Functional differences between low- and high-affinity CD8(+) T cells in the tumor environment

Rinke Bos, Kristi L Marquardt, Jocelyn Cheung, Linda A Sherman, Rinke Bos, Kristi L Marquardt, Jocelyn Cheung, Linda A Sherman

Abstract

Weak T-cell antigen receptor (TCR)-ligand interactions are sufficient to activate naïve CD8(+) T cells, but generally do not result in tumor eradication. How differences in TCR affinity affect the regulation of T-cell function in an immunosuppressive tumor environment has not been investigated. We have examined the functional differences of high- vs. low-affinity CD8(+) T cells and we observed that infiltration, accumulation, survival and cytotoxicity within the tumor are severely impacted by the strength of TCR-ligand interactions. In addition, high-affinity CD8(+) T cells were found to exhibit lower expression of inhibitory molecules including PD-1, LAG-3 and NKG2A, thus being less susceptible to suppressive mechanisms. Interferon γ and autocrine interleukin-2 were both found to influence the level of expression of these molecules. Interestingly, although high-affinity CD8(+) T cells were superior to low-affinity CD8(+) T cells in their ability to effect tumor eradication, they could be further improved by the presence of tumor specific CD4(+) T cells. These findings illustrate the importance of both TCR affinity and tumor-specific CD4 help in tumor immunotherapy.

Figures

https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g1.jpg
Figure 1. Antitumor efficacy of high and low-affinity CD8+ T cells. (A and B) 8–9 week old RIP-Tag2-HA mice were immunized with cognate peptide and polyI:C in IFA and Clone 1 (A) or Clone 4 cells (B) (2 × 105) were injected i.v. Glucose levels in the blood were measured at the indicated time points, and each line represents one mouse. Data are representative of two independent experiments.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g2.jpg
Figure 2. High-affinity CD8+ T cells are superior to low-affinity CD8+ T cells in the tumor milieu. 8–9 week old RIP-Tag2-HA mice were immunized with peptide and polyI:C in IFA and Clone 1 or Clone 4 cells (3 × 104) were injected i.v. (A–H) Pancreata and spleens were analyzed at day 7 by flow cytometry to assess percentage of CD8+Thy1.1+ cells, the percentage of cells exhibiting granzyme B, percentage of dividing cells and the expression level of Bim. Data are cumulative from 2 independent experiments with 3 mice per group. I-J, Pancreas derived CD8+Thy1.1+ cells were analyzed by qRT-PCR for perforin and Bcl-2 mRNA levels. Delta Ct values were compared using actin as the normalization control. Data are from 1 experiment with 3 independent samples per group. *p < 0.05, **p < 0.005, ***p < 0.0005.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g3.jpg
Figure 3. Expression of inhibitory molecules in the spleen and tumor microenvironment. 8–9 week old RIP-Tag2-HA mice were treated as described in Figure 2. Pancreata and spleens were analyzed at day 7 by flow cytometry to assess the expression of PD-1, LAG-3 and NKG2A on CD8+Thy1.1+ cells. Histograms are representative of 3 independent experiments with 3 mice per group. Isotype control = gray line, Clone 4 = black line, Clone 1 = dashed line. Mean MFI ± SD, PD-1: Clone 4 2428.0 ± 216.4, Clone 1 2784.0 ± 234.7. LAG-3: Clone 4 446.5 ± 98.2, Clone 1 664.0 ± 135.76. NKG2A: Clone 4 724.5 ± 151.0, Clone 1 1001.0 ± 186.9.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g4.jpg
Figure 4. Infiltration of high- vs. low-affinity CD8+ T cells. (A) Purified CD8+ Clone 1 or Clone 4 cells (3 × 105) were incubated with HA110–119 peptide pulsed splenocytes for 15 h. Cells were analyzed for the expression of integrins and lectins by flow cytometry. Data shown are representative of 2 independent experiments. (B–E) Clone 1 or Clone 4 cells were activated in vitro with HA110–119 peptide and after 6 d cells (5 × 106) were injected into InsHA (B and C) or RIP-Tag2-HA mice (14 weeks old, D,E). Pancreata, tumors and spleens were isolated 40 h after injection. In panels B and C, cumulative data are shown from 5 experiments with 15 mice total. Data shown in D and E are representative of 2 independent experiments with 3 mice per group. **p < 0.005, ***p < 0.0005.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g5.jpg
Figure 5. Effects of autocrine IL-2 and IFNγ on function of Clone 4 cells in the tumor microenvironment. 8–9 week old RIP-Tag2-HA mice were immunized with peptide, polyI:C in IFA and Clone 4 or Clone 4 IL-2−/− (3x104) were injected i.v.. One group receiving Clone 4 cells was also injected with IFNγ neutralizing antibodies at days 4,5 and 6. (A–D) CD8+Thy1.1+ cells from pancreata were analyzed at day 7 by flow cytometry to assess the number of cells in the pancreas, the percentage of cells exhibiting granzyme B, the expression level of Bim and the percentage of dividing cells. Cumulative data are shown from 3 experiments with 2–3 mice per group per experiment. (E) Histograms are representative of 3 independent experiments with 3 mice per group. Isotype control = grey line, Clone 4 = black line, Clone 4 IL2-/- = dashed line, Clone 4 + anti-IFNγ = dotted line.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g6.jpg
Figure 6. High-affinity CD8+ T cells benefit from CD4 help in the tumor milieu. 8–9 week old RIP-Tag2-HA mice were immunized with peptide, polyI:C in IFA and 3 x 104 Clone 4 cells with or without 2 × 105 SFE cells were injected i.v.. (A–D) Pancreata were analyzed at day 7 by flow cytometry to assess the number of cells in the pancreas, the percentage of cells exhibiting granzyme B, the expression level of Bim and the percentage of dividing cells. Data are cumulative of 2 experiments with 2–3 mice per group and are representative of 4 independent experiments. (E) Pancreata were analyzed at day 7 by flow cytometry to assess the expression of PD-1, LAG-3 and NKG2A on CD8+Thy1.1+ cells. Histograms are representative of 3 independent experiments with 2–3 mice per group. Isotype control = gray line, Clone 4 = black line, Clone 4 + SFE = dashed line. Mean MFI ± SD, PD-1: Clone 4 2428.0 ± 216.4; Clone 4+SFE 1569.0 ± 72.1. LAG-3: Clone 4 446.5 ± 98.2; Clone 4 + SFE 353.0 ± 33.9. NKG2A: Clone 4 724.5 ± 151.0; Clone 4 + SFE 681.0 ± 84.4.
https://www.ncbi.nlm.nih.gov/pmc/articles/instance/3518496/bin/onci-1-1239-g7.jpg
Figure 7. Improvement of antitumor efficacy of high-affinity CD8+ T cells by the presence of tumor-specific CD4+ T cells. 8–9 week old RIP-Tag2-HA mice were immunized with peptide and polyI:C in IFA and Clone 4 cells (A–B: 2 × 105, C–D: 3 × 104) with or without 2 × 105 SFE cells were injected i.v.. Glucose levels in the blood were measured at the indicated time points, and each line represents one mouse. Data are representative of two independent experiments. (A) compared with (B): p < 0.05, (C) compared with (D): p < 0.0005.

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