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Pediatric-onset systemic lupus erythematosus arises in humans and mice lacking the endonuclease Dnase1L3. When Dnase1L3 is absent, DNA from circulating apoptotic bodies is not cleared, leading to anti-DNA antibody production. Compared to early anti-DNA and anti-chromatin responses, other autoantibody responses and general immune activation in Dnase1L3 -/- mice are greatly delayed. We investigated the possibility that immune activation, specifically inflammasome activation, is regulated by Dnase1L3. Here, we report that Dnase1L3 inhibition blocked both NLR family, pyrin domain containing 3 (NLRP3) and NLRC4 inflammasome-mediated release of high-mobility group box 1 protein and IL-1β. In contrast to IL-1β release, Dnase1L3 inhibition only mildly impaired NLRP3-dependent pyroptosis, as measured by propidium iodide uptake or LDH release. Mechanistically, we found that Dnase1L3 was needed to promote apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) nuclear export and speck formation. Our results demonstrate that Dnase1L3 inhibition separates cytokine secretion from pyroptosis by targeting ASC. These findings suggest that Dnase1L3 is necessary for cytokine secretion following inflammasome activation.

MUC1 is a glycoprotein expressed on the apical surface of ductal epithelial cells. Malignant transformation results in loss of polarization and overexpression of hypoglycosylated MUC1 carrying truncated carbohydrates known as T or Tn tumor antigens. Tumor MUC1 bearing Tn carbohydrates (Tn-MUC1) represent a potential target for immunotherapy. We evaluated the Tn-MUC1 glycopeptide in a human phase I/II clinical trial for safety that followed a preclinical study of different glycosylation forms of MUC1 in rhesus macaques, whose MUC1 is highly homologous to human MUC1. Either unglycosylated rhesus macaque MUC1 peptide (rmMUC1) or Tn-rmMUC1 glycopeptide was mixed with an adjuvant or loaded on autologous dendritic cells (DC), and responses were compared. Unglycosylated rmMUC1 peptide induced negligible humoral or cellular responses compared with the Tn-rmMUC1 glycopeptide. Tn-rmMUC1 loaded on DCs induced the highest anti-rmMUC1 T-cell responses and no clinical toxicity. In the phase I/II clinical study, 17 patients with nonmetastatic castrate-resistant prostate cancer (nmCRPC) were tested with a Tn-MUC1 glycopeptide-DC vaccine. Patients were treated with multiple intradermal and intranodal doses of autologous DCs, which were loaded with the Tn-MUC1 glycopeptide (and KLH as a positive control for immune reactivity). PSA doubling time (PSADT) improved significantly in 11 of 16 evaluable patients (P = 0.037). Immune response analyses detected significant Tn-MUC1-specific CD4 + and/or CD8 + T-cell intracellular cytokine responses in 5 out of 7 patients evaluated. In conclusion, vaccination with Tn-MUC1-loaded DCs in nmCRPC patients appears to be safe, able to induce significant T-cell responses, and have biological activity as measured by the increase in PSADT following vaccination. Cancer Immunol Res; 4(10); 881-92. ©2016 AACR., (©2016 American Association for Cancer Research.)

The ability of dendritic cells (DC) to mediate CD4(+) T cell help for cellular immunity is guided by instructive signals received during DC maturation, as well as the resulting pattern of DC responsiveness to the Th signal, CD40L. Furthermore, the professional transfer of antigenic information from migratory DC to lymph node-residing DC is critical for the effective induction of cellular immune responses. In this study we report that, in addition to their enhanced IL-12p70 producing capacity, human DC matured in the presence of inflammatory mediators of type 1 immunity are uniquely programmed to form networks of tunneling nanotube-like structures in response to CD40L-expressing Th cells or rCD40L. This immunologic process of DC reticulation facilitates intercellular trafficking of endosome-associated vesicles and Ag, but also pathogens such HIV-1, and is regulated by the opposing roles of IFN-γ and IL-4. The initiation of DC reticulation represents a novel helper function of CD40L and a superior mechanism of intercellular communication possessed by type 1 polarized DC, as well as a target for exploitation by pathogens to enhance direct cell-to-cell spread., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

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.

Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1β secretion and pyroptosis. The structural determinants of pore-forming toxins required for NLRP3 activation remain unknown. Here, we demonstrate using streptolysin O (SLO) that pore-formation controls IL-1β secretion and direct toxicity. An SLO mutant incapable of pore-formation did not promote direct killing, pyroptosis or IL-1β production. This indicated that pore formation is necessary for inflammasome activation. However, a partially active mutant (SLO N402C) that was less toxic to macrophages than wild-type SLO, even at concentrations that directly lysed an equivalent number of red blood cells, enhanced IL-1β production but did not alter pyroptosis. This suggests that direct lysis may attenuate immune responses by preventing macrophages from successfully repairing their plasma membrane and elaborating more robust cytokine production. We suggest that mutagenesis of pore-forming toxins represents a strategy to enhance adjuvant activity.

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors.

Aberrant activation of macrophages in arterial walls by oxidized lipoproteins can lead to atherosclerosis. Oxidized lipoproteins convert macrophages to foam cells through lipid uptake and TLR signaling. To investigate the relative contributions of lipid uptake and TLR signaling in foam cell formation, we established an in vitro assay using liposomes of defined lipid compositions. We found that TLRs signaling through Toll/IL-1R domain-containing adapter inducing IFN-β promoted foam cell formation by inducing both NF-κB signaling and type I IFN production, whereas TLRs that do not induce IFN, like TLR2, did not enhance foam cell formation. Addition of IFN-α to TLR2 activator promoted robust foam cell formation. TLR signaling further required peroxisome proliferator-activated receptor α, as inhibition of peroxisome proliferator-activated receptor α blocked foam cell formation. We then investigated the ability of endogenous microparticles (MP) to contribute to foam cell formation. We found that lipid-containing MP promoted foam cell formation, which was enhanced by TLR stimulation or IFN-α. These MP also stimulated foam cell formation in a human skin model. However, these MP suppressed TNF-α production and T cell activation, showing that foam cell formation can occur by immunosuppressive MP. Taken together, the data reveal novel signaling requirements for foam cell formation and suggest that uptake of distinct types of MP in the context of activation of multiple distinct TLR can induce foam cell formation.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type(1). In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines(2). Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms. Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism(3,4). This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation(4). In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines(5,6,7). A link between IL-1β release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes(8). To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

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)., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Immunization against beta-amyloid (Aβ) is a promising approach for the treatment of Alzheimer's disease, but the optimal timing for the vaccination remains to be determined. Preventive immunization approaches may be more efficacious and associated with fewer side-effects; however, there is only limited information available from primate models about the effects of preclinical vaccination on brain amyloid composition and the neuroinflammatory milieu., Methods: Ten non-human primates (NHP) of advanced age (18-26 years) and eight 2-year-old juvenile NHPs were immunized at 0, 2, 6, 10 and 14 weeks with aggregated Aβ42 admixed with monophosphoryl lipid A as adjuvant, and monitored for up to 6 months. Anti-Aβ antibody levels and immune activation markers were assessed in plasma and cerebrospinal fluid samples before and at several time-points after immunization. Microglial activity was determined by [(11)C]PK11195 PET scans acquired before and after immunization, and by post-mortem immunohistochemical and real-time PCR evaluation. Aβ oligomer composition was assessed by immunoblot analysis in the frontal cortex of aged immunized and non-immunized control animals., Results: All juvenile animals developed a strong and sustained serum anti-Aβ IgG antibody response, whereas only 80 % of aged animals developed detectable antibodies. The immune response in aged monkeys was more delayed and significantly weaker, and was also more variable between animals. Pre- and post-immunization [(11)C]PK11195 PET scans showed no evidence of vaccine-related microglial activation. Post-mortem brain tissue analysis indicated a low overall amyloid burden, but revealed a significant shift in oligomer size with an increase in the dimer:pentamer ratio in aged immunized animals compared with non-immunized controls (P < 0.01). No differences were seen in microglial density or expression of classical and alternative microglial activation markers between immunized and control animals., Conclusions: Our results indicate that preventive Aβ immunization is a safe therapeutic approach lacking adverse CNS immune system activation or other serious side-effects in both aged and juvenile NHP cohorts. A significant shift in the composition of soluble oligomers towards smaller species might facilitate removal of toxic Aβ species from the brain.

Toxins secreted by bacteria can impact the host in a number of different ways. In some infections, toxins play a crucial and central role in pathogenesis (i.e., anthrax), while in other bacterial infections, the role of toxins is less understood. The cholesterol-dependent cytolysins (CDCs), of which streptolysin O is a prototype, are a class of pore-forming toxins produced by many gram-positive bacteria and have only been studied in a few experimental infection models. Our laboratory has demonstrated that CDCs have effects on macrophages that are both pro- and anti-inflammatory. Here, we review evidence that CDCs promote inflammation by driving secretion of IL-1β and HMGB-1 from macrophages in a NLRP3-dependent manner, while also causing shedding of membrane microvesicles from cells that can interact with macrophages and inhibit TNF-α release. CDCs thus impact macrophage function in ways that may be both beneficial and detrimental to the host.

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.

Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Viral and bacterial infections of the lower respiratory tract are major causes of morbidity and mortality worldwide. Alveolar macrophages line the alveolar spaces and are the first cells of the immune system to respond to invading pathogens. To determine the similarities and differences between the responses of mice and macaques to invading pathogens we profiled alveolar macrophages from these species following infection with two viral (PR8 and Fuj/02 influenza A) and two bacterial (Mycobacterium tuberculosis and Francisella tularensis Schu S4) pathogens. Cells were collected at 6 time points following each infection and expression profiles were compared across and between species. Our analyses identified a core set of genes, activated in both species and across all pathogens that were predominantly part of the interferon response pathway. In addition, we identified similarities across species in the way innate immune cells respond to lethal versus non-lethal pathogens. On the other hand we also found several species and pathogen specific response patterns. These results provide new insights into mechanisms by which the innate immune system responds to, and interacts with, invading pathogens.

ATP-mediated activation of the purinergic receptor P2X(7) 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-1beta and IL-18 following priming. In this study, we demonstrate the activation potential of P2X(7)-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 microm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-alpha secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-kappaB 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. P2X(7) activation is known to induce calcium-independent phospholipase A(2), calcium-dependent phospholipase A(2), 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.

The P2X(7) receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-of-function polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X(7) cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X(7) agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-of-function change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X(7) variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X(7) receptor. Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X(7) is critical for regulation of P2X(7)-mediated pore formation.

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-1beta from LPS-primed murine BMDM. IL-1beta 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-1beta and caspase-1 occurs only at lower TLO doses, whereas at a higher concentration, cells release the biologically inactive proforms. IL-1beta release at a low TLO dose requires potassium efflux, calcium influx, and the activities of calcium-independent PLA(2), caspase-1, and cathepsin B. Additionally, mature IL-1beta release induced by a low TLO dose is dependent on the NLRP3 inflammasome, and pro-IL-1beta release induced by a high TLO dose occurs independently of NLRP3. These results further elucidate a mechanism of CDC-induced IL-1beta release and suggest a novel, immune evasion strategy in which IL-1beta-containing macrophages might release primarily inactive cytokine following exposure to high doses of these toxins.

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Ca2+-driven responses in dendritic cells (DCs) are less well characterized than in lymphocytes. When DCs undergo a sequence of activation/maturation events, typically beginning with exposure to pathogens in the periphery, Ca2+ entry into the cytosol from stores in the endoplasmic reticulum or from outside the cell can occur at various steps and participate in intracellular signaling. However, not all cellular processes identified in these cells are Ca2+ dependent. While immigration of precursor DCs into the peripheral tissues as well as emigration to secondary lymphoid sites following microbial challenge depend on processes that involve Ca2+, other processes such as DC maturation in response to Toll-like receptor agonist stimulation appear not to. Certain microbial stimuli and host-derived chemokines induce Ca2+ entry that is important for the induced responses. In this article, we review the current state of our understanding of the role of Ca2+ in DC biology and argue that homeostatic control of Ca2+ levels in these cells is critical for maintaining their proper function. We also consider evidence for intercellular transmission of Ca2+ signals between DCs that are physically linked by thin membranous extensions termed tunneling nanotubules.

Immunization with particulate Ag effectively induces antitumor and antiviral T cell-mediated immunity. Immature dendritic cells (DCs) efficiently internalize, process, and present a variety of particulate Ags; however, previously published data suggest that both the uptake of soluble Ag through micropinocytosis, and phagocytosis of particulates are significantly curtailed in activated DC populations. In this study, we demonstrate that although macropinocytosis of soluble Ag is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags. Live cell imaging of activated DCs reveals that phagocytosis of particulates can result in cytoskeletal remodeling and perinuclear lysosome cluster disruption in a time-dependent manner. Interestingly, our results suggest that in activated DC populations, presentation of phagocytosed particulate Ags is dependent on the nature of the activation signal. These results provide direct evidence of functional heterogeneity in DC populations and contribute to the development of particle-based immunization strategies.

The University of Pittsburgh School of Medicine has a long tradition of excellence in immunology research and training. Faculty, students, and postdoctoral fellows walk through hallways that are pictorial reminders of the days when Dr. Jonas Salk worked here to develop the polio vaccine, or when Dr. Niels Jerne chaired the Microbiology Department and worked on perfecting the Jerne Plaque Assay for antibody-producing cells. Colleagues and postdoctoral fellows of Professor Salk are still on the faculty of the University of Pittsburgh Medical School as are graduate students of Professor Jerne. A modern research building, the 17 story high Biomedical Science Tower, is a vivid reminder of the day when Dr. Thomas Starzl arrived in Pittsburgh and started building the most prominent solid-organ-transplant program in the world. The immunology research that developed around the problem of graft rejection and tolerance induction trained numerous outstanding students and fellows. Almost 20 yr ago, the University of Pittsburgh founded the University of Pittsburgh Cancer Institute (UPCI) with the renowned immunologist Dr. Ronald Herberman at its helm. This started a number of new research initiatives in cancer immunology and immunotherapy. A large number of outstanding young investigators, as well as several well-established tumor immunologists, were recruited to Pittsburgh at that time.

We have examined mechanisms of antigen uptake by dendritic cells (DC). While multiple experimental approaches have been used, live cell imaging has been especially informative, and is reviewed here. DC were shown to bind to and internalize bacteria, but not inert particles, demonstrating an unexpected selectivity that included sensing of secreted bacterial products and subsequent cell activation. This led to the observation that induced calcium fluxes in these cells could be propagated to other cells via thin membrane connections termed tunneling nanotubules that were shown to form an extensive network between cells, and are also present in other cell types. These data are discussed in terms of DC function, and also demonstrate the power of live cell imaging in elucidating complex biological phenomena that are not readily revealed using static imaging approaches or by biochemical analyses.

Intercellular signals can be transmitted through neuronal synapses or through gap junctions, with the latter mediating transmission of calcium fluxes and small molecules between cells. We show here that a third form of communication between cells can be mediated by tunneling nanotubules (TNT). When myeloid-lineage dendritic cells and monocytes are triggered to flux calcium by chemical or mechanical stimulation, the signal can be propagated within seconds to other cells at distances hundreds of microns away via TNT. A complex and transient network of TNT is seen in live cells, with individual tubules exhibiting substantial variation in length and diameter. In addition to calcium fluxes, microinjected dye tracers can be transferred through these connections. Following TNT-mediated stimulation, spreading of lamellipodia occurs in dendritic cells characteristic of that seen during the phagocytic response to bacteria. These results demonstrate that nonneuronal cells can transmit signals to distant cells through a physically connected network.

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-gamma 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.

Using live cell imaging, we demonstrate that immature dendritic cells (DC) derived from human peripheral blood monocytes undergo pronounced morphologic changes in vitro within minutes of exposure to unopsonized Escherichia coli, developing extensive membrane veils that efficiently capture additional bacteria. Internalization does not occur in the veils, but instead, bacteria are transported to the central region of the cell, where they sink directly into the plasma membrane. In contrast, exposure to polystyrene beads does not induce notable changes in cell morphology, and DC do not efficiently capture beads when introduced alone or mixed with bacteria. Long dendritic processes were also visualized in some cells that allowed capture of clumps of bacteria at a distance of more than 100 microm. These results demonstrate that immature DC can distinguish between inert particles and bacteria and alter their shape and phagocytic capacity in response to the latter.

We describe a B-cell line, BDCM, which arose spontaneously from culture of a pheresis product from a patient with M5a myeloid leukaemia. Cell growth was associated with autocrine activation of the signal transducer and activator of transcription 1 (Stat-1) transcription factor. Although the cells expressed several B-cell surface markers and had a rearranged immunoglobin J region, they also exhibited several characteristics associated with dendritic cells. These included extensive surface projections, cross-priming ability and strong T cell-stimulating capability. In addition, the cytokine production profile of BDCM cells was nearly identical to that of mature monocyte-derived dendritic cells.

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-gamma 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., (Copyright 2002 Wiley-Liss, Inc.)

MHC class I tetramers containing peptide epitopes are sensitive tools for detecting antigen-specific CD8(+) T-cell responses. We demonstrate here that binding of HLA-A2 tetramers to CD8(+) T cells specific for the melanoma-associated antigen Melan-A/MART-1 can be fine-tuned by altering either the bound peptide epitope or residues in the alpha 3 domain of HLA-A2, which is important for CD8 binding. Antigen-specific T cells expressing high levels of CD8 could be detected using HLA-A2 tetramers containing the peptide AAGIGILTV, an epitope which is naturally processed and presented from Melan-A/MART-1. In contrast, low CD8-expressing, antigen-specific T cells could be detected efficiently only by using a mutated HLA-A2 tetramer with an altered CD8 binding site or, less efficiently, using the wild-type HLA-A2 tetramer loaded with the peptide analogue ELAGIGILTV, which is superior in stimulating antigen-specific T-cell responses. Our results suggest ways to optimize the identification and expansion of antigen-specific T cells with different requirements for the costimulatory CD8 molecule in facilitating T-cell receptor binding to peptide variants., (Copyright 2002 Wiley-Liss, Inc.)

Dendritic cells and human B cell lines were compared for ability to present synthetic peptides corresponding to residues 145-159 and 188-203 of human Ig kappa-chains to peptide-specific mouse T cell hybridomas restricted by HLA-DR4Dw4. B cell lines presented both peptides, but dendritic cells could only efficiently present the latter epitope. In this paper, we show that dendritic cells degrade the 145-159 peptide, removing four residues from the amino terminus. Binding of the peptide to the class II restriction element is not required for this process. The degradation product is resistant to further cleavage, accumulates in the culture supernatant, and does not bind to HLA-DR4Dw4 or stimulate T cell reactivity. Cleavage can be blocked with bestatin, but not with other protease inhibitors tested, or by a mAb directed against aminopeptidase N (CD13). Addition of an acetyl group to the amino terminus of peptide 145-159 also blocks degradation, and allows dendritic cells to present the peptide to specific T cells with greatly increased efficiency. These results demonstrate that CD13 on dendritic cells is able to selectively and efficiently degrade exogenously provided peptide Ags, in a process that can be blocked by addition of an acetyl group to the amino terminus of the peptide. Modification of the amino terminus of peptide epitopes susceptible to degradation may prove to be useful as a general strategy for enhancing their immunogenicity.

Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection.

We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.

Increased levels of agalactosyl IgG (G0 IgG) are found in several autoimmune diseases, including rheumatoid arthritis, in which they are correlated with severity of the disease. To investigate whether structural alteration of IgG may lead to aberrant processing and presentation of IgG peptides as autoantigens, we have studied uptake of G0 IgG by human dendritic cells and macrophages cultured from PBMC. We found that enzymatic removal of terminal galactose residues, which exposes N-acetylglucosamine residues, increases uptake of soluble IgG mediated by mannose receptor on macrophages and dendritic cells. Efficient uptake appears to require recycling of the receptor, can be blocked by saccharides or Abs reactive with mannose receptor, and is dependent upon the state of maturation of the dendritic cells. No differences between IgG isotypes in ability to be internalized by APC were identified, suggesting that uptake would not be limited to a particular subset of Abs. These results suggest a novel pathway by which Abs or Ag-Ab complexes can be taken into dendritic cells and macrophages, and potentially generate epitopes recognized by T cells. These findings may have particular relevance for autoimmune disorders characterized by high levels of G0 IgG.

Calnexin is a lectin-like chaperone that binds to class I MHC molecules soon after their synthesis, retaining unassembled heavy chains and also assisting their folding. Following association with beta2-microglobulin (beta2m) in the endoplasmic reticulum, a large proportion of human class I molecules release from calnexin, whereas mouse class I molecules do not. We asked whether addition of a second N-glycan to the human class I molecule A*0201 at position 176, a site present in mouse, would affect its binding to calnexin. The 176dg mutant with N-glycans at positions 86 and 176, when transfected into CIR cells, demonstrated increased binding to calnexin, detectable both before and after association with beta2m, and reduced interaction with calreticulin and TAP relative to wild-type protein bearing a single N-glycan at position 86. Cell surface levels of the mutant were decreased only slightly relative to the wild type, suggesting that the protein is not misfolded or grossly altered structurally. A subpopulation of mutant molecules was retained in the endoplasmic reticulum, and surprisingly, these molecules reacted with w6/32, which recognizes an epitope present on transport-competent class I HLA complexes. Transfection into Daudi cells demonstrated that 176dg reacts with w6/32 in the absence of beta2m, suggesting that the Ab epitope can be induced by binding of calnexin. These data may explain previously noted differences between mouse and human class I MHC proteins and demonstrate that the location of N-oligosaccharides within proteins can influence their folding and interactions with chaperones such as calnexin and calreticulin.

Prostate-specific antigen (PSA) has been demonstrated to release the active form of insulin-like growth factor I in vitro (P. Cohen et al., J. Clin. Endocrinol. & Metab., 75: 1046-1053, 1992; P. Cohen et al., J. Clin. Endocrinol. & Metab., 79: 1410-1415, 1994; P. Cohen et al., Horm. Metab. Res., 26: 81-84, 1994) and has significant mitogenic activity on osteoblast cells, fibroblasts, and other cultured cells (C. S. Killian et al., Biochem. Biophys. Res. Commun., 192: 940-947, 1993). Recently, PSA has been found not only in prostate tissues but also in breast, colon, ovarian, and other tissues (E. P. Diamandis and H. Yu, J. Clin. Endocrinol. & Metab., 80: 1515-1517, 1995; E. P. Diamandis and H. Yu, Clin. Chem., 41: 204-210, 1995; A. Clements and A. Mukhtar, J. Clin. Endocrinol. & Metab., 78: 1536-1539, 1994). Therefore, PSA has been proposed as a candidate growth factor, cytokine, or growth factor regulator. In this setting, knowing how to manipulate or block the secretion of PSA by the prostate cancer cells could be a useful approach to controlling the progression of human prostate cancers. Using metabolic labeling experiments, we have studied the biosynthesis and secretion of PSA in LNCaP cells. We have also examined the effects of DTT, tunicamycin, 1-deoxymannojirimycin, pilocarpine, and testosterone on PSA biosynthesis and secretion. The results indicate that the secretion of PSA in LNCaP cells is constitutive instead of regulated and that the disruption of intramolecular disulfide bonds affects the transport of PSA from the endoplasmic reticulum to the Golgi apparatus. The biosynthesis of PSA is potentiated by testosterone and inhibited by brefeldin A and DTT. These results will help us understand PSA biosynthesis and secretion in human prostate cancers.

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.

Natural killer (NK) cells recognize class I HLA molecules via a family of related receptors composed of two or three Ig-like domains. Using neighbor-joining analysis of available sequences, groups of these receptors were identified that are likely to share specificity in HLA binding, which in some cases had been previously determined for individual group members. Subgroups or clades were further identified which did not appear to correlate with ligand binding, but instead reflect differences in the cytoplasmic region of the proteins. The Ig-like domains, which form the extracellular segment responsible for specificity of HLA recognition, were individually shown to be characteristic of specific groups of receptors, and do not appear to have been assorted between groups. Thus it does not appear that recombination of domains played a major role in generating diversity within regions of these proteins important for HLA binding. Finally, the Ig-like domains of KIR proteins are shown to be between 35-45% identical to those of CD89, a receptor for IgA on myeloid cells. This level of homology, combined with their shared localization on chromosome 19q13.4, suggests a common evolutionary origin.

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

Class I major histocompatibility complex heavy chains bind to calnexin before associating with beta 2-microglobulin (beta 2m) and peptides. Calnexin has been shown to retain in the endoplasmic reticulum those class I heavy chains which have not assembled properly and, thus, to serve as a quality control mechanism. In addition, calnexin may direct the folding of class I subunits or their subsequent assembly. We asked whether calnexin plays a role in the initial folding of HLA-B*0702 heavy chains by assessing disulfide bond formation in vivo. Our results show that class I heavy chains form intrachain disulfide bonds very soon after translation, and that calnexin is bound to both reduced and oxidized forms during this process. When a cell-permeable reducing agent, dithiothreitol, was added to cells, disulfide bond formation in newly synthesized heavy chains was substantially blocked, as was their association with calnexin. The reducing agent appeared to affect calnexin directly, since binding was similarly abolished to a subset of proteins which do not contain internal disulfide bonds. Addition of the glucosidase inhibitor castanospermine to cells, shown previously to disrupt calnexin binding to ligands, slowed formation of disulfide bonds but did not decrease the amount of assembled heavy chain-beta 2m complexes that formed. Our data suggest that calnexin can promote disulfide bond formation in class I heavy chains but does not directly facilitate subsequent binding to beta 2m.

Proper folding of nascent polypeptides is essential for their function and is monitored by intracellular "quality control" elements. The molecular chaperone calnexin participates in this process by retaining in the endoplasmic reticulum a variety of unfolded proteins, including class I major histocompatibility complex molecules. We transfected human B cell lines with genes encoding either wild-type HLA-A2 heavy chains or mutant heavy chains lacking sites for glycosylation or deficient in binding to beta 2-microglobulin (beta 2m). In CIR cells, calnexin did not associate detectably with wild-type heavy chains but bound strongly to mutant heavy chains unable to bind beta 2m. Removal of the glycosylation addition site by further mutagenesis prevented binding of mutant heavy chains to calnexin. In Daudi cells, deficient in synthesis of beta 2m, wild-type HLA-A2 heavy chains, but not a non-glycosylated mutant, bound calnexin. Castanospermine, which blocks trimming of glucose residues from asparagine-linked glycans, inhibited association of calnexin with heavy chains encoded by a second class I gene, HLA-B*0702. Although initiation of calnexin binding appears to depend on the presence of oligosaccharide on the substrate, removal of the glycan from calnexin-associated heavy chains by digestion with endoglycosidase H did not disrupt the interaction. These results suggest that calnexin first recognizes carbohydrate on substrate proteins and then binds more stably to peptide determinants, which disappear upon folding.

Class I major histocompatibility complex (MHC) molecules are heterotrimers of heavy chains, beta 2-microglobulin, and 8-10 amino acid-long peptides. Assembly of class I MHC molecules into complexes which are stable and can be transported to the cell surface occurs soon after insertion of individual subunits into the endoplasmic reticulum (ER). To identify subcellular compartments required for class I MHC assembly, we studied class I biosynthesis in human cell lines treated with several inhibitors of intracellular transport. We found that HLA-B701 molecules do not assemble in CIR transfectants in which a block in protein transport from the ER is established by treatment with phosphatase inhibitors. In contrast, stable HLA-B701 complexes form in cells in which the ER becomes mixed with the Golgi after treatment with brefeldin A. Neither treatment impaired binding of HLA-B701 to the ER-resident protein calnexin, and unassembled heavy chains in phosphatase-inhibited cells showed prolonged association with calnexin. In addition, the mouse class I molecule H-2Db, which binds beta 2-microglobulin in human T2 cells in the absence of transporter of antigenic peptides, formed complexes in CIR cell transfectants treated with phosphatase inhibitors. Taken together, these data demonstrate that phosphatase inhibitors do not prevent assembly of class I heavy chain beta 2-microglobulin dimers, but instead interfere with peptide loading. These results are consistent with the possibility that class I MHC molecules are transported from their initial site of insertion into the rough ER before binding peptides, or alternatively that peptide loading mediated by transporter of antigenic peptides is blocked by phosphatase inhibitors.

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.

Intracellular transport of class I MHC complexes is dependent on assembly of class I heavy chains with beta 2-microglobulin (beta 2m) and peptides. This suggests that amino acid residues of individual class I molecules which are important for their stability and transport are likely to include those which contribute to binding of a majority of the cleft-associated peptides. To identify such critical residues, substitutions at polymorphic positions within the peptide binding cleft were introduced into a mutant HLA-A*0201 molecule bearing an additional gly > lys substitution at position 242 (242K). The 242K mutation weakens association of the HLA-A*201 heavy chain with beta 2m and was used to enhance potential effects of substitutions in the peptide binding groove on class I stability. Critical in choosing which binding cleft positions to mutate was the observation that HLA-A*6801 was less sensitive to the effects of 242K mutation than HLA-A*0201 and A*6901. This suggested that one or more of the six residues in the alpha 2 domain differing between HLA-A*6901 and A*6801 were likely to affect class I complex stability. Positions 95, 97, 107, 114, 116, and 156 in either 242K or wild-type HLA-A*0201 molecules were therefore each converted to those residues found in HLA-A*6801. One of the second-site substitutions, arg > met at position 97, increased stability and restored surface expression of the 242K molecule. Five other substitutions either had no additional effect or further impaired 242K stability. Substitution of his > arg at position 114 blocked surface expression of both 242K and wild-type HLA-A*0201 molecules. These results demonstrate that polymorphic residues in the binding cleft influence the stability of class I complexes, and suggest that position 97 plays a critical role in stabilizing class I molecules for transport.

Using a newly described pH 3.3 acid elution technique, peptides were extracted by denaturation of class I molecules on the surface of human melanomas. HPLC fractionation of this material revealed six T cell epitopes (termed P1-P6) recognized by HLA-A2-restricted, melanoma-specific tumor infiltrating lymphocyte (TIL) lines. Three of these fractions (P1, P2, and P4) appeared to represent shared/immunodominant melanoma Ag recognized in the context of HLA-A2 because they were expressed by 4/4 HLA-A2+ melanoma cell lines and were each recognized by all four oligoclonal HLA-A2-restricted TIL lines examined. Interestingly, P1 and P2 (but not P3-P6) could also be recognized by these same TIL when presented by the HLA-Aw69 class I molecule, which is closely related to HLA-A2. P3, P5, and P6 displayed more restricted expression and were differentially recognized by the four oligoclonal TIL lines. These results suggest that synthetic peptide derived from P1, P2, and P4 sequences (when deduced) may form the basis of effective prophylactic or therapeutic melanoma vaccines by stimulating CD8+ CTL in HLA-A2+ individuals. This approach of identifying T cell epitopes presented by class I molecules should prove generally applicable to the study of other tumors recognized by class I-restricted CTL.

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.