Dissection of stromal and cancer cell-derived signals in melanoma xenografts before and after treatment with DMXAA.

BACKGROUND: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. METHODS: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. RESULTS: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. CONCLUSION: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Analysis of successful immune responses in persons infected with hepatitis C virus.

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-gamma, a "stunned" phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes.

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Detailed characterisation of CB2 receptor protein expression in peripheral blood immune cells from healthy human volunteers using flow cytometry.

It is commonly accepted from gene expression studies that the CB2 receptor is expressed by most cell types of the rodent and human immune system. However, the exact identity of cells expressing CB2 receptor protein in human blood or the abundance of receptors expressed by each immune subset is not well characterised. We conducted a detailed analysis of CB2 protein levels expressed by blood-derived immune cells from healthy human donors. Flow-cytometry was conducted using 4 commercially available anti-CB2 polyclonal antibodies in conjunction with a selection of immune cell specific markers. Across multiple healthy subjects we observed that NK cells, B-lymphocytes and monocytes expressed a higher level of CB2 receptor than CD4+ or CD8+ T-lymphocytes. Neutrophils also expressed a low level of CB2 receptor. NK cells had the greatest variation in CB2 expression levels, whereas for each of the other cell types CB2 levels were relatively similar between subjects. In contrast to other methods, the high sensitivity of flow-cytometry revealed that CB2 receptors are present on resting T-lymphocytes at low abundance in some healthy subjects. These data provide the first detailed analysis of CB2 protein levels in blood leukocyte subsets from healthy donors and identifies the cell types which could be targeted with CB-mimetic drugs in humans.

Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.

Although cytotoxic T lymphocytes (CTLs) are thought to be involved in the control of human immunodeficiency virus-type 1 (HIV-1) infection, it has not been possible to demonstrate a direct relation between CTL activity and plasma RNA viral load. Human leukocyte antigen-peptide tetrameric complexes offer a specific means to directly quantitate circulating CTLs ex vivo. With the use of the tetrameric complexes, a significant inverse correlation was observed between HIV-specific CTL frequency and plasma RNA viral load. In contrast, no significant association was detected between the clearance rate of productively infected cells and frequency of HIV-specific CTLs. These data are consistent with a significant role for HIV-specific CTLs in the control of HIV infection and suggest a considerable cytopathic effect of the virus in vivo.

Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood.

Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.

Use of fluorogenic histocompatibility leukocyte antigen-A*0201/HPV 16 E7 peptide complexes to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous human papillomavirus antigens.

Cervical cancer (CaCx) is the second most common female malignancy worldwide and remains a clinical problem despite improvements in early detection and therapy. CaCx and preinvasive cervical intraepithelial neoplasia (CIN3) are strongly associated with infection by human papillomavirus (HPV), particularly types 16 and 18. Two nonstructural viral proteins, E6 and E7, are constitutively expressed in cervical tumors and are crucial for the maintenance of the transformed phenotype. These proteins thus provide attractive targets for immunotherapy of CaCx mediated by CD8+ CTLs. However, reliable detection and generation of HPV-specific CTLs in humans has been difficult. Recently, soluble fluorogenic MHC-peptide complexes (tetramers) have greatly increased the sensitivity of antiviral and antitumor CTL detection. To examine the feasibility of this approach for detecting HPV-specific CTLs, we constructed a tetramer consisting of HLA-A*0201 and the best studied HPV CTL peptide epitope, HPV 16 E711-20. Between 2 and 12% of short-term HPV 16 E711-2 CTL lines derived from CaCx patients stained highly with the tetramer. Direct ex vivo staining of peripheral blood mononuclear cells revealed CD8+ tetramer+ high cells at low frequencies in both CIN3 patients (1 of 1,260 to 1 of 19,073) and normal controls (1 of 1,855 to 1 of 42,004). However, short-term in vitro stimulation with the HPV 16 E711-20 peptide expanded CD8+tetramer+ cells to a greater extent in the peripheral blood mononuclear cells from CIN3 patients. Furthermore, the tetramer provided a powerful tool to isolate polyclonal and clonal peptide-specific CTLs from an established HPV 16 E7,11-20-specific CTL line. These purified CTLs were able to lyse both peptide-pulsed targets and targets expressing endogenously processed HPV antigens. This tetramer may therefore be useful for selecting rare high-affinity HPV-specific CTLs for the immunotherapy of CaCx.

Serological Evidence of Immune Priming by Group A Streptococci in Patients with Acute Rheumatic Fever.

Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to 'prime' the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host.

Efficient expression of the tumor-associated antigen MAGE-3 in human dendritic cells, using an avian influenza virus vector.

Dendritic cells (DCs) are the most potent inducers of immune reactions. Genetically modified DCs, which express tumor-associated antigens (TAA), can efficiently induce antitumor immunity and thus have a high potential as tools in cancer therapy. The gene delivery is most efficiently achieved by viral vectors. Here, we explored the capacity of influenza virus vectors to transduce TAA genes. These viruses abortively infect DCs without interfering with their antigen-presenting capacity. In contrast to other viruses used for DC transduction, influenza viruses can be efficiently controlled by antiviral pharmaceuticals, lack the ability to integrate into host chromosomes, and fail to establish persistent infections. Genes encoding a melanoma-derived TAA (MAGE-3), or the green fluorescence protein (GFP), were introduced into a high-expression avian influenza virus vector. Monocyte-derived mature DCs infected by these recombinants efficiently produced GFP or MAGE-3. More than 90% of the infected DCs can express a transduced gene. Importantly, these transduced DCs retained their characteristic phenotype and their potent allogeneic T cell stimulatory capacity, and were able to stimulate MAGE-3-specific CD8(+) cytotoxic T cells. Thus influenza virus vectors provide a highly efficient gene delivery system in order to transduce human DCs with TAA, which consequently stimulate TAA-specific T cells.

Exploiting retrograde transport of Shiga-like toxin 1 for the delivery of exogenous antigens into the MHC class I presentation pathway.

Shiga-like toxin 1 (SLT) from Escherichia coli O157:H7 enters mammalian cells by endocytosis from the cell surface to the endoplasmic reticulum before translocating into the cytosol. Here, SLT was engineered at its N- or C-terminus to carry a peptide derived from influenza virus Matrix protein for delivery to major histocompatibility complex (MHC) class I molecules. We show that SLT N-Ma was capable of sensitising cells for lysis by appropriate cytotoxic T-lymphocytes whilst no killing of SLT-resistant cells was observed. Our results demonstrate that peptide was liberated intracellularly and that retrograde transport of a disarmed cytotoxic protein can intersect the MHC class 1 presentation pathway.

Measurement of immune markers in the serum and cerebrospinal fluid of multiple sclerosis patients during clinical remission.

Magnetic resonance imaging of multiple sclerosis (MS) patients often shows active inflammatory lesions despite clinical remission. No immunological marker of disease activity has been identified in these patients. Concentrations of neopterin, interleukin-2 (IL-2), soluble interleukin-2 receptor (sIL-2R) and tumour necrosis factor-alpha (TNF-alpha) were measured in the serum and cerebrospinal fluid of 19 clinically-inactive MS patients and compared with those of 19 non-inflammatory controls. Cerebrospinal fluid (CSF) neopterin concentrations were significantly higher in the MS group than in controls (mean 9.1 mM vs 3.4 nM, P < 0.01) and 10 of 19 MS patients had levels above the control range. This finding provides evidence of ongoing T-cell-directed and interferon-gamma-mediated macrophage activation in the central nervous system. Analysis of IL-2, sIL-2R and TNF-alpha concentrations revealed no significant differences between MS patients and controls. We conclude that CSF neopterin concentration may correlate with disease activity in asymptomatic patients.

Urinary neopterin quantification indicates altered cell-mediated immunity in healthy subjects under psychological stress.

In an effort to quantify changes in cell-mediated immunity (CMI) in healthy subjects under stress, we measured levels of neopterin, a well-validated marker of CMI activation, in the urine of medical students undergoing academic examinations. Neopterin/creatinine ratios measured on the first day of examinations (mean 46 mumol/mol) were significantly lower than those measured two weeks before (mean 78 mumol/mol, p = .004). Minimum neopterin production coincided with maximum subjective stress, as measured by a visual analogue scale. After examinations, neopterin/creatinine ratios rose (means 62 mumol/mol immediately after, and 65 mumol/mol two weeks after examinations), and these levels were not statistically different from those two weeks before examinations. Over this post-examination period, subjective distress was significantly lower than at either time point before examinations. We conclude that urinary neopterin/creatinine ratios may change significantly during periods of psychological stress, indicating concomitant alterations in CMI activation.

Immunoassay of platelet-derived growth factor in the blood of patients with diabetes mellitus.

Platelet-derived growth factor (PDGF) is a powerful mitogen for many cell types, and is believed to play a major role in wound healing when released from platelets at sites of injury. In diabetes mellitus, it has been proposed that premature release of PDGF from platelets impairs the ability of platelets to initiate healing, and also accelerates the development of diabetic complications such as angiopathy by increasing plasma-borne PDGF. However, plasma samples from diabetic patients have not previously been assayed for PDGF using suitable techniques. A sensitive monoclonal enzyme-linked immunoassay for PDGF was applied to plasma and serum samples from 18 healthy control subjects and 60 diabetic patients. Neither plasma nor serum PDGF concentrations differed significantly between control subjects, insulin-dependent, and non-insulin-dependent diabetic patients. However, 23% of the diabetic subjects had serum PDGF levels above the control range. Limited joint mobility, which is characterised by joint contractures and collagen deposition in the skin, and is associated with microvascular disease, was used as a marker of diabetic complications. Limited joint mobility affected 43% of the diabetic subjects. Patients with moderate limited joint mobility had had diabetes significantly longer than those without limited joint mobility (means 17 years and 9 years, respectively, p = 0.008). However, limited joint mobility was not associated with elevated serum or plasma PDGF in insulin-dependent or non-insulin-dependent diabetes. We conclude that complications of diabetes are unlikely to be caused by changes in systemic levels of PDGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Neopterin measurement provides evidence of altered cell-mediated immunity in patients with depression, but not with schizophrenia.

Neopterin is a validated marker of the activation of cell-mediated immunity in a variety of disease states. We measured neopterin and creatinine concentrations in the plasma and urine of 22 schizophrenic and 26 depressed patients admitted acutely to hospital, and compared results with those in a large group of normal controls. Neopterin/creatinine ratios were normal in the schizophrenic patients, but significantly elevated in the plasma of depressed patients. In each diagnostic group, the use of psychotropic drugs before admission had no effect on the neopterin ratios observed. Our findings indicate altered cell-mediated immunity in depression.

Immunoassay of platelet-derived growth factor in the plasma of patients with scleroderma.

It has been postulated that platelet-derived growth factor (PDGF) is responsible for the abnormal fibroblast proliferation observed in scleroderma. In one previous study, plasma samples from patients with scleroderma caused increased mitogenesis in cultured fibroblasts, suggesting that the pathogenesis of scleroderma is related to increased plasma PDGF concentrations. To test this hypothesis, we used a sensitive, monoclonal antibody-based ELISA to measure PDGF in the plasma of 12 scleroderma patients. A rigorous sampling protocol prevented false elevations in plasma PDGF levels from ex vivo platelet degranulation: beta-thromboglobulin concentrations were measured in each plasma sample to monitor platelet lysis. Plasma PDGF concentrations in the scleroderma patients were not statistically different from those observed in age- and sex-matched normal controls, and patients with RA. While it is possible that changes in PDGF activity at a local level alter fibroblast function, we cannot conclude that elevated plasma concentrations of PDGF play a role in the pathogenesis of scleroderma.