Bernd Schneider

Inhibiting effects on the induction of cytotoxic T lymphocytes by dendritic cells pulsed with lysates from acute myeloid leukemia blasts

Dendritic cells (DCs) were established from 25 patients in complete remission of acute myeloid leukemia (AML). In patients during hematopoietic regeneration following chemotherapy the yield of DC was comparable to that of healthy donors. In patients, more than 2 months after chemotherapy, significantly less DC were generated. Comparison of the antigen-presenting capacity using tetanus toxoid of six AML patients and six healthy volunteers did not show significant differences. In six AML patients, lymphocytes stimulated with blast cell lysate pulsed DC were analyzed for cytotoxic activity against autologous blast cells. 8.4-35.6% of autologous blast cells were lysed by DC stimulated lymphocytes. In three of the six patients maximum lysis of target cells was achieved by unpulsed DC. Thus, it seems that in some patients blast cell lysates mediate inhibitory effects, which may explain to some extend immune escape mechanisms in AML.

Induction of Apoptosis by Cladribine (2-CdA), Gemcitabine and Other Chemotherapeutic Drugs on CD34 + /CD38 + and CD34 + /CD38 - Hematopoietic Progenitor Cells: Selective Effects of Doxorubicin and 2-CdA with Protection of Immature Cells

Due to the emerging role of high dose chemotherapy with stem cell rescue and ex vivo purging in hematological diseases, we examined the effect of chemotherapeutic drugs on the rate of apoptosis in more mature CD34 + /CD38 + and less differentiated CD34 + /CD38 - stem cells. CD34 + cells were obtained by cell apheresis from healthy donors (n = 25) or patients (n = 25) prepared for high dose chemotherapy and stem cell transplantation. Cells were incubated with different concentrations of doxorubicin, mitoxantrone, mafosphamide, cladribine or gemcitabine. Apoptosis was determined after 24 and 48 h. Generally, the percentage of apoptotic cells was higher in the more mature CD34+/CD38+ progenitor population than in the less differentiated CD34 + /CD38 - cells. By analysis of variance (ANOVA) significantly (p < 0:05) more apoptotic cells within the CD34 + /CD38 + progenitors were calculated after incubation with mafosphamide, doxorubicine and cladribine. Mafosphamide induced the highest rate of apoptosis on CD34 + /CD38 - cells, whereas doxorubicine had nearly no effect on this immature population. Dose effect plots for mafosphamide and doxorubicin were steep, suggesting a large therapeutic index. The dose response of cladribine showed a flat course. Furthermore we found a selective induction of apoptosis by doxorubicin and cladribine on more mature CD34 + /CD38 + progenitors in contrast to simultaneous protection of CD34 + /CD38 - progenitors. From these findings, in particular the demonstrated low stem cell toxicity, we conclude that doxorubicin and cladribine might be efficient alternatives in ex vivo purging of autologous grafts, as well as safe components in primary treatment schedules of lymphomas or prior to stem cell harvest with respect to stem cell toxicity.

T-large granular lymphocyte leukaemia with natural killer cell-like cytotoxicity and expression of two different α- and β-T-cell receptor chains

We describe a case of cytotoxic T‐large granular lymphocyte leukaemia showing typical morphological features, expressing antigens characteristic for cytotoxic T cells and exhibiting marked natural killer‐like cytotoxicity towards different target cells. Moreover, characterization of the T‐cell receptors revealed simultaneous expression of two different types of β‐chains as well as α‐chains by the malignant cell clone. The patient had an 8 year history of indolent disease, before progressing to an aggressive clinical course hardly responsive to chemotherapeutic treatment. This is the first description of a peripheral T‐cell neoplasm expressing four distinct types of T‐cell receptor molecules.

The tyrosine kinase inhibitor AMN107 (Nilotinib) exhibits off-target effects in lymphoblastic cell lines

The aminopyrimidine inhibitor AMN107 (Nilotinib) was rationally designed to antagonize the aberrant tyrosine kinase activity of Bcr-Abl-positive cells. We here evaluated, whether AMN107 is also able to induce apoptosis in Bcr-Abl-negative cells of lymphatic origin. The B-cell lines DOHH-2 and WSU-NHL and the T-cell lines Jurkat and HUT78 were incubated with increasing amounts of AMN107 corresponding to clinically achievable dosages. Subsequently, induced molecular changes were assessed by FACS analysis, Western blot, and enzyme activity assays. Although AMN107 exhibited only a minor apoptosis-inducing effect in the T-cell lines, it exerted a considerable, dose-dependent cytotoxicity in the B-cell lines. Using selective caspase-inhibitors, we show that apoptosis in responder cell lines critically relies on activation of caspase-6 and caspase-9. Cell lines sensitive and resistant towards AMN107 can be discriminated by their differential expression of Src-kinases. Although the AMN107-sensitive cell lines DOHH-2 and WSU-NHL exhibited low or no expression of the Src-kinases Lck, phosphorylated Lck, and Yes with a concomitant high expression of Hck, Lyn, and phosphorylated Lyn, the expression pattern of these kinases was inverse in the AMN107-resistant T-cell lines. In conclusion, this is the first report providing evidence that activity of AMN107 is not restricted to Bcr-Abl, c-Kit, or PDGFR-positive cells, but also extends to lymphatic cell lines of B-cell origin.

Host factors and disease severity in two patients with SARS Wirtsfaktoren und Erkrankungsschwere bei zwei Patienten mit SARS

Abstract Infection with the SARS (Severe Acute Respiratory Syndrome)-associated coronavirus results in respiratory failure probably by immunological mechanisms in 10% of patients. Laboratory markers that predict subsequent respiratory failure would therefore be useful in patient management. We describe the clinical course, hematologic parameters, lymphocyte subpopulations and markers of inflammation in two patients with SARS, i.e., one man with diabetes mellitus and one pregnant woman, infected by the same viral isolate. The patient with underlying diabetes mellitus developed respiratory failure after admission in the second week of the illness while the second patient developed only a mild disease without respiratory failure. Subsequent respiratory dysfunction was associated with low numbers of Natural Killer (NK) cells at presentation and elevated CRP levels during the illness. NK cells and CRP levels at the end of the first week of the disease might be related to subsequent respiratory dysfunction and may link underlying conditions to disease severity.

Dendritic Cells from AML Patients in Complete Remission are Capable to Present Antigen to Autologous T Lymphocytes

Evidence for immunogenicity of AML blasts is supported by graft versus leukemia reaction, the effect of donor lymphocyte transfusion and in vitrostudies with cytotoxic T cells. Because of the potency of dendritic cells (DC) to present antigens, it seems feasible to study, whether functional DC can be generated in AML patients with the perspective of future vaccination strategies. Therefore, peripheral blood was obtained from 19 patients in complete remission of AML and 13 healthy controls (HC). DC were established from monocytes by culturing in the presence of GM-CSF and IL-4 for 8 days. To collect mature DC GM-CSF and TNF-α were added to cultures until day 12. At days 8 and 12 cell numbers were counted and expression of surface markers typical for DC was analyzed by flowcytometry. The ability of established DC to present antigens was tested using tetanus toxoid (TT) as a recall antigen. Furthermore day 8 DC were incubated with lysates of leukemic blasts and cocultured with autologous lymphocytes. Cytotoxicity against autologous blast cells was measured by a PKH67 assay. The numbers of DC were significantly lower in AML patients >2 months after chemotherapy as compared to HC (p=0,04) or AML patients during hematopoietic regeneration following chemotherapy (p=0,01). There was no statistical difference in expression of surface markers by DC from AML patients and HC. CD83, the most specific marker for DC, was comparable in both groups (day 8: HC 11-62%, AML 10-67% p=0,07; day 12: HC 42-91%, AML 23-78% p=0,08). TT was presented efficiently to autologous lymphocytes by DC from HC and AML patients. At an effector:target-ratio of 20:1 lymphocytes from one patient stimulated with DC pulsed with blast lysates killed 20,3% of autologous blasts compared to 13,6% lysis by effector cells from HC. Cloning of these T cells is currently in progress. In conclusion dendritic cells can be efficiently generated in AML patients especially during hematopoietic regeneration following chemotherapy. Preliminary results indicate the activation of autologous T cells after stimulation by DC pulsed with blast lysates.

Immunological Response to IL-2 and α-IFN-Treatment After Autologous BMT in Patients with BCR-ABL-positive ALL

Patients with Ph1+/BCR-ABL+ALL have an extremely poor prognosis after treatment with conventional chemotherapy [19, 31]. Bone marrow transplantation, however, offers a chance of cure for a proportion of these patients [1, 25]. Evidence from preclinical [9] and clinical studies at various institutions including our own [3, 4] suggests that maintenance immunotherapy with IL-2 may exert an additional “GvL”-like antileukemic effect after chemotherapy or autologous BMT in patients with AML [5, 6, 11, 15, 24, 28]. Less data are published on posttransplant therapy with IL-2 in patients with high risk ALL [5, 10, 24, 30]. Alternatively, some patients with Ph1-pos ALL were reported to receive maintenance therapy with α-IFN [14, 16, 26, 27]. We combined these approaches and initiated a pilot phase II study for patients with BCR-ABL-positive ALL to receive sequential cycles of rIL-2 and α-rIFN after autologous BMT as part of an ongoing ABMT-program [23].