Biomedical subjects

Diana Metes

Publications and source records attributed to Diana Metes.

7 recordsLinked to original sources

PubMed2006

Detection of CD8+ T cells sensitized to BK virus large T antigen in healthy volunteers and kidney transplant recipients.

BK virus (BKV) infections after renal transplantation are increasingly recognized. Development of immune monitoring strategies against BKV requires definition of antigenic epitopes. Hence, T cells from HLA-A02-positive healthy subjects and kidney transplant recipients were stimulated by BKV lysate pulsed on mature autologous dendritic cells and screened against four different T antigen peptides or against BKV lysate. IFN-gamma production was measured by ELISPOT assays. The peptide BKV362-371 (MLTERFNHIL) was naturally processed and recognized by five of six healthy subjects (39 +/- 11 IFN-gamma spots/100,000 cells) and five of seven kidney transplant recipients (21 +/- 12 IFN-gamma spots). Less frequent and weaker CD8+ T-cell responses were detected against three other peptides. Thus, BKV large T antigen is a target for CD8+ T-cell immunity. T-antigen-specific T-cytotoxic cells circulate in healthy blood donors, implying that transient expression of T antigen presumably occurs at sites of viral latency and helps maintain a constant pool of circulating CD8+ T memory cells.

Adult↗

PubMed2005

EBV-specific memory CD8+ T cell phenotype and function in stable solid organ transplant patients.

Immune responses to EBV in immunosuppressed (IS) solid organ transplant (SOTx) recipients have not been well characterized. Here we evaluate the phenotype and function of EBV-specific CD8+ T cells in peripheral blood isolated from "stable" IS SOTx recipients. The EBV-specific CD8+ T cell memory subset distribution in the peripheral blood of patients was examined by flow cytometric analysis using HLA-A2 tetramers incorporating BMLF1 (lytic), and LMP2 and EBNA3A (latent)-derived peptides, in conjunction with mAbs against the CD45RO, CD45RA, and CD62L markers. The ability of CD8+ T cells to produce IFN-gamma in response to the same EBV-derived peptides was measured by ELISPOT assay. Patients and healthy normal donors exhibited similar anti-EBV CD8+ T cell frequencies and specificities against the EBV epitopes evaluated. When compared to healthy normal donors, an overall significant expansion of the CD8+ T cell "effector memory" (CD45RO+/CD62L-) pool, including that of EBV "latent" (LMP2 and EBNA3A)-specific CD8+ T cells was detected in IS SOTx patients. However, the patients' EBV-specific CD8+ T cells showed decreased IFN-gamma production to the EBV-peptide stimulation. These results indicate that the impairment of EBV-specific CD8+ T cell activity is not due to clonal depletion, but is mainly due to impaired functional activation.

Antigens, Viral↗

PubMed2005

Augmentation of type-1 polarizing ability of monocyte-derived dendritic cells from chronically immunosuppressed organ-transplant recipients.

BACKGROUND: Chronic immunosuppressive (IS) therapy impairs normal T-cell immune surveillance and may predispose to opportunistic infections and malignancies that represent life-threatening complication of solid-organ transplantation (SOTx). Our study was designed to ascertain the impact of chronic in vivo administration of IS on the ability of monocyte-derived dendritic cells (MoDC) to differentiate, mature, and function ex vivo. The potential of these cells to be implemented for DC-based adoptive immunotherapy was also considered. METHODS: MoDCs were propagated by conventional procedures, their phenotype was analyzed by flow cytometry, and their function was assessed by mixed leukocyte reaction, enzyme-linked immunoadsorbent assay, and ELISPOT assays. Nuclear translocation of nuclear factor (NF)-kB was analyzed by electrophoretic mobility shift assay. RESULTS: Circulating DC1s in peripheral blood were reduced in SOTx patients. MoDCs generated from patients displayed higher endocytic activity versus normal DCs, indicating their comparative immaturity. Patients' DCs exposed to pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin [IL]-1beta, and IL-6) were less able to mature, to stimulate recall antigen (Ag)- or allo-Ag-induced proliferation responses, or to secrete IL-12p70. These deficiencies were associated with a decrease in NF-kB translocation. In contrast, combination of pro-inflammatory cytokines and interferon (IFN)-gamma (a Th1-polarizing factor) augmented patients' DC1-type function and IL-12p70 production by way of an NF-kB-independent mechanism. CONCLUSIONS: Chronic IS restrains DC differentiation, maturation, and function at a transcriptional level; however, type-1 polarizing potential of patients' DC1 can be augmented ex vivo by a two-signal stimulation provided by pro-inflammatory cytokines and IFN-gamma. These results may have implications for DC-based immunotherapy of malignancies in the transplantation setting.

Active Transport, Cell Nucleus↗

PubMed2005

Kidney transplantation under minimal immunosuppression after pretransplant lymphoid depletion with Thymoglobulin or Campath.

BACKGROUND: Multiple drug immunosuppression has allowed the near elimination of rejection, but without commensurate improvements in longterm graft survival and at the cost of quality of life. We have suggested that transplantation outcomes can be improved by modifying the timing and dosage of immunosuppression to facilitate natural mechanisms of alloengraftment and acquired tolerance. STUDY DESIGN: Two therapeutic principles were applied for kidney transplantation: pretransplant recipient conditioning with antilymphoid antibody preparations (Thymoglobulin [Sangstat] or Campath [ILEX Pharmaceuticals]), and minimal posttransplant immunosuppression with tacrolimus monotherapy including "spaced weaning" of maintenance doses when possible. The results in Thymoglobulin- (n = 101) and Campath-pretreated renal transplantation recipients (n = 90) were compared with those in 152 conventionally immunosuppressed recipients in the immediately preceding era. RESULTS: Spaced weaning was attempted in more than 90% of the kidney transplant recipients after pretreatment with both lymphoid-depleting agents, and is currently in effect in two-thirds of the survivors. Although there was a much higher rate of acute rejection in the Thymoglobulin-pretreated recipients than in either the Campath-pretreated or historic control recipients, patient and graft survival in both lymphoid depletion groups is at least equivalent to that of historic control patients. In the Thymoglobulin-conditioned patients for whom followups are now 24 to 40 months, chronic allograft nephropathy (CAN) progressed at the same rate as in historic control patients. Selected patients on weaning developed donor-specific nonreactivity. CONCLUSIONS: After lymphoid depletion, kidney transplantation can be readily accomplished under minimal immunosuppression with less dependence on late maintenance immunosuppression and a better quality of life. Campath was the more effective agent for pretreatment. Guidelines for spaced weaning need additional refinement.

Analysis of Variance↗

PubMed2003

Four-color flow cytometric analysis of peripheral blood donor cell chimerism.

Passenger leukocytes have been demonstrated to play significant roles in initiating and also regulating immune reactions after organ transplantation. Reliable techniques to detect donor leukocytes in recipients after organ transplantation are essential to analyze the role, function, and behavior of these leukocytes. In this report we describe a simple, reliable method to detect donor cells with low frequencies using peripheral blood samples. Detection of small numbers of major histocompatibility complex (MHC) mismatched cells was first studied using four-color flow cytometry in artificially created cell mixtures. By selecting the CD45(+) population and simultaneous staining with several leukocyte lineage markers (CD3, CD4, CD8, CD56, and CD19), MHC-mismatched leukocytes were consistently detected in cell suspensions prepared from directly stained whole blood samples with a threshold sensitivity as low as 0.1%-0.2%. When the fresh peripheral blood mononuclear cells were separated by conventional Ficoll gradient purification, similar, but slightly lower levels of donor cells were detected. Blood samples obtained 1-5 months after liver, kidney, and intestine transplants revealed that the kind of organ allograft influenced levels and lineage pattern of the circulating donor cells. This procedure provided a simple and reliable method in determining early chimerism in transplant recipients. However, the detection of MHC-mismatched leukocytes of all lineages was much lower when frozen peripheral blood mononuclear cells were used.

Antibodies, Monoclonal↗

PubMed2003

Ex vivo priming of naïve T cells into EBV-specific Th1/Tc1 effector cells by mature autologous DC loaded with apoptotic/necrotic LCL.

Posttransplant lymphoproliferative disorders (PTLDs) represent life-threatening complications of bone marrow and solid organ transplantation (SOTx). These are B-cell malignancies triggered by Epstein-Barr Virus (EBV) infection in chronically immunosuppressed (IS) recipients. Immunosuppressed EBV seronegative (EBV(-)) organ recipients are at highest risk of developing PTLD owing to the lack of anti-EBV memory T cells to control subsequent EBV challenges. Our aim is to establish effective anti-EBV T-cell generation protocols for prevention or treatment of PTLD encountered in SOTx. We have used autologous dendritic cells (DCs) loaded with apoptotic/necrotic lymphoblastoid cell lines (LCLs) to evaluate the ability of such an approach to activate naïve T cells in vitro. In EBV(-) individuals, both CD8+ and CD4+ T-cell responses were amplified by this approach, as detected by IFN-gamma ELISPOT and cytotoxicity assays. The CD8+ T cells were poly-specific anti-EBNA3 A, -LMP2 and -BMLF1, with uniform reversion to a CD45RO+/RA-phenotype, decreased CD62L expression, and up-regulation of the activation markers CD28 and CD69. Addition of rhIL-12 improved anti-viral T-cell responses and reduced the functional differences observed between EBV(+) and EBV(-) responders. In conclusion, the DC/LCL method promotes cross-presentation of EBV-associated epitopes and may serve as an effective protocol for the adoptive immunotherapy of PTLD in EBV(-) SOTx patients.

Antigen Presentation↗

PubMed2001

Allelic polymorphisms in the FcgammaRIIC gene can influence its function on normal human natural killer cells.

Natural killer (NK) cells are important in host defense against viruses and tumors and can induce death of virally infected cells following engagement of cell surface receptors. Human NK cells express receptors for the Fc portion of IgG which stimulate antibody-dependent cell-mediated cytotoxicity and induce cytokine production. We have shown that NK cells from certain individuals can express, in addition to CD16 (FcgammaRIIIa), isoforms of CD32 (FcgammaRIIc1-4). Expression of CD32 on NK cells is dependent on an allelic polymorphism of the FcgammaRIIC gene. We analyzed the expression and function of CD32 on NK cells from 31 normal donors. Fourteen of the 31 (45%) donors expressed CD32 on their NK cells. Molecular characterization of FcgammaRIIc isoforms expressed by the CD32+ donors revealed that the majority of donors expressed the FcgammaRIIc1 isoform. Interestingly, 3 of the 14 positive donors did not express FcgammaRIIc1, and we identified a novel isoform, FcgammaRIIc5, expressed by these individuals. The expression of this isoform was correlated to a second allelic polymorphism that controls exon splicing. One of the three was found to express FcgammaRIIb on the NK cells. Biochemical analysis revealed that CD32+ donors of both types expressed a 40-kDa protein, specifically immunoprecipitated by anti-CD32 monoclonal antibodies. Functionally, only individuals expressing the FcgammaRIIc1 isoform were able to trigger reverse antibody-dependent cell-mediated cytotoxicity via CD32 whereas a CD32+ individual expressing the FcgammaRIIb isoform was unable to trigger this function. These results demonstrate that the presence of multiple allelic polymorphisms in the FcgammaRIIC gene determine the expression and function of CD32 on NK cells.

Adult↗