Figure 1.
Generation of FVB Kbα1α2Db transgenic mice.
(A) Segments of H-2Kb EcoRI and H-2Db HindIII were used to generate a chimeric genomic construct with an H-2Db Sal I/XbaI fragment on an H-2Kb backbone. (B) Expression of the construct yields a chimeric MHC class I molecule composed of the α1α2 domain from H-2Db and the α3 domain from H-2Kb. (C) Verification of H-2Db and Kbα1α2Db transgene expression in 293T cells by flow cytometry. Data are mean fluorescence intensity of phycoerythrin labeled cells.
Figure 2.
Spinal cord demyelination and persistent virus infection in FVB Kbα1α2Db transgenic mice.
(A) Transgenic expression of H-2Kb fails to protect from TMEV induced demyelination, similar to non-transgenic control (B). (C) Expression of H-2Db transgene protects FVB mice from TMEV induced demyelination present in littermate controls (D). (E) Expression of a chimeric Kbα1α2Db molecule fails to protect from demyelination, similar to non-transgenic (F). (G) Relative TMEV RNA levels in spinal cords from transgenic mice infected for 21 days (* p<0.05 by ANOVA). (H) Relative TMEV RNA levels in the brains of transgenic mice after 24 days of infection.
Table 1.
Spinal cord pathology.
Figure 3.
FVB Kbα1α2Db transgenic mice generate functional CTL responses against the immunodominant VP2121–130 peptide.
(A) Control H-2Db/E749–57 and TMEV specific H-2Db/VP2121–130 tetramer and CD8 staining of acute CD45+ lymphocytes isolated from the brains of transgenic and non-transgenic mice infected with TMEV for 6 days (* p<0.05 by ANOVA). (B) VP2121–130 specific killing of peptide pulsed targets by acute brain infiltrating lymphocytes isolated from H-2Db and H-2Kbα1α2Db transgenic mice compared to killing of non-pulsed EL-4 cells.
Figure 4.
Non-equivalent mRNA expression of MHC class I genes due to elements outside of the α1α2 coding region of the transgenic D locus.
(A) FVB skin fibroblast mRNA levels for H-2Db and H-2Dq with or without treatment with IFNγ (**p<0.01 comparing Db versus Kbα1α2Db without or with IFNγ). (Third panel) Transgenic mRNA stability as determined by degradation of mRNA transcripts after actinomycin D inhibition of transcription. qRT-PCR amplification was used to detect H-2Db and TNFα specific transcripts from FVB Db and FVB Kbα1α2Db transgenic fibroblasts previously treated with IFNγ. (B) H-2Dq mRNA expression in TMEV infected brain tissue from H-2Db and FVB Kb α1α2Db transgenic mice (** p<0.05). (C) Competitive RT-PCR and sequence identity for MHC specific transcripts from the brain and spleen of TMEV infected mice (* p = 0.091, Fisher Exact Test comparing the ratio of Kq and Lq sequences recovered). (D) mRNA expression of transgenic and endogenous H-2 genes (** p<0.01 by Two-way ANOVA).
Figure 5.
Surface expression of H-2 transgenes is regulated by genomic elements outside of sequences encoding peptide binding domains.
(A) The mean fluorescence intensity (MFI) for peripheral blood lymphocytes stained with the H-2Db specific antibody B22.249 from H-2b haplotype mice (white) and transgenic mice (gray); (* Line 1 v. B10 or FVB Db, p<0.05, a Line 2 v. B10 or FVB Db, p<0.05). (B) Splenocytes from mice infected with TMEV for 21 days were isolated and tested by flow cytometry for H-2Db expression levels using B22.249.R1 labeled with FITC. Data are expressed as the MFI of the FITC labeled cells (* p<0.05, Two-way ANOVA). (C) The same splenocytes in (B) were co-stained with AF6-88.5 labeled phycoerythrin to determined expression of the H-2Kb present on the chimeric Kbα1α2Db molecule. Data are expressed as MFI of PE labeled cells (* p<0.001, Two-way ANOVA). (D) Brain resident cells from FVB, FVB Db and FVB Kbα1α2Db mice were isolated from naïve mice and analyzed by flow cytometry. Side-scatter and forward-scatter were analyzed for the presence of mononuclear resident cells using a lymphocyte gate. Resident antigen-presenting cells were further analyzed by a CD45 mid-level staining gate and assessed for expression of MHC class I. FVB (green), FVB Db (black) and FVB Kbα1α2Db (blue) brain cells were assessed for H-2Db expression by FACS (* p<0.001, t-test). (E) Brain cells isolated from 6 day TMEV infected mice were gated as in (D) and assessed for changes in H-2Db expression by FACS. Data are the MFI for H-2Db-FITC.