamma to splenocytes from either wild-type or IFN-gamma-knockout mice,or the addition of recombinant IFN-gamma to purified T-cells,was expected to stimulate IRF-1 expression. However,robust expression of IRF-1 in cells from estrogen-treated mice was not seen,unlike what was observed in cells from placebo-treated mice. Diminished IFN-gamma induction of IRF-1 in cells from estrogen-treated mice was noticed despite comparable phosphorylated STAT-1 activation. These studies are the first to show that estrogen regulates IFN-gamma-inducible IRF-1 in lymphoid cells,a finding that may have implications to IFN-gamma-regulated immune and vascular diseases. View Publication

Although natural killer (NK) cells are well known for their ability to kill tumors,few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here,we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1,ULBP2,and ULBP3. In line with the NK receptor ligand expression profiles,antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. View Publication

The presence of persistent,latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models,which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses,which comprise the vast majority of all infected cells,and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here,we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA,but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus,ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus,our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection. View Publication

Priming of human NK cells with IL-2 is necessary to render them functionally competent upon NKG2D engagement. We examined the underlying mechanisms that control NKG2D responsiveness in NK cells and found that IL-2 upregulates expression of the amino acid transporters SLC1A5 and CD98. Using specific inhibitors to block SLC1A5 and CD98 function,we found that production of IFN-γ and degranulation by CD56bright and CD56dim NK cells following NKG2D stimulation were dependent on both transporters. IL-2 priming increased the activity of mTORC1,and inhibition of mTORC1 abrogated the ability of the IL-2-primed NK cells to produce IFN-γ in response to NKG2D-mediated stimulation. This study identifies a series of IL-2-induced cellular changes that regulates the NKG2D responsiveness in human NK cells. View Publication

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity,which links a given individual stimulus to a unique activated state. Here,we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells. View Publication

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1),suggesting a role in neutrophil migration. However,CD177pos neutrophils exhibit no clear migratory advantage in vivo,despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system,we found that CD177pos and CD177neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177pos neutrophils,an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly,CD177 ligation enhanced its interaction with β2 integrins,as revealed by fluorescence lifetime imaging microscopy,leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity,impaired internalization of integrin attachments,and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. View Publication