Investigation of product-derived lymphoma following infusion of piggyBac-modified CD19 chimeric antigen receptor T cells.

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381.

The role of neuraminidase 1 and 2 in glycoprotein Ibα-mediated integrin αIIbß3 activation.

Upon vascular injury, platelets adhere to von Willebrand Factor (VWF) via glycoprotein Ibα (GPIbα). GPIbα contains many glycans, capped by sialic acid. Sialic acid cleavage (desialylation) triggers clearance of platelets. Neuraminidases (NEU) are responsible for desialylation and so far, NEU1-4 have been identified. However, the role of NEU in healthy platelets is currently unknown. Aim of the study was to study the role of NEU1 and NEU2 in platelet signalling. Membrane association of platelet attached glycans, NEU1 and NEU2 was measured following activation with agonists using flow cytometry. Adhesion on fibrinogen, aggregation and fibrinogen-binding were assessed with/without the NEU-inhibitor, 2-deoxy-2-3-dide-hydro-N-acetylneuraminic acid. Cellular localisation of NEU1 and NEU2 was examined by fluorescence microscopy. Desialylation occurred following GPIbα-clustering by VWF. Basal levels of membrane NEU1 were low; glycoprotein Ibα-clustering induced a four-fold increase (n=3, P<0.05). Inhibition of αIIbß3-integrin prevented the increase in NEU1 membrane-association by ~60%. Membrane associated NEU2 increased two-fold (n=3, P<0.05) upon VWF-binding, while inhibition/removal of GPIbα reduced the majority of membrane associated NEU1 and NEU2 (n=3, P<0.05). High shear and addition of fibrinogen increased membrane NEU1 and NEU2. NEU-inhibitior prevented VWF-induced αIIbß3-integrin activation by 50% (n=3, P<0.05), however, promoted VWF-mediated agglutination, indicating a negative feedback mechanism for NEU activity. NEU1 or NEU2 were partially co-localised with mitochondria and α-granules respectively. Neither NEU1 nor NEU2 co-localised with lysosomal-associated membrane protein 1. These findings demonstrate a previously unrecognised role for NEU1 and NEU2 in GPIbα-mediated and αIIbß3-integrin signalling.

Early expansion of CD38+ICOS+ GC Tfh in draining lymph nodes during influenza vaccination immune response.

T follicular helper (Tfh) cells provide critical help to B cells during the germinal center (GC) reaction to facilitate generation of protective humoral immunity. Accessing the human lymph node (LN) to study the commitment of CD4 T cells to GC Tfh cell differentiation during in vivo vaccine responses is difficult. We used ultrasound guided fine needle biopsy to monitor recall responses in axillary LNs to seasonal influenza vaccination in healthy volunteers. Specific expansion of GC cell subsets occurred exclusively within draining LNs five days postvaccination. Draining LN GC Tfh and precursor-Tfh cells express higher levels of CD38, ICOS, and Ki67, indicating they were significantly more activated, motile, and proliferating, compared to contralateral LN cells. These observations provide insight into the early expansion phase of the human Tfh lineage within LNs during a vaccine induced memory response and highlights early LN immune responses may not be reflected in the periphery.