Mice lacking all conventional MHC class II genes

Abstract

MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCII(Delta/Delta) mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aalpha and Abeta knockout animals, notably a dearth of CD4(+) lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCII(Delta/Delta) mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering "humanized" mouse models of human MHC-II-associated autoimmune disorders.

Figure 1

Strategy for genomic deletion of all classical MHC-II genes. (A and B) Genomic organization of the mouse MHC around and in the class II region. Arrows indicate the transcriptional orientation of the different genes. (C) Mutation strategy. (C1) The wild-type Aβ and Eα genes. Exons are shown as filled boxes. The triangle represents the promoter deletion present in the Eα gene of the H2^b haplotype. H, HindIII; Bs, BstEII; Bsp, BspEI; E, EcoRI; B, BglII; X, XbaI; S, SpeI; Sm, SmaI; Xh, XhoI. (C2) The mutated Aβ and Eα loci after homologous recombination. The homologous recombination event introduced a TK-neo cassette into exon 2 of the Aβ gene and a loxP-hygromycin cassette into exon 3 of the Eα gene. The targeting DNA fragments used for electroporation are indicated by a bold line, the loxP sites as triangles. The probes used for the screening are shown under the targeted loci. (C3) Map of the deleted locus after Cre-mediated recombination. The Southern blot probe and PCR primers used to identify the deleted locus are shown under the map. (D) Southern blot analysis of genomic DNA isolated from wild-type embryonic stem (ES) cells and the double-targeted ES clone before and after the deletion event (BglII digest; Aβ1 probe).

Figure 2

Expression of MHC-II molecules in MHCII^Δ/Δ mice. (A) Flow-cytometry profiles of anti-class-II staining on IgM⁺ splenocytes in MHCII^+/Δ and MHCII^Δ/Δ littermates. Upper and Lower panels present staining with Y3P or M5/114 anti-class-II antibodies, respectively (thin lines). Plain lines represent staining with an irrelevant primary antibody. (B) Anti-class-II (Y3P) staining of thin sections from thymus and spleen of MHCII^+/Δ and MHCII^Δ/Δ littermates. (C) Absence of Aα transcripts in MHCII^Δ/Δ mice. Serial dilutions (1 in 10) of cDNA prepared from spleen RNA were amplified by PCR with Aα-specific oligonucleotides; the total reaction run on an agarose gel is visualized by ethidium bromide staining. Amplification with HPRT-specific primers is shown as a control for cDNA quality.

Figure 3

Expression of nonconventional class II genes and MHC-I molecules in MHCII^Δ/Δ mice. (A) Semiquantitative RT-PCR for expression of H2-Oα, H2-Oβ, and H2-Mα. RT-PCR was performed on 10-fold serial dilutions of cDNA from spleen RNA of MHCII^+/Δ and MHCII^Δ/Δ littermates. (B) Flow-cytometric analysis of MHC-I expression on IgM⁺ splenocytes. Plain lines represent control staining with an irrelevant primary antibody; dotted lines represent staining with the anti-K^b antibody K9/178.

Figure 4

T lymphocyte populations in MHCII^Δ/Δ mice. (A) Anti-CD4/CD8 staining of thymocytes from MHCII^+/Δ and MHCII^Δ/Δ littermates and an Aβ^o/o mouse. The values represent the percentage of thymocytes that fall within the gates in this representative experiment. (B) Profiles of CD3 and peanut agglutinin receptor (PNAr) expression on CD4⁺CD8⁻ thymocytes from the same mice described in A. (C) CD4/CD8 profiles of lymph node lymphocytes.

Figure 5

B lymphocyte lineage in MHCII^Δ/Δ mice. (A) B lymphocyte differentiation in the bone marrow; anti-B220 and anti-IgM staining of bone marrow cells from MHCII^+/Δ and MHCII^Δ/Δ littermates (gated on lymphocytes by scatter). (B and C) B cell populations in the spleen: B220/IgM profiles on whole splenocytes and IgM/IgD profiles gated on B220⁺ B cells. (D) Serum Ig levels in naive MHCII^+/+ and MHCII^Δ/Δ mice. Serum concentration for various Ig isotypes determined by ELISA (arbitrary units; 1 is defined as the average of control sera in the experiment). Each bar represents an individual mouse.