Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons.

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 647-672, 2013.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.

Role of the REST/RE-1 system in beta-cell life, function and differentiation

SUMMARYDiabetes is characterized by insulin deficiency that results from the destruction of insulin-secreting pancreatic beta-cells (Type 1), or in part from beta-cell death and insulin secretion defects (Type 2). Therefore, understanding the mechanisms of beta cell neogenesis (to generate unlimited supply of beta cells for T1D transplantation] or identifying the specific genes that favors insulin secretion or beta-cell survival is of great importance for the management of diabetes. The transcriptional repressor RE-1 Silencing Transcription Factor (REST) restricts the expression of a large number of genes containing its binding element, called Repressor Element-1 (RE-1), to neurons and beta cells. To do so, REST is ubiquitously expressed but in neurons and beta cells. To identify these essential genes and their functional significance in beta cells, we have generated transgenic mice that express REST specifically in beta cells under the control of the rat insulin promoter (RIP-REST mice). This resulted in the repression of the RE-1- containing genes in beta cells, and we analyzed the consequences.We first showed that RIP-REST mice were glucose-intolerant because of a defective insulin secretion. To explain this defect, we identified that a subset of the REST target genes were necessary for insulin exocytosis, such as Snap25, Synaptotagmin (Syt) IX, Complexin II, and Ica512, and we further demonstrated that among the identified REST targets, Syt IV and VII were also involved in insulin release. We next analyzed a novel RIP-REST mouse line that featured diabetes and we showed that this defect was due to a major loss of beta-cell mass. To explain this phenotype, we identified REST target genes that were involved in beta-cell survival, such as Ibl, Irs2, Ica512 and Connexin36, and revealed that another REST target, Cdk5r2 is also involved in beta-cell protection. In a third part, we finally suggest that REST may be important for pancreatic endocrine differentiation, since transgenic mice expressing constitutive REST in pancreatic multipotent progenitors show impaired formation of Ngn3-expressing endocrine- committed precursors, and impaired formation of differentiated endocrine cells. Mapping the pattern of REST expression in wild type animals indicates that it is expressed in multipotent progenitors to become then excluded from endocrine cells. Preliminary results suggest that a downregulation of REST would result in relieved expression of at least the Mytl target, favoring subsequent acquisition of the endocrine competence by endocrine precursor cells.Thus, we propose that the REST/RE-1 system is an important feature for beta-cell neogenesis, function and survivalRESUMELe diabète se caractérise par une déficience en insuline qui résulte d'une destruction des cellules bêta (β) pancréatiques sécrétant l'insuline [Type 1], ou à un défaut de sécrétion d'insuline qui peut être associé à la mort des cellules β (Type 2). La compréhension des mécanismes de néogenèse des cellules β, ainsi que l'identification de gènes impliqués dans leur survie et dans le contrôle de la sécrétion d'insuline est donc importante pour le traitement du diabète. Le facteur de transcription de type répresseur, RE-1 Silencing Transcription Factor [REST], contribue à la spécificité d'expression dans les neurones et les cellules β, d'un grand nombre de gènes portant son motif de fixation, le Repressor Element-1 (RE-1). Pour cela, REST est exprimé dans toutes les cellules, sauf dans les neurones et les cellules β. Afin d'identifier les gènes cibles de REST ainsi que leur fonction au sein de la cellule β, nous avons généré des souris transgéniques qui expriment REST spécifiquement dans ces cellules, sous la dépendance du promoteur de l'insuline (souris RIP-REST]. Cette expression ectopique de REST a permis de diminuer l'expression des gènes contrôlés par REST, et d'en analyser les conséquences. Nous avons montré que les souris RIP-REST étaient intolérantes au glucose et que ceci était du à un défaut de sécrétion d'insuline. Pour expliquer ce phénotype, nous avons mis en évidence le fait que des gènes cibles de REST codent pour des protéines importantes pour l'exocytose de l'insuline, comme SNAP25, Synaptotagmin (Syt) IX, Complexin II ou ICA512. De plus, nous avons découvert deux nouvelles cibles de REST impliquées dans la sécrétion d'insuline, Syt IV et Syt VII. Par la suite, nous avons démontré qu'une nouvelle lignée de souris RIP-REST étaient atteintes d'un diabète sévère à cause d'une perte massive des cellules β. La disparition de ces cellules a été expliquée par l'identification de gènes cibles de REST impliqués dans la survie des cellules β, comme Ibl, Irs2, Ica512 ou la Connexine36. De plus, nous avons découvert qu'une nouvelle cible, Cdk5r2, était aussi impliquée dans la survie des cellules β. Dans une dernière partie, nous suggérons, grâce à l'analyse de nouvelles souris transgéniques exprimant constitutivement REST dans les cellules progénitrices du pancréas embryonnaire, que REST empêche la formation des précurseurs de cellules endocrines ainsi que la différenciation de ces cellules. L'analyse de l'expression de REST au cours du développement embryonnaire du pancréas indique que la diminution de l'expression de REST conduit en partie, à l'induction d'un de ses gènes cible Mytl, qui favorise la formation de précurseurs endocrines. Nous proposons donc que le système REST/RE-1 est important pour la génération, la fonction et la survie des cellules β.

An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

Auditory Neurons with Transitory Axons to Visual Areas Form Short Permanent Projections.

The kitten's auditory cortex (including the first and second auditory fields AI and AII) is known to send transient axons to either ipsi- or contralateral visual areas 17 and 18. By the end of the first postnatal month the transitory axons, but not their neurons of origin, are eliminated. Here we investigated where these neurons project after the elimination of the transitory axon. Eighteen kittens received early (postnatal day (pd) 2 - 5) injections of long lasting retrograde fluorescent traces in visual areas 17 and 18 and late (pd 35 - 64) injections of other retrograde fluorescent tracers in either hemisphere, mostly in areas known to receive projections from AI and AII in the adult cat. The middle ectosylvian gyrus was analysed for double-labelled neurons in the region corresponding approximately to AI and AII. Late injections in the contralateral (to the analysed AI, AII) hemisphere including all of the known auditory areas, as well as some visual and 'association' areas, did not relabel neurons which had had transient projections to either ipsi- or contralateral visual areas 17 - 18. Thus, AI and AII neurons after eliminating their transient juvenile projections to visual areas 17 and 18 do not project to the other hemisphere. In contrast, relabelling was obtained with late injections in several locations in the ipsilateral hemisphere; it was expressed as per cent of the population labelled by the early injections. Few neurons (0 - 2.5%) were relabelled by large injections in the caudal part of the posterior ectosylvian gyrus and the adjacent posterior suprasylvian sulcus (areas DP, P, VP). Multiple injections in the middle ectosylvian gyrus relabelled a considerably larger percentage of neurons (13%). Single small injections in the middle ectosylvian gyrus (areas AI, AII), the caudal part of the anterior ectosylvian gyrus and the rostral part of the posterior ectosylvian gyrus relabelled 3.1 - 7.0% of neurons. These neurons were generally near (<2.0 mm) the outer border of the late injection sites. Neurons with transient projections to ipsi- or contralateral visual areas 17 and 18 were relabelled in similar proportions by late injections at any given location. Thus, AI or AII neurons which send a transitory axon to ipsi- or contralateral visual areas 17 and 18 are most likely to form short permanent cortical connections. In that respect, they are similar to medial area 17 neurons that form transitory callosal axons and short permanent axons to ipsilateral visual areas 17 and 18.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Human alveolar macrophages infected by virulent bacteria expressing SipB are a major source of active interleukin-18.

Recent publications have demonstrated that the protease caspase-1 is responsible for the processing of pro-interleukin 18 (IL-18) into the active form. Studies on cell lines and murine macrophages have shown that the bacterial invasion factor SipB activates caspase-1, triggering cell death. Thus, we investigated the role of SipB in the activation and release of IL-18 in human alveolar macrophages (AM), which are the first line of defense against inhaled pathogens. Under steady-state conditions, AM are a more important source of IL-18 than are dendritic cells (DC) and monocytes. Cytokine production by AM and DC was compared after both types of cells had been infected with a virulent strain of Salmonella enterica serovar Typhimurium and an isogenic sipB mutant, which were used as an infection model. Infection with virulent Salmonella led to marked cell death with features of apoptosis while both intracellular activation and release of IL-18 were demonstrated. In contrast, the sipB mutant did not induce such cell death or the release of active IL-18. The specific caspase-1 inhibitor Ac-YVAD-CMK blocked the early IL-18 release in AM infected with the virulent strain. However, the type of Salmonella infection did not differentially regulate IL-18 gene expression. We concluded that the bacterial virulence factor SipB plays an essential posttranslational role in the intracellular activation of IL-18 and the release of the cytokine in human AM.

Nedd4-2 ubiquitin ligase: a contributor to experimental neuropathic pain?

Background: Neuropathic pain is associated with altered expression of voltage-gated sodium channels (VGSCs) leading to peripheral nerve hyperexcitability. Interestingly, in cell expression systems, the ubiquitin ligase Nedd4-2 regulates the cell membrane density of the most abundant peripheral and pain-related VGSC, namely Nav1.7, and decreases its sodium current. Yet nothing is known about the involvement of Nedd4-2 in nociception and chronic pain. Therefore, the goal of this study is (i) to characterize Nedd4-2 and Nav1.7 expression in an experimental model of neuropathic pain (ii) to design by viral vector-mediated gene therapy an approach to depict the implication of Nedd4-2 in chronic pain. Methods: Western Blot and immunohistochemistry experiments detecting Nav1.7 and Nedd4-2 were performed in rodent DRGs 7 days after spared nerve injury (SNI). For the viral vector-mediated gene therapy, a recombinant Adeno-Associated Virus (rAAV2/6) was generated expressing the Nedd4-2 gene. Intrathecal injection of rAAV2/6 was followed 2 weeks after by the SNI surgery. Data are expressed in mean ± SEM, n = 4 in each condition. Results: Immunofluorescence on DRGs neurons reveals a decreased number of positive Nedd4-2 cells in the SNI model (27.0 ± 1.2%) versus sham group (43.4 ± 3.5%; p <0.005), as well as an increase in positive Nav1.7 cells in SNI (50.1 ± 2.9%) versus Sham (41.6 ± 1.8%; p <0.05). The change of Nedd4-2 expression was confirmed by western-blot analysis. In addition, we show that Nedd4-2 and Nav1.7 are largely expressed in overlapping cell populations, chiefly colocalizing with markers of small nociceptive neurons. Furthermore, we report that intrathecal injection of rAAV is able to counteract the reduction of Nedd4-2 expression in SNI animals. Conclusion: Our results indicate that Nedd4-2 is mainly expressed in nociceptors and downregulated after nerve injury. Moreover, our data suggest that the reduction of Nedd4-2, after nerve injury, may modulate Nav1.7 activity and contribute to hyperexcitability in neuropathic pain. A normal level of Nedd4-2 can be restored using a viral vector and we will further assess its functional effect on pain sensitivity.

Glutamine Synthetase is Expressed by Primary Sensory Neurons from Chick Embryos In Vitro but not In Vivo: Influence of Skeletal Muscle Extract.

Glutamine synthetase (GS) catalyses the ATP-dependent formation of glutamine from glutamate and ammonia. To determine whether dorsal root ganglion (DRG) cells from chick embryos express the enzyme in vivo or in vitro, GS was detected by immunocytochemical reaction either in vibratome sections of DRG or in dissociated DRG cell cultures. The immunocytochemical detection of GS showed that in vivo the DRG taken from chick embryos at day 10 (E10), E14, E18 or from chickens after hatching were free of any GS-positive ganglion cells; in contrast, in neuron-enriched cultures of DRG cells grown in vitro at E10, virtually all the neuronal cells (98.6 +/- 1.0%) express GS at 3, 5 or 7 days of culture. In mixed DRG cell cultures, only 83.6+/-4.6% of the neurons displayed a GS-immunoreactivity. In both culture conditions, neither the presence of horse serum nor the age of the culture appeared to affect the percentage of neurons which displayed a GS-immunoreactivity. After [3H]glutamine uptake, radioautographs revealed that only 80% of the neurons were labelled in neuron-enriched DRG cell cultures while 96% of the neurons were radioactive in mixed DRG cell cultures. Furthermore the most heavily [3H]glutamine-labelled neurons were exclusively found in mixed DRG cell cultures. Combination of both immunocytochemical detection of GS and radioautography after [3H]glutamine uptake showed that strongly GS-immunostained neurons corresponded to poorly radioactive ones and vice versa. When skeletal muscle extract (ME) was added to DRG cell cultures, the number of GS-positive neurons was reduced to 77.5 +/- 2.5% in neuron-enriched cultures or to 43.6 +/- 3.8% in mixed DRG cell cultures; in both types of culture, the intensity of the neuronal immunostaining was depressed. Furthermore, combined action of ME and non-neuronal cells potentiates the enzyme repression exerted separately by ME or non-neuronal cells. Since GS-immunoreactivity is expressed in DRG cells grown in vitro, but not in vivo, it is suggested that microenvironmental factors influence the expression of GS. More specifically, the repression of GS by primary sensory neurons grown in vitro may be strongly induced by soluble factors present in skeletal muscle, and to a lesser extent in brain, and potentiated by non-neuronal cells.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.