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 L(d) in association with peptides derived from alpha-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 L(d) 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 L(d) 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 (125)I, (131)I, (14)C, and (3)H.

Anti-melanoma cytotoxic T lymphocytes (CTL) recognize numerous antigenic peptides having 'self' sequences: autoimmune nature of the anti-melanoma CTL response.

A line of tumor-infiltrating lymphocytes (660TIL) specifically lysed the autologous HLA-A2+ melanoma (660MEL) and also most A2+ melanoma cell lines. We immunoprecipitated A2 from a large number (>10(12)) of 660MEL cells, extracted naturally processed peptides, fractionated them by HPLC, screened the fractions for recognition by 660TIL, and found a single predominant and a minor peak of activity. Although too little was recovered of the major 660MEL peptide to establish its sequence, HPLC fingerprinting showed that it did not correspond to any of the known A2-associated melanoma peptides recognized by T cells, including peptides from tyrosinase, MART-1/Melan-A, gp100 and MAGE-3. The major 660MEL antigenic peptide appears to be derived from MART-1/Melan-A but is neither AAGIGILTV nor ILTVILGVL nor any other MART-1/Melan-A peptide containing the A2 consensus motif. The multiplicity of melanoma peptides recognized by CD8+ T cells, most of which are non-mutated (including most likely the present 660MEL peptide), suggests the existence of unknown mechanisms, perhaps similar to those operating in autoimmune disorders, whereby T cells that recognize normal 'self' sequences become activated.

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).

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.

Maxadilan binds to membrane fractions of brain tissue.

Maxadilan is a potent vasodilator peptide isolated from salivary glands extracts of the hematophagous sand fly. Besides effects on the cutaneous vasculature, it has also been shown to relax rabbit aortic rings while elevating levels of cAMP. As a result of the effects on the skin and aorta, it was elected to undertake an examination of the tissue distribution of binding sites for maxadilan. In addition to specific binding in rabbit aorta and spleen, binding was detected in brain from various species including human, bovine, rabbit, rat and mouse with a kD of between 85 and 201 pM. Competitive displacement of [125I] maxadilan by a number of known vasoconstrictor peptides, vasodilator peptides and small molecule receptor ligands did not occur in the rabbit brain preparation. These results suggest the presence of specific binding sites in mammalian tissue for maxadilan whose endogenous ligand remains unknown.

Tissue distribution of natural peptides derived from a ubiquitous dehydrogenase, including a novel liver-specific peptide that demonstrates the pronounced specificity of low affinity T cell reactions.

The peptides recognized by CD8+ CTL normally arise by proteolysis of intracellular proteins. To learn whether these peptides are generated similarly in diverse cell types, we examined the variety and abundance of naturally processed peptides that derive from a ubiquitous enzyme, alpha-ketoglutarate dehydrogenase, and are recognized in association with the class I MHC protein, Ld, by a CTL clone (2C). A characteristic set of three peptides was found in diverse tissues, but their abundance varied greatly, apparently unrelated to differences in class I MHC expression, e.g., they were surprisingly abundant in liver. We also found in liver a fourth naturally processed peptide (p2Ca-Y4, LSPYPFDL) that differs by one oxygen atom from a previously characterized natural peptide (p2Ca, LSPFPFDL). CTL discrimination between these peptides in association with the same class I MHC protein, Kb, demonstrates the striking specificity that can be exhibited by low affinity T cell reactions.

Variations in the number of peptide-MHC class I complexes required to activate cytotoxic T cell responses.

We determined equilibrium constants for the binding of 16 peptides (based on four T cell epitopes) to three MHC class I proteins (A2, Kb, and Ld) on intact cells and estimated the number of accessible peptide-binding sites on these cells. From these results, and the concentrations of peptides required to sensitize target cells for lysis by CD8+ CTL, we conclude that the critical number of peptide-MHC complexes required per target cell for the activation of CTL responses varies with different combinations of peptide-MHC complexes and CTL clones from several thousand complexes to fewer than ten per target cell.

High-affinity reactions between antigen-specific T-cell receptors and peptides associated with allogeneic and syngeneic major histocompatibility complex class I proteins.

We report here that the intrinsic affinities of the antigen-specific T-cell receptors (TCR) of two unrelated CD8+ T-cell clones for their respective peptide-major histocompatibility complex (MHC) ligands are higher than the values generally thought to prevail for TCR. The TCR of one clone (2C) binds an allogeneic class I MHC protein (Ld) in association with an alpha-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL, termed QL9) with an intrinsic affinity (intrinsic equilibrium association constant) of 1-2 x 10(7) M-1. The TCR of the other clone (4G3) binds a syngeneic class I MHC protein (Kb) in association with an ovalbumin octapeptide (SIINFEKL, termed pOV8) with an intrinsic affinity of 1.5 x 10(6) M-1. A comparison of the two clones, combined with current views of T-cell repertoire selection in the thymus, leads us to propose that TCR affinities are generally likely to be higher for allogeneic MHC-peptide complexes than for syngeneic MHC-peptide complexes.

Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.

A cytotoxic T lymphocyte clone can recognize the same naturally occurring self peptide in association with a self and 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 peptide's 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.

Effects of peptide length and composition on binding to an empty class I MHC heterodimer.

Class I major histocompatibility complex (MHC) proteins present peptide antigens to T cells during the immune response against viruses. Peptides are loaded into newly synthesized class I heterodimers in the endoplasmic reticulum such that most or all cell surface class I molecules contain peptides derived from endogenous or foreign proteins. We previously reported the assembly of empty heterodimers of the murine class I MHC molecule H-2Kd, from denatured heavy and light chains from which endogenous peptides had been removed [Fahnestock et al. (1992) Science 258, 1658-1662]. Here we measure thermal stability profiles of empty versus peptide-filled molecules and compare the effects of human versus murine light chains on the overall stability of the Kd heterodimer. The majority of empty heterodimers are stable at 37 degrees C regardless of the species of light chain, indicating that our previous report of the unexpectedly high thermal stability was an intrinsic property of the Kd molecule and not due to use of a murine/human chimeric protein. Binding constants are derived for a series of peptides interacting with empty Kd heterodimers. The dissociation constants of four known Kd-restricted peptides range from 2.3 x 10(-7) to 3.4 x 10(-8) M. Using a series of 24 analog peptides, the effects of length and peptide composition on binding affinity of one Kd-restricted peptide are explored, and the results are interpreted with reference to the known three-dimensional structures of class I MHC protein/peptide complexes.

A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD8+ T-cell clone.

We previously isolated from mouse spleen an octapeptide (LSPFPFDL) that in association with the class I major histocompatibility complex protein Ld is recognized by the antigen-specific receptor of an alloreactive CD8+ T-cell clone (2C). Guided by an assay dependent upon the same 2C T-cell receptor, we have now isolated from the same source another naturally occurring peptide. The second peptide (VAITRIEQLSPFPFDL) includes the entire octapeptide sequence and preliminary evidence suggests that it may be a natural precursor of the octapeptide. On finding extensive sequence homology between the 16-mer and part of human 2-oxoglutarate dehydrogenase, we determined the cDNA sequence of mouse 2-oxoglutarate dehydrogenase and found that the deduced amino acid sequence matches precisely the two naturally occurring peptides, indicating their origin by cellular processing of this ubiquitous self protein.

Stoichiometric labeling of peptides by iodination on tyrosyl or histidyl residues.

Radioiodination with 125I or 131I is a favored technique for labeling biologically active peptides or proteins because of high specific radioactivities and convenience in counting gamma-emissions. Previous studies used trace labeling, in which fewer than 1% of the molecules are iodinated. We describe procedures for obtaining stoichiometrically iodinated and therefore chemically homogeneous peptides with specific activities exceeding 10(7) cpm/micrograms (approximately 10 Ci/mmol). By analyzing the pH dependence of iodination on tyrosyl and histidyl residues, we show that the method described can be applied to many short peptides and optimized for labeling on tyrosine and/or histidine. The power of reverse-phase HPLC is exploited to resolve multiple products substituted with different molar equivalents of iodine from each other and from unlabeled peptide. Specific radioactivity ratios can be used to identify the products, as confirmed by Edman sequence analysis under conditions that separate iodinated tyrosine and histidine derivatives from all other amino acids. We also show that the biological activities of iodinated and uniodinated peptides can differ by several orders of magnitude in a T cell assay and demonstrate the usefulness of stoichiometric labeling to overcome ambiguities inherent in studying biological activities with trace-labeled peptides.

Sample preparation for HPLC by Centricon ultrafiltration.

Peptides and other bioactive materials can be purified from complex biological sources by reverse-phase high-performance liquid chromatography (RP-HPLC), provided the mixture is suitably prepared before injection onto an HPLC system. Ultrafiltration offers a convenient and rapid sample preparation technique with numerous advantages over alternative methods such as conventional gel filtration chromatography. We demonstrate the use of ultrafiltration as an HPLC sample preparation step in the purification of peptides bound to class I major histocompatibility complex (MHC-I) membrane proteins. When ultrafiltration was performed with a Centricon-10 ultrafiltration device, peptides were efficiently separated from the alpha (45 kDa) and beta 2m (12 kDa) chains of MHC-I proteins and could be subjected to HPLC without further treatment. Furthermore, even samples as crude as whole cell lysates or supernatants could be prepared for HPLC in a single ultrafiltration step, affording a remarkably straightforward route to the purification of biologically important peptides.

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.

An optimal viral peptide recognized by CD8+ T cells binds very tightly to the restricting class I major histocompatibility complex protein on intact cells but not to the purified class I protein.

CD8+ cytotoxic T lymphocytes recognize cell surface complexes formed by class I major histocompatibility complex (MHC-I) glycoproteins and antigenic peptides. We have identified a peptide nonamer (termed IV9) derived from the human immunodeficiency virus that is over a millionfold more active (at subpicomolar concentrations) than peptide analogues longer or shorter by one or two amino acid residues. Although IV9 does not detectably bind to isolated MHC-I molecules as measured by equilibrium dialysis, we quantitated its specific binding in unaltered form to MHC-I on intact cells. Less than 1% of cell surface MHC-I forms complexes with IV9, which suffices to trigger maximal cytotoxic T-lymphocyte activity. By contrast, a peptide dodecamer that includes the IV9 sequence and is active at micromolar concentrations does not bind to MHC-I on intact cells, raising the possibility that this longer peptide undergoes processing. Using stoichiometrically iodinated IV9 to obviate the ambiguities associated with trace labeling methods, we measured the dissociation kinetics of purified peptide/MHC-I complexes isolated by affinity chromatography and found these complexes to be exceedingly stable (t1/2 = 200-600 hr).