Fig 1.
Scheme of the experimental protocol and numbers of live exhausted CTL generated by in vitro exhaustion.
(A) Scheme of the experimental set up. IR: Inhibitory receptors, TF: Transcription factors, IC: Intracellular (B) Purified CD8+ T cells (0.5x106 cells per well) were cultured either unstimulated, stimulated once with peptide and repeat peptide stimulated. Live cells were counted on day 5. Pooled data showing absolute cell numbers (left) and fold expansion (right). Data are from n = 13 and 10 independent experiments.
Fig 2.
Repeat peptide stimulated CD8+ T cells show reduced cytokine production capacity and lose polyfunctionality.
Purified OT-I CD8+ T cells were cultured either without peptide (no peptide), stimulated one time for 2 days with OVA peptide (single peptide) or stimulated with OVA peptide daily (repeat peptide). On day 5 cells were harvested and re-stimulated with OVA peptide. (A) Representative flow cytometry plots showing frequency of cytokine producing live CD8+ T cells after re-stimulation. (B) Pooled data showing the frequency of cytokine producing cells after re-stimulating with OVA peptide. (C) SPICE figures depicting the frequency of cells producing one, two or three cytokines in different combinations. Each symbol represents one animal (n = 11–12), 9 independent experiments performed. Line depicts mean ± SE. Between the groups, Student’s t test with Welch’s correction was performed except for % IFN-γ+ (ANOVA with Tukey’s post hoc test). *<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 3.
Repeat peptide stimulated CD8+ T cells have decreased cytotoxic function.
On day 5, cells were harvested and re-stimulated with OVA peptide. (A) Median fluorescence intensity (MFI) of the degranulation marker, CD107a, shown (left panel). Fold change of CD107a MFI induced by peptide re-stimulation depicted on the right panel. (B) MFI of Granzyme B (GzmB) depicted for the different culture conditions. Each symbol represents one animal (n = 11–12), 9 independent experiments performed. (C) No peptide, single peptide and repeat peptide stimulated cells were co-culture with target cells (OVA-pulsed AE-17 cells) at different ratios. Percentage of specific killing is depicted. One of five independent experiments shown. Line depicts mean ± SE. Between the groups, Student’s t test with Welch’s correction was performed except for CD107a MFI (Wilcoxon signed rank test). *<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 4.
Repeat peptide stimulated cells express multiple inhibitory receptors.
The expression of the inhibitor receptors PD-1, CD244, CD160, Lag3 were determined by flow cytometry on day 5 of in vitro culture of OT-I CD8+ T cells. Cells were cultured either without peptide (no peptide), one time OVA peptide stimulation (single) or daily peptide stimulations (repeat peptide). (A) Representative flow cytometry plots depicting the frequency of live CD8+ T cells expressing inhibitory receptors. (B) Pooled data of frequency of CTL expressing individual inhibitory receptors within the different culture conditions. (C) SPICE figures depicting the frequency of CD8+ T cells expressing one, two, three or four inhibitor receptors (PD-1, CD244, CD160, Lag3) simultaneously. Each symbol representative one animal (n = 7–11), with 7–9 independent experiments performed. Line depicts mean ± SE. Between the groups, Wilcoxon signed rank test was performed with exception of % Tim-3+ and % CD160+ for which a Student’s t test with Welch’s correction was used. *<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 5.
Repeat peptide stimulation induces a distinct pattern of transcription factor expression.
Transcription factors (TF) were analyzed in OT-I CD8+ T cells on day 5 of culture. Cells either unstimulated (no peptide), stimulated one time (single peptide) or daily (repeat peptide) shown. (A) Representative flow cytometry plots of the frequency of TF-expressing live CD8+ T cells shown for the differently treated cultures. (B) Pooled data depicting the MFI of TCF1 and TOX in live CD8+ T cells. (C) Pooled data showing the frequency of EOMES and T-bet expressing live CD8+ T cells. Each symbol represents one animal (n = 9–12), with n = 7–9 independent experiments performed. Line depicts mean ± SE. ANOVA with Tukey’s post hoc test was performed on TCF1 MFI, Student’s t test with Welch’s correction was used for TOX MFI and Wilcoxon signed rank test for data in (C). *<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 6.
Repeat peptide stimulation decreases in vivo CTL expansion capacity.
OT-I CD8+ T cells were cultured without peptide stimulation (no peptide), one-time stimulation (single peptide) or daily stimulation (repeat peptide) and sorted on day 5. Live CD8+ T cells were adoptively transferred into wild type mice which were then infected with the OVA(257–264)-expressing influenza virus WSN-OVA. Freshly isolated OT-I CD8+ T cells from a naïve mouse were also transferred (naïve OT-I). Lungs were harvested on day 10 post infection. (A) Frequency of donor CD45.1+ OT-I CD8+ T cells within total lung CD8+ T cells shown. (B) Absolute number of lung donor CD45.1+ OT-I CD8+ T cells depicted. (C) Frequency of endogenous CD45.2+ OVA-specific CD8+ T cells within the total lung CD8+ T cells shown. (D) Absolute number of endogenous CD45.2+ OVA-specific CD8+ T cells shown. Each symbol represents one animal (n = 8–9) from n = 3 independent experiments. Line depicts mean ± SE. To determine significant differences between the different animal groups, a Mann-Whitney U test was used except for data in (A) (ANOVA with Tukey’s post hoc test). ***P<0.001, ****P<0.0001.
Fig 7.
Repeat peptide stimulated cells have distinct transcriptional profile.
RNAseq analysis was performed on live CD8+ T cells sorted from 5 day culture of OT-I CD8+ T cells. Cells were either without peptide (no peptide), one-time stimulation (single peptide) or daily peptide stimulations (repeat peptide). (A) PCA plot of RNA-seq data show that cells from identical culture conditions cluster together and away from each other. (B) Heatmap shown for the top 50 differentially expressed genes within CTL from the different culture conditions. (C) Heatmaps for the individual genes clustered based on function presented. (D) Repeat peptide stimulated CTL transcriptomes were enriched in gene signatures of LCMV-Cl13 exhausted CTL. Differentially expressed genes in repeat peptide stimulated CTL were analyzed by GSEA for their enrichment in gene sets found in exhausted CTL from LCMV-Cl13 infected mice. Enrichment in upregulated (left) and downregulated (right) genes shown. (E) Ingenuity pathway analysis was performed on the differentially expressed genes in repeat peptide stimulated cells (compared to single peptide). Significantly upregulated pathways (orange) and downregulated pathways (blue) in repeat peptide stimulated cells are depicted. P values (hypergeometric test) are presented as -log10.
Fig 8.
Genome wide DNA methylation changes during T cell stimulation reveal Tcf7 promotor methylation.
Sorted live CD8+ T cells were processed and whole genome methylated DNA sequencing (MeD-seq) was performed. (A) Hierarchical clustering on DMRs (differentially methylated regions) found between the three different peptide exposure conditions are shown. (B) Boxplot of DNA methylation read count data in a 2kb window surrounding the TSS of mentioned genes are shown. The samples were collected from three independent experiments. (C) Representative histogram of TCF1 expression (left) and pooled data showing TCF1 MFI on live CD8 cells (right) in the presence or absence of 20μM DNMT inhibitor during the last 3 days of repeat peptide stimulated cells shown. Data are from n = 6 performed in 3 independent experiments, paired Student’s t test performed. **P<0.01.