Relaxed DM Requirements During Class II Peptide Loading and CD4+ T Cell Maturation in BALB/c Mice

Abstract

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.

DM Loss in k Haplotype Mice Reveals Isotype-Specific Chaperone Requirements

“…These findings strongly argue that DM is strictly required to mediate CLIP release and promote occupancy by diverse peptide ligands. Considerable data suggest that DM plays a less critical role during peptide acquisition by other allelic products (22)(23)(24)(25)(26)43), but the structural framework of these selective DM requirements remains ill defined.…”

“…These 5Ј and 3Ј arms were placed in between positive (neo) and negative (DT-A) drug selection cassettes to create the isogenic CBA/J-based targeting vector, introduced into H-2 bxk F 1 TT2 ES cells (44). From 1089 drug-resistant colonies analyzed, we recovered 10 recombinant clones carrying the desired 1.7-kb deletion spanning exon 1, intronic sequences, and most of exon 2, as judged by diagnostic Southern blots with restriction digests and probes as described (43). Because the wild-type k allele contains a unique SacI site mapped to intron 1, absent from the C57BL/6 locus, the 5Ј probe distinguishes SacI fragments derived from wild-type k (4.6 kb), wild-type b (8 kb), or the targeted locus (7.5 kb).…”

“…In contrast to gain-of-function approaches used previously, this strategy has allowed us to examine functional consequences in professional APC expressing appropriate ratios of class II and Ii chain isoforms under control of endogenous regulatory elements. Recent experiments demonstrate relaxed DM requirements during class II peptide loading and CD4 ϩ T cell maturation in BALB/c mice (43 To further investigate allele-and isotype-specific DM actions, here we engineered a novel mutant allele in k haplotype mice. DM loss in this context offers the opportunity for side-by-side comparisons of activities mediated by A k and E k molecules encoded by tightly linked loci.…”

“…The targeting strategy for disruption of the DM␣ locus has been described previously (43). Briefly, we used long-range PCR to isolate genomic subclones comprised of a 4.5-kb BamHI-ScaI fragment immediately 5Ј to exon 1 and a HindIII-EcoRI fragment containing 30 nt of exon 2 and extending 4 kb downstream.…”

DM, but not cathepsin L, is required to control an aerosol infection withMycobacterium tuberculosis

“…I-A b forms unusually stable complexes with CLIP and thus, relies heavily on DM-mediated CLIP-peptide exchange-at least in late endocytic compartments [15,24]. Other class II alleles, with weaker affinities for CLIP, vary to the degree at which they require DM to mediate CLIP release and exchange for antigenic peptides [41,42]. Nonetheless, these alleles are still dependent on the function of DM as a peptide editor [13,14].…”