Allogeneic Bone Marrow Transplantation and Role of CD44 in T cell Maturation

Allogene Knochenmarktransplantation nach nicht-myeloablativer Konditionierung wird in der Klinik in zunehmendem Maße als letzte Möglichkeit einer kurativen Therapie bei Tumorpatienten eingesetzt. Man weiß inzwischen, dass diese Therapieoption durch eine aktive Vakzinierung deutlich verbessert werden kann. Eine aktive Vakzinierung erfordert jedoch, dass die im Wirt gereiften Doner T-Zellen gegenüber dem Wirt tolerant sind. Hierzu müssen sie im Wirtsthymus reifen. Da bei älteren Menschen nur noch ein Thymusrudiment vorliegt, konnte dieses Problem in der klinik bisher nicht hinreichend gelöst werden. Daher habe ich im Rahmen meiner Dissertation versucht im Tiermodell Möglichkeiten aufzuzeigen, die die T-Zell-rekonstitution nach allogener Knochenmarktransplantation unterstützen. Erste Untersuchungen zeigten auf, dass nach myeloreduktiver Konditionierung des allogenen Wirts eine verlässliche Rekonstitution mit T-Zell-depletierten und NK-Zell-depletierten Knochenmarkzellen ohne schwerwiegende Graft versus Host (GvH) Erkrankung erzielt werden kann. Dies war jedoch im tumortragenden Tier nicht der Fall. Progenitor T-Zellen wanderten nicht in den Thymus und ein erheblicher Prozentsatz der Tiere entwickelte schwere GvH Störungen. Die Repopulation des Thymus konnte durch stringentere Myeloreduktion, Applikation von IL-7 und insbesondere durch die Applikation von unreifen CD4+ CD8+ T-Zellen verbessert werden. Die CD4+ CD8+ Vorläufer-T-Zellen wanderten präferentiell in den Thymus. Nach der Reifung tolerierten die tumortragenden Tiere eine Vakzinierung, welche die Überlebenszeit und Überlebensrate der Tiere signifikant verbesserte. Basierend auf diesen Ergebnissen und früheren Befunden, die eine Beteiligung von CD44 am Prozess der Immigration von Progenitor-T-zellen in den Thymus nahe-legten, untersuchte ich, inwieweit CD44 am homing der Progenitor-T-Zellen und an deren Reifung beteiligt ist. Untersuchungen mit blockierenden Antikorper, sowie mit Mäusen mit einer targetierten Deletion von CD44v6/v7 oder CD44v7 belegten, dass die CD44 Standardform maßgeblich am Einwandern hämatopoetischer Progenitorzellen in das Knochenmark und den Thymus beteiligt ist. Das Einwandern in das Knochenmark wird darüber hinaus durch CD44v7 auf Stromazellen begünstigt. Hingegen ist die Wanderung in den Thymus CD44v6/CD44v7 unabhängig. Allerdings verstärkt CD44v6 die Expansion und Apoptoseresistenz von insbesondere frühen, sogenannten doppel-negativen Thmyozyten. Die gesteigerte Apoptoseresistenz geht mit einer Aktivierung von Akt einher. Für die CD44v6-induzierte Proliferation doppel-negativer Thymozyten ist die Aktivierung der MAPK Signalkaskade verantwortlich. Dies gilt nicht für Thymozyten in einem späteren Reifestadium, in dem CD44 nur noch als akzessorisches Molekül die Aktivierung über den T-Zell-Rezeptor unterstützt. Diese Befunde belegen, dass CD44 nicht nur das Einwandern von Progenitor-T-Zellen in den Thymus fördert, sondern darüber hinaus CD44v6 die frühen Stadien der Thymozytenreifung beschleunigt und somit eine wesentliche Rolle bei der Rekonstitution und Toleranzinduktion nach allogener Knochenmarkrekonstitution einnimmt

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1331-4) contains supplementary material, which is available to authorized users

Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these \u27hotspot\u27 sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer

Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these 'hotspot' sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.This is a manucript of an article published as Shukla, Sachet A., Michael S. Rooney, Mohini Rajasagi, Grace Tiao, Philip M. Dixon, Michael S. Lawrence, Jonathan Stevens et al. "Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes." Nature biotechnology 33, no. 11 (2015): 1152. doi: 10.1038/nbt.3344. Posted with permission.</p

Putative Biomarkers of Clinical Benefit With Pembrolizumab in Advanced Urothelial Cancer: Results from the KEYNOTE-045 and KEYNOTE-052 Landmark Trials

Purpose: In an exploratory analysis, we investigated the association between programmed death ligand 1 (PD-L1), tumor mutational burden (TMB), T-cell–inflamed gene expression profile (TcellinfGEP), and stromal signature with outcomes of pembrolizumab in urothelial carcinoma (UC). Patients and Methods: Patients with advanced UC received first-line pembrolizumab 200 mg every 3 weeks in the single-arm phase II KEYNOTE-052 trial (NCT02335424) and salvage pembrolizumab 200 mg every 3 weeks or chemotherapy (paclitaxel/docetaxel/vinflunine) in the randomized phase III KEYNOTE-045 trial (NCT02256436). The association of each biomarker (continuous variable) with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) was evaluated using logistic regression (ORR) and Cox PH (PFS, OS), adjusted for ECOG PS; nominal P values were calculated without multiplicity adjustment (one-sided, pembrolizumab; two-sided, chemotherapy). Significance was prespecified at a ¼ 0.05. Results: In KEYNOTE-052, PD-L1, TMB, and TcellinfGEP were significantly associated with improved outcomes; stromal signature was significantly associated with worse outcomes. In KEYNOTE-045, although findings for TMB and TcellinfGEP with pembrolizumab were consistent with those of KEYNOTE-052, PD-L1 was not significantly associated with improved outcomes, nor was stromal signature associated with worse outcomes with pembrolizumab; chemotherapy was not associated with outcomes in a consistent manner for any of the biomarkers. Hazard ratio (HR) estimates at prespecified cutoffs showed an advantage for pembrolizumab versus chemotherapy regardless of PD-L1 or TMB, with a trend toward lower HRs in the combined positive score ≥10 and the TMB ≥175 mutation/exome subgroup. For TcellinfGEP, PFS and OS HRs were lower in the TcellinfGEP-nonlow subgroup regardless of treatment. Conclusions: Multiple biomarkers characterizing the tumor microenvironment may help predict response to pembrolizumab monotherapy in UC, and potential clinical utility of these biomarkers may be context-dependent