The PD-1/PD-L1 Pathway Affects the Expansion and Function of Cytotoxic CD8(+) T Cells During an Acute Retroviral Infection

David, Paul; Megger, Dominik A.; Kaiser, Tamara; Werner, Tanja; Liu, Jia; Chen, Lieping; Sitek, Barbara; Dittmer, Ulf; Zelinskyy, Gennadiy

The in vivo CTL assay described by Barber et al. (23) was modified to measure cytotoxicity in FV-infected mice ( Figure 4A ). Splenocytes from naïve CD45.1 mice were loaded with 1-5 µM D b GagL peptide (18, 22) . The peptide loaded cells were stained with 200 nM of CFSE (Molecular Probes). As a reference, splenocytes isolated from naïve CD45.1 mice were used. Splenocytes (1 × 10 7 cells of each population) were transferred i.v. into naïve or 10 day FV-infected mice. One hour after adoptive transfer, the spleens and bone marrows from recipient mice were harvested and cell suspensions were prepared. Cell suspensions were stained with anti CD45.1 antibodies and measured by LSR II. Donor cells were distinguished from recipient cells and from one another based on CFSE positivity and on the expression of CD45.1. The percentage of killing was calculated as follows: 100 -([(% peptide pulsed in infected/% unpulsed in infected)/(% peptide pulsed in uninfected/% unpulsed in uninfected)] × 100).