Michael D. Roth

Dendritic Cell Vaccination in Glioblastoma Patients Induces Systemic and Intracranial T-cell Responses Modulated by the Local Central Nervous System Tumor Microenvironment

Purpose: We previously reported that autologous dendritic cells pulsed with acid-eluted tumor peptides can stimulate T cell–mediated antitumor immune responses against brain tumors in animal models. As a next step in vaccine development, a phase I clinical trial was established to evaluate this strategy for its feasibility, safety, and induction of systemic and intracranial T-cell responses in patients with glioblastoma multiforme. Experimental Design: Twelve patients were enrolled into a multicohort dose-escalation study and treated with 1, 5, or 10 million autologous dendritic cells pulsed with constant amounts (100 μg per injection) of acid-eluted autologous tumor peptides. All patients had histologically proven glioblastoma multiforme. Three biweekly intradermal vaccinations were given; and patients were monitored for adverse events, survival, and immune responses. The follow-up period for this trial was almost 5 years. Results: Dendritic cell vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. One patient had an objective clinical response documented by magnetic resonance imaging. Six patients developed measurable systemic antitumor CTL responses. However, the induction of systemic effector cells did not necessarily translate into objective clinical responses or increased survival, particularly for patients with actively progressing tumors and/or those with tumors expressing high levels of transforming growth factor β2 (TGF-β2). Increased intratumoral infiltration by cytotoxic T cells was detected in four of eight patients who underwent reoperation after vaccination. The magnitude of the T-cell infiltration was inversely correlated with TGF-β2 expression within the tumors and positively correlated with clinical survival (P = 0.047). Conclusions: Together, our results suggest that the absence of bulky, actively progressing tumor, coupled with low TGF-β2 expression, may identify a subgroup of glioma patients to target as potential responders in future clinical investigations of dendritic cell–based vaccines.

Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 2

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-α, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-γ secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.

Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells.

The diverse roles of interferon‐α (IFN‐α) in regulating the immune response to infectious agents suggested that it might affect dendritic cell (DC) development. Peripheral blood mononuclear cells cultured with IFN‐α and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) developed a dendritic morphology and expressed high levels of the class I and II human leukocyte antigens (HLA), B7 costimulatory molecules, adhesion proteins, and CD40. Elevated DC expression of B7‐2 and HLA‐DR was observed with increasing IFN‐α concentrations up to 5000 U/mL. The effects of IFN‐α on DC immunophenotype were not reversed by adding neutralizing antibodies against interleukin‐4 (IL‐4) or tumor necrosis factor α to the cell cultures or by eliminating lymphocytes from the cultures. The addition of IFN‐α to cultures containing optimal concentrations of IL‐4 and GM‐CSF significantly increased the B7‐2 and HLA‐DR levels above those present on DCs grown in two cytokines. The DCs generated with IFN‐α and GM‐CSF were potent antigen‐presenting cells in allogeneic mixed leukocyte reactions. They also were capable of taking up, processing, and presenting tetanus toxin to autologous T lymphocytes. These results demonstrate an important role for IFN‐α in the generation of DCs with potent antigen‐presenting capabilities from peripheral blood monocytes. J. Leukoc. Biol. 64: 358–367; 1998.

Activation of Toll-Like Receptor 2 on Human Dendritic Cells Triggers Induction of IL-12, But Not IL-10

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1a+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.

Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells.

Tumors produce a number of immunosuppressive factors that block the maturation of CD34+ stem cells into dendritic cells (DC). We hypothesized that tumors might also interfere with the maturation and/or function of human monocyte-derived DC. In contrast to stem cells, we found that CD14+ cells responded to tumor culture supernatant (TSN) by increasing expression of APC surface markers, up-regulating nuclear translocation of RelB, and developing allostimulatory activity. Although displaying these characteristics of mature DC, TSN-exposed DC lacked the capacity to produce IL-12, did not acquire full allostimulatory activity, and rapidly underwent apoptosis. The effects of TSN appeared to be specific for maturing DC, and were not reversed by Abs against known DC regulatory factors including IL-10, vascular endothelial growth factor, TGF-β, or PGE2. Supernatants collected from nonmalignant cell sources had no effect on DC maturation. The altered maturation and early apoptosis of monocyte-derived DC may represent another mechanism by which tumors evade immune detection.

HUMAN CD14+ LEUKOCYTES ACQUIRE THE PHENOTYPE AND FUNCTION OF ANTIGEN-PRESENTING DENDRITIC CELLS WHEN CULTURED IN GM-CSF AND IL-4

The combination of granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) induces the growth of antigen‐presenting cells (APC) from adherent peripheral blood leukocytes. These cells have been characterized as dendritic cells (DC), yet many questions exist regarding their relationship to other DC populations and the nature of their progenitors. To address these issues, we utilized a combination of immunomagnetic depletion, cell sorting, and cell culture to isolate four distinct APC populations; macrophages expressing high levels of CD14 (CD14bright macrophages), DC produced by culturing adherent cells in GM‐CSF and IL‐4 (cultured DC), and two different subsets of fresh DC that express low levels of CD14 (CD14dim DC). Each population exhibited a unique morphology and a unique profile of cell surface markers. In contrast to macrophages, all three DC populations expressed the DC marker CD83, as well as high levels of MHC molecules and the costimulatory molecules B7‐1 (CD80) and B7‐2 (CD86). In addition, all three DC populations presented soluble tetanus toxin antigen and stimulated T cell proliferation to levels far superior to that of macrophages. Blocking studies demonstrated a costimulatory role for B7‐1, B7‐2, and CD40 in antigen presentation, although B7‐2 expression was the single most important factor. To identify the progenitors of cultured DC, we sorted the adherent fraction of PBMC into discrete subpopulations prior to exposure to GM‐CSF and IL‐4. DC activity derived entirely from CD14+ precursors and was equally demonstrable using either the CD14dim or CD14bright subsets. Although these DC precursors lost expression of CD14 in culture, they maintained most of their other myeloid features. We conclude that human CD14+ leukocytes acquire the phenotype and function of DC when cultured in GM‐CSF and IL‐4.

Δ-9-Tetrahydrocannabinol Inhibits Antitumor Immunity by a CB2 Receptor-Mediated, Cytokine-Dependent Pathway

In this study, we show that Δ-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, suppresses host immune reactivity against lung cancer. In two different weakly immunogenic murine lung cancer models, intermittent administration of THC (5 mg/kg, four times/wk i.p. for 4 wk) led to accelerated growth of tumor implants compared with treatment with diluent alone. In contrast to our findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing SCID mice. The immune inhibitory cytokines, IL-10 and TGF-β, were augmented, while IFN-γ was down-regulated at both the tumor site and in the spleens of THC-treated mice. Administration of either anti-IL-10- or anti-TGF-β-neutralizing Abs prevented the THC-induced enhancement in tumor growth. Both APC and T cells from THC-treated mice showed limited capacities to generate alloreactivity. Furthermore, lymphocytes from THC-treated mice transferred the effect to normal mice, resulting in accelerated tumor growth similar to that seen in the THC-treated mice. THC decreased tumor immunogenicity, as indicated by the limited capacity for tumor-immunized, THC-treated mice to withstand tumor rechallenge. In vivo administration of a specific antagonist of the CB2 cannabinoid receptor also blocked the effects of THC. Our findings suggest the THC promotes tumor growth by inhibiting antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.

Δ9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells

Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and Delta 9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625-5 microg/ml). THC suppressed T cell proliferation, inhibited the production of interferon-gamma and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-gamma, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway.

High-Resolution CT Scan Findings in Patients With Symptomatic Scleroderma-Related Interstitial Lung Disease

BACKGROUND Lung disease has become the leading cause of mortality and morbidity in scleroderma (SSc) patients. The frequency, nature, and progression of interstitial lung disease seen on high-resolution CT (HRCT) scans in patients with diffuse SSc (dcSSc) compared with those with limited SSc (lcSSc) has not been well characterized. METHODS Baseline HRCT scan images of 162 participants randomized into a National Institutes of Health-funded clinical trial were compared to clinical features, pulmonary function test measures, and BAL fluid cellularity. The extent and distribution of interstitial lung disease HRCT findings, including pure ground-glass opacity (pGGO), pulmonary fibrosis (PF), and honeycomb cysts (HCs), were recorded in the upper, middle, and lower lung zones on baseline and follow-up CT scan studies. RESULTS HRCT scan findings included 92.9% PF, 49.4% pGGO, and 37.2% HCs. There was a significantly higher incidence of HCs in the three zones in lcSSc patients compared to dcSSc patients (p = 0.034, p = 0.048, and p = 0.0007, respectively). The extent of PF seen on HRCT scans was significantly negatively correlated with FVC (r = - 0.22), diffusing capacity of the lung for carbon monoxide (r = - 0.44), and total lung capacity (r = - 0.36). A positive correlation was found between pGGO and the increased number of acute inflammatory cells found in BAL fluid (r = 0.28). In the placebo group, disease progression was assessed as 30% in the upper and middle lung zones, and 45% in the lower lung zones. No difference in the progression rate was seen between lcSSc and dcSSc patients. CONCLUSIONS PF and GGO were the most common HRCT scan findings in symptomatic SSc patients. HCs were seen in more than one third of cases, being more common in lcSSc vs dcSSc. There was no relationship between progression and baseline PF extent or lcSSc vs dcSSc. TRIAL REGISTRATION Clinicaltrials.gov Identifier: NCT00004563.

Respiratory and Immunologic Consequences of Marijuana Smoking

Habitual smoking of marijuana has a number of effects on the respiratory and immune systems that may be clinically relevant. These include alterations in lung function ranging from no to mild airflow obstruction without evidence of diffusion impairment, an increased prevalence of acute and chronic bronchitis, striking endoscopic findings of airway injury (erythema, edema, and increased secretions) that correlate with histopathological alterations in bronchial biopsies, and dysregulated growth of the bronchial epithelium associated with altered expression of nuclear and cytoplasmic proteins involved in the pathogenesis of bronchogenic carcinoma. Other consequences of regular marijuana use include ultrastructual abnormalities in human alveolar macrophages along with impairment of their cytokine production, antimicrobial activity, and tumoricidal function. Cannabinoid receptor expression is altered in leukocytes collected from the blood of chronic smokers, and experimental models support a role for Δ9‐tetrahydrocannabinol in suppressing T cell function and cell‐mediated immunity. The potential for marijuana smoking to predispose to the development of respiratory malignancy is suggested by several lines of evidence, including the presence of potent carcinogens in marijuana smoke and their resulting deposition in the lung, the occurrence of premalignant changes in bronchial biopsies obtained from smokers of marijuana in the absence of tobacco, impairment of antitumor immune defenses by Δ9‐tetrahydrocannabinol, and several clinical case series in which marijuana smokers were disproportionately overrepresented among young individuals who developed upper or lower respiratory tract cancer. Additional well‐designed epidemiological and immune monitoring studies are required to determine the potential causal relationship between marijuana use and the development of respiratory infection and/or cancer.