Alaap B. Shah

Liver Dendritic Cells Are Less Immunogenic Than Spleen Dendritic Cells because of Differences in Subtype Composition

The unique immunological properties of the liver may be due to the function of hepatic dendritic cells (DC). However, liver DC have not been well characterized because of the difficulty in isolating adequate numbers of cells for analysis. Using immunomagnetic bead and flow cytometric cell sorting, we compared freshly isolated murine liver and spleen CD11c+ DC. We found that liver DC are less mature, capture less Ag, and induce less T cell stimulation than spleen DC. Nevertheless, liver DC were able to generate high levels of IL-12 in response to CpG stimulation. We identified four distinct subtypes of liver DC based on the widely used DC subset markers CD8α and CD11b. Lymphoid (CD8α+CD11b−) and myeloid (CD8α−CD11b+) liver DC activated T cells to a similar degree as did their splenic DC counterparts but comprised only 20% of all liver DC. In contrast, the two more prevalent liver DC subsets were only weakly immunostimulatory. Plasmacytoid DC (B220+) accounted for 19% of liver DC, but only 5% of spleen DC. Our findings support the widely held notion that liver DC are generally weak activators of immunity, although they are capable of producing inflammatory cytokines, and certain subtypes potently activate T cells.

Natural Killer Dendritic Cells Have Both Antigen Presenting and Lytic Function and in Response to CpG Produce IFN-γ via Autocrine IL-12

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-γ than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-γ via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.

Liver Sinusoidal Endothelial Cells Are Insufficient to Activate T Cells

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45−) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcγRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4+ or CD8+ TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.

Murine Flt3 Ligand Expands Distinct Dendritic Cells with Both Tolerogenic and Immunogenic Properties

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c+ and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-γ, and TNF-α, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4+ T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.

Endogenous Granulocyte-Macrophage Colony-Stimulating Factor Overexpression In Vivo Results in the Long-Term Recruitment of a Distinct Dendritic Cell Population with Enhanced Immunostimulatory Function

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 × 108 cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8α+CD4− or CD8α−CD4− but not CD8α+CD4+ or CD8α−CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-α compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-γ. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-α. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells.

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)

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.

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.)

Natural killer cell depletion confounds the antitumor mechanism of endogenous IL‐12 overexpression

IL‐12 gene transfer to hepatocytes using a recombinant adenovirus vector (AdIL‐12) has been shown to protect against primary and metastatic liver tumors in mice. However, the mechanism of protection has been elusive and studies using depleting monoclonal antibodies or transgenic mice have purported it to be independent of T and NK cells. We postulated that depletion of NK cells may distort the experimental model and misrepresent the antitumor mechanism by altering the magnitude and duration of transgene expression. We show in mice treated with AdIL‐12 that NK depletion increased serum IL‐12 levels by more than 250‐fold and prolonged transgene expression by nearly 2 weeks compared to nondepleted mice. To determine the contribution of NK cells to tumor protection after AdIL‐12 treatment, we analyzed NK cells from treated animals. Isolated NK cells were markedly activated in terms of their lytic activity and IFN‐γ secretion. Adoptive transfer of NK cells from mice that had been treated with AdIL‐12 to naive mice was sufficient to confer protection against colorectal hepatic metastases. This protection was mediated in part by NK‐cell production of IFN‐γ. Our findings indicate that NK‐cell depletion distorts the model of systemic AdIL‐12 administration by markedly altering transgene expression, which then may potentiate other antitumor mechanisms, and that endogenous IL‐12 overexpression activates NK cells, rendering them sufficient to protect against liver metastases. These data have critical implications for investigating the immunologic mechanisms of experimental models that utilize gene transfer.