Judy Peng

Cross-Regulation between Type I and Type II NKT Cells in Regulating Tumor Immunity: A New Immunoregulatory Axis

Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Abnormal NF-κB function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-κB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in ∼60% of patients. The monocyte and DC NF-κB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-κB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-κB-mediated signalling, moderated by TGF-β

Human papillomavirus‐like particles (HPV‐VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll‐like receptor 4 (TLR4) and nuclear factor (NF)‐κB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL‐10 production by DC but not by LC. The lack of LC activation was reversible when TGF‐β was removed from the LC medium. VLP‐induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF‐κB activation, heparin or TLR4 mAb. The data provide evidence that HPV‐VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF‐κB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF‐β, but not in situations in which TGF‐β concentrations are reduced.

Generation and maturation of dendritic cells for clinical application under serum-free conditions

Monocyte-derived dendritic cells (MoDCs) in clinical use for cancer immunotherapy are ideally generated in serum-free medium (SFM) with inclusion of a suitable maturation factor toward the end of the incubation period. Three good manfacturing practice (GMP) grade SFMs (AIM-V, X-VIVO 15, and X-VIVO 20) were compared with RPMI-1640, supplemented with 10% fetal bovine serum or 10% human serum. DCs generated for 7 days in SFM were less mature and secreted less interleukin (IL) 12p70 and IL-10 than DCs generated in 10% serum. DC yield was comparable in SFMs, and a greater proportion of cells was viable after maturation. Toll-like receptor (TLR) ligands were compared for their ability to induce cytokine secretion under serum-free conditions in the presence of interferon (IFN) γ. With the exception of Poly I:C, TLR ligands stimulated high levels of IL-10 secretion. High levels of IL-12p70 were induced by two TLR4-mediated stimuli, lipopolysaccharide and Ribomunyl, a clinical-grade bacterial extract. When T-cell responses were compared in allogeneic mixed leukocyte reaction, DCs stimulated with Ribomunyl induced higher levels of IFNγ than DCs stimulated with the cytokine cocktail: tumor necrosis factor-α, IL-1β, IL-6, and prostaglandin E2. In the presence of IL-10 neutralizing antibodies, DC IL-12p70 production and T-cell IFNγ were increased in vitro. Similarly, DCs stimulated with Ribomunyl, IFNγ, and anti-IL-10 induced high levels of tetanus toxoid-specific T-cell proliferation and IFNγ secretion. Thus, MoDCs generated in SFM efficiently stimulate T-cell IFNγ production after maturation in the presence of a clinical-grade TLR4 agonist and IL-10 neutralization.

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL‐12p70, through which they play a direct role in the development of IFN‐γ‐secreting Th1 cells, costimulation of CTL differentiation and NK‐cell activation. In contrast, IL‐10, which is also produced by DC, negatively regulates IL‐12 production. IL‐12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL‐12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL‐12p70 secretion by monocyte‐derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL‐12p70, presence of the IL10 high genotype restricted IL‐12p70 production in these individuals. These observations provide a further genetic control of IL‐12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function.

Sequence variation in the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen‐presenting cell (APC) function of infected B cells and immune escape by EBV‐specific T cells and thus contribute to the development of malignancy. Normal B cell‐associated LMP1 (B‐LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)‐κB subunit, RelB. We examined the ability of B‐LMP1 and a nasopharyngeal carcinoma‐associated LMP1 (NPC‐LMP1) to modulate B cell APC function and T‐cell responses. B lymphoma cells transfected with NPC‐LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B‐LMP1 transfectants. Unexpectedly, antigen presentation to CD4+ T helper cells was reduced owing to potentiation of regulatory T‐cell function by NPC‐LMP1 transfectants, which produce increased levels of interleukin‐10, rendering CD4+ T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC‐LMP1. These observations have important implications for the establishment of EBV‐associated malignancy in the context of infection with tumour‐associated EBV LMP1 variants.

Monocyte-derived Dc Primed With Tlr Agonists Secrete Il-12p70 in a Cd40-dependent Manner Under Hyperthermic Conditions

Fever is an evolutionarily conserved mechanism to improve survival during infection. Previous studies have shown that feverlike temperatures directly enhance the function of murine bone marrow-derived dendritic cells (DCs). In the present study, we examined the response of human monocyte-derived DC to 39.5°C hyperthermia. When primed with toll-like receptor agonists or bacterial extract but not proinflammatory cytokines, hyperthermia specifically enhanced secretion of interleukin (IL)-12p70 by DC, without altering the secretion of IL-10, tumor necrosis factor α or IL-1β. These DC induced significantly higher levels of T-cell proliferation and interferon γ production in assays of antigen presentation and MLR. Endogenous heat-sock protein 70 colocalized with CD40 in DC exposed to hyperthermic conditions. Recombinant CD40-Fc fusion protein blocked the increase in IL-12p70 secretion by DC primed with bacterial extract and hyperthermia. Thus, DC primed with toll-like receptor-agonists respond to hyperthermia with increased IL-12p70 secretion, mediated by heat-shock protein binding and activation of CD40. The data have important applications for clinical immunotherapy and the mechanism of fever.

Dendritic cell immunotherapy for melanoma

Dendritic cells (DC) are the most potent antigen-presenting cells that initiate T cell-mediated immune responses against cancer. It has been almost a decade since the first trial of DC-based cancer immunotherapy was published. Despite the many clinical trials conducted since, few solid conclusions have been reached, and no specific-immunotherapy has routinely demonstrated meaningful anti-tumour responses. Clinical-grade DC can be obtained from three distinct cell populations in the blood - monocytes, CD34(+) progenitors or direct isolation of circulating blood DC. This review discusses the science behind DC-based cancer immunotherapy, with a particular emphasis on the use of monocyte-derived DC in melanoma clinical trials, and the various potential avenues for improvement of patient clinical response rates.

Identification and isolation of synovial dendritic cells.

In rheumatoid arthitis patients, three compartments need to be considered: peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST). Dendritic cells (DC) characterized from each compartment have different properties. The methods given are based on cell sorting for isolation of cells, and flow cytometry and immunohistochemical staining for analysis of cells. Myeloid non-T cells are first enriched by density gradient centrifugation, sheep erythocyte rosetting, and, in some cases, magnetic immunodepletion. By flow cytometry, DC can then be analyzed or sorted based on two- or three-color immunofluorescence. Some variations on this basic theme are also outlined. The basic protocol for two-color immunohistochemistry of formalin-fixed ST is then given. This is based on the localization of the NFκB family member, RelB, to the nucleus of differentiated DC, and exclusion of B cells, macrophages, and follicular DC by double staining. Some variations that are particularly useful in frozen sections follow.

Defective NF-kappaB Activation in Response to LPS in Type 1 Diabetes Dendritic Cells

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.