Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2b haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived Ab/class II-associated invariant chain-derived peptide (CLIP) complexes, mature Ad and Ed molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4+ T cells fail to undergo superantigen-mediated negative selection, as judged by TCR Vβ usage. Ag presentation assays reveal consistent differences for Ad- and Ed-restricted T cells. Indeed, the mutation leads to decreased peptide capture by Ad molecules, and in striking contrast causes enhanced peptide loading by Ed molecules. Thus, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.

Abstract. Gastric papillomas were diagnosed in flaky skin (fsn/fsn) and steel-Dickie (S l / S l d ) mutant mice but not littermate controls. Both mutants suffer from severe anemia of differing causes. Immunohistochemical screening and Southern blot analyses failed to detect any evidence of a papillomavirus in the gastric lesions. Phenotypic expression of the fsn and Sl d mutant genes may play an essential role in the spontaneous development of forestomach papillomas in these mouse mutants.The stomachs of rodents are divided into the forestomach (squamous portion) and the glandular portion that are separated by a limiting ridge. ), 11 and recently we have observed this lesion in the new mouse mutation called flaky skin (fsn/fsn). The cause of forestomach hyperplasia or neoplasia in mutant mice is unknown; however, many stocks suffer from various forms of anemia. 2,17,23 necropsy and bisected longitudinally; half was frozen in liquid nitrogen and stored at -80 C for DNA extraction. The remaining tissue was fixed in Fekete's acid-alcohol-formalin solution for 12 hr, transferred to 70% ethanol, embedded by routine methods in paraffin, and serially sectioned at 5 µm. Sections were stained with hematoxylin and eosin or placed on gelatin-chrome alum-coated slides for immunohistochemical evaluation. 25Measurements of gastric forestomach epithelium. The thickness of the stratified squamous epithelium that lined the forestomachs of the mice in this study was determined using a computerized image analyzer.c,d Full thickness measurements from the basement membrane to the upper layer to the stratum corneum were made in the affected areas of the mutant mouse forestomach and comparable areas in the littermate controls. A minimum of 25 measurements were taken for each section. Data were analyzed using Student's t-test.The purpose of this project was to compare the lesions and to determine if papillomaviruses were involved in the etiology of gastric papillomas in flaky skin or steel-Dickie mutant mice. Materials and methodsImmunohistochemistry. Immunohistochemical evaluation was performed on paraffin sections using a rabbit polyclonal antiserum directed against papillomavirus group-specific antigens' prepared by detergent disruption of purified bovine papillomavirus type-1 (BPV-1). This antiserum is broadly cross-reactive with mammalian and avian papillomavirus antigens.26 the avidin-biotin complex (ABC) techInbred and mutant laboratory mice. Fourteen male flaky skin (BALB/cBy.A N1F2-fsn/fsn) and 11 littermate controls (?/+), and 11 steel-Dickie (WCB6F1-Sl/Sl d ) and 6 littermate controls (sl/+) were utilized in this study. Mice were 2-3 mo of age and were obtained from research or animal resources colonies maintained at The Jackson Laboratory, Bar Harbor, Maine. Mice were fed 2 commercial rodent diets a,b and provided chlorinated water (10-20 ppm residue chlorine) ad libitum. Packed cell volumes (PCV) were determined on blood obtained via the ventral coccygeal artery following transitory warming of each mouse to dilate tail ...

Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii−DM− doubly deficient mice. As for Ii chain mutants, their mature AαbAβb dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy AαbAβb dimers is TAP independent. In comparison with Ii chain mutants, Ii−DM− doubly deficient cell populations exhibit increased peptide binding activities and consistently greater presentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii−DM− double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal.

Mutant mouse strains expressing either p31 or p41 Ii chain appear equally competent with respect to their class II functional activities including Ag presentation and CD4+ T cell development. To further explore possibly divergent roles provided by alternative Ii chain isoforms, we compare class II structure and function in double mutants also carrying a null allele at the H2-DM locus. As for DM mutants expressing wild-type Ii chain, AαbAβb dimers present in DM-deficient mice expressing either Ii chain isoform appear equally occupied by class II-associated Ii chain-derived peptides (CLIP). Surprisingly, in functional assays, these novel mouse strains exhibit strikingly different phenotypes. Thus, DM-deficient mice expressing wild-type Ii chain or p31 alone are both severely compromised in their abilities to present peptides. In contrast, double mutants expressing the p41 isoform display markedly enhanced peptide-loading capabilities, approaching those observed for wild-type mice. The present data strengthen evidence for divergent class II presentation pathways and demonstrate for the first time that functionally distinct roles are mediated by alternatively spliced forms of the MHC class II-associated Ii chain in a physiologic setting.

Allelic differences are known to influence many important aspects of class II biosynthesis, including subunit assembly, Ii chain associations, and DM-mediated peptide loading. Mutant mouse strains lacking Ii chain expression have been previously studied on mixed genetic backgrounds. The present experiments describe cellular and functional characteristics of congenic BALB/c Ii chain mutants. As expected, class II surface expression was markedly decreased, but in contrast to I-Ad-transfected cell lines, serological analysis of BALB/c Ii chain-deficient spleen cells gave no evidence for discordant expression of class II conformational epitopes. Thus, we conclude that properly folded class II molecules are exported via the Ii chain-independent pathway. Functional assays demonstrate consistently superior peptide-loading capabilities, suggesting that these I-Ad molecules are empty or occupied by an easily displaced peptide(s). Defective B cell development was observed for three mutant strains established on diverse genetic backgrounds. Ii chain function is also essential for optimal class II surface expression by mature splenic dendritic cells. Surprisingly, we observe in BALB/c Ii chain mutants, relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. The milder phenotype displayed by BALB/c Ii chain mutants in comparison with class II functional defects previously described for mouse strains lacking Ii chain is likely to have an effect on disease susceptibility.