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.

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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 aflogeneic class I MHC protein (Ld) in association with an a-ketoglutarate dehydrogenase nonapeptide (QLSPFPFDL, termed QL9) with an intrinsic anity (intrinsic equilibrium association constant) of 1-2 x 107 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 inins affinity of 1.5x 106 M-l. A compain of the two clones, combined with current views of T-cell repertoire selection in the thymus, leads us to propose that TCR afities are generally likely to be higher for algeneic MHC-peptide complexes than for syngeneic MHC-peptide complexes.Because the somatic hypermutation that underlies the generation of high-affinity antibodies is not evident in T cells, it has long been surmised that the affinities of antigen-specific T-cell receptors (TCR) for their natural ligands, now known to be peptide-major histocompatibility complex (MHC) complexes, are likely to be low and in the narrow range (104 to 105 M-1) exhibited by antibodies made early in immune responses, before the onset of somatic hypermutation. Previously, however, we showed (1) that one of the two murine T-cell clones studied here (clone 2C) has a relatively high intrinsic affinity (2 x 106 M-1) for its ligand: Ld, a class I MHC protein, in association with an octapeptide (LSPF-PFDL, termed p2Ca) that derives from murine a-ketoglutarate dehydrogenase (a-KGDH) (2, 3). This clone also recognizes Ld in association with several other peptides from a-KGDH. One of them, the nonapeptide QLSPFPFDL (termed QL9), proved to be exceptionally active in sensitizing Ld+ target cells (T2-Ld) for lysis by 2C cells. We show here that the affinity of the TCR on 2C cells for the complex formed by this peptide with Ld (QL9-Ld) is about 10 times greater than for p2Ca-Ld and, to our knowledge, is the highest affinity value found so far for any TCR.Since the 2C clone arose in a mouse of the H-2b haplotype, its high-affinity binding of the peptide-Ld complex is an allogeneic reaction (alloreaction). To compare it with a CD8+ cytotoxic T lymphocyte (CTL) clone that is also highly reactive cytolytically but recognizes a syngeneic MHCpeptide complex, we took advantage of clone 4G3. This clone, which also arose in an H-2b mouse, reacts specifically with the ovalbumin octapeptide pOV8 (SIINFEKL) in association with Kb. In this syngeneic reaction, the TCR affinity for its peptide-MHC complex was about 10-fold lower than in the alloreaction. Though the comparison involves only two clones, when the findings are considered in light of recent studies ofT-cell repertoire selection in the thymus (4-6), they lead us to propose that the affinity ofa TCR for peptide-MHC complexes will ge...

SurrtmaryWe have established long-term cultures of several cell lines stably and uniformly expressing human immunodeliciency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-l-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 pr6cessed 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 and =12 molecules per infected Jurkat-A2 cell, respectivdy. 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 peptidetreated target cells.

A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD8+ T-cell clone (T-ceil ABSTRACTWe previously isolated from mouse spleen an octapeptide (LSPFPFDL) that in association with the class I major histocompatibiity complex protein Ld is recognized by

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 (antigen Contributed by Herman N. Eisen, September 17, 1991 ABSTRACT CD81 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 millionfd 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 maxmal 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

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).

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