MERTK in the rat trigeminal system: a potential novel target for cluster headache?

The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.

Progesterone distribution in the trigeminal system and its role to modulate sensory neurotransmission: influence of sex.

BACKGROUND: Women are disproportionately affected by migraine, representing up to 75% of all migraine cases. This discrepancy has been proposed to be influenced by differences in hormone levels between the sexes. One such hormone is progesterone. Calcitonin gene-related peptide (CGRP) system is an important factor in migraine pathophysiology and could be influenced by circulating hormones. The purpose of this study was to investigate the distribution of progesterone and its receptor (PR) in the trigeminovascular system, and to examine the role of progesterone to modulate sensory neurotransmission. METHODS: Trigeminal ganglion (TG), hypothalamus, dura mater, and the basilar artery from male and female rats were carefully dissected. Expression of progesterone and PR proteins, and mRNA levels from TG and hypothalamus were analyzed by immunohistochemistry and real-time quantitative PCR. CGRP release from TG and dura mater were measured using an enzyme-linked immunosorbent assay. In addition, the vasomotor effect of progesterone on male and female basilar artery segments was investigated with myography. RESULTS: Progesterone and progesterone receptor -A (PR-A) immunoreactivity were found in TG. Progesterone was located predominantly in cell membranes and in Aδ-fibers, and PR-A was found in neuronal cytoplasm and nucleus, and in satellite glial cells. The number of positive progesterone immunoreactive cells in the TG was higher in female compared to male rats. The PR mRNA was expressed in both hypothalamus and TG; however, the PR expression level was significantly higher in the hypothalamus. Progesterone did not induce a significant change neither in basal level nor upon stimulated release of CGRP from dura mater or TG in male or female rats when compared to the vehicle control. However, pre-treated with 10 µM progesterone weakly enhanced capsaicin induced CGRP release observed in the dura mater of male rats. Similarly, in male basilar arteries, progesterone significantly amplified the dilation in response to capsaicin. CONCLUSIONS: In conclusion, these results highlight the potential for progesterone to modulate sensory neurotransmission and vascular responses in a complex manner, with effects varying by sex, tissue type, and the nature of the stimulus. Further investigations are needed to elucidate the underlying mechanisms and physiological implications of these findings.

Lasmiditan and 5-Hydroxytryptamine in the rat trigeminal system; expression, release and interactions with 5-HT1 receptors.

BACKGROUND: 5-Hydroxytryptamine (5-HT) receptors 1B, 1D and 1F have key roles in migraine pharmacotherapy. Selective agonists targeting these receptors, such as triptans and ditans, are effective in aborting acute migraine attacks and inhibit the in vivo release of calcitonin gene-related peptide (CGRP) in human and animal models. The study aimed to examine the localization, genetic expression and functional aspects of 5- HT1B/1D/1F receptors in the trigeminal system in order to further understand the molecular sites of action of triptans (5-HT1B/1D) and ditans (5-HT1F). METHODS: Utilizing immunohistochemistry, the localization of 5-HT and of 5-HT1B/1D/1F receptors was examined in rat trigeminal ganglion (TG) and combined with quantitative polymerase chain reaction to quantify the level of expression for 5-HT1B/1D/1F receptors in the TG. The functional role of these receptors was examined ex vivo with a capsaicin/potassium induced 5-HT and CGRP release. RESULTS: 5-HT immunoreactivity (ir) was observed in a minority of CGRP negative C-fibres, most neuron somas and faintly in A-fibres and Schwann cell neurolemma. 5-HT1B/1D receptors were expressed in the TG, while the 5-HT1F receptor displayed a weak ir. The 5-HT1D receptor co-localized with receptor activity-modifying protein 1 (RAMP1) in Aδ-fibres in the TG, while 5-HT1B-ir was weakly expressed and 5-HT1F-ir was not detected in these fibres. None of the 5-HT1 receptors co-localized with CGRP-ir in C-fibres. 5-HT1D receptor mRNA was the most prominently expressed, followed by the 5-HT1B receptor and lastly the 5-HT1F receptor. The 5-HT1B and 5-HT1D receptor antagonist, GR127935, could reverse the inhibitory effect of Lasmiditan (a selective 5-HT1F receptor agonist) on CGRP release in the soma-rich TG but not in soma-poor TG or dura mater. 5-HT release in the soma-rich TG, and 5-HT content in the baseline samples, negatively correlated with CGRP levels, showing for the first time a physiological role for 5-HT induced inhibition. CONCLUSION: This study reveals the presence of a subgroup of C-fibres that store 5-HT. The data shows high expression of 5-HT1B/1D receptors and suggests that the 5-HT1F receptor is a relatively unlikely target in the rat TG. Furthermore, Lasmiditan works as a partial agonist on 5-HT1B/1D receptors in clinically relevant dose regiments.

Dual action of the cannabinoid receptor 1 ligand arachidonyl-2'-chloroethylamide on calcitonin gene-related peptide release.

BACKGROUND: Based on the current understanding of the role of neuropeptide signalling in migraine, we explored the therapeutic potential of a specific cannabinoid agonist. The aim of the present study was to examine the effect of the synthetic endocannabinoid (eCB) analogue, arachidonyl-2'-chloroethylamide (ACEA), on calcitonin gene-related peptide (CGRP) release in the dura and trigeminal ganglion (TG), as cannabinoids are known to activate Gi/o-coupled cannabinoid receptors type 1 (CB1), resulting in neuronal inhibition. METHODS: The experiments were performed using the hemi-skull model and dissected TGs from male Sprague-Dawley rats. CGRP release was induced by either 60 mM K+ (for depolarization-induced stimulation) or 100 nM capsaicin (for transient receptor potential vanilloid 1 (TRPV1) -induced stimulation) and measured using an enzyme-linked immunosorbent assay. The analysis of CGRP release data was combined with immunohistochemistry in order to study the cellular localization of CB1, cannabinoid receptor type 2 (CB2), CGRP and receptor activity modifying protein 1 (RAMP1), a subunit of the functional CGRP receptor, in the TG. RESULTS: CB1 was predominantly expressed in neuronal somas in which colocalization with CGRP was observed. Furthermore, CB1 exhibited colocalization with RAMP1 in neuronal Aδ-fibres but was not clearly expressed in the CGRP-immunoreactive C-fibres. CB2 was mainly expressed in satellite glial cells and did not show substantial colocalization with either CGRP or RAMP1. Without stimulation, 140 nM ACEA per se caused a significant increase in CGRP release in the dura but not TG, compared to vehicle. Furthermore, 140 nM ACEA did not significantly modify neither K+- nor capsaicin-induced CGRP release. However, when the TRPV1 blocker AMG9810 (1 mM) was coapplied with ACEA, K+-induced CGRP release was significantly attenuated in the TG and dura. CONCLUSIONS: Results from the present study indicate that ACEA per se does not exhibit antimigraine potential due to its dual agonistic properties, resulting in activation of both CB1 and TRPV1, and thereby inhibition and stimulation of CGRP release, respectively.

Biological and small molecule strategies in migraine therapy with relation to the calcitonin gene-related peptide family of peptides.

Migraine is one of the most common of neurological disorders with a global prevalence of up to 15%. One in five migraineurs have frequent episodic or chronic migraine requiring prophylactic treatment. In recent years, specific pharmacological treatments targeting calcitonin gene-related peptide (CGRP) signalling molecules have provided safe and effective treatments, monoclonal antibodies for prophylaxis and gepants for acute therapy. Albeit beneficial, it is important to understand the molecular mechanisms of these new drugs to better understand migraine pathophysiology and improve therapy. Here, we describe current views on the role of the CGRP family of peptides - CGRP, calcitonin, adrenomedullin, amylin - and their receptors in the trigeminovascular system. All these molecules are present within the trigeminovascular system but differ in expression and localization. It is likely that they have different roles, which can be utilized in providing additional drug targets. LINKED ARTICLES: This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

Neuropeptides and the Nodes of Ranvier in Cranial Headaches.

The trigeminovascular system (TGV) comprise of the trigeminal ganglion with neurons and satellite glial cells, with sensory unmyelinated C-fibers and myelinated Aδ-fibers picking up information from different parts of the head and sending signals to the brainstem and the central nervous system. In this review we discuss aspects of signaling at the distal parts of the sensory fibers, the extrasynaptic signaling between C-fibers and Aδ-fibers, and the contact between the trigeminal fibers at the nerve root entry zone where they transit into the CNS. We also address the possible role of the neuropeptides calcitonin gene-related peptide (CGRP), the neurokinin family and pituitary adenylyl cyclase-activating polypeptide 38 (PACAP-38), all found in the TGV system together with their respective receptors. Elucidation of the expression and localization of neuropeptides and their receptors in the TGV system may provide novel ways to understand their roles in migraine pathophysiology and suggest novel ways for treatment of migraine patients.

Estrogen receptors α, ß and GPER in the CNS and trigeminal system - molecular and functional aspects.

BACKGROUND: Migraine occurs 2-3 times more often in females than in males and is in many females associated with the onset of menstruation. The steroid hormone, 17ß-estradiol (estrogen, E2), exerts its effects by binding and activating several estrogen receptors (ERs). Calcitonin gene-related peptide (CGRP) has a strong position in migraine pathophysiology, and interaction with CGRP has resulted in several successful drugs for acute and prophylactic treatment of migraine, effective in all age groups and in both sexes. METHODS: Immunohistochemistry was used for detection and localization of proteins, release of CGRP and PACAP investigated by ELISA and myography/perfusion arteriography was performed on rat and human arterial segments. RESULTS: ERα was found throughout the whole brain, and in several migraine related structures. ERß was mainly found in the hippocampus and the cerebellum. In trigeminal ganglion (TG), ERα was found in the nuclei of neurons; these neurons expressed CGRP or the CGRP receptor in the cytoplasm. G-protein ER (GPER) was observed in the cell membrane and cytoplasm in most TG neurons. We compared TG from males and females, and females expressed more ER receptors. For neuropeptide release, the only observable difference was a baseline CGRP release being higher in the pro-estrous state as compared to estrous state. In the middle cerebral artery (MCA), we observed similar dilatory ER-responses between males and females, except for vasodilatory ERß which we observed only in female arteries. CONCLUSION: These data reveal significant differences in ER receptor expression between male and female rats. This contrasts to CGRP and PACAP release where we did not observe discernable difference between the sexes. Together, this points to a hypothesis where estrogen could have a modulatory role on the trigeminal neuron function in general rather than on the acute CGRP release mechanisms and vasomotor responses.

C-fibers may modulate adjacent Aδ-fibers through axon-axon CGRP signaling at nodes of Ranvier in the trigeminal system.

BACKGROUND: Monoclonal antibodies (mAbs) towards CGRP or the CGRP receptor show good prophylactic antimigraine efficacy. However, their site of action is still elusive. Due to lack of passage of mAbs across the blood-brain barrier the trigeminal system has been suggested a possible site of action because it lacks blood-brain barrier and hence is available to circulating molecules. The trigeminal ganglion (TG) harbors two types of neurons; half of which store CGRP and the rest that express CGRP receptor elements (CLR/RAMP1). METHODS: With specific immunohistochemistry methods, we demonstrated the localization of CGRP, CLR, RAMP1, and their locations related to expression of the paranodal marker contactin-associated protein 1 (CASPR). Furthermore, we studied functional CGRP release separately from the neuron soma and the part with only nerve fibers of the trigeminal ganglion, using an enzyme-linked immunosorbent assay. RESULTS: Antibodies towards CGRP and CLR/RAMP1 bind to two different populations of neurons in the TG and are found in the C- and the myelinated Aδ-fibers, respectively, within the dura mater and in trigeminal ganglion (TG). CASPR staining revealed paranodal areas of the different myelinated fibers inhabiting the TG and dura mater. Double immunostaining with CASPR and RAMP1 or the functional CGRP receptor antibody (AA58) revealed co-localization of the two peptides in the paranodal region which suggests the presence of the CGRP-receptor. Double immunostaining with CGRP and CASPR revealed that thin C-fibers have CGRP-positive boutons which often localize in close proximity to the nodal areas of the CGRP-receptor positive Aδ-fibers. These boutons are pearl-like synaptic structures, and we show CGRP release from fibers dissociated from their neuronal bodies. In addition, we found that adjacent to the CGRP receptor localization in the node of Ranvier there was PKA immunoreactivity (kinase stimulated by cAMP), providing structural possibility to modify conduction activity within the Aδ-fibers. CONCLUSION: We observed a close relationship between the CGRP containing C-fibers and the Aδ-fibers containing the CGRP-receptor elements, suggesting a point of axon-axon interaction for the released CGRP and a site of action for gepants and the novel mAbs to alleviate migraine.

Exercise training during rehabilitation of patients with COPD: a current perspective.

Patients with chronic obstructive pulmonary disease (COPD) suffer frequently from physiologic and psychological impairments, such as dyspnea, peripheral muscle weakness, exercise intolerance, decreased health-related quality of life (HRQOL) and emotional distress. Rehabilitation programmes have shown to result in significant changes in perceived dyspnea and fatigue, utilisation of healthcare resources, exercise performance and HRQOL. Exercise training, which consists of whole-body exercise training and local resistance training, is the cornerstone of these programmes. Regrettably, the positive effects of respiratory rehabilitation deteriorate over time, especially after short programmes. Hence, attention should be given to the aftercare of these patients to prevent them to revert again to a sedentary lifestyle. On empirical basis three possibilities seem to be clinically feasible: (1) continuous outpatient exercise training; (2) exercise training in a home-based or community-based setting; or (3) exercise training sessions in a group of asthma and COPD patients.

Ewing's tumour: novel recurrent chromosomal abnormalities demonstrated by molecular cytogenetic analysis of seven cell lines and one primary culture.

Conventional cytogenetics has led to the identification of the primary t(11;22)(q24;q12) translocation in the Ewing's family of tumours, and to the demonstration of certain recurring secondary aberrations that may contribute to neoplastic progression. Other important cytogenetic abnormalities may previously have been overlooked due to the limited resolution of chromosome banding. Here, we have applied the molecular cytogenetic techniques of spectral karyotyping, multiplex-fluorescence in situ hybridisation and comparative genomic hybridisation to the characterisation of seven Ewing's tumour cell lines and one primary culture. These complementary techniques have enabled us to produce a detailed description of the karyotypes of the cell lines and to demonstrate recurring numerical and structural abnormalities. In particular, we have identified a novel, unbalanced translocation involving chromosomes 16 and 17 in three of eight samples, including the primary culture. The unbalanced translocation was associated with comparative genomic hybridisation evidence of loss of 16q and 17p, copy number imbalances that were seen in five and four of the eight samples respectively. Recurrent breakpoints at 16p11.2, 16q11.1, 17p11.2 and 17q11.2 were identified. Our findings indicate that chromosomes 16 and 17 should be investigated further in the search for genes involved in the development of Ewing's family tumours.

Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression.

Group I Burkitt lymphoma (BL) lines retaining the original BL tumor cell phenotype are unable to present endogenously expressed antigens to HLA class I-restricted cytotoxic T cells (CTL) but can be recognized if the relevant HLA class I/peptide epitope complex is reconstituted at the cell surface by exogenous addition of synthetic target peptide. Endogenous antigen-processing function is restored in BL lines that have undergone Epstein-Barr virus (EBV)-induced drift in culture to the group III phenotype typically displayed by EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin. We compared group I versus group III cells for their expression of proteasome components, transporter proteins and HLA-class I antigens, all of which are thought to be involved in the endogenous antigen processing pathway. By Western blot analysis, there were not consistent differences in the low molecular mass protein subunits of proteasomes (lmp)-2, lmp-7 and delta, although the mb-1 proteasome subunit was regularly present at higher levels in group I BL lines relative to group III lines or LCL. By contrast there were marked differences in the expression of peptide transporter-associated proteins (Tap), with down-regulation of Tap-1 and Tap-2 in 8/8 and 7/8 group I BL lines, respectively. Surface levels of HLA class I antigens were also consistently lower in group I cells; this was not associated with an intracellular accumulation of free HLA heavy chains, such as is seen in the Tap-deficient T2 processing-mutant line, but instead reflected a reduced rate of HLA class I synthesis in group I cells. Analysis of EBV gene transfectants of the B lymphoma lines BJAB and BL41 showed that the virus-encoded latent membrane protein-1 (LMP1), which is one of several EBV antigens expressed in group III but not in group I cells, was uniquely able to up-regulate expression both of the Tap proteins and HLA class I. Furthermore, this was accompanied by a restoration of antigen-processing function as measured by the ability of these cells to present an endogenously expressed viral antigen to CTL. These effects of LMP1 were similar to those induced in the same cell lines by interferon-gamma treatment. The results implicate both Tap and HLA class I expression as factors limiting the antigen-processing function of BL cells, and suggest that the accessibility of other EBV-associated malignancies to CTL surveillance may be critically dependent upon their LMP1 status.

Toxocariasis simulating hepatic recurrence in a patient with Wilms' tumor.

We report the case of a 3-year-old girl with stage I Wilms' tumor of favorable histology. During the course of chemotherapy 5 months post-diagnosis, an abdominal ultrasonogram revealed hypoechoic areas consistent with hepatic tumor recurrence. A liver biopsy performed to rule out recurrence of the malignancy was suggestive of toxocariasis and the diagnosis was confirmed by serologic testing. Although the patient had few classic signs of visceral larva migrans, her eosinophilia and family social history should have suggested this possibility. This case demonstrates that hepatic toxocariasis should be considered in evaluating hepatic hypoechoic lesions in a child, even when features typical of the disease are absent.

Presentation of endogenous viral peptide epitopes by anti-CD40 stimulated human B cells following recombinant vaccinia infection.

Cytotoxic T lymphocytes (CTLs) recognising viral antigens are an important host defence mechanism in restricting the proliferation of virus-infected cells. Previously, lymphoblastoid cell lines (LCLs) infected with vaccinia recombinants encoding viral proteins have been used to identify specific CTL epitopes. However, to localise the EBV CTL epitopes encoded by Epstein-Barr virus (EBV) transformants, LCLs are an inappropriate host for vaccinia recombinants. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-CD40 stimulated human B cells replicated vaccinia virus and expressed EBV nuclear antigen(s) (EBNA) following infection with recombinant vaccinia encoding the appropriate region of the EBV genome. Recombinant vaccinia-infected anti-CD40 stimulated B cell lines were then used to localise target epitopes for a panel of EBV-specific CTL clones. Most importantly, in vitro stimulation of unfractionated mononuclear cells with recombinant vaccinia-infected anti-CD40 B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 defined the epitope for the EBNA6-specific CTL clones to the sequence KEHVIQNAFRK. This work clearly demonstrates that anti-CD40 stimulated B cell lines not only provide an efficient tool for localising CTL epitopes but also presents an alternative mechanism of reactivating a memory T cell response to any gene product expressed by recombinant vaccinia.

EBV peptide epitope sensitization restores human cytotoxic T cell recognition of Burkitt's lymphoma cells. Evidence for a critical role for ICAM-2.

The pathogenesis of EBV+ Burkitt's lymphoma (BL) suggests evasion of the CTL response against EBV. Two important features of this tumor have been previously suggested to explain this immune evasion, (a) absence/low expression of cellular adhesion molecules and (b) restricted expression of EBV latent Ag. To determine the relative importance of these features in relation to evasion of EBV-specific CTL, a group of BL cell lines with variable expression of the aforementioned phenotypic characteristics were assayed for specific CTL lysis after exogenous addition of EBV peptide epitopes. In spite of down-regulated expression of the adhesion molecules LFA-1, LFA-3, and/or ICAM-1, peptide-sensitized BL cells were recognized and lysed by EBV-specific CTL. Moreover, there was no significant difference between the CTL lysis of the BL cells and that of adhesion molecule-positive control cells over a wide range of peptide epitope concentrations. Blocking experiments with mAb to individual adhesion molecules suggested that virus-specific CTL recognition of lymphoblastoid cell lines was dependent on an intact LFA-3/CD2 pathway. In contrast, the CTL recognition of peptide-sensitized BL cells was critically dependent on the LFA-1/ICAM pathway, with an insignificant contribution by CD2/LFA-3. The consistently high expression of ICAM-2 on all BL cell lines suggests that the accessory function in CTL recognition of these cells is mediated by the LFA-1/ICAM-2 pathway. Thus, down-regulation of LFA-1, LFA-3, and/or ICAM-1 expression on BL cells does not provide an absolute barrier to tumor cell recognition by virus-specific CTL. The ability of virus-specific CTL to recognize peptide epitope-sensitized BL cells as efficiently as normal cells has demonstrated the importance of latent Ag expression in the CTL control of EBV+ tumors.

Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.

Expression of Epstein-Barr virus nuclear antigens in anti-IgM-stimulated B cells following recombinant vaccinia infection and their recognition by human cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6-specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia.