Immune deficiency due to high copy numbers of an Ask transgene ( class i / transgenic mice )

Because allelic polymorphism of the major histocompatibility complex class II antigens affects the immune response at several levels, we wished to characterize the contribution of a particular a or 13 chain in vivo using transgenic mice. We have established and characterized 12 lines of H-2rs mice carrying from 1 to 65 copies of an Apk transgene. The transgene was coexpressed with the endogenous allele in a tissue-specific manner, and AP mRNA expression correlated well with transgene copy number. High copy number (extreme overexpression) of the transgene was associated with a variety of defects, including a significant reduction in Ia cell-surface expression, a severe decrease in B-cell number, abnormal extramedullary granulopoiesis, and an increased susceptibility to infection. In this paper we describe in detail the phenotype associated with high copy numbers of the Ak transgene. The defects we have observed may be relevant to similar phenomena seen in other transgenic mice. In addition, these mice have fortuitously provided a system in which to assess the effect of various levels of class II cell-surface expression in the thymus on selection of the T-cell repertoire. The major histocompatibility complex class II (Ia) antigens are highly polymorphic, and the alleles expressed within an individual animal largely determine its ability to respond to particular antigens (1). Two class II isotypes are expressed in the mouse, I-A and I-E. Each is a cell-surface glycoprotein composed of noncovalently associated a (33-35 kDa) and 13 (27-29 kDa) chains (2, 3). With the exception of Ea, all chains are polymorphic, and most of the allelic sequence differences are clustered in the membrane-distal domain of each chain (al and f31). A structural model for the class TI antigens (4) has been derived from the class I HLA-A2 crystal structure (5), which suggests that the al and 131 domains interact to form a peptide-binding groove on the outer surface of the molecule. Functionally, these domains are important for T-cell recognition and a//3 chain pairing (6-8). In addition, the level of any particular class II heterodimer expressed on the surface of antigen-presenting cells is critical in that a minimum threshold is required for T-cell activation (9-12). Because the class II antigens affect the immune response at several levels, the contributions of specific amino acids or epitopes to peptide binding, T-cell recognition, and a/P chain pairing have been the subject of intense research. To extend these studies to an in vivo system, we introduced an A4 genomic clone into the germ line of H-2'/1 mice (which do not express I-E) and have established 12 independent lines carrying from 1 to -65 copies ofthe A4 transgene. The transgene was coexpressed with the endogenous allele, and A4 mRNA expression correlated well with transgene copy number. High copy number (extreme overexpression) of the transgene was deleterious in these mice and interfered with cell-surface expression of I-A and with B-cell differentiation at two distinct stages. This B-cell depletion was accompanied by a variety of defects, including increased numbers of granulocytes, abnormal extramedullary hematopoiesis (primarily granulopoiesis), and an increased susceptibility to infection. The defects we have observed can clearly be attributed to overexpression of the Ad transgene, and the phenotype described may be relevant to similar phenomena observed in other transgenic mice. The effect of increasing levels of A4 mRNA expression on a/P chain pairing and cell-surface expression will be described in detail elsewhere (13). Because cell-surface levels of Ia can be modulated by breeding appropriate pairs, these mice also provide a well-controlled system in which to assess the effect of extreme variation in la cell-surface expression in the thymus on the selection of the T-cell repertoire. MATERIALS AND METHODS Mice. SJL, BALB/c, and 129 strain mice were obtained from The Jackson Laboratories, and B10.S mice from Olac (Bicester, U.K.). B10.A(4R) and F1 offspring from these strains were bred and maintained in our colony. Ak transgenic mice (from the Ak-26 line; ref. 14) were provided by D. Mathis and C. Benoist. Production of Transgenic Mice. An 11.6-kilobase (kb) fragment of the p-A -1 genomic clone (15) containing 2-3 kb of 5' sequence and 1-2 kb of 3' sequence was purified and microinjected into the pronuclei of (B10.S x SJL)F2 embryos using standard techniques (16, 17). Founders carrying the transgene were identified by blotting (18) Bgl II-digested genomic DNA extracted from tail tissue. Filters were hybridized with a full-length 32P-labeled A4 cDNA clone. Copy number was determined in representatives from each line of mice (13). Monoclonal Antibodies (mAbs). mAb 10-3.6 (anti-Ask f ru. ref. 19), mAb 40.N (anti-Afru, ref. 20), and mAb MKS4 (anti-Afu, ref. 21) were grown in ascites and purified by ammonium sulfate precipitation followed by protein A affinity chromatography. Preparations were directly biotinylated (22). mAb RA3-6b2 (anti-B220, ref. 23), mAb MI/70 (antiMac 1, ref. 24), anti-CD5 (Lyl, ref. 25), mAb AF6-122 (anti-IgD, 5b; ref. 26), and mAb 33.12 (anti-IgM, ref. 27) antibodies were provided by A. Stall and N. Uchida (Stanford University). Anti-CD4 (L3T4) and anti-CD8 (Ly2) were purchased from Becton Dickinson. Fluorescence-Activated Cell Sorter (FACS) Analysis. FACS analysis was done as described by Hayakawa et al. (28). Cells were analyzed on a dual-laser FACS IV (Becton Dickinson Immunocytometry) equipped with a logarithmic amplifier. Dead cells were excluded by propidium iodide staining. Immunohistochemistry. Tissue samples were cut into small pieces, embedded in OCT-compound (Tissue-Tek; Miles Scientific), and frozen in dry ice/isopentane without fixation. Abbreviations: NT, nontransgenic; 4RxNT, [B1O.A(4R) x NT]F1; FACS, fluorescence-activated cell sorter; SPF, specific pathogen free; mAb, monoclonal antibody; (H,G)-A--L, (histidine, glutamic acid)-alanine--lysine. 7319 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) Cryosections 4 Am thick were cut and air dried for 1 hr, fixed with cold acetone for 10 min, and washed three times in phosphate-buffered saline (PBS). For staining, the sections were first blocked with 1% bovine serum albumin (fraction V; Sigma) and then incubated for 30 min in an appropriate dilution of a biotinylated antibody. Slides were washed with PBS, then incubated with avidin-peroxidase for 30 min, washed, and developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma). Slides were then rinsed in H20 and mounted under coverslips.