Limits...
Targeting HER-3 to elicit antitumor helper T cells against head and neck squamous cell carcinoma.

Kumai T, Ohkuri T, Nagato T, Matsuda Y, Oikawa K, Aoki N, Kimura S, Celis E, Harabuchi Y, Kobayashi H - Sci Rep (2015)

Bottom Line: In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition.To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors.Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Asahikawa Medical University, Asahikawa, Japan.

ABSTRACT
HER-3 expression has been reported to act as an important oncoprotein in head and neck squamous cell carcinoma. This protein is known to control tumor proliferation and acquisition of resistance by tumor cells towards EGFR inhibitors, therefore, development of a HER-3-targeted therapy is desirable. In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition. To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors. This epitope induced potent cytolytic activity of CD4 T cells against such tumor cells. Moreover, pan HER-family tyrosine kinase inhibitor augmented the responses of HER-3-reactive CD4 T cells via upregulation of HLA-DR protein on the surface of tumor cells. Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.

No MeSH data available.


Related in: MedlinePlus

Induction of HER-3-reactive CD4 T cells.(A) HER-3-reactive CD4 T cells were elicited in 4 healthy individuals (H16 is from donor1: DR4/9, DR53; T11 is from donor2: DR9/12, DR53; O3 is from donor3: DR9/13, DR53; n24 is from donor4: DR9/13, DR53) and then tested for their ability to produce IFN-γ in response to HER-3 peptides. Peptides (0–30 μg/ml) were loaded on irradiated autologous PBMCs. Points: means of triplicate measurements, bars: SEM. The results shown are representative of 3 experiments that were performed on the same samples. (B) We used an IFN-γ assay to assess the responses of HER-3-reactive CD4 T cells to HER-3 peptide (3 μg/ml)-loaded irradiated autologous PBMCs. L243 (10 μg/ml, an anti-HLA-DR antibody) or W6/32 (10 μg/ml, an anti-HLA Class I antibody) was added to confirm the HLA-DR restriction of the T-cell responses. (C) L-DR4, L-DR9 or L-DR53 cells with or without HER-3 peptide (3 μg/ml) were used as antigen-presenting cells (APCs) to evaluate the specific HLA-DR restriction of each HER-3-reactive CD4 lines by means of an IFN-γ assay. Columns: means of triplicate measurements, bars: SEM. The results shown are representative of 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4633732&req=5

f2: Induction of HER-3-reactive CD4 T cells.(A) HER-3-reactive CD4 T cells were elicited in 4 healthy individuals (H16 is from donor1: DR4/9, DR53; T11 is from donor2: DR9/12, DR53; O3 is from donor3: DR9/13, DR53; n24 is from donor4: DR9/13, DR53) and then tested for their ability to produce IFN-γ in response to HER-3 peptides. Peptides (0–30 μg/ml) were loaded on irradiated autologous PBMCs. Points: means of triplicate measurements, bars: SEM. The results shown are representative of 3 experiments that were performed on the same samples. (B) We used an IFN-γ assay to assess the responses of HER-3-reactive CD4 T cells to HER-3 peptide (3 μg/ml)-loaded irradiated autologous PBMCs. L243 (10 μg/ml, an anti-HLA-DR antibody) or W6/32 (10 μg/ml, an anti-HLA Class I antibody) was added to confirm the HLA-DR restriction of the T-cell responses. (C) L-DR4, L-DR9 or L-DR53 cells with or without HER-3 peptide (3 μg/ml) were used as antigen-presenting cells (APCs) to evaluate the specific HLA-DR restriction of each HER-3-reactive CD4 lines by means of an IFN-γ assay. Columns: means of triplicate measurements, bars: SEM. The results shown are representative of 3 separate experiments.

Mentions: We previously identified a CD4 T cell peptide epitope in the EGFR protein that was effective in inducing antitumor responses11. Interestingly, CD4 T cells induced with this EGFR peptide epitope, (EGFR875−889, KVPIKWMALESILHR) were capable of recognizing the corresponding HER-3872−886 peptide (KTPIKWMALESIHFG), which bears 73% amino acid homology. The CD4 T cells also recognized EGFR-negative tumors that expressed HER-3. In view of these findings, we proceeded to test whether the HER-3872−886 peptide itself could induce antigen-specific, tumor-reactive CD4 T cells. Stimulation of CD4 T cells, purified from PBMCs of 4 healthy donors with HER-3872−886-pulsed autologous DCs resulted in the induction of peptide-reactive CD4 T cell lines that were subsequently cloned. As shown in Fig. 2A, four T cell lines secreted IFN-γ when stimulated with the peptide antigen in a dose dependent manner. The analysis of T cell receptor Vβ usage showed that these cell lines were heterogenous populations but the majority of each cell line was consisted of single Vβ positive cells (Supplementary Fig. 1). Production of IL-4, IL-5, IL-10 and IL-17A was not detected in the peptide-stimulated T cell lines (not shown). The addition of an anti-HLA-DR antibody to the peptide stimulation assays resulted in the inhibition of IFN-γ production by HER-3 reactive CD4 T cell lines indicating that peptide HER-3872−886 was recognized in the context of MHC class II molecules and specifically by HLA-DR since this antibody (L243) does not react with the HLA-DQ and -DP proteins (Fig. 2B). As expected, peptide recognition by the CD4 T cell lines was not affected by the addition of anti-HLA class I antibody (W6/32). To determine the HLA-DR alleles restricting the responses in these T cell lines, we used a panel of mouse fibroblasts expressing single HLA-DR molecules (L-DR4, L-DR9, L-DR53) as APCs. The results shown in Fig. 2C indicate that CD4 T cell lines H16, O3, and n24 recognized the HER-3872−886 in the context of HLA-DR53, whereas T11 was restricted by HLA-DR9. Because the frequency of HLA-DR53-linked HLA-DR alleles has been reported to be 39% in the HNSCC patients12, these results indicate that peptide HER-3872−886 is able to elicit CD4 responses in broad population of HNSCC patients.


Targeting HER-3 to elicit antitumor helper T cells against head and neck squamous cell carcinoma.

Kumai T, Ohkuri T, Nagato T, Matsuda Y, Oikawa K, Aoki N, Kimura S, Celis E, Harabuchi Y, Kobayashi H - Sci Rep (2015)

Induction of HER-3-reactive CD4 T cells.(A) HER-3-reactive CD4 T cells were elicited in 4 healthy individuals (H16 is from donor1: DR4/9, DR53; T11 is from donor2: DR9/12, DR53; O3 is from donor3: DR9/13, DR53; n24 is from donor4: DR9/13, DR53) and then tested for their ability to produce IFN-γ in response to HER-3 peptides. Peptides (0–30 μg/ml) were loaded on irradiated autologous PBMCs. Points: means of triplicate measurements, bars: SEM. The results shown are representative of 3 experiments that were performed on the same samples. (B) We used an IFN-γ assay to assess the responses of HER-3-reactive CD4 T cells to HER-3 peptide (3 μg/ml)-loaded irradiated autologous PBMCs. L243 (10 μg/ml, an anti-HLA-DR antibody) or W6/32 (10 μg/ml, an anti-HLA Class I antibody) was added to confirm the HLA-DR restriction of the T-cell responses. (C) L-DR4, L-DR9 or L-DR53 cells with or without HER-3 peptide (3 μg/ml) were used as antigen-presenting cells (APCs) to evaluate the specific HLA-DR restriction of each HER-3-reactive CD4 lines by means of an IFN-γ assay. Columns: means of triplicate measurements, bars: SEM. The results shown are representative of 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4633732&req=5

f2: Induction of HER-3-reactive CD4 T cells.(A) HER-3-reactive CD4 T cells were elicited in 4 healthy individuals (H16 is from donor1: DR4/9, DR53; T11 is from donor2: DR9/12, DR53; O3 is from donor3: DR9/13, DR53; n24 is from donor4: DR9/13, DR53) and then tested for their ability to produce IFN-γ in response to HER-3 peptides. Peptides (0–30 μg/ml) were loaded on irradiated autologous PBMCs. Points: means of triplicate measurements, bars: SEM. The results shown are representative of 3 experiments that were performed on the same samples. (B) We used an IFN-γ assay to assess the responses of HER-3-reactive CD4 T cells to HER-3 peptide (3 μg/ml)-loaded irradiated autologous PBMCs. L243 (10 μg/ml, an anti-HLA-DR antibody) or W6/32 (10 μg/ml, an anti-HLA Class I antibody) was added to confirm the HLA-DR restriction of the T-cell responses. (C) L-DR4, L-DR9 or L-DR53 cells with or without HER-3 peptide (3 μg/ml) were used as antigen-presenting cells (APCs) to evaluate the specific HLA-DR restriction of each HER-3-reactive CD4 lines by means of an IFN-γ assay. Columns: means of triplicate measurements, bars: SEM. The results shown are representative of 3 separate experiments.
Mentions: We previously identified a CD4 T cell peptide epitope in the EGFR protein that was effective in inducing antitumor responses11. Interestingly, CD4 T cells induced with this EGFR peptide epitope, (EGFR875−889, KVPIKWMALESILHR) were capable of recognizing the corresponding HER-3872−886 peptide (KTPIKWMALESIHFG), which bears 73% amino acid homology. The CD4 T cells also recognized EGFR-negative tumors that expressed HER-3. In view of these findings, we proceeded to test whether the HER-3872−886 peptide itself could induce antigen-specific, tumor-reactive CD4 T cells. Stimulation of CD4 T cells, purified from PBMCs of 4 healthy donors with HER-3872−886-pulsed autologous DCs resulted in the induction of peptide-reactive CD4 T cell lines that were subsequently cloned. As shown in Fig. 2A, four T cell lines secreted IFN-γ when stimulated with the peptide antigen in a dose dependent manner. The analysis of T cell receptor Vβ usage showed that these cell lines were heterogenous populations but the majority of each cell line was consisted of single Vβ positive cells (Supplementary Fig. 1). Production of IL-4, IL-5, IL-10 and IL-17A was not detected in the peptide-stimulated T cell lines (not shown). The addition of an anti-HLA-DR antibody to the peptide stimulation assays resulted in the inhibition of IFN-γ production by HER-3 reactive CD4 T cell lines indicating that peptide HER-3872−886 was recognized in the context of MHC class II molecules and specifically by HLA-DR since this antibody (L243) does not react with the HLA-DQ and -DP proteins (Fig. 2B). As expected, peptide recognition by the CD4 T cell lines was not affected by the addition of anti-HLA class I antibody (W6/32). To determine the HLA-DR alleles restricting the responses in these T cell lines, we used a panel of mouse fibroblasts expressing single HLA-DR molecules (L-DR4, L-DR9, L-DR53) as APCs. The results shown in Fig. 2C indicate that CD4 T cell lines H16, O3, and n24 recognized the HER-3872−886 in the context of HLA-DR53, whereas T11 was restricted by HLA-DR9. Because the frequency of HLA-DR53-linked HLA-DR alleles has been reported to be 39% in the HNSCC patients12, these results indicate that peptide HER-3872−886 is able to elicit CD4 responses in broad population of HNSCC patients.

Bottom Line: In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition.To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors.Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Asahikawa Medical University, Asahikawa, Japan.

ABSTRACT
HER-3 expression has been reported to act as an important oncoprotein in head and neck squamous cell carcinoma. This protein is known to control tumor proliferation and acquisition of resistance by tumor cells towards EGFR inhibitors, therefore, development of a HER-3-targeted therapy is desirable. In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition. To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors. This epitope induced potent cytolytic activity of CD4 T cells against such tumor cells. Moreover, pan HER-family tyrosine kinase inhibitor augmented the responses of HER-3-reactive CD4 T cells via upregulation of HLA-DR protein on the surface of tumor cells. Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.

No MeSH data available.


Related in: MedlinePlus