SummaryThe T lymphocytes mediating autoimmune destruction of pancreatic [3 cells in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes mellitus (IDDM) may be generated due to functional defects in hematopoietically derived antigen-presenting cells (APC). However, it has not been clear which particular subpopulations ofAPC (B lymphocytes, macrophages, and dendritic cells) contribute to the development and activation of diabetogenic T cells in NOD mice. In the current study we utilized a functionally inactivated immunoglobulin (Ig)l.* allele (Ig/x ''a) to generate a "speed congenic" stock of B lymphocyte-deficient NOD mice that are fixed for linkage markers delineating previously identified diabetes suscepnbility (Ida") genes, These B lymphocyte NOD.Igi.,, ''tt mice had normal numbers of T cells but were free of overt IDDM and insulitis resistant, while the frequency of disease in the B lymphocyte intact segregants was equivalent to that of standard NOD mice in our colony. Thus, B lymphocytes play a heretofore unrecogmzed role that is essential for the initial development and/or activation of [3 cell autoreactive T cells in NOD mice.

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately two-fold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we demonstrate that engineered haplodeficiency of IL-2 gene expression not only reduces T cell IL-2 production by two-fold but also mimics the autoimmune dysregulatory effects of the naturally-occurring susceptibility alleles of IL-2. Reduced IL-2 production achieved by both genetic mechanisms correlates with fewer and less functional CD4+CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.Multifactorial diseases with high population prevalence develop as a result of interactions between multiple genetic and environmental factors. Since the early 1990s, several loci have been mapped by genetic linkage and association analyses in humans and in rodent models of autoimmune disease, including type 1 diabetes (T1D). T1D is caused by the destruction of the insulin-producing pancreatic beta cells by various immune cell types, including CD8+ cytotoxic T-cells. In human T1D, four loci, in addition to the HLA region, have been identified: the genes encoding insulin, the negative immunoregulatory molecules CTLA-4 and LYP, and, most recently, the alpha chain of the interleukin-2 receptor (CD25) 1 . All of these common variants or haplotypes support the concept that autoimmunity is a part of normal physiology, and that the balance between immune responses to foreign antigens and

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic ␤ cells. CD8 ؉ T cells are essential for this ␤ cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8 ؉ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.T he nonobese diabetic (NOD) mouse is a widely studied model of human type 1 diabetes, an autoimmune disease characterized by inflammation of pancreatic islets (insulitis) followed by T cell-mediated destruction of insulin (INS)-producing ␤ cells (1). Both CD4 ϩ and CD8 ϩ T cells are required for this pathogenic process (1); however, CD8 ϩ T cells appear to be responsible for the initial ␤ cell insult (1-3). Whereas the pathogenicity of B cells and autoantibodies is less clear, the autoantigens currently believed to contribute to autoimmune diabetes pathogenesis in NOD mice and humans all were originally identified based on the presence of specific autoantibodies rather than by T cell recognition (4-6). Little is known of the ␤ cell antigens targeted by the pathogenic CD8 ϩ T cells. Although one study identified an INS peptide as the antigenic target of the majority of islet-infiltrating CD8 ϩ T cells in NOD mice (7), the prevalence of these INS-reactive CD8 ϩ T cells was not confirmed in subsequent studies (8,9).A substantial proportion of ␤ cell-autoreactive CD8 ϩ T cells isolated from NOD islets express a shared T cell receptor ␣ (TCR␣) chain (V␣17-J␣42), suggesting recognition of a common ␤ cell peptide (3, 10). These T cells do not recognize the antigenic INS peptide mentioned above (11,12). The pathogenicity of this prevalent T cell population has been well established through studies of the 8.3 T cell clone (a representative T cell clone of the V␣17-J␣42-expressing T cell population) (13, 14). 8.3-Like T cells are present in the earliest islet infiltrates of NOD mice (3) and undergo avidity maturation as islet inflammation progresses to overt disease (8). At any given time, 8.3-like T cells can constitute up to 30-40% of the islet-associated CD8 ϩ T cells (9). Strikingly, quantification of 8.3-like T cells in peripheral blood predicts diabetes development in individual NOD mice (9), unlike any other single immune indicator identified to date. Although the prevalence and pathogenicity of 8.3-like T cells has been clearly established, the identity of their ligand has remained elusive. Materials and MethodsMice. NOD͞Lt mice were maintained by brother-sister mating. 8.3-TCR␣␤-transgenic NOD mice, designated 8.3-NOD, ha...

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually Xat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long Wngerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proofof-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

The Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms. We have examined the in vivo role of the Jagged2 (Jag2) gene, which encodes a ligand for the Notch family of transmembrane receptors, by making a targeted mutation that removes a domain of the Jagged2 protein required for receptor interaction. Mice homozygous for this deletion die perinatally because of defects in craniofacial morphogenesis. The mutant homozygotes exhibit cleft palate and fusion of the tongue with the palatal shelves. The mutant mice also exhibit syndactyly (digit fusions) of the fore-and hindlimbs. The apical ectodermal ridge (AER) of the limb buds of the mutant homozygotes is hyperplastic, and we observe an expanded domain of Fgf8 expression in the AER. In the foot plates of the mutant homozygotes, both Bmp2 and Bmp7 expression and apoptotic interdigital cell death are reduced. Mutant homozygotes also display defects in thymic development, exhibiting altered thymic morphology and impaired differentiation of ␥␦ lineage T cells. These results demonstrate that Notch signaling mediated by Jag2 plays an essential role during limb, craniofacial, and thymic development in mice.