Background: Graft-versus-host disease (GvHD) is a severe complication after hematopoietic stem cell transplantation, in which donor T-cells attack the patient's tissue. GvHD is mediated by escalated ...secretion of pro-inflammatory cytokines such as TNFα and imbalance of effector and regulatory T-cells. Further, CCR7-mediated migration of naive and regulatory donor T-cells into secondary lymphoid organs is crucial in the pathogenesis of GvHD. Although mesenchymal stem cells and their extracellular vesicles (MSC-EVs) contain immune-modulatory capabilities, the strength of the immune-modulatory effects and thus the efficacy of corresponding clinical products may vary between individual preparations. To warrant a certain quality, it is the aim of our study to establish a functional in vitro assay allowing testing for the immunemodulatory capacities of MSC-EV preparations considered as GvHD therapeutics. Methods: Peripheral blood lymphocytes in presence/absence of two different MSC-EV preparations (MSC-EV1 and MSC-EV2) were either stimulated with PMA/Ionomycin for 4 h or with CD3/CD28 for 48 h to monitor cytokine response and T-cell subsets, respectively. GvHD relevant MSC-EV modulations were evaluated by 12-colour (CD45RA, CCR7, CCR4, FOXP3, CD25, CD38, CD39, Ki67, TNFα, IFNg, IL-10 and live/dead) flow cytometric analysis. Results: Upon PMA/Ionomycin stimulation, MSC-EV1 increased the frequencies of IFNg and TNFα secretion of different T-cell subsets, whereas MSC-EV2 decreased the frequencies. Upon CD3/CD28 stimulation, MSC-EV1 decreased the frequency of Ki67- naive T-cells (CD3 +CD45RA+CCR7+) while the frequency of Ki67- effector memory cells (CD3+CD45RA-CCR7-) was increased. Interestingly, the effect of MSCEV2 was vice versa. Summary/Conclusion: This demonstrates that we are able to determine differences in the immune-modulatory capacity of different MSC-EVs towards T-cell cytokine response and towards composition and activation/regulatory status of T-cell subsets.
T-cells play a crucial role in the Graft-versus-Leukemia (GvL)-effect, since T-cells depleted grafts are associated with a higher relapse risk. Unfortunately, the GvL-effect is often associated with ...Graft-versus-Host-Disease (GvHD). T-cells can be divided into two phenotypic sub-groups by the expression of specific alpha/beta- and gamma/delta T-cell receptors. Gamma/delta-T-cells might provide a useful source for T-cell-immunotherapy since they may exert a GvL-effect without inducing a GvHD-risk.
Only few studies have been carried out investigating the possible impact of gamma/delta-T-cell recovery following allogeneic hematopoietic stem cell transplantation (HSCT). A recent prospective study (Godder et al., BMT 2007) indicates a survival advantage for patients (pts) recovering with higher gamma/delta-T-cell numbers following HSCT. The data presented here emerge from a single-centre analysis evaluating the possible impact of higher gamma/delta-T-cell numbers following HSCT in pts with hematologic malignancies. We included all patients who had at least three consecutive analyses of alpha/beta- and gamma/delta-T-cell numbers within the first year after HSCT. This cohort of 107 patients includes the following haematological malignancies: AML (n=40), CML (n=19), ALL (n=13), MDS (n=11), OMF (n=9), NHL (n=7), and other diseases (n=8). Median patient age was 41 years (range 16 – 67 years). Median donor age was 38 years (range 18 – 70 years). HSCT was performed with related donors in 37 pts (35%) and with unrelated donors in 70 pts (65%).
We defined the threshold for “high” gamma/delta-T-cell recovery as three ore more absolute gamma/delta-T-cell numbers above the absolute median gamma/delta-T number in the peripheral blood within the first 12 months after HSCT. According to this threshold 29 pts (27%) recovered with “high” gamma/delta-T-cells. These pts achieved a significantly higher overall survival with lower gamma/delta-T-cell numbers (log-rank p .029). This resulted from a lower relapse risk and a lower risk for acute GvHD. In multivariate analysis including other prognostic factors of overall survival (patient age, disease status, donor type, grades of acute GvHD and relapse), the beneficial effect of “high” gamma/delta-T-cell recovery could be confirmed. In contrast, recovery of alpha/beta T-cell numbers in peripheral blood had no significant influence on HSCT endpoints and were further not associated with the recovery of gamma/delta T-cells.
This analysis supports the hypothesis of a beneficial effect of high gamma/delta-T-cells recovery following HSCT regarding overall survival. Further analyses and research are warranted to determine more accurately the importance of increased recovery of gamma/delta-T-cells to possibly develop new therapeutic options in HSCT as e.g. graft engineering.
Posttransplant lymphoproliferative disease (PTLD) is a life-threatening complication after allogeneic hematopoietic stem cell (HSCT) or solid organ transplantation. The majority of PTLD are ...associated with the Epstein-Barr virus (EBV). PTLD may involve several organs like liver, lungs, kidney, spleen, and small intestine. PTLD with central nervous system (CNS) involvement only has been reported in very few cases so far.
We here describe 2 cases who 7 and 12 months after HSCT developed EBV PTLD with CNS lymphomas only. The efficacy of therapy was monitored by magnetic resonance imaging, EBV replication, light chain detection, and the clinical course. Both patients responded very well to the intravenous administration of the monoclonal anti-CD20 antibody rituximab.
These 2 case reports illustrate that in EBV PTLD with CNS lymphomas, the systemic administration of rituximab only may prove effective.
Abstract 1203
Poster Board I-225
HHCT using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion is of low toxicity and enables fast engraftment even with grafts of low ...CD34 content. Immune reconstitution may be improved compared to graft CD34 selection due to the cotransplantation of facilitating cells, CD34- progenitors, dendritic cells and natural killer (NK)-cells. A prospective multicenter phase II study of HHCT using CD3/CD19-depleted grafts after RIC with fludarabine (150-200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) was initiated. No post grafting immunosuppression was applied if the graft contained <5×104 CD3+ cells/kg. 60 adults with median age of 45 years (range, 19-61) have been enrolled from 2002 to 2009. Diagnoses were AML (n=37), ALL (n=8), NHL (n=6), myeloma (n=4), CML (n=3), CLL (n=1) and MDS (n=1). Patients were “high risk” because of refractory disease (n=6), cytogenetics (n=5), chemosensitive relapse (n=26) or relapse after prior HCT (allo=16, auto=6, both=1). At HHCT, 30 patients were in CR and 30 in PR. Grafts contained median of 6.8×106 (range, 3.5-22) CD34+ cells/kg, 4.0×104 (range, 0.9-44) CD3+ T cells/kg and 2.8×107 (range, 0.00-37.3) CD56+ cells/kg. Engraftment was rapid with median of 12 days to >500 granulocytes/μL (range, 9-50) and 11 days to >20.000 platelets/μL (range, 7-38). Incidence of grade II-IV acute GVHD and chronic GvHD (cGvHD) was 47% and 15%, respectively. Non-relapse-mortality on day 100 was 25% and 44% for the whole study period. Current overall survival (OS) is 18 of 60 patients (30%) with median follow-up of 525 days (range, 21-1542) of patients alive resulting in a Kaplan-Meier estimate 1-year OS of 41% and 2-year OS of 24%. Kaplan-Meier estimate 1-year OS was 40 % in AML, 38 % in ALL and 67% in NHL patients. No positive impact of KIR-mismatch on survival even in the subgroup of patients with AML was observed. Patients with cGVHD had a trend toward better survival with 56% vs. 39% (p=0.09). 14 patients died because of infections. Detailed immune reconstitution was analyzed in 24 patients. NK-cell engraftment was fast with median of 248 CD16+56+CD3- cells/μl (range, 1-886) on day 20. Increased natural cytotoxicity receptors (NKP30, NKP44, NKP46) and NKG2A, but decreased NKG2D and KIR-expression was observed on NK-cells in the first 3 months. T-cells regenerated delayed with median of 191 CD3+ cells/μl (range, 38-799) on day 100. A slow reconstitution of CD8+ and CD4+ T-cells with median of 66 CD8+ (range, 8-170) vs. 70 CD4+ cells/μl (range, 12-301) on day 100 and 157 (range, 32-379) vs. 181 cells/μl (range, 19-980) on day 400 was observed. The subset of memory T-cells regenerated faster compared to naïve T-cells with median of 25 CD4+45RA+ (range, 4-109) vs. 80 CD4+45RO+ cells/μl (range, 0 to 255) and 46 (range 7-191) vs. 164 cells/μl (range, 66-323) on days 80 and 250, respectively. T-cell repertoire was skewed with oligoclonal T-cell expansion to day 100 and normalization after day 200. B-cell reconstitution was slow with median of 8 (range, 0-407) CD19+20+ cells/μl on day 100. 6 of these 24 patients received donor-lymphocyte-infusions for relapse or mixed chimerism resulting in acceleration of immune recovery in T- and NK-cells. In conclusion, HHCT using RIC and CD3/CD19 depleted grafts is of low toxicity and allows fast engraftment even with low doses of CD34 cells. Overall survival seems promising in a high risk patient cohort. Recovery of NK cells occurs early and fast. KIR receptor expression remains low in the first 3 months. T- and B-cell reconstitution is delayed but seems faster compared to published data after CD34 selection. To evaluate the treatment protocol earlier during the course of disease a new study has just been initiated.
Off Label Use: The use of Fludarabine, Thiotepa, Melphalan and OKT-3 in the conditioning is off-label-use.
Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with a megadose of CD34-selected stem cells has been complicated by regimen related toxicities, slow engraftment ...and delayed immune reconstitution leading to high treatment related mortality (TRM). A new regimen using reduced intensity conditioning (RIC) and immunomagnetic CD3/CD19 graft depletion may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also graft-facilitating cells, CD34- progenitors, dendritic and natural killer cells which may allow stable engraftment even without a megadose of CD34+ cells.
A multicenter phase I/II study of HHCT using RIC with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day -5 to +14) and CD3/CD19 graft depletion has been initiated. No post grafting immunosuppression was applied if the graft contained <5x104 CD3+ cells/kg.
To date, 51 patients with a median age of 45 years (range, 19–65) have been enrolled in this study. Diagnosis were AML (n=34), ALL (n=7), NHL (n=6), MM (n=2), and CML (n=2). Patients were “high risk” because of relapsed or refractory disease (n=30), or relapse after preceding HCT (auto=7, allo=14). Stage at HCT was complete remission (n=25) and partial remission (n=26). The CD3/CD19 depleted haploidentical grafts contained a median of 7.1 x 106 (range, 3.4–18x106) CD34+cells/kg, 3.9 x104 (range, 0.6–44x104) CD3+T cells/kg and 2.8x107 (range, 0.02–37.3x107) CD56+cells/kg. The regimen was well tolerated with maximum acute toxicity being CTC-grade 1–2 mucositis. Five cases of reversible peripheral neuropathy and three cases of progressive multifocal leukencephalopathy (PML) occurred posttransplant in heavily pretreated patients. Engraftment was rapid with median time to >500 granulocytes/μL of 12 days (range, 9–50) and to >20000 platelets/μL of 11 days (range, 7–38). Full donor chimerism was reached after 2–4 weeks in all but four patients (median of 14 days (range, 7–215)). Four patients experienced rejection/non-engraftment, two were rescued by a second CD3/CD19 depleted graft from another haploidentical donor. Incidence of grade II-IV acute GVHD was 51% with grade II=16, III=6 and IV=4. So far there are six cases of limited chronic GVHD and one case of extensive chronic GVHD. TRM in the first 100 days was 11/51 (22%) and overall 20/51 (39%). Overall survival is 17/51 patients (33%) with deaths due to relapse (n=14), infection (n=15), PML (n=2), GVHD (n=2) and cardiac failure (n=1), with a median follow-up of alive patients of 397 days (range, 62–1180). This results in a Kaplan-Meier estimate 1-year survival of 37%. So far, we did not observe a statistical significant survival advantage for patient transplanted from a KIR-mismatched donor (28/51 patients).
This regimen with low toxicity as well as fast and sustained engraftment is promising in high risk patients lacking a suitable donor. To investigate the treatment protocol earlier in course of disease a new study for high-risk patients with acute leukemia in first complete remission is in preparation.
Texte remanié de: Diss.--Medizinische Fakultät--Tübingen--Eberhard-Karls-Universität, 1997.
La couverture porte le titre: "Hegels kritische Analyse der Schädellehre Galls in der "Phänomenologie ...des Geistes"" Bibliogr. p. 235-243. Index.
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