Denise Kühn

High Diversity in the TCR Repertoire of GAD65 Autoantigen-Specific Human CD4+ T Cells

Autoreactive CD4+ T cells are an essential feature of type 1 diabetes mellitus. We applied single-cell TCR α- and β-chain sequencing to peripheral blood GAD65-specific CD4+ T cells, and TCR α-chain next-generation sequencing to bulk memory CD4+ T cells to provide insight into TCR diversity in autoimmune diabetes mellitus. TCRs obtained for 1650 GAD65-specific CD4+ T cells isolated from GAD65 proliferation assays and/or GAD65 557I tetramer staining in 6 patients and 10 islet autoantibody-positive children showed large diversity with 1003 different TCRs identified. TRAV and TRBV gene usage was broad, and the TRBV5.1 gene was most prominent within the GAD65 557I tetramer+ cells. Limited overlap (<5%) was observed between TCRs of GAD65-proliferating and GAD65 557I tetramer+ CD4+ T cells. Few TCRs were repeatedly found in GAD65-specific cells at different time points from individual patients, and none was seen in more than one subject. However, single chains were often shared between patients and used in combination with different second chains. Next-generation sequencing revealed a wide frequency range (<0.00001–1.62%) of TCR α-chains corresponding to GAD65-specific T cells. The findings support minor selection of genes and TCRs for GAD65-specific T cells, but fail to provide strong support for TCR-targeted therapies.

Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Abundant CMV reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation

Allogeneic stem cell transplantation is potentially curative, but associated with post‐transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR‐α repertoire and its clinical relevance in patients following stem cell transplantation. Using next‐generation sequencing we examined the TCR‐α repertoire of CD8+ T cells and CMV‐specific CD8+ T cells in four patients. Additionally, we performed single‐cell TCR‐αβ sequencing of CMV‐specific CD8+ T cells. The TCR‐α composition of human leucocyte antigen (HLA)‐A*0201 CMVpp65– and CMVIE‐specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8+ T cells and half of the total CD8+ T cell repertoire was dominated by few CMV‐reactive clonotypes. Some TCR‐α clonotypes were shared between patients. Gene expression of the circulating CMV‐specific CD8+ T cells was consistent with chronically activated effector memory T cells. The CD8+ T cell response to CMV reactivation resulted in an expansion of a few TCR‐α clonotypes to dominate the CD8+ repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti‐viral T cell response in this setting.

Compromised immune response in infants at risk for type 1 diabetes born by Caesarean Section

Children born by Caesarean Section have a higher risk for type 1 diabetes. We aimed to investigate whether Caesarean Section leads to alterations of the immune response in children with familial risk for type 1 diabetes. We examined measures of innate and adaptive immune responses in 94 prospectively followed children, including 40 born by Caesarean Section. Proinflammatory serum cytokine concentrations were determined at age 6 months. As a measure of vaccine response, IgG1, IgG2, and IgG4 tetanus antibody titers and CD4(+) T cell proliferation against tetanus toxoid were quantified. Compared to infants born by vaginal delivery, infants born by Caesarean Section had lower concentrations of the cytokines IFN-ɣ (p=0.014) and IL-8 (p=0.005), and weaker CD4(+) T cell responses to tetanus measured in the first (p=0.007) and second year (p=0.047) of life. Overall, our findings provide evidence that the mode of delivery influences the immune status and responsiveness during childhood.

CD8 + T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage

CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and β-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen.

Generation of high-avidity, WT1-reactive CD8+ cytotoxic T cell clones with anti-leukemic activity by streptamer technology

Antje Tunger, Rebekka Wehner, Malte von Bonin, Denise K€ uhn, Falk Heidenreich, Sarah Matko, Magdalena Nauerth, Elke R€ ucker-Braun, Sevina Dietz, Cornelia S. Link, Anne Eugster, Marcus Odendahl, Dirk H. Busch, Torsten Tonn, Ezio Bonifacio, Lothar Germeroth, Johannes Schetelig, Michael P. Bachmann, Martin Bornh€auser and Marc Schmitz Institute of Immunology, Medical Faculty, TU Dresden, Dresden, Germany; National Center for Tumor Diseases, University Hospital Carl Gustav Carus, TU Dresden, Germany; Department of Medicine I, University Hospital of Dresden, Dresden, Germany; German Cancer Consortium (DKTK), Dresden, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Center for Regenerative Therapies Dresden (CRTD), Medical Faculty, TU Dresden, Dresden, Germany; Institute of Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany; Institute for Medical Microbiology, Immunology and Hygiene, TU Munich, Munich, Germany; Juno Therapeutics GmbH, G€ottingen, Germany; Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz Center Dresden-Rossendorf, Dresden, Germany

Underground Degradation of Lignite Coal Spoil Material by a Mixed Microbial Community under Acid Mine Drainage Conditions

In a field study, biogeochemical processes in a large lignite coal spoil area with moderate AMD generation were investigated. Underneath this area, large amounts of groundwater are impacted by degradation and transformation processes of coal remainders in the former open pit mining area. An investigation was performed to find out the sources for the ground and surface water contaminations of larger areas. Samples were taken from different places and different depths of the coal spoil area and were investigated for different metabolic groups of microorganisms. As a result, fungi are able to degrade humic matter in coal spoil heaps in a first step to oligomers. Other microorganisms do a further degradation of first intermediates in a commensalic community. Streptomycetes do a cleavage of lignocelluloses, strepto- and other actinomycetes also degrade cellobiose and xylose related parts of the humic coal spoil matter. The different members of the microbial community exist in different “floors” of the spoil area: fungi and most Actinomycetes prefer the oxic zone, whereas degraders of aromatic and heterocyclic compounds can also exist in the capillary and ground water zones; here more frequently Arthrobacter, Pseudomonas, Rhodococcus and Mycobacterium strains were detected. Ferric iron formed in biooxidation of pyrite seems to play an important role as a catalyst for oxic as well as anaerobic degradation of complex organic matter in the underground. A complex linkage between microbial Fe-, S-, C- and N-cycles was figured out on this site that induces a high and long-term impact on ground water contamination in this area.

Vagaries of the ELISpot assay: specific detection of antigen responsive cells requires purified CD8(+) T cells and MHC class I expressing antigen presenting cell lines.

Quantification of antigen-specific CD8(+) T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFNγ ELISpot assays occurred independently of CD8(+) T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8(+) T cell IFNγ production was attributed to the CD4(+) T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8(+) T cells and demonstrated that it specifically detected CD8(+) T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8(+) ELISpot assay.

Mass spectrometry-based identification of a naturally presented receptor tyrosine kinase-like orphan receptor 1-derived epitope recognized by CD8+ cytotoxic T cells

Despite recent treatment improvements with the approval of cell signaling pathway inhibitors, additional treatment options are needed for patients with relapsed/refractory high-risk chronic lymphocytic leukemia (CLL). An emerging immunotherapeutic strategy for CLL is based on the adoptive transfer