Various kinetic studies on nucleotide incorporation by DNA polymerases have established that a rate-limiting step occurs that is crucial in the mechanism of discrimination between correct versus incorrect nucleotide. Crystallographic studies have indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. However, there is no direct evidence to support this hypothesis. In order to investigate whether or not the open-to-closed conformational transition affecting the fingers subdomain is rate limiting, we have developed a fluorescence resonance energy transfer (FRET) system, which monitors motions of the fingers subdomain. We establish that the closing of the fingers subdomain is significantly faster than the kinetically determined rate-limiting step. We propose that the rate-limiting step occurs after the closing of the fingers subdomain and is caused by local reorganization events in the active site.

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden . Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8 + T cells from WNV-infected mice induced IFN-c production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope-and nonstructural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells—particularly when the antigenic peptide has relatively weak affinity for the MHC.

MHC class I peptide complexes (pMHC) are routinely used to enumerate T cell populations and are currently being evaluated as vaccines to tumors and specific pathogens. Herein, we describe the structures of three generations of single-chain pMHC progressively designed for the optimal presentation of covalently associated epitopes. Our ultimate design employs a versatile disulfide trap between an invariant MHC residue and a short C-terminal peptide extension. This general strategy is nondisruptive of native pMHC conformation and T cell receptor engagement. Indeed, cell-surface-expressed MHC complexes with disulfide-trapped epitopes are refractory to peptide exchange, suggesting they will make safe and effective vaccines. Furthermore, we find that disulfide-trap stabilized, recombinant pMHC reagents reliably detect polyclonal CD8 T cell populations as proficiently as conventional reagents and are thus well suited to monitor or modulate immune responses during pathogenesis.

Somatic mutations in JAK2 are frequently found in myeloproliferative diseases, and gain-of-function JAK3 alleles have been identified in M7 acute myeloid leukemia (AML), but a role for JAK1 in AML has not been described. We screened the entire coding region of JAK1 by total exonic resequencing of bone marrow DNA samples from 94 patients with de novo AML. We identified 2 novel somatic mutations in highly conserved residues of the JAK1 gene (T478S, V623A), in 2 separate patients and confirmed these by resequencing germ line DNA samples from the same patients. Overexpression of mutant JAK1 did not transform primary murine cells in standard assays, but compared with wild-type JAK1, JAK1 T478S , and JAK1 V623A expression was associated with increased STAT1 activation in response to type I interferon and activation of multiple downstream signaling pathways. This is the first report to demonstrate somatic JAK1 mutations in AML and suggests that JAK1 mutations may function as disease-modifying mutations in AML pathogenesis. IntroductionThe Janus kinase (JAK) genes encode nonreceptor tyrosine kinases, including 4 family members JAK1, JAK2, JAK3, and TYK2. 1 Ligand binding to cytokine receptor results in transphosphorylation of JAK kinases, and activated JAK kinases in turn phosphorylate receptor intracellular domain tyrosines to create docking sites to recruit SH2-domain-containing downstream signaling molecules-especially the signal transducers and activators of transcription (STATs). 2 The first genetic evidence implicating constitutive JAK-STAT activation in oncogenesis was derived from studies of signal transduction in the fruit fly. Fruit flies expressing constitutively activated Drosophila melanogaster JAK homolog, hopscotch, develop a hematopoietic neoplasia resembling leukemia. 3,4 Dysregulation of the JAK-STAT signaling pathway has been described in a variety of malignancies, including hematopoietic neoplasms. 5 Direct evidence for aberrant JAK activation in human tumorigenesis was first confirmed by the cloning of the t(9;12) translocation breakpoint in acute lymphocytic leukemia and the identification of the TEL-JAK2 fusion protein. 6 The somatic JAK2 mutation V617F has been reported in most patients with polycythemia vera (PV) as well as in approximately one third of patients with essential thrombocythemia and or idiopathic myelofibrosis. 7-10 The JAK2 V617F protein demonstrates constitutively activated kinase activity in vitro and coexpression of JAK2 V617F with type I cytokine receptors in Ba/F3 cells resulted in cytokine-independent JAK-STAT signaling and growth factor-independent cell growth. 11Additional JAK2 gain-of-function mutations have been identified in PV, idiopathic erythrocytosis, and acute leukemia. 12,13 Furthermore, activating alleles of JAK3 have been reported in association with acute megakaryoblastic leukemia. 14 JAK1 plays important roles in cytokine signal transduction 15 but has not been implicated in leukemia development. We sought to evaluate the frequency and the possible contributi...