Based on these findings, we propose that the presence of infectious MLVs in mouse cancer models should be considered as biological contamination. expression of retroviral nucleic acids and proteins in mouse cancers. Infectious ERV-derived retroviruses produced by mouse cancer cells could directly infect tumor-infiltrating host immune cells and fundamentally modified the hosts immune defenses to cancer, as well as the outcome of immunotherapy. Therefore, infectious retroviruses, variably arising in mouse cancer models, VU0134992 but not in human cancer, have the potential to confound many immunological studies and should be considered as a variable, if not altogether avoided. more frequently than previously estimated. Although ERV expression is upregulated in human cancer and certain human cancer lines produce retroviral particles, no evidence for replication of an ERV-derived retrovirus in humans has yet been reported (12,13). Thus, ERV-derived infectious retroviruses are restricted to mouse models, where diseases may be unwittingly studied in the setting of a retroviral infection. Here, we examined how frequently ERV infectivity is restored in mouse cancers and found an extensive, but not universal presence, of infectious retroviruses in transplantable, as well as genetic mouse cancer models. We found that ERV-derived infectious retroviruses produced by cancer cells can infect tumor-infiltrating immune cells and fundamentally alter disease outcome. Thus, potential production of infectious retroviruses in mouse cancer models is a variable that should be considered in outcome interpretation. Materials and Methods Mice Inbred C57BL/6J (B6), CBA/J, and CD45.1+ congenic B6 (B6.SJL-(the gene encoding Blimp1), induced in B cells at early stages of the germinal center reaction by expression of a C1-Cre transgene, have been previously described (15). Mice bearing the conditional allele of or the C1-Cre transgene were provided by Drs. Alexander Tarakhovsky (The Rockefeller University, New York, USA) and Klaus Rajewsky (Max Delbrck Center for Molecular Medicine, Berlin Germany), respectively (15) and were intercrossed in the Calado lab. Eight to twelve-week old male and female gender-matched recipient mice were used for all experiments. All animal experiments were approved by the ethical committee of the Francis Crick Institute and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986 (ASPA). Retroviral vectors Open reading frames encoding either the wild-type (WT) envelope glycoprotein (WT (encoding Blimp1) in class-switching B cells (15) and were passaged once in a WT B6 host. A1 and G7 cells were isolated from progenitor B-cell lymphomas that developed in IL7-overexpressing mice, as previously described VU0134992 (19), and were kindly provided by Dr. Amanda Fisher. Cells were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 5%-10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA USA), 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (0.1 mg/mL). JAWSII cells were grown in the same medium additionally supplemented with recombinant GM-CSF (20 ng/mL; Peprotech Ltd, Rocky Hill, NJ, USA). HCmel cell lines were grown Rabbit Polyclonal to MARCH2 in Minimum Essential Media (MEM) supplemented with non-essential amino acids (Gibco). All cell lines (Supplementary Table S1) were verified as murine and were mycoplasma free. Species identification was carried out by the Cell Services facility at the Francis Crick Institute, using previously established multiplex PCR assays (20,21). Transplantable cancer cell lines were passaged for a maximum 18-24 times or kept for a maximum of 8 weeks in culture. MCA-38 and MCA-205 cells producing GFP-encoding transducing retroviral particles were generated by transduction of the replication-defective XG7 retroviral vector, which encompasses a neomycin resistance gene (fibroblast cells (M. dunni cells; CRL-2017), kindly provided by the Stoye lab, were used for the viral infectivity assays as previously described (10). Infections and transductions were carried out by adding serial dilutions of the viral stocks to target cells in the presence of polybrene (4 g/mL). EL4 sublines transduced with WT open reading frame of was sequenced from virion genomes as previously described (10). Briefly, RNA was extracted from culture supernatant of the EL4.BU, MCA-205 MCA-38 and HCmel12 tumor cell lines using the QIAamp viral RNA mini Kit (Qiagen, Hilden, Germany) and then reverse-transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher). The first half of retroviral genomes was amplified using Ranger DNA polymerase (Bioline) and the following primers: Forward 5-GCGCCAGTCCTCCGATAGACT-3; Reverse 5-CCGGGAGAGGGAGTAAGGTGGC-3. Amplicons were purified with the QIAquick PCR purification kit (Qiagen) and then subjected to Sanger sequencing at the Francis Crick Institute using the same primers. Sequence analyses, comparisons, and alignments were performed with Vector NTI v11.5 (Invitrogen). Tumor challenge and immunotherapy Tumor challenge was initiated by subcutaneous inoculation of tumor cell suspensions into the right flank of recipient mice. One million XG7-transduced MCA-38 or MCA-205 cells were inoculated into (CBAB6) F1 hosts, which were monitored three times per week for up to 21 VU0134992 days. Two million EL4 or EL4.BU cells were inoculated into CD45.2+ or CD45.1+ congenic B6 or.
- kinase assays showed the fact that N-terminal p57 fragment was phosphorylated towards the same level because the full-length protein whereas the C-term fragment had not been phosphorylated in any way (Body 1C)
- As the CD-associated variants of NOD2 are localized possibly in or close to the LRRs,13 one consequence of these mutations may be the stabilization of the inactive conformation, producing a NOD2 even more refractory to activation by MDP