CTL activation using the natural low-affinity epitope 222-229 from tyrosinase-related protein 1 leads to tumor rejection

Abstract

Vaccine strategies for cancer immunotherapy have focused on peptide ligands with high affinity for MHC class I. Largely, these vaccines have not been therapeutic. We have examined the peptide specificity of a strongly protective T-cell response that eradicates established B16 melanoma and find that the recognized epitope is generated by a low-affinity MHC class I ligand from tyrosinase-related protein 1 (TRP1). Cytotoxic T-cell responses are induced against TRP1(222-229) by several vaccination schemes using a Toll-like receptor agonist, T regulatory cell depletion, or the immune modulator B7-DCXAb to drive immunity. TRP1(222) CTL are generated from multiple antigen sources, including antigens expressed by tumors growing in situ, tumor cell lysates, and peptide vaccines. The key finding in this study is that protection from freshly implanted or established B16 tumors is primarily mediated by TRP1(222)-specific CTL and not by CTL specific for more traditional melanoma antigens such as TRP2 or gp100. This finding challenges the assumption that the optimal peptide antigens for cancer vaccines are high-affinity MHC ligands. We propose that when administered appropriately, native low-affinity MHC ligands are optimal inducers of immunotherapeutic CTL.

Figure 1. B16 challenge or B16-CL immunization induces CTL recognizing an epitope from TRP1

Cytotoxicity data assessing CTL antigen specificity are expressed as percent lysis of peptide-pulsed target cells. Error bars represent the standard deviation of triplicate measurements. (A) B7-DCXAb induced CTL using live B16 tumor cells (white bars) or B16-CL (black bars). (B) Antigen specificity of CTL induced with CpG/B16-CL was assessed without (white bars) or with CD25+ T cell depletion (black bars). (C) Peptide specific CTL responses induced with Titermax and B16-CL after in vitro re-stimulation with peptide-pulsed stimulators.

Figure 2. B16 challenge induces CTL that recognize a low affinity MHC class I ligand presented by the B16 tumor line

Cytotoxicity assays were performed as in Fig. 1. (A) EL4 target cells were pulsed with H-2K^b-binding peptides from TRP1 and CTL were from draining lymph nodes of mice treated with B7-DCXAb using live B16 (black bars) or B16-CL (gray bars) as antigen. (B) CTL generated from the spleens of TRP1₂₂₂-immunized mice were used to assess presentation of the TRP1₂₂₂ epitope. Targets were TRP1₂₂₂ and VSV₅₂ pulsed EL4 or B16 tumor cells (left panel) or C57SV fibroblasts transfected with a full length TRP1, a TRP1 H-2K^b anchor residue mutant, or non-transfected C57SV (right panel). (C) Peptide binding to H-2K^b was assessed by the change in mean fluorescence intensity (MFI) from peptide versus non-peptide pulsed RMAS cells. Data are representative of 3 independent trials. Symbols represent the change in MFI over a range of peptide concentrations for the indicated peptides.

Figure 3. TRP1₂₂₂ specific depletion inhibits killing of B16 melanoma in vitro and in vivo

(A) Lysis of peptide-pulsed EL-4 targets from mice pre-treated with TRP1₂₂₂ or VSV₅₂ prior to vaccination with B7-DCXAb and live B16 tumor cells (left) or B16-CL (right). (B) Percent of B16-challenged animals surviving after TRP1₂₂₂ depletion and B7-DCXAb treatment compared to treatment with control peptide and B7-DCXAb or control antibody (p<0.001 TRP1 vs. VSV treated animals). (C) Percent of surviving animals immunized with B16-CL and depleted prior to immunization as in (B) are shown (p<0.001 TRP1 versus VSV depletion).

Figure 4. Gamma irradiation and in vivo growth affects B16 immunogenecity and antigen presentation

(A) CTL induced with irradiated B16 cells (γIR-B16) were assessed for target specificity using B16 or peptide-pulsed C57SV targets. (B) TRP1_222-229 (left) and TRP2_180-188 (right) CTL were used to assess antigen presentation by unmanipulated B16, γIR-B16, and control C57SV targets. (C) CTL as in (B) were used to determine antigen presentation in unmanipulated B16, IFNγ treated B16 and control C57SV targets. (D) B16 tumors grew in situ for 5 days before B7-DCXAb treatment. Six days later CTL were tested for specificity using B16 and EL4 (left panel) and TRP1₂₂₂, TRP2₁₈₀, or gp100₂₅-pulsed EL4 (right panel).

Figure 5. TRP1₂₂₂ specific CTL induced with B7-DCXAb eradicate established B16 tumors

(A) Animals were depleted with TRP1₂₂₂, TRP2₁₈₀ or VSV₅₂ one day before challenge with B16. On day 4, the depleted animals were treated with B7-DCXAb; CTL activity was assessed 6 days later for specificity using B16 and TRP1₂₂₂, TRP2₁₈₀-pulsed or unpulsed EL4 targets. (B) Tumor challenged animals were monitored for survival as in Fig. 2 (p<0.001 TRP1₂₂₂ versus VSV₅₂ or TRP2₁₈₀ depletion). (C) Tumor growth in individual mice treated with TRP1₂₂₂, TRP2₁₈₀, gp100₂₅ or VSV₅₂ pulsed DC treated with B7-DCXAb (DC^XAb). Peptide-pulsed DC^XAb vaccine was administered 4 days after B16 tumor challenge. (D) Tumor free survival in groups treated with DC^XAb or control IgM treated DC (DC^Ctrl) pulsed with TRP1₂₂₂, TRP2₁₈₀, gp100₂₅ or VSV₅₂ before transfer into mice, challenged 4 days prior with B16 (p<0.001 DC^XAb TRP1₂₂₂ versus DC^XAb VSV₅₂).

Figure 6. TRP1₂₂₂-specific CTL are sufficient for clearing established B16 tumors

(A) TRP1₂₂₂ and TRP2₁₈₀-specific effector CTL were depleted of CD4 or CD8 T cells before assessing CTL specificity to TRP1₂₂₂ or TRP2₁₈₀-pulsed EL4. (B) Blocking antibodies to H-2K^b (B8-24-3) and H-2D^b (B22-249.R1 and 28-14-8) were added to peptide pulsed targets prior to TRP1₂₂₂ and TRP2₁₈₀ effector CTL. (C) In vitro generated TRP1₂₂₂ or TRP2₁₈₀ CTL were infused into mice challenged with B16 5 days prior. Mice receiving no vaccine were used as controls (p<0.001 TRP1₂₂₂ versus TRP2₁₈₀ or None).