Gp33 peptide

To activate naïve CD8⁺ T cells in
vitro, P14⁺ CD8⁺ T cells (with or without
Tcf7^GFP/+ reporter) were purified with the CD8⁺ T Cell
Isolation Kit (Miltenyi Biotec), labeled with one of the cell proliferation dyes
and activated with gp33 peptide (1µg/ml) (Anaspec) and recombinant IL-2
(100 IU) in the presence of congenic naïve splenocytes. All cells were
cultured in complete lymphocyte media (Iscove's DMEM, 10% FBS,
Pen-Strep, L-Glutamine, 55µM 2-mercaptoethanol). In some experiments,
pre-activated P14 CD8⁺ T cells of designated phenotypes were sorted
from infected mice or in vitro cultures and re-stimulated with or without gp33
peptides (1µg/ml) and in the presence of rIL-2 and congenic
naïve splenocytes. Pharmacological inhibitors of G1 phase (Mimosine
250µM, Sigma), S phase (Hydroxyurea 200µM, Sigma) or G2/M phase
(Nocodazole 1µg/ml, Sigma) and were added to the cells at the time of
re-stimulation where indicated.

Inguinal, axillary, brachial and mesenteric lymph nodes (LN) or spleens were isolated from CD45.1 OT-I or P14 LCMV mice. LN and spleens were meshed through 70μm filters and treated with ACK red blood cell lysis buffer. CD8+ T cells were purified using EasySep CD8 negative selection kits (Stemcell Technologies). 1×105 (for >14 day read-out) – 2×106 T cells (for day 4 read-out) were adoptively transferred through retro-orbital injection in 100μl PBS.
For the comparison of OT-I T cells and p14 LCMV T cells, mice received a 1:1 mix of both T cells in 100μl of PBS through retro-orbital injection. The following day mice were inoculated with a bolus of CFA containing gp33-peptide (50μg/mouse; Anaspec) and SL8/SIINFEKL peptide (50μg/mouse; Anaspec) subcutaneously, to sustain both T cell populations.