Page 194 - 2021_03-Haematologica-web

R. Li et al.
Table 1. Potential Dickkopf-1 peptides for different MHC molecules. MHC class I
HLA Name Sequence Position
A0201 P3 ALGAAGATRV 3
Predictive bindinga.b.c
Py3 Py11v P20 Py20 Py25 Py32v P36 P36v P66v
A1 Py35v A0205 Py11v
P13 A24 P12 B62 P66 B7 P24 P59
B5101 P68
B5102 P68
B5103 P5 P51
B5201 P4 CW0401 P12 P24
MHC class II
YLGAAGATRV RVFVAMVAA ALGGHPLLGV YLGGHPLLGV YLLGVSATL YLNSVLNSNV VLNSNAIKNL VLNSNAIKNV ILYPGGNKV YVLNSNAIV YVFVAMVAV FVAMVAAAL VFVAmVAAAL ILYPGGNKY HPLLgVSATL AVSAAPGIL YPGGnKYQTI YPGGnKYQTI GAAGaTRVFV GAAGhPGSAV LGAAgATRVF VFVAmVAAAL HPLLgVSATL
70 3 320 11 238 20 160 20 736 25 364 32 320
36 84 36 226 66 378 35 42.5 11 72 13 42 12 42 66 124.8 24 80 59 60 68 692 68 1064.8 5 121 51 110
4 74.3 12 240 24 96
10 33 26 25 30 26 26 30
7 24
DRB1*0101 P10 TRVFVAMVAAALGGH DRB1*0301 P26 LLGVSATLNSVLNSN DRB1*0401 P30 SATLNSVLNSNAIKN DRB1*0701 P26 LLGVSATLNSVLNSN
DRB1*1501 P7 AGATRVFVAMVAAAL
ahttp://www.bimas.cit.nih.gov/molbio/hla_bind.bhttp://www.imtech.res.in/raghava/propred/. chttp://www.immuneepitope.org.
DR1, -DR4, or -DR7) (Table 1 and Figure 1). DKK13-76-LP contains our previously identified HLA-A*0201-restricted T-cell epitopes DKK1-P20 and DKK1-P66v.19
In vivo immunogenicity of the Dickkopf-13-76-long peptide in activating Dickkopf-1-specific CD8+ cytotoxic T lymphocytes
To assess the immunity of the DKK13-76-LP in inducing CD8+ CTL response in vivo, we used HLA-A*0201-trans- genic mice and immunized them four times with either DKK13-76-LP or DKK1-P20 short peptide. The capacity of DKK13-76-LP to prime DKK1-specific CTL response was examined using HLA-A*0201-P20- (Figure 2A) or HLA- A*0201-P66v tetramer staining (Figure 2B). The results clearly showed that mice immunized with DKK1-P20 short peptide (Figure 2A and B, top panels) had increased percentages of DKK1-P20 (P<0.01, compared with con- trol), but not DKK1-P66v, tetramer+ CD8+ T cells in the spleen after each round of immunization, whereas mice immunized with DKK13-76-LP (Figure 2A and B, bottom panels) generated CD8+ T-cell response against both
DKK1-P20 and DKK1-P66v (P<0.01, compared with con- trol). Moreover, CD8+ T cells isolated from mice immu- nized with DKK13-76-LP were not only able to kill syngene- ic DC pulsed with DKK13-76-LP, but also DC pulsed with DKK1-P20 short peptide (P<0.01, compared with controls) (Figure 2C). Furthermore, these CD8+ T cells also killed HLA-A*0201+ U266, but not HLA-A*0201 ARP-1 myelo- ma cells or K562 cells (to exclude natural killer [NK]-cell activity) (P<0.01, compared with controls) (Figure 2D). Hence, these results demonstrate that DC, after uptake of DKK13-76-LP, efficiently cross-present T-cell epitopes on the DKK13-76-LP and activate CTL specific for various T- cell epitopes, including DKK1-P20- and DKK1-P66v, in vivo.
In vivo immunogenicity of the Dickkopf-13-76-long peptide in activating Dickkopf-1-specific CD4+ T-helper cells and antibody production
Next, we assessed whether DKK13-76-LP could also elicit DKK1-specific CD4+ Th cell response. HLA-DR*4-trans- genic mice were available commercially and immunized
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