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.

Figures

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 1st vaccine. C). One week after 2nd vaccine. D). One week after 3rd vaccine. E). One week after 4th vaccine. F). One week after 5th vaccine. G). One week after 6th vaccine. H). Twelve weeks after 6th 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.