Polyomavirus

The EO771 cell line was derived from a spontaneous mammary tumour in a C57BL/6 mouse (Casey et al., 1951 (link)) and was stored in liquid nitrogen vapour phase. Early-passage parental EO771 cells were transduced with the pMSCV (murine stem cell virus) retroviral vector expressing the mCherry fluorescent protein (Denoyer et al., 2011 (link)). The lungs from a mouse orthotopically implanted with EO771_mCherry cells in our laboratory were excised and sorted by flow cytometry for mCherry-positive cells that were expanded in culture. This sequence of orthotopic growth in vivo followed by recovery of mCherry-positive cells from the lung was repeated. Upon the second round of mammary fat-pad injections of these mCherry-positive cells, visible lung nodules were detected. One of these nodules was designated Lung Metastasis nodule B and, after being returned to culture, became the EO771.LMB cell line. 67NR and 4T1 cell lines were derived from a subpopulation of a single mammary tumour that arose in a BALB/c/C3H mouse (Aslakson and Miller, 1992 (link)), with the 4T1.2 cell line being derived from a single-cell clone of the 4T1 population (Lelekakis et al., 1999 (link)). EMT6.5 (Ellis et al., 2000 (link)) is a single-cell clone derived from the EMT6 mammary tumour (Rockwell and Kallman, 1973 (link)). NMuMG immortal murine mammary epithelial cells were obtained from ATCC. The Pik3ca-mutant murine mammary tumour line MH248 was a kind gift from Dr Wayne Phillips (Tikoo et al., 2012 (link)). The murine mammary tumour line AT3, derived from polyoma-middle T antigen transgenic mice (Stewart and Abrams, 2007 (link)), was a kind gift from Dr Trina Stewart (Griffith University, Queensland, Australia). 67NR and 4T1.2 mammary adenocarcinoma cells were maintained in Eagle’s minimum essential medium (alpha modification) supplemented with 5% (v/v) fetal bovine serum (FBS) (SAFC Biosciences, Brooklyn, Victoria, Australia) and 1% (v/v) penicillin-streptomycin, whereas EO771, EO771.LMB, EMT6.5, AT3, MH248 and NMuMG cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing HEPES (20 mM) supplemented with 10% (v/v) FBS, penicillin (100 IU/ml) and streptomycin (100 μg/ml). All cells were cultured at 37°C in 5% CO₂ (v/v) in air and were maintained in culture for a maximum of 4-5 weeks.

To generate the Byrne et al data set, wild-type C57BL/6 mice were purchased from The Jackson Laboratory and housed at the University of Pennsylvania. Animal protocols were reviewed and approved by the Institute of Animal Care and Use Committee at the University of Pennsylvania. Mice were euthanized in a CO2 chamber using a flow meter to ensure CO2 was displaced at a rate of 30–70% of the chamber volume per minute and maintained for at least 1 minute after the loss of righting reflex is observed. Euthanasia was confirmed by bilateral thoracotomy. Animal handling information regarding tumor injections, drug prep and injection are included in Byrne et al (18 (link)).
To generate transgenic TCR data sets, spleen tissue from P14 and OT1 TCR transgenic mice (Nolz lab) were obtained. The spleen tissue was homogenized, treated with RBC lysis buffer and the resulting single-cell suspension was pelleted. Genomic DNA was extracted from the cell pellet using DNeasy blood and tissue kit (Qiagen).
To generate all the other data using animals, wild-type C57BL/6 mice were purchased from The Jackson Laboratory and maintained within the UCSF or OHSU laboratory for animal care barrier facility according to IACUC procedures. To generate the ST dilution series data set with mammary tumor and the Mesothelioma data set, we used mammary tumors from mouse mammary tumor virus (MMTV)-Polyomavirus middle T (PyMT) transgenic FVB/N mice and 40L orthotopic mesothelioma tumors respectively. Mammary tumors were resected from day 95 mice. For 40L orthotopic mesothelioma tumors, 2 x 10⁶ cancer cells were injected i.p. into wild-type male C57BL/6 that were 6–12 weeks of age. For both tumor models, all mice were euthanized at a pre-defined end-stage for tissue harvest by cardiac puncture followed by cervical dislocation. These mice were cardiac perfused, under anesthesia using 1 –5% isoflurane, with 20 mL solution of heparin in PBS to clear tissues of residual blood followed by tissue harvest for further analysis including TCR sequencing. Tumor tissue was excised and flash frozen in liquid nitrogen and stored at −80 degree Celsius until further use for extracting genomic DNA for TCR sequencing. Murine SCC tumors were obtained from a previously published study, Medler et al (19 (link)).

This study was approved by the University of Pittsburgh IRB (protocol 10110393). STA kidney specimens and biopsies diagnosed as TCMR or BKPyVN were selected from weekly clinical conferences conducted immediately prior to commencement of the study. Diabetes mellitus, hypertension, and glomerulonephritis were the three most common causes of end-stage kidney disease in these subjects. All patients received Thymoglobulin induction followed by dual maintenance immunosuppressive therapy consisting of mycophenolate mofetil and tacrolimus. Corticosteroids were tapered over the first 7 days and then discontinued. Histologic diagnoses were based on the Banff classification for kidney allograft pathology (17 (link)). Diagnostically relevant Banff scores for the TCMR patients were g0, v0, i2, ptc0, cg0, ci1, ct1 for all biopsies. The t -score was 2 in all biopsies, except for 1 biopsy in which it was t3. The core needle biopsy specimens (18 gauge) were fixed in formalin immediately and paraffin embedded within 24 h.
The patients (eight males, seven females) in the discovery cohort ranged in age from 32 to 84 years with mean values of 60.8, 56.2, and 51.6 in the STA, BKPyVN, and TCMR groups, respectively. Biopsies had been performed 23-526 days post-transplant (mean 263) and showed renal cortex with mild interstitial fibrosis and tubular atrophy. For the BKPyVN biopsies, the concentration of viral loads ranged from 2.38E+08 to 6.67E+10 copies per mL in the urine and 8.11E+03 to 3.85E+05 copies per mL in the plasma. All biopsies showed polyomavirus antigens on immunohistochemistry.
The patients (two males, seven females) in validation cohort ranged in age from 27 to 73 years with mean values of 52.7. Biopsies had been performed 74-106 days post-transplant (mean 88).

Age- and sex-matched non-obese diabetic (NOD) mice born and bred within our animal facility were used for all experiments. Mice were housed in OptiMouse cages with corn cob bedding and up to 5 cage-mates. Mice had ad libitum access to chow and reverse osmosis chlorinated (2-3ppm)-purified water. Housing rooms were kept on a 14.5/9.5-hour light/dark cycle with temperature maintained at 22-25°C and 50-70% humidity. Sentinel mice were placed on dirty bedding and nesting material in experimental rooms and subsequently tested on a quarterly basis for presence of parasites (pinworms and fur/follicular mites, Pneumocystis spp. (carinii, murina)), bacteria (Mycoplasma pulmonis), and viruses (RADIL Comprehensive Panel) including Mouse Hepatitis Virus (MHV), Mouse Minute Virus (MMV), Mouse Parvovirus (NS1 – Generic Parvovirus & MPV 1-5), Theiler’s Murine Encephalitis Virus (TMEV), Epizootic Diarrhea of Infant Mice (EDIM), Sendai Virus, Pneumonia Virus of Mice (PVM), Reo3 virus (REO3), Lymphocytic Choriomeningitis Virus (LCMV), Ectromelia virus, Murine Adenovirus I/II (MAV1/MAV11), and Polyomavirus. All experiments were performed in compliance with protocols approved by the Animal Care Committee of The University of British Columbia.
Virus stocks for coxsackievirus B4 Edwards strain 2 (CVB4) were prepared as previously described (15 (link)). Normoglycemic NOD mice at 11-12 weeks old were injected intraperitoneally with either 400 plaque-forming units (pfu) CVB4 or DMEM vehicle. Non-fasting blood glucose was monitored using a OneTouch LifeScan monitor. Mice were considered diabetic with two consecutive readings above 16.2 mg/dL separated by 24 hours.