Yuri Sykulev

Evidence that a Single Peptide–MHC Complex on a Target Cell Can Elicit a Cytolytic T Cell Response

Using a chemically homogeneous radiolabeled peptide of high specific activity (125I-QLSPYPFDL, 3.5 x 10(18) cpm per mole) we show that at a peptide concentration (5 pM) causing half-maximal lysis of target cells by a cytolytic T lymphocyte (CTL) clone that recognizes the peptide in association with Ld, a class I MHC protein, only 3 peptide molecules on average are bound by Ld per target cell. From the distribution of Ld on the target cells, we suggest that a single peptide-MHC complex per target cell can trigger activation of the T cell cytolytic response.

Kinetics and affinity of reactions between an antigen-specific T cell receptor and peptide-MHC complexes

We show here that the net rate of accumulation of complexes formed by the antigen-specific receptor of T cells (TCR) of a T cell clone with its natural ligand, an octapeptide in association with Ld, a class I protein of the major histocompatibility complex (MHC), approaches the maximal value determined by the affinity of the TCR for this peptide-MHC ligand in 1-2 min, which is well within the lifetime of transient T cell-target cell conjugates. Consistent with this finding, we also found that the widely divergent affinity values (equilibrium constants) of this TCR for six related peptide-MHC complexes correlate well with the extent of specific lysis of target cells bearing various level of these complexes.

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.

Cytotoxic T lymphocytes form an antigen-independent ring junction.

Immunological synapses are organized cell-cell junctions between T lymphocytes and APCs composed of an adhesion ring, the peripheral supramolecular activation cluster (pSMAC), and a central T cell receptor cluster, the central supramolecular activation cluster (cSMAC). In CD8(+) cytotoxic T lymphocytes, the immunological synapse is thought to facilitate specific killing by confining cytotoxic agents to the synaptic cleft. We have investigated the interaction of human CTLs and helper T cells with supported planar bilayers containing ICAM-1. This artificial substrate provides identical ligands to CD4(+) and CD8(+) T cells, allowing a quantitative comparison. We found that cytotoxic T lymphocytes form a ring junction similar to a pSMAC in response to high surface densities of ICAM-1 in the planar bilayer. MICA, a ligand for NKG2D, facilitated the ring junction formation at lower surface densities of ICAM-1. ICAM-1 and MICA are upregulated in tissues by inflammation- and stress-associated signaling, respectively. Activated CD8(+) T cells formed fivefold more ring junctions than did activated CD4(+) T cells. The ring junction contained lymphocyte function associated antigen-1 and talin, but did not trigger polarization and granule translocation to the interface. This result has specific implications for the mechanism of effective CTL hunting for antigen in tissues. Abnormalities in this process may alter CTL reactivity.

Quantum dot/peptide-MHC biosensors reveal strong CD8-dependent cooperation between self and viral antigens that augment the T cell response

Cytotoxic T lymphocytes (CTL) can respond to a few viral peptide-MHC-I (pMHC-I) complexes among a myriad of virus-unrelated endogenous self pMHC-I complexes displayed on virus-infected cells. To elucidate the molecular recognition events on live CTL, we have utilized a self-assembled biosensor composed of semiconductor nanocrystals, quantum dots, carrying a controlled number of virus-derived (cognate) and other (noncognate) pMHC-I complexes and examined their recognition by antigen-specific T cell receptor (TCR) on anti-virus CD8+ T cells. The unique architecture of nanoscale quantum dot/pMHC-I conjugates revealed that unexpectedly strong multivalent CD8–MHC-I interactions underlie the cooperative contribution of noncognate pMHC-I to the recognition of cognate pMHC-I by TCR to augment T cell responses. The cooperative, CD8-dependent spread of signal from a few productively engaged TCR to many other TCR can explain the remarkable ability of CTL to respond to virus-infected cells that present few cognate pMHC-I complexes.

Kinetics of Early T Cell Receptor Signaling Regulate the Pathway of Lytic Granule Delivery to the Secretory Domain

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

Multiple effector functions mediated by human immunodeficiency virus-specific CD4(+) T-cell clones.

ABSTRACT Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4+ T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-γ) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.

ANTIGEN-SPECIFIC T-CELL RECEPTORS AND THEIR REACTIONS WITH COMPLEXES FORMED BY PEPTIDES WITH MAJOR HISTOCOMPATIBILITY COMPLEX PROTEINS

The chapter gives an overview of an antigen-specific T-cell receptors and their reactions with complexes formed with major histocompatibility complex proteins. The receptors on the two major classes of lymphocytes, B and T cells, are similar structurally but profoundly different functionally. On B cells, the receptors are immunoglobulins (Ig) embedded in the cell surface as integral membrane proteins. The antigen-specific receptors on T cells (T-cell receptors or TCR) are also Ig-like cell surface integral membrane proteins; their recognition of antigens triggers T cells to exercise a great variety of functions but not to secrete the receptors. The enormous diversity of B- and T-cell receptors arises from the many germline gene segments that encode them; as each lymphocyte matures, different combinations of these segments are joined (combinatorial diversity) and additional variations in sequence are introduced at the junctures (junctional diversity), leading to an immense number of variable domain sequences. The TCR reviewed in this chapter normally recognizes and responds only to the complexes formed between small peptides and a specialized set of proteins encoded by the major histocompatibility complex (MHC). The chapter also discusses the T-Cell Receptor genes, proteins, ligand: peptide MHC complexes and TCR accessory proteins (CD3, CD4 and CD8).

Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.

Destruction of virus-infected cells by CTL is an extremely sensitive and efficient process. Our previous data suggest that LFA-1-ICAM-1 interactions in the peripheral supramolecular activation cluster (pSMAC) of the immunological synapse mediate formation of a tight adhesion junction that might contribute to the sensitivity of target cell lysis by CTL. Herein, we compared more (CD8+) and less (CD4+) effective CTL to understand the molecular events that promote efficient target cell lysis. We found that abrogation of the pSMAC formation significantly impaired the ability of CD8+ but not CD4+ CTL to lyse target cells despite having no effect of the amount of released granules by both CD8+ and CD4+ CTL. Consistent with this, CD4+ CTL break their synapses more often than do CD8+ CTL, which leads to the escape of the cytolytic molecules from the interface. CD4+ CTL treatment with a protein kinase Cθ inhibitor increases synapse stability and sensitivity of specific target cell lysis. Thus, formation of a stable pSMAC, which is partially controlled by protein kinase Cθ, functions to confine the released lytic molecules at the synaptic interface and to enhance the effectiveness of target cell lysis.

Enteric alpha defensins in norm and pathology

Microbes living in the mammalian gut exist in constant contact with immunity system that prevents infection and maintains homeostasis. Enteric alpha defensins play an important role in regulation of bacterial colonization of the gut, as well as in activation of pro- and anti-inflammatory responses of the adaptive immune system cells in lamina propria. This review summarizes currently available data on functions of mammalian enteric alpha defensins in the immune defense and changes in their secretion in intestinal inflammatory diseases and cancer.