Umer I. Chaudhry

Combined Stimulation with Interleukin-18 and CpG Induces Murine Natural Killer Dendritic Cells to Produce IFN-γ and Inhibit Tumor Growth

Natural killer dendritic cells (NKDC) are a novel subtype of dendritic cells with natural killer (NK) cell properties. IFN-gamma is a pleiotropic cytokine that plays an important role in the innate immune response to tumors. Based on our previous finding that the combination of Toll-like receptor 9 ligand CpG and interleukin (IL)-4 stimulates NKDC to produce IFN-gamma, we hypothesized that NKDC are the major IFN-gamma-producing dendritic cell subtype and may play a significant role in the host antitumor response. We found that under several conditions in vitro and in vivo NKDC accounted for the majority of IFN-gamma production by murine spleen CD11c(+) cells. IL-18 alone induced NKDC to secrete IFN-gamma, and the combination of IL-18 and CpG resulted in a synergistic increase in IFN-gamma production, both in vitro and in vivo. NK cells made 26-fold less IFN-gamma under the same conditions in vitro, whereas dendritic cells produced a negligible amount. The mechanism of IFN-gamma secretion by NKDC depended on IL-12. NKDC selectively proliferated in vitro and in vivo in response to the combination of IL-18 and CpG. Systemic treatment with IL-18 and CpG reduced the number of B16F10 melanoma lung metastases. The mechanism depended on NK1.1(+) cells, as their depletion abrogated the effect. IL-18 and CpG activated NKDC provided greater tumor protection than NK cells in IFN-gamma(-/-) mice. Thus, NKDC are the major dendritic cell subtype to produce IFN-gamma. The combined use of IL-18 and CpG is a viable strategy to potentiate the antitumor function of NKDC.

Murine liver plasmacytoid dendritic cells become potent immunostimulatory cells after Flt-3 ligand expansion

The liver has unique immunological properties. Although dendritic cells (DCs) are central mediators of immune regulation, little is known about liver DCs. Plasmacytoid DCs (pDCs) are a recently identified subtype of murine liver DC. We sought to define the function of freshly isolated murine liver pDCs. We found that normal liver pDCs were weak in stimulating T cells, yet they possessed a proinflammatory cytokine profile with high tumor necrosis factor‐α and low IL‐10 secretion. To facilitate the investigation of murine liver pDCs, we expanded them in vivo with fms‐like tyrosine kinase 3 ligand (Flt3L). After Toll‐like receptor‐9 ligation, expanded liver pDCs secreted high levels of IFN‐α and were able to stimulate NK cells, NKT cells, and antigen‐specific CD8+ T cells in vitro. In addition, Flt3L expansion alone generated pDCs capable of activating antigen‐specific CD8+ T cells in vivo. Conclusion: Unstimulated liver pDCs exist in a latent state with the potential to become potent activators of the innate and adaptive immune systems through their interactions with other immune effectors. Our findings have implications for understanding the role of the liver in tolerance and immunity. (HEPATOLOGY 2007;45:445–454.)

In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells

Natural killer dendritic cells (NKDC) are a unique class of murine immune cells that possess the characteristics of both natural killer (NK) cells and dendritic cells (DC). Because NKDC are able to secrete IFN‐γ, directly lyse tumor cells, and present antigen to naïve T cells, they have immunotherapeutic potential. The relative paucity of NKDC, however, impedes their detailed study. We have found that in vivo, overexpression of the hematopoietic cytokine Flt3 ligand (Flt3L) expands NKDC in various organs from 2–18 fold. Flt3L expanded splenic NKDC retain the ability to lyse tumor cells and become considerably more potent at activating naïve allogeneic and antigen‐specific T cells. Compared to normal splenic NKDC, Flt3L‐expanded splenic NKDC have a more mature phenotype, a slightly increased ability to capture and process antigen, and a similar cytokine profile. In vivo, we found that Flt3L‐expanded splenic NKDC are more effective than normal splenic NKDC in stimulating antigen‐specific CD8 T cells. Additionally, we show that NKDC are able to cross‐present antigen in vivo. The ability to expand NKDC in vivo using Flt3L will facilitate further analysis of their unique biology. Moreover, Flt3L‐expanded NKDC may have enhanced immunotherapeutic potential, given their increased ability to stimulate T cells.—Chaudhry, U. I., Katz, S. C., Kingham, T. P., Pillarisetty, V. G., Raab, J. R., Shah, A. B., and DeMatteo, R. P. In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells. FASEB J. 20, E108–E117 (2006)

NK Dendritic Cells Are Innate Immune Responders to Listeria monocytogenes Infection

NK dendritic cells (NKDC) are recently described immunologic cells that possess both lytic and Ag-presenting function and produce prolific quantities of IFN-γ. The role of NKDC in innate immunity to bacterial infection is unknown. Because IFN-γ is important in the immune response to Listeria monocytogenes (LM), we hypothesized that NKDC play a critical role during LM infection in mice. We found that LM increased the frequency and activation state of NKDC in vivo. Using in vivo intracellular cytokine analysis, we demonstrated that NKDC are a major source of early IFN-γ during infection with LM. Adoptive transfer of wild-type NKDC into IFN-γ-deficient recipients that were subsequently infected with LM decreased bacterial burden in the liver and spleen and prolonged survival. In contrast, NK cells were depleted early during LM infection, produced less IFN-γ, and conferred less protection upon adoptive transfer into IFN-γ-deficient mice. In vitro, LM induction of IFN-γ secretion by NKDC depended on TLR9, in addition to IL-18 and IL-12. Our study establishes NKDC as innate immune responders to bacterial infection by virtue of their ability to secrete IFN-γ.

Obstructive Jaundice Expands Intrahepatic Regulatory T Cells, Which Impair Liver T Lymphocyte Function but Modulate Liver Cholestasis and Fibrosis

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4+CD25+Foxp3+ regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4+CD25− T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

Management of Resectable Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is a rare neoplasm that recently has become an intense focus of scientific investigation, as it serves as a model for the molecular therapy for cancer. Although surgery remains the principle treatment of primary localized GIST, imatinib mesylate, a selective inhibitor of KIT protein, achieves dramatic responses in metastatic GIST. Multimodality therapy integrating surgery and molecular therapy has shown promise. This article summarizes the epidemiology, clinicopathologic features, natural history, and clinical management of GIST.

Biliary Obstruction Selectively Expands and Activates Liver Myeloid Dendritic Cells

Obstructive jaundice is associated with immunologic derangements and hepatic inflammation and fibrosis. Because dendritic cells (DCs) play a major role in immune regulation, we hypothesized that the immunosuppression associated with jaundice may result from the functional impairment of liver DCs. We found that bile duct ligation (BDL) in mice expanded the myeloid subtype of liver DCs from 20 to 80% of total DCs and increased their absolute number by >15-fold. Liver myeloid DCs following BDL, but not sham laparotomy, had increased Ag uptake in vivo, high IL-6 secretion in response to LPS, and enhanced ability to activate T cells. The effects of BDL were specific to liver DCs, as spleen DCs were not affected. Expansion of liver myeloid DCs depended on Gr-1+ cells, and we implicated monocyte chemotactic protein-1 as a potential mediator. Thus, obstructive jaundice selectively expands liver myeloid DCs that are highly functional and unlikely to be involved with impaired host immune responses.

NK Dendritic Cells Expanded in IL-15 Exhibit Antitumor Responses In Vivo

NK dendritic cells (NKDC) are a novel subtype of DC with NK cell properties. IL-15 is a pleiotropic cytokine that plays an obligate role in the proliferation and survival of NK cells. We hypothesized that IL-15 is also essential for NKDC development. NKDC were nearly absent in IL-15−/− mice, but restored by administration of exogenous IL-15. Treatment of wild-type mice with IL-15 caused a 2- to 3-fold expansion of both NK cells and NKDC. After 7 days of culture with IL-15, sorted splenic NKDC expanded 10-fold while NK cells increased 5-fold. NKDC expanded in IL-15 retained their cytolytic capacity but lost the ability to stimulate naive T cells. Meanwhile, NKDC expanded in IL-15 produced 10 times more IFN-γ as fresh NKDC and conferred protection in a tumor prevention model. Thus, IL-15 is essential to the proliferation and survival of NKDC and IL-15 expanded NKDC possess antitumor properties.

Conventional liver CD4 T cells are functionally distinct and suppressed by environmental factors.

The contribution of intrahepatic conventional T cells to the unique immunologic properties of the liver has not been clearly defined. We isolated bulk and CD4 T cells from mouse liver and compared their functions with each other and with their splenic counterparts. Unlike bulk spleen T cells, bulk liver T cells reacted minimally to allogeneic or antigen‐loaded syngeneic dendritic cells. However, after exclusion of natural killer T cells (NKTs) and γδ T cells by FACS, liver and spleen CD4 T cells actually proliferated to a similar extent upon allogeneic or antigen‐specific stimulation. Liver CD4 T cells were more sensitive to interleukin 2 (IL‐2) than were spleen CD4 T cells, but had a similar proliferative potential based on their response to CD3 ligation. In addition, activated liver CD4 T cells produced higher levels of IL‐4, IL‐5, IL‐10, and interferon gamma (IFN‐γ) than did splenic CD4 T cells. Therefore, liver CD4 T cells are intrinsically different from spleen CD4 T cells. In vitro, liver or spleen NKTs and γδ T cells suppressed liver and spleen CD4 T‐cell proliferation in a dose‐dependent fashion. In conclusion, unconventional T cells constrain liver CD4 T‐cell function. Our findings have implications for pathological conditions of the liver that involve the response of conventional CD4 T lymphocytes. (HEPATOLOGY 2005;42:293–300.)