Eduardo Guillen

A Proposed Mechanism for the Induction of Cytotoxic T Lymphocyte Production by Heat Shock Fusion Proteins

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 partners 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.

Screening individual hybridomas by microengraving to discover monoclonal antibodies

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).

Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers

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-2Kb and H-2Db with full retention of their structural and functional integrity. We ranked all possible H-2Kb octameric and H-2Db nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from ≈2,000 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.

A Role for Toll-Like Receptor 4 in Dendritic Cell Activation and Cytolytic CD8+ T Cell Differentiation in Response to a Recombinant Heat Shock Fusion Protein

Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp’s ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.

Multiple intracellular routes in the cross-presentation of a soluble protein by murine dendritic 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.

IgG1+ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells

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 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin 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.

Ubiquitin-dependent control of class II MHC localization is dispensable for antigen presentation and antibody production.

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production.