Differential miRNA expressions in peripheral blood mononuclear cells for diagnosis of lung cancer.

Tremendous efforts have been made to develop cancer biomarkers by detecting circulating extracellular miRNAs directly released from tumors. Yet, none of the cell-free biomarkers has been accepted to be used for early detection of non-small cell lung cancer (NSCLC). Peripheral blood mononucleated cells (PBMCs) act as the first line of defense against malignancy in immune system, their dysfunction may occur as an early event in cancer immunogenicity or immune evasion. We proposed to investigate whether analysis of miRNA expressions of PBMCs has diagnostic value for NSCLC. We first used a microarray to analyze PBMCs of 16 stage I NSCLC patients and 16 cancer-free smokers, and identified seven PBMC miRNAs with a significantly altered expression level in NSCLC patients. In a training set of 84 NSCLC patients and 69 cancer-free smokers, a panel of two miRNAs (miRs-19b-3p and -29b-3p) were developed from the seven PBMC miRNAs, producing 72.62% sensitivity and 82.61% specificity in identifying NSCLC. Furthermore, the miRNAs could identify squamous cell lung carcinoma (SCC), a major type of NSCLC, with 80.00% sensitivity and 89.86% specificity. The expression levels of the miRNAs were independent of disease stage. In a testing set of 56 NSCLC patients and 46 controls, the performance of the biomarkers was reproducibly confirmed. The study presents the first in-depth analysis of PBMC miRNA profile of NSCLC patients. The assessment of PBMC miRNAs may provide a new diagnostic approach for the early detection of NSCLC.

Vγ2Vδ2 T cell Costimulation Increases NK cell Killing of Monocyte-derived Dendritic Cells.

Interactions between NK and dendritic cells (DC) affect maturation and function of both cell populations, including NK killing of DC (editing) that is important for controlling the quality of immune responses. We also know that antigen-stimulated Vγ2Vδ2 T cells costimulate NK cells via 4-1BB to enhance killing of tumor cell lines but we do not know what regulates 4-1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK:γδ T cell cross talk. Here we show that antigen-stimulated γδ T cells costimulate NK through ICOS:ICOSL and this signal increases NK killing of autologous DC. Effects of NK:γδ T cell co-culture, which could be reproduced with soluble ICOS-Fc fusion protein, included increased CD69 and 4-1BB expression, IFN-γ, TNF-α, MIP-1ß, I-309, RANTES and sFasL production, as well as elevated mRNA levels for costimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Thus, ICOS/ICOSL costimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK γδ T cell cross talk links innate and antigen-specific lymphocyte responses in the control of cytotoxic effector function and dendritic cell killing. This article is protected by copyright. All rights reserved.

Clinical response of advanced cancer patients to cellular immunotherapy and intensity-modulated radiation therapy.

Patients afflicted with advanced cancers were treated with the intratumoral injection of autologous immature dendritic cells (iDCs) followed by activated T-cell infusion and intensity-modulated radiation therapy (IMRT). A second round of iDCs and activated T cells was then administered to patients after the last radiation cycle. This complete regimen was repeated for new and recurring lesions after 6 weeks of follow-up. One year post therapy, outcome analyses were performed to evaluate treatment efficacy. Patients were grouped according to both the number and size of tumors and clinical parameters at treatment initiation, including recurrent disease after standard cancer therapy, Stage IV disease, and no prior therapy. Irrespective of prior treatment status, 23/37 patients with ≤ 5 neoplastic lesions that were ≤ 3 cm in diameter achieved complete responses (CRs), and 5/37 exhibited partial responses (PRs). Among 130 individuals harboring larger and more numerous lesions, CRs were observed in 7/74 patients that had received prior SCT and in 2/56 previously untreated patients. Some patients manifested immune responses including an increase in CD8+CD56+ lymphocytes among circulating mononuclear cells in the course of treatment. To prospectively explore the therapeutic use of these cells, CD8+ cells were isolated from patients that had been treated with cellular immunotherapy and IMRT, expanded in vitro, and injected into recurrent metastatic sites in 13 individuals who underwent the same immunoradiotherapeutic regimens but failed to respond. CRs were achieved in 34 of 58 of such recurrent lesions while PRs in 17 of 58. These data support the expanded use of immunoradiotherapy in advanced cancer patients exhibiting progressive disease.

Therapeutic response in patients with advanced malignancies treated with combined dendritic cell-activated T cell based immunotherapy and intensity-modulated radiotherapy.

Successful cancer immunotherapy is confounded by the magnitude of the tumor burden and the presence of immunoregulatory elements that suppress an immune response. To approach these issues, 26 patients with advanced treatment refractory cancer were enrolled in a safety/feasibility study wherein a conventional treatment modality, intensity modulated radiotherapy (IMRT), was combined with dendritic cell-based immunotherapy. We hypothesized that radiation would lower the tumor burdens, decrease the number/function of regulatory cells in the tumor environment, and release products of tumor cells that could be acquired by intratumoral injected immature dendritic cells (iDC). Metastatic lesions identified by CT (computed tomography) were injected with autologous iDC combined with a cytokine-based adjuvant and KLH (keyhole limpet hemocyanin), followed 24 h later by IV-infused T-cells expanded with anti-CD3 and IL-2 (AT). After three to five days, each of the injected lesions was treated with fractionated doses of IMRT followed by another injection of intratumoral iDC and IV-infused AT. No toxicity was observed with cell infusion while radiation-related toxicity was observed in seven patients. Five patients had progressive disease, eight demonstrated complete resolution at treated sites but developed recurrent disease at other sites, and 13 showed complete response at various follow-up times with an overall estimated Kaplan-Meier disease-free survival of 345 days. Most patients developed KLH antibodies supporting our hypothesis that the co-injected iDC are functional with the capacity to acquire antigens from their environment and generate an adaptive immune response. These results demonstrate the safety and effectiveness of this multimodality strategy combining immunotherapy and IMRT in patients with advanced malignancies.

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity.

BACKGROUND: The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i) the small number of NK cells in peripheral blood, (ii) the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii) the need to activate the NK cells in order to induce NK cell mediated killing and (iv) the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i) if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii) their ability to kill allogeneic and autologous tumor targets by direct cytotoxicity and by antibody-mediated cellular cytotoxicity and (iii) defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. METHODS: Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. RESULTS: Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. CONCLUSION: These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical evidence necessary to support clinical trials utilizing autologous expanded NK cells, both directly and in combination with monoclonal antibodies in future cell-based immunotherapy in select solid tumors.

Monocytes differentiated with IL-15 support Th17 and Th1 responses to wheat gliadin: implications for celiac disease.

Interleukin (IL)-15 contributes to the immunopathogenesis of Celiac disease (CD). However, it is not clear how IL-15 affects APC that shape adaptive immune responses to the dietary antigen, gliadin. Using PBMC from healthy individuals, we show that monocytes differentiated with IL-15 (IL15-DC) produced IL-1beta, IL-6, IL-15, IL-23, TNFalpha and CCL20 in response to pepsin-trypsin digested gliadin (PTG) and activated contact-dependent Th17 and Th1 responses from autologous CD4(+) T cells. Lower concentrations of IL-15 augmented IFNgamma responses to PTG in PBMC from CD patients compared to controls. Thus, IL-15 supports Th17 and Th1 responses to a dietary antigen that is normally well-tolerated in healthy individuals by generating IL15-DC. These potentially pathogenic immune responses may result in CD patients and not healthy individuals as a consequence of IL-15 hypersensitivity. Therefore, genetic and/or environmental factors that control IL-15 expression and responsiveness in the intestine likely participate in the pathogenesis of CD.

Combining conventional therapies with intratumoral injection of autologous dendritic cells and activated T cells to treat patients with advanced cancers.

Dendritic cells (DCs) are potent antigen-presenting cells that have been used in cancer immunotherapy. To take advantage of the ability of DCs to acquire antigenic materials from their environment and generate primary as well as recall immune responses, 37 patients with advanced cancers were enrolled in a series of protocols based on direct intratumoral injection of immature DCs. To augment antigen uptake and antitumor immune response, DC injection was combined with radiotherapy or chemotherapy and/or injection of activated T cells. Treatments were well tolerated with no adverse reactions. Clinical responses were based on Response Evaluation Criteria in Solid Tumors, with the majority of patients showing stable disease. One of two patients who also received local radiation achieved a sustained complete response at injected and metastatic sites. The clinical responses observed in cancer patients with advanced disease suggest potential effectiveness of combination strategies and establish the basis for the current treatment protocol that is underway.

Epidemiologic characteristics and natural history of HIV-1 natural viral suppressors.

OBJECTIVE: The objective of this study was to detail the epidemiologic characteristics and natural history of HIV-1 natural viral suppressors (NVSs), a cohort of HIV-1-infected individuals who are able to suppress viral replication to undetectable levels in the absence of therapy. DESIGN AND METHODS: HIV-1 patients who met the NVS criteria were enrolled into a prospective study. The incidence and prevalence of NVS were calculated by performing a chart review on all patients seen in 1 clinic in a 10-year period. Cumulative probability of progression-free survival was calculated by Kaplan-Meier product limit method. RESULTS: Forty individuals enrolled in the study. The median year of diagnosis was 1994, and individuals demonstrated a median 6.7 years of HIV-1 viral suppression and CD4 count of 795 cells per microliter. NVS had an incidence of 1.1% [95% confidence interval (CI), 0.0 to 2.1] and prevalence of 1.5% (95% CI, 0.8 to 2.1). Only 1 patient (2.5%) has progressed. Within the first 10 years for follow-up having met the definition of NVS, 95.1% (95% CI 86.5% to 100%) of the NVS continued to control their viral loads to undetectable levels. CONCLUSIONS: The NVS cohort has demonstrated remarkable stability and a low rate of progression over many years. Detailed evaluations of viral-host immune regulatory factors associated with persistent HIV-1 natural viral suppression, and loss of such suppression, has the potential to provide important new insight in HIV pathogenesis and future immune regulatory targeted preventive and therapeutic research.

Cutting edge: IL-1 controls the IL-23 response induced by gliadin, the etiologic agent in celiac disease.

IL-23 has been implicated in the pathogenesis of several tissue-specific autoimmune diseases. Currently, celiac disease (CD) is the only autoimmune disease in which both the major genetic (95% HLA-DQ2(+)) and etiologic factors (dietary glutens) for susceptibility are known. We demonstrate that wheat gliadin induces significantly greater production of IL-23, IL-1beta, and TNF-alpha in PBMC from CD patients compared with HLA-DQ2(+) healthy controls, strongly advocating a role for IL-23 in the pathogenesis of CD. Moreover, IL-1beta alone triggered IL-23 secretion and the IL-1R antagonist inhibited this response in PBMC and purified monocytes. This sequence of events was replicated by beta-glucan, another substance known to induce IL-23 production. Our results suggest that gliadin and beta-glucan stimulate IL-23 secretion through induction of the IL-1 signaling pathway and reveal for the first time that the IL-1 system regulates IL-23 production. These findings may provide therapeutic targets for this disease and other inflammatory conditions mediated by IL-23.

Products of anti-CD3/anti-CD28 activated lymphocytes induce differentiation and maturation of dendritic cells and have adjuvant-like activity in vitro and in vivo.

Dendritic cells (DC) orchestrate immune responses under direction of cytokines/chemokines in their microenvironment. To investigate the influence of that generated during T cell activation, we stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 and anti-CD28 coated beads and tested cell-free culture supernatants (lymphocyte conditioned medium, LCM) for cytokine/chemokine composition and biologic activity. LCM contained a battery of mediators important in the biology of myeloid (mDC) and plasmacytoid (pDC) DC. LCM differentiated monocytes into functional immature mDC, and induced maturation of immature mDC. LCM also augmented maturation and IFNalpha-production of CpG-treated pDC. Functional activity of LCM-derived DC was confirmed by their ability to enhance in vitro recall T cell responses and substantially augment in vivo cellular and humoral immune responses to various vaccines in non-human primates. These results demonstrate that products of anti-CD3/anti-CD28 stimulated PBMC generate biologically active DC in vitro and function as a vaccine adjuvant in vivo.

Detection of CD4(+) T-cell antibodies in a patient with idiopathic CD4 T lymphocytopenia and cryptococcal meningitis.

Idiopathic CD4(+) T lymphocytopenia (ICL) is defined as a CD4(+) T-cell count <0.3 x 10(9)/l or <20% of the total T-cell count on two occasions in the absence of any immunodeficiency disorder or therapy associated with reduced CD4(+) T-cell count. Although several mechanisms of ICL have been reported, the pathophysiology is still largely unknown. This case report describes a patient who presented with cryptococcal meningitis and was subsequently discovered to meet the criteria for ICL. Flow cytometric analysis of the patient's peripheral blood mononuclear cells revealed antibodies coating a much larger proportion of his CD4(+) T cells (33.61%) than the CD4(+) T cells of normal donors (3.94 +/- 1.77%). The reasons behind the development of these autoantibodies are explored.

Activation of innate immunity by prostate specific antigen (PSA).

BACKGROUND: Prostate specific antigen (PSA) is a serine protease secreted by the prostatic epithelium. The only known function of the protein is to cleave seminogelin. We wished to determine if PSA activated peripheral blood mononuclear cells (PBMC). METHODS: PBMC and selected sub-populations were cultured with purified PSA. Secretion of IFNgamma was measured by cytokine capture flow cytometry and enzyme-linked immunosorbent assay. RESULTS: We observed secretion of IFNgamma and a proliferative response in PBMC cultured with PSA. We found that NK cells were the source of the IFNgamma but NK cells were not directly stimulated by PSA. Rather, a soluble factor secreted primarily by CD14 monocytes in response to PSA stimulated NK cells to secrete IFNgamma. DISCUSSION: PSA induces a pro-inflammatory response that results in the secretion of INFgamma by NK cells. The presence of large amounts of PSA could contribute to the common finding of inflammatory infiltrates in the prostate.

Granulomatous prostatitis linked to HLA-DRB1*1501.

PURPOSE: Granulomatous prostatitis is characterized by a pattern of granulomatous inflammation in the prostate. In most cases the etiology is unknown. Based on the hypothesis that granulomatous prostatitis may be an autoimmune disease we performed intermediate and selective high resolution typing of HLA-DR in a group of patients with the disease and compared the frequency of class II HLA phenotypes to that in a control group of volunteer marrow donors in the military. MATERIALS AND METHODS: Histological records from 1 institution from 1990 to 2000 revealed 12 patients with diffuse granulomatous prostatitis. Three patients were dead and 1 refused blood drawing. Peripheral blood from the remaining 8 patients was typed along with blood from an additional 3 identified at the practice of one of us from 1999 through 2002. All slides were reviewed by 1 pathologist. Intermediate resolution typing of HLA-A, B and DR was performed by polymerase chain reaction-sequence specific oligonucleotide probe. High resolution, allele specific identification of HLA DR15 was performed if patients were DR15 positive by intermediate resolution typing. RESULTS: There were 3 black and 8 white individuals identified with diffuse nonspecific granulomatous prostatitis. Six of 8 white patients (75%) were HLA-DR15 by intermediate resolution typing. One of the 3 black American patients (33%) was HLA-DR15. In the control group 127 of 451 white (28.2%) and 23 of 89 black (25.8%) volunteer marrow donors were HLA-DR15. The case-control comparison of white patients was significantly different (Fisher's exact test p = 0.0086). There were no statistically significant differences between case-control comparisons for any other HLA-DR phenotype. High resolution DR15 typing showed that the white patients were HLA-DRB1*1501 and the black patient was HLA-DRB1*1503. CONCLUSIONS: The data suggest an association between HLA-DRB1*1501 and granulomatous prostatitis. HLA-DR15 is strongly associated with other autoimmune diseases, notably multiple sclerosis. The data are consistent with an autoimmune etiology for nonspecific granulomatous prostatitis.

CD4 and CD8 T-lymphocyte recognition of prostate specific antigen in granulomatous prostatitis.

In order to develop immunotherapies for prostate cancer, many groups are exploring vaccination strategies to induce an immune response against prostate specific antigen (PSA). To determine if T-cell recognition of PSA might be a feature of a naturally occurring human disease, we have studied patients with prostatitis, a poorly understood clinical syndrome of men in which there is evidence that an immune response directed against the prostate may be occurring. We wished to determine if a T-cell response to PSA might be occurring in these patients. We generated long-term T-cell lines from peripheral blood mononuclear cells (PBMC) of one patient with granulomatous prostatitis using purified PSA as an antigen. Several CD4+ and CD8+ TcR alpha/beta+ T-cell lines were selected for PSA reactivity as measured by at least a threefold increase in IFN-gamma secretion in response to PSA presented by irradiated autologous PBMC. CD4 and CD8 T-cell lines recognized PSA in the context of HLA-DRbeta1*1501 and HLA-B*0702, respectively. The specificity and HLA restriction of the lines was confirmed using EBV-B cell lines infected with a recombinant PSA-expressing vaccinia virus and also engineered to express PSA by retroviral transfection. HLA-matched targets infected by control vector as well as HLA-mismatched PSA-expressing targets did not induce the response. The data demonstrate that PSA-specific T cells are present in the PBMC of this patient with granulomatous prostatitis, who may be manifesting naturally the type of immune response directed at the prostate that is the goal of prostate cancer immunotherapy. However, the Class I-restricted epitope has not yet been demonstrated to be expressed on the surface of prostate cancer cells. To our knowledge, this is the first demonstration of HLA-DRB1*1501- or HLA-B*0702-restricted responses to PSA and extends the number of HLA molecules accommodating the use of PSA antigen as a candidate vaccine for prostate cancer immunotherapy.

Timed sequential therapy of acute leukemia with flavopiridol: in vitro model for a phase I clinical trial.

PURPOSE: The survival of adults with acute leukemias remains unsatisfactory and requires new treatment approaches. Flavopiridol modulates cell cycle progression, inhibits transcription, and induces apoptosis. We designed an in vitro model of timed sequential therapy for acute leukemia to determine whether flavopiridol can: (a). trigger apoptosis in fresh acute leukemia; and (b). recruit surviving leukemic cells to a proliferative state, thereby priming such cells for the S-phase-related cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C). EXPERIMENTAL DESIGN: Bone marrow cells from 20 adults with relapsed and refractory acute leukemias were enriched for blasts by Ficoll Hypaque sedimentation. Blasts were cultured on day 0 in flavopiridol 250 nM for 24 h, removed from flavopiridol for 24 h, and then cultured in ara-C 1 microM for an additional 72 h (F(250)A(1)). Apoptosis and cell cycle phase distribution were estimated from cells stained with propidium iodide. Cell survival was determined after the 72 h ara-C exposure by double cytofluorescence assay with fluorescein diacetate and propidium iodide. RESULTS: Flavopiridol induced a 4.3-fold increase in apoptosis in human leukemia samples within the first 24 h of culture. Subsequent removal of flavopiridol led to a 1.7-fold increase in the proportion of cells in S phase by day 2. Mean survival in F(250)A(1) cultures after 72 h exposure to ara-C was 35.6% compared with flavopiridol alone (F(250)A(0), 56.1%; P = 0.0003) and ara-C alone (F(0)A(1), 65.2%; P < 0.00001). CONCLUSIONS: Flavopiridol induces apoptosis in marrow blasts from patients with refractory acute leukemias. Furthermore, flavopiridol pretreatment increases the proapoptotic and cytotoxic effects of ara-C. The advantage of sequential FP(250)A(1) over either agent alone is seen for both acute myelogenous leukemia and acute lymphoblastic leukemia. These findings support a clinical trial of timed sequential therapy where flavopiridol is given for cytoreduction and subsequent priming of remaining leukemic cells for enhanced cycle-dependent drug cytotoxicity.

MIC expression in renal and pancreatic allografts.

BACKGROUND: MHC class I chain-related antigen A (MICA) and MHC class I chain-related antigen B (MICB) are HLA class I related products of polymorphic MHC genes. Constitutive expression in normal tissue is limited to gut epithelium but can be induced in other epithelial cells by stress. Specific antibodies against MICA have been reported in the serum of patients who had rejected kidney allografts, suggesting a potential role for these molecules in transplant immunopathology. However, expression of MICA and MICB in transplanted organs has not been demonstrated. In this study, we report the expression of MICA and MICB in renal and pancreatic allograft biopsies, which were obtained due to clinical signs of rejection. METHODS: A monoclonal antibody directed against MICA and MICB was used to perform indirect immunohistochemistry on formalin fixed, paraffin embedded needle biopsies of kidney and pancreas allografts. The results of staining were then compared to the standard light microscopic evaluation of the biopsies for rejection. RESULTS: A total of 53 individual renal transplant biopsies and 19 pancreas transplant biopsies were assayed for expression of MIC. Histologically, renal biopsies were diagnosed as no rejection, acute tubular necrosis (ATN), acute rejection (AR), chronic rejection (CR), and acute and chronic rejection (ACR). No staining was observed in 7 of 10 kidneys showing no rejection. All 11 of the kidney biopsies with AR were positive, as were the 11 ATN cases, 9 of the 11 kidney biopsies with CR, and 7 of the 10 with ACR. The acini of normal, nontransplanted, pancreas, control specimen were consistently negative; however, islets were positive in all specimens. The acini and islets of five histologically normal pancreas biopsies were positive, as were the four biopsies with AR, seven biopsies with CR, and two with ACR. CONCLUSIONS: MICA and MICB are expressed in epithelial cells in allografted kidney and pancreas that show histologic evidence of rejection and/or cellular injury. In addition to previous findings of alloantibodies against MICA, expression of these gene products may play a role in allograft rejection.