[Show abstract][Hide abstract] ABSTRACT: In adults, one-haplotype-mismatched haematopoietic SCT (haploidentical HSCT) is associated with slow immune recovery due to decaying thymic function and extensive T-cell depletion of the graft. Although essential for preventing GVHD, T-cell depletion underlies the major reasons for transplant failure: leukemia relapse and infections, with infection-related mortality accounting for about 40% of non-leukemic deaths. Adoptive T-cell therapy would be helpful for these patients but to administer it without causing GVHD, alloreactive T cells need to be eliminated from donor T lymphocytes before infusion. In a preclinical study, to address this problem, we determined the efficacy of photodynamic purging of alloreactive T cells, by investigating combinations of parameters in order to achieve maximum allodepletion, preservation of T-regulatory cells and of pathogen and leukemia-specific T-cell responses in donor-vs-recipient MLR. We also needed to identify an optimal method to quantify the Ag-specific T-cell repertoires. Optimal procedures were identified. In particular, we compared limiting-dilution analyses (LDA) of proliferating T cells with H(3)-thymidine incorporation by bulk T cells and with flow cytometry CD25 expression, which is accepted as a T-cell activation marker. This study demonstrated that LDA is a reliable, predictable and sensitive method for measuring alloreactive, pathogen- and leukemia-specific T-cell frequencies.
Bone marrow transplantation 12/2011; 47(9):1196-200. · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We designed a phase I/II clinical study to determine safety and efficacy of thymosin alpha1 (Talpha1) administration in recipients of one HLA haplotype (haploidentical) stem cell transplants for hematologic malignancies. Talpha1 administration did not cause acute or chronic graft versus host disease and was associated with significant improvement in polymorphonuclear (phagocytosis) and dendritic cell (phagocytosis, expression of costimulatory molecules, and cytokine production) functions. It was also associated with increased T-cell counts and earlier appearance of functional pathogen-specific T cell responses (by a sensitive limiting dilution assay that detects frequency of T cells specific for Aspergillus, Candida, CMV, ADV, VZV, HSV, Toxoplasma). Five of six haploidentical transplant recipients who received Talpha1 are alive and disease free at a median follow-up of 10 months after transplantation (range: 5-20). They experienced only a single nonlethal infectious episode and one patient developed fatal immune hemolytic anemia. At this very early stage of the clinical trial, we conclude Talpha1 administration is safe and may impact favorably on immune function. Larger numbers of patients and longer follow-up are, of course, needed to assess its impact on survival.
Annals of the New York Academy of Sciences 04/2010; 1194:153-61. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After haploidentical stem cell transplantation immune recovery is inevitably slow and infectious related mortality is about 30-40%. Immune reconstitution could be improved by infusing donor T cells, but the obstacle is graft-versus-host disease. In a mixed lymphocyte reaction, alloantigen-stimulated T cells uptake 4,5-dibromorhodamine methyl ester (TH9402), a compound that is structurally similar to rhodamine. TH9402 preferentially localizes in mitochondria and when exposed to 500- to 600-nm wavelength visible light delivered through the Theralux device (Kiadis Pharma, Amsterdam, The Netherlands), it becomes highly cytotoxic through oxidative damage. This study investigated a range of parameters, and combinations thereof, with the aim of achieving optimal T cell allodepletion and preservation of pathogen-specific responses. We report on 11 clinical scale dry runs which reproducibly yielded the following results. Blood mononuclear cells were stimulated with haploidentical irradiated (20 Gy). Blood mononuclear cells in a mixed lymphocyte reaction. Cells were then incubated with TH9402 and exposed to light delivered through the Theralux device. Optimal conditions for T cell allodepletion emerged as (1) duration of mixed lymphocyte reaction: 24 h; (2) responder cell concentration: 3-5x10(6)/ml; (3) TH9402 concentration: 5 microM; (4) quantity of internalized TH9402, as measured by mean fluorescence intensity (MFI): 20,000-25,000 MFI; (5) energy delivered: 0.1 J/cm(2). Only under these conditions were the frequencies (by limiting dilution analyses) of alloantigen-specific T cells maximally reduced, i.e., 2467+/-639 (mean+/-SD) times, when compared with non-TH9402-treated cells. Pathogen-specific responses to pathogen antigens such as Cytomegalovirus, Adenovirus, Varicella Zoster Virus, Herpes Simplex Virus, Aspergillus fumigatus, Candida albicans, Toxoplasma gondii were retained, although with a 19+/-9.7 times reduction in frequency. This remarkable drop in frequency of alloreactive T cells is expected to allow safe infusion of relatively large numbers of T cells across histocompatibility barriers for adoptive transfer of donor immunity. Consequently, a clinical trial is planned to incorporate infusion of photo-allodepleted donor T cells after haploidentical stem cell transplantation with the aim of decreasing infection-related mortality.
[Show abstract][Hide abstract] ABSTRACT: We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse), who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I, killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P > .003), better event-free survival in patients transplanted in relapse (34% versus 6%, P = .04) and in remission (67% versus 18%, P = .02), and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor, 0.48; 95% CI, 0.29-0.78; P > .001). In all patients we tested the "missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus and cytomegalovirus are major causes of morbidity/mortality after haploidentical hematopoietic transplantation. The high degree of mismatching makes cell immunotherapy impossible as it would result in lethal graft-versus-host disease (GvHD). We generated large numbers of donor T-cell clones specific for Aspergillus or cytomegalovirus antigens. We identified clones potentially responsible for causing GvHD by screening them for cross-reactivity against recipient mononuclear cells. Non-recipient reactive, pathogen-specific clones were infused soon after transplantation. They were CD4+ and produced high levels of interferon-gamma and low levels of interleukin-10. In 46 control transplant recipients who did not receive adoptive therapy, spontaneous pathogen-specific T cells occurred in low frequency 9 to 12 months after transplantation and displayed a non-protective low interferon-gamma/high interleukin-10 production phenotype. In the 35 recipients who received adoptive therapy, one single infusion of donor alloantigen-deleted, pathogen-specific clones in the dose range of 10(5) to 10(6) cells/kg body weight did not cause GvHD and induced high-frequency T-cell responses to pathogens, which exhibited a protective high interferon-gamma/low interleukin-10 production phenotype within 3 weeks of infusion. Frequencies of pathogen-specific T cells remained stable over time, and were associated with control of Aspergillus and cytomegalovirus antigenemia and infectious mortality. This study opens new perspectives for reducing infectious mortality after haploidentical transplantations.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cell-mediated, donor-vs.-recipient alloresponses occur following transplantation of human leukocyte antigen (HLA) haplotype-mismatched hematopoietic stem cells (HSCs). NK cell alloreactivity reduced the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-vs.-host disease (GvHD). NK cells are primed to kill by several activating receptors. NK killing of autologous cells is prevented because NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) MHC class I alleles. As KIRs are clonally distributed, the NK population in any individual is constituted of a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and in some cases, functional assessment of donor NK clones will identify haploidentical donors who are able to mount donor-vs.-recipient NK alloreactions.
[Show abstract][Hide abstract] ABSTRACT: Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, beta-tubulin and EF 1-alpha genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.
[Show abstract][Hide abstract] ABSTRACT: Tuber spp. are fungi that establish symbiosis with several trees and shrubs. Some of these fungal species produce edible ascomata, also known as truffles, which are highly appreciated for their taste and odour. We isolated and characterized eight polymorphic microsatellite loci from Tuber magnatum, the finest white truffle species, and assessed their variability in 370 individuals collected from all over the species range of distribution. Although two to 18 alleles per locus were found, no heterozygous individuals were observed. The availability of simple sequence repeat loci provides valuable tools for assessment of the genetic structure and population dynamics in this species.