Simon Völkl

Hyperactive mTOR pathway promotes lymphoproliferation and abnormal differentiation in autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder characterized by defective Fas signaling, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ(+) CD4(-) CD8(-) double-negative T (DNT) cells. Although their phenotype resembles that of terminally differentiated or exhausted T cells, lack of KLRG1, high eomesodermin, and marginal T-bet expression point instead to a long-lived memory state with potent proliferative capacity. Here we show that despite their terminally differentiated phenotype, human ALPS DNT cells exhibit substantial mitotic activity in vivo. Notably, hyperproliferation of ALPS DNT cells is associated with increased basal and activation-induced phosphorylation of serine-threonine kinases Akt and mechanistic target of rapamycin (mTOR). The mTOR inhibitor rapamycin abrogated survival and proliferation of ALPS DNT cells, but not of CD4(+) or CD8(+) T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation by DNT cells. Importantly, increased mitotic activity and hyperactive mTOR signaling was also observed in recently defined CD4(+) or CD8(+) precursor DNT cells, and mTOR inhibition specifically reduced these cells in vivo, indicating abnormal programming of Fas-deficient T cells before the DNT stage. Thus, our results identify the mTOR pathway as a major regulator of lymphoproliferation and aberrant differentiation in ALPS.

Suppressive effects of tumor cell-derived 5′-deoxy-5′-methylthioadenosine on human T cells

ABSTRACT The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5′-deoxy-5′-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

A new flow cytometric assay for the simultaneous analysis of antigen-specific elimination of T cells in heterogenous T cell populations

Novel immunosuppressive strategies are targeting for an antigen-specific deletion of T cells responsible for organ damage in autoimmunity and allograft rejection. Here, we present a new flow cytometry-based assay that allows the reliable and efficient detection of T cells that were eliminated in an antigen-specific fashion. A stable cell-labelling technique utilizing the two membrane dyes PKH26 and PKH67 has been combined with annexin V and 7-aminoactinomycin (7-AAD) staining to detect apoptotic cells. A differential gating strategy enabled us to determine the viability/apoptosis for each PKH-stained T cell subpopulation independently. The capability to simultaneously analyze apoptosis within T cell mixtures of different antigen specificities establishes this assay as a superior tool for the further development of novel antigen-specific immunosuppressive approaches.

IL-7 Abrogates the Immunosuppressive Function of Human Double-Negative T Cells by Activating Akt/mTOR Signaling

Recently, a novel subset of TCRαβ+ CD4− CD8− double-negative (DN) T cells was described to suppress immune responses in both mice and humans. Moreover, in murine models, infusion and/or activation of DN T cells specifically suppressed alloreactive T cells and prevented the development of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. We demonstrated that human DN T cells, like their murine counterparts, are highly potent suppressor cells of both CD4+ and CD8+ T cell responses. After hematopoietic stem cell transplantation and other lymphopenic conditions, IL-7 plays an important role in the reconstitution, survival, and homeostasis of the T cell compartment. Because IL-7 was shown to interfere with T cell functionality, we asked whether IL-7 affects the functionality of human DN T cells. Intriguingly, IL-7 diminished the suppressive activity of DN T cells toward allogeneic CD4+ effector T cells. Of interest, our studies revealed that IL-7 activates the Akt/mechanistic target of rapamycin (mTOR) pathway in human DN T cells. Importantly, selective inhibition of the protein kinases Akt or mTOR reversed the IL-7 effect, thereby restoring the functionality of DN T cells, whereas inhibition of other central T cell signaling pathways did not. Further analyses suggest that the IL-7/Akt/mTOR signaling cascade downregulates anergy-associated genes and upregulates activation- and proliferation-associated factors that may be crucial for DN T cell functionality. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T cell functionality in graft-versus-host disease.

BATF-dependent IL-7RhiGM-CSF+ T cells control intestinal graft-versus-host disease

Acute graft-versus-host disease (GVHD) represents a severe, T cell–driven inflammatory complication following allogeneic hematopoietic cell transplantation (allo-HCT). GVHD often affects the intestine and is associated with a poor prognosis. Although frequently detectable, proinflammatory mechanisms exerted by intestinal tissue–infiltrating Th cell subsets remain to be fully elucidated. Here, we show that the Th17-defining transcription factor basic leucine zipper transcription factor ATF-like (BATF) was strongly regulated across human and mouse intestinal GVHD tissues. Studies in complete MHC-mismatched and minor histocompatibility–mismatched (miHA-mismatched) GVHD models revealed that BATF-expressing T cells were functionally indispensable for intestinal GVHD manifestation. Mechanistically, BATF controlled the formation of colon-infiltrating, IL-7 receptor–positive (IL-7R+), granulocyte-macrophage colony-stimulating factor–positive (GM-CSF+), donor T effector memory (Tem) cells. This T cell subset was sufficient to promote intestinal GVHD, while its occurrence was largely dependent on T cell–intrinsic BATF expression, required IL-7–IL-7R interaction, and was enhanced by GM-CSF. Thus, this study identifies BATF-dependent pathogenic GM-CSF+ effector T cells as critical promoters of intestinal inflammation in GVHD and hence putatively provides mechanistic insight into inflammatory processes previously assumed to be selectively Th17 driven.

Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein-Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.

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.

Advances in cellular therapy: 5th International Symposium on the clinical use of cellular products, 19 and 20 March 2009, Nürnberg, Germany

“Cellular Therapy 2009”: The 5th International Symposium on the Clinical Use of Cellular Products was held in Nuremberg, Germany on 19–20 March 2009. Cellular therapy encompasses a well-established treatment modality exempliWed by the curative potential of hematopoietic stem cell transplantation (HSCT). Combinatorial use of cell types such as T cells, natural killer (NK) cells, and antigen-presenting cells (APCs) provides opportunities for a fascinating, emerging concept in the treatment of cancer, autoimmune, and infectious diseases. The 2-day meeting was attended by approximately 350 international guests, and included 40 oral presentations and almost 100 selected poster presentations. The plenary sessions covered hot topics in the Weld of immune eVector cells (T cells, regulatory T cells, NK cells, APC) as well as adoptive T cell transfer, cancer vaccination, and HSCT. Each scientiWc session included state-of-the-art lectures on pre-clinical and clinical studies. This meeting report will highlight the main issues discussed and summarize the presentations from the plenary sessions.