The demand for monoclonal antibodies (mAbs) in biomedical research is significant, but the current methodologies used to discover them are both lengthy and costly. Consequently, the diversity of antibodies available for any particular antigen remains limited. Microengraving is a soft lithographic technique that provides a rapid and efficient alternative for discovering new mAbs. This protocol describes how to use microengraving to screen mouse hybridomas to establish new cell lines producing unique mAbs. Single cells from a polyclonal population are isolated into an array of microscale wells (~105 cells per screen). The array is then used to print a protein microarray, where each element contains the antibodies captured from individual wells. The antibodies on the microarray are screened with antigens of interest, and mapped to the corresponding cells, which are then recovered from their microwells by micromanipulation. Screening and retrieval require approximately 1–3 d (9–12 d including the steps for preparing arrays of microwells).

Summary The widely-conserved natural resistance associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a—absent from a previous Nramp structure. Mapping our cysteine accessibility scanning results onto this structure, we identified the metal permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations, which impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells.

Class I MHC tetramers allow direct phenotypic identification of CD8 ؉ T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8 ؉ T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2K b and H-2D b with full retention of their structural and functional integrity. We ranked all possible H-2K b octameric and H-2D b nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from Ϸ2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8 ؉ T cells.conditional ligands ͉ crystal structure ͉ consensus epitope prediction

Transporters in the Golgi apparatus membrane translocate nucleotide sugars from the cytosol into the Golgi lumen before these can be substrates for the glycosylation of proteins, lipids, and proteoglycans. We have cloned the mammalian Golgi membrane transporter for uridine diphosphate-N-acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized Kluyveromyces lactis mutant deficient in Golgi transport of the above nucleotide sugar. Phenotypically corrected transformants were separated from mutants in a f luorescent-activated cell sorter after labeling of K. lactis cells with f luorescein isothiocyanate (FITC) conjugated to Griffonia simplicifolia II lectin, which binds terminal N-acetylglucosamine. A 2-kb DNA fragment was found to restore the wild-type cell lectin binding phenotype, which reverted to the mutant one upon loss of the plasmid. The DNA fragment contained an ORF encoding a hydrophobic, multitransmembrane spanning protein of 326 aa that had only 22% amino acid sequence identity with the corresponding transporter from K. lactis but showed 53% amino acid sequence identity to the mammalian UDPgalactose transporters and 40% to the CMP-sialic acid transporter. Golgi vesicles from the transformant regained their ability to transport UDP-GlcNAc in an assay in vitro. The above results demonstrate that the mammalian Golgi UDPGlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane.

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1 + ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1−/− mice have exclusively IgG1 antiovalbumin in their serum, OBI mice show elevated levels of antiovalbumin of nearly all isotypes 3′ of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM + IgG1 + double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3.allelic exclusion | natural antibodies | TN mice B cells exist as a polyclonal pool such that an antibody response may be mounted against any possible pathogen. When a B-cell recognizes its cognate antigen through its B-cell receptor (BCR), the B cell proliferates and differentiates into antibody-secreting plasma cells and a smaller population of memory B cells. Clonal selection theory rests on the idea that a B cell expresses a BCR of a single specificity; harmful consequences could ensue if a B cell activated in the normal course of an immune response also produced a second antibody that reacted with self-antigen. Several mechanisms ensure that a B cell produces only a single specificity BCR, including monoallelic initiation of recombination, restricted access of the RAG proteins, rapid entry into the cell cycle, chromatin remodeling, and subunit pairing constraints (1, 2). Thus, nearly all B cells express a BCR encoded by single alleles at the IgH and Igκ or λ loci. Allelic exclusion, however, is not perfect, and ∼0.01% of B cells express two rearranged IgH genes (3), whereas 1-7% of B cells express two rearranged Igκ genes (4, 5).B-cell development begins in the bone marrow when B-cell progenitors express RAG1/2 and rearrange the Ig heavy chain locus (6). D to J rearrangement occurs first, often on both chromosomes, followed by V to DJ rearrangements. A productive, inframe VDJ results in cell surface expression of the Ig heavy chain paired with VpreB and λ5 surrogate light chains. Pre-BCR signaling induces proliferation and prevents further rearrangements on the other chromosome. After several rounds of cell division, pre-B cells re-express the RAG genes and engage in V to J rearrangement of the Igκ light chain locus. Surface expression of the BCR marks transition to the immature B-cell stage.Once in the periphery, B cells engage a wider array of antigens, including those from food and commensal microbes. Transitional B cells further differentiate into one of three major B-cell populations: long-lived marginal zone (MZ) B cells that reside in the marginal zone sinus of the spleen; follicular B cells that form the B-cell zones of spleen and lymph nodes; and B-1 B cells that reside mainly in the peritoneal cavity and are a major source of natural...

The UDP-galactose (UDP-Gal) transporter present in the Golgi apparatus is a member of a transporter family comprising hydrophobic proteins with multiple transmembrane domains. Co-immunoprecipitation experiments showed that the full-length UDP-Gal transporter protein forms oligomeric structures in the MDCK cell. A ricin-resistant mutant of the MDCK cell line (MDCK-RCA(r)) is deficient in galactose linked to macromolecules because of a lower UDP-Gal transport rate into the Golgi apparatus. We cloned this mutated protein and found that it contains a stop codon close to the 5' terminus of its open reading frame. We also detected a shorter splicing variant of the UDP-Gal transporter which contains a 183-nt in-frame deletion in both the wild-type and the mutant mRNA. We showed that the protein, when overexpressed, is localized in the Golgi apparatus and could partially correct the phenotype of the MDCK-RCA(r) and CHO-Lec8 mutant cell lines. The level of mRNA of the UDP-Gal transporter is much lower (25-30 copies per cell) than those of the CMP-sialic acid transporter (100 copies per cell), UDP-N-acetylglucosamine transporter (80 copies per cell), and GDP-fucose transporter (65 copies per cell). The transcript level of the shorter splicing variant of the UDP-Gal transporter is extremely rare in wild-type MDCK cells (a few copies per cell), but it is significantly increased in the mutant, RCA-resistant cells.

Soluble heat shock fusion proteins (Hsfp) stimulate mice to produce CD8+ CTL, indicating that these proteins are cross-presented by dendritic cells (DC) to naive CD8 T cells. We report that cross-presentation of these proteins depends upon their binding to DC receptors, likely belonging to the scavenger receptor superfamily. Hsfp entered DC by receptor-mediated endocytosis that was either inhibitable by cytochalasin D or not inhibitable, depending upon aggregation state and time. Most endocytosed Hsfp was transported to lysosomes, but not the small cross-presented fraction that exited early from the endocytic pathway and required access to proteasomes and TAP. Naive CD8 T cell (2C and OT-I) responses to DC incubated with Hsfp at 1 μM were matched by incubating DC with cognate octapeptides at 1–10 pM, indicating that display of very few class I MHC-peptide complexes per DC can be sufficient for cross-presentation. With an Hsfp (heat shock protein-OVA) having peptide sequences for both CD4+ (OT-II) and CD8+ (OT-I) cells, the CD4 cells responded far more vigorously than the CD8 cells and many more class II MHC-peptide than class I MHC-peptide complexes were displayed.