Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose

Abstract

In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.

Fig. 1

Similar frequencies and differentiation of Melan-A-specific CD8+ T cells after vaccination with low and high peptide doses. Percentages of circulating Melan-A-specific cells in CD8+ T cells after vaccination with 0.1 mg peptide (low dose; n = 16 patients) and 0.5 mg peptide (high dose; n = 5) determined directly ex vivo by flow cytometry. Shown are a representative dot plot of ex vivo Melan-A-specific CD8+ T cells in patient blood after four vaccinations (a), percentages of Melan-A-specific T cells at time of peak response (b), the number of vaccinations needed to reach this peak (c) and the differentiation status of the specific CD8+ T cells, determined as fraction of naïve (CCR7+ CD45RA+), central memory (CM; CCR7+ CD45RA−), effector memory (EM; CCR7−, CD45RA−) and effector RA+ (EMRA; CCR7−, CD45RA+) cells within Melan-A-specific CD8+ T cells (d) and fraction of CD28-cells (e) in Melan-A-specific EM or EMRA subpopulations. Data points for each patient and mean ± SEM are shown. N.S. not significant

Fig. 2

Equal capacity of cytokine production by Melan-A-specific CD8+ T cells from patients vaccinated with low and high doses of Melan-A peptide. CD8+ T cells from low (n = 6) and high (n = 5) dose-vaccinated patients were analyzed directly ex vivo, upon triggering with unpulsed or Melan-A peptide-pulsed T2 cells for 4 h, followed by intracellular staining for TNF-α (a, b) and IL-2 (a, c). Dot plots of Melan-A-specific CD8+ T cells are shown from two representative patients (a) and percentages of cytokine positive cells for each patient, with mean ± SEM (b, c)

Fig. 3

Superior capacity of degranulation and target cell lysis by Melan-A-specific CD8+ T cells from low peptide dose-vaccinated patients. Melan-A-specific CD8+ clones were assessed for ability to lyse Melan-A peptide-pulsed T2 target cells (a, n = 323 for low, n = 94 for high peptide dose) or melanoma cell lines (b, n = 14 for low, n = 15 for high peptide dose) positive (Me290) or negative (Na8) for Melan-A expression, with (+ELA) and without additional peptide. Percent specific lysis (a) or lytic units within 10⁷ effector cells (b) are shown as individual data points for each clone, and mean ± SEM for the two patient groups. Ability of ex vivo Melan-A-specific CD8+ T cells (c–e, n = 6 for low, n = 5 for high peptide dose) or clones (f, n = 22 for low, n = 17 for high peptide dose) to degranulate was measured as surface expression of CD107a during peptide stimulation. Shown are representative histograms for CD107a expression by Melan-A-specific CD8+ T cells analyzed ex vivo (c), after stimulation with T2 cells without or with Melan-A peptide. Percentages of positive cells (d) or expression per cell (e, mean MFI ± SEM) by ex vivo Melan-A-specific CD8+ T cells, or by Melan-A-specific clones (f, mean MFI ± SEM) for each specified group are depicted

Fig. 4

Similar steady-state expression of perforin/granzyme B, but increased capacity of perforin release by Melan-A-specific CD8+ T cells from patients vaccinated with low peptide dose. Intracellular staining of perforin (a, c, d) and granzyme B (b, e) was performed in ex vivo Melan-A-specific CD8+ T cells (a–e, n = 6 for low, n = 5 for high peptide dose) or Melan-A-specific CTL clones (f, g) from vaccinated patients. Depicted are percentages of perforin (a) or granzyme B (b) positive Melan-A-specific CD8+ T cells for the two patient groups, or percentages of specific cells that lost intracellular expression of perforin upon 4 h stimulation with peptide-loaded target cells (c). Results are shown per patient/clone as well as mean ± SEM. Also shown are mean MFI ± SEM for specific cells (d, e) or clones (f, g; n = 43 for low, n = 20 for high peptide dose)

Fig. 5

Decreased CD8 dependence but similar avidity of Melan-A-specific CD8+ T cells from low peptide dose-vaccinated patients. Functional avidity of Melan-A-specific clones was determined as logEC50 (M) of Melan-A peptide (a). Values are depicted as separate data points and mean ± SEM for the specified group (low peptide dose: n = 247, high peptide dose: n = 60). Ex vivo CD8+ T cells (b, c; n = 6 for low, n = 5 for high peptide dose) or Melan-A-specific CD8+ T cell clones (d–f; CD8: n = 43 for low, n = 20 for high peptide dose, tetramers: n = 29 for low, n = 20 for high peptide dose) from vaccinated patients were stained with wild-type Melan-A tetramers (b, e) or for CD8 (c, f). Clones were also stained with mutated Melan-A tetramer unable to bind CD8 (d). Mean MFI ± SEM is depicted for clones from each specified group