Phenotypic and functional maturation of tumor antigen-reactive CD8+ T lymphocytes in patients undergoing multiple course peptide vaccination - PubMed (original) (raw)

Phenotypic and functional maturation of tumor antigen-reactive CD8+ T lymphocytes in patients undergoing multiple course peptide vaccination

Daniel J Powell Jr et al. J Immunother. 2004 Jan-Feb.

Abstract

Successful immunotherapy with peptide vaccines depends on the in vivo generation of sufficient numbers of anti-tumor T cells with appropriate phenotypic and functional characteristics to mediate tumor destruction. Herein, we report the induction of high frequencies of circulating CD8+ T cells (4.8% to 38.1%) directed against the native gp100:209-217 peptide derived from the gp100 melanoma-melanocyte tumor antigen in five HLA-A*0201 patients at high risk of recurrence of melanoma after multiple courses of immunization with modified gp100:209-217(210M) peptide in IFA. Longitudinal peripheral blood mononuclear cell (PBMC) analysis revealed a phenotypic shift of native peptide-specific CD8+ T cells from an early effector to an effector memory (CD27- CD28- CD62L- CD45RO+) phenotype with repeated immunizations and functional maturation that correlated with gp100:209-217 peptide-specific T-cell precursor frequencies. Postimmunization PBMC exhibited direct ex vivo recognition of melanoma cell lines in ELISPOT analysis, showed lytic capability against peptide-pulsed target cells, and proliferated in response to native peptide stimulation. One year after final immunization, circulating vaccine-specific CD8+ T cells persisted in patients' PBMC with a maintained effector memory phenotype. The results herein demonstrate the efficacy of a multiple course peptide-immunization strategy for the generation of high frequencies of tumor antigen-specific T cells in vivo, and further show that continued peptide immunization results in the escalating generation of functionally mature, tumor-reactive effector memory CD8+ T lymphocytes.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

Longitudinal ex vivo evaluation of peptide vaccine-induced CD8+ T-cell responses in the peripheral blood of patients 5 and 3 before (PRE) and 3 weeks after each vaccination course (POST 1-4). PBMC from patients 5 and 3 were stained with either gp100:209-217 peptide/HLA-A*0201 or HIV gag/HLA-A*0201 tetramers and anti-CD8FITC antibody after overnight incubation in CM and immediately analyzed by flow cytometric analysis. Dot plots are shown for propidium iodide-negative gated PBMC. Values correspond to the percentage of total CD8bright T cells that are g209/A2 tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8bright T cells minus the CD8[H11502] tetramer+ background [H11503]100.

FIGURE 2

FIGURE 2

Phenotypic maturation of native peptide-specific CD8+ T cells in vivo after multiple course peptide vaccination. PBMC from vaccinated patients were incubated overnight in culture media and stained for gp100:209-217 peptide/HLA-A*0201 tetramer, CD8, and either CD28 (A), CD27 (B), perforin (C), CD45RA (D), CD45RO (E), HLA-DR (F), CD62L (G), or CCR7 (H) using multiparameter flow cytometry. Propidium iodide staining was performed for viable cell gating in all cases with the exception of anti-perforin antibody-stained cells requiring fixation and permeabilization. Values correspond to the percentage of g209-specific CD8+ T cells positively expressing the given marker after each vaccination course. (I) Values represent the average percentage of tetramer-positive CD8+ T cells expressing the marker from patients with detectable g209/A2 tetramer-positive PBMC after the first vaccination course. (J) and (K), Values represent the percentage of the CD8+ T-cell population that are g209/A2+ and express the indicated marker from patients 4 and 5 PBMC, respectively.

FIGURE 3

FIGURE 3

Functional maturation of tetramer-positive CD8+ T cells in the peripheral blood of g209-2M peptide-vaccinated patients. The absolute number of gp100:209-217 peptide/HLA-A*0201 tetramer-positive CD8+ T cells per 105 PBMC was deduced by applying the percentage of tetramer-positive CD8+ PBMC in the circulation (total number of tetramer-positive CD8+ PBMC/mL divided by total number of PBMC/mL [H11503] 100) to 105 PBMC for patient PBMC collected over the immunization course and compared with the number of IFNγ-secreting cells/105 PBMC after overnight incubation with 1 μM g209 peptide as measured by ELISPOT assay. Circles correspond to paired results for each PBMC sample tested. (P < 0.001).

FIGURE 4

FIGURE 4

Cytolytic capacity of PBMC after multiple courses of g209-2M peptide vaccination. Pre- (left panels) and postimmunization course 4 PBMC (right panels) from patients 1-5 were rested overnight in CM and analyzed for cytolytic activity against T2 target cells alone (triangles) or pulsed with 1 μM native g209 (squares) or g280 (circles) in a 4-hour 51Cr-release assay. Values represent the mean percent lysis from triplicate wells [H11506] SEM at the indicated effector-to-target-cell ratio. Asterisk indicates a significant difference (P < 0.05) between g209-specific and g280 control lysis determined by two-sided Kruskal-Wallis test.

FIGURE 5

FIGURE 5

Phenotypic analysis of g209-specific CD8+ T cells from 1-year postimmunization PBMC. PBMC from vaccinated patients 1-year after final peptide vaccination were incubated overnight in culture media and were stained using gp100:209-217 peptide/HLA-A*0201 tetramer, anti-CD8 antibody, and antibodies specific for either CD27, CD28, CD45RA, CD45RO, CD62L, or CCR7 molecules for multiparameter flow cytometric analysis. Propidium iodide staining was performed for viable cell gating in all cases. Values correspond to the average percentage of g209-specific CD8+ T cells from patients 1, 2, 3, and 5 PBMC that positively express the indicated markers.

Similar articles

Cited by

References

    1. Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature. 2001;411:380–384. - PubMed
    1. Kawakami Y, Eliyahu S, Delgado CH, et al. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A. 1994;91:6458–6462. - PMC - PubMed
    1. Skipper JC, Gulden PH, Hendrickson RC, et al. Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART- 1 and gp100. Int J Cancer. 1999;82:669–677. - PubMed
    1. Kawakami Y, Eliyahu S, Jennings C, et al. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression. J Immunol. 1995;154:3961–3968. - PubMed
    1. Salgaller ML, Afshar A, Marincola FM, et al. Recognition of multiple epitopes in the human melanoma antigen gp100 by peripheral blood lymphocytes stimulated in vitro with synthetic peptides. Cancer Res. 1995;55:4972–4979. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources