Translate this page to:
Russian
In JoVE (1)
Other Publications (18)
- Nature
- Biochemistry
- Annual Review of Biochemistry
- Molecular Cell
- Seminars in Immunology
- Autoimmunity
- Journal of Immunological Methods
- Biophysical Journal
- Nature Immunology
- Nature
- Proceedings of the National Academy of Sciences of the United States of America
- Annual Review of Immunology
- Seminars in Immunology
- Journal of Immunology (Baltimore, Md. : 1950)
- American Journal of Translational Research
- The Journal of Experimental Medicine
- PLoS Biology
- Nature
Automatic Translation
This translation into Russian was automatically generated.
English Version | Other Languages
Articles by Michelle Krogsgaard in JoVE
JoVE Immunology and Infection
Ретровирусные трансдукция Т-клеточных рецепторов в мышь Т-клеток
1NYU Cancer institute, New York University School of Medicine, 2Program in Structural Biology, New York University School of Medicine
Мы представляем протокол производить антиген-специфические Т-мышь клеток с использованием ретровирусной трансдукции
Other articles by Michelle Krogsgaard on PubMed
Direct Observation of Ligand Recognition by T Cells
The activation of T cells through interaction of their T-cell receptors with antigenic peptide bound to major histocompatibility complex (MHC) on the surface of antigen presenting cells (APCs) is a crucial step in adaptive immunity. Here we use three-dimensional fluorescence microscopy to visualize individual peptide-I-E(k) class II MHC complexes labelled with the phycobiliprotein phycoerythrin in an effort to characterize T-cell sensitivity and the requirements for forming an immunological synapse in single cells. We show that T cells expressing the CD4 antigen respond with transient calcium signalling to even a single agonist peptide-MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present. This sensitivity is highly dependent on CD4, because blocking this molecule with antibodies renders T cells unable to detect less than about 30 ligands.
Distinct Molecular Mechanisms Account for the Specificity of Two Different T-cell Receptors
Analysis of the thermodynamics of the interactions between the D3 T-cell receptor (TCR) and its natural ligand, an HIV peptide bound to a HLA-A0201 (HLA-A2) major histocompatibility complex (MHC) protein, shows both similarities and striking differences when compared with the 2B4 TCR binding to its peptide-MHC ligand. The equilibrium thermodynamic parameters of both reactions are consistent with a conformational adjustment at the binding interface during the formation of specific TCR-peptide-MHC complexes. However, osmolytic reagents that dehydrate protein surfaces have profoundly different effects on the strength of the two reactions, indicating that water molecules make very different contributions-enhancing the binding of D3 TCR but weakening the binding of 2B4 TCR. The use of these different mechanisms by TCRs to recognize ligands might be an important means augmenting their inherent cross-reactivity.
Dynamics of Cell Surface Molecules During T Cell Recognition
Recognition of foreign antigens by T lymphocytes is a very important component of vertebrate immunity-vital to the clearance of pathogenic organisms and particular viruses and necessary, indirectly, for the production of high affinity antibodies. T cell recognition is mediated by the systematic scanning of cell surfaces by T cells, which collectively express many antigen receptors. When the appropriate antigenic peptide bound to a molecule of the major histocompatibility complex is found-even in minute quantities-a series of elaborate cell-surface molecule and internal rearrangements take place. The sequence of events and the development of techniques required to observe these events have significantly enhanced our understanding of T cell recognition and may find application in other systems of transient cell:cell interactions as well.
Evidence That Structural Rearrangements And/or Flexibility During TCR Binding Can Contribute to T Cell Activation
While in many cases the half-life of T cell receptor (TCR) binding to a particular ligand is a good predictor of activation potential, numerous exceptions suggest that other physical parameter(s) must also play a role. Accordingly, we analyzed the thermodynamics of TCR binding to a series of peptide-MHC ligands, three of which are more stimulatory than their stability of binding would predict. Strikingly, we find that during TCR binding these outliers show anomalously large changes in heat capacity, an indicator of conformational change or flexibility in a binding interaction. By combining the values for heat capacity (DeltaCp) and the half-life of TCR binding (t(1/2)), we find that we can accurately predict the degree of T cell stimulation. Structural analysis shows significant changes in the central TCR contact residue of the peptide-MHC, indicating that structural rearrangements within the TCR-peptide-MHC interface can contribute to T cell activation.
Linking Molecular and Cellular Events in T-cell Activation and Synapse Formation
The complex sequence of events in which T cells recognize foreign entities on other cells is not well understood. However, the development of new techniques and approaches in both the molecular and cellular aspects of this problem have provided significant insights into the mechanisms of T-cell recognition and synapse formation. In particular, we have a clearer picture of T-cell sensitivity, the role of co-stimulation in formation of the immunological synapse, and how TCR signaling acts to maintain synapse structure and potentiate the T cells over many hours of engagement. We also are aware of new complexities in the way T-cell receptor molecules bind peptide-MHC (pMHC) ligands and what that may mean for TCR scanning, cross-reactivity, and activation. Ultimately, we want to integrate these cellular aspects of T-cell recognition with key features of the molecular interactions that drive specific events.
Selection of Aberrant Class II Restricted CD8+ T Cells in NOD Mice Expressing a Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgene
We previously described the generation of non-obese diabetic (NOD) mice expressing a transgenic T cell receptor (TCR) specific for peptide epitope 286-300 of the diabetes related self antigen, glutamic acid decarboxylase (GAD)65 in the context of I-A(g7) class II MHC, that are paradoxically protected from diabetes. In this report, we examine the atypical CD8+ cells in these mice. Unlike typical class II restricted TCR transgenic mice, GAD286 mice have normal numbers of CD8+ cells, half of which express high levels of the transgenic TCR. These MHC mismatched CD8+ cells persist in the periphery and proliferate to GAD286-300 peptide in vitro and in vivo in a class II restricted fashion. Interestingly, the CD8+ tetramer(-) T cells that are expressing endogenous TCR can delay diabetes induction in a transfer model, as we previously showed for CD4+ tetramer+ T cells in these mice. The MHC mismatched CD8+ cells appear to be positively selected in an atypical fashion, in that they do not upregulate CD69 or reexpress CD44, and they escape negative selection. We find that production of these CD8+ cells is not dependent on NOD thymus or high affinity of the TCR, but is dependent on the atypical TCR transgenic thymic environment.
High-level Bacterial Secretion of Single-chain Alphabeta T-cell Receptors
While numerous antibody-antigen systems have been structurally characterized, studies of structurally analogous T-cell receptor MHC systems have lagged behind largely due to the lack of a general TCR expression system. Efforts to develop bacterial systems have resulted in low yields (< 0.5 mg/l) of active material which is prone to proteolysis and aggregation. Here we report a strategy to secrete folded, soluble single chain T-cell receptors (scTCR) in the Escherichia coli periplasm using three representative alphabeta TCRs (172.10, 1934.4/c19 and 2B4). Shake flask yields between 0.5 and 30 mg/l active, purified material were attained for all TCRs studied and found to depend on the introduction of solubility-increasing amino acid substitutions, skp chaperone co-expression and C-terminal fusion to a human kappa constant domain in the context of a tightly regulated expression vector. This system will greatly enable crystallographic, thermodynamic and other biophysical analyses of TCRs which require large quantities of homogeneous material.
Quantitative Imaging of Lymphocyte Membrane Protein Reorganization and Signaling
Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4+ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture.
How T Cells 'see' Antigen
T lymphocytes bearing alphabeta T cell receptors are pivotal in the immune response of most vertebrates. For example, helper T cells orchestrate antibody production by B cells as well as stimulating other cells, whereas cytotoxic T cells kill virally infected or abnormal cells. Regulatory T cells act to dampen responsiveness, and natural killer-like T cells monitor lipid metabolism. The specificity of these cells is governed by the alphabeta T cell receptors - antibody-like heterodimeric receptors that detect antigenic fragments (peptides) or lipids bound to histocompatibility molecules. Intriguing clues as to how these peculiar ligands are recognized have gradually emerged over the years and tell a remarkable story of biochemical and cellular novelty. Here we summarize some of the more recent work on alphabeta T cell receptor recognition and discuss the implications for activation.
Agonist/endogenous Peptide-MHC Heterodimers Drive T Cell Activation and Sensitivity
Alphabeta T lymphocytes are able to detect even a single peptide-major histocompatibility complex (MHC) on the surface of an antigen-presenting cell. This is despite clear evidence, at least with CD4+ T cells, that monomeric ligands are not stimulatory. In an effort to understand how this remarkable sensitivity is achieved, we constructed soluble peptide-MHC heterodimers in which one peptide is an agonist and the other is one of the large number of endogenous peptide-MHCs displayed by presenting cells. We found that some specific combinations of these heterodimers can stimulate specific T cells in a CD4-dependent manner. This activation is severely impaired if the CD4-binding site on the agonist ligand is ablated, but the same mutation on an endogenous ligand has no effect. These data correlate well with analyses of lipid bilayers and cells presenting these ligands, and indicate that the basic unit of helper T cell activation is a heterodimer of agonist peptide- and endogenous peptide-MHC complexes, stabilized by CD4.
Molecular Flexibility Can Influence the Stimulatory Ability of Receptor-ligand Interactions at Cell-cell Junctions
Direct cell-cell communication is crucial for many processes in biology, particularly embryogenesis, interactions between hematopoetic cells, and in the nervous system. This communication is often mediated by the binding of receptors to cognate ligands at a cell-cell junction. One such interaction that is very important for the development of many immune responses is the binding of the alphabeta T cell receptor for antigen (TCR) on T lymphocytes with peptide-MHC complexes on other cells. In general, the stability (e.g., half-life) of TCR-peptide-MHC binding measured in solution correlates with functional responses. Several anomalies have been reported, however. For example, for some anomalous ligands, large changes in heat capacity can apparently substitute for a lack of stability in TCR-ligand interactions. Here, we show that, when there are significant conformational changes during receptor-ligand binding and the receptor/ligand have relatively rigid molecular subdomains, the difference between the half-life of this receptor-ligand complex at a cell-cell junction and that measured using soluble molecules is large. Thus, receptors/ligands with these specific molecular features do not follow correlations between stimulatory potency in the cellular environment and half-lives measured with soluble molecules. Our "first-principles" prescription for correcting the half-life measured in solution to obtain the pertinent value at a cell-cell junction illuminates the origin of correlations of T cell response with thermodynamic properties. Application of our ideas to diverse systems where receptor-ligand interactions occur across juxtaposed cells may help avoid debates about "anomalies" that may simply arise from receptor/ligand-specific differences between half-lives in solution and in the cellular environment.
T Cells As a Self-referential, Sensory Organ
In light of recent data showing that both helper and cytotoxic T cells can detect even a single molecule of an agonist peptide-MHC, alphabeta T cells are clearly a type of sensory cell, comparable to any in the nervous system. In addition, endogenous (self) peptides bound to MHCs are not just important for thymic selection, but also play an integral role in T cell activation in the response to foreign antigens. With the multitude of specificities available to most T cells, they can thus be considered as a sensory organ, trained on self-peptide-MHCs and primed to detect nonself.
A Role for "self" in T-cell Activation
The mechanisms by which alphabeta T-cells are selected in the thymus and then recognize peptide MHC (pMHC) complexes in the periphery remain an enigma. Recent work particularly with respect to quantification of T-cell sensitivity and the role of self-ligands in T-cell activation has provided some important clues to the details of how TCR signaling might be initiated. Here, we highlight recent experimental data that provides insights into the initiation of T-cell activation and also discuss the main controversies and uncertainties in this area.
In Vivo Enhancement of Peptide Display by MHC Class II Molecules with Small Molecule Catalysts of Peptide Exchange
Rapid binding of peptides to MHC class II molecules is normally limited to a deep endosomal compartment where the coordinate action of low pH and HLA-DM displaces the invariant chain remnant CLIP or other peptides from the binding site. Exogenously added peptides are subject to proteolytic degradation for extended periods of time before they reach the relevant endosomal compartment, which limits the efficacy of peptide-based vaccines and therapeutics. In this study, we describe a family of small molecules that substantially accelerate the rate of peptide binding to HLA-DR molecules in the absence of HLA-DM. A structure-activity relationship study resulted in analogs with significantly higher potency and also defined key structural features required for activity. These compounds are active over a broad pH range and thus enable efficient peptide loading at the cell surface. The small molecules not only enhance peptide presentation by APC in vitro, but are also active in vivo where they substantially increase the fraction of APC on which displayed peptide is detectable. We propose that the small molecule quickly reaches draining lymph nodes along with the coadministered peptide and induces rapid loading of peptide before it is destroyed by proteases. Such compounds may be useful for enhancing the efficacy of peptide-based vaccines and other therapeutics that require binding to MHC class II molecules.
Developing a Multidisciplinary Prospective Melanoma Biospecimen Repository to Advance Translational Research
Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic.
Peptide-MHC Heterodimers Show That Thymic Positive Selection Requires a More Restricted Set of Self-peptides Than Negative Selection
T cell selection and maturation in the thymus depends on the interactions between T cell receptors (TCRs) and different self-peptide-major histocompatibility complex (pMHC) molecules. We show that the affinity of the OT-I TCR for its endogenous positively selecting ligands, Catnb-H-2Kb and Cappa1-H-2Kb, is significantly lower than for previously reported positively selecting altered peptide ligands. To understand how these extremely weak endogenous ligands produce signals in maturing thymocytes, we generated soluble monomeric and dimeric peptide-H-2Kb ligands. Soluble monomeric ovalbumin (OVA)-Kb molecules elicited no detectable signaling in OT-I thymocytes, whereas heterodimers of OVA-Kb paired with positively selecting or nonselecting endogenous peptides, but not an engineered null peptide, induced deletion. In contrast, dimer-induced positive selection was much more sensitive to the identity of the partner peptide. Catnb-Kb-Catnb-Kb homodimers, but not heterodimers of Catnb-Kb paired with a nonselecting peptide-Kb, induced positive selection, even though both ligands bind the OT-I TCR with detectable affinity. Thus, both positive and negative selection can be driven by dimeric but not monomeric ligands. In addition, positive selection has much more stringent requirements for the partner self-pMHC.
Attenuated T Cell Responses to a High-potency Ligand in Vivo
αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4(+) T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.
Polyreactivity Increases the Apparent Affinity of Anti-HIV Antibodies by Heteroligation
During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV) disfavours homotypic bivalent antibody binding. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV.
