Limits...
EGFR-specific T cell frequencies correlate with EGFR expression in head and neck squamous cell carcinoma.

Schuler PJ, Boeckers P, Engers R, Boelke E, Bas M, Greve J, Dumitru CA, Lehnerdt GF, Ferris RL, Andrade Filho PA, Brandau S, Lang S, Whiteside TL, Hoffmann TK - J Transl Med (2011)

Bottom Line: Patients' results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections.Proliferation and anti-tumor activity of peptide-specific CTL was demonstrated by in vitro stimulation with dendritic cells pulsed with the peptides.EGFR-specific CTL from cancer patients were expanded ex vivo and produced IFN-γ upon recognition of EGFR+ target cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Universität Duisburg-Essen, Hals-Nasen-Ohrenklinik Essen, Germany. patrick.schuler@uk-essen.de

ABSTRACT

Background: In head and neck squamous cell carcinoma (HNSCC), expression levels of the epidermal growth factor receptor (EGFR) correlate with poor prognosis and decreased survival rates. As the mechanisms responsible for cellular immune response to EGFR in vivo remain unclear, the frequency and function of EGFR-specific cytotoxic T cells (CTL) was determined in HNSCC patients.

Methods: The frequency of CTL specific for the HLA-A2.1-restricted EGFR-derived YLN peptide (YLNTVQPTCV) and KLF peptide (KLFGTSGQKT) was determined in 16 HLA-A2.1+ HNSCC patients and 16 healthy HLA-A2.1+ individuals (NC) by multicolor flow cytometry. Patients' results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections. Proliferation and anti-tumor activity of peptide-specific CTL was demonstrated by in vitro stimulation with dendritic cells pulsed with the peptides.

Results: Frequency of EGFR-specific CTL correlated significantly with EGFR expression in tumor sections (p = 0.02, r2 = 0.6). Patients with elevated EGFR scores (> 7) had a significantly higher frequency of EGFR-specific CTL than NC and patients with low EGFR scores (< 7). EGFR-specific CTL from cancer patients were expanded ex vivo and produced IFN-γ upon recognition of EGFR+ target cells.

Conclusion: EGFR expressed on HNSCC cells induces a specific immune response in vivo. Strategies for expansion of EGFR-specific CTL may be important for future immunotherapy of HNSCC patients.

Show MeSH

Related in: MedlinePlus

EGFR-specific CTL recognize tumor cells. CTL were generated from PBMC of HNSCC patients in co-cultures with autologous DC loaded with EGFR-peptides KLF or YLN. T cells were tested against the EGFR-positive cell line UD-SCC-8 and the EGFR-negative cell line HLac79, which were either unpulsed or pulsed with the EGFR peptides. The T cell: target cell ratio was 1:1. The data show the mean number of counted spots for unpulsed and pulsed cell lines obtained using CTL generated from PBMC of HNSCC patients. Pulsing cell lines with peptide significantly increased IFN-γ production in CTL (* p = 0.002). Background in the unpulsed EGFR-negative cell line HLac79 was not subtracted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198929&req=5

Figure 5: EGFR-specific CTL recognize tumor cells. CTL were generated from PBMC of HNSCC patients in co-cultures with autologous DC loaded with EGFR-peptides KLF or YLN. T cells were tested against the EGFR-positive cell line UD-SCC-8 and the EGFR-negative cell line HLac79, which were either unpulsed or pulsed with the EGFR peptides. The T cell: target cell ratio was 1:1. The data show the mean number of counted spots for unpulsed and pulsed cell lines obtained using CTL generated from PBMC of HNSCC patients. Pulsing cell lines with peptide significantly increased IFN-γ production in CTL (* p = 0.002). Background in the unpulsed EGFR-negative cell line HLac79 was not subtracted.

Mentions: After in vitro expansion, PBMC of HLA-A2.1+ patients were tested for reactivity against the EGFR-positive cell line (UD-SCC-8) and the EGFR-negative cell line (HLac79) in INF-γ ELISPOT experiments (n = 3). Both target cell lines were used unpulsed or after pulsing with EGFR-peptides. INF-γ-secretion was significantly increased by pulsing target cells with EGFR-peptides (p = 0.002). Consequently, for both peptides, the highest IFN-γ-secretion was observed in the EGFR-positive cell line UD-SCC-8 which was additionally pulsed with EGFR-peptide (32 ± 3 spots for YLN, 41 ± 3 spots for KLF/1 × 10^5 cells). Pulsing target cells with the KLF-peptide increased INF-γ-production by 14 ± 1 spots/1 × 10^5 cells. The increase was 15 ± 1 spots/1 × 10^5 cells, when cells were pulsed with the YLN-peptide. Specificity of CTL for the EGFR-peptides was confirmed by the observation that IFN-γ-secretion was almost undetectable in the unpulsed EGFR negative cell line HLac79, and only pulsing the target cells with the EGFR peptides increased IFN-γ-secretion by 6-fold. The results of ELISPOT assays are shown in Figure 5. Frequencies of EGFR-peptide specific CTL were compared before and after in vitro expansion. For the KLF-peptide, the frequency before expansion was 0.02 - 0.04% of CD8+ T cells in HNSCC patients with high EGFR score (>7). After in vitro expansion, the frequency of EGFR-peptide specific CTL was 20 spots/10,000 cells, corresponding to 0.2 ± 0.03% of PBMC. The effect of expansion was similar for YLN-peptide specific CTL.


EGFR-specific T cell frequencies correlate with EGFR expression in head and neck squamous cell carcinoma.

Schuler PJ, Boeckers P, Engers R, Boelke E, Bas M, Greve J, Dumitru CA, Lehnerdt GF, Ferris RL, Andrade Filho PA, Brandau S, Lang S, Whiteside TL, Hoffmann TK - J Transl Med (2011)

EGFR-specific CTL recognize tumor cells. CTL were generated from PBMC of HNSCC patients in co-cultures with autologous DC loaded with EGFR-peptides KLF or YLN. T cells were tested against the EGFR-positive cell line UD-SCC-8 and the EGFR-negative cell line HLac79, which were either unpulsed or pulsed with the EGFR peptides. The T cell: target cell ratio was 1:1. The data show the mean number of counted spots for unpulsed and pulsed cell lines obtained using CTL generated from PBMC of HNSCC patients. Pulsing cell lines with peptide significantly increased IFN-γ production in CTL (* p = 0.002). Background in the unpulsed EGFR-negative cell line HLac79 was not subtracted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198929&req=5

Figure 5: EGFR-specific CTL recognize tumor cells. CTL were generated from PBMC of HNSCC patients in co-cultures with autologous DC loaded with EGFR-peptides KLF or YLN. T cells were tested against the EGFR-positive cell line UD-SCC-8 and the EGFR-negative cell line HLac79, which were either unpulsed or pulsed with the EGFR peptides. The T cell: target cell ratio was 1:1. The data show the mean number of counted spots for unpulsed and pulsed cell lines obtained using CTL generated from PBMC of HNSCC patients. Pulsing cell lines with peptide significantly increased IFN-γ production in CTL (* p = 0.002). Background in the unpulsed EGFR-negative cell line HLac79 was not subtracted.
Mentions: After in vitro expansion, PBMC of HLA-A2.1+ patients were tested for reactivity against the EGFR-positive cell line (UD-SCC-8) and the EGFR-negative cell line (HLac79) in INF-γ ELISPOT experiments (n = 3). Both target cell lines were used unpulsed or after pulsing with EGFR-peptides. INF-γ-secretion was significantly increased by pulsing target cells with EGFR-peptides (p = 0.002). Consequently, for both peptides, the highest IFN-γ-secretion was observed in the EGFR-positive cell line UD-SCC-8 which was additionally pulsed with EGFR-peptide (32 ± 3 spots for YLN, 41 ± 3 spots for KLF/1 × 10^5 cells). Pulsing target cells with the KLF-peptide increased INF-γ-production by 14 ± 1 spots/1 × 10^5 cells. The increase was 15 ± 1 spots/1 × 10^5 cells, when cells were pulsed with the YLN-peptide. Specificity of CTL for the EGFR-peptides was confirmed by the observation that IFN-γ-secretion was almost undetectable in the unpulsed EGFR negative cell line HLac79, and only pulsing the target cells with the EGFR peptides increased IFN-γ-secretion by 6-fold. The results of ELISPOT assays are shown in Figure 5. Frequencies of EGFR-peptide specific CTL were compared before and after in vitro expansion. For the KLF-peptide, the frequency before expansion was 0.02 - 0.04% of CD8+ T cells in HNSCC patients with high EGFR score (>7). After in vitro expansion, the frequency of EGFR-peptide specific CTL was 20 spots/10,000 cells, corresponding to 0.2 ± 0.03% of PBMC. The effect of expansion was similar for YLN-peptide specific CTL.

Bottom Line: Patients' results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections.Proliferation and anti-tumor activity of peptide-specific CTL was demonstrated by in vitro stimulation with dendritic cells pulsed with the peptides.EGFR-specific CTL from cancer patients were expanded ex vivo and produced IFN-γ upon recognition of EGFR+ target cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Universität Duisburg-Essen, Hals-Nasen-Ohrenklinik Essen, Germany. patrick.schuler@uk-essen.de

ABSTRACT

Background: In head and neck squamous cell carcinoma (HNSCC), expression levels of the epidermal growth factor receptor (EGFR) correlate with poor prognosis and decreased survival rates. As the mechanisms responsible for cellular immune response to EGFR in vivo remain unclear, the frequency and function of EGFR-specific cytotoxic T cells (CTL) was determined in HNSCC patients.

Methods: The frequency of CTL specific for the HLA-A2.1-restricted EGFR-derived YLN peptide (YLNTVQPTCV) and KLF peptide (KLFGTSGQKT) was determined in 16 HLA-A2.1+ HNSCC patients and 16 healthy HLA-A2.1+ individuals (NC) by multicolor flow cytometry. Patients' results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections. Proliferation and anti-tumor activity of peptide-specific CTL was demonstrated by in vitro stimulation with dendritic cells pulsed with the peptides.

Results: Frequency of EGFR-specific CTL correlated significantly with EGFR expression in tumor sections (p = 0.02, r2 = 0.6). Patients with elevated EGFR scores (> 7) had a significantly higher frequency of EGFR-specific CTL than NC and patients with low EGFR scores (< 7). EGFR-specific CTL from cancer patients were expanded ex vivo and produced IFN-γ upon recognition of EGFR+ target cells.

Conclusion: EGFR expressed on HNSCC cells induces a specific immune response in vivo. Strategies for expansion of EGFR-specific CTL may be important for future immunotherapy of HNSCC patients.

Show MeSH
Related in: MedlinePlus