ALBERT

Beschreibung: B-chronic lymphocytic leukemia (BCLL) is a lymphoproliferative disease that is characterized by clonal expansion of CD5+ B cells. BCLL is associated with secondary immunodeficiency and hypogammaglobulinemia. It has been suggested that T-cell dysregulation may play a role in the hypogammaglobulinemia and in the increased incidence of autoimmunity in BCLL patients. We attempted to transfer human peripheral blood mononuclear cells (PBMC) from BCLL patients in different stages of the disease into immunodeficient mice. PBMC from BCLL patients in stage 0, stages I to II, and stages III to IV were transplanted into the peritoneal cavity of lethally irradiated Balb/c or beige/nude/Xid (BNX) mice radioprotected with bone marrow (BM) from severe combined immunodeficiency (SCID) mice. Different engraftment profiles were found in the chimeric mice 2 weeks after transplantation of PBMC according to the disease stage of the BCLL donors. Infusion of PBMC from donors in stage 0 led to marked engraftment of human T cells, whereas the human tumor cells could hardly be detected. In contrast, chimeric mice receiving PBMC from patients in stage III to IV disease exhibited engraftment with a dominance of tumor cells, compared with a miniscule level of T cells. The ability of the engrafted cells to produce human Ig was also found to be correlated with the disease stage of the donor, although all donors had the same magnitude of hypogammaglobulinemia. Total human Ig production in the chimeric mice was normal in mice receiving PBMC from donors in stage 0, whereas in chimeric mice engrafted with PBMC from donors in stages III to IV almost no human Igs could be detected. This differential reconstitution of antibody production in the mouse model according to the stage of the patient's disease will allow further studies on possible cellular interactions between malignant and immune cells in BCLL.

Beschreibung: CD74 is an integral membrane protein that was thought to function mainly as an MHC class II chaperone. However, CD74 was recently shown to have a role as an accessory-signaling molecule. Our studies demonstrated that CD74 regulates B-cell differentiation by inducing a pathway leading to the activation of transcription mediated by the NF-κB p65/RelA homodimer and its coactivator, TAFII105. Here, we show that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κB activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These studies therefore demonstrate that surface CD74 functions as a survival receptor.

Beschreibung: Induction of donor type chimerism under reduced intensity conditioning in the absence of GVHD represents a major challenge in transplantation biology. Despite their remarkable potent veto activity, CD8 CTLs could not be used to enhance engraftment due to their GVH reactivity. To address this problem we developed a new approach for the generation of human host-nonreactive CTLs, based on stimulation of donor CD8 T cells against 3rd party stimulators under IL-2 deprivation. A major attribute of such anti-3rd party CTLs reported recently by our group is their ability to eradicate pathological B-CLL cells. This B-CLL killing was shown to be independent of TCR recognition and it was found to be mediated both by autologous and by allogeneic anti-3rd party CTLs. In the present study we demonstrate that anti-3rd party CTLs can also efficiently eradicate primary mantle lymphoma cells and Burkitt and mantle cell-lines. Furthermore, the use of lymphoma cell lines has enabled to define several aspects of the mechanism leading to their apoptosis. Thus, we found that anti-3rd party CTLs avidly bind to target lymphoma cells and form stable aggregates within the first minutes of contact. Conjugate formation as well as the actual killing of the tumor cells were effectively inhibited by blocking antibodies against LFA1 or ICAM1 but not against ICAM3. Moreover, effective inhibition was attained only when applying anti-LFA1 to the CTLs and anti-ICAM1 to the tumor cells (100%±10% and 72%±7% inhibition, respectively) but not vice-versa. Given the role of CD8-MHC class I interaction in veto mechanisms, we sought to evaluate its role in the killing of lymphoma cells by human anti-3rd party CTLs. Indeed, in 10 experiments survival of target lymphoma cells was increased from 50%±5% to 82%±1% upon addition of anti HLA-ABC Ab to the incubation with anti 3rd party CTLs. A similar survival increase to 77.5%±1% was found upon the addition of anti-CD8 Ab, suggesting that CD8 binding to MHC class I constant region is required for lymphoma cell killing. The important role of MHC class I was further established by comparing CTL-mediated killing of Daudi lymphoma cells lacking cell surface MHC class I expression and Daudi cells with reconstituted surface MHC class I due to stable β2-microglobulin overexpression (β2m-Daudi). Thus in contrast to the poor killing of Daudi cells which did not exceed 20%, the β2m-Daudi cell killing was comparable to that exhibited with burkitt lymphoma line expressing MHC class1 (56%±1% and 63%±3%, respectively in 4 experiments). Importantly, FACS analysis showed that, MHC I expression did not influence conjugate formation, but was rather involved in the induction of apoptosis (annexin V). Furthermore, a similar pattern was also found in vivo when β2m-Daudi or Daudi cells were infused into the peritoneum of SCID mice in the presence or absence of a 2.5-fold excess of anti 3rd party CTLs. In 3 experiments, FACS analysis of the cells recovered from the peritoneum showed that the CTLs killed 91% of the β2m-Daudi cells as opposed to 13% killing of the Daudi cells. Taken together, our results suggest that TCR independent killing of B cell malignancies by anti-3rd party CTLs is mediated via a rapid adhesion through ICAM1-LFA1 binding, followed by slow induction of apoptosis upon a critical interaction between CD8 on the CTL and MHC class I on the tumor cell.

Beschreibung: Rituximab is a chimerical human/mouse monoclonal anti-CD20 antibody successfully applied in B-cell lymphoproliferative disorders. The suppression of B-lymphocytes may induce hypogammaglobulinemia and increased risk for infection. These complications are rarely observed as a result of monotherapy with rituximab but have been often noted following combination with aggressive chemotherapy or in the setting of autologous stem cell transplantation (ASCT). In an attempt to study the role of rituximab in induction of hypogamma, we analyzed the frequency of this phenomenon, its possible causes, infectious complications and infection-related mortality in lymphoma patients treated with different chemotherapy regimens with or without rituximab. We analyzed 136 patients who received rituximab concurrently with chemotherapy (R-Chemo) and 46 patients who were treated with chemotherapy only, with follow-up period of up to five years. Following R-Chemo, 17 cases (12.5%) of severe hypogamma (defined as 6 doses of rituximab were given. Moreover, rituximab in 〉8 doses leaded to severe hypogamma in 50% of patients compared to 11.8% in patients treated with

Beschreibung: Abstract 3437 Poster Board III-325 Previously, we have shown that Allicin, the highly active compound of freshly crushed garlic, produced by the reaction of the enzyme Alliinase with its substrate Alliin, induced the apoptotic killing of B-CLL cells in vitro. In addition, we also reported that generation of Allicin in situ on the surface of B-CLL cells by targeting Alliinase to the cell surface of the CD20+ cells by Rituximab, resulted in the eradication of primary B-CLL in a human-mouse chimeric model, denoting the marked anti-CLL potential of combining these two different molecules, with different mechanism of action, into a single drug entity (Arditti et al., Mol Cancer Ther 2005;4(2)325-331). Indeed, monotherapeutic approaches, even if effective, are usually not sufficient to fully eradicate B-CLL and the most effective therapeutic protocols require the utilization of more than one agent. With this in mind, we took advantage of the high reactivity of Allicin, with SH-containing compounds, and created novel chimeric compounds by the combination of Allicin with 6-Mercapto-Purine (6MP) and 6MP-riboside (6MPR), both SH-containing purine analogs used for decades for the treatment of hematologic malignancies. The resulting novel compounds, S-Allyl-6MP (SA-6MP) and S-Allyl-6MPR (SA-6MPR), were examined against primary B-CLL cells obtained from the peripheral blood of patients at Binnet stage C. In our in vitro assays, Annexin-V staining indicated that SA-6MP acted in a dose dependent manner, inducing the apoptotic death of 37.9% and 95.2% of plated CD19+CD5+ B-CLL cells (10.9% in untreated cells) incubated for 16 h at 37 °C in the presence of 50 uM or 100 uM, respectively. As expected, the original 6MP compound had no impact on the viability of plated B-CLL cells (9.7% and 8.7%) at doses of up to 150 uM. In preliminary in vivo experiments, we compared the anti-BCLL activity of SA-6MP with that of SA-6MPR and the original 6MP compound on primary B-CLL cells from 5 different patients (Binnet stage C) in a human-SCID/Beige mouse model. Following the engraftment of the human B-CLL cells, mice were treated with i.p. injections of 2.5 mg/kg body weight of SA-6MP, SA-6MPR, or 6MP on a daily basis throughout 7 consecutive days, after which, the engraftment of primary B-CLL cells was examined by the recovery of CD45+CD19+CD5+ from injected mice. An additional group of mice injected with vehicle (1% DMSO) was also examined as a control. In close similarity to our in vitro results, engraftment of primary B-CLL cells was considerably reduced following treatment with SA-6MP (〉90% reduction), as compared with treatment with the original 6MP drug. In addition, the chimeric riboside 6MP derivative, SA-6MPR, induced a potent anti-BCLL effect comparable to that of SA-6MP. In summary, our results in vitro and in vivo suggests that combining the pro-apoptotic effects of Allicin with the antiproliferative effects of 6MP or 6MPR is superior to the effect of either of the purine analogs alone. This approach may be evaluated at first instance in B-CLL patients with refractory disease. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Scientific background: Increasing evidence points to the crucial role of malignant lymphoma interaction with the tumor microenvironment. Our previous investigations indicated that the stromal cytokine activin A induces apoptotic death of myeloma cells due to its antagonism with the growth promoting effect of interleukin-6. Activin A is a homodimer of the ßA inhibin polypeptide chain. Like other transforming growth factor (TGF)ß superfamily members, it is a pleiotropic molecule, which is widely expressed. It has initially been studied in the reproductive system, but has also been implicated in the regulation of hemopoiesis; it is an erythroid differentiation factor and is expressed within the bone marrow microenvironment. Activin A functions are tightly regulated by the competitive inhibitor inhibin A (a heterodimer, /ßA) and the binding inhibitors, follistatins. We previously showed that abundance of activin A was restrictive for B cell production in vitro and that within human nasal polyps activin A expression was widespread but absent from foci of B lineage cells. We were therefore interested to find out whether activin A plays a role in the occurrence of BM (bone marrow) involvement in malignant lymphomas. Materials and methods: The patient population consisted of 17 patients with lymphoma and 3 patients without lymphoma served as controls. Paraffin embedded sections were prepared and immunohistochemical staining was performed using an antibody to the bA chain. The slides were reviewed by team of 5 investigators and graded separately. Results: Out of 17 lymphoma cases, 10 patients showed BM involvement. In patients with BM involvement the level of activin A was significantly decreased in the area surrounding the lymphoid infiltrate (Fig 1a). This was seen uniformly in all the patients except for one. The level of activin A in the rest of the BM was similar to the level seen in specimens of reactive BM. In all 7 patients that had no BM involvement we found a diffuse staining for activin A (similar to what we saw in patients with reactive BM) (Fig.1b). Discussion: It is interesting that only some of the patients with malignant lymphomas have BM involvement. This could stem from a difference in the migratory abilities of the lymphoid cells, which is unlikely, or from a difference in their ability to home and flourish in the BM microenvironment. We have demonstrated that activin A, is significantly down regulated in the vicinity of the lymphoid infiltrate in the BM, as opposed to what occurs in normal inflammatory BM. This suggests that an interaction between the lymphoma cell and the BM microenvironment leads to down-regulation of activin A expression and possibly promotes the survival of the lymphoid cells. Figure 1: low power view of activin staining in the vicinity of the involved BM (a) and uninvolved BM (b). Figure 1:. low power view of activin staining in the vicinity of the involved BM (a) and uninvolved BM (b).

Beschreibung: In the last few years, point mutation of the Janus Kinase 2 (JAK2) gene (V617F) was found in the majority of patient with Polycythemia Vera and two related diseases ET and MMM. This mutation initiates constitutive activation of JAK2 transduction gene, inducing growth factor- independent hemopoiesis and plays a dominant role in origin of myeloproliferative disorder. Inactivation of this mutated gene may restore the normal hemopoiesis. Based on the evidence that the inhibition of the Jak-Stat axis by AG490 induced apoptosis in leukemic compared with normal cell, we attempt to analyzed the AG490 effect on the survival of MPD cells carrying JAK2 mutation in cultures. Peripheral blood (PB) samples were collected from 49 MPD patients (PV n=31, ET n=14, MMM n=4) at the time of diagnosis or during the follow up. The JAK2-V617F mutation tested by an allele-specific, semi-quantitative PCR was detected in 35 patients. MNPBC’s from four patients homozygous for the JAK2 mutation and from two normal volunteers were cultured in methylcellulose with 50 ng/ml SCF, 10ng/ml GM-CSF, 10ng/ml IL-3 and 3U/ml EPO, and in the same medium without growth factors. On the sixth day, 50 micromole of AG490 were added to the cultures and dishes with no addition of AG490 were kept for control. Colonies were counted on 6th day and on 14th day, and we checked the growth patterns on 14th day. Spontaneous colonies without growth factors were found only in the four MPD patients. Although, the number of the colonies was not significantly decreased after the addition of the AG490 inhibitor, we observed an obvious change in the normal growth pattern. The appearance of the colonies became darker reflecting an apoptotic process and death. Such a phenomenon was not seen in normal controls or in MPD cells without addition of the inhibitor. In conclusion our experiment clearly demonstrated preferential inhibition of MPD cells with JAK2 mutation by AG490, probably by induction of apoptosis. Our results suggest a therapeutic potential of inhibitors of JAK2 for the future.

Beschreibung: Previous studies suggest that cells within the CD34+ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34+ cells.