Tox reinforces the phenotype and longevity of dysfunctional T cell populations during chronic viral infection [WT vs KO +/- Tim3]

Chronic CD8 T-cell stimulation in persisting infections or tumors can induce a stable gene expression program, known as T-cell dysfunction or exhaustion, that limits the cell's effector functions and anti-viral and anti-tumor immunity. Thus far, the underlaying molecular mechanisms that induce and stabilize this phenotype are vaguely understood. We report here that establishing this program requires the thymocyte selection-associated high mobility group-box protein (Tox). Genetic disruption of Tox augments effector function, decreases the expression of PD-1, and significantly enhances immunopathology. These changes are linked to a failure in fixing the dysfunctional phenotype in the critical Tcf1+ progenitor population and to impaired epigenetic programing. Surprisingly, the gains in effector function co-incide with declining numbers of Tcf1+ cells and result ultimately in reduced total numbers of pathogen-specific T-cells. Thus, we establish Tox as a critical factor for the development of T-cell dysfunction and establish a clear link between CD8 T-cell intrinsic suppression of effector function and protection against immune-pathology. Overall design: 2*10^3 wt or Tox? P14 T-cells were transferred into C57BL6 host, infected with 5x10^6 PFU LCMV-c13 and collected on day 8. Tim3+ and Tim3- P14 T-cells were isolated and FACS-sorted, then lysed for the following RNA extraction using the Agencourt RNAdvance Cell v2 kit (A47942, Beckman Coulter). Samples with RIN>8 were used for cDNA synthesis and library preparation. 1 ng of total RNA was used for cDNA synthesis and PCR using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (634891, Takara/Clontech). 150 pg of the amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024, Illumina). The fragmented libraries were amplified and the samples purified with (0.6x) Agencourt AMPure XP beads. The quality of the library was checked with the use of Agilent High Sensitivity DNA Kit (5067-4626, Agilent). Library quantification was performed with the use of KAPA SYBR FAST qPCR Master Mix (KK4600, Kapa Biosystems). The sequencing of the samples was performed with the Illumina HiSeq 2500 system at rapid run, 100 base pairs single-end read, dual-indexed sequencing resulting in 20 million reads per sample.