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Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8+ CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2–restricted TAL 168–176–specific CD8+ T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28–337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8+ T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.

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.

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.

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

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.

Human autoimmune diseases are thought to develop through a complex combination of genetic and environmental factors. Genome-wide linkage searches of autoimmune and inflammatory/immune disorders have identified a large number of non-major histocompatibility complex loci that collectively contribute to disease susceptibility. A comparison was made of the linkage results from 23 published autoimmune or immune-mediated disease genome-wide scans. Human diseases included multiple sclerosis, Crohn’s disease, familial psoriasis, asthma, and type-I diabetes (IDDM). Experimental animal disease studies included murine experimental autoimmune encephalomyelitis, rat inflammatory arthritis, rat and murine IDDM, histamine sensitization, immunity to exogenous antigens, and murine lupus (systemic lupus erythematosus; SLE). A majority (≈65%) of the human positive linkages map nonrandomly into 18 distinct clusters. Overlapping of susceptibility loci occurs between different human immune diseases and by comparing conserved regions with experimental autoimmune/immune disease models. This nonrandom clustering supports a hypothesis that, in some cases, clinically distinct autoimmune diseases may be controlled by a common set of susceptibility genes.

Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.

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.

Identification of the targets of autoreactive T cells is important for understanding the pathogenesis of many autoimmune diseases. In multiple sclerosis, myelin proteins are thought to be the targets of autoreactive T-cell responses. To date only major histocompatibility complex class II-restricted CD4+ T-cell responses to myelin proteins have been investigated. In the present study, the ability of self peptides derived from human myelin proteins to induce autoreactive CD8+ T-cell responses has been assessed. Peptide sequences from human myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin oligodendrocyte glycoprotein have been identified that bind to and form stable complexes with HLA-A2. MBP 110-118, PLP 80-88, MAG 287-295, MAG 509-517, and MAG 556-564 were all able to induce peptide-specific HLA-A2-restricted CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro in HLA-A2+ individuals. CTLs specific for MBP 110-118 and MAG 556-564 could recognize endogenously processed antigens presented by HLA-A2. CTL clones reactive to MBP 110-118 and MAG 556-564 produced tumor necrosis factor alpha and a subset of these clones also produced interferon gamma. These results demonstrate that (i) self peptides derived from human myelin proteins can induce autoreactive CD8+ CTLs and (ii) these CD8+ T cells produce cytokines thought to be important in mediating demyelinating disease. These studies provide an experimental approach for the assessment of CD8+ T-cell responses in such autoimmune diseases.

An alternative pathway for class I-restricted antigen presentation has been suggested on the basis of peptides bound to HLA-A2 molecules in cells lacking the transporter for antigen presentation (TAP). Most of these peptides were derived from signal sequences for translocation into the endoplasmic reticulum (ER). However, it is not known whether these peptides can be presented to T cells. The hydrophobic nature of an HLA-A2-restricted T cell epitope (M1 58-66) was exploited to test whether it could be presented to T cells when derived from a signal sequence. Replacing the signal sequence of the influenza virus hemagglutinin molecule H3 with an artificial sequence containing that HLA-A2-restricted T cell epitope resulted in efficient translocation of H3 molecules into the ER and transport to the cell surface. This signal sequence-derived epitope was presented to HLA-A2-restricted T cells. Involvement of cytosolic processing for this presentation is very unlikely, because (a) presentation occurred in cells lacking TAP; (b) expression of H3 molecules with the artificial signal sequence did not produce a detectable cytosolic form of H3; and (c) presentation of the same epitope expressed in cytosolic forms of antigen required TAP. Thus, a peptide derived from a signal sequence cleaved in the ER can provide an epitope for HLA-A2-restricted T cell recognition.

The pathogenesis of multiple sclerosis (MS) is thought to involve a T-cell-mediated autoimmune process. Experimental allergic encephalomyelitis (EAE), an animal model resembling MS, can be induced by immunization with myelin antigens such as myelin basic protein. The T-cell antigen receptor (TCR) usage in EAE is highly restricted in some strains of animals and experimental treatments targeting the TCR have been successful in EAE. Examination of the TCR beta-chain variable-region (V beta) usage of MBP-specific T-cell lines in MS patients has produced conflicting results. Our previous studies of TCR alpha-chain variable-region usage in monozygotic twins demonstrated a general skewing of the TCR repertoire in individuals with MS. This skewing became apparent only after stimulation with antigens; in peripheral blood lymphocyte preparations from individuals with MS V alpha 8-bearing T cells were preferentially selected by stimulation with myelin basic protein. In the present study we examined complementarity-determining region 3 of those V alpha 8-positive TCRs. Marked sequence heterogeneity was found in all individuals with severe MS. In contrast, restricted areas of complementarity-determining region 3 were found in healthy control individuals and in individuals with a mild form of MS. Sequences from tetanus toxoid-specific V alpha 8-positive T cells generated from the same individuals were relatively homogeneous within individuals regardless of disease activity and were distinct from the sequences of complementarity-determining region 3 in myelin basic protein-stimulated lines. These findings suggest that disease severity may be associated with increased heterogeneity of myelin antigen-specific T cells and could reflect an impaired ability of the immune system to down-regulate these anti-self responses.

Although the cause of multiple sclerosis (MS) is unknown, it is thought to involve a T cell-mediated autoimmune mechanism. Susceptibility to the disease is influenced by genetic factors such as genes of the HLA and T-cell receptor (TCR) complex. Other evidence for a genetic influence includes the low incidence in certain ethnic groups, the increased risk if there are affected family members and the increased concordance rate for disease in monozygotic twin pairs (26%), compared to dizygotic twins. Epidemiological studies indicate that there may be an additional role for environmental factors. Although the target antigen(s) are not yet identified, several myelin or myelin-associated proteins have been suspected, among them myelin basic protein. A lack of genetically comparable controls has impaired the analysis of the T-cell response in MS patients and caused disagreement on TCR usage in the disease. Here we analyse the role of TCR genes in MS by comparing TCR usage in discordant versus concordant monozygotic twins in response to self and foreign antigens. We find that after stimulation with myelin basic protein or tetanus toxoid, control twin sets as well as concordant twin sets select similar V alpha chains. Only the discordant twin sets select different TCRs after stimulation with antigens. Thus exogenous factors or the disease shape the TCR repertoire in MS patients, as seen by comparison with unaffected genetically identical individuals. This skewing of the TCR repertoire could contribute to the pathogenesis of MS and other T-cell-mediated diseases.

A motif specific to peptides that bind to the human class I major histocompatibility complex molecule HLA-A3 was identified by sequence analysis of HPLC fractions containing endogenous peptides. Twenty-six different sequences were obtained, 19 of which were nonamers. The majority of these endogenous peptide sequences contained Leu at position (P)2, while most sequences contained Tyr or Lys at P9. In addition, Phe was shared by 16 sequences at P3. Synthetic peptides corresponding to endogenous peptide sequences were shown to bind to HLA-A3. The importance of Leu at P2 and Tyr or Lys at P9 ("anchor" residues) for peptide binding to HLA-A3 was demonstrated by the following results: (i) peptides GLFGGGGGY, GLFGGGGGK, and GLGGGGFGY, but not GLFGGGGGV, specifically bound to HLA-A3 and (ii) six nonapeptides from within the influenza A nucleoprotein, matrix, and polymerase proteins, selected for synthesis based upon their possession of P2 and P9 anchor residues, were shown to bind HLA-A3. In contrast, none of a set of eight peptides that bound to HLA-A2, or six that bound to HLA-B27, bound detectably to HLA-A3. These findings provide a rationale for the design and selection of peptides that can be recognized by HLA-A3-restricted T cells.

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.

T-cell recognition of peptides that are bound and presented by class I major histocompatibility complex molecules is highly specific. At present it is unclear what role class I peptide binding plays relative to T-cell receptor specificity in determination of immune recognition. A previous study from our group demonstrated that the HLA-A2.1 molecule could bind to 25% of the members of a panel of unrelated synthetic peptides as assessed by a functional peptide competition assay. To determine the peptide-binding specificity of another HLA class I molecule, we have examined the capacity of this panel of peptides to compete for the presentation of influenza virus nucleoprotein peptide NP-(335-350) by HLA-B37 to NP-peptide-specific HLA-B37-restricted cytotoxic T-lymphocyte lines. Forty-two percent of peptides tested were capable of inhibiting NP-(335-350) presentation by HLA-B37. Remarkably, none of these HLA-B37-binding peptides belong to the subset that was previously shown to bind to the HLA-A2.1 molecule. Only the NP-(335-350) peptide was capable of binding to both HLA-A2.1 and HLA-B37. These findings demonstrate that the peptide-binding specificities of HLA-B37 and HLA-A2.1 are largely nonoverlapping and suggest that, from the universe of peptides, individual HLA class I molecules can bind to clearly distinct subsets of these 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.

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 Valpha 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 Valpha 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.

T cell recognition by peptide-specific αβ TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the α1 and α2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.

Antigen-specific CD8+ T cells acquire peptide-major histocompatibility complex (MHC) clusters through T-cell receptor (TCR)-mediated endocytosis after specific antigen stimulation. We generated an antigen-presenting cell (APC) expressing human leukocyte antigen (HLA)-A*201 coupled to the enhanced green fluorescent protein (GFP), which delivered GFP to an antigen-specific T cell when pulsed with antigenic peptide. We quantitatively identified human T-cell lymphotropic virus type I (HTLV-I) Tax(11-19) peptide-specific T-cell populations in peripheral blood mononuclear cells (PBMCs) from patients with HTLV-I-associated neurologic disease and defined a new CD8+ T-cell epitope in the HTLV-I envelope region. Acquisition of peptide-HLA-GFP complexes by antigen-specific T cells could distinguish, with respect to phenotype and perforin production, T cells from the chronic viral infections cytomegalovirus and HTLV-I. This approach will be a powerful tool in understanding the role of antigen-specific T-cell responses in health and disease.

The structures of αβ TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the α1 and α2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the α1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.

Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 Å) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, ΔTm = 16°C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a “closed conformation” of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.

We describe a novel human zinc finger cNDA, C2H2-171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger-like domains are found in the C-terminal end of the protein. At the N-terminus, C2H2-171 contains a POZ/tramtrack-like domain similar to that found in the tumor associated zinc finger proteins LAZ-3/BCL-6 and PLZ-F, as well as in non-zinc finger proteins. C2H2-171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2-171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2-171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2-171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44-ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2-171 may play an important role in vertebrate brain development and function.

Down-regulation of the initial burst of viremia during primary HIV infection is thought to be mediated predominantly by HIV-specific cytotoxic T lymphocytes, and the appearance of this response is associated with major perturbations of the T cell receptor repertoire. Changes in the T cell receptor repertoire of virus-specific cytotoxic T lymphocytes were analyzed in patients with primary infection to understand the failure of the cellular immune response to control viral spread and replication. This analysis demonstrated that a significant number of HIV-specific T cell clones involved in the primary immune response rapidly disappeared. The disappearance was not the result of mutations in the virus epitopes recognized by these clones. Evidence is provided that phenomena such as high-dose tolerance or clonal exhaustion might be involved in the disappearance of these monoclonally expanded HIV-specific cytotoxic T cell clones. These findings should provide insights into how HIV, and possibly other viruses, elude the host immune response during primary infection.

To identify genes that are expressed in MS pathogenesis, we have analyzed a normalized cDNA library made from mRNA obtained from CNS lesions of a patient with primary progressive MS. Complementary DNA clones obtained from this library were subjected to automated DNA sequencing to generate expressed sequence tags. Analysis of this MS cDNA library revealed the presence of 54 cDNAs that were associated with immune activation and indicated the presence of an ongoing inflammatory response with evidence of both cell-mediated and humoral immune responses. The surprising finding was that 16 of the cDNAs encoded autoantigens associated with seven other autoimmune disorders, while only three of these 16 autoantigen cDNAs were present in a similarly constructed adult brain library. Such aberrant autoantigen expression could provide a source of secondary autoimmune stimulation that could contribute to the ongoing inflammatory response in MS. In addition, two cDNAs were found that mapped to a known MS susceptibility locus (5p14-p12): one encoded an excitatory amino acid transporter and the other a human homologue of the Drosophila disabled gene. This approach to the molecular biology of MS pathogenesis may help to illuminate previously unappreciated aspects of this disease.

We report here the nucleotide sequence of a clone, C2H2-34.10 , isolated from a human brain cDNA library using degenerate oligodeoxyribonucleotide hybridization. C2H2-34.10 has extensive homology to the Xenopus laevis 5S DNA/ RNA-binding protein, TFIIIA. The deduced amino acid (aa) sequence of the human clone gives a protein of 363 aa with identity to TFIIIA from both X. laevis (57%) and Rana pipiens (59%). This human clone contains nine C2H2-type zinc fingers like frog TFIIIA. Northern blot analysis indicates that the C2H2-34.10 RNA is expressed in human ovary, as well as human neuronal cell lines.

Recognition by natural killer (NK) cells of major histocompatibility complex (MHC) class I molecules on target cells inhibits NK-mediated lysis. Here, inhibition of NK clones by HLA-B*2705 molecules mutated at single amino acids in the peptide binding site varied among HLA-B*2705-specific NK clones. In addition, a subset of such NK clones was inhibited by only one of several self peptides loaded onto HLA-B*2705 molecules expressed in peptide transporter-deficient cells, showing that recognition was peptide-specific. These data demonstrate that specific self peptides, complexed with MHC class I, provide protection from NK-mediated lysis.

A HLA-A3.1-restricted CD8+ cytotoxic T-cell clone, E7.20, that lyses cells infected with human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 768-778 (RLRDLLLIVTR, NL43 env sequence) of the cytoplasmic domain of gp41 by successive use of a panel of recombinant vaccinia viruses that express truncated env genes and synthetic peptides. The epitope is conserved on 7 (NL43, BRU, HXB2, BRVA, SC, JH3, and JFL) of 13 human immunodeficiency virus type 1 isolates from North America. Synthetic peptides of this region of strains RF and CDC4 are also recognized by E7.20 despite a nonconservative Thr----Val or Thr----Ala change at amino acid 777; however, an MN peptide, which has four amino acid substitutions, was not reactive. The epitope recognized by E7.20 has a predicted hydrophobic alpha-helical structure, with three contiguous Leu residues followed by Ile and Val at amino acids 772-776. Cytotoxicity was restricted by HLA-A3.1 using allogeneic target cells that shared HLA class I antigens with the donor and an HLA-A and -B negative human plasma cell line transfected with the HLA-A3.1 gene. The transfected cells were infectable by human immunodeficiency virus type 1 strains IIIB and MN but only the former virus sensitized them to killing by E7.20. The ability of E7.20 to specifically lyse a human lymphocyte line infected with a human immunodeficiency virus type 1 strain carrying the conserved epitope is consistent with an important role for cytotoxic T cells in controlling infection.

Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu-self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS.

A recently described HLA gene, SB, which maps between GLO and HLA-DR, codes for Ia-like molecules that are similar to but distinct from HLA-DR molecules. Cytotoxic T lymphocytes (CTL) specific for SB1, SB2, SB3, and SB4 were compared with HLA-A2-specific CTL with respect to their surface expression of the T cell differentiation antigens OKT3, OKT4, and OKT8. All CTL activity was eliminated by treatment with OKT3 and C'. The SB-specific cytotoxicity was eliminated by OKT4 plus C' but not by OKT8 plus C'. In contrast, HLA-A2-specific killing was completely susceptible to treatment with OKT8 plus C' but not with OKT4 plus C'. Cytotoxicity was analyzed in the presence of OKT8 and a series of monoclonal antibodies (OKT4A, 4B, 4C, and 4D) that react with distinct epitopes on the OKT4 molecule. SB1-, SB3-, and SB4-specific CTL were partially inhibited by OKT4A and 4B (45-75%), whereas HLA-A2-specific CTL were partially inhibited by OKT8 (48-63%) but not by OKT4. SB2-specific CTL were not inhibited (less than 26%) by OKT8 or by any of the OKT4-related antibodies. These results suggest that the OKT4 marker may be expressed on most T cells that recognize allogeneic Ia or self Ia plus foreign antigens; OKT4+ cells do not appear to be functionally homogeneous in that they can act both as helper/inducer and cytotoxic cells. Models are proposed for the functional involvement of the OKT4 molecule in T cell-Ia antigen interactions.

Major histocompatibility complex-restricted and alloreactive cytotoxic T lymphocytes (CTL) can discriminate between the HLA-A3.1 and HLA-A3.2 antigens. HLA-A3.1 and the rare variant HLA-A3.2 have been shown to differ by two amino acids in the alpha 2 domain at positions 152 (A3.1, glutamic acid; A3.2, valine) and 156 (A3.1, leucine; A3.2, glutamine). To determine the structural basis for the ability of CTL to differentiate A3.1 from A3.2, two site-directed mutants of the HLA-A3.2 gene were produced, 152A3.1-156A3.2 and 152A3.2-156A3.1, that have the indicated codons for positions 152 and 156. These mutated HLA-A3 genes, as well as the nonmutated HLA-A3.1 and HLA-A3.2 genes, were then transfected into the murine cell line P815-HTR and used as targets for human CTL. Influenza virus-specific HLA-A3.1-restricted CTL lysed virus-infected P815 cells transformed with the HLA-A3.1 and 152A3.1-156A3.2 genes, but not P815 cells transformed with the HLA-A3.2 and 152A3.2-156A3.1 genes. HLA-A3.2-allospecific CTL lysed the P815 cells transformed with the HLA-A3.2 and 152A3.2-156A3.1 genes but did not lyse P815 cells transformed with the HLA-A3.1 or 152A3.1-156A3.2 genes. Thus, a single amino acid change at position 152, substituting valine for glutamic acid and thereby introducing a charge difference, produces major structural changes in the epitopes recognized by major histocompatibility complex-restricted and alloreactive CTL.

We have investigated elements of the genetic control of human in vitro cytotoxic T-cell responses to influenza virus-infected autologous cells by studies of a large family. The pattern of virus-immune cytotoxicity among siblings demonstrated T-cell recognition of influenza virus predominantly (greater than 90%) in association with determinants which are coded by genes linked to HLA (P less than 0.0002). Many family members consistently generated cytotoxic activity against influenza predominantly in association with antigens coded by genes of only one of their HLA haplotypes. Such haplotype preferences were consistent among HLA-identical siblings, indicating that the specificity of the T-cell response to influenza virus in association with HLA-A and -B antigens is controlled by genes linked to HLA.

Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.

To determine whether healthy homosexual men are immunologically impaired, peripheral blood leukocytes (PBL) from 20 male homosexuals were compared prospectively with PBL from 14 age-matched male heterosexual donors with respect to: (a) the capacity of their PBL to generate functional T cell immune responses in vitro; and (b) the content of total T cells and T cell subsets in their peripheral blood. The homosexual donors studied indicated moderate sexual life styles in that all but one of the donors had less than five current sexual partners. The percentages of OKT3+, OKT4+, and OKT8+ T cells were similar to those of heterosexual controls. T cell function was assessed by measuring cytotoxic T cell responses to influenza virus and to allogeneic cells. Approximately one-third of the homosexual donors consistently exhibited weak cytotoxic T lymphocyte (CTL) responses to influenza virus, whereas all of the heterosexual donors generated strong CTL responses to influenza. There was no correlation between the strength of CTL responsiveness to influenza virus and the strength of CTL responses to allogeneic cells. These results suggest that the influenza-specific CTL response may be a sensitive indicator of immunologic defects in asymptomatic homosexuals. If acquired immune deficiency syndrome results from an infectious agent, it remains to be seen if such immunosuppression predisposes to the infection, or if it reflects early consequences of infection.

To dissect the molecular basis for T-cell recognition of class I major histocompatibility complex antigens, we have examined the ability of human cytotoxic T lymphocytes (CTL) to recognize murine L cells transformed with a human class I gene. Three transformed L-cell lines were generated that expressed the human HLA-A3 gene from donor E1 at levels comparable to those of the endogenous L-cell H-2Kk molecules. CTL were generated in secondary and tertiary mixed lymphocyte culture against the HLA-A3 subtype of donor E1 by culturing irradiated E1 peripheral blood lymphocytes with the peripheral blood lymphocytes of responder donor M3 (M3 shares all defined class I antigens with E1 but expresses a different HLA-A3 subtype). Each of the HLA-A3-transformed L cells was lysed by M3 anti-E1 CTL in a short-term 51Cr release assay and this recognition was blocked by a monoclonal anti-HLA-A3 antibody. Antibodies specific for the human T8 and LFA-1 molecules on the CTL effectors (but absent from the transformed targets) also blocked lysis of each of the HLA-A3 transformed L-cell targets. Antibodies to other T-cell molecules failed to block lysis. The present results demonstrate that human CTL can recognize human class I molecules on targets that do not express any other human gene product and further suggest that effector T-cell molecules T8 and LFA-1 are functionally involved in this recognition process.

The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 10(3) tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.

By using a panel of HLA-D-defined subtypes of HLA-DR2 HCL with known beta chain structural variabilities, we have demonstrated that HLA-DR2, OKT4+ cytotoxic T lymphocyte (CTL) clones specific for measles virus are apparently restricted to a distinct DR beta chain. The presence of this DR beta 2 molecule correlated precisely with the susceptibility of measles virus-infected HLA-DR2 HCL to lysis by these CTL clones. These studies demonstrate that delineation of HLA-DR2 into various subgroups can have a functional significance that parallels the structural differences within the HLA-D region. These results are discussed in the context of the possible association of HLA class II-restricted, measles virus-specific CTL and multiple sclerosis.

Cytotoxic thymus-derived lymphocytes generated after interaction with trinitrophenyl (TNP)-substituted or virus-infected cells only lyse H-2 compatible target cells modified with the component used to immunize (TNP or virus). Prior saturation of TNP-reactive sites inhibits neither the infectivity of influenza A viruses, nor the capacity of infected cells to develop antigenic changes recognized by influenza-immune T cells. The two antigens are distinct entities on the cell membrane and do not obviously compete to form interactions with H-2 molecules.

The interactions of three singly substituted peptide variants of the HTLV-1 Tax peptide bound to HLA-A2 with the A6 T cell receptor have been studied using T cell assays, kinetic and thermodynamic measurements, and X-ray crystallography. The three peptide/MHC ligands include weak agonists and antagonists with different affinities for TCR. The three-dimensional structures of the three A6-TCR/peptide/HLA-A2 complexes are remarkably similar to each other and to the wild-type agonist complex, with minor adjustments at the interface to accommodate the peptide substitutions (P6A, V7R, and Y8A). The lack of correlation between structural changes and the type of T cell signals induced provides direct evidence that different signals are not generated by different ligand-induced conformational changes in the alphabeta TCR.

The three-dimensional structure of a human αβ T cell receptor (TCR), B7, bound to the HLA-A2 molecule/HTLV-1 Tax peptide complex was determined by x-ray crystallography. Although different from the A6 TCR, previously studied, in 16 of the 17 residues that contact HLA-A2/Tax, the B7 TCR binds in a similar diagonal manner, only slightly tipped and rotated, relative to the A6 TCR. The structure explains data from functional assays on the specificity differences between the B7 and A6 TCRs for agonist, partial agonist, and null peptides. The existence of a structurally similar diagonal binding mode for TCRs favors mechanisms based on the formation of geometrically defined supramolecular assemblies for initiating signaling.

The H-2L locus is closely linked to H-2D and codes for antigenic specificities present on a 45,000 mol wt glycoprotein that is distinct from the molecule which bears the D region private specificity. It was found that BALB/c-H-2db mice, which lack detectable cell-surface H-2L gene products, were able to generate influenza- and vaccinia-immune cytotoxic T cells which lyse D region-compatible target cells, although they have been reported to be incapable of making a similar response to ectromelia virus (7). Thus, the lack of H-2L antigenic specificities does not produce a general loss of responsiveness for other viruses even when a highly cross-reactive pox virus (vaccinia) was studied. Antisera-blocking experiments utilizing sera specific for either L or D molecules indicated that BALB/c mice generate influenza virus-immune cytotoxic T-cell subsets which independently recognize H-2L and H-2D gene products in association with viral antigens. These results are the first indication that products of the H-2L locus can operate analogously to H-2K/D gene products in virus-immune T-cell recognition.

The present study examines the potential role of the T4 molecule in functional cell-cell interactions between target cells and human cytotoxic T lymphocyte (CTL) clones that are specific for HLA class II alloantigens encoded by the SB locus. There were marked differences (greater than 30-fold) between the seven SB-specific clones studied with respect to their susceptibility to inhibition by anti-T4 as well as anti-T3 antibodies. We wished to test the hypothesis that such variation among the clones would be due to differences in clonal "affinity" for antigen. To quantitate differences among the CTL clones in the tightness with which they bind target cells, the clones were analyzed using a previously published assay of susceptibility of CTL-target cell conjugates to dissociation in the presence of unlabeled targets. The results revealed that the clones that were most susceptible to inhibition by anti-T4 and anti-T3 were the weakest target cell binders, and vice versa. Anti-T4 antibody could partially induce dissociation of functional CTL-target cell conjugates in the absence of any added cold targets. For the "highest affinity" clone such anti-T4 antibody-induced dissociation could be observed at 4 degrees C but not 23 degrees C. These results indicate that the T4 molecule is functionally involved in target cell binding by CTL, and raise the possibility that although it is easiest to demonstrate the function of the T4 molecule in "low affinity" clones, that function may also be operative in the "high affinity" clones.

Antisera to the type-specific internal influenza virus matrix (M) protein of a type A influenza virus were produced in goats. In the presence of complement, anti-M serum was cytotoxic for target cells which were infected with a variety of serologically distinct type A influenza viruses, but did not react with type B influenza virus-infected cells. Absorption experiments indicated that anti-M serum detected a common antigen(s) on the surface of type A-infected cells. This serological cross-reactivity parallels the cross-reactivity observed for the cytotoxic T-cell response to type A viruses.

Most antigens must be processed intracellularly before they can be presented, in association with major histocompatibility complex (MHC) molecules at the cell surface, for recognition by the antigen-specific receptor of T cells. This processing appears to involve cleavage of protein antigens to smaller peptides. Only certain fragments of any protein can serve as T-cell epitopes and this is, at least in part, determined by the requirement that peptides be able to bind the MHC molecules. Class I restricted antigens are derived from proteins, such as viral antigens, that are synthesized within the presenting cell. Many of these antigens are cytosolic proteins and recent evidence suggests that it is in the cytosol that these proteins are processed to produce either the antigenic peptides or processed intermediates. How and where these processed cytosolic antigens cross the membrane of the vacuolar system and bind to the extracellular domain of the class I molecule is not known but one obvious site for this process is the endoplasmic reticulum (ER), because this organelle is specialized to translocate proteins across the membrane from the cytosol into the secretory system. Based on this model, we reasoned that if we could pharmacologically block the movement of proteins out of the ER, endogenous antigen presentation would cease. An agent which causes such an effect is available--the fungal antibiotic Brefeldin A (BFA). Consistent with the above hypothesis, we report that BFA completely abolishes the ability of a cell to present endogenously synthesized antigens to class I restricted cytotoxic T cells.