ALBERT

Description: Background: Reactivation of CMV and EBV negatively impacts on outcome after allogeneic stem cell transplantation (aSCT). Specific antiviral therapy is only available for CMV. With the exception of ganciclovir all drugs are being used off-label. 40-50% of patients reactivate CMV following aSCT. For the 20-30% of patients reactivating EBV, only rituximab is available to control EBV. Rituximab leads to long term B-cell depletion requiring frequent administration of immunoglobulins. To cover the unmet medical need of CMV and EBV control after aSCT, we investigate a somatic cell therapy approach by means of CMV- and EBV-specific peptide-stimulated T cells. We have set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the preventive and preemptive adoptive transfer of this ATMP in patients after aSCT (EudraCT number 2012-004240-30). The multi-center trial is currently recruiting. Methods: For manufacturing of the cell product two peptide pools (CMV and EBV) each covering 17 well-defined HLA class I and class II epitopes for stimulation of donor derived PBMC are used. PBMC collected by leukapheresis of mobilized or non-mobilized donors can be used as starting material. To avoid a second leukapheresis of the donor, CMV- and EBV-specific T cells are preferentially expanded from a small fraction of the stem cell graft. A strong expansion of virus-specific T cells could be observed in the cell products as determined by HLA class I multimer analysis. Reconstitution and cell counts of leukocytes after aSCT are monitored for both treatment and control group. To obtain further insights in the expansion of transferred T cells, the TCR beta (TCRb) repertoire of the T-cell product before and after adoptive transfer in the patient is monitored by high throughput sequencing. Specificities of TCRb sequences can be assigned by determining the repertoire of HLA/peptide-multimer-sorted CD8+ T cells. New virus-specific TCRb sequences can be identified thereby. Furthermore, TCR sequences within the T-cell product can be tracked in the patient. Study design: After recruitment patients are randomized in intervention or control group. Patients of the intervention group receive three applications of virus specific T cells (5x10e4/kg bodyweight) starting the first adoptive transfer 30 days after allogeneic stem cell transplantation. Cells are transferred as preventive, preemptive, or also as therapeutic treatment. Patients are monitored for occurrence of GvHD, for viral load, as well as for immune reconstitution, especially of virus-specific T cells. Results: So far, 10 patients have been randomized. The reconstitution of virus-specific T cells of treated patients looks encouraging after transfer so far. The immunomonitoring of six included patients is completed. New CMV- and EBV-specific TCRb sequences could be identified and tracked. Conclusion: Our first observations show promising results regarding feasibility and efficacy of our approach under clinical trial conditions. Disclosures Hennig: HS Diagnomics GmbH: Employment, Equity Ownership.

Description: Down-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4-CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide–HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2–restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A–HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag–specific T cells after transplantation.

Description: Key Points ALPS DNT cells and their putative precursors reveal high proliferative activity in vivo, which is associated with hyperactive mTOR signaling. Rapamycin therapy controls mitotic activity and abnormal differentiation of ALPS DNT cells and reduces CD4+ or CD8+ precursor DNT cells.

Description: Key Points Lack of KLRG1 and T-bet expression is a unique feature of DNT and subsets of single positive T cells in ALPS patients. Genetic, phenotypic, and transcriptional evidence indicates that DNT in ALPS patients derive from both CD4+ and CD8+ T cells.

Description: It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14-progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen-presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens.

Description: Abstract 584 Specific T-cell responses are initiated by T-cell receptor (TCR) recognition of peptide-MHC-complexes on antigen presenting cells (APCs). Upon specific interaction of T cells with APCs T cells capture membrane fragments and surface molecules of APCs in a process termed trogocytosis. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. Here, we demonstrate that human antigen-specific CD8+ T cells do acquire the co-inhibitory molecule programmed death ligand 1 (PD-L1) from mature monocyte-derived dendritic cells (mDC) and tumor cells in an antigen-specific manner. The kinetics of PD-L1 transfer revealed a maximal PD-L1 expression on antigen-specific T cells within 3–4 hours after co-incubation with antigen-pulsed APCs, being detectable up to 72 hours. Antigen-pulsed immature DCs were less effective in transfering surface molecules such as PD-L1 onto CD8+ T cells after antigen-specific recognition. Using a transwell system we could show that the acquisition of PD-L1 requires cell-cell contact. Furthermore, PD-L1 cannot be acquired by T cells from a lysate of mDCs. The transfer process is impaired after pretreatment of T cells with concanamycin A, a specific inhibitor of vacuolar ATPases, playing an important role in membrane trafficking. Moreover, fixation of DCs with glutaraldehyde completely abrogated the acquisition of PD-L1 on T cells suggesting that an active interaction between APCs and T cells is required for trogocytosis. Of importance, CD8+ T cells which acquired PD-L1 complexes, were able to induce apoptosis of neighbouring PD-1 expressing CD8+ T cells, that could be completely blocked by an anti-PD-L1 antibody. In summary our data demonstrate for the first time that human antigen-specific CD8+ T cells take up functionally active PD-L1 from APCs in an antigen-specific fashion, leading to apoptosis of PD-1 expressing T cells. The transfer of functionally active co-inhibitory molecules from APCs onto human CD8+ T cells may serve to limit clonal expansion of antigen-specific T-cell responses but may also play a major role for T-cell exhaustion in chronic infection and tumor immunosurveillance. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3491 Novel methods to quantify chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) based on single-nucleotide polymorphism (SNP) specific real-time quantitative polymerase chain reaction (qPCR) require high amounts of input DNA. The applicability of these methods in cases where DNA quantity is limiting, however, is currently unclear. Here, we demonstrate that SNP typing performed with just 5ng of input DNA still allowed for the distinction of positive (mean ct 28.05) and negative (ct 〉 36) signals. In addition, we confirm the high informative value of a set of 19 SNP markers, with 12 markers exceeding 20% informativity in our population (n=74). Of interest, a four-fold reduction of input DNA for qPCR standard curves did not alter PCR efficiencies. Most importantly, in 7 out of 16 allogeneic HSCT patients who experienced disease relapse, a retrospective analysis using the SNP-qPCR method revealed re-appearance of recipient chimerism earlier (mean 95 days) compared to previous results obtained by a short tandem repeat (STR) specific PCR. Taken together our data clearly demonstrate that SNP-qPCR is more sensitive compared to the widely used STR-PCR even with reduced amounts of input DNA. We therefore recommend that SNP-qPCR is preferable to STR-PCR for chimerism analysis in cases where DNA quantity is a limiting factor. Disclosures: No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is considered a chronic and incurable disease due to its highly complex and heterogeneous molecular abnormalities and the support from myeloma microenvironment factors. Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies and can directly eliminate tumor cells. However, myeloma-associated macrophages (MAMs) regularly fail to exert direct effector functions. Given their abundance in MM, an attractive therapeutic approach would be to stimulate their tumoricidal activity in order to promote antitumor immunity. Lenalidomide, an immunomodulatory agent that enhances antibody dependent cell mediated cytotoxicity (ADCC), has the potential to synergize with MOR202, an anti-CD38 monoclonal IgG1 antibody currently in phase I/IIa for the treatment of MM. Furthermore, vitamin D plays a key role in regulating effector functions of human macrophages. This is closely linked to the expression of the vitamin D-1-hydroxylase CYP27B1, which catalyzes the conversion of 25-hydroxy-vitamin D (25D) to the bioactive form 1,25-di-hydroxy-vitamin D (1,25D). We have previously shown, that vitamin D promotes tumoricidal activity of macrophages and improves the efficacy of rituximab-dependent cytotoxicity (Bruns et al., Sci. Transl. Med., 2015; Bittenbring et al., JCO, 2014). Therefore, we hypothesized that the combination of MOR202 with lenalidomide and MOR202 with 1,25D would enhance the MOR202- dependent macrophage-mediated effector functions against myeloma cells. Here we report that MAMs exhibit an altered vitamin D metabolism with a reduced expression of the vitamin D receptor (VDR) and CYP27B1, while the expression of CYP24A1, which catabolizes 1,25D, is increased. As a consequence MAMs cannot convert 25D into bioactive 1,25D. Given the importance of the vitamin D pathway for antibody mediated cytotoxicity, we screened several drugs for their ability to restore the vitamin D pathway in human macrophages. We found, by RNA-sequencing, that lenalidomide treatment modulates the phenotype of monocyte-derived macrophages and isolated MAMs, and that lenalidomide significantly increases the expression of the VDR, CYP27B1 and CAMP (cathelicidin antimicrobial peptide) in these macrophages. Furthermore, we demonstrate that isolated MAMs regularly fail to eliminate primary myeloma cells, and that the lack of effector functions can be overcome by treatment with lenalidomide and vitamin D. Moreover, we show that MOR202-dependent elimination of myeloma cells is enhanced by pre-treatment of isolated MAMs with lenalidomide and supplementation of vitamin D. In summary, these data show that the bioactive form of vitamin D (1,25D) is essential for the effector functions of MAMs and that the therapeutic activation of the vitamin D pathway by lenalidomide may restore their tumoricidal effector mechanisms. Furthermore, these findings highlight that stimulating the tumoricidal activity of MAMs, an abundant component of the stromal cell compartment, with lenalidomide and 1,25D is an attractive therapeutic approach in combination with the anti-CD38 monoclonal IgG1 antibody MOR202. Disclosures Bruns: Morphosys: Research Funding; Celgene: Research Funding.

Description: In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma membrane leaflet. This lipid asymmetry which is maintained by the concerted action of phospholipid transport proteins is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTL) expose PS upon T-cell receptor (TCR)-mediated antigen recognition: antigen-specific CTL, recognizing the HLA-A2 binding Melan-A peptide, demonstrated a marked exposure of PS as determined by annV-FITC staining, a highly specific PS-binding protein, after 4 h of stimulation with Melan-A-loaded antigen presenting cells (APC). PS exposure was Ag-specific (〉80% annV-positive T cells) but control peptide (gp100)-pulsed and unpulsed APC also induced a slight background PS exposure on the CTL. To follow more precisely the fate of annV-positive CTL after antigen-specific stimulation, we labeled Melan-A-specific CTL with the membrane dye PKH-26 and isolated all annV+ PKH-26+ CTL 4 h after stimulation with peptide-pulsed APC cells by cell sorting. Results demonstrate that the annV-positive T-cell population is heterogenous: while the annV-high T-cell population retained the annV-high phenotype and consisted of apoptotic T cells, the annV-intermediate(int) T-cell population revealed a constant decrease of annV binding and became annV-negative at 54 to 72 h after stimulation. Using three independent assays for apoptosis we found that annV-int T cells are propidium iodide negative, do not exhibit DNA strand brakes and contain no active caspase 3. In contrast, annV-int CTL revealed a strongly activated phenotype indicated by an upregulation of CD69 expression and downregulation of the TCRαβ chain expression. Fluorescence microscopic analysis demonstrated that PS is distributed inhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunological synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we determined a constitutive outward transfer of PS molecules in Melan-A-specific CTL. The constitutive PS outward transport was not further accelerated after antigen re-stimulation. In sharp contrast, the inward transporting flippase was strongly inhibited in stimulated CTL resulting in an accumulation of PS molecules on the cell surface. Shielding of exposed PS by annexinV protein during antigen recognition diminished cytokine secretion, activation and cell-cell clustering of antigen-specific CTL. In summary, our data demonstrate for the first time that externalized PS on antigen-stimulated CTL is linked to T-cell activation and probably involved in cell-cell contact formation at the immunological synapse.

Description: It is well established, that the curative potential of allogeneic peripheral blood stem cell transplantation (allo PBSCT) is due to immunocompetent donor T cells inducing potent anti-neoplastic effects against host tumor cells. This reaction, which is termed graft-versus-leukemia (GVL) effect, is clinically effective against a number of different hematologic malignancies such as myeloid and lymphoid leukemias. Despite great efforts of allo PBSCT in treatment of CML, the 5-year survival rate of AML patients after allo PBSCT is only about 30% due to relapsing disease. The recurrent disease is inefficiently controlled by the immune system, due most likely to the various immune escape mechanisms described for AML blasts including upregulation of anti-apoptotic molecules. Since cytotoxic T lymphocytes (CTL) and natural killer cells are the cells responsible for eliminating leukemic blasts, the most important effector molecule is Granzyme B (GrB). Misdirected GrB is quenched by its specific physiological inhibitor Protease Inhibitor-9 (PI-9) leading to inactivation of GrB. PI-9 expression by tumour cells can be used to escape immune surveillance and its presence has been shown for different tumors e.g. melanoma, colon carcinoma and lymphoma. Despite other regulators, interferon-γ (IFN-γ) has been shown to upregulate PI-9 expression in hepatocytes. Here, we wanted to investigate the expression of PI-9 in primary AML blasts and its regulation by IFN-γ. Using CD34+ positive magnetic selection, we isolated primary blasts with a purity of 〉90% from 20 AML patients with different FAB subtypes. For detection of PI-9 expression by Western Blotting, whole cell lysates were made from freshly purified blasts and after 24 h +/− 200 IU/ml IFN-γ. In some patients, PI-9 expression was confirmed by FACS analysis with an anti- PI-9 specific monoclonal antibody. Here we describe for the first time, that PI-9 is constitutively expressed in 16/20 (80%) of AML blasts. Treatment of AML blasts with IFN-γ could upregulate PI-9 expression in a dose-dependent manner (2–2,000 IU/ml) and strong expression of PI-9 was detectable in 6/18 patients within 4–5 h after IFN-γ exposure. Of note, a mild upregulation of PI-9 upon 24 h incubation w/o IFN-γ could be detected in 4/18 (22%) patients. We conclude, that cytokines such as IFN-γ which are secreted during the cytokine storm of acute graft-versus-host disease can contribute to the development of immune escape mechanisms in AML blasts.