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M M Davis

Publications and source records attributed to M M Davis.

At least 37 records · Page 2Linked to original sources
PubMed2000

Thirty-six views of T-cell recognition.

While much is known about the signalling pathways within lymphocytes that are triggered during activation, much less is known about how the various cell surface molecules on T cells initiate these events. To address this, we have focused on the primary interaction that drives T-cell activation, namely the binding of a particular T-cell receptor (TCR) to peptide-MHC ligands, and find a close correlation between biological activity and off-rate; that is, the most stimulatory TCR ligands have the slowest dissociation rates. In general, TCRs from multiple histocompatibility complex (MHC) class-II-restricted T cells have half-lives of 1-11s at 25 degrees C, a much narrower range than found with antibodies and suggesting a strong selection for an optimum dissociation rate. TCR ligands with even faster dissociation rates tend to be antagonists. To observe the effects of these different ligands in their physiological setting, we made gene fusions of various molecules with green fluorescent protein (GFP), transfected them into the relevant lymphocytes, and observed their movements during T-cell recognition using multicolour video microscopy. We find that clustering of CD3zeta-GFP and CD4-GFP on the Tcell occurs concomitantly or slightly before the first rise in calcium by the T cell, and that various GFP-labelled molecules on the B-cell side cluster shortly thereafter (ICAM-1, class II MHC, CD48), apparently driven byT-cell molecules. Most of this movement towards the interface is mediated by signals through the co-stimulatory receptors, CD28 and LFA-1, and involves myosin motors and the cortical actin cytoskeleton. Thus, we have proposed that the principal mechanism by which co-stimulation enhances T-cell responsiveness is by increasing the local density of T-cell activation molecules, their ligands and their attendant signalling apparatus. In collaboration with Michael Dustin and colleagues, we have also found that the formation and stability of the TCR-peptide-MHC cluster at the centre of the interaction cap between T and B cells is highly dependent on the dissociation rate of the TCR and its ligand. Thus, we are able to link this kinetic parameter to the formation of a cell surface structure that is linked to and probably causal with respect to T-cell activation.

Animals↗
PubMed2000

Differential clustering of CD4 and CD3zeta during T cell recognition.

Whereas T helper cells recognize peptide-major histocompatibility complex (MHC) class II complexes through their T cell receptors (TCRs), CD4 binds to an antigen-independent region of the MHC. Using green fluorescent protein-tagged chimeras and three-dimensional video microscopy, we show that CD4 and TCR-associated CD3zeta cluster in the interface coincident with increases in intracellular calcium. Signaling-, costimulation-, and cytoskeleton-dependent processes then stabilize CD3zeta in a single cluster at the center of the interface, while CD4 moves to the periphery. Thus, the CD4 coreceptor may serve primarily to "boost" recognition of ligand by the TCR and may not be required once activation has been initiated.

Animals↗
PubMed2000

Determination of the relationship between T cell responsiveness and the number of MHC-peptide complexes using specific monoclonal antibodies.

We describe the generation of three mAbs that recognize the complex of the class II MHC molecule IEk bound to a peptide derived from the carboxyl terminus of moth cytochrome c (residues 95-103). Reactivities of these mAbs are sensitive to single alterations in the sequence of both helices of the MHC molecule and to the bound peptide. The epitopes of these reagents are distinct but overlap substantially. One of these mAbs specifically blocks lymphokine release by T cells responsive to this complex but not others. We have used another to examine how the number of complexes on an APC is related to its ability to stimulate T cells. We find that 200-400 complexes per cell are necessary and sufficient to induce a degree of stimulation, whereas maximum stimulation is achieved only if more than 5000 complexes are present. The analysis indicates that T cell activation is a stochastic process.

Amino Acid Sequence↗
PubMed2000

Kinetics of peptide binding to the class II MHC protein I-Ek.

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.

Amino Acid Sequence↗
PubMed2000

The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire.

p53 is an attractive target for cancer immunotherapy since it is overexpressed in half of all tumors. However, it is also expressed in normal lymphoid tissue, and self tolerance leaves a p53-specific repertoire purged of high avidity CTL. To better understand the mechanism of tolerance and the basis for such low avidity interaction, p53-specific CTL from p53 deficient (p53-) and sufficient (p53+) A2.1/Kb transgenic mice were compared with respect to their ability to bind HLA-A2.1 tetramers containing cognate murine p53 peptide Ag, p53 261-269. Since the murine CD8 molecule cannot interact with human HLA-A2.1, this tests the ability of the TCR to bind the A2.1/peptide complex tetramer. CTL from p53- mice demonstrated strong binding of such A2.1/p53 261-269 tetramers; however, the CTL from tolerant p53+ mice were devoid of tetramer-binding CD8+ T cells. Examination of TCR expression at the clonal level revealed that CTL from p53+ and p53- mice each expressed comparable levels of the p53-specific TCR. These results indicate that normal expression of p53 promotes elimination of T cells expressing TCRs with sufficient affinity to achieve stable binding of the A2.1/p53 261-269 tetramers.

Amino Acid Sequence↗
PubMed2000

Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities.

All rearranging antigen receptor genes have one or two highly diverse complementarity determining regions (CDRs) among the six that typically form the ligand binding surface. We report here that, in the case of antibodies, diversity at one of these regions, CDR3 of the V(H) domain, is sufficient to permit otherwise identical IgM molecules to distinguish between a variety of hapten and protein antigens. Furthermore, we find that somatic mutation can allow such antibodies to achieve surprisingly high affinities. These results are consistent with a model in which the highly diverse CDR3 loops are the key determinant of specificity in antigen recognition in both T cell receptors (TCR) and antibodies, whereas the germline-encoded CDR1 and CDR2 sequences are much more cross-reactive.

Amino Acid Sequence↗
PubMed2000

Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia.

Although the immune system has long been implicated in the control of cancer, evidence for specific and efficacious immune responses in human cancer has been lacking. In the case of chronic myelogenous leukemia (CML), either allogeneic bone marrow transplant (BMT) or interferon-alpha2b (IFN-alpha2b) therapy can result in complete remission, but the mechanism for prolonged disease control is unknown and may involve immune anti-leukemic responses. We previously demonstrated that PR1, a peptide derived from proteinase 3, is a potential target for CML-specific T cells. Here we studied 38 CML patients treated with allogeneic BMT, IFN- alpha2b or chemotherapy to look for PR1-specific T cells using PR1/HLA-A*0201 tetrameric complexes. There was a strong correlation between the presence of PR1-specific T cells and clinical responses after IFN-alpha and allogeneic BMT. This provides for the first time direct evidence of a role for T-cell immunity in clearing malignant cells.

Blood Circulation↗
PubMed1999

Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning.

Antigen-dependent activation of T lymphocytes requires T cell receptor (TCR)-mediated recognition of specific peptides, together with the MHC molecules to which they are bound. To achieve this recognition in a reasonable time frame, the TCR must scan and discriminate rapidly between thousands of MHC molecules differing from each other only in their bound peptides. Kinetic analysis of the interaction between a TCR and its cognate peptide-MHC complex indicates that both association and dissociation depend heavily on the temperature, indicating the presence of large energy barriers in both phases. Thermodynamic analysis reveals changes in heat capacity and entropy that are characteristic of protein-ligand associations in which local folding is coupled to binding. Such an "induced-fit" mechanism is characteristic of sequence-specific DNA-binding proteins that must also recognize specific ligands in the presence of a high background of competing elements. Here, we propose that induced fit may endow the TCR with its requisite discriminatory capacity and suggest a model whereby the loosely structured antigen-binding loops of the TCR rapidly explore peptide-MHC complexes on the cell surface until some critical structural complementarity is achieved through localized folding transitions. We further suggest that conformational changes, implicit in this model, may also propagate beyond the TCR antigen-binding site and directly affect self-association of ligated TCRs or TCR-CD3 interactions required for signaling.

Binding Sites↗
PubMed1999

Negative selection of T cells occurs throughout thymic development.

Thymic positive and negative selections govern the development of a self-MHC-reactive, yet self-tolerant, T cell repertoire. Whether these processes occur independently or sequentially remains controversial. To investigate these issues, we have employed tetrameric peptide-MHC complexes to fluorescently label and monitor polyclonal populations of thymocytes that are specific for moth cytochrome c (MCC)/I-Ek. In TCR beta mice tetramer-positive thymocytes are detectable even in the most immature TCR-expressing cells. In the presence of MCC peptide, thymocytes that bind strongly to MCC/I-Ek tetramers are deleted earlier in development and more extensively than cells that bind weakly. This negative selection of the MCC/I-Ek-specific cells occurs continuously throughout development and before any evidence of positive selection. Thus, positive and negative selections are independent processes that need not occur sequentially.

Amino Acid Sequence↗
PubMed1999

The immunological synapse: a molecular machine controlling T cell activation.

The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation.

Animals↗
PubMed1999

Catalysis of peptide dissociation from class II MHC-peptide complexes.

Certain peptides such as dynorphin A [dynA-(1-13)] enhance the release of antigenic peptides bound to class II MHC molecules at neutral pH. This enhanced release has been termed push off. Previous work has shown that the antigenic pigeon cytochrome c peptide PCC-(89-104) has at least two conformational isomers when bound to the class II MHC protein I-Ek. We have accordingly studied the push off of PCC-(89-104) from the complex PCC-(89-104)/I-Ek to see whether these isomeric conformations are distinguished by the push-off effect. A comparison of the association and dissociation kinetics of PCC-(89-104)/I-Ek in the presence of dynA-(1-13) shows that dynA-(1-13) does not simply replace PCC-(89-104) but rather acts catalytically. The major product is peptide-free I-Ek, which is receptive to further peptide binding. Evidence is presented that a two peptide-one MHC complex is formed in solution. This ternary complex represents the first step of the mechanism of push off. 19F NMR data are presented that indicate that dynA-(1-13) interacts specifically with only one of the two isomeric complexes of PCC-(89-104) and I-Ek. A push-off mechanism is proposed in which dynA-(1-13) binds outside the peptide binding groove. In a second step, the dissociation of one of the two isomers is specifically enhanced. Thus the push-off effect may be useful for identifying conformational isomers and for separating them.

Animals↗
PubMed1999

A PR1-human leukocyte antigen-A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia.

We previously showed (E. Clave et al., J. Immunother., 22: 1-6, 1999; J. Molldrem et al., Blood, 88: 2450-2457, 1996) that PR1, a human-lymphocyte-antigen (HLA)-A2.1-restricted peptide from proteinase 3, could be used to elicit CTLs from normal individuals. These CTLs showed HLA-restricted cytotoxicity and colony inhibition of myeloid leukemia cells that overexpress proteinase 3. In this study, we constructed a phycoerythrin-labeled PR1-HLA-A2 tetramer to identify PR1-specific CTLs by flow cytometry. No peripheral blood lymphocytes from three HLA-2.1+ donors stained with the tetramer, but, after 20 days in culture with weekly PR1 stimulation, 2-8% became tetramer+. Tetramer staining identified up to 40-fold more PR1-specific CTLs than were identified by limiting dilution analysis and correlated better with lysis of PR1-coated T2 cells (R2 = 0.95 versus R2 = 0.76). Tetramer+ CTLs were memory phenotype (91% CD45RO+), and most (58% CD95+) were activated. Tetramer-sorted allogeneic CTLs produced 83% lysis of HLA-A2.1+ chronic myelogenous leukemia (CML) blasts at an E:T ratio of 2.5:1, compared with 23% lysis by nonsorted CTLs, with no background lysis of HLA-A2.1+ normal cells. Cytoplasmic proteinase-3 expression was one log greater in CML blasts than in normal granulocytes. These results show that a PR1-HLA-A2 tetramer can be used to identify and select CTLs from normal donors that preferentially lyse CML cells, which could be used for leukemia-specific adoptive immunotherapy.

Flow Cytometry↗
PubMed1999

Quantitative analysis of hepatitis C virus-specific CD8(+) T cells in peripheral blood and liver using peptide-MHC tetramers.

It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.

Antigens, CD↗
PubMed1999

Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.

Immunogenic peptides of human tumor Ag have been used to generate antigen-specific CTL. However, the vast majority of these peptide-specific CTL clones are of low avidity and are peptide, but not tumor, reactive. Peptide-MHC tetramers have been shown to bind specific TCRs with sufficient affinity to be useful reagents for flow cytometry. In this paper we demonstrate that peptide-MHC tetramers can also be used to selectively identify high avidity tumor-reactive CTL and enrich, from a heterogeneous population, the subpopulation of peptide-reactive T cells that can lyse tumor targets. The melanoma proteins, MART-1 and gp100, were used to induce potentially tumor-reactive T cells, and the intensity of T cell staining by TCR binding of specific peptide-MHC tetramers was assessed. A range of fluorescence intensity was detected, and the magnitude of tetramer binding was correlated with T cell avidity. The population of peptide-reactive T cells was phenotypically similar with regard to expression of TCR and adhesion molecules, suggesting that this differential avidity for tumor cells reflected differential affinity of the TCR for its peptide-MHC ligand. Sorting, cloning, and expansion of tetramerhigh CTL from a heterogeneous population of peptide-stimulated PBMCs enabled rapid selection of high avidity tumor-reactive CTL clones, which retained their functional and tetramerhigh phenotype on re-expansion. These results demonstrate that the avidity of a T cell for its tumor target is due to the specific affinity of the TCR for its peptide-MHC ligand, that this interaction can be described using peptide-MHC tetramers and used to isolate high avidity tumor-reactive CTL.

Antigen-Presenting Cells↗
PubMed1999

Conformational isomers of a class II MHC-peptide complex in solution.

A number of kinetic measurements of peptide dissociation from class II MHC-peptide complexes provide compelling evidence for the existence of conformational isomers in solution. There is evidence that T-lymphocytes can distinguish such isomers. However, virtually nothing is known about the structure of these isomers. Accordingly, we have investigated a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c residues 89-104 (PCC) by 19F-NMR. Two fluorine labels were placed on the PCC peptide; one fluorine label was placed at a MHC contact site, the other at a position involved in T-cell receptor (TCR) recognition. Introduction of these labels did not alter the observed kinetics of the PCC/I-Ek complex. The NMR data show two conformational isomers of this immunogenic complex. The presence of conformational isomers at a TCR contact site suggests that these structures may be recognized differently by the TCR. The agreement between the dissociation kinetics and the 19F-NMR data demonstrate that kinetic heterogeneity is correlated with structural counterparts observed by NMR. Dissociations in the presence of dimethyl sulfoxide were used to show that the rate of interconversion of these conformational isomers at pH 7.0 is low, with a lifetime on the order of hours or more. Modification of a peptide residue of PCC occupying the minor MHC binding pocket P6 alters the 19F-NMR spectra of both labels. This demonstrates that distant changes of amino acid residues can influence the conformation of the whole antigenic peptide inside the MHC binding cleft.

Animals↗
PubMed1999

Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART).

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77-85) and reverse transcriptase (amino acids 464-472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50-1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals

Amino Acid Sequence↗