MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

BACKGROUND: Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients. METHODS: miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets. RESULTS: Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology. CONCLUSION: This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

EBMT risk score predicts outcome of allogeneic hematopoietic stem cell transplantation in patients who have failed a previous transplantation procedure.

Increasing numbers of allogeneic hematopoietic stem cell transplantation (allo-SCT) are being performed for patients who have failed a previous allogeneic or autologous SCT. We investigated whether the EBMT risk score could predict outcome after a subsequent allo-SCT. We analyzed prognostic factors in 124 consecutive patients who underwent a second transplantation using an allogeneic donor at our institution. Patients with either a first autologous (N = 64) or first allogeneic (N = 60) SCT were included. Age, disease stage, time interval from diagnosis to transplantation, donor type, and donor-recipient sex combination were used to establish a score from 0 to 7 points, from which 3 groups were identified. The 5-year survival probability decreased from 51.7% for risk scores 0-3 (low, n = 25), to 29.3% for risk score 4 (intermediate, n = 42), and only 10.4% for risk scores 5-7 (high, n = 57), P = .001. We propose that the EBMT risk score can identify patients most likely to benefit from a second transplantation.

Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts.

Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

Negative immunomagnetic selection of T cells from peripheral blood of presentation AML specimens.

To date, studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation, block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens, we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique, unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia, was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33, CD34, CD123, CD11c and CD36, to deplete myeloblasts.

E(mu)-TCL1 mice represent a model for immunotherapeutic reversal of chronic lymphocytic leukemia-induced T-cell dysfunction.

Preclinical animal models have largely ignored the immune-suppressive mechanisms that are important in human cancers. The identification and use of such models should allow better predictions of successful human responses to immunotherapy. As a model for changes induced in nonmalignant cells by cancer, we examined T-cell function in the chronic lymphocytic leukemia (CLL) Emu-TCL1 transgenic mouse model. With development of leukemia, Emu-TCL1 transgenic mice developed functional T-cell defects and alteration of gene and protein expression closely resembling changes seen in CLL human patients. Furthermore, infusion of CLL cells into young Emu-TCL1 mice induced defects comparable to those seen in mice with developed leukemia, demonstrating a causal relationship between leukemia and the T-cell defects. Altered pathways involved genes regulating actin remodeling, and T cells exhibited dysfunctional immunological synapse formation and T-cell signaling, which was reversed by the immunomodulatory drug lenalidomide. These results further demonstrate the utility of this animal model of CLL and define a versatile model to investigate both the molecular mechanisms of cancer-induced immune suppression and immunotherapeutic repair strategies.

Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug.

Cancer is associated with immune deficiency, but the biologic basis of this is poorly defined. Here we demonstrate that impaired actin polymerization results in CD4+ and CD8+ T cells from patients with chronic lymphocytic leukemia (CLL) exhibiting defective immunological synapse formation with APCs. Although this synapse dysfunction was in part a result of the CLL cells having poor APC function, defective actin polymerization was also identified in T cells from patients with CLL. We further demonstrate that, following contact with CLL cells, defects in immune synapse formation were induced in healthy allogeneic T cells. This required direct contact and was inhibited by blocking adhesion molecules on CLL B cells. In T cells from patients with CLL and in T cells from healthy individuals that had been in contact with CLL cells, recruitment of key regulatory proteins to the immune synapse was inhibited. Treatment of autologous T cells and CLL cells with the immunomodulating drug lenalidomide resulted in improved synapse formation. These results define what we believe to be a novel immune dysfunction in T cells from patients with CLL that has implications for both autologous and allogeneic immunotherapy approaches and identifies repair of immune synapse defects as an essential step in improving cancer immunotherapy approaches.

Transplantation in chronic lymphocytic leukemia.

Although there have been no randomized trials comparing the outcome of stem cell transplantation (SCT) with standard chemotherapy for patients with chronic lymphocytic leukemia (CLL), increasingly, both autologous and allogeneic SCT approaches are being explored in this disease. Clinical trials have demonstrated that these approaches are feasible, but current data suggest that autologous transplantation is not curative and myeloablative SCT, although offering the potential for cure, is associated with high treatment-related mortality. There is a clear demonstration of a graft-versus-leukemia effect in CLL, with encouraging results seen after SCT with reduced-intensity conditioning. Because no other treatment modalities are currently capable of improving survival in this disease, the treatment of choice for younger patients with poor-risk CLL may well be SCT, but continued enrollment of appropriate patients into well-designed clinical trials is vital to compare advances in SCT with the advances occurring in chemoimmunotherapy in CLL.

Vaccine- and immune-based therapy in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) would appear to be an ideal target of T-cell-mediated responses against the cancer cell. The cancer arises in cells that can act as antigen-presenting cells (APCs), CLL cells express tumor antigens, and the cells can be a target of the allogeneic T cells in a graft-versus-leukemia effect. Despite these potential benefits, immune responses against CLL cells have been difficult to elicit. CLL induces immune defects in the host, the tumor cells are inefficient APCs, and therapies given to patients with CLL are themselves immunosuppressive. Successful vaccination approaches in this disease will require steps to overcome these difficulties, including steps to improve the immune defects in this disease, identification of the targets of the immune response to monitor immunologic responses, and improved presentation of antigen.

CD40 activation: lessons for HIV immunotherapy from malignancies?

In HIV, the immune defects seen are due not only to a decrease in T-cell numbers, but also to qualitative impairment in T-cell function as well as decreased antigen-presenting cell (APC) function. These defects in cell-mediated immunity lead to increased level of infection, contributing to inability to clear the HIV virus, and an increased incidence of tumours. One of the major defects in HIV appears to be the failure of CD4 T cells to provide CD 154 (CD40 ligand)-mediated help, which is required for APC function. In lymphomas, activation through CD40 leads to increased APC activity and induction of immune responses against tumours. Such an effect may also be useful in HIV to increase response against the virus and improve immune surveillance of tumours.