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Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.

Summary: Donor lymphocyte infusion (DLI) is an effective method to establish full donor chimerism or to prevent and treat relapse after allogeneic haematopoietic cell transplantation (allo‐HCT). Usually, DLIs are collected from naïve donors as steady‐state lymphocytes. When donor lymphocytes are collected during stem cell apheresis, donors are pre‐treated with granulocyte colony‐stimulating factor (G‐CSF). However, the impact of G‐CSF stimulation and the resulting composition of DLIs on beneficial anti‐leukaemic responses and survival remain elusive. Therefore, we performed a retrospective analysis to evaluate the role of G‐CSF‐DLIs: 44 patients received either steady‐state DLIs or G‐CSF‐DLIs to prevent and treat relapse or establish full donor chimerism after allo‐HCT. The G‐CSF‐DLI patient cohort showed an improved conversion to full donor chimerism and a lower cumulative incidence of relapse or disease progression without a significantly increased cumulative incidence of graft‐versus‐host disease (GVHD). CD34+ cells, monocytic myeloid‐derived suppressor cells and monocytes as well as donor age and the subsequent occurrence of chronic GVHD were identified as risk factors that significantly improve overall survival after DLI administration. In conclusion, our data suggest that administration of G‐CSF‐DLIs results in graft‐versus‐leukaemia effects without exacerbating GVHD, therefore, improving survival after DLIs. [ABSTRACT FROM AUTHOR]

Poor graft function ( PGF) is a severe complication of haematopoietic stem cell transplantation ( HSCT) and administration of donor stem cell boosts ( SCBs) represents a therapeutic option. We report 50 paediatric patients with PGF who received 61 boosts with CD34+ selected peripheral blood stem cells ( PBSC) after transplantation from matched unrelated ( n = 25) or mismatched related ( n = 25) donors. Within 8 weeks, a significant increase in median neutrophil counts (0·6 vs. 1·516 × 109/l, P < 0·05) and a decrease in red blood cell and platelet transfusion requirement (median frequencies 1 and 7 vs. 0, P < 0·0001 and <0·001), were observed, and 78·8% of patients resolved one or two of their cytopenias. 36·5% had a complete haematological response. Median lymphocyte counts for CD3+, CD3+ CD4+, CD19+ and CD56+ increased 8·3-, 14·2-, 22.- and 1·6-fold. The rate of de novo acute graft-versus-host disease (Gv HD) grade I-III was only 6% and resolved completely. No Gv HD grade IV or chronic Gv HD occurred. Patients who responded to SCB displayed a trend toward better overall survival ( OS) ( P = 0·07). Thus, administration of CD34+ selected SCBs from alternative donors is safe and effective. Further studies are warranted to clarify the impact on immune reconstitution and survival. [ABSTRACT FROM AUTHOR]

Graft failure is a life-threatening complication after allogeneic haematopoietic stem cell transplantation ( HSCT). We report a cohort of 19 consecutive patients (median age: 8·5 years) with acute leukaemias ( n = 14) and non-malignant diseases ( n = 5) who experienced graft failure after previous HSCT from matched ( n = 3) or haploidentical donors ( n = 16) between 2003 and 2012. After total nodal irradiation ( TNI)-based reconditioning combined with fludarabine, thiotepa and anti-T cell serotherapy, all patients received T cell-depleted peripheral blood stem cell grafts from a second, haploidentical donor. Median time between graft failure and retransplantation was 14 d (range 7-40). Sustained engraftment (median: 10 d, range 9-32) and complete donor chimerism was observed in all evaluable patients. 5 patients additionally received donor lymphocyte infusions. Graft-versus-host disease (Gv HD) grade II and III occurred in 1 patient each (22%); no Gv HD grade IV was observed. 2 patients had transient chronic Gv HD. The regimen was well tolerated with transient interstitial pneumonitis in one patient. Treatment-related mortality after one year was 11%. Event-free survival and overall survival 3 years after retransplantation were 63% and 68%. Thus, a TNI-based reconditioning regimen followed by transplantation of haploidentical stem cells is an option to rescue patients with graft failure within a short time span and with low toxicity. [ABSTRACT FROM AUTHOR]

Objective: CD3/CD19 depletion may improve engraftment and immune reconstitution after haploidentical hematopoietic cell transplantation (HHCT) as grafts not only contain CD34+ stem cells but also CD34− progenitors and natural killer, dendritic, and facilitating cells. Patients and Methods: Ten consecutive patients received HHCT with CD3/CD19-depleted grafts. Reduced-intensity conditioning was performed with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), and OKT-3 (5 mg/day, day −5 to +14) without additional posttransplant immunosuppression. Diagnoses were AML (n = 4), ALL (n = 3), NHL (n = 2), and multiple myeloma (n = 1). All patients were “high risk” with refractory disease or relapse after preceding HCT. The CD3/CD19-depleted haploidentical grafts contained a median of 7.8 × 106 (range, 5.2–17 × 106) CD34+ cells/kg, 5.5 × 107 (range, 0.02–8.6 × 107) CD56+ cells/kg, and 2.0 × 104 (range, 0.006–44 × 104) CD3+ T cells/kg. Engraftment was rapid with median time to greater than 500 granulocytes/μL of 13 (range, 11–17) days, greater than 20,000 platelets/μL of 11 (range, 8–16) days, and full donor chimerism after 2 weeks in all patients. Six cases of grade II GVHD occurred. One patient, who received the highest T cell dose, developed lethal grade IV GVHD. Treatment-related mortality in the first 100 days was 3/10 (30%) with one death each due to idiopathic pneumonia syndrome, GVHD, and CMV disease. Two patients died after day 100, one due to relapse and one with systemic adenoviral infection. Overall survival is 5/10 patients (50%) with a median follow-up of 435 (range, 229–814) days. Conclusion: This regimen is promising in high-risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing. [Copyright &y& Elsevier]

: ObjectiveIn patients with lymphopenia following allogeneic stem cell transplantation adenovirus (ADV) infection is associated with high morbidity and mortality despite aggressive antiviral drug therapy. Virus-specific T cells seem to be essential for virus elimination. The aim of this study was to isolate and expand donor-derived human ADV-specific T lymphocytes for adoptive transfer of sufficient cell numbers to restore protective immunity after allogeneic stem cell transplantation.: Materials and methodsA clinical-grade strategy to generate ADV-specific T cells using the interferon-γ (IFN-γ) secretion assay, followed by expansion to numbers sufficient for clinical application with interleukin-2 (IL-2) and feeder cell stimulation, is described.: ResultsA mean number of 3.4×106 (±3 SD) ADV antigen-specific T lymphocytes were isolated from 0.1 to 2×109 mononuclear cells from peripheral blood (n = 5) or leukapheresis products (n = 6). Characterization of ADV-specific T cells after isolation revealed a mean purity of 85.1% (±12% SD) using antigen-specific intracellular cytokine staining. Isolated cells were expanded ex vivo for a median of 18 days (range 7–29 days; n = 5) to greater than 108 total cells using IL-2 and autologous feeder cell stimulation. ADV-specific response to adenovirus antigen was confirmed in the generated T cell lines, using intracellular cytokine staining, IFN-γ Elispot assay, and 3H-thymidine uptake. Generated T-cell lines showed specific killing of ADV-infected B-LCL (n = 4). Alloreactive proliferation of generated T-cell lines in mixed lymphocyte cultures was significantly reduced when compared to unmanipulated PBMCs.: ConclusionGeneration of adenovirus-specific T cells in a simple and rapid clinical-grade protocol was established, using IFN-γ secretion assay with short expansion times, leading to sufficient numbers of ADV-specific T cells that can be used for adoptive immunotherapy. [Copyright &y& Elsevier]