Gabriele Niedermann

Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum

The generation of many HLA class I peptides entails a final trimming step in the endoplasmic reticulum that, in humans, is accomplished by two candidate aminopeptidases. We show here that one of these, ERAP1, was unable to remove several N-terminal amino acids that were trimmed efficiently by the second enzyme, ERAP2. This trimming of a longer peptide required the concerted action of both ERAP1 and ERAP2, both for in vitro digestion and in vivo for cellular antigen presentation. ERAP1 and ERAP2 localized together in vivo and associated physically in complexes that were most likely heterodimeric. Thus, the human endoplasmic reticulum is equipped with a pair of trimming aminopeptidases that have complementary functions in HLA class I peptide presentation.

The ZEB1/miR‐200 feedback loop controls Notch signalling in cancer cells

Notch signalling is important for development and tissue homeostasis and activated in many human cancers. Nevertheless, mutations in Notch pathway components are rare in solid tumours. ZEB1 is an activator of an epithelial–mesenchymal transition (EMT) and has crucial roles in tumour progression towards metastasis. ZEB1 and miR‐200 family members repress expression of each other in a reciprocal feedback loop. Since miR‐200 members target stem cell factors, ZEB1 indirectly induces stemness maintenance and associated drug resistance. Here, we link ZEB1 and its cancer promoting properties to Notch activation. We show that miR‐200 members target Notch pathway components, such as Jagged1 (Jag1) and the mastermind‐like coactivators Maml2 and Maml3, thereby mediating enhanced Notch activation by ZEB1. We further detected a coordinated upregulation of Jag1 and ZEB1, associated with reduced miR‐200 expression in two aggressive types of human cancer, pancreatic adenocarcinoma and basal type of breast cancer. These findings explain increased Notch signalling in some types of cancers, where mutations in Notch pathway genes are rare. Moreover, they indicate an additional way how ZEB1 exerts its tumour progressing functions.

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector Txa0cells, but memory Txa0cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon Txa0cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for Txa0cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory Txa0cells. Ourxa0data show that initial CD28 signals during Txa0cell activation prime mitochondria with latent metabolic capacity that is essential for future Txa0cell responses.

Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

T cell-recruiting bispecific antibodies (bsAb) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133×CD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by BIM and FAS as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers, and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor regrowth, reminiscent of tolerance induction by CD3 mAb-mediated T-cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors. Cancer Res; 76(16); 4673-83. ©2016 AACR.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms

Oncogenic JAK mutation sensitizes myeloproliferative neoplasms to immune checkpoint inhibition. Cancers JAK up an immune checkpoint Myeloproliferative neoplasms, a group of hematologic cancers, are associated with mutations activating the JAK2 oncogene. JAK2 is located on chromosome 9, in the vicinity of the immunosuppressive PD-L1 gene, and Prestipino et al. discovered that myeloproliferative cancers with overactive JAK2 generally have increased PD-L1 as well. Although PD-L1 helps cancers evade the immune system, immune checkpoint inhibitors developed in recent years provide a way to block its function and turn PD-L1 expression into a therapeutic vulnerability for the tumors, as the authors demonstrate in this study. Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Molecular radiobiology meets clinical radiation oncology

Background:u2003The 2nd Langendorff Congress in Freiburg in Breisgau (Germany) gathered basic and translational scientists as well as clinicians interested in recent developments in molecular and clinical radiobiology. The topics ranged from the most recent insight into the organisation of the DNA damage response and radiotherapeutically relevant cell death mechanisms to biological imaging for treatment planning and advances in the understanding of the molecular biological effects of particle beams. Clinical aspects of stem cell and tumour stem cell biology as well as of angiogenesis and hypoxia, the search for novel molecular radiosensitisers and potential strategies for exploitation of the immune system to further improve tumour radiotherapy were also discussed. Results and conclusion:u2003This report surveys the presentations at the meeting, considering their significance in light of the literature, and documents the increasing importance of molecular radiobiology for clinical radiooncology.

Abscopal effects with hypofractionated schedules extending into the effector phase of the tumor-specific T cell response

PURPOSEnHypofractionated radiation therapy (hRT) combined with immune checkpoint blockade can induce T-cell-mediated local and abscopal antitumor effects. We had previously observed peak levels of tumor-infiltrating lymphocytes (TILs) between days 5 and 8 after hRT. Because TILs are regarded as radiosensitive, hRT schedules extending into this period might be less immunogenic, prompting us toxa0compare clinically relevant, short and extended schedules with equivalent biologically effective doses combined with anti-programmed cell death 1 (PD1) antibody treatment.nnnMETHODS AND MATERIALSnIn mice bearing 2 B16-CD133 melanoma tumors, the primary tumor was irradiated with 3xa0×xa09.18xa0Gy in 3 or 5xa0days or with 5xa0×xa06.43xa0Gy in 10xa0days; an anti-PD1 antibody was given weekly. The mice were monitored for tumor growth and survival. T-cell responses were determined on days 8 and 15 of treatment. The role of regional lymph nodes was studied by administering FTY720, which blocks lymph node egress of activated T cells. Tumor growth measurements after combination treatment using short or extended hRT and control treatment were also performed in the wild-type B16 melanoma and 4T1 breast carcinoma models.nnnRESULTSnIn the B16-CD133 model, growth inhibition of irradiated primary andxa0nonirradiated secondary tumors and overall survival were similar with all 3xa0hRT/anti-PD1 combinations, superior to hRT and anti-PD1 monotherapy, and was strongly dependent on CD8+ T cells. TIL infiltration and local and systemic tumor-specific CD8+ T-cell responses were also similar, regardless of whether short or extended hRT was used. Administration of FTY720 accelerated growth of both primary and secondary tumors, strongly reduced their TIL infiltration, and increased tumor-specific CD8+ T cells in the lymph nodes draining the irradiated tumor. In the 4T1 model, local and abscopal tumor control was also similar, regardless of whether short or extended hRT was used, although the synergy between hRT and anti-PD1 was weaker. No synergies were found in the B16 wild-type model lacking an exogenous antigen.nnnCONCLUSIONSnOur data suggest that combination therapy with hRT schedules extending into the period during which treatment-induced T cells infiltrate the irradiated tumor can provoke local and systemic antitumor effects similar to those with therapy using shorter schedules, if the regional lymph nodes supply sufficient tumor-specific T cells. This has implications for planning clinical RT/immune checkpoint blockade trials.

Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.