Hgp10025 33 peptide

Thy1.1 and Ly5.1 Pmel-1TCR-transgenic (Pmel) mice have been described previously(20 (link)). NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice and C57BL/6 (B6) mice were purchased from The Jackson Laboratory. Mice were housed in the National Institutes of Health (NIH) Clinical Research Center vivarium and maintained in compliance with the NIH Animal Care and Use Committee. Mice were excluded from analysis if less than 6 weeks old and not age- and gender-matched with experimental cohort. Mice were randomized to treatment group and investigators blinded when measuring outcomes of: tumor size, survival after adoptive transfer, and histopathological analysis. Splenocytes from Pmel mice were stimulated with hgp100_25-33 peptide (1μM) and 1000 IU/mL recombinant human interleukin-2 (rhIL-2; Novartis) in the presence or absence of 1μM AktI-1/2 (Akti; Calbiochem) and CD8⁺ T cells were harvested at day 5. Secondary stimulation was performed using peptide-pulsed irradiated B6 feeder cells. The human SK23 melanoma tumor line and B16F10 tumor line (B16) was obtained from the National Cancer Institute tumor repository and tested for mycoplasma contamination.

After 30 days expansion, TIL were enriched using Miltenyi magnetic column CD8+ separation (order no. 130-096-495) according to manufacturer’s instructions. Five replicates per treatment group (Akti vs. vehicle) were analyzed on multiple platforms including gas and liquid chromatography-mass spectrometry with electron ionization (see supplemental methods for additional detail). For murine analysis, splenocytes from Pmel mice were stimulated with hgp100_25-33 peptide (1μM) and 100 IU/mL recombinant human interleukin-2 (rhIL-2; Novartis) in the following treatment groups: 0 μM, 1 μM, and 2.5 μM AktI-1/2 (Akti; Calbiochem). Splenocytes were harvested on day 10 and CD8+ T cells were enriched using a MACS negative selection kit (Miltenyi Biotech).