children often continue to shed virus in their urine for several years. In congenital infections, a defect of specific cell-mediated immunity to. CMV has been ...
Vol. 35, No. 3
INFECTION AND IMMUNITY, Mar. 1982, p. 1162-1164
0019-9567/82/031162-03$02.00/0
Cell-Mediated Immunity in Human Cytomegalovirus Infection M. FIORILLI,l* M. C. SIRIANNI,1 P. IANNETTI,2 A. PANA,3 M. DIVIZIA,3 AND F. AIUTI' Departments of Clinical Immunology,' Pediatrics,2 and Institute of Hygiene,3 University of Rome, Rome 00161, Italy
Received 17 July 1981/Accepted 27 October 1981
The direct leukocyte migration inhibition test, in response to cytomegalovirus stimulation, was used to study cell-mediated immunity in a group of children with cytomegalovirus infection. The test was impaired in children with chronic disease associated with cytomegaloviruria. In those cases with no viruria at the moment of the test, leukocyte migration inhibition was normal. Our data suggest that the acquired chronic cytomegalovirus infection may be sustained by a state of specific cellular desensitization, as already demonstrated for congenital infection. The mechanism of host virus interaction in human chronic viral infections is still poorly elucidated. Certain viruses, such as cytomegalovirus (CMV), can be retained in an inactive form for years or even for life after the primary infection (5). In some cases, the infection remains active for a long time. For example, after a congenital (5) or acquired (6) CMV infection, children often continue to shed virus in their urine for several years. In congenital infections, a defect of specific cell-mediated immunity to CMV has been demonstrated (3, 4, 7). A similar state of cellular immune desensitization has been hypothesized (5), but not demonstrated, in the case of acquired infections. We had previously used the direct leukocyte migration inhibition test (LMIT) to demonstrate a defect of cell-mediated immunity against CMV in patients with congenital infections (2, 3). We report here additional data showing that longlasting prolonged specific cellular unresponsiveness may also follow acquired CMV infections. Our study was carried out on a group of nine children (cases 3 to 11) in whom cytomegaloviruria had been revealed during routine virological studies for febrile convulsions of unknown etiology and on two children (cases 1 and 2) with congenital CMV infection. Cases 3 to 11 lacked the clinical signs of congenital CMV infection (low birthweight, microcephaly, cerebral calcifications, mental or motor retardation, etc.), and they had been symptomless until the age of 2 years or more when they presented febrile convulsions. To our knowledge, this latter manifestation has never been related to congenital CMV infection. The direct leukocyte migration inhibition test in response to CMV antigen (CMV LMIT) was performed as previously described (2). Briefly, leukocyte-rich plasma was obtained from heparinized blood; the cells were washed twice in
buffered saline and suspended at 6 x 106 cells per ml in RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) with 10% fetal calf serum (Flow Laboratories, Inc., Rockville, Md.) and antibiotics. The cell suspension (20 Iul) was aspirated into siliconized capillary tubes which were subsequently heat sealed at one end, centrifuged at 800 x g for 10 min, and cut at the cellfluid interface. Capillary stumps were fixed on the floor of plastic migration chambers (Sterilin Ltd., Middlesex, England), which were filled with medium alone or with the same medium containing living CMV (strain AD 169) at a concentration of 300 mean tissue cytopatic doses per ml. Each assay was set up in quadruplicate. After an incubation of 20 h at 37°C in 5% C02, the migration areas were measured, and migration indexes (MI) were calculated as follows: MI = (average areas in presence of CMV)/ (average areas in control medium) x 100. For comparison, the relationship of CMV LMIT and the levels of serum complement-fixing antibodies was studied in 16 healthy donors. The values of MI (mean ± 2 SD) were 48 ± 28.2 in seven healthy seropositive (complement-fixing antibody titer of 1:32 to 1:160) subjects, with no CMV excretion in urine and 95.2 ± 20.6 in nine normal seronegatives. Thus, an MI lower than 75 is considered to express a significant migration inhibition response to CMV antigen. For CMV isolation, urine samples were cultured on human embryonic fibroblasts and observed for at least 40 days for the typical cytopathic effect. Serum antibodies to CMV early antigens were detected by the method of Ten Napel and The (8). In 6 of the 11 cases studied, viruria was no longer present 11 months to 4 years, 1 month after it had been first revealed. In the other five cases, CMV was still excreted in the urine after intervals ranging from 13 months to 6 years, 7
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TABLE 1. LMIT in response to CMV in patients with CMV infection CMV immunity' Cytomegaloviruria First evaluation Last evaluation Serum antibodies" LMIT (% MI) Case no. EA CF Viruria Viruria Age Age 1:16 40 1:30 + 4 yr, 7 mo 1 6 mo 1:24 5 mo + 7 yr + 75 1:60 2 + 48 1:80 Neg llyr,llmo 9yr,9mo 3 + 5 yr, 2 mo + 75 1:80 1:16 4 3 yr, 7 mo 1:8 + 5 yr, 5 mo 65 1:80 5 3 yr, 4mo 4 yr, 1 mo + 57 1:20 Neg 3 yr, 4 mo 6 + 4 yr, 1 mo 57 1:20 Neg 7 3 yr, 2 mo + + 54 1:20 1:20 8 3yr,1 mo 2yr + 80 1:40 ND 5yr,11mo 9 ND 38 1:20 6 yr, 6 mo 100 1:160 ND + 10 4yr, 10 mo 1:8 87 1:60 + 6yr, 2 mo + 100 ND ND 3 yr, 4 mo 11 1:5 100 1:30 + 5yr,1mo aAt the time of the last evaluation for cases 1 to 8. b CF, complement fixing; EA, early antigen, Neg, negative response; and ND, not done.
months after the first isolation. An overall comparison of the data (Table 1), including those obtained in the same patients at different times, shows that all of the children without viruria had positive CMV LMIT (i.e., MI c 75%), whereas only one of eight children excreting virus reacted (P 2 0.01, x2 test with Yate's correction). In cases 9, 10, and 11, tests were repeated at intervals of several months. In case 9, the cessation of viruria was accompanied by positive CMV LMIT, whereas in cases 10 and 11, viruria persisted and CMV LMIT responses remained negative after 16 and 21 months, respectively. All of the children had normal serum immunoglobulin and T lymphocyte (sheep rosette-forming cells) levels. No correlation could be found between serum antibodies against early CMV antigens and viral excretion. Chronic viruria after congenital CMV infection is accompanied, and perhaps sustained, by a state of specific cellular unresponsiveness to the virus (3, 4, 7). Our findings show that in this condition viral excretion and immunological impairment can persist up to 7 years after birth (to our knowledge, the oldest reported child with congenital CMV still presenting viruria and specific impairment of cell-mediated immunity was 3 years, 10 months old [4]). Starr and Gold (6) have shown that even infections acquired during childhood are often followed by prolonged viral excretion. No data on the immunological basis of this latter condition are available at present. Among the nine children with acquired CMV infection described in this paper, three continued to excrete virus in
urines and to have negative responses in CMV LMIT 13 to 21 months after the infection had been first recognized, whereas in five children the cessation of viruria was accompanied by a positive LMIT. In one instance, viruria persisted despite significant migration inhibition in response to CMV antigen. The association between positive CMV LMIT and the cessation of viruria suggests a cause-effect relationship. This correlation is supported by the results of a follow-up study of three children, which showed a positive conversion of CMV LMIT in the only patient whose viruria subsided. CMV infection itself might induce an impairment of specific cellular immune responses, perhaps via the activation of suppressor cells (1). Immunological unresponsiveness would in turn allow the persistence of the virus. Ten Napel and The (8) studied cell-mediated immunity after acquired CMV infection in adults and found that significant blastogenic responses of lymphocytes to CMV antigen usually develop within a matter of weeks after infection concomitant with the cessation of viruria. They reported that lymphocytes of two patients, who still excreted virus many months after infection, failed to respond; however, since in some other patients the blastogenic assay was positive while viral excretion was demonstrable, they argued against a causal relation between persistence of infection and lymphocyte responsiveness. We conclude that a mechanism of specific cellular desensitization may operate not only in the congenital form of the disease but also after acquired CMV infections. Further studies are
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needed to establish whether this is true only for infections acquired during childhood and not for those contracted during adult life. LITERATURE CITED 1. Carney, W. P., R. H. Rubln, R. A. Hoffman, W. P. Hansen, R. Healey and M. S. HIrsch. 1981. Analysis of T lymphocyte subsets in Cytomegalovirus mononucleosis. J. Immunol. 126:2114-2116. 2. Florilli, M., A. Pana, M. C. Sirianni, A. Spazban, and F. AlutI. 1978. Specific cellular unreactivity during Cytomegalovirus infection in man. Boll. Ist. Sieroter. Mil. 57:452457. 3. FlorIlli, M., M. C. Siranni, A. Spaziani, F. AiutI, and A. Pan&. 1978. Immune responses in Cytomegalovirus infection. Lancet 1:780. 4. Gehrz, R., S. 0. Knorr, S. C. Parker, J. M. Kalls, and H.
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H. Balfour, Jr. 1977. Specific cell-mediated immune defect in active Cytomegalovirus infection of young children and their mothers. Lancet l:844-847. Hanshaw, J. B. 1971. Congenital Cytomegalovirus infection: a fifteen-year perspective. J. Infect. Dis. 123:555-561. Starr, J., and E. Gold. 1970. Prevalence and duration of postnatally acquired human Cytomegalovirus infection. J. Chronic Dis. 22:603-607. Starr, J., M. D. Tolpin, H. M. Friedman, S. A. Plotkin, and H. Paucker. 1977. Immune responses in children with congenital Cytomegalovirus and their mothers. Lancet 1:1357. Ten Napel, H. H., and T. H. The. 1980. Acute Cytomegalovirus infection and the host immune response. I. Development and maintenance of Cytomegalovirus (CMV) induced in vitro lymphocyte reactivity and its relationship to the production of CMV antibodies. Clin. Exp. Immunol. 39:263-271.
