Activated T Lymphocytes in the Cerebrospinal Fluid of a Patient With Epstein-Barr Virus-Associated Meningoencephalitis

Infectious mononucleosis (IM) is usually an uncomplicated, self-limiting lymphoproliferative disease caused by Epstein-Barr virus (EBV). However, neurologic complications such as meningoencephalitis, Guillain-Barré syndrome and cranial nerve palsy occur in 1 to 5% of the patients.1 The pathogenesis of central nervous system (CNS) involvement in IM is not yet well-understood.2 We report a patient with IM-associated meningoencephalitis. Flow cytometric analysis revealed that the majority of lymphocytes in CNS pleocytosis were CD8+ T lymphocytes positive for CD45RO and for the activation antigens CD29 and HLA-DR. Our findings suggest that this subset of lymphocytes may contribute to the immunopathogenesis of CNS complications associated with EBV infection. Case report. Within 2 weeks of the onset of acute mononucleosis characterized by fever, tonsillitis and lymphadenopathy, the patient, a 16-year-old boy, was admitted with generalized tonic-clonic seizures and became comatose. Physical examination at the time of admission showed an agitated patient responding only to painful stimuli. Kernig and Brudzinski signs were present and deep tendon reflexes were symmetrically brisk. The liver was very enlarged (15 cm below the costal margin) and the spleen was not palpable. The initial white blood cell count was 11 300/mm3 with 55% granulocytes, 31% lymphocytes, 11% atypical lymphocytes, 2% monocytes and 1% plasma cells. Other abnormal laboratory tests included an aspartate aminotransferase of 72 units/l, alanine aminotransferase of 20 units/l, creatine phosphokinase of 1052 units/l and lactate dehydrogenase of 372 units/l. Serum IgG, IgA and IgM values were elevated at 1670, 394 and 253 mg/dl, respectively. A lumbar puncture revealed a pleocytosis with 37 white blood cells/mm3 including 52% lymphocytes (most of which were atypical lymphocytes) and 42% monocytes; the glucose was 74 mg/dl and protein was 729 mg/dl. Oligoclonal bands and myelin basic protein were not detected in cerebrospinal fluid (CSF). On admission a Monospot® test was positive. Viral serology for EBV was consistent with acute primary infection; serum IgG antibodies against viral capsid antigen and early antigen were both 1:32 as determined by indirect immunofluorescence. IgM serum antibodies against viral capsid antigen could not be detected, probably because of a short term period of IgM antibody production before hospital admission. No antibodies against Epstein-Barr nuclear antigen were measured. EBV-specific antibodies were absent in CSF. PCR revealed EBV type 1 DNA in peripheral blood but not in CSF. At follow-up 10 months later anti-viral capsid antigen (IgG) was 1:160, anti-early antigen was negative and anti-Epstein-Barr nuclear antigen 1:32. At the time of admission the patient required intubation with mechanical ventilation. He received parenteral acyclovir (3 × 400 mg/m2) for 10 days and parenteral dexamethasone (3 × 2.5 mg/m2) for 20 days. An electroencephalogram disclosed generalized slowing, but no focal abnormalities. On the seventh day T2-weighted sequences of cranial magnetic resonance imaging showed areas of high density in the right hemisphere consistent with the findings of Bale et al.3 in a girl with EBV meningoencephalitis. The patient's clinical condition improved during the next days and mechanical ventilation was stopped at the 19th hospital day. At that time the CSF and the peripheral blood showed normal values. Twenty-three days after admission the patient was discharged in good clinical condition with only minor neurologic deficits. His left sided cranial nerve IX and XII palsies resolved during the next 10 months. Methods.Antibodies. Fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies directed against the antigens CD4 (IgG1), CD8 (IgG1), CD29 (IgG2a), CD45RO (IgG2a), CD14 (IgG2a), CD19 (IgG1) and HLA-DR (IgG2b) and biotinylated monoclonal antibodies against CD3 (IgG2a) were purchased from Dianova (Hamburg, Germany). Biotinylated CD3 antibodies were labeled by Streptavidin Red 670 obtained from Life Technologies (Eggenstein, Germany). Cells. Peripheral blood mononuclear cells were isolated from heparinized venous blood by standard procedures. Peripheral blood mononuclear cells and cerebrospinal cells collected by spinal lumbar puncture were cryoconserved in the presence of 20% fetal calf serum and 10% dimethyl sulfoxide in liquid nitrogen. Immunofluorescence analysis. This was done after triple color staining with monoclonal antibodies on a FACScan (Becton Dickinson, Mountain View, CA; FACScan 2.1. software, Becton Dickinson). Cells were gated by forward scatter and 90-degree side scatter and for CD3+ expression. Results. The absolute number of CD8+ T cells measured in the peripheral blood of the patient at the onset of CNS complications of EBV infection was elevated 3-fold compared with values in healthy controls.4 Most of the patient's CD8+ lymphocytes expressed CD45RO and activation antigens such as HLA-DR and CD29. In contrast the absolute number of activated peripheral CD4+ T cells varied only slightly from control values; consequently the ratio was markedly tipped in favor of activated CD8+ cells (Table 1).4 Immunophenotyping of the CNS sample revealed a ratio of lymphocytes similar to that observed in the periphery (Table 1). The CD4+/CD8+ T cell ratio was reversed in both the CSF and blood, at 0.3 and 0.18, respectively. CD8+ T cells expressing the surface molecules HLA-DR, CD29 and CD45RO quantitatively comprised the majority of lymphocytes in CNS pleocytosis, whereas only a small percentage of activated CD4+ T cells was demonstrated (Table 1). T cells not in activated form were found in the peripheral blood only and not in the CSF (data not shown). CD19+ B cells showed a normal absolute number in the patient's peripheral blood and were not detected in CSF (Table 1). The absolute number of peripheral blood monocytes expressing the surface molecule CD14+ was comparable to those of healthy controls. Discussion. In accordance with previously reported data, we found that the majority of the patient's peripheral blood lymphocytes were CD8+ T cells expressing the surface molecules HLA-DR, CD45RO and CD29.5-7 HLA-DR, also termed Class II histocompatibility antigen, is composed of two noncovalently associated glycoproteins and is expressed on B cells, monocytes and activated T cells.8 The surface molecule CD45 can be found on human T cells in various isoforms. After activation CD45RA+ cells down-regulate CD45RA expression and concomitantly gain high density expression of CD45RO.9 CD29 is the marker for the beta chain of the very late activation antigens integrin subfamily. These heterodimers are made up of various pairings of alpha and beta subunits and play a role in the trafficking of lymphocytes.10 HLA-DR and the adhesion molecule CD29 are known as transient markers of activation whereas the expression of CD45RO is stably increased after activation.7, 11 In some patients with neurologic complications of IM, atypical lymphocytes have been found previously in CSF and cerebral parenchyma, but to date no data have been published on their immunophenotypic analysis.12, 13 Here we demonstrate that pleocytosis in CSF of a patient with EBV-associated meningoencephalitis consists of CD8+ and to a lesser extent of CD4+ T cells expressing CD45RO and the activation antigens HLA-DR and CD29. Furthermore we found a similar distribution of activated CD4+ and CD8+ T cells in peripheral blood. A small percentage of T cells not in activated form was demonstrated in the patient's peripheral blood but not in CSF. This is in agreement with animal studies that imply that only activated T cells can enter the CNS in a random manner.14 In addition the trafficking of lymphocytes into the CNS is controlled by adhesion molecules such as CD29 which was clearly increased in expression in T cells seen in CSF and peripheral blood of our patient.11, 15 In contrast to the findings of Tsutsumi et al.,16 we detected EBV DNA and virus-infected B cells in peripheral blood only, possibly because of a previous rapid clearance by EBV-specific cytotoxic T cells in CSF. Our findings suggest that activated T cells and/or their secreted lymphokines play an immunopathogenic role in the development of IM-associated CNS complications such as meningoencephalitis. Because in animals a trafficking of lymphocytes from the CSF along cranial nerves into peripheral blood and lymph has been shown, activated T cells may additionally play a potential role in the development of cranial nerve palsies.15 Further studies are warranted to elucidate the pathogenesis of IM-associated CNS complications to provide a rationale for therapeutic intervention. Acknowledgements. We thank Stephen Chanock, M.D., National Institutes of Health, for critically reviewing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Schu 560/2-3). Thomas Lehrnbecher, M.D.; Bernd Chittka; Ralph Nanan, M.D.; Silvia Seidenspinner; Hans Wolfgang Kreth, M.D.; Volker Schuster, M.D. Department of Pediatrics University of Wurzburg Wurzburg, Germany