Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic ␤ cells. Although both major histocompatibility complex class I-restricted CD8؉ and class II-restricted CD4 ؉ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8؉ T cells responsible, we isolated and propagated in vitro CD8 ؉ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2K d , and showed a diverse T cell receptor ␤ chain repertoire. In contrast, their ␣ chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of V␣17 family members frequently joined to the J␣42 gene segment. These results suggest that a number of the CD8 ؉ T cells participating in the initial phase of autoimmune ␤ cell destruction recognize a common structural component of K d ͞peptide complexes on pancreatic ␤ cells, possibly a single peptide.Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by T cell-mediated destruction of pancreatic islet ␤ cells (1). The nonobese diabetic (NOD) mouse (2) constitutes a widely studied model system for IDDM, as it shares many of the characteristics of the human disease. For example, human patients and NOD mice both develop lymphocytic infiltration of islets (insulitis) and subsequent ␤ cell destruction mediated by T lymphocytes. Cell-surface ␣␤ T cell receptors (TCRs) enable such T lymphocytes to recognize specific antigens on the surfaces of target cells in the form of peptides complexed with major histocompatibility complex (MHC) molecules, with CD8 ϩ cytotoxic T lymphocytes being restricted to class I MHC molecules and CD4 ϩ T cells to class II.Certain unusual MHC class II alleles provide the strongest genetic component of IDDM susceptibility in both humans and NOD mice (3). Thus, it is not surprising that autoreactive CD4 haplotype (15). However, although the requirement for CD8 ϩ T cells in IDDM development is clear, whether they are only needed to initiate the earliest events of ␤ cell destruction or are critical to all stages of diabetogenesis remained unknown and is one of the questions addressed in this study.It is also necessary to define the characteristics and specificities of MHC class I-restricted effectors participating in the earliest initiative phases of IDDM. In view of the importance of understanding the nature of these initiating T cells, we have developed a technique for the isolation of monoclonal and oligoclonal populations of NOD ␤ cell-cytotoxic CD8 ϩ T cells that uses islets from a newly developed stock of NODscid.RIPB7 mice as a potent source of stimulating antigen. This approach has enabled us to isolate cytotoxic CD8 ϩ T cells from pred...

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Minor histocompatibility Ags (minor H Ags) are substantial impediments to MHC-matched solid tissue and bone marrow transplantation. From an antigenic standpoint, transplantation between MHC-matched individuals has the potential to be remarkably complex. To determine the extent to which the immune response is simplified by the phenomenon of immunodominance, we used peptide/MHC tetramers based on recently discovered minor H Ags (H60, H13, and HY) and monitored in vivo CD8 T cell responses of female C57BL/6 mice primed with MHC-matched, but background-disparate, male BALB.B cells. CD8 T cells against H60 overwhelmed responses to the H13 and HY throughout primary and secondary challenge. H60 immunodominance was an inherent quality, overcoming a lower memory precursor frequency compared with that of H13 and evoking a T cell response with diverse TCRVβ usage. IFN-γ staining examining congenically defined minor H Ags extended H60 dominance over additional minor H Ags, H28, H4, and H7. These four minor H Ags accounted for up to 85% of the CD8 T cell response, but H60 stood out as the major contributor. These findings show that immunodominance applies to antigenically complex transplantation settings in vivo and that the responses to the H60 minor H Ag dominates in this model. We suggest that immunodominant minor H Ags are those that result from the absence of a self analog.

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-α rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-κB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-κB (IκBα; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-κB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-κB binding sequences, respectively. The intronic NF-κB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-α stimulation or overexpression of NF-κB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-κB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-α stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-κB signaling via intronic sequences regulates FcRn expression and function.