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The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, β2m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg(65) and Lys(66) and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg(65)-Lys(66) region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.

T cell receptors (TCR) adopt a similar orientation when binding with major histocompatibility complex (MHC) molecules, yet the biological mechanism that generates this similar TCR orientation remains obscure. We show here the cocrystallographic structure of a mouse TCR bound to a human MHC molecule not seen by the TCR during thymic development. The orientation of this xenoreactive murine TCR atop human MHC deviates from the typical orientation more than any previously determined TCR/MHC structure. This unique orientation is solely due to the placement of the TCR Vα domain on the MHC. In light of new information provided by this structure, we have reanalyzed the existing TCR/MHC cocrystal structures and discovered unique features of TCR Vα domain position on class I MHC that correlate with CD8 dependence. Finally, we propose that the orientation seen in TCR recognition of MHC is a consequence of selection during T cell development.

Binding of the TCR to class I or class II MHC molecules ever, a poorer correlation was observed between activity complexed with peptide is necessary for T cell activation and dissociation rate, as the TCR/peptide/MHC comand initiation of a cell-mediated immune response. Sim- plex with one of the antagonist peptides had a half-life ple alterations to antigenic peptides can result in a range almost twice as long as that observed with the weak of effects from loss of TCR signaling to weak or partial agonist. This prompted the authors to suggest that corsignaling or to the phenomenon of TCR antagonism relations observed between activity and dissociation (Sloan-Lancaster and Allen, 1996). Antagonist peptides rate may be coincidental, reflecting an underlying corredo not normally produce any of the outcomes associ- lation with affinity. ated with activation but result in an inhibition of the T X-ray crystallographic structures of abTCR/peptide/ cell response when the cell is subsequently challenged MHC complexes suggest that the entire range of TCRwith an activating agonist peptide.

The purpose of TCR ligand binding is to initiate signal transduction that results in the activation of functional programs within the cell. How does binding of ligand on the extracellular V domains of the TCR translate into a functional message inside the cell? One model to explain these events is that ligand binding by the TCR V domains induces a conformational change in the C domains that, in turn, changes the dynamics of the interactions with CD3 chains and/or ζ chains associated with the receptor and thus transduces a signal through these associated components. However, a comparison of the liganded and unliganded forms of 2C showed that the conformational changes that occurred in the TCR as a result of antigen binding did not appear to involve the constant domains of the molecule (Garcia et al. 1998xStructural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Garcia, K.C, Degano, M, Pease, L.R, Huang, M, Peterson, P.A, Teyton, L, and Wilson, I.A. Science. 1998; 279: 1166–1172CrossRef | PubMed | Scopus (517)See all ReferencesGarcia et al. 1998). In addition, comparison of the Cα-Cβ interfaces of the liganded B7 TCR with unliganded 2C and N15 showed that they were nearly identical (Ding et al. 1998xTwo human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Ding, Y.-H, Smith, K.J, Garboczi, D.N, Utz, U, Biddison, W.E, and Wiley, D.C. Immunity. 1998; 8: 403–411Abstract | Full Text | Full Text PDF | PubMed | Scopus (359)See all ReferencesDing et al. 1998). Thus, the current structural evidence does not support the conformational change model.An alternate model for signal transduction by the TCR postulates that ligand binding induces receptor aggregation and/or oligomerization that segregates the bound receptors into cell–cell attachment patches (Davis et al. 1998xLigand recognition by αβ T cell receptors. Davis, M.M, Boniface, J.J, Reich, Z, Lyons, D, Hampl, J, Arden, B, and Chien, Y.-H. Annu. Rev. Immunol. 1998; 16: 523–544CrossRef | PubMed | Scopus (632)See all ReferencesDavis et al. 1998). Such receptor aggregation would facilitate clustering of intracellular components required for the recruitment and phosphorylation of signal transduction components. The adoption of a uniform orientation in TCR binding to peptide/MHC complexes may provide a mechanism that facilitates TCR aggregation and interactions with the coreceptors (CD3s, CD4/CD8) that can generate a T cell signal (Davis et al. 1998xLigand recognition by αβ T cell receptors. Davis, M.M, Boniface, J.J, Reich, Z, Lyons, D, Hampl, J, Arden, B, and Chien, Y.-H. Annu. Rev. Immunol. 1998; 16: 523–544CrossRef | PubMed | Scopus (632)See all ReferencesDavis et al. 1998). At this juncture, no evidence for dimerization or oligomerization of liganded TCRs is provided from the crystallographic data. However, the conditions used to produce these complexes may not favor multimerization, whereas the conditions of cell surface TCRs associated with CD3s/ζ chains could be much more favorable. Future structural studies of unliganded and liganded TCRs in association with the CD3 chains and ζ chains could provide new insights into the mechanisms of the initiation of signal transduction by the TCR.‡E-mail: web@helix.nih.gov and dgarboczi@atlas.niaid.nih.gov.

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is in part due to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. In this study, we have identified a peptide sequence derived from the CNS-specific myelin protein proteolipid protein (PLP) which could bind to HLA-A3 and induce a HLA-A3-restricted CD8+ CTL response from HLA-A3+ donors. These PLP peptide-specific CTL could lyse HLA-A3+ target cells pulsed with a homologous peptide derived from the CRM1 protein of Saccharomyces cerevisae. These findings demonstrate the immunogenic potential of a PLP-derived peptide for generation of autoreactive HLA-A3-restricted CD8+ CTL, and further show that these CTL can be activated by a peptide derived from a common environmental microorganism.

Most neural cells constitutively lack major histocompatibility complex (MHC) class I and beta 2-microglobulin gene expression. Cytokines and viruses may, however, induce expression of these genes in some neural cells, and this correlates with factor binding to the NF-kappa B and interferon stimulated response elements of these genes. Here, we demonstrate that NF-kappa B is capable of inducing MHC class I and beta 2-microglobulin gene expression when transiently co-transfected into CHP-126 neuroblastomas, and that IRF-2 represses this induction. Interferon regulatory factor-2 (IRF-2) repression of MHC class I and beta 2-microglobulin gene expression in CHP-126 neuroblastomas may demonstrate a mechanism by which virus persists in neural cells. We show here that IRF-2 physically interacts in vitro with NF-kappa B. This interaction may contribute to the repression of the expression of these genes. Our demonstration that IRF family members, in addition to IRF-2, physically interact in vitro with NF-kappa B (p50 and p65), provides a general mechanism by which these transcription factors may, in concert, regulate the expression of a variety of genes involved in immune responses in the brain.

Most peptides that bind to a particular major histocompatibility complex class I molecule share amino acid residues important for binding at one or two positions. Sequence analyses of peptides bound to HLA-B14 revealed at least four candidates for these so-called anchor residues: Arg at P2, Tyr at P3, Arg at P5, and Leu at P9. Combinations of any three of these amino acids sufficed for binding to HLA-B14 in vitro. Using this information, we identified an antigenic peptide critical for cytotoxic T lymphocyte recognition of virus-infected cells. Molecular models of HLA-B14 peptide complexes were constructed to investigate how the potential anchor residues might function. By using binding data to calculate the contribution to binding of each amino acid at anchor positions and predicting the stability of all possible nonapeptide complexes that could be formed from antigenic proteins, we estimate that three known antigenic nonapeptides are in the highest affinity cohort of peptides. Thus, even when multiple combinations of anchor residues contribute to binding, antigenic peptides are routinely identifiable.

CARE-LASS is a highly sensitive, fast, simple and safe fluorometric microassay. Target cells are loaded with acetoxymethyl ester of calcein (calcein-AM) that passively crosses the cell membrane. Intracellular esterases convert the molecule to calcein, a polar fluorochrome which, in cells with intact plasma membranes, displays good retention characteristics and low pH sensitivity. In analogy to standard 51Cr release assays, the CARE-LASS system is based on the release of a marker into the supernatant that is measured by an automated fluorescence scanner and correlates with the number of lysed cells. We tested the CARE-LASS system by measuring cytotoxicity in major histocompatibility complex (MHC) class I and MHC class II restricted cytotoxic T lymphocyte (CTL) assays as well as lymphokine activated killer (LAK) mediated cytotoxicity. We applied a small set of target cell lines at various effector to target (E:T) ratios, at different antigen concentrations and compared CARE-LASS CTL data to data resulting from conventional 51Cr release assays. The CARE-LASS system provides a reliable and sensitive method to measure cell-mediated cytotoxicity.

Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58–66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58–66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58–66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58–66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58–66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

To understand better the specificity of peptide binding by MHC class I molecules, we have evaluated the capacity of a panel of unrelated peptides to compete for the presentation of viral peptides presented by HLA-A3 and HLA-B27. The HIV-Nef7F peptide (74-82) was presented by HLA-A3 to Nef-specific HLA-A3-restricted CTL lines, and the influenza nucleoprotein peptide NP(380-393) was presented by HLA-B27 to NP(380-393)-specific HLA-B27-restricted CTL lines. In addition, we have extended studies from our group that have evaluated the capacity of a similar panel of peptides to inhibit presentation of an influenza nucleoprotein peptide NP (335-349) by HLA-B37 and a matrix peptide, M1 (57-68), by HLA-A2 to the appropriate peptide-specific CTL lines. Out of 41 peptides tested, only five bound to more than one of the MHC molecules analyzed. Pairwise comparisons of the peptide binding specificities among these four different class I molecules revealed no common competitor peptides in four of the six possible comparisons. Thus, each class I molecule appears to have a functionally distinct peptide binding site, as reflected by the ability to bind largely non-overlapping sets of peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.

C2H2 zinc finger proteins constitute the largest family of transcription factors known with an estimated 300-500 genes encoding these proteins in the human genome. These proteins possess two conserved cysteines that are part of an antiparallel beta-sheet and two conserved histidines that are part of an alpha-helix, coordinated by a central zinc atom to form a globular domain. The protein family is typified by the RNA polymerase II transcription factor TFIIIA and the gap gene product Kruppel. 13 refs., 1 fig.

The self-specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to identify the relationship between the self-determinants which they recognize and the serologically defined HLA-A and -B antigenic determinants. Virus-immune T cells were generated in vitro by culture of normal adult peripheral blood lymphocytes with A/HK influenza virus. Virus-immune effectors from HLA-A2 positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for the HLA-A2 specificity. Virus-immune T cells from 11/11 A2-positive donors lysed all 2A-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cell populations consistently failed to lyse the virus-infected target cells from ove A2-positive donor (designated M7). Although the A2 antigen of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serological analyses. Results of iscelectric focusing of A2 molecules from three individuals plus M7 demonstrated that the M7 A2 heavy polypeptide chain is structurally distinct. These results indicate that: 1) there is a strong but incomplete associatione between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity; and 2) there may be at least two structurally and functionally distinct epitopes on the same A2 molecule: one is the serologically defined HLA-A2 antigenic determinant; the other is the self determinant recognized by T cells on HLA-A2 molecules.

The present report attempts to determine if there are distinct epitopes on the T8 molecule that are involved in class I-restricted cytotoxic T lymphocyte (CTL) function. A panel of 9 monoclonal antibodies (OKT8A,B,C,D,E,F,G,H,I, and OKT5) was produced and all antibodies were shown to bind to the T8 molecule. This panel of antibodies was employed to characterize the distribution of distinct epitopes on the T8 molecule and to block the activity of class I-specific influenza virus-immune and allo-immune CTL effector function. Significant differences in the ability of the anti-T8 antibodies to block CTL function were observed: OKT8C and T8F blocked best (49 and 55% respectively); OKT8A,E,G,H,I, and OKT5 blocked less well (24–31%); and OKT8B blocked marginally (11%). There was no correlation between the capacity of the antibodies to block CTL function and their heavy chain isotype. Competitive binding of the different OKT8 antibodies to the cell surface and differential trypsin sensitivity of the epitopes recognized by the antibodies indicated that OKT8C and T8F were located on topographically distinct regions of the T8 molecule. These results indicate that specific epitopes on the T8 molecule are involved in CTL function, and that there could be more than one functional site on the molecule.

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.