Figure 1.
Transgenes encoding chimeric HLA-DR4/I-Ed molecule.
(A) Schematic diagram of mouse T cell receptor (mTCR, green), mouse CD4 (mCD4, pale green) and HLA-DR4/I-Ed chimeric molecules (orange and yellow). (B) To avoid inter-species interactions between mCD4 and HLA-DR4, the second exons of I-Edα and I-Edβ genes encoding α1 and β1 domains were substituted with those of HLA-DRA and HLA-DR4B genes (red boxes), respectively. The transgenes contain the endogenous I-Edα and I-Edβ promoter regions spanning 3.2 kb and 5.2 kb of 5'-untranslated regions, respectively. (C) WT1332-347 peptide-pulsed L-cells expressing HLA-DR4/I-Ed stimulated IFN-γ production by the WT1332-347 peptide-specific and HLA-DR4-restricted human Th clone (gated on CD4).
Figure 2.
Selection of transgene-positive founder mice (F0).
Twenty five F0 mice were arbitrarily numbered #1~#25. PBMCs were analyzed for HLA-DR4/I-Ed expression by flow-cytometric analyses (gated on lymphocytes) using anti-HLA-DRα and β mAbs (A) and genomic PCR analyses (B). A typical dot blot of PBMCs from other mice negative for transgenes is shown (null (#9) in A) and results from representative 10 mice from 25 F0 mice are shown in B.
Figure 3.
Chromosomal localization of transgene insertion site and cell-type specific expression of HLA-DR4/I-Ed.
(A) Mapping of transgene insertion by fluorescence in situ hybridization (FISH) revealed integration in chromosome 3, H2-H4 region in line #5 Tgm. (B) PBMCs from line #5 Tgm were stained with anti-HLA-DR and anti-B220 mAbs (left) or anti-HLA-DR and anti-I-Ab mAbs (right). Numbers indicate the percentage of HLA-DR4/I-Ed-positive cells in B220-positive cells and MHC-II-positive cells indicated by the red boxes, respectively (gated on lymphocytes).
Figure 4.
Chimeric HLA-DR4/I-Ed molecules induced the differentiation of mouse CD4+ cells.
(Upper panels) PBMCs (gated on lymphocytes) from MHC-II knock-out mice (DR-/-MHC-II-/-), Tgm (DR+/-MHC-II+/+) and Tgm that lack MHC-II (DR+/-MHC-II-/-) were stained with anti-HLA-DR and anti-mouse CD4 mAbs. (Lower panels) PBMC (gated on lymphocytes) from each mouse was stained with anti-B220 mAb and anti-mouse MHC-II mAb.
Figure 5.
Immunization of TAA-derived peptides induced peptide-specific and HLA-DR4-restricted Th-cell responses in Tgm.
Peptides emulsified in CFA and IFA were injected into the tail base of Tgm or C57BL/6 mice (WT) on day 0 and day 7. On day 14, the splenocytes were harvested and cultured in vitro for 7 days with the peptides (1 μg/ml). (A) Purified CD4+ cells (1 × 105/well) were co-cultured with L-DR4 (3 × 104/well) pulsed with or without CMV-egH290–302 peptide for 72 h and 3H-thymidine uptake was measured. (B) Purified CD4+ cells (1 × 105/well) were co-cultured with L cells (3 × 104/well) or L-DR4 (3 × 104/well) pulsed with or without CDCA155-78 peptide for 72 h and 3H-thymidine uptake was measured. (C, D) Immunization of Tgm with syngeneic BM-DCs (5 × 105) pulsed with WT1332-347 peptide (C) or KIF20A494-517 peptide (D) followed by a booster shot of the peptide in CFA successfully induced IFN-γ production by Th cells (1 × 105/well) in response to the peptide-pulsed L-DR4 cells (5 × 104/well), but not to unpulsed L-DR4 cells.
Figure 6.
DEPDC1191-213 peptide induced peptide-specific and HLA-DR4-restricted Th-cell responses in both Tgm and human PBMCs.
(A) Tgm and C57BL/6 mice (WT) were immunized with DEPDC1191-213 peptide or control peptide (DEPDC160-85) in CFA and IFA as described in Figure 6 legend. On day 14, inguinal lymph node cells were harvested and ex vivo 3H-thymidine incorporation was measured. (B) Induced CD4+ T cells (1 × 104/well) were co-cultured with peptide-pulsed L-DR4 (5 × 104/well) or L-DR53 cells (5 × 104/well) or unpulsed L-DR4 in the presence or absence of anti-HLA-DR blocking mAb L243. The CD4+ T cells cultured with peptide only did not produce IFN-γ.