The crystal structures of K(bm1) and K(bm8) reveal that subtle changes in the peptide environment impact thermostability and alloreactivity.

The K(bm1) and K(bm8) natural mutants of the murine MHC class I molecule H-2K(b) were originally identified by allograft rejection. They also bind viral peptides VSV8 and SEV9 with high affinity, but their peptide complexes have substantially decreased thermostability, and the K(bm1) complexes do not elicit alloreactive T cell responses. Crystal structures of the four mutant complexes at 1.7-1.9 A resolution are similar to the corresponding wild-type K(b) structures, except in the vicinity of the mutated residues, which alter the electrostatic potential, topology, hydrogen bonding, and local water structure of the peptide binding groove. Thus, these natural K(b) mutations define the minimal perturbations in the peptide environment that alter antigen presentation to T cells and abolish alloreactivity.

Differences in antigen recognition and cytolytic activity of CD8(+) and CD8(-) T cells that express the same antigen-specific receptor.

CD8(+) and CD8(-) T cell lines expressing the same antigen-specific receptor [the 2C T cell receptor (TCR)] were compared for ability to bind soluble peptide-MHC and to lyse target cells. The 2C TCR on CD8(-) cells bound a syngeneic MHC (K(b+))-peptide complex 10-100 times less well than the same TCR on CD8(+) cells, and the CD8(-) 2C cells lysed target cells presenting this complex very poorly. Surprisingly, however, the CD8(-) cells differed little from CD8(+) cells in ability to bind an allogeneic MHC (L(d+))-peptide complex and to lyse target cells presenting this complex. The CD8(+)/CD8(-) difference provided an opportunity to estimate how long TCR engagements with peptide-MHC have to persist to initiate the cytolytic T cell response.

T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells.

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.

The major histocompatibility complex-encoded class I-like HFE abrogates endocytosis of transferrin receptor by inducing receptor phosphorylation.

The major histocompatibility complex-encoded gene, Hfe, has been implicated to play a pivotal role in hereditary hemochromatosis, a common autosomal recessive disorder of iron metabolism. The recent finding that a physical interaction between HFE and transferrin receptor establishes a functional link between HFE and transferrin receptor-mediated iron metabolism in the pathophysiology of hereditary hemochromatosis. To elucidate the underlying mechanisms by which HFE interacts with and affects transferrin receptor function, we have systematically investigated the consequences of the HFE-transferrin receptor interaction in cellular iron homeostasis. Herein we show that in HFE-expressing cells, the amount of intracellular transferrin is decreased by approximately 28%, despite a approximately 40% increase in surface-expressed transferrin receptor. Kinetic analysis of receptor-bound transferrin endocytosis reveals that HFE expression not only reduces transferrin binding but also abrogates transferrin receptor endocytosis. As a result, HFE expression leads to an accumulation of non-functional transferrin receptors at the cell surface, and a decrease in iron uptake. Moreover, HFE expression induces hyper-serine phosphorylation of the transferrin receptor. Taken together, these results suggest that HFE negatively modulates cellular iron uptake by impairing transferrin receptor endocytosis via HFE-induced receptor phosphorylation.

Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells.

Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.

Requirements for stimulating naive CD8+ T cells via signal 1 alone.

In the absence of costimulation, TCR recognition of peptide/MHC complexes is generally considered to be nonimmunogenic. In agreement with this view, naive TCR transgenic CD8+ cells failed to respond to specific peptides presented by MHC class I (Ld) molecules bound to mouse RBC. However, peptide/Ld complexes presented by cell-sized beads or bound to plastic led to overt proliferative responses in the absence of added cytokines. Significantly, equivalent strong proliferative responses occurred when mouse RBC were fixed with glutaraldehyde before Ld coupling. The implication therefore is that the intensity of signaling via the TCR is a reflection of the mobility of the ligand being recognized; TCR signaling is weak when the ligand can move laterally on the cell membrane but strong when the ligand is immobilized.

Peptide antagonism and T cell receptor interactions with peptide-MHC complexes.

We describe antagonist peptides that specifically inhibit cytolytic activity of T cell clones and lines that express the antigen-specific receptor of CD8+ T lymphocyte clone 2C, which recognizes peptides in association with syngeneic (Kb) and allogeneic (Ld) MHC proteins. Addition of an antagonist peptide that can bind to Kb on 2C cells decreased the tyrosine phosphorylation of CD3 zeta chains elicited by prior exposure of the cells to an agonist peptide-Kb complex. Contrary to previous agonist-antagonist comparisons, the 2C T cell receptor had higher affinity for an antagonist peptide-Kb complex than for a weak agonist peptide-Kb complex. This difference is considered in light of evidence that antigen-specific receptor affinity values can be substantially higher when determined with the receptor on live cells than with the receptor in cell-free systems.

Application of an artificial neural network to predict specific class I MHC binding peptide sequences.

Computational methods were used to predict the sequences of peptides that bind to the MHC class I molecule, K(b). The rules for predicting binding sequences, which are limited, are based on preferences for certain amino acids in certain positions of the peptide. It is apparent though, that binding can be influenced by the amino acids in all of the positions of the peptide. An artificial neural network (ANN) has the ability to simultaneously analyze the influence of all of the amino acids of the peptide and thus may improve binding predictions. ANNs were compared to statistically analyzed peptides for their abilities to predict the sequences of K(b) binding peptides. ANN systems were trained on a library of binding and nonbinding peptide sequences from a phage display library. Statistical and ANN methods identified strong binding peptides with preferred amino acids. ANNs detected more subtle binding preferences, enabling them to predict medium binding peptides. The ability to predict class I MHC molecule binding peptides is useful for immunolological therapies involving cytotoxic-T cells.

Structural basis of 2C TCR allorecognition of H-2Ld peptide complexes.

MHC class I H-2Ld complexed with peptide QL9 (or p2Ca) is a high-affinity alloantigen for the 2C TCR. We used the crystal structure of H-2Ld with a mixture of bound peptides at 3.1 A to construct a model of the allogeneic 2C-Ld/QL9 complex for comparison with the syngeneic 2C-Kb/dEV8 structure. A prominent ridge on the floor of the Ld peptide-binding groove, not present in Kb, creates a C-terminal bulge in Ld peptides that greatly increases interactions with the 2C beta-chain. Furthermore, weak electrostatic complementarity between Asp77 on the alpha1 helix of Kb and 2C is enhanced in the allogeneic complex by closer proximity of QL9 peptide residue AspP8 to the 2C HV4 loop.

Biomagnetic isolation of antigen-specific CD8+ T cells usable in immunotherapy.

Isolating antigen-specific T lymphocytes is hampered by the low frequency of the cells and the low affinity between T-cell receptors (TCR) and antigen. We describe the isolation and purification of antigen-specific CD8+ T lymphocytes from mixed T-cell populations. Magnetic beads coated with major histocompatibility complex class I molecules loaded with specific peptide were used as a substrate for T-cell capture. Low-frequency T cells, as well as T cells with TCR of low affinity for the antigen were captured on the beads. Following isolation and expansion, recovered cells specifically killed target cells in vitro, and displayed antiviral effect in vivo.

Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

Altered antigen presentation in mice lacking H2-O.

HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.

Augmentation of mature CD4+ T cell responses to isolated antigenic class II proteins by fibronectin and intercellular adhesion molecule-1.

Mature CD4+ T cells can undergo stable adhesion to isolated antigenic MHC complexes, and upon TCR engagement exhibit up-regulated adhesion to the integrin ligands fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1). Here, we have examined T cell responses to purified antigenic class II complexes, alone or coimmobilized in the presence of FN or ICAM-1. T cell adhesion to immobilized peptide-MHC complexes alone stimulated suboptimal, but marked levels of IFN-gamma and IL-2 secretion, and this was accompanied by cell proliferation. T cell adhesion to both FN and ICAM-1 strongly augmented cytokine release and T cell proliferation. Activation of Vbeta3+ and Vbeta8+ T cell lines by isolated staphylococcal enterotoxin-MHC complexes was also examined, and surprisingly, a Vbeta8+ T cell line displayed significant cell adhesion or later response to staphylococcal enterotoxin B-MHC complexes only when Ag was coimmobilized with ICAM-1 or FN. The results demonstrate that adhesion of CD4+ T cells to ICAM-1 or FN activated by natural TCR ligands can strongly augment T cell signaling and downstream responses. Moreover, for some Ags, T cell interaction with accessory ligands may be critical in attaining a threshold level of receptor occupancy for cell activation.

Peptide-independent recognition by alloreactive cytotoxic T lymphocytes (CTL).

We have isolated several H-2K(b)-alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2K(b). These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2K(b) molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2K(b) on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2K(bm8) molecule. These findings suggested that the clones recognized determinants on H-2K(b) that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2K(b) produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2K(b) molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent.

Requirements for peptide-induced T cell receptor downregulation on naive CD8+ T cells.

The requirements for inducing downregulation of alpha/beta T cell receptor (TCR) molecules on naive major histocompatibility complex class I-restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent requirements did not apply to TCR downregulation. Thus, TCR downregulation seemed to depend solely on TCR/peptide/interaction and did not require either CD8 or B7-1 expression; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides. TCR downregulation was most prominent with high affinity peptides and appeared to be neither obligatory nor sufficient for T cell activation. In marked contrast to T cell activation, TCR downregulation was resistant to various metabolic inhibitors. The biological significance of TCR downregulation is unclear, but could be a device for protecting T cells against excessive signaling.

Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization.

Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells.

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes.

T-cell antigen receptors (TCR) generally interact with moderate affinity with the complex formed by major histocompatibility complex (MHC) molecules and foreign peptides. MHC/TCR recognition is followed by the generation of a signal to the T cell through a monomorphic multicomponent system that includes the CD3 complex and accessory molecules such as CD4 and CD8. The interaction between the extracellular domains of MHC and TCR molecules, and the interaction of MHC and CD4/CD8 molecules, have been considered to occur independently of one another. We report here that the affinity of CD8 dimers for MHC class I molecules is independent of haplotype and peptide content, and that the affinity of the TCR for its specific ligand is enhanced through a reduced 'off' rate in the presence of either CD8alpha alpha homo- or CD8alpha beta heterodimers. Moreover, CD8 seems to help recognition of the specific MHC-peptide complex either by guiding an energetically favourable docking of TCR onto MHC, or by inducing conformational changes in the MHC complex that can augment the TCR/MHC-peptide interaction. CD8 should therefore be considered as an active participant in the T-cell recognition complex, rather than simply as an accessory molecule.