Addition of GM-CSF to a peptide/KLH vaccine results in increased frequencies of CXCR3-expressing KLH-specific T cells

Abstract

T-cell trafficking is determined by expression patterns of chemokine receptors. The chemokine receptor CXCR3 is expressed on a subpopulation of type 1 T cells and plays an important role for migration of T cells into inflamed and tumor tissues. Here, we studied the chemokine receptor expression on specific T cells generated against the neoantigen keyhole limpet hemocyanin (KLH) in patients who had been immunized in the context of a tumor peptide vaccination trial with or without the adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF). In patients immunized in the presence of GM-CSF the fraction of CXCR3(+) KLH-specific T cells was significantly higher than in patients immunized in the absence of GM-CSF (median 45 vs. 20%, P = 0.001). In contrast, the chemokine receptor CCR4, associated with migration to the skin was found in both cohorts on less than 10% of KLH-specific T cells. These results show that CXCR3 expression on vaccine-induced T cells can be modulated by modifying the local vaccine milieu.

Fig. 1

CXCR3 is downregulated in vitro following antigen stimulation and incubation with brefeldin A. a CXCR3/IFNγ profile of CD3⁺CD8⁺ gated lymphocytes stimulated with influenza peptide, phorbol myristate acetate/Ionomycin or tyrosinase peptide are shown. CXCR3 was stained after antigen stimulation and brefeldin A incubation (left dot plots) or prior to antigen stimulation (respective right dot plots). T cells reactive with tyrosinase peptide were from a HLA-A2+ melanoma patient who exhibited a spontaneous high-frequency specific T-cell response [28]. b PBMCs of a HLA-A2+ healthy donor were stimulated with HIV (left dot plots) and influenza (respective right dot plots) peptides. The CXCR3/IFNγ profile of CD3⁺CD8⁺ gated lymphocytes are shown. CXCR3 staining was either performed before antigen stimulation (b, upper dot plots), or after antigen stimulation but before addition of brefeldin A (b, middle dot plots) or after antigen stimulation and addition of brefeldin A (b, lower dot plots). MFI mean fluorescence intensity of CXCR3 expression of influenza-specific IFNγ-producing T cells, ab antibody

Fig. 2

Chemokine receptor expression by KLH-specific T cells in patients immunized in the presence or absence of GM-CSF. a CXCR3/IFNγ profile of CD3⁺CD4⁺ gated lymphocytes in unstimulated (left) or KLH-exposed (right) PBMCs in two patients immunized in the absence (upper dot plots) or presence of GM-CSF (lower dot plots) are shown. The percentages of total CD3⁺CD4⁺ T cells and KLH-specific CD3⁺CD4⁺ T cells are displayed expressing CXCR3 (b), CCR4 (c) or CCR9 (d) in patients immunized in the presence (n = 8, filled cycles) or absence (n = 7, open squares) of GM-CSF. In each patient, chemokine receptor expression of all CD3⁺CD4⁺ T cells was compared with the respective chemokine receptor expression of the KLH-specific CD3⁺CD4⁺ T cells (connecting line). CCR4 and CCR9 were only determined in a subset of samples (GM-CSF cohort: CCR4 n = 5; CCR9: n = 4; non GM-CSF cohort: CCR4 n = 6, CCR9 n = 7) due to paucity of material. Bars indicate the median

Fig. 3

Course of CXCR3 profile of KLH-specific T cells in two patients. Analysis of PBMCs was performed in two patients (patient A: GM-CSF cohort, patient B: non GM-CSF cohort) 1, 6/4 and 17/18 months after immunization. The percentages of CXCR3⁺ T cells among the KLH-specific CD4⁺ T-cell fraction (black columns) and the total CD4⁺ T-cell fraction (hatched columns) are shown