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David M Kranz

Publications and source records attributed to David M Kranz.

9 recordsLinked to original sources
PubMed2003

A yeast display system for engineering functional peptide-MHC complexes.

In a cellular immune response, antigenic peptides derived by intracellular processing of foreign pathogens are bound to the class I major histocompatability complex (MHC I) and presented to CD8(+) cytotoxic T cells. Although the crystal structures of several different MHC products have been solved, many MHC molecules, including some associated with diseases, have not been amenable to biochemical and structural studies. The variability in this success is based largely on the fact that peptide-MHC complexes vary extensively in their stability. These properties also are intimately tied to the biological activity of the complexes. The ability to apply the techniques of directed evolution to this system in order to engineer stable complexes has been complicated by the trimeric structure of peptide-MHC complexes, requiring association of three polypeptides: the heavy chain, beta2-microglubulin (beta2m), and a short peptide. We show here that single-chain forms of peptide-MHC complexes can be expressed as Aga-2 fusions on the surface of yeast. Three different complexes, SIYRYYGL-K(b)-beta2m (SIYR-K(b)), EQYKFYSV-K(b)-beta2m (dEV8-K(b)), and SIINFEKL-K(b)-beta2m (OVA-K(b)), were expressed on yeast and detected by flow cytometry with a conformation-specific anti-K(b) antibody (B.8.24.3). In addition, yeast displaying K(b) loaded with exogenous SIYR and OVA peptides were recognized by a high-affinity T cell receptor that is specific for SIYR-K(b) and by an antibody (25.D1-16) that is specific for OVA-K(b), respectively. Finally, yeast that display the SIYRYYGL-K(b) also directly stimulated CD69 up-regulation on naive 2C T cells. Hence, yeast display represents a technology that can be used for directed evolution of any of the components of the trimeric pep-MHC complex.

Animals↗
PubMed2003

Allogeneic and syngeneic class I MHC complexes drive the association of CD8 and TCR on 2C T cells.

In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8(+) T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L(d) exhibits a less significant role for CD8 than recognition of the syngeneic MHC K(b). Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.

Animals↗
PubMed2003

Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation.

The relative roles of CD8, TCR:pepMHC affinity, and TCR:pepMHC dissociation rate in T cell activation have remained controversial. To determine the relationships among these factors, we used T cells transfected with normal and in vitro engineered alphabeta TCRs, in the presence or absence of CD8. The TCRs exhibited a wide range of affinities (K(D) values of 80 microM to 5 nM). T cells with the highest affinity TCRs were efficiently stimulated by peptide, with or without CD8. In contrast, CD8 was required for T cells that expressed TCRs with affinities typical of syngeneic reactions (K(D) values above approximately 3 microM). The results suggest that virtually all normal syngeneic interactions require CD8, which enhances peptide sensitivity by one million-fold or more.

Animals↗
PubMed2003

A model T-cell receptor system for studying memory T-cell development.

When T-cell clones were first grown in long-term cell culture, each clone was considered to be capable of displaying a limited range of functional activities, constrained by the clones' coreceptor, CD4 or CD8, and the specificity of its antigen-specific receptor (TCR) for one or a few peptides in association with a class I or class II MHC molecule. Subsequent studies, especially with transgenic mice, have shown, however, that T cells expressing the same receptor can be obtained in a variety of differentiated states, including naïve cells, activated effector cells, memory cells, and anergic or tolerant cells, as well as cells with or without a coreceptor. In each of these states T cells can display distinctly different responses to the peptide-MHC (pepMHC) complexes the TCR recognizes. Recently, memory T cells have received considerable attention, in part because of the likelihood that they confer long-term protective immunity against diverse infectious agents and possibly against some forms of cancer. Here we review some recent studies that our colleagues and we have carried out on memory CD8(+) T cells. These studies have made extensive use of cells that express the TCR called 2C. The diverse set of cells expressing the 2C TCR, in mice and in cultured clones and cell lines, are referred to as the 2C system. Before reviewing the studies of memory cells, we summarize the 2C system's main features, including evidence that a large and diverse array of pepMHC complexes, involving at least four class I MHC proteins, can stimulate TCR-mediated responses of 2C cells.

Amino Acid Sequence↗
PubMed2003

Directed evolution of a single-chain class II MHC product by yeast display.

Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.

Amino Acid Sequence↗
PubMed2002

TCRs with high affinity for foreign pMHC show self-reactivity.

T cells with high-affinity T cell receptors (TCRs) for a foreign peptide-major histocompatibility complex (pMHC) appear to be negatively selected, even though they have never seen the foreign antigen. To examine how this process operates, we used in vitro yeast display to isolate high-affinity TCRs from the T cell clone 2C. The TCRs showed fast on-rates, which were consistent with reduced CDR (complementarity determining region) flexibility, and cross-reactivity with other cognate pMHCs. T cell hybridomas transfected with a high-affinity TCR were stimulated by endogenous self-pMHC, which suggested that T cells bearing the TCR would be negatively selected. The immune system appears to maintain a repertoire of flexible, low-affinity TCRs at the expense of more effective high-affinity TCRs.

Animals↗
PubMedDate not supplied

Rapid method for measuring ScFv thermal stability by yeast surface display.

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.

Amino Acid Sequence↗