ALBERT

Description: Key Points Lowest numbers of ex vivo–selected CD8+ memory T cells can reconstitute pathogen-specific immunity in immunocompromised hosts.

Description: The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral gene-transfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.

Description: Background: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). For the clearance of CMV, CD8+ T cells are pivotal. Patients after allo-PBSCT with recurrent CMV reactivation usually lack such CMV specific T cells. Conventional antiviral therapy of CMV reactivation characteristically results in myelosuppression and further suppression of CMV specific T cells. Adoptive transfer of CMV specific T cells may help to overcome this problem. A novel technology designated “streptamers” allows the selection of CMVpp65 specific CD8+ T cell up to 98% purity without altering the functional properties of the selected T cells and without requiring cumbersome and time consuming T cell cultures. Materials and Methods: Here, the novel streptamer technology was used for adoptive transfer of CMV specific T cells into two acute leukemia patients with recurrent high CMV antigenemia after allo-PBSCT. Standard peripheral blood mononuclear cell apheresis was performed on the former stem cell donors of two patients with acute leukemia. Isolation of CMV specific donor lymphocytes was performed using a Good Manufacturing Product (GMP)-grade Streptamer selection kit on a CliniMacs™ device. Briefly, MHC-Streptamers (CMVpp65/HLA-B7 for patient 1; CMVpp65/HLA-A2 for patient 2) were labeled with beads overnight to obtain MHC-streptamer-bead complexes. Subsequently CMV specific T-lymphocytes were immunomagnetically labeled by incubating mononuclear cells with MHC-Streptamer-bead complexes. Cells were run on a CliniMacs™ device. The positive fraction was then incubated with biotin to detach the steptamers from the T cells. Results: A single specific donor lymphocyte infusion (sDLI) of 0.4 or 2.2 ×105 CMVpp65 specific T cells per kg body weight was performed in an AML or ALL patient respectively, after allogeneic PBSCT developing a CMVpp65 antigenemia with a maximum of 959 or 716 CMVpp65 positive/500,000 cells and treatment with foscarnet, ganciclovir and valganciclovir. After sDLI, the CMV antigenemia was cleared and remained persistently controlled even after discontinuation of valganciclovir therapy in both patients. No acute or chronic toxic side effect, particularly no aggravation of graft-versus-host disease (GvHD) was observed. A strong and sustained increase of the absolute count of CMV-specific CD8+ T cells in concordance with the increase of CD3+CD8+ T cells up to 440/μl was detected. CMV-specific CD8+ T cells showed no significant expression of CCR7, CD62L or CD107, but stained increasingly positive for CD45RA, indicating a preferential effector T cell phenotype. Results from stimulation experiments of CD3+ T cells with HLA-B7 versus HLA-A2 restricted CMVpp65 derived peptides demonstrate late reconstitution of HLA-A2-restricted CMV-specific T cells, whereas the adoptively transferred HLA-B7-restricted CMV-specific T-cell response augmented very early und was maintained over time. The chimerism analysis of the in vivo expanded CMV-specific CD8+ T cells demonstrated a 100% donor chimerism. T cell receptor excision circle (sjTRECs) analysis revealed a frequency of sjTRECs two logs lower than expected, indicating peripheral expansion rather than thymic proliferation of CMV specific CD8+ T cells. cDNA generated from FACS-purified donor-derived CMV B7 pp65-specific CD8+ T cells was probed with the indicated 5′ Vß14-specific and 3′ CDR3-specific primers for the presence of clonotypic T cells. The respective CDR3 region sequence was identical for both donor T cells and CMVpp65 specific T cells in the patients at different time points after the adoptive T cell transfer, thus clearly indicating that the expanded CMV specific T cell were of clonogenic donor origin. Conclusion: Streptamer technology offers the advantage of selecting CMV specific CD8+ T cells at GMP level for adoptive T cell transfer. Two CMVpp65 specific T cell transfers resulted in a marked increase of CMV-specific CD8+ T cells and induced long-lasting CD8+ T cell responses, which allowed the patients to discontinue toxic antiviral drug therapy without further high level reactivation of CMV.

Description: The continuously growing Natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origin without affecting normal human cells. It is the only NK cell line that has entered clinical trials to date. Acute lymphoblastic leukemias (ALL) show variable sensitivity towards NK cells including NK-92-lysis. All T-ALL cell lines tested (MOLT-3, MOLT-4 and CEM-T) display moderate sensitivity to NK cytotoxicity with approximately 22±2 – 24±2% killing at an effector to target (E:T) ratio of 10:1, respectively. Comparable results were obtained with primary patient derived T-ALLs (n=4), displaying specific killing rates of 17±5 – 20±2%. In contrast, B lineage ALL cell lines (NALM-6, SEM, REH, JKB, BV173, SupB15, TOM-1, TMD5) and cells derived from patients (n=11) were resistant to NK-92-mediated lysis, with specific killing rates ranging from 2±1% to 13±3% at an E:T ratio of 10:1. Approaches to overcome resistance of B-precursor ALL by re-targeting may shed light on the specific reasonable mechanism conferring resistance and lead to novel therapeutic strategies. Here we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the pan B cell antigen CD19 which is universally expressed by B lineage leukemia cells. This receptor fragment kindly provided by H. Zola (Child Health Research Institute North Adelaide, Australia) consists of the CD19 specific scFv(CD19) antibody fragment, a flexible hinge region, the CD3 ζ chain and a Myc-tag. Transduced cells were selected with G418, and surface expression of the chimeric scFv(CD19)-ζ construct was verified by FACS analysis using the Myc-tag-specific mAb 9E10. No difference in cytotoxic activity of NK-92 and transduced NK-92-scFv(CD19)-ζ cells towards CD19 negative targets (K562, MOLT-4) was found. In contrast, NK-92-scFv(CD19)-ζ cells specifically and efficiently lysed CD19 expressing B precursor leukemia cells (NALM-6, SEM, B173, SupB15, TOM-1, TMD5) including cells that were completely resistant to cytolytic activity of parental NK-92 cells. Killing of SupB15 and BV173 cells was 10,0±0,8% and 12,0±8,5% with parental NK92 vs. 58,2±14,8% and 52,3±4,1% lysis with NK92 cells expressing the CD19 specific scFv(CD19) antibody fragment. These results demonstrate that efficient retargeting of NK-cell cytotoxicity can be achieved, and might allow the generation of potent cell-based therapeutics. Since the widespread resistance of B precursor ALL blasts to NK cell cytotoxicity is not caused by mechanisms commonly relevant in tumors, this model may allow dissection of possible mechanisms of resistance.

Description: More than 100 ABO subgroup-related variations were detected in the coding region of glycosyltransferases, which may be causative for a weak blood group antigen A or B expression. Most variation in expression is explained genetically by mutations that reduce transferase activity. However, a substantial number of weak variants have not yet been explained by current methods. We recently postulated a role of miRNA in the regulation of blood group A antigen expression levels. By using different approaches, including gene array analysis, luciferase reporter assay and overexpression of glycosyltransferase specific miRNAs in primary hematopoietic stem cells (HSCs), we found that miR-331-3p directly targets glycosyltransferase A and B mRNA. Now we have been further embarking on the underlying mechanisms of miRNA and glycosyltransferase interactions and show that the effects of miR-331-3p are mediated by inhibition of transcription factor SP1, which is a major regulator of the ABO gene, thereby resulting in downregulation of blood group A antigen expression in A2O individuals by up to 80% and in A1O individuals by up to 50%. Using microRNA target prediction tools we also identified Sp1 as a potential target gene for miR-331-3p. Western blot analyses of glycosyltransferase protein expression showed that overexpression of miR-331 led to a decreased glycosyltransferase and SP1 protein expression. Further approaches with the SP1 inhibitor, mithramycin A, revealed similar results. Analysis of Aw04/O1v genotypes revealed elevated miRNA levels, thus confirming microRNAs as regulators of blood group glycosyltransferase expression. Our findings extend our understanding of blood group regulation in carriers of weak blood group variants. Involvement of miRNAs in the downregulation of ABO blood group antigen expression may also provide an explanation for observed changes of ABO antigen expression in abnormal processes such as tumorigenesis, pregnancy and aging. Furthermore, this pathway may play a role in the regulation of other blood group variants (for instance Rhesus, KELL, amongst others). Disclosures No relevant conflicts of interest to declare.

Description: In clinical transfusion and transplantation medicine, AB0 is the most important blood group system. A and B antigens are synthesized by glycosyltransferases A and B encoded by co-dominantly inherited A and B genes. The 0 phenotype derives from a single nucleotide deletion; the resulting stop codon in the exon 7 of glycosyltransferase A leads to a lack of glycosylation activity to A and/or B. The DNA structure of the three major alleles of the human blood group AB0 system was first described in 1990. Besides the 4 major AB0 blood groups (A, B, AB, 0), weak expression variants commonly occur. Beside introns and exons, two regions were found to be critical for transcriptional activity of AB0 genes: a promotor sequence upstream of the translation start site and a CBF/NF-Y-binding enhancer element. However, the molecular basis for the weak expression levels of AB0 blood group receptors is not fully understood. Here we asked whether microRNA´s may play a role in the regulation of blood group A and B antigens. MiRNA's are small, non-protein encoding RNA sequences that bind to the 3' untranslated region (UTR) of target mRNAs resulting in translation repression or mRNA degradation. By distinct complementary approaches, including gene array analysis and overexpression of glycosyltransferase specific micro-RNAs (miRNAs) in primary hematopoietic stem cells, we identify here that miR-331-3p and miR-1908-5p directly target glycosyltransferase A and B. Consistently, overexpression of miR-331-3p and -1908-5p in hematopoietic stem cells (HSCs) leads to a 30-50% reduction of blood group A antigens per cell in differentiated RBCs. Downregulation of endogenous miR-1908-5p but not miR-331-3p leads to increased amount of blood group A antigen per red blood cell compared to control cells. This discrepancy between the effects of downregulation of miR-1908-5p and of miR-331-3p is likely explained by the constitutive expression levels of both miRNAs during erythropoiesis. In particular, miR-1908-5p is expressed throughout erythroid differentiation, while miR-331 displays low expression, thus, further downregulation of miR-331-3p could not further affect blood group A antigen levels. Furthermore miR-331-3p and -1908-5p were enhanced in red blood cells of Aweak variants. Sequencing of the 3'UTR of 6 Aweak variants revealed the presence of more miRNA binding sites for miR-1908-5p compared to normal controls. These data identify microRNAs as regulators of blood group glycosyltransferase expression, thereby extending our understanding of blood group regulation in carriers of weak blood group variants. Regulating blood groups by microRNAs could pave the way for the development of universal blood. Disclosures No relevant conflicts of interest to declare.

Description: While adoptive transfer of virus antigen specific T cells has shown to be effective in therapy of resistant recurrent viremia which is frequently associated with the lack of protective immunity following hematopoietic stem cell transplantation, the transfer of leukemia associated antigen specific (LAA) T cells is less implemented and appears to depend on factors that hamper a successful translation into the clinic. Among them are low frequencies and low antigen affinity of LAA specific T cells which currently mandate laborious in vitro expansion protocols. Moreover, screening of healthy individuals with regard to the presence of LAA specific T cells revealed contradictory results. Since we failed to detect LAA specific T cells in healthy donors using single peptide specificities to known LAA epitopes coupled to MHC Streptamers, here we asked if the use of peptide mixes comprising 15mers overlapping by 11 amino acids and spanning the entire LAA protein could elicit in vitro T cell responses in healthy donors, otherwise undetectable by single peptide staining. A cohort of 48 HLA A*0201 healthy individuals was screened using intracellular cytokine staining (ICS) after stimulation with tumor specific peptide mixes representing well known LAAs (WT1, PRAME, NY-ESO, Survivin and p53). While distinct T-helper cell responses were not observed in either of the specimen tested, cytotoxic T lymphocytes could be elicited and measured after incubation with peptide mixes for 5 hours and subsequent CD8+ IFNγ+ staining in 12 out of 48 healthy subjects. Only one individual displayed specifies against multiple antigens (WT1:0,1%; PRAME:0,5%; NY-ESO:0,1%; p53:0.06%), while the remaining responses were directed to one single antigen per individual. Most prevalent and highest T cell frequencies were found against PRAME in 5 out of all screened subjects (mean 0.4±0.3%; max. 0.8%), followed by WT1 in 4 (mean 0.07±0.03%; max. 0.1%) and NY-ESO in 3 individuals (mean 0,07±0,04%; max. 0,1%); one showed CD8 T cells specific against Survivin (0,03%) and 2 individuals had CD8 frequencies specific against p53 (0,05±0,01; max. 0,06%), respectively. The calculated limit of detection (LOD) for the enumeration of LAA specific T cells was 0,02%. In contrary, testing LAA positive individuals with according MHC Streptamers presenting single peptides of previously described epitopes showed no frequencies exceeding LOD. Further analysis showed LAA specific CD8+ IFNγ+ T cells exhibit mainly a less differentiated phenotype (CD45RA+, CCR7+/-, TNFα+, IL-2+/-) and could be immune-magnetically isolated to purities of 94.5±0.7% using a PRAME-specific IFN-γ capture assay yielding 1*104 antigen specific T cells out of 4*107 PBMCs. Simultaneous enrichment of helper T cells to a purity of 73.0±7.6% proofed their existence, despite no CD4+ response could be detected via ICS in the first place. The cytotoxic potential of the cell product was confirmed in an Europium assay using T2 cells loaded with PRAME peptide mix. The specific lysis accounted to 19.3% at an E:T ratio of 1:1 after 90 minutes of co-incubation. In conclusion, using LAA specific peptide mixes in combination with ICS we were able to show a relatively high prevalence of LAA specific T cells, especially for PRAME, in healthy donors. These LAA specific T cells can be enriched without the need of in vitro expansion culturing ex vivo using the IFN-γ capture assay with regard to achieving a functional LAA specific T cell product for adoptive T cell transfer. Furthermore, a less differentiated phenotype exhibited by a large proportion of LAA specific T cells might contribute to their long term survival in a patient after transplantation. Disclosures No relevant conflicts of interest to declare.

Description: Introduction CMV-infection is a serious complication in patients after allogeneic stem cell transplantation (SCT) where immunosuppressive therapy and impaired T cell reconstitution result in a high risk for viral infections. Monitoring of CMV-virus load by PCR and preemptive therapy are important tools to prevent CMV disease. However, CMV specific cytotoxic T cells (CMV-CTLs) are needed to successfully control CMV-infections. CMV-specific multimers composed of the patients HLA Class I molecule bound to CMV pp65 epitopes give the possibility to monitor CMV-CTLs. Here, we present the case of CMV-reactivation following SCT for AML. Methods The percentage of CMV-specific CD8+ T cells was determined by flow cytometry and mapped to clinical and laboratory parameters of the patient. CD8+ T cells were detected using CD8-fluorescein isothiocyanate (FITC, Beckman Coulter) antibody and CD3 as a T-cell marker was labeled with CD3-allophycocyanin (APC, MACS Miltenyi Biotec) antibody. CMV-specific CD8+ T cells were detected using the CMV major histocompatibility complex (MHC) with Strep-Tactinphycoerythrin (PE) conjugate (Streptamers, IBA GmbH). Case A 60 years old male patient was diagnosed with acute myeloid leukemia (AML) with 95% myeloid blasts in the bone marrow and extramedullary AML manifestations at the time of diagnosis. Following induction therapy the patient was transplanted from a matched unrelated donor. The stem cell recipient as well as his donor had been tested sero-positive for CMV prior to SCT. Within the first month following transplantation, the patient developed an effective CMV specific immunity as seen by high levels of CMV-specific T cells (Figure 1). About three months following transplantation the patient was diagnosed with intestinal GVHD requiring high-dose glucocorticoid treatment. Following steroid exposure, levels of CMV-CTLs dropped and shortly thereafter rising CMV-copy numbers were observed which was accompanied by clinical signs of CMV enteritis. With the administration of antiviral treatment the CMV specific virus load decreased. However, levels of CMV-CTLs remained low, presumably as a result of ongoing steroid exposure. Discussion High levels of CMV-CTLs appeared to control CMV, as seen by a non-detectable virus load in standard PCR testing. The close correlation between the drop in CMV-CTL count and CMV activation highlights the potential of this method to monitor and understand immune responses to CMV following SCT. Of note, early presence of high frequencies of CMV-CTLs did not guarantee CMV-control under steroid exposure as seen in our case. Previous reports have suggested that high dose glucocorticoids may impact CMV-CTLs survival. This is supported by our case, where we see a rapid drop in CMV-CTLs following glucocorticoid exposure. However, the exact molecular mechanisms and more importantly, the predictive value of this finding remain elusive. Furthermore, these data suggest, that patients with ongoing high steroid exposure may not benefit from a transfer of CMV-specific T-cells to control CMV disease. Conclusion Further investigations to clarify the potential of CMV-CTL measurements and to understand the effect of steroid exposure at the functional level are warranted. Studies to correlate CMV-CTL counts with the level of immunosuppression and their influence on controlling CMV-disease will follow. In future, this tool could provide a chance to select patients at high risk of CMV reactivation who could profit from an individualized monitoring and early treatment. Disclosures: No relevant conflicts of interest to declare.