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Understanding CD8 + T-cell responses toward the native and alternate HLA-A * 02:01-restricted WT1 epitope

View Article: PubMed Central - PubMed

ABSTRACT

Ry: The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (MFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant MFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B)), can induce WT1-specific CD8+ T cells, although WT1B is more stably bound to HLA-A*02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8+ T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105–106) to other naive CD8+ T-cell pools (for example, A2/HIV-Gag77-85), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Importantly, only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8+ T-cell responses to the native tumor epitope following re-stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique T-cell receptor-αβ gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.

No MeSH data available.


Polyfunctional WT1A-specific CD8+ T cells induced following WT1A- or WT1B vaccination. In one experiment, cells isolated from iLN and spleen were stimulated with WT1A, WT1B or the SV4 control peptide in a 6-h intracellular cytokine staining (ICS)-CD107a assay to measure IFN-γ, TNF-α and CD107a responses following vaccination. Representative functional profiles from individual mice in the WT1A/CpG (n=4) or WT1B/CpG (n=4) are shown for iLN (a) and spleen (b) as a percentage of CD8+ T cells. Polyfunctionality was assessed using Boolean gate analysis to determine the proportion of functional CD8+ T cells expressing 1, 2 or 3 of the functional markers in iLN (c) and spleen (d). Bar graphs below the pie charts show the percentages (mean±s.d.) of total functional cells based on the CD8+ T-cell population. Each bar graph dot point represents individual mice, except for dot points in the iLN HPV/CpG group (c), which represent WT1A and WT1B peptide responses from pooled mice (n=3). (e) Mean fluorescence intensity (MFI, geometric mean) of functionally positive CD8+ T cells (that is, CD107a+, IFN-γ+ or TNF-α+) of iLN are shown.
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fig4: Polyfunctional WT1A-specific CD8+ T cells induced following WT1A- or WT1B vaccination. In one experiment, cells isolated from iLN and spleen were stimulated with WT1A, WT1B or the SV4 control peptide in a 6-h intracellular cytokine staining (ICS)-CD107a assay to measure IFN-γ, TNF-α and CD107a responses following vaccination. Representative functional profiles from individual mice in the WT1A/CpG (n=4) or WT1B/CpG (n=4) are shown for iLN (a) and spleen (b) as a percentage of CD8+ T cells. Polyfunctionality was assessed using Boolean gate analysis to determine the proportion of functional CD8+ T cells expressing 1, 2 or 3 of the functional markers in iLN (c) and spleen (d). Bar graphs below the pie charts show the percentages (mean±s.d.) of total functional cells based on the CD8+ T-cell population. Each bar graph dot point represents individual mice, except for dot points in the iLN HPV/CpG group (c), which represent WT1A and WT1B peptide responses from pooled mice (n=3). (e) Mean fluorescence intensity (MFI, geometric mean) of functionally positive CD8+ T cells (that is, CD107a+, IFN-γ+ or TNF-α+) of iLN are shown.

Mentions: To assess the functional quality of WT1A-specific CD8+ T-cell responses elicited following either WT1A or WT1B vaccination, cells from iLN and spleens were incubated with either peptide for 6 h and examined for their capacity to induce cytotoxicity, via cell surface expression of the degranulation marker CD107a, and for their cytokine production of IFN-γ and tumor necrosis factor-α (TNF-α). All WT1A- and WT1B-vaccinated mice showed a robust functional CD8+ T-cell response toward their cognate peptide stimulus (Figures 4a and b). In the iLN of WT1A/CpG-vaccinated mice stimulated with the cognate WT1A peptide, 22.50±12.10% of the total CD8+ T-cell population expressed 1, 2 or 3 of the functional markers (CD107a, IFN-γ or TNF-α), with 38.00±10.95% of total functional cells expressing all three functional markers (Figure 4c). Interestingly, WT1A peptide stimulation in WT1B/CpG-vaccinated mice showed comparable polyfunctionality to its cognate WT1B peptide stimulation, with 23.52±7.34% and 23.76±6.39% of total functional iLN CD8+ T cells being IFN-γ+CD107a+TNF-α+, respectively. Notably, similar trends were observed in the spleen (Figure 4d), whereby total functional WT1A-specific CD8+ T-cell responses were at least twice times higher following WT1A peptide stimulation in WT1A/CpG-vaccinated mice compared with WT1B/CpG-vaccinated mice, which was statistically significant (P=0.0286).


Understanding CD8 + T-cell responses toward the native and alternate HLA-A * 02:01-restricted WT1 epitope
Polyfunctional WT1A-specific CD8+ T cells induced following WT1A- or WT1B vaccination. In one experiment, cells isolated from iLN and spleen were stimulated with WT1A, WT1B or the SV4 control peptide in a 6-h intracellular cytokine staining (ICS)-CD107a assay to measure IFN-γ, TNF-α and CD107a responses following vaccination. Representative functional profiles from individual mice in the WT1A/CpG (n=4) or WT1B/CpG (n=4) are shown for iLN (a) and spleen (b) as a percentage of CD8+ T cells. Polyfunctionality was assessed using Boolean gate analysis to determine the proportion of functional CD8+ T cells expressing 1, 2 or 3 of the functional markers in iLN (c) and spleen (d). Bar graphs below the pie charts show the percentages (mean±s.d.) of total functional cells based on the CD8+ T-cell population. Each bar graph dot point represents individual mice, except for dot points in the iLN HPV/CpG group (c), which represent WT1A and WT1B peptide responses from pooled mice (n=3). (e) Mean fluorescence intensity (MFI, geometric mean) of functionally positive CD8+ T cells (that is, CD107a+, IFN-γ+ or TNF-α+) of iLN are shown.
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fig4: Polyfunctional WT1A-specific CD8+ T cells induced following WT1A- or WT1B vaccination. In one experiment, cells isolated from iLN and spleen were stimulated with WT1A, WT1B or the SV4 control peptide in a 6-h intracellular cytokine staining (ICS)-CD107a assay to measure IFN-γ, TNF-α and CD107a responses following vaccination. Representative functional profiles from individual mice in the WT1A/CpG (n=4) or WT1B/CpG (n=4) are shown for iLN (a) and spleen (b) as a percentage of CD8+ T cells. Polyfunctionality was assessed using Boolean gate analysis to determine the proportion of functional CD8+ T cells expressing 1, 2 or 3 of the functional markers in iLN (c) and spleen (d). Bar graphs below the pie charts show the percentages (mean±s.d.) of total functional cells based on the CD8+ T-cell population. Each bar graph dot point represents individual mice, except for dot points in the iLN HPV/CpG group (c), which represent WT1A and WT1B peptide responses from pooled mice (n=3). (e) Mean fluorescence intensity (MFI, geometric mean) of functionally positive CD8+ T cells (that is, CD107a+, IFN-γ+ or TNF-α+) of iLN are shown.
Mentions: To assess the functional quality of WT1A-specific CD8+ T-cell responses elicited following either WT1A or WT1B vaccination, cells from iLN and spleens were incubated with either peptide for 6 h and examined for their capacity to induce cytotoxicity, via cell surface expression of the degranulation marker CD107a, and for their cytokine production of IFN-γ and tumor necrosis factor-α (TNF-α). All WT1A- and WT1B-vaccinated mice showed a robust functional CD8+ T-cell response toward their cognate peptide stimulus (Figures 4a and b). In the iLN of WT1A/CpG-vaccinated mice stimulated with the cognate WT1A peptide, 22.50±12.10% of the total CD8+ T-cell population expressed 1, 2 or 3 of the functional markers (CD107a, IFN-γ or TNF-α), with 38.00±10.95% of total functional cells expressing all three functional markers (Figure 4c). Interestingly, WT1A peptide stimulation in WT1B/CpG-vaccinated mice showed comparable polyfunctionality to its cognate WT1B peptide stimulation, with 23.52±7.34% and 23.76±6.39% of total functional iLN CD8+ T cells being IFN-γ+CD107a+TNF-α+, respectively. Notably, similar trends were observed in the spleen (Figure 4d), whereby total functional WT1A-specific CD8+ T-cell responses were at least twice times higher following WT1A peptide stimulation in WT1A/CpG-vaccinated mice compared with WT1B/CpG-vaccinated mice, which was statistically significant (P=0.0286).

View Article: PubMed Central - PubMed

ABSTRACT

Ry: The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (MFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant MFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B)), can induce WT1-specific CD8+ T cells, although WT1B is more stably bound to HLA-A*02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8+ T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105–106) to other naive CD8+ T-cell pools (for example, A2/HIV-Gag77-85), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Importantly, only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8+ T-cell responses to the native tumor epitope following re-stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique T-cell receptor-αβ gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.

No MeSH data available.