ALBERT

Description: The needs of a growing human population require rapid and efficient development of improved cultivars by plant breeders. The doubled haploid (DH) technology enables generating completely homozygous lines in a single step and, thus, is central to modern genetics and breeding approaches. Rapid and reliable identification of seeds with a haploid embryo after in vivo haploid induction is elementary in the method utilized in maize but current systems have severe shortcomings preventing their use in many germplasm types. Here, we describe an alternative method for discrimination of haploid from diploid seeds based on differences in their oil content stemming from pollination with high oil inducers. After presenting some fundamental theory, we provide a proof-of-concept with experimental results, demonstrating acceptable error rates across different germplasm. Our approach represents a breakthrough in DH technology in maize, because it is amenable to automated high-throughput screening and applicable to any maize germplasm worldwide. Scientific Reports 3 doi: 10.1038/srep02129

Description: Key Points Cav-1–deficient T cells preferentially differentiate into Tregs, which translates into lower GVHD severity in mice. Reduced TCR:Lck clustering in Cav-1–deficient T cells is responsible for reduced TCR downstream signaling events promoting Treg differentiation.

Description: Abstract 2545 Steroid refractory Graft versus Host Disease (GvHD) is a serious complication following allogeneic haematopoietic stem cell transplantation (HCT). Recently, novel therapeutic strategies involving multipotent stromal cell (MSC) transfusions have shown promising clinical results. Nevertheless, little is known on the interaction between MSC and immunosuppressive agents currently used for GvHD treatment and prophylaxis. Here we investigated the effects of mammalian target of rapamycin (mTOR) and calcineurin inhibitors on MSC mediated allogeneic T cell suppression. Since both of these drugs inhibit protein translation and exert potent antiproliferative effects, we assumed that MSC immunomodulation would be abrogated following mTOR or calcineurin inhibitor treatment. In an experimental in vitro suppression assay human MSC were pre-incubated with either everolimus or cyclosporine at concentrations currently used in the clinical practice. After 3 hours, MSC were washed several times to remove any immunosuppressive drug in the supernatant and cultured with magnetically sorted allogeneic human CD4+ or CD8+ T cells at a ratio of 1 MSC to 10 T cells prior to T cell activation with anti-CD3/CD28 coated beads. Surprisingly, MSC treated with mTOR inhibitors exerted significantly enhanced suppression of allogeneic CD4+ T cell proliferation (76%+/−12%) as determined by thymidine incorporation in comparison to MSC pre-incubated with cyclosporine (59%+/−12%) or untreated MSC (39%+/−10%). Similar results were obtained when MSC were cultured with CD8+ T cells. High pressure liquid chromatography (HPLC) did confirm that no remaining immunosuppressive drug in the culture supernatant was responsible for this observation. Subsequently we investigated, whether regulatory T cells (Treg) expansion would account for this enhanced MSC mediated immunosuppression. When everolimus treated MSC were added to CD4+ T cells in the suppression assay, significantly more lymphocytes expressed a regulatory CD4+CD25high FOXP3high phenotype (22%) in short and long term cultures while cyclosporine pre-treatment of MSC induced a Treg population (10%) comparable to untreated MSC (7,3%). When neutralizing antibodies against transforming growth factor-beta (TGF-β) were added, lower numbers of Tregs were induced by mTOR treated MSC (15,7%), cyclosporine treated MSC (8,8%) and untreated MSC (7,3%). In addition, several reports proposed indoleamine 2,3-dioxygenase (IDO) as a potent mediator of MSC dependent immunosuppression, since IDO expression results in depletion of tryptophan that is essential for cell proliferation. In the presence of interferon-gamma (IFN-γ), MSC up-regulate IDO and exert enhanced suppression of alloreactive T cell proliferation. However, when MSC pre-treated with everolimus were stimulated with IFN-γ, IDO expression was reduced. In addition IFN-γ signalling abrogated Treg expansion induced by everolimus pre-treated MSC. Thus, combined effects of mTOR inhibitors and IFN-γ signalling reduced MSC mediated T cell suppression. Collectively, these data suggest that MSC pre-treated with mTOR inhibitors induce enhanced immunosuppressive capacity towards allogeneic T cells due to induction and expansion of Tregs in a, at least partially, TGF-β dependent way. In contrast, mTOR inhibitors and IFN-γ enhance MSC immunomodulation by independent mechanisms. However, when combined they antagonize each others effects. In conclusion, our results support the combined use of mTOR inhibitors and MSC for the treatment of steroid refractory GvHD. This combination may induce Treg expansion that can treat GvHD without limiting graft versus tumor effects. Additionally, determination of IFN-γ serum levels may predict the outcome of this combined therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4870 Since the succesful treatment of T cell lymphoma remains to be problematic, identification of new pharmacological targets in this malignancies are desperately needed. The AMPK-Rheb-mTOR signaling pathway plays an important role in regulating processes such as proliferation and proteinsynthesis according to energy and nutrient levels in normal and malignant T cells. Inhibitors of mTOR have shown promising results in clinical trials in several lymphoma types. Similarly, recent data could prove inhibitors of farnesyltransferase (FTI) to be effective as a single agent in certain subtypes of T cell lymphoma. Despite divergent data regarding the molecular target of FTI action, recently published work suggest inhibition of prenylation of the GTPase Rheb as putative mechanism for the antineoplastic effects of FTI (Basso et al., J Biol Chem, 2005). Therefore, combining inhibition of mTOR and Rheb might result in increased inhibition of T cell lymphoma proliferation. To investigate this hypothesis, human T cell lymphoma cell lines DERL-2 (originated from hepatosplenic gamma-delta T cell lymphoma), Karpas-299 (originated from anaplastic large cell T cell lymphoma) and normal human CD4+ T cells were incubated with a combination of everolimus as mTOR inhibitor and FTI (lonafarnib, SCH-66336) or the single agents. While both substances showed an additive combined anti-proliferative effect in DERL-2 cells, proliferation of Karpas cells were more susceptible to inhibition by FTI. On a molecular level, despite substantial growth inhibition in both cell lines by everolimus alone, phosphorylation of 4EBP1 and p70S6K remained unaffected, while FTI mediated reduction of Karpas cell proliferation was associated with a substantial decrease in AMPK phosphorylation together with an overexpression of p27kip, which could not be observed in DERL-2 cells. In contrast, incubation of stimulated human CD4+ T cells with the drugs alone or in combination did not result in changes in the phosporylation status of AMPK. Nevertheless, in contrast to everolimus, FTI induced a reduction of total protein expression of AMPK and other proteins, e.g. AKT. In addition, contrary to the observations in the malignant T cells, FTI treatment of unstimulated human CD4+ T cells resulted even in an increase of AMPK-phosphorylation. A hint for the explanation of these conflicting data came from analyses of Rheb expression in the examined cell types. While Rheb was easily detectable in the malignant T cell lines and the stimulated CD4+ T cells, it was almost absent in unstimulated CD4+ T cells. A model derived from this findings is that FTI effects depend on different targets available for inhibition of prenylation according to the activation or differentiation status of the T cells. While Rheb might be the target in malignant or activated T cells, another target, e.g. phosphatases, might be responsible for the FTI effect in resting T cells where Rheb is not available. In Karpas cells a particular connection between Rheb and AMPK might exist, as described for other cell lines (Lacher et al., Oncogene, 2010). Inhibition of this Rheb-AMPK axis might explain the particular gowth inhibiting effect of FTI in this model of anaplastic large T cell lymphoma. Nevertherless, the presented data show a combined effect of mTOR inhibitors and FTI for the potent treatment of T cell lymphoma involving different molecular mechanisms according to the lymphoma subtype. Disclosures: Finke: Fresenius Biotech GmbH: Honoraria, Research Funding.

Description: Introduction The outcome of patients with T cell lymphoma treated with standard chemoterapeutic substances remain poor, making the search for new active substances a highly medical need in this hematologic neoplasia. Recent phase II clinical trials showed very promising activity of farnesyltransferaseinhibitors (FTI) in relapsed/refractory T-NHL patients (Witzig et al. 2011). Regarding the molecular mechanisms behind this therapeutic effect, conflicting data regarding Ras as the initially proposed intracellular target of FTI and the involvement of MAP kinases in cellular effects of FTI in T cells exist (Marks et al. 2007, Ding et al. 2011). Together with observations in breast and ovarian cancer cells suggesting the GTPase Rheb as target for inhibition of farnesylation (Basso et al. 2005), the targets of FTI might vary according to the examined cell type. Interestingly, in breast cancer cells FTI mediatied inhibition of Rheb action resulted in reduced mTOR signaling. Nevertheless, as a putative additional targeted treatment approach in T-NHL, incubation with mTOR inhibitors showed not only substantial antiproliferative effects in normal T cells but also in malignant human T cell lymphoma lines in vitro (Huang et al. 2010). Since further clinical trials with both substances did not show severe side effects, adding everolimus as combination partner might even enhance clinical activity of FTI in T cell lymphomas. Therefore, in order to test this hypothesis and to analyse if both substances differ in their molecular mechanisms of action, FTI and everolimus were tested in vitro in T cell lymphoma lines (Karpas, Derl-2, Jurkat) to evaluate potential synergistic modes of action. Methods and Results Incubation of human T cell lymphoma lines Karpas and Derl-2 with the FTI SCH66336 (lonafarnib) or the mTOR inhibitor everolimus showed a reduction in proliferation in a dose dependent manner (EC50 for everolimus: 0.1nM, EC50 for lonafarnib: 0.5 µM). Combining both drugs resulted in synergistic inhibition of proliferation. This inhibitory effect correlated with increased p27KIP1 expression. In our experiments, Rheb appeared to be highly expressed in all examined T cell lymphoma lines with even additional increase of protein expression in Karpas cells after FTI incubation. Comparing FTI action to inhibition of mTOR by everolimus on a molecular level, in our experiments lonafarnib treatment of Karpas cells resulted in an unexpected reduction in AMPK-phosphorylation, implicating involvement of this metabolic pathway in FTI mediated inhibition of proliferation in malignant T cells. This effect could not be observed in everolimus treated Karpas cells. In contrast, naive human CD4+ T cells showed very little Rheb protein expression, which could be significantly increased after TCR stimulation by induction of Rheb mRNA transcription. While everolimus treatment of TCR-activated normal human CD4+ T cells resulted in AKT-hyperphosphorylation, FTI did not induce any changes in AKT. Contrary to the malignant T cells, FTI treatment had no impact of AMPK phosphorylation in activated T cells. Actually, naive T cells treated with FTI showed an hyperphosphorylated AMPK status. Conclusion Lonafarnib and everolimus show synergistic antiproliferative effects in T cell lymphoma lines, most likely by interfering with mTOR and AMPK signalling, making this combination therapy interesting for clinical trials. In contrast, FTI does not mediate AMPK in activated normal T cells. This observations are in accordance with a differential targeting of Rheb by FTI in malignant or normal human T cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1264 Tolerance induction together with sustained disease control remain to be critical for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are involved in the regulation of graft versus host disease (GvHD) and graft versus leukemia effect (GvL). Although calcineurin inhibitors are frequently being used for the prevention of GvHD, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. Data from animal models of HCT suggest that inhibition of the mammalian target of rapamycin (mTOR) results not only in suppressed T cell alloreactivity but also in sustained Treg function. On the other hand severe reduction of T cell alloreactivity might result in diminished control of especially advanced malignant disease. To explore the clinical efficacy of a calcineurin-free GvHD prophylaxis regimen, we initiated a phase I/II monocenter trial using everolimus and mycophenolate-sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after reduced toxicity and standard conditioning. 28 patients were included (median age: 49,2 years, range: 21–65). The diagnoses were: AML/MDS (n=16), ALL (n=3), CML/MPS (n=3), T-PLL (n=1), NHL/CLL (n=6). At the time of transplantation 22/28 (78%) patients were at high risk (not in CR1/CP, untreated) for early relapse. Conditioning included fludarabin based reduced intensity (n=24) or standard regimens containing busulfan (n=2) or clofarabine (n=2). Four CR1 patients received additional alemtuzumab (total 10mg) for GvHD prophylaxis. PBSC grafts were obtained from unrelated (n=20) and related (n=8) HLA-matched donors. No graft failure occurred. Engraftment kinetics for myeloid cells were normal, patients without alemtuzumab showed rapid reconstitution of the T cell compartment with median cell counts of 〉200 CD4+ cells/μl at day +30 together with complete donor chimerism in 15/18 evaluable patients. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow up of 9 months, 3 relapses of 24 patients with CR after HCT (12,5%) occurred, of those one AML patient could be salvaged with withdrawal of immunosuppression. Nine patients have died. The causes were underlying malignant disease (n=2), GvHD (n=2), viral infections (n=3 with two cases of HHV6 associated encephalitis), post-surgery thrombembolism (n=1), and one unknown. Treatment related mortality after 100 days is 14,2%, after 1 year 21,4%. Due to the early recovery of T cell immunity mild forms of acute skin GvHD were common early after reconsitution, while acute GvHD Grade III-IV could be observed in 8/26 patients. Chronic GvHD occurred in 15/22 patients (68%) with moderate and severe forms in n=10 and n=3 patients, respectively. Cytomegalovirus (CMV) reactivation could be seen in 5/20 patients at risk, while no CMV disease developed. Importantly, in the first year after HCT no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. The whole cohort experienced a median overall survival of 20 months, median progression free survival was 19 months. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of moderate chronic GvHD without major bacterial and fungal infectious complications. Since this sustained alloreactivity might reduce the risk of relapse this regimen could be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures: No relevant conflicts of interest to declare.