Russell D. Salter

Identification of T-cell epitopes : rapid isolation of class I-presented peptides from viable cells by mild acid elution

A novel method was developed to isolate immunogenic peptides (CD8+ T-cell epitopes) from class I complexes expressed at the cell surface of viable cells. Cells treated at pH 3.3 with citrate-phosphate buffer for periods as short as 15 s remained viable and became phenotypically class I deficient. Qualitative loss of class I determinants was verified both serologically and by the incapacity of acid-treated cells to be lysed by class I-restricted cytolytic T lymphocytes (CTLs) in contrast to non-acid-treated controls. Flow cytometric analysis of acid-treated cells suggests that class I heavy chains remain associated with the cell membrane, while the class I light chain (beta 2-microglobulin) is absent. Since the physical dissociation of beta 2-microglobulin from class I heavy chain is correlated with the release of previously class I-bound peptides, we examined acid-eluted cell-free supernatants for the presence of immunogenic peptides. Peptides were acid eluted from an influenza A strain-infected, HLA-A2+ cell line and were subsequently fractionated by reverse-phase high performance liquid chromatography (RP-HPLC). These fractionated peptides were examined for their capacity to sensitize an HLA-A2+ B cell line to lysis mediated by an influenza A matrix peptide- (Flu M1 57-68) specific, HLA-A2-restricted CTL line. A single peak of biologic activity was identified in HPLC fractions 47 and 48 derived from influenza-infected cells. These fractions contained a peptide of M(r) 968 with a sequence similar to the Flu M1 58-66 sequence GILGFVFTL. The application of this technique to other T-cell-based systems may aid in the definition of peptide epitopes relevant to viral, autoimmune, or neoplastic disorders.

Mitochondrial Reactive Oxygen Species Induces NLRP3-Dependent Lysosomal Damage and Inflammasome Activation

The nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome drives many inflammatory processes and mediates IL-1 family cytokine release. Inflammasome activators typically damage cells and may release lysosomal and mitochondrial products into the cytosol. Macrophages triggered by the NLRP3 inflammasome activator nigericin show reduced mitochondrial function and decreased cellular ATP. Release of mitochondrial reactive oxygen species (ROS) leads to subsequent lysosomal membrane permeabilization (LMP). NLRP3-deficient macrophages show comparable reduced mitochondrial function and ATP loss, but maintain lysosomal acidity, demonstrating that LMP is NLRP3 dependent. A subset of wild-type macrophages undergo subsequent mitochondrial membrane permeabilization and die. Both LMP and mitochondrial membrane permeabilization are inhibited by potassium, scavenging mitochondrial ROS, or NLRP3 deficiency, but are unaffected by cathepsin B or caspase-1 inhibitors. In contrast, IL-1β secretion is ablated by potassium, scavenging mitochondrial ROS, and both cathepsin B and caspase-1 inhibition. These results demonstrate interplay between lysosomes and mitochondria that sustain NLRP3 activation and distinguish cell death from IL-1β release.

Flow-cytometric determination of peptide-class I complex formation identification of p53 peptides that bind to HLA-A2

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.

Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane

Cells survive exposure to bacterial pore-forming toxins, such as streptolysin O (SLO), through mechanisms that remain unclear. Previous studies have suggested that these toxins are cleared by endocytosis. However, the experiments reported here failed to reveal any evidence for endocytosis of SLO, nor did they reveal any signs of damage to endosomal membranes predicted from such endocytosis. Instead, we illustrate that SLO induces a characteristic form of plasma membrane blebbing that allows cells to shed SLO by the process known as ectocytosis. Specifically, ‘deep-etch’ electron microscopy of cells exposed to SLO illustrates that the toxin is rapidly sequestered into domains in the plasmalemma greatly enriched in SLO pores, and these domains bleb outwards and bud from the cell surface into the medium. Such ectocytosis is even observed in cells that have been chemically fixed before exposure to SLO, suggesting that it is caused by a direct physical action of the toxin on the cell membrane, rather than by an active cellular reaction. We conclude, therefore, that ectocytosis is an important means for SLO clearance and hypothesize that this is a primary method by which cells defend themselves generally against pore-forming toxins.

Cholesterol-dependent cytolysins induce rapid release of mature IL-1β from murine macrophages in a NLRP3 inflammasome and cathepsin B-dependent manner

CDC are exotoxins secreted by many Gram‐positive bacteria that bind cholesterol and oligomerize to form pores in eukaryotic cell membranes. We demonstrate that CDC TLO induces caspase‐1 cleavage and the rapid release of IL‐1β from LPS‐primed murine BMDM. IL‐1β secretion depends on functional toxin pore formation, as free cholesterol, which prevents TLO binding to cell membranes, blocks the cytokine release. Secretion of the mature forms of IL‐1β and caspase‐1 occurs only at lower TLO doses, whereas at a higher concentration, cells release the biologically inactive proforms. IL‐1β release at a low TLO dose requires potassium efflux, calcium influx, and the activities of calcium‐independent PLA2, caspase‐1, and cathepsin B. Additionally, mature IL‐1β release induced by a low TLO dose is dependent on the NLRP3 inflammasome, and pro‐IL‐1β release induced by a high TLO dose occurs independently of NLRP3. These results further elucidate a mechanism of CDC‐induced IL‐1β release and suggest a novel, immune evasion strategy in which IL‐1β‐containing macrophages might release primarily inactive cytokine following exposure to high doses of these toxins.

Peptide-specific CD8+ T-cell evolution in vivo: Response to peptide vaccination with Melan-A/MART-1

Monitoring of CD8+ T‐cell responses in cancer patients during peptide vaccination is essential to provide useful surrogate markers and to demonstrate vaccine efficacy. We have longitudinally followed CD8+ T‐cell responses in 3 melanoma patients who were immunized with peptides derived from Melan‐A/MART‐1. Recombinant HLA‐A2 tetramers loaded with the naturally presented Melan‐A/MART‐1 nonamer peptide (AAGIGILTV) and the Melan‐A/MART‐1 analog (ELAGIGILTV) were used in combination with phenotypical analysis for different T‐cell subsets including naive T cells, effector T cells, “true memory” T cells and “memory effector” T cells, based on CD45RA/RO and CCR7‐expression. At least in a single patient, T cells binding to the AAGIGILTV epitope were detected in naive, precursor (CD45RA+/CCR7+) CD8+ T cells, and CD8+ T cells binding to the analog ELAGIGILTV peptide were identified in the terminally differentiated (CD45RA+/CCR7−) T‐cell subset. Molecular and functional analysis of tetramer‐binding T cells revealed that the T‐cell receptor (TCR) repertoire was oligo/polyclonal in AAGIGILTV‐reactive T cells, but different and restricted to a few TCR clonotypes in ELAGIGILTV‐reactive T cells prior to vaccination. The TCR repertoire reactive with Melan‐A/MART‐1 peptide epitopes was broadened during vaccination and exhibited a different profile of cytokine release after specific stimulation: tetramer‐binding T cells from 2/3 patients secreted granulocyte/macrophage colony‐stimulating factor (GM‐CSF) and interferon‐γ but not interleukin‐2 (IL‐2) in response to Melan‐A/MART‐1 peptides. In the third patient, tetramer‐binding T cells secreted IL‐2 exclusively. Our results show that T‐cell responses to peptide vaccination consist of different T‐cell subsets associated with different effector functions. Complementary analysis for TCR CDR3 and cytokine profiles may be useful to define the most effective CD8+ T‐cell population induced by peptide vaccination.

Genetically Encoded pH Sensor for Tracking Surface Proteins through Endocytosis

Traffic cam: a tandem dye prepared from a FRET acceptor and a fluorogenic donor functions as a cell surface ratiometric pH indicator, which upon internalization serves to follow protein trafficking during endocytosis. This sensor was used to analyze agonist-dependent internalization of β(2)-adrenergic receptors. It was also used as a surrogate antigen to reveal direct surface-to-endosome antigen transfer between dendritic cells (not shown).

β2-Microglobulin and calnexin can independently promote folding and disulfide bond formation in class I histocompatibility proteins

Class I histocompatibility proteins fold and assemble with beta 2-microglobulin (beta 2m) into heterodimers before binding short peptides in the endoplasmic reticulum. Here, we show that class I proteins rapidly form disulfide bonds, and that the process is highly reversible in Daudi cells lacking beta 2m. Three distinct class I protein conformations are present in equal amounts in these cells, each associated with the molecular chaperone calnexin. When binding of calnexin is inhibited by the glucosidase inhibitor castanospermine, fully oxidized class I proteins are no longer detected, suggesting that calnexin is required for completion of folding. However, in Daudi cells transfected to express beta 2m, castanospermine decreases only slightly the levels of fully oxidized class I proteins, indicating that folding is much less dependent on calnexin in the presence of beta 2m. Furthermore, calreticulin, a chaperone with functional similarities to calnexin, associates with class I molecules in beta 2m-positive cells. but not in Daudi cells, consistent with completion of folding and disulfide bond formation of class I heavy chains before binding to calreticulin occurs. This study demonstrates that calnexin and beta 2m can function independently to promote folding of class I heavy chains prior to formation of stable class I dimers.

Escherichia coli Expressing Recombinant Antigen and Listeriolysin O Stimulate Class I-Restricted CD8+ T Cells following Uptake by Human APC

Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8+ T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-γ ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8+ T cell responses against pathogens or tumors.

Activation of Macrophages by P2X7-Induced Microvesicles from Myeloid Cells Is Mediated by Phospholipids and Is Partially Dependent on TLR4

ATP-mediated activation of the purinergic receptor P2X7 elicits morphological changes and proinflammatory responses in macrophages. These changes include rapid shedding of microvesicles (MV) and the nonconventional secretion of cytokines, such as IL-1β and IL-18 following priming. In this study, we demonstrate the activation potential of P2X7-induced MV isolated from nonprimed murine macrophages. Cotreatment of nonprimed macrophages with ATP and calcium ionophore induced a rapid release of MV that were predominantly 0.5–1 μm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-α secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-κB dependent, and partially dependent on TLR4 activity, but was high-mobility group box 1 independent. Biochemical fractionation of the MV demonstrated that the phospholipid fraction, not the protein fraction, mediated macrophage activation through a TLR4-dependent process. P2X7 activation is known to induce calcium-independent phospholipase A2, calcium-dependent phospholipase A2, and phospholipase D activities, but inhibition of these enzymes did not inhibit MV generation or shedding. However, blocking phospholipase D activity resulted in release of MV incapable of activating recipient macrophages. These data demonstrate a novel mechanism of macrophage activation resulting from exposure to MV from nonprimed macrophages, and identifies phospholipids in these MV as the biologically active component. We suggest that phospholipids delivered by MV may be mediators of sterile inflammation in a number of diseases.