Theodore J. Tsomides

Evidence that a Single Peptide–MHC Complex on a Target Cell Can Elicit a Cytolytic T Cell Response

Using a chemically homogeneous radiolabeled peptide of high specific activity (125I-QLSPYPFDL, 3.5 x 10(18) cpm per mole) we show that at a peptide concentration (5 pM) causing half-maximal lysis of target cells by a cytolytic T lymphocyte (CTL) clone that recognizes the peptide in association with Ld, a class I MHC protein, only 3 peptide molecules on average are bound by Ld per target cell. From the distribution of Ld on the target cells, we suggest that a single peptide-MHC complex per target cell can trigger activation of the T cell cytolytic response.

A naturally occurring peptide recognized by alloreactive CD8+ cytotoxic T lymphocytes in association with a class I MHC protein

The antigenic structures that initiate T cell responses to foreign (allogeneic) cells have long attracted considerable interest. We have purified and sequenced a peptide from mouse spleen that is recognized in association with the class I MHC protein H-2Ld by 2C, an alloreactive CD8+ T cell clone. The peptide (LSP-FPFDL) greatly enhances the susceptibility of Ld+ cells to lysis by 2C, and this activity is completely blocked by a clonotypic antibody against the 2C T cell receptor. Thus, this study characterizes the naturally occurring peptide moiety of an MHC-I/peptide complex recognized by alloreactive CD8+ T cells. The peptide, which occurs in the thymus of MHC-disparate mice, can be used to study T cell development in mice expressing transgenes for the 2C T cell receptor.

Correlation Between the Number of T Cell Receptors Required for T Cell Activation and TCR–Ligand Affinity

The number of T cell receptors on CTL clone 2C that are required for recognition of various peptide-MHC or superantigen-MHC ligands were measured as a function of both the ligand density on target cells and the binding affinity of the TCR. Quantitative inverse correlations were determined between the number of TCRs required for recognition and the number of ligands on target cells, and the number of TCR required and the Ka of the TCR for the ligand. We propose and test predictive uses of these relationships to determine the number of endogenous peptide-MHC complexes on a target cell (when TCR affinity is known) or to determine the affinity of the TCR (when the number of ligands is known).

ANTIGEN-SPECIFIC T-CELL RECEPTORS AND THEIR REACTIONS WITH COMPLEXES FORMED BY PEPTIDES WITH MAJOR HISTOCOMPATIBILITY COMPLEX PROTEINS

The chapter gives an overview of an antigen-specific T-cell receptors and their reactions with complexes formed with major histocompatibility complex proteins. The receptors on the two major classes of lymphocytes, B and T cells, are similar structurally but profoundly different functionally. On B cells, the receptors are immunoglobulins (Ig) embedded in the cell surface as integral membrane proteins. The antigen-specific receptors on T cells (T-cell receptors or TCR) are also Ig-like cell surface integral membrane proteins; their recognition of antigens triggers T cells to exercise a great variety of functions but not to secrete the receptors. The enormous diversity of B- and T-cell receptors arises from the many germline gene segments that encode them; as each lymphocyte matures, different combinations of these segments are joined (combinatorial diversity) and additional variations in sequence are introduced at the junctures (junctional diversity), leading to an immense number of variable domain sequences. The TCR reviewed in this chapter normally recognizes and responds only to the complexes formed between small peptides and a specialized set of proteins encoded by the major histocompatibility complex (MHC). The chapter also discusses the T-Cell Receptor genes, proteins, ligand: peptide MHC complexes and TCR accessory proteins (CD3, CD4 and CD8).

A cytotoxic T lymphocyte clone can recognize the same naturally occurring self peptide in association with a self and a nonself class I MHC protein

The alloreactive CD8+ cytotoxic T lymphocyte (CTL) clone 2C was previously shown to recognize complexes made up of the class I MHC (MHC-I) molecule Ld and an octapeptide (LSPFPFDL, termed p2Ca) isolated from tissues of H-2d mice. Because peptide p2Ca has also been found in BALB.B (H-2b) mice, the strain from which clone 2C originated, the question arises as to whether these T cells can recognize peptide p2Ca in association with a self MHC protein of the H-2b haplotype. Here we show that 2C CTL do indeed recognize peptide p2Ca in association with Kb on the surface of H-2b cells or on transfected cells expressing Kb, but that an approximately 1000-fold higher concentration of this peptide is required to sensitize Kb+ than Ld+ target cells for lysis by 2C cells. However, the peptides binding to Kb was not much weaker than to Ld, with only an approximately 10-fold difference in the respective equilibrium constants. These results predict that the T cell receptor (TcR) of clone 2C has a much lower intrinsic affinity for p2Ca-Kb complexes than for p2Ca-Ld complexes, and they provide some quantitative limits on the requirements for triggering T cell-mediated autoimmune reactivity.

Potent Cytolytic Response by a CD8+ CTL Clone to Multiple Peptides from the Same Protein in Association with an Allogeneic Class I MHC Molecule

CTL clone 2C recognizes the allogeneic class I MHC molecule Ld in association with peptides derived from α-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface Ld than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with Ld as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.

In Vitro Radiolabeling of Peptides and Proteins

Radiolabeling of peptides or proteins is often performed to enhance the sensitivity of detection, to quantitate the binding of peptides to other molecules, or for radioimmunoassays. This unit presents a variety of assays for radiolabeling peptides and proteins with 125I, 131I, 14C, and 3H.