Johannes Clausen

Evidence from a leukaemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells

BACKGROUND Vascular endothelial cells lost from the blood-vessel endothelium through necrosis or apoptosis must be replaced. We investigated in a leukaemia model whether bone-marrow-derived endothelial cells contribute to this maintenance angiogenesis. METHODS We studied six patients with chronic myelogenous leukaemia (CML) carrying the BCR/ABL fusion gene in their bone-marrow-derived cells. We screened endothelial cells generated in vitro from bone-marrow-derived progenitor cells and vascular endothelium in myocardial tissue for the BCR/ABL fusion gene by in-situ hybridisation. For detection of donor-type endothelial cells after transplantation of haemopoietic stem cells, recipient tissue was stained with monoclonal antibodies against donor-type HLA antigens. FINDINGS We identified the BCR/ABL fusion gene in variable proportions (0-56%) of endothelial cells generated in vitro. Endothelial cells expressing the fusion gene were found in the vascular endothelium of a patient. In a recipient of an allogeneic stem-cell transplant, normal donor-type endothelial cells were detected in the vascular endothelium. INTERPRETATION These findings suggest that CML is not solely a haematological disease but originates from a bone-marrow-derived haemangioblastic precursor cell that can give rise to both blood cells and endothelial cells. Moreover, normal bone-marrow-derived endothelial cells can contribute to the maintenance of the blood vascular endothelium. The integration of bone-marrow-derived endothelial cells into the vascular endothelium provides a rationale for developing vascular targeting strategies in vasculopathies, inflammatory diseases, and cancer.

Thrombocytes Are the Major Source for Soluble Vascular Endothelial Growth Factor in Peripheral Blood

Serum levels of vascular endothelial growth factor (VEGF-S) have been reported to correlate with tumor stage and prognosis in various human malignancies. The source of soluble VEGF in peripheral blood remains obscure. We therefore measured the concentration of immunoreactive VEGF in 241 serum samples and 61 plasma samples (VEGF-P) from 20 subjects undergoing myeloablative chemotherapy and from 3 normal platelet donors. A significant correlation between the peripheral blood platelet count (PC) and VEGF-S (r = 0.86) but not VEGF-P was found. VEGF-S levels were 58.43 ± 42.50 pg/ml (mean ± SD) in patients with a PC < 50 × 109/l, 203.29 ± 176.56 pg/ml for a PC of 50–150 × 109/l, and 457.42 ± 475.41 pg/ml for a PC > 150 × 109/l. Interestingly, VEGF-P levels were substantially lower than the corresponding VEGF-S values, namely below the detection limit in most cases. Supernatants from platelet-rich plasma contained no VEGF, but after in vitro lysis of the platelets very high VEGF levels were found. The VEGF content per 109 platelets was calculated at 2.51 ± 2.39 pg and was dependent on the mean platelet volume. In summary, VEGF release from platelets during blood clotting was found to be the main source of VEGF in serum samples. Cancer patients in clinical remission have negligible amounts of soluble VEGF in peripheral blood, and myeloablative chemotherapy causes a significant drop in VEGF-S levels corresponding to the decrease in PC. Thus, studies addressing the diagnostic and prognostic value of VEGF-S in cancer patients must be interpreted with caution. Our data provide the basis for predicting VEGF-S in relation to PC in vivo, and for reevaluating former studies of VEGF-S in patients with malignant or nonmalignant disease.

Functional significance of the activation-associated receptors CD25 and CD69 on human NK-cells and NK-like T-cells.

The application of autologous ex-vivo expanded cytotoxic lymphocytes to cancer patients may help to control minimal residual disease. However, the number of effector cells and the resulting antitumoral activity that can be generated in vitro are remarkably variable. Thus, we separately assessed the proliferative and cytotoxic potential of CD56+ CD3- natural killer (NK) and CD56+ CD3+ T-cells in relation to their expression of CD25, CD69, and CD16 in vitro. Two-week lymphocyte cultures from peripheral blood (n = 51) and from G-CSF-mobilized progenitor cell harvests (n = 11) were performed repeatedly from 14 women with breast cancer throughout conventional- and high-dose chemotherapy. A large proportion of CD25+ cells on day 7 of the culture predicted high expandability (r = 0.69, p < 0.00001), while elevated expression of CD69 predicted augmented cytotoxicity (r = 0.72; p = 0.00001) and low expandability (r = -0.69, p < 0.00001). CD25 and CD69 expression were inversely correlated (r = -0.8, p < 0.0001). CD16 expression was not suited to predict functional properties. Additionally, NK-cells were sorted by FACS according to CD25 versus CD69 expression. In a [3H]thymidine incorporation assay the CD25+ NK-cell fraction exhibited a higher proliferation rate than did the CD69+ fraction in all of three experiments. Together, our data suggest that CD69 is a useful marker for cytotoxic activity of NK cells, whereas proliferative potential is indicated by CD25 expression. These findings should help optimizing the ex-vivo generation of large numbers of cytotoxic effector cells for immunotherapy.

Regulatory T-cells in the graft and the risk of acute graft-versus-host disease after allogeneic stem cell transplantation

Background. FOXP3+ regulatory T-cells (Treg) are important regulators of allo-reactivity and may therefore represent an important predictor for the risk of graft versus-host disease (GVHD) after allogeneic stem cell transplantation. Methods. To determine the clinical significance of Treg-content in stem cell grafts, we analyzed 58 human leukocyte antigen (HLA)-identical sibling donors (34 patients received myeloablative and 24 patients reduced intense conditioning regimens) and correlated the Treg frequency with clinical outcome after stem cell transplantation (SCT). Results. A mean value of 9.1×106 CD4+FOXP3+ Treg per kg body weight (bw) of the recipient was transplanted (ranging from 0.7 to 33.7×106 Treg/kg bw). Graft content of Treg correlated with mononuclear cells and CD3+ T-cells. Patients receiving low numbers of Treg (Treglow) after myeloablative conditioning for SCT had a significantly increased cumulative incidence of 76% for acute GVHD when compared with 23% for individuals receiving high numbers of Treg (Treghigh). This observation, however, was not made in patients after reduced intense conditioning-SCT. Notably, relapse rate was not significantly different between Treglow and Treghigh patients in either patient group and overall survival was even increased in Treghigh patients after myeloablative SCT. Finally, low Treg graft levels represent an independent prognostic factor in multivariate analysis for the appearance of acute GHVD. Conclusion. Donor-derived Treg might be of particular significance for the development of acute GVHD after myeloablative SCT using HLA-identical sibling donors.

Progressive multifocal leukoencephalopathy after allogeneic stem cell transplantation and posttransplantation rituximab.

Progressive multifocal leukoencephalopathy (PML) is a rare but fatal demyelinating disease of the brain caused by the JC papovavirus (JCV), affecting mainly immunocompromised patients. Recently, an association between PML and the application of rituximab after autologous stem cell transplantation (S

Optimal timing for the collection and in vitro expansion of cytotoxic CD56(+) lymphocytes from patients undergoing autologous peripheral blood stem cell transplantation.

To identify the optimal time for the collection of CD56(+) cytotoxic lymphocytes for adoptive immunotherapy in patients undergoing high-dose chemotherapy (HDCT) and peripheral blood stem cell (PBSC) transplantation, 18 breast cancer patients receiving either three cycles of epirubicin/paclitaxel (CT x 3) followed by HDCT and PBSC transplantation (n = 12) or CTx6 (n = 6) were studied. Blood samples were obtained before each CT/HDCT cycle, from PBSC collections, and repeatedly after autografting for up to 12 months. The number of CD56(+)3(-) and CD56(+)3(+) lymphocytes, their in vitro expandability with interleukin-2, and their cytotoxicity against MCF-7 and Daudi cells were analyzed. Six healthy females served as controls. CD56(+) cell counts in both treatment groups were subnormal but stable during the observation period. The cytotoxicity of the expanded CD56(+) cells was normal and unaffected by the treatment. The in vitro CD56(+) cell expandability (controls, 100 +/- 31-fold, mean +/- SEM) was normal before CT1 and CT2, but reduced in PBSC harvests performed after CT2 and application of G-CSF (21 +/- 6-fold; p < 0.01). After PBSC harvesting, the CD56(+) cell expandability increased to 185 +/- 74-fold and 170 +/- 69-fold (before CT3 and HDCT). This increase was not observed in those patients who did not undergo PBSC mobilization. Two weeks after autografting, the CD56(+) cell expandability was minimal (6 +/- 1-fold), and recovered to 34 +/- 6-fold. Thus, CT, HDCT and autografting do not alter the frequency and inducible cytotoxicity of CD56(+) cells in breast cancer patients. However, the proliferative capacity of CD56(+) cells obtained from PBSC harvests and after autografting is impaired. Therefore, instead of the PBSC graft, maximally expandable CD56(+) cells obtained at least 1 week after PBSC collection should be considered for adoptive immunotherapy after PBSC autografting.

HLA-C KIR-Ligands Determine the Impact of Anti-Thymocyte Globulin (ATG) on Graft versus Host and Graft versus Leukemia Effects Following Hematopoietic Stem Cell Transplantation

Rabbit anti-thymocyte globulins (ATGs) are widely used for the prevention of acute and chronic graft versus host disease (aGVHD, cGVHD) following allogeneic hematopoietic stem cell transplantation (HSCT). However, most prospective and retrospective studies did not reveal an overall survival (OS) benefit associated with ATG. Homozygosity for human leukocyte antigen (HLA)-C group 1 killer-cell immunoglobulin-like receptor ligands (KIR-L), i.e. C1/1 KIR-L status, was recently shown to be a risk factor for severe aGVHD. Congruously, we have previously reported favorable outcomes in C1/1 recipients after ATG-based transplants in a monocentric analysis. Here, within an extended cohort, we test the hypothesis that incorporation of ATG for GVHD prophylaxis may improve survival particularly in HSCT recipients with at least one C1 KIR-ligand. Retrospectively, 775 consecutive allogeneic (excluding haploidentical) HSCTs were analyzed, including peripheral blood and bone marrow grafts for adults with hematological diseases at two Austrian HSCT centers. ATG-Fresenius/Grafalon, Thymoglobuline, and alemtuzumab were applied in 256, 87, and 7 transplants, respectively (subsequently summarized as “ATG”), while 425 HSCT were performed without ATG. Median follow-up of surviving patients is 48 months. Adjusted for age, disease-risk, HLA-match, donor and graft type, sex match, cytomegalovirus serostatus, conditioning intensity, and type of post-grafting GVHD prophylaxis, Cox regression analysis of the entire cohort (n = 775) revealed a significant association of ATG with decreased non-relapse mortality (NRM) (risk ratio (RR), 0.57; p = 0.001), and overall mortality (RR, 0.71; p = 0.014). Upon stratification for HLA-C KIR-L, the greatest benefit for ATG emerged in C1/1 recipients (n = 291), by reduction of non-relapse (RR, 0.34; p = 0.0002) and overall mortality (RR, 0.50; p = 0.003). Less pronounced, ATG decreased NRM (RR, 0.60; p = 0.036) in HLA-C group 1/2 recipients (n = 364), without significantly influencing overall mortality (RR, 0.70; p = 0.065). After exclusion of higher-dose ATG-based transplants, serotherapy significantly improved both NRM (RR, 0.54; p = 0.019; n = 322) and overall mortality (RR, 0.60; p = 0.018) in C1/2 recipients as well. In both, C1/1 (RR, 1.70; p = 0.10) and particularly in C1/2 recipients (RR, 0.94; p = 0.81), there was no statistically significant impact of ATG on relapse incidence. By contrast, in C2/2 recipients (n = 121), ATG neither reduced NRM (RR, 1.10; p = 0.82) nor overall mortality (RR, 1.50; p = 0.17), but increased the risk for relapse (RR, 4.38; p = 0.02). These retrospective findings suggest ATG may provide a survival benefit in recipients with at least one C1 group KIR-L, by reducing NRM without significantly increasing the relapse risk.

Expansion of Mobilized Peripheral Blood Progenitor Cells under Defined Culture Conditions Using CD34+ CD71- CD45- Cells as a Starting Population

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions supporting the expansion of transplantable hematopoietic stem cells. We assessed the expansion potential of CD34+CD71-CD45- cells derived from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood under recently defined serum-free culture conditions. The CD34+CD71-CD45- cells in mobilized peripheral blood were found to contain the majority (92%+/-5.6) of primitive long-term culture initiating cells (LTCIC) and 53.5%+/-16.7 of the more committed colony-forming cells (CFC). Furthermore, this population represents 23.3%+/-4.1 of the total CD34+ cells and allows reduction of the cell density important for maintenance/expansion of primitive progenitor cells. CD34+ CD71- CD45- cells were cultured in defined serum-free media supplemented with 300 ng each of Flt-3 ligand and stem cell factor (SCF), 60 ng of interleukin (IL)-3, and 20 ng each of IL-6 and G-CSF. Mononuclear cells (MNC) and CFC were expanded 50-fold and 200-fold, respectively; primitive progenitor cells (LTC-IC) were maintained at input values after a total of 10 days of expansion. The addition of IL-15 to our cytokine cocktail expanded LTC-IC 2- to 3-fold and CFC to >500-fold. The data presented should allow clinical manipulation (purging) and expansion procedures with mobilized PBPC harvests without the loss of primitive progenitor cells and could be made applicable for large-scale clinical expansion.

HLA‐A*0201 is associated with a better outcome after donor lymphocyte infusion for recurrent malignancy

To the Editor: Donor lymphocyte infusion (DLI) from the original stem cell donor is an established treatment option for leukemic relapse following allogeneic stem cell transplantation (1, 2). Donor T cells recognizing host-derived minor histocompatibility antigens presented on the cell surface by MHC class I and II molecules have been implicated in both graft-versus-host disease as well as graft-versus-leukemia reactions (3, 4). In the present study, we aimed to analyze whether certain HLA alleles are associated with long-term outcome after DLI by defining risk factors for death in 30 consecutive patients receiving DLIs for the treatment of relapse at our institution. Nineteen patients had acute myeloid leukemia, six patients had chronic myelogenous leukemia, and five patients had other diagnoses. Twentytwo patients were grafted from HLA-identical sibling donors and eight patients were transplanted from 12 ⁄ 12 HLA-antigen-matched volunteer unrelated donors. Twenty patients received unmanipulated granulocytecolony-stimulating factor-mobilized peripheral blood stem cells and ten patients received bone marrow stem cells. At the time of DLI (median T cell dose 0.99 · 10 ⁄kg, range 0.05–2.0), all patients had documented disease relapse ⁄progression according to previously published criteria (5). The complete remission rate was highest in patients with chronic myelogenous leukemia (67%), followed by patients with acute myeloid leukemia (47%), and patients with other diagnoses (40%). The overall survival rate was 30% for the entire cohort, 67% for patients with chronic myelogenous leukemia, 14% for patients with acute leukemia, and 60% for patients with other diagnoses (P = 0.049, log rank test). Achieving complete remission or graft-versus-host disease after DLI was associated with a significantly better survival. HLAA2-positive patients, who were not significantly different to HLA-A2-negative patients with regard to recipient age, donor age, diagnosis, donor type, time interval between transplantation and relapse, time interval from relapse to DLI, CD3 cell dose, disease-specific salvage therapy in combination with DLI, or immunosuppression at the time of relapse, had a significantly higher complete remission rate (79% vs. 25%, P = 0.0034, chisquare test) resulting in a significantly better survival (52% vs. 9%, P = 0.0223, log rank test; Fig. 1). Among HLA-A2 positive patients, four patients had CML, eight patients had AML, and two patients had other diagnoses. HLA-A2 positive patients also experienced a significantly higher rate of graft-versus-host disease following DLI (57% vs. 19%, P = 0.029, chi-square test). All HLA-A2-positive patients expressed the HLA-A*0201 allele as determined by high resolution typing (Olerup SSP HLA-A*02 lot; QIAGEN Vertriebs GmbH, Vienna, Austria). By multivariate Cox regression analysis HLA-A2 positivity (RR 0.03, P = 0.02), being off immunosuppression at the time of relapse (RR 0.03, P = 0.01), and diseasespecific chemotherapy in combination with DLI (RR 0.06, P = 0.007) were associated with a significantly lower risk for death. These preliminary data provide first evidence that besides several well known clinical parameters long-term outcome following DLI might be influenced by genetic determinants such as the expression of certain HLA alleles (5–8). HLA-A*0201 is an important minor histocompatibility antigen-presenting molecule presenting HA-1, HA-2, HA-8, and HY A2 (3, 4). HA-1 as well as HA-2-specific cytotoxic T cells have been identified being responsible for graft-versus-leukemia reactions following DLI in individual cases (9, 10). Although a minor histocompatibility antigen (including HY) disparity in 0.0 0.2 0.4 0.6 0.8 1.0

Enrichment of CD8+ CD28- cytotoxic T cells in circulating lymphocytes of patients with ankylosing spondylitis

In patients with ankylosing spondylitis (AS), HLA-B27 restricted cytotoxic T lymphocytes (CTLs) exist with specificity for arthritogenic bacteria, viral peptides or autoantigens. These MHC-class I restricted CTLs could maintain the inflammatory process even after the bacterial pathogen itself had been eradicated by antibacterial immune responses and thus be directly involved in the pathogenesis of spondylarthropathies. Phenotypically they are characterized by CD28-negativity and CD57/CD11a high-positivity. This study was performed to directly compare the relative number of CD8+ CTLs from AS patients with age-matched healthy controls. Until now only few data are available for the incidence of CD8+CD28- T cells in autoimmune diseases.