Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors.

BackgroundThe adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6.MethodsTo obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay.ResultsStarting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes.ConclusionsThe generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.

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Whereas the CD34 CE was significantly different with the AS104 and the Spectra, the CD34 CE of both machines correlated inversely with peripheral blood CD34+ cell counts, showing a significant decline with increasing numbers of circulating CD34+ cells. Nevertheless, at > or 40 preapheresis CD34+ cells per microL, sufficient hematopoietic autografts of > or =2.5 x 10(6) CD34+ cells per kg were harvested by a single conventional-volume (11 L) leukapheresis on both cell separators.

In contrast to the non-survivors, we observed a significant decrease in the C-reactive protein levels shortly after initiation of the GTX treatment in the surviving patients. A clear-cut benefit of GTX for children with neutropenic sepsis cannot be concluded from these data, but in children with (severe) bacterial sepsis refractory to antibiotic treatment, GTX were feasible, safe and could reduce mortality rates in this subgroup of patients.

Objectives: Microbiological cultures and posttransplantation course were analyzed in order to investigate the incidence and clinical significance of bacterial contamination of autologous bone marrow (BM) grafts. Methods: Cultures were obtained from BM after collection, BM concentrate after processing, contaminated/cryopreserved BM at thawing, and from peripheral blood (PB) following autologous BM transplantation (ABMT). The posttransplantation course of patients grafted with culture-positive BM was recorded and compared with patients who underwent ABMT with noncontaminated BM grafts. Results: In 239 BM grafts processed, the incidence of microbiological contamination was 26.4% (n = 63). Fifty marrow grafts were contaminated by bacteria from the skin flora: coagulase-negative Staphylococcus (CNSC), Propionibacterium, and Corynebacterium species (79%). Thirty-eight patients underwent ABMT (day 0) with cryopreserved culture-positive BM, and 32 patients were evaluable for microbiological cultures at thawing: in 10 of 32 BM grafts CNSC was found prior to reinfusion. Following ABMT, PB cultures revealed CNSC in 5 of 38 patients between days +4 and +12. However, the late occurrence of positive PB cultures after BM reinfusion made a relationship between BM CNSC and PB CNSC unlikely. In 33 of 38 patients, no graft-contaminating bacteria were detected in PB. Comparison of the posttransplantation course of patients who received contaminated BM with that of patients grafted with noncontaminated BM showed no significant differences concerning time to engraftment, febrile days, and days on antibiotics. Conclusion: (1) Collection and/or ex vivo processing can result in microbiological contamination of BM grafts predominantly with bacteria from the skin flora, and (2) only CNSC can be cultured at thawing from previously contaminated/cryopreserved BM. Since patients undergoing ABMT usually receive oral antibiotics from beginning of the conditioning regimen which are active against CNSC, no further administration of antibiotics is recommended for the reinfusion of bacterially contaminated BM grafts.

At least in donors with short interdonation intervals, most suspected CMV seroconversions are due to false-positive results of the screening test. As window period donations are rare and contain less CMV DNA than the first seropositive donation, avoidance of blood products from primarily seropositive donors is especially helpful to avoid TT-CMV if donors with short interdonation intervals are concerned.

The COM.TEC machine makes it possible to obtain WBC-reduced SDPs that comply with current standards.