Armenian hamster igg isotype

Spleens were harvested and single cell suspensions created by filtration through 70 μm mesh in RPMI1640 culture media containing 10% fetal bovine serum and antibiotics. Splenocytes were pelleted and erythrocytes lysed using 0.84% NH₄Cl (J.T. Baker) in H₂O. For T cell repertoire profiling, splenocytes were labeled with anti-CD45, anti-CD3, and anti-CD8 antibodies (eBioscience) and MHC class I K^b tetramers provided by the NIH Tetramer Core Facility for identification of HSV-1 specific CD8⁺ T cells. Tetramer-labeled cells were analyzed using a MacsQuant flow cytometer (Miltenyi Biotec) as previously described.^{23 (link)} For adoptive transfers into Ifnar1^−/− mice, bulk preparations of CD3⁺ splenocytes were obtained by immunomagnetic isolation using anti-CD3 microbeads (Miltenyi Biotec) according to the manufacturer’s directions. Alternatively, splenocytes were labeled with anti-CD4 or anti-CD8 antibodies (eBioscience) and sorted using an S3e cell sorter (Biorad) for adoptive transfers into TCRα^−/− mice. Adoptive transfer of isolated cells was mediated by intravenous retroorbital injection. In some experiments, TCRα^−/− mice were injected i.p. with 250 μg anti-mouse CD154 (MR1 clone) or Armenian hamster IgG isotype (both from BioXcell) at days 0, 3, and 6 p.i. to evaluate CD40-dependent immune activation following adoptive transfer of CD4 T cells.

Mice were injected with MC38 colon adenocarcinoma (5×10⁵ cells s.c.), B16-F10 or B16-gp100 melanoma (1.25×10⁵ cells i.d.). Tumors were measured every 3 days with digital calipers and tumor volume calculated blinded and randomized. Mice were removed from study when tumor growth reached a mean diameter of 1.5cm or when necrosis was observed. A cohort of mice received a secondary MC38 injection (2.5×10⁵ cells subcutaneously) on d42, following primary MC38 tumor injection and resection at d12 or sham control animals.
Therapeutic anti-PD1 was administered when tumors were palpable (6 days) and mice received 200μg anti-PD1 (clone G4) or Armenian hamster IgG isotype (Bioxcell) i.p. on day 6, 9 and 12. Tumors, draining and non-tumor draining lymph nodes were collected for analysis on the indicated time-point. TILs were prepared with enzymatic (collagenase IV (Worthington Biochemical) and dispase (StemCell Technologies); both 1mg/ml) and mechanical disruption.
For Amph-vax experiments, mice were injected with B16-gp100 or B16-F10 melanoma (1.25×10⁵ cells i.d.). On days 4 and 11, mice were immunized with 20μg Amph-gp100, or Amph-E7 as a control, s.c. at the base of the tail. Mice also received anti-PD1 (clone G4) or Armenian hamster IgG as an isotype control as the therapeutic regimen detailed above. Tumor area was measured with digital calipers over time.