Elizabeth K. Bikoff

Mice lacking Bmp6 function.

Bmp6, a member of the 60A subgroup of bone morphogenetic proteins (BMPs), is expressed in diverse sites in the developing mouse embryo from preimplantation stages onwards. To evaluate roles for Bmp6 signaling in vivo, gene targeting was used to generate a null mutation at the Bmp6 locus. The resulting Bmp6 mutant mice are viable and fertile, and show no overt defects in tissues known to express Bmp6 mRNA. The skeletal elements of newborn and adult mutants are indistinguishable from wild-type. However, careful examination of skeletogenesis in late gestation embryos reveals a consistent delay in ossification strictly confined to the developing sternum. In situ hybridization studies in the developing long bones and sternum show that other BMP family members are expressed in overlapping domains. In particular we find that Bmp2 and Bmp6 are coexpressed in hypertrophic cartilage, suggesting that Bmp2 may functionally compensate in Bmp6 null mice. The defects in sternum development in Bmp6 null mice are likely to be associated with a transient early expression of Bmp6 in the sternal bands, prior to ossification. These sternal defects are slightly exacerbated in Bmp5/6 double mutant animals.

The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse

Blimp1, a zinc-finger containing DNA-binding transcriptional repressor, functions as a master regulator of B cell terminal differentiation. Considerable evidence suggests that Blimp1 is required for the establishment of anteroposterior axis formation and the formation of head structures during early vertebrate development. In mouse embryos, Blimp1 is strongly expressed in axial mesendoderm, the tissue known to provide anterior patterning signals during gastrulation. Here, we describe for the first time the defects caused by loss of Blimp1 function in the mouse. Blimp1 deficient embryos die at mid-gestation, but surprisingly early axis formation, anterior patterning and neural crest formation proceed normally. Rather, loss of Blimp1 expression disrupts morphogenesis of the caudal branchial arches and leads to a failure to correctly elaborate the labyrinthine layer of the placenta. Blimp1 mutant embryos also show widespread blood leakage and tissue apoptosis, and, strikingly, Blimp1 homozygous mutants entirely lack PGCs. At the time of PGC allocation around 7.25 days post coitum, Blimp1 heterozygous embryos exhibit decreased numbers of PCGs. Thus Blimp1 probably acts to turn off the default pathway that allows epiblast cells to adopt a somatic cell fate, and shifts the transcriptional program so that they become exclusively allocated into the germ cell lineage.

Allelic differences affecting invariant chain dependency of MHC class II subunit assembly.

The conserved invariant chain associates with highly polymorphic alpha and beta subunits guiding class II transport through the secretory pathway. Early associations of these three polypeptides inside antigen-presenting cells are poorly understood. The present experiments provide a detailed picture of the structure and fate of class II alpha and beta subunits in invariant chain mutants possessing different MHC haplotypes. In the absence of invariant chain, A alpha bA beta b is predominantly expressed as free A alpha b and A beta b chains by both splenocytes and activated LPS/IL-4 blasts, confirming that A alpha bA beta b assembly is strongly dependent on invariant chain coexpression. A quite different situation exists with respect to other allelic products. In the absence of invariant chain, A alpha kA beta k, E alpha kE beta k, and A alpha dA beta d molecules assemble efficiently and are conformationally similar to mature wild-type heterodimers. The contribution of invariant chain to subunit assembly thus differs for allelic variants, suggesting that sequential associations of alpha, beta, and invariant chain may be affected by polymorphic differences.

Requirement for endocytic antigen processing and influence of invariant chain and H-2M deficiencies in CNS autoimmunity

The role of processing in antigen (Ag) presentation and T cell activation in experimental allergic encephalomyelitis (EAE) was evaluated in wild-type mice, mice that selectively express either Ii p31 or p41, and mice completely deficient in Ii or H-2M. We demonstrate that processing of myelin oligodendrocyte glycoprotein (MOG) is required for presentation of the dominant encephalitogenic MOG epitope, p35-55. Ii p31- and p41-expressing mice developed EAE with similar incidence to wild-type mice, although p41 mice had a more severe course. Ag-presenting cells (APCs) from Ii- or H-2M-deficient mice could present p35-55, but not MOG, demonstrating that these APCs could not process native MOG. Ii- and H-2M-deficient mice were not susceptible to EAE by immunization with p35-55 or MOG or by adoptive transfer of encephalitogenic T cells. However, CD4+ T cells from p35-55-immunized H-2M-deficient mice proliferated, secreted IFN-gamma, and transferred EAE to wild-type, but not H-2M-deficient, mice. Thus, EAE resistance in H-2M-deficient mice is not due to an inability of APCs to present p35-55, or an intrinsic defect in the encephalitogenic T cell repertoire, but reflects a defect in APC function. Our results indicate that processing is required for initial Ag presentation and CNS T cell activation and suggest that autopathogenic peptides of CNS autoantigen may not be readily available for presentation without processing.

T Cells Distinguish MHC-Peptide Complexes Formed in Separate Vesicles and Edited by H2-DM

The peptide spanning residues 48-61 of hen egg white lysozyme (HEL) presented by I-A(k) gives rise to two T cell populations, referred to as type A and B, that distinguish the complex generated intracellularly upon processing of HEL from that formed with exogenous peptide. Here, we ascribe this difference to recognition of distinct conformers of the complex and show that formation of the two complexes results from antigen processing in different intracellular compartments and is dependent upon H2-DM. While the type A complex preferentially formed in a lysosome-like late vesicle, the type B complex failed to form in this compartment; this distinction was abolished in antigen-presenting cells lacking DM. Experiments in vitro indicated that H2-DM acts directly on the complex to eliminate the type B conformation. We conclude that different antigen-processing pathways generate distinct MHC-peptide conformers, priming T cells with distinct specificity that may play unique roles in immunity.

The p41 isoform of invariant chain is a chaperone for cathepsin L

The p41 splice variant of major histocompatibility complex (MHC) class II‐associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II‐restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41–CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow‐derived antigen‐presenting cells that lack p41. This defect mainly concerns the mature two‐chain forms of CatL, which depend on p41 to be expressed at wild‐type levels. Indeed, pulse–chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late‐endocytic compartments of antigen‐presenting cells.

Invariant Chain Controls the Activity of Extracellular Cathepsin L

Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p4165aa) of major histocompatibility complex class II–associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs). Here we show that interaction of p4165aa with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p4165aa in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-γ–treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p4165aa is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p4165aa stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response.

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

DM actions as a class II chaperone promote capture of diverse peptides inside the endocytic compartment(s). DM mutant cells studied to date express class II bound by class II-associated invariant chain-derived peptide (CLIP), a short proteolytic fragment of the invariant chain, and exhibit defective peptide-loading abilities. To evaluate DM functional contributions in k haplotype mice, we engineered a novel mutation at the DMa locus via embryonic stem cell technology. The present experiments demonstrate short-lived Ak/CLIP complexes, decreased Ak surface expression, and enhanced Ak peptide binding activities. Thus, we conclude that DM loss in k haplotype mice creates a substantial pool of empty or loosely occupied Ak conformers. On the other hand, the mutation hardly affects Ek activities. The appearance of mature compact Ek dimers, near normal surface expression, and efficient Ag presentation capabilities strengthen the evidence for isotype-specific DM requirements. In contrast to DM mutants described previously, partial occupancy by wild-type ligands is sufficient to eliminate antiself reactivity. Mass spectrometry profiles reveal Ak/CLIP and a heterogeneous collection of relatively short peptides bound to Ek molecules. These experiments demonstrate that DM has distinct roles depending on its specific class II partners.

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

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 peptide-editing function leads to immunodominance in CD4 T cell responses in vivo

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158–173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM−/−) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33–48 and 261–276). DM−/− B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.