Regina Gary

IL-33 Shifts the Balance from Osteoclast to Alternatively Activated Macrophage Differentiation and Protects from TNF-α–Mediated Bone Loss

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2−/− bone marrow led to more bone loss compared with the chimeras with ST2+/+ bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206+ AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Characterization of the immunoregulatory function of human TCR-αβ+ CD4−CD8− double-negative T cells

Regulatory T cells (Tregs) play an important role in the maintenance of immune tolerance to self‐antigens and are involved in modulating immune responses in autoimmunity, transplant rejection, and tumor immunity. Recently, a novel subset of TCR‐αβ+ CD4−CD8− (double negative, DN) T cells has been described to specifically suppress T‐cell responses in mice. Here, we demonstrate that human DN T cells are highly potent suppressors of both CD4+ and CD8+ T‐cell responses. In contrast to naturally occurring CD4+CD25+ Tregs, DN T cells have to be activated by antigen‐presenting cells (APCs) to induce their regulatory potential. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T‐cell growth factors. Furthermore, DN T‐cell‐mediated suppression toward responder T cells is TCR dependent and requires novel protein synthesis. In contrast to murine DN T cells, which eliminate effector T cells via Fas/FasL or perforin/granzyme, human DN T cells suppress proliferation of responder T cells by cell contact‐dependent mechanisms. Taken together, our data indicate that human DN T cells exert strong immunosuppressive effects on both CD4+ and CD8+ T cells and may serve as a new therapeutic approach to treat autoimmunity and transplant rejection.

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I–dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8+ cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

Antigen-Specific Transfer of Functional Programmed Death Ligand 1 from Human APCs onto CD8+ T Cells via Trogocytosis

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8+ T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8+ T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell–cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8+ T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1–expressing CD8+ T cells. In summary, our data demonstrate that human CD8+ T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1–expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8+ T cells could have a regulatory role in immune responses.

Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRβ) gene rearrangements of flow cytometry–sorted CD4+ and CD8+ T cells from the peripheral blood of 23 allogeneic donors before G‐CSF administration and on the day of apheresis. For this purpose, TCRβ rearrangements were amplified by multiplex PCR followed by high‐throughput amplicon sequencing. Overall, CD4+ T cells displayed a significantly higher TCRβ diversity compared to CD8+ T cells irrespective of G‐CSF administration. In line, no significant impact of G‐CSF treatment on the TCR Vβ repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4+ TCRβ diversity after G‐CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein–Barr virus. By contrast, no protecting correlation was observed for CD8+ T cells. In essence, our deep TCRβ analysis identifies the importance of the CD4+ T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Advances in cellular therapy: 6th international symposium on the clinical use of cellular products, March 24 and 25, 2011, Erlangen, Germany

‘‘Cellular Therapy 2011’’: The 6th International Symposium on the Clinical Use of Cellular Products was held in Erlangen, Germany, on March 24–25, 2011. Twelve years ago, the conference took place for the first time in Regensburg and was originally initiated by Andreas Mackensen. Over the past years, it gained reputation and attracted leading researchers in the field from all over the world. This year’s conference was jointly organized at the University of Erlangen by the Department of Hematology & Oncology (Andreas Mackensen), the Department of Dermatology (Gerold Schuler), Erlangen, and the Department of Hematology & Oncology, Regensburg (Reinhard Andreesen). In principal, the ‘‘Cellular Therapy Meeting’’ aims at providing a multidisciplinary forum for basic and clinical researchers to communicate and to learn about the broad scope of cell therapy from different perspectives. The two-day meeting was attended by approximately 400 international participants and included sessions in the field of immune effector cells (T cells and NK cells), regulatory cells (Treg and MSC), antigen-presenting cells, cancer vaccination, gene-based cellular therapy, metabolism as well as regulatory issues and clinical applications of cellular therapy. These topics were presented by talks from 23 invited international experts. In addition, 20 short talks selected from abstracts of highest scientific quality and almost 130 posters were presented at the meeting. In this report, we summarize the most important research findings and highlight the main discussion topics of the plenary sessions.

Impact of collection programs for the generation of monocyte apheresis products on product quality and composition as starting material for the generation of cellular therapeutics: Apheresis Programs for Cell Therapy

With the discontinuation of the last generation of apheresis machines, new options for monocyte apheresis became available. As apheresis products play a crucial role in the generation of new cellular therapeutics (e.g., generation of dendritic cells [DCs] or precursor for T‐cell experiments) we sought to compare two different collection programs for potential benefits or disadvantages due to different composition of the cellular products.

Abstract CT028: Adoptive transfer of CMV- and EBV- specific peptide-stimulated T cells after allogeneic stem cell transplantation: A Phase I/IIa clinical trial

Allogeneic stem cell transplantation (ASCT) to date is the only permanent curative treatment for many hematological cancers. Besides the development of Graft versus Host disease (GvHD), infections are the major adverse effect of ASCT. Specifically, reactivation of viruses is highly problematic in the aftermath of ASCT. Reactivation of human CMV and EBV negatively impacts on outcome after ASCT. 40-50% of patients reactivate CMV following ASCT, while the only CMV specific antiviral therapy available is ganciclovir, with other drugs being used off-label. 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 this unmet medical need of CMV and EBV control after ASCT, we investigate a somatic cell therapy approach by adoptive transfer of CMV- and EBV-specific peptide-stimulated T cells. We specifically are scrutinizing the effect of application of the cells to prevent virus reactivation before its onset or preemptively at the early stages of viral infection. We set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the safety and efficacy of preventive and preemptive adoptive transfer of this ATMP in patients after ASCT (EudraCT number 2012-004240-30). The design of the trial allows to applicate low numbers of activated T cells, thereby reducing the potential risks of GvHD for the recipient of adoptively transferred T cells. The multi-center trial is currently recruiting, so far, 13 patients have been randomized. ASCT patients are randomly assigned to the intervention or control group. Subjects of the control group receive standard of care. For all subjects of the intervention group, a personalized cell product (ATMP) containing a standardized number of virus specific T cells is manufactured from an aliquot of the leukapheresis product previously used for ASCT and cryopreserved until being administered. Subjects receive the cell product in intervals of at least 30 days, starting with the first adoptive transfer 30 days after ASCT at the earliest. Cells are transferred as preventive, preemptive, or also as therapeutic treatment. Subjects are monitored for occurrence of GvHD, for viral load, as well as for immune reconstitution and composition of the TCR pool, especially of virus-specific T cells. Citation Format: Michael Aigner, Regina Gary, Andreas Moosmann, Stefanie Maas, Julian Strobl, Robert Zimmermann, Jurgen Zingsem, Anita Kremer, Andreas Mackensen, Armin Gerbitz. Adoptive transfer of CMV- and EBV- specific peptide-stimulated T cells after allogeneic stem cell transplantation: A Phase I/IIa clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT028. doi:10.1158/1538-7445.AM2017-CT028

Abstract 3201: Generation of tumor-antigen specific T cells under highly controlled environmental conditions by utilization of a novel cGMP compliant cell culture reactor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Large scale in vitro generation of tumor-antigen specific T cells for adoptive transfer under good manufacturing procedures (GMP) conditions is one of the major challenges for future clinical applications. The novel Z(R)RP cell culture system consists of high precision control units for gas and fluidics in combination with a class A micro laminar flow unit and a GMP conform culturing unit which allows for maximal control of cell culture conditions. This system is - to our knowledge - the only commercially available product that fully complies with all regulatory specifications required for advanced therapy medical products (ATMP) production in the European Union. In addition, the Z(R)RP bioreactor system also implements the guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q10 and the requirements from 21 Code of federal regulations (CFR) parts 210, 211, 600 and 1271, making this system a potential candidate for the cGMP compliant production of human cellular products for individualized medicine approaches. We studied the potency of the Z(R)RP system for the generation of MART-1 antigen-specific T cells. Mononuclear cells (MNC) from healthy donors were cultured in GMP grade media supplemented with 1% of autologous serum. MNC were repeatedly pulsed with MART-1 peptide and culture outcome was analysed with respect to cellular composition, relative expansion and expression of cytokines of MART-1 specific T cells by flow cytometry. In short term cultures of 5x10E8 cells for up to 2 weeks medium pH as well as CO2 and O2 saturation was kept constant throughout culture duration. Glucose and lactate levels were monitored daily and continuous influx of fresh medium was adapted accordingly to match nutrient depletion and metabolite production. Media glucose concentration was kept at >500mg/ml and lactate concentrations at <1500mg/ml. Antigen specific T cells as identified by tetramer staining were expanded up to 350 fold. In comparison, standard control closed system cultures in bags resulted in 10 to 40% lower yield of specific T cells. In addition, overall cell yield was 25 - 90% higher from bioreactor cultures than from closed bag cultures. Analysis of MART-1 specific CD8+ T cells revealed a more naive phenotype as shown by CD45RA staining (65% in reactor vs. 20% in bag cultures). Production of cytokines as demonstrated by intracellular FACS staining of interferon-gamma and interleukin-4 was not significantly different in T cell populations from bioreactor or bag cultures. Our results show the superior potential of this bioreactor principle for expansion of high numbers of tumor-antigenspecific T cells under fully cGMP compliant conditions, which is pre-requisite for future individualized anti-tumoral cellular therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3201. doi:1538-7445.AM2012-3201