Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail.

Vitiligo is an autoimmune disease of the skin mediated by CD8 T cells that kill melanocytes and create white spots. Skin lesions in vitiligo frequently return after discontinuing conventional treatments, supporting the hypothesis that autoimmune memory is formed at these locations. We found that lesional T cells in mice and humans with vitiligo display a resident memory (T) phenotype, similar to those that provide rapid, localized protection against reinfection from skin and mucosal-tropic viruses. Interleukin-15 (IL-15)-deficient mice reportedly have impaired T formation, and IL-15 promotes T function ex vivo. We found that both human and mouse T express the CD122 subunit of the IL-15 receptor and that keratinocytes up-regulate CD215, the subunit required to display the cytokine on their surface to promote activation of T cells. Targeting IL-15 signaling with an anti-CD122 antibody reverses disease in mice with established vitiligo. Short-term treatment with anti-CD122 inhibits T production of interferon-γ (IFNγ), and long-term treatment depletes T from skin lesions. Short-term treatment with anti-CD122 can provide durable repigmentation when administered either systemically or locally in the skin. On the basis of these data, we propose that targeting CD122 may be a highly effective and even durable treatment strategy for vitiligo and other tissue-specific autoimmune diseases involving T.

Antibody therapy is coming of age, with 15 monoclonal antibodies approved for therapeutic use in the United States and many others currently undergoing clinical trials (1). The advent of antibody engineering over the past two decades has contributed to the recent clinical success of therapeutic antibodies. The development of chimeric (2) and humanized (3) antibodies not only reduced the potent immunogenicity of rodent antibodies in humans but also improved the serum halflives and efficacy of such therapeutics compared with rodent antibodies. Phage display (4) and other display technologies have led to the ability to increase the affinity of antibodies for their target antigens. More recently, antibody engineering has been used to modify the effector functions of antibodies by altering their binding to C1q (5) and various Fc␥ receptors (6).The neonatal Fc receptor (FcRn) 1 is a heterodimer that comprises a transmembrane ␣ chain with structural homology to the extracellular domains of the ␣ chain of major histocompatibility complex class I molecules, and a soluble light chain consisting of ␤2-microglubulin (␤2m) (7). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (8). Evidence for the latter role initially came from studies indicating an unusually short serum halflife for IgG antibodies in ␤2m-deficient mice (9 -11). This observation led to the generation of mutant mouse hinge-Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (12). Recently, several studies have identified human IgG 1 mutants with enhanced FcRn binding (6, 13), although no improvement in the serum half-lives of these mutants was observed in mice (13) or reported in primates.The binding of IgG to FcRn is sharply pH-dependent; IgG binds to FcRn under mildly acidic conditions and is released under slightly basic conditions (14). It has been hypothesized that pinocytosed IgG antibodies are captured by FcRn in acidified endosomes, rescued from degradation in lysosomes, recycled back to the cell surface, and returned to the circulation (8). Mutagenesis studies have identified both the mouse (15, 16) and human (17) Fc residues believed to be important in mediating pH-dependent binding. The results of the mutagenesis studies are consistent with the interpretation of a crystallographic study of the Fc⅐FcRn interaction (18). In the current study, molecular modeling was used to identify residues in the human IgG Fc near the FcRn binding site that, when mutated, might alter binding to FcRn without affecting the pH dependence of this interaction. Following exhaustive mutagenesis at these positions, several IgG 2 mutants were identified with improved binding to FcRn at pH 6.0 that retained the property of pH-dependent release. A pharmacokinetics study in rhesus monkeys showed that two mutant IgG 2 antibodies with increased FcRn binding affinity had considerably longer serum half-lives than the wild-type antibody. EXPERIMENTAL PROCEDURESMolecular Modeling-Molecular models o...

Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including ζ chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of ζ (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cγ-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 × anti-CD4 F(ab)′2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3.