Peptide vaccination in Montanide adjuvant induces and GM-CSF increases CXCR3 and cutaneous lymphocyte antigen expression by tumor antigen-specific CD8 T cells

Abstract

T cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules--CXCR3 and CLA - on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T cell infiltration of melanoma. We demonstrate that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3(+)CLA(+) cells. Addition of GM-CSF significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of Tbet, IFN-γ, and IL-12Rβ1. Collectively, these studies demonstrate that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma.

Trial registration: ClinicalTrials.gov NCT00089193.

Keywords: CLA; CXCR3; GM-CSF; IL-12R; adjuvant; melanoma; peptide vaccines.

Fig. 1. Peptide vaccination with MELITAC 12.1 in Montanide adjuvant induces CXCR3 and CLA expression in tetramer-positive CD8⁺ T cells

Representative flow cytometry of patient-derived peripheral blood samples. Each panel represents an individual patient. PBMC were collected one week after a single vaccination with MELITAC 12.1, either with (A, B) or without (C, D) GM-CSF. All populations are gated on CD8⁺ CD45RO⁺ tetramer⁺ cells and shows i.c. CXCR3 and surface CLA staining. Each panel is from an individual patient.

Fig. 2. Peptide vaccine enhances the proportion of tetramer-positive CD8 T cells expressing CXCR3 and CLA

Percentage of tetramer-positive CD45RO⁺ CD8⁺ cells expressing CXCR3 and CLA. A). CXCR3 expression in tetramer-positive CD8 cells, with and without GM-CSF in vaccine. B). CLA expression in tetramer-positive CD8 cells, with and without GM-CSF in vaccine. C). Expression levels (mean fluorescence intensity) of CXCR3 on tetramer-positive CD8 cells, with and without GM-CSF in vaccine. D). Expression levels (mean fluorescence intensity) of CLA on tetramer-positive CD8 cells, with and without GM-CSF in vaccine.

Fig. 3. Peptide vaccine and adjuvant enhances Tbet (TBX21) and IFN-γ production in CXCR3⁺, but not CXCR3⁻, tetramer-positive CD8 T cells

Open histogram, unstained control; light gray histogram, no GM-CSF in vaccine; dark gray histogram, GM-CSF in vaccine. A). Representative Tbet staining in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. B). Representative Tbet staining in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine. C). Representative IFN-γ staining in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. D). Representative IFN-γ staining in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine.

Fig. 4. Peptide vaccine and adjuvant enhances the proportion of tetramer-positive CD8 T cells expressing the transcription factor Tbet (TBX21) and IFN-γ in CXCR3⁺, but not CXCR3⁻, cells

A). Tbet expression in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. B). Tbet expression in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine. C). IFN-γ expression in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. D). IFN-γ expression in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine.

Fig. 5. Peptide vaccine and adjuvant enhances the proportion of tetramer-positive CD8 T cells expressing the transcription factor IL-12Rβ1

Open histogram, unstained control; light gray histogram, no GM-CSF in vaccine; dark gray histogram, GM-CSF in vaccine. A). Representative IL-12Rβ1 staining in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. B). Representative IL-12Rβ1 staining in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine. C). IL-12Rβ1 expression in CXCR3⁺ tetramer-positive cells, with or without GM-CSF in vaccine. D). IL-12Rβ1 expression in CXCR3⁻ tetramer-positive cells, with or without GM-CSF in vaccine.

Fig. 6. CLA, but not CXCR3, expression fluctuates over the course of peptide vaccination in adjuvant

Percentage of tetramer-positive CD45RO⁺ CD8⁺ cells expressing the indicated homing molecule over the course of vaccination. A). Pre-vaccine. B) One week after 1^st vaccine. C). One week after 2^nd vaccine. D). One week after 3^rd vaccine. E). One week after 4^th vaccine. F). One week after 5^th vaccine. G). One week after 6^th vaccine. H). Twelve weeks after 6^th vaccine.

Fig. 7. GM-CSF in vaccine enhanced CLA expression on tetramer-positive CD8 T cells

Percentage of tetramer-positive CD45RO⁺ CD8⁺ cells expressing the indicated homing molecule over the course of vaccination. A). CXCR3 expression over the course of vaccination. B). CLA expression over the course of vaccination.