T cells specific for a TAP-independent self-peptide remain naïve in tumor-bearing mice and are fully exploitable for therapy

Figures & data

Figure 1. High antigen and MHC-I levels are required for TEIPP T cell priming. CFSE labelled TCR-transgenic TEIPP T cells were transferred to recipient mice and T cell activation and proliferation were measured after challenge with indicated irradiated tumor cells. (A) Blood samples were analysed for the presence and activation status of T cells by flow cytometry five days after the second injection of MHC-I^low RMA-S cell lines. ‘Trh4’ indicates cells transfected with full length cDNA of the cognate antigen and ‘B7’ indicated RMA-S cells transfected with the mouse CD80 gene. IFNγ production by TEIPP T cells was measured in blood after the second injection, upon overnight stimulation with short Trh4 peptide. (B) Blood samples from mice challenged with MHC-I^high RMA cell lines. Means and SEM are plotted from one (RMA-S.B7, RMA-S.Trh4.B7), two (RMA and RMA-S.Trh4) or five experiments (RMA.Trh4). Student T-test compared to T cells only: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. TEIPP T cell activation is mediated by direct priming on tumor cells. Mice received naïve LnB5 tg T cells and were injected with irradiated tumor cells. (A) Analysis of phenotype of T cells in blood of mice injected with allogeneic P815 or P815.Trh4 cells, five days after the second injection. IFNγ production by TEIPP T cells was measured by overnight stimulation with short Trh4 peptide. Data pooled from two independent experiments, with 4 mice per group, shown as mean and SEM. (B) Expression of H2-D^b and H2-K^b molecules on RMA.Trh4 cells generated by Crispr/CAS9 technology: wildtype (wt), D^b-/− or K^b-/− cells. Plots representative for at least two experiments. (C) IFNγ release by the LnB5 T cell clone upon in vitro co-culture with the decreasing amounts of cells from the RMA.Trh4 cell panel. Data shown as mean and SD, from one of two experiments with comparable results. (D) Naïve LnB5 tg T cells were transferred to recipient mice that were then injected twice with irradiated RMA.Trh4, RMA.Trh4 D^b-/− or RMA.Trh4 K^b-/− cells. LnB5 T cell activation was measured in blood after the second injection. IFNγ production by TEIPP T cells in blood, upon overnight stimulation with short Trh4 peptide. Data pooled from two independent experiments, with 4 mice per group, shown as mean and SEM. Student T-test: n.s. = not significant, **P < 0.01, ***P < 0.001.

Figure 3. Tumor infiltration by tumor-specific TCR-transgenic T cells. LnB5 transgenic T cells (CD90.1⁺) were labelled with CFSE and transferred to (CD90.1⁻) mice bearing palpable RMA-S (A), RMA-S.Trh4 (D) or RMA.S.B7 (E) tumors. (B) Melanoma-specific pmel transgenic T cells (CD90.1⁺) were transferred to B16F10-tumor bearing mice. (C) OVA-specific OT-I transgenic T cells (CD90.1⁺) were transferred to B16F10.OVA-tumor bearing mice. After seven days, frequency and phenotype of transferred T cells was analysed in tumor, tumor-draining lymphnode (dLN) and non-draining lymphnodes (ndLN). Representative dot plots are shown, gated on live cells (upper row), or gated on CD90.1⁺ T cells (lower row). Data is pooled from 2 or 3 independent experiments with 3 mice per group. Student T-test: *P<0.05, **P < 0.01, ***P < 0.001, n.s.: non-significant.

Figure 4. TEIPP T cell activation promotes tumor infiltration and protection against tumor outgrowth. (A and B) Naïve mice received LnB5 transgenic T cells and injection with irradiated RMA.Trh4 cells, and were inoculated with a subcutaneous RMA-S tumor. At day 20 after T cell injection, tumor, tumor-draining lymphnode (dLN) and non-draining lymphnodes (ndLN) were analysed. Data pooled from two independent experiments with four mice per group. Student t-test: **P < 0.01. (C) Mice received T cells and were immunized twice with irradiated RMA.Trh4 cells and one week after the second injection challenged with a RMA-S tumor. (D) Individual tumor outgrowth curves and (E) Kaplan-Meier survival plot. Pooled means and SEM from three independent experiments are shown. Log-rank test: *P < 0.05, ***P < 0.001.