Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes.

A melanoma vaccine composed of HLA-A2-restricted peptide YLEPGPVTA (gp100(280)), with or without a modified T-helper epitope from tetanus toxoid AQYIKANSKFIGITEL, has been evaluated in a Phase I trial to assess safety and immunological response. The vaccines were administered s.c. in either of two adjuvants, Montanide ISA-51 or QS-21, to 22 patients with high-risk resected melanoma (stage IIB-IV). Local and systemic toxicities were mild and transient. We detected CTL responses to the gp100(280) peptide in peripheral blood in 14% of patients. Helper T-cell responses to the tetanus helper peptide were detected in 79% of patients and had a Th1 cytokine profile. One patient with a CTL response to gp100 had a recurrence in a lymph node 2 years later; her nodes contained CD8+ cells reactive to gp100(280) (0.24%), which proliferated in response to peptide. The overall survival of patients is 75% (95% confidence interval, 57-94%) at 4.7 years follow-up, which compares favorably with expected survival. Four of 14 patients who completed at least six vaccines subsequently developed metastases, all of which were solitary and surgically resectable. They remain alive and clinically free of disease at last follow-up. Data from this trial demonstrate immunogenicity of the gp100(280) peptide and suggest that immune responses may persist long-term in some patients. The frequency and magnitude of the CTL response may be improved with more aggressive vaccination regimens. Although this Phase I study was not intended to evaluate clinical benefit, the excellent survival of patients on this protocol suggests the possibility of a benefit that should be assessed in future studies.

Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients.

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients.

Analysis of a natural immune response against tumor antigens in a melanoma survivor: lessons applicable to clinical trial evaluations.

The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.

How automatically is meaning accessed: a review of the effects of attention on semantic processing.

This chapter attempts to review electrophysiological data that stand at the intersection of two large domains of research: selective attention and semantic processing. Analogous behavioral studies will be discussed as a way of introducing the reader to some of the more complex paradigms, and providing convergent evidence. The focus of this review will be on data that add special insight into the issue of semantic processing in the absence of attention. However, paradigms which allow semantic activation in the absence of awareness will also be examined as these studies have also yielded data concerning automatic semantic processing. It is concluded that a great deal of semantic processing can occur in the absence of attention as well as in the absence of awareness.

Effect of frequency separation and stimulus rate on the mismatch negativity: an examination of the issue of refractoriness in humans.

Refractoriness of the generators of the mismatch negativity (MMN) was examined in two experiments in which two deviant tones occurred in a row. In Experiment 1, the size of the MMN elicited by the first deviant was manipulated by using deviants that were close to or far from the standard in frequency. In Experiment 2, the time between two identical deviants was varied. It was found that neither the size of the MMN elicited by the first deviant, nor the time between two deviants, affected the amplitude of the MMN elicited by the second deviant. It was concluded that refractoriness played no role in the amplitude of the MMN for the parameters used.

Event-related potential indices of semantic priming using masked and unmasked words: evidence that the N400 does not reflect a post-lexical process.

Several authors have contended that the N400 is a reflection of a post-lexical event such as that proposed by Neely and Keefe [J.H. Neely, D.E. Keefe, Semantic context effects on visual word processing: a hybrid prospective/retrospective processing theory, in: G.H. Bower (Ed.), The Psychology of Learning and Motivation: Advances in Research and Theory, Vol. 23, Academic Press, New York, 1989, pp. 207-248.], whereby the subject compares the word on the current trial to the "context" provided by the word on the preceding trial [M. Besson, M. Kutas, The many facets of repetition: A cued-recall and event-related potential analysis of repeating words in same versus different sentence contexts, Journal of Experimental Psychology: Learning, Memory and Cognition, 19 (5) (1993), 1115-1133; C. Brown, P. Hagoort, The processing nature of the N400: Evidence from masked priming. Journal of Cognitive Neuroscience, 5(1) (1993), 34-44; P.J. Holcomb, Semantic priming and stimulus degradation: Implications for the role of the N400 in language processing, Psychophysiology 30 (1993), 47-61; M.D. Rugg, M.C. Doyle, Event-related potentials and stimulus repetition in indirect and direct tests of memory, in: H. Heinze, T. Munte, G.R. Mangun (Eds), Cognitive Electrophysiology, Birkhauser Boston, Cambridge, MA, 1994]. A study which used masked primes to directly test this possibility has been reported by Brown and Hagoort [C. Brown, P. Hagoort, The processing nature of the N400: evidence from masked priming. Journal of Cognitive Neuroscience, 5(1) (1993), 34-44]. When the primes were masked, no priming effect was observed on the N400. When behavioral data were collected in the same paradigm, from another group of subjects, the usual priming effect on RT was obtained. Considered together, the data from the two groups of subjects indicated that activation of semantic representations had occurred without conscious awareness. As no N400 priming effect was observed, it was suggested that N400 must reflect a post-lexical process. This interpretation, however, is at odds with the findings of other studies which have reported N400 priming effects under conditions where post-lexical processes would not be thought to operate[J. Anderson, P. Holcomb, Auditory and visual semantic priming using different stimulus onset asynchronies: an event-related brain potential study. Psychophysiology 32 (1995), 177-190; J. Boddy, Event-related potentials in chronometric analysis of primed word recognition with different stimulus onset asynchronies, Psychophysiology 23 (1986), 232-245; D. Deacon, T. Uhm, W. Ritter, S. Hewitt, The lifetime of automatic priming effects may exceed two seconds, Cognitive Brain Research 7 (1999), 465-472; P.J. Holcomb, Automatic and attentional process: an event-related brain potential analysis of semantic priming. Brain and Language 35 (1998) 66-85]. The present study replicated Brown and Hagoort using a repeated measures design, a shorter SOA (stimulus onset asynchrony), and a slightly different threshold setting procedure. Significant priming effects were obtained on the mean amplitude of the N400 regardless of whether the words were masked or unmasked. The findings imply that the processing subserving the N400 is not postlexical, since the N400 was manipulated without the subjects being aware of the identity of the words.

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Two cognitive systems simultaneously prepared for opposite events.

Event-related potentials (ERPs) were recorded during a go/no-go reaction time (RT) task in which subjects responded to rare target tones and withheld response to frequent tones. In a predictable condition, a rare visual stimulus signalled the impending occurrence of a rare tone, whereas a frequent visual stimulus signalled that a frequent tone would be presented. The rare visual stimuli elicited P3, associated with violations of expectations, whereas the rare tones, being predictable, did not. The rare tones elicited the mismatch negativity (MMN), a component associated with preattentive deviance detection, despite the fact that they were expected. RT was faster in the predictable condition than in another condition in which the tones were not predictable. The P3 and RT data indicate, respectively, that subjects anticipated and were ready to respond to the target tones. The MMN result indicates that immediately before target tones, the preattentive system underlying the MMN was set for frequent tones, being unaffected by the information available to the higher order system. Thus, the higher order cognitive system associated with P3 and the lower order cognitive system associated with the MMN were prepared simultaneously for opposite events.

The lifetime of automatic semantic priming effects may exceed two seconds.

The N400 component of event-related potentials (ERPs) was obtained in a modified version of the Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254.] paradigm which permits unconfounding of semantic priming effects due to automatic and attentional processes. It was found that a short stimulus onset asynchrony (SOA) of 250 ms between the prime and the target was associated with automatic but not expectancy effects on the amplitude of N400. At a long SOA of 2000 ms between prime and target, semantic priming effects on N400 were obtained associated with both automatic and expectancy processes. Moreover, there was no significance difference in the magnitude of the automatic effects at the two SOAs, suggesting that automatic processing had not decayed within the 2000 ms interval between the prime and target. The results support the two-processing interpretation of semantic priming advanced by Posner and Synder [M.I. Posner, C.R.R. Snyder, Attention and cognitive control, in: R.L. Solso (Ed.), Information Processing and Cognition: The Loyola Symposium, Erlbaum, Hillsdale, NJ (1975).] and concur with the results of Neely [J.H. Neely, Semantic priming and retrieval from lexical memory: Roles of inhibitionless spreading activation and limited-capacity attention, Journal of Experimental Psychology: General, Vol. 106 (1977), pp. 226-254], with the exception of indicating a longer persistence of automatic processes.

The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene.

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.

Automatic change detection: does the auditory system use representations of individual stimulus features or gestalts?

The effects of global and feature-specific probabilities of auditory stimuli were manipulated to determine their effects on the mismatch negativity (MMN) of the human event-related potential. The question of interest was whether the automatic comparison of stimuli indexed by the MMN was performed on representations of individual stimulus features or on gestalt representations of their combined attributes. The design of the study was such that both feature and gestalt representations could have been available to the comparator mechanism generating the MMN. The data were consistent with the interpretation that the MMN was generated following an analysis of stimulus features.

Event-related potential indices of semantic priming following an unrelated intervening item.

Interposing an unrelated word between related primes and targets often disrupts priming. This finding has been used to support the view that semantic information is represented in a distributed fashion, rather than locally. In some studies where unrelated items intervened between the prime and target, however, significant priming was nevertheless obtained. The discrepant results of these studies has been attributed to differences in speed-accuracy tradeoff, post-lexical checking, conscious rehearsal of the prime and differences in the depth to which the prime and target were processed. The present study was designed in such a way as to minimize variability associated with post-lexical influences. The N400 component of the human event-related potential was used as a physiological index of the extent to which priming occurred with and without the interposition of an unrelated item. Priming effects on both the amplitude and latency of the N400 were rendered non-significant by the presence of an intervening unrelated word. The results are interpreted as tentative evidence that semantic representations are distributed.

Activated alpha 2-macroglobulin reverses the immunosuppressive activity in human breast cancer cell-conditioned medium by selectively neutralizing transforming growth factor-beta in the presence of interleukin-2.

The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). A human breast cancer cell line derived from malignant ascites (BRC 173) secreted TGF-beta, but not IL-10, into tissue culture supernatant (TCS). BRC 173 TCS suppressed natural killer (NK) and lymphokine-activated killer (LAK) cell activity and also blocked the generation of HLA-A*0201-restricted tumor-reactive cytotoxic T-lymphocyte (CTL) lines in vitro. Human alpha 2-macroglobulin (alpha 2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-beta family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. alpha 2M was converted to its activated conformation by reaction with methylamine (alpha 2M-MA) and then incubated with normal human peripheral blood lymphocytes (PBL) in the presence of IL-2 and BRC 173 TCS. Lysis of NK targets (K562) and LAK cell targets (DM6 melanoma) by the PBL was examined after 6 days of culture. PBL cultured in IL-2, without TCS or alpha 2M-MA, were lytic for both target cells. BRC 173 TCS substantially suppressed the lytic activity of the PBL in the presence of IL-2. When TGF-beta-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. alpha 2M-MA (280 nM) neutralized almost all of the immunosuppressive activity in the TCS, restoring 80-100% of the lytic activity without any apparent effect on the activity of IL-2. The ability of alpha 2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate the activated alpha 2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.

Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp100.

Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.

Storage of information in transient auditory memory.

This study concerns the manner in which features of auditory stimuli are stored in acoustic memory. Event-related potentials (ERPs) were recorded to sequences of tones in which sequential, infrequent deviant tones were presented in a row, each of which differed from the frequent standard tones along a different stimulus dimension. The object was to determine whether a change in a single feature of a stimulus would have an effect on the entire representation of the standard tone in memory, or only on the representation of the stimulus dimension by which the first deviant differed from the standards. It was found that the amplitude of the mismatch negativity elicited by subsequent deviants was not reduced by the presence of the first deviant, supporting independent storage of features.

Variation in the latencies and amplitudes of N400 and NA as a function of semantic priming.

The purpose of this study was to determine whether the latencies of two event-related potential (ERP) components, the NA and N400, were sensitive to semantic priming. Subjects performed a semantic judgment task, which was designed in such a way that the N400 could be examined without overlap from the P3. Priming effects on the latencies of both NA and N400 were most apparent at frontocentral sites. The amplitude of NA was not significantly affected by priming. The amplitude of N400 was smaller for primed than for unprimed words, but the effect was significant only at centroposterior sites. Current source density (CSD) analyses performed on the ERP data suggested the activation of multiple generators in the N400 time region. The ERP and CSD data were consistent with the existence of two types of N400, a frontal N400 that varies in latency as a function of semantic priming, and a posterior N400 that varies in amplitude.

The ski/sno protooncogene family in hematopoietic development.

The ski/sno protooncogenes encode nuclear proteins that may act as transcription factors. We examined ski and sno mRNA expression in hemolymphopoietic lineages. The ski protooncogene is expressed in B- and T-lineage cells, mature macrophages, and mast cells. In normal murine marrow-derived progenitors analyzed by single-cell reverse transcription-polymerase chain reaction (RT-PCR), ski expression is limited to dual-lineage megakaryocyte/erythrocyte colony-starts. Expression of sno is more limited than ski in mature cells; it is expressed in T lymphopoietic cells, but not in B-lineage cells. The sno protooncogene is expressed more widely than ski in myeloid progenitors, as it is found consistently in tri-, dual-, and single-lineage progenitors. Both ski and sno are cell cycle-regulated in synchronized factor-dependent mouse myeloid cells. Expression of ski mRNA peaks in mid G1 in cells synchronized by isoleucine deprivation in the presence of growth factor, but falls off rapidly when growth factor is withdrawn. Expression of sno mRNA is maximal in early to mid G1 and then oscillates as the cells continue through cycle. These results suggest that the ski/sno protooncogenes play a role in hematopoiesis, growth factor responses, and cell cycle-regulation, with the two members of the family showing differing properties.

The high-proliferative-potential megakaryocyte mixed (HPP-Meg-Mix) cell: a trilineage murine hematopoietic progenitor with multiple growth factor responsiveness.

Megakaryocyte progenitors are notable for their response to synergistic cytokine combinations and apparently early position in the hematopoietic differentiation pathway. Although high-proliferative-potential murine megakaryocyte progenitors have been described, they have previously been primarily observed as unilineage colonies. We describe a subclass of agar-based high-proliferative-potential colony forming cells (HPP-CFC) derived from populations enriched for early hematopoietic progenitors which, when stimulated with the combination of CSF-1, G-CSF, GM-CSF, IL-1 alpha, IL-3 and SCF, produces colonies with trilineage potential for macrophages, granulocytes, and megakaryocytes. The high proliferative potential megakaryocyte mixed (HPP-Meg-Mix) colony-forming cell is defined as a cell meeting traditional criteria for HPP-CFC but additionally containing 20 or more megakaryocytes per colony. The combination of high-proliferative-potential, multilineage differentiation, and megakaryocytic lineage capacity suggests that the HPP-Meg-Mix marks a very early hematopoietic progenitor cell.

The mismatch negativity of event-related potentials as a probe of transient auditory memory: a review.

The use of the mismatch negativity as a probe to study the memory upon which it depends is reviewed. Topics about the memory include its duration, its capacity, the kind of information that can be stored in the memory, how the information is stored, whether the memory is accessed in parallel or in series, whether it is hard-wired or can be modified by experience, and its relationship to sensory memory.