A sequential regimen of chemotherapy, reduced-intensity conditioning (RIC) for allogeneic stem cell transplantation (SCT), and prophylactic donor lymphocyte transfusion (pDLT) was studied in 103 patients with refractory acute myeloid leukemia (AML). According to published criteria, refractoriness was defined by primary induction failure (PIF; n ‫؍‬ 37), early (n ‫؍‬ 53), refractory (n ‫؍‬ 8), or second (n ‫؍‬ 5) relapse. Chemotherapy consisted of fludarabine (4 ؋ 30 mg/m 2 ), cytarabine (4 ؋ 2 g/m 2 ), and amsacrine (4 ؋ 100 mg/ m 2 ), followed 4 days later by RIC, comprising 4 Gy total body irradiation (TBI), cyclophosphamide, and antithymocyte globulin. Patients without graft-versus-host disease (GvHD) at day ؉120 received pDLT in escalating doses. Patients' median age was 51.8 years. Before conditioning, 99 patients had active disease, 3 were aplastic, 1 was in second complete remission (CR2). Forty-one patients had family donors, 62 had unrelated donors. With a 25-month median follow-up, overall survival (OS) at 1, 2, and 4 years was 54%, 40%, and 32%; the respective leukemiafree survival (LFS) was 47%, 37%, and 30%. Patients with PIF showed a 2-year OS of 62.5%. OS was 87% in 17 patients receiving pDLT. One-year cumulative incidence of leukemic death and non-relapsemortality was 28.7% and 17.2%. In a multivariate analysis, more than 2 courses of prior chemotherapy were the strongest predictor for poor outcome (P ‫؍‬ .007; HR ‫؍‬ 3.01 [OS]; P ‫؍‬ .002; HR ‫؍‬ 3.25 [LFS]). These results indicate a high activity of the regimen in refractory AML.

In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

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.

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed “young” CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.

Tetramer monitoring can help to predict (recurrent) CMV reactivation and is a useful approach to monitor individual patients with increased risk for recurrent reactivation post HSCT; thus, it could help to identify patients in need of adoptive transfer of CMV-CTL or to optimize the use of antiviral drugs.

Platzbecker U on behalf of the German MDS and Cooperative Transplant Study Group (GCTSG). Lenalidomide maintenance after allogeneic HSCT seems to trigger acute graft-versus-host disease in patients with : results of the LENAMAINT trial. Haematologica 2012;97(9):e34-35. doi:10.3324/haematol.2012 LETTERS TO THE EDITOR© F e r r a t a S t o r t i F o u n d a t i o n © F e r r a t a S t o r t i F o u n d a t i o n

GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.