Lymphocyte Subsets and Natural Killer Cell Responses

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1 INFECTION AND IMMUNITY, May 1983, p /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Vol. 40, No. 2 Lymphocyte Subsets and Natural Killer Cell Responses During Cytomegalovirus Mononucleosis CHARLES R. RINALDO, JR.,.12* MONTO HO,1-3 WALID H. HAMOUDI,1 XI-EN GUI,1 AND ROBBIN L. DEBIASIO1 Department of Microbiology,1 University of Pittsburgh Graduate School of Public Health, and Departments ofpathology2 and Medicine,3 Presbyterian-University Hospital and University ofpittsburgh School of Medicine, Pittsburgh, Pennsylvania Received 28 December 1982/Accepted 4 February 1983 Cell-mediated immunity in seven patients with cytomegalovirus mononucleosis was assessed by monoclonal antibody to lymphocyte subsets, lymphocyte blastogenesis, and natural killer cell activity. The patients had decreased ratios of helper (Leu 3a) to suppressor-cytotoxic (Leu 2a) T cells as compared with normal donors. Leu 2a+ T cells from the patients were uniquely sensitive to overnight culturing, which resulted in a 2.6-fold increase in the helper/suppressor cell ratio and the appearance of an unusual third Leu 1+ 2a- 3a- T cell subset. This correlated directly with enhanced blastogenesis to concanavalin A after preculture. Base-line and interferon-boosted natural killer cell responses versus K562 cell targets during cytomegalovirus mononucleosis did not differ from those of normal donors. This suggests that certain cellular immune functions are depressed during cytomegalovirus mononucleosis in correlation with the emergence of a culture-sensitive subset of suppressor T cells. In contrast, cytotoxic lymphocytes appear to function normally and may aid in the resolution of the illness. Cytomegalovirus (CMV) mononucleosis is a self-limiting illness in normal, healthy individuals (14). The acute infection results in depressed cellular immune function as evidenced by decreased lymphocyte blastogenesis to mitogens and viral antigens (18, 24, 25, 29, 30) and diminished reactivity to skin test antigens (22). This immunosuppressive effect of the virus infection diminishes with time, as cells from convalescent patients gradually respond as well as do those from normal donors. Further studies have shown that decreased blastogenesis to the mitogen concanavalin A (ConA) can be abrogated by overnight preculturing of the mononuclear leukocytes at 37 C before treatment with the mitogen (3, 25). This was related to a loss of suppressor cell activity in the plastic-adherent, mononuclear leukocyte subpopulation (monocytes). Immunosuppression in patients with fungal disease (27) and neoplasia (10, 21, 32) has similarly been reported to be associated with culture-sensitive suppressor cells in peripheral blood. Recent evidence from Carney et al. (5) has shown that CMV mononucleosis patients have elevated levels of suppressor-cytotoxic T lymphocytes (OKT8+) and inverted ratios of helper to suppressor T cells (OKT4:OKT8) as compared with normal donors. We were interested in examining whether the suppressor T cells induced during CMV mononucleosis could be more directly related to functional suppressor activity by the preculture assay and the effect of preculture on cell surface markers. In the present study, we analyzed immunoregulatory T cells of CMV mononucleosis patients by in vitro culturing and immunofluorescence with monoclonal antibodies to lymphocyte differentiation antigens for evidence of unique functional and cell surface characteristics. We show that CMV infection results in the emergence of an unusual suppressor T lymphocyte subset that is not found in normal donors. We also found that natural killer (NK) cell activity is functionally normal during CMV mononucleosis, in the face of this immunosuppression. (This investigation was part of a thesis submitted by W.H.H. in partial fulfillment of the requirements for the M.S. Hyg. degree in microbiology from the Graduate School of Public Health, University of Pittsburgh. These data were presented at the 15th International Leucocyte Culture Conference, December 1982, and published in abstract form [Immunobiology 163:233, 1982].) MATERIALS AND METHODS Patients. Seven patients (three females and four males) with a mean age of 35 years (range, 20 to 57 years) were studied during the acute phase of CMV 472

2 VOL. 40, 1983 mononucleosis (mean, 22.3 days; range, 10 to 34 days after the onset of illness). All of the patients were considered to have community-acquired illness and had no recent history of blood transfusions or surgery. One of these seven patients had insulin-dependent diabetes mellitus. All of the patients had protracted fever (mean, 13.0 days; range, 10 to 15 days) and a mononucleosis with atypical lymphocytes (peak levels noted: mean, 34.3%; range, 12 to 55%) and elevated liver enzymes in peripheral blood (peak levels noted: serum aspartate aminotransferase: mean, 201 IU/liter; range, 80 to 530 IU/liter [normal values, <34 IU/liter]; alkaline phosphatase: mean, 369 IU/liter; range, 115 to 1,065 IU/liter [normal values, '100 IU/liter]). Four of these patients were also studied during convalescence (mean, 162 days; range, 87 to 279 days after the onset of illness) when they were clinically asymptomatic. Control donors were three females and seven males with a mean age of 28.4 years (range, 22 to 51 years). Viral cultures. Urine, throat washings, and peripheral blood mononuclear and polymorphonuclear leukocyte-rich fractions were cultured on human foreskin cells (originated from primary tissue obtained from MaGee Women's Hospital, Pittsburgh, Pa.; passage 9 to 30) in medium 199 (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 2 mm glutamine, 250 U of penicillin per ml, 250 plg of streptomycin per ml, g of NaHCO3 per ml, and 10lo heat-inactivated (56 C for 30 min) fetal calf serum (MA Bioproducts, Bethesda, Md.) at 37 C in 5% C02-humidified atmosphere. Cultures were observed for characteristic CMV-mediated cytopathic effect as previously described (24). Lymphocyte blastogenesis assay. Venous blood anticoagulated with heparin (15 U/ml) was separated on Isolymph gradients (Gallard-Schlesinger Corp., Carle Place, N.Y.) into mononuclear and polymorphonuclear leukocyte-rich fractions (1). Mononuclear leukocytes averaged 89%o lymphocytes (including atypical lymphocytes in the patient samples), 8% monocytes, and 3% polymorphonuclear leukocytes by Giemsa and esterase staining and averaged 92% viable by trypan blue dye exclusion. Mononuclear leukocytes were diluted to a concentration of 1.5 x 106 viable cells per ml in medium 199 with the standard supplements and 10%o human AB' serum (negative for CMV antibody) instead of fetal calf serum. The cells were placed into flat-bottom microwells (3 x 105 viable cells in 0.2 ml; 0.32-cm2 wells; Costar, Cambridge, Mass.) and replicate samples were treated with 25,ug of ConA (crystallized three times; Miles Laboratories, Elkhart, Ind.) that was known to induce maximal blastogenesis in lymphocytes from normal donors. Lymphocyte blastogenesis was measured at the time of peak incorporation on day 4 by the addition of 1 ACi of [methyl- 3H]thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear Corp., Boston, Mass.) 4 h before harvesting. Results were expressed as counts per minute (cpm) ± the standard error of the mean (SEM) per sample and as the difference in cpm (Acpm) between untreated and mitogen-stimulated cultures. The blastogenic responses were analyzed for statistical significance by comparison of the mean Acpm among patient and control samples by Student's t test. Assay of suppressor cell function. Mononuclear leukocytes were assayed for suppressor function by the preliminary culturing method (25). Mononuclear cells SUPPRESSOR AND NK CELLS IN CMV MONONUCLEOSIS 473 from freshly donated blood were cultured for 18 h in medium o human AB' serum at 37 C in 5% CO2 and humidity (3 x 105 viable cells per microwell). Nonadherent and plastic-adherent cells were washed three times each with medium 199. The total recovery of cells after preculture averaged 55.7 ± 6.5% (72.4% viable) for acute-phase patients, 65.8 ± 7.3% (91.7% viable) for convalescent-phase patients, and 58.5 ± 5.1% (84.2% viable) for normal donors. The nonadherent cells were 98% lymphocytes, 2% monocytes, and -l% polymorphonuclear leukocytes. The precultured, nonadherent cells were added to microwells (3 x 10' viable cells per well) containing the washed, precultured adherent cells. The samples were then assayed for lymphocyte blastogenesis to ConA in the same manner as the freshly donated cells. T lymphocyte identification. T lymphocytes in freshly donated and precultured mononuclear cells were enumerated by direct immunofluorescence with fluorescein-conjugated monoclonal antibodies anti-leu 1 (8) and -Leu 4 (16) (total, pan T cells), anti-leu 2a (suppressor-cytotoxic T cells) (7, 9), and anti-leu 3a (helper-inducer T cells) (7, 9) (Becton, Dickinson & Co., Sunnyvale, Calif.) with a fluorescence-activated cell sorter (FACS IV; Becton Dickinson). A 5SO-l amount of cell suspension (2.5 x 105 to 5 x 105 cells) in medium 199 without serum was mixed with 50,ul (1,ug) of the appropriate monoclonal antibody in roundbottom microwells (Linbro Scientific, McLean, Va.). The cell mixtures were incubated on ice for 30 min, with agitation at 10-min intervals. After incubation, 100 p.l of salt solution was added to the wells, and the plates were centrifuged at 75 x g for 3 min at 4 C. The cells were washed three times with 100 ILI of salt solution by centrifugation at 75 x g for 3 min at 4 C. Results were expressed as the percent lymphocyte subtype (±SEM) per total amount of viable cells counted. The data were analyzed for statistical significance by a comparison of the mean percent lymphocyte subtypes among patient and control samples by Student's t test. NK cell assay. K562 cells, a myeloerythroid cell line (19) obtained from G. V. Quinnan, were used as targets in the NK cell assay. The cells were cultured in medium RPMI 1640 (GIBCO) with standard supplements and 1O0o fetal calf serum. The cells were negative for mycoplasmal contamination by culture on standard mycoplasma media and by stain with DNAbinding dye. Immediately before use in the assay, 1 x 106 target cells were labeled with 50 p.ci of 51Cr (Na2-51CrO4; New England Nuclear Corp.), incubated for 1 h at 37 C, washed three times with serum-free medium, and suspended to 5 x 104 cells per ml in complete medium. A total of 100 p.l containing 5 x 103 labeled target cells was added in triplicate to round-bottom microwells (Linbro Scientific, Hamden, Conn.). Effector cells were added in 100-pAl volumes to each well to give effector-to-target (E:T) cell ratios of 50:1, 25:1, and 12.5:1. Wells containing either target cells and medium or target cells and 0.5% Triton X-100 (Packard Instrument Co., Downers Grove, Ill.) were included to obtain values for spontaneous 51Cr release and total lysis. After 16 to 18 h of incubation at 37 C in a humidified 5% CO2 incubator, the supernatant fluid in each well was harvested with a supernatant collection system (Titertek; Flow Laboratories, McLean, Va.).

3 474 RINALDO ET AL. Samples were counted in a gamma counter (Packard Instrument Co.). Spontaneous release averaged 25%. Cytotoxicity from NK cell activity was expressed as the percent specific release of 51Cr or % specific release = 5tCr release from wells with effector cells - spontaneous release total release - spontaneous release x 100 To determine the effect of in vitro incubation with interferon on natural cytotoxicity, we added 500 U of human leukocyte interferon (lot M-76 P-IF; K. Cantell, Helsinki, Finland) to 2.5 x 106 to 5 x 106 effector cells (12). Interferon-treated and untreated cells were incubated at 37 C for 2.5 h. They were then washed three times, counted, and suspended to an appropriate concentration of viable cells in complete medium before the assay of cytotoxicity. The difference in cytotoxicity (percent specific release) between the sample incubated with interferon and the control represented enhancement. Student's t test was used for the calculation of significance. TABLE 1. RESULTS Virus isolation. CMV was isolated from the polymorphonuclear leukocyte-rich fraction of peripheral blood from all seven patients during their acute illness. Virus was not isolated from the mononuclear leukocyte-rich fraction of any patient during acute CMV mononucleosis. CMV was isolated from three of six throat washes and four of eight urines tested. Virus was not isolated from the blood of the four patients studied during the convalescent phase of CMV mononucleosis. During convalescence, virus was present in the throat wash of one of the four patients (87 days after the onset) and in the urine of all four patients tested. Response of mononuclear leukocytes to ConA. Mononuclear leukocytes from acute-phase CMV mononucleosis patients displayed significantly depressed blastogenic reactivity (Table 1) in response to ConA as compared with convalescent-phase patients (P < 0.05) or normal donors (P < 0.005). During convalescence, the response to ConA was similar to that of normal donors. Preliminary culture resulted in a 2.5-fold enhancement of the blastogenic response to ConA during acute CMV mononucleosis (P < 0.06, freshly donated compared with precultured cells). This preculture effect was less evident in the patients during convalescence. It should be noted that the one patient with diabetes mellitus was similar to the other patients in that the lymphocyte blastogenic response to ConA during convalescence was not significantly different from normal or enhanced by preculture. These data suggest that the depressed response to ConA during acute CMV mononucleosis is related to a suppressor effect that is eliminated by culture for 18 h at 370C (25). Alteration of lymphocyte subsets during CMV mononucleosis. Studies were done to delineate the role of T and B lymphocytes in immunosuppression during CMV mononucleosis. Our initial experiments showed that the total percentage of peripheral blood T lymphocytes as identified by active E rosettes (13) was elevated during acute CMV mononucleosis (74.0 ± 1.6% in five patients, 66.1 ± 2.1% in 26 controls; P < 0.01). B cell levels in these patients were equivalent to those of normal controls as demonstrated by T lymphocyte subsets and response to ConA before and after preculture INFECT. IMMUN. Mean % positively stained cells (±SEM) Leu 3a/ ConA Donor Cells Leu 2a response Leu 1 Leu 4 Leu 2a Leu 3a (±SEM) (Scpm)- SEM) Acute CMV Before preculture ,447 mononucleosis (±4.0) (±3.5) (±4.6) (±3.6) (±0.1) (±14,977) patients (n = 6) (n = 3) (n = 6) (n = 6) (n = 6) (n = 6) After preculture ,263 (±8.3) (±5.2) (±2.5) (±0.4) (±20,201) (n = 4) (n = 1) (n = 5) (n = 3) (n = 3) (n = 6) Convalescent CMV Before preculture ,827 mononucleosis (±9.8) (±4.4) (±6.6) (±2.7) (±0.3) (±18,792) patients (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) After preculture ,426 (±5.0) (±5.0) (±4.7) (±6.0) (±0.7) (±27,957) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) Normal donors Before preculture ,169 (±1.4) (±1.6) (±2.0) (±1.9) (±0.3) (+13,823) (n = 10) (n = 9) (n = 10) (n = 10) (n = 10) (n = 10) After preculture ,504 (±2.2) (±1.9) (+1.5) (±2.4) (±0.2) (+15,034) (n = 10) (n = 9) (n = 10) (n = 10) (n = 10) (n = 10)

4 VOL. 40, 1983 rosetting with immunoglobulin-coated polyacrylamide beads (6) (12.5 ± 1.3% in five patients, 12.2 ± 0.8% in 20 controls). Further studies with monoclonal antibodies to T cell differentiation antigens confirmed that there was a significant increase in the percentage of T (Leu 1 + and Leu 4+) cells during acute CMV mononucleosis as compared with normal donors (P < 0.001) (Table 1). The total increase in T lymphocytes during CMV mononucleosis was accompanied by an inversion of the normal ratio of helper to suppressor T cells (Leu 3a/Leu 2a). This was due to a nearly three-fold increase in suppressor Leu 2a+ T cells, with a concurrent two-fold decrease in helper Leu 3a+ T cells as compared with normal donors (P < 0.001). Levels of T lymphocytes remained elevated during convalescence, although the percentages of T suppressor and helper cells were approaching normal levels (P < for suppressor T cells, P, not significant for total T or helper T cells). Thus, the enhanced immunosuppression during CMV mononucleosis correlates with increased levels of T cells and an inverted ratio of T helper to T suppressor cells, in agreement with the previous report of Carney et al. (5), using the OKT series of monoclonal antibodies. Of greater significance is that we noted a direct correlation between enhanced blastogenesis to ConA after preculture and a selective loss of Leu 2a+ suppressor T cells (Table 1). The percentage of Leu 2a+ cells from acute CMV mononucleosis patients decreased 2.3-fold after preculture (P < 0.001). The percentage of Leu 3a+ helper T cells remained stable after preculture, resulting in a 2.6-fold increase in the ratio of helper to suppressor T cells in cultures from acute CMV mononucleosis patients. Interestingly, an unusual third subset of Leu 1+ T cells emerged after the 18-h incubation in TABLE 2. SUPPRESSOR AND NK CELLS IN CMV MONONUCLEOSIS 475 cell cultures from acute CMV mononucleosis patients. Approximately 33% of Leu 1+ T cells did not label with anti-leu 2a or anti-leu 3a after preliminary culture, i.e., were Leu 1+ 2a- 3a-. A similar but less pronounced decrease in Leu 2a+ cells after preculture (1.5-fold; P, not significant), an increase in the Leu 3a/Leu 2a ratio, and the appearance of Leu 1+ 2a- 3acells after preculture were observed during the convalescent phase of CMV mononucleosis. However, the preculture of normal donor cells did not result in any of these alterations in T lymphocyte subsets. NK cell responses. NK cell activity during CMV mononucleosis did not differ from that noted in normal donors (Table 2). The cells were responsive to treatment with interferon, with a comparable significant enhancement of cytotoxic reactivity. No relationship was found between base-line or interferon-enhanced NK cell responses and elapsed time after the onset of symptoms. DISCUSSION CMV mononucleosis patients had increased amounts of circulating T lymphocytes as shown by both E-rosetting and immunofluorescence with anti-pan T cell monoclonal antibodies Leu 1 and Leu 4. The level of immunoglobulin-bearing B lymphocytes in the patients was equal to that of normal donors. As in normal donors, freshly donated T lymphocytes from CMV mononucleosis patients consisted of suppressor/cytotoxic (Leu 2a+) and helper (Leu 3a+) T cell subsets. However, the percentage of suppressor T (Leu 2a+) lymphocytes in the acute-phase patients was nearly threefold above normal, with a concurrent, twofold decrease in helper T (Leu 3a+) lymphocytes. These data are in agreement with those of Carney et al. (5), who reported similar NK cell response during CMV mononucleosis Mean % specific release (±SEM) Subjects No.' Untreatedb Interferon-treatedb 50:1 25:1 12.5:1 50:1 25:1 12.5:1 Acute CMV mononucleosis 8 (7) patients (±3.2) (±5.5) (±5.7) (±4.2) (±4.4) (±4.6) Convalescent CMV mononucleosis 4 (4) patients (±10.9) (±7.8) (±8.9) (±8.7) (±8.0) (±11.4) Normal donors 16 (11) (±6.5) (±4.4) (±4.7) (±5.0) (±4.4) (±4.5) a Number of samples (number of donors). b The values for interferon-treated samples from acute CMV mononucleosis patients and normal donors were significantly higher than those for untreated samples at 50:1 and 25:1 ratios (P < 0.05) and 12.5:1 (P < 0.05 for acute-phase patients only). Values for untreated and interferon-treated samples from convalescent patients were not significantly different. Values for each group of subjects (untreated and interferon treated) were not significantly different. I Effector target cell ratio.

5 476 RINALDO ET AL. alterations in T cell levels during CMV mononucleosis with the OKT series of monoclonal antibodies. We found that suppressor Leu 2a+ T lymphocytes induced during CMV mononucleosis are sensitive to culture at 37 C for 18 h. The percentage of helper Leu 3a+ T cells in the patients was similar before and after preculture, resulting in a greater than twofold increase in the Leu 3a/Leu 2a cell ratio. This directly correlated with enhanced lymphocyte blastogenesis to the polyclonal activator ConA after preculture. Suppressor T lymphocytes of CMV mononucleosis patients were still sensitive to preliminary culture during convalescence. The proportions of normal-donor T cell subsets were not altered by preculture. After the submission of this manuscript, other investigators reported that Leu 2a-equivalent OKT8+ suppressor T cells from acute CMV mononucleosis patients were selectively sensitive to in vitro culture for 7 days (4). They also noted that freshly donated OKT4 (helper)-depleted cells were unresponsive to ConA, whereas OKT8-depleted cells were responsive to the mitogen. Our results further indicate that a new T cell subset emerges after the preculture of mononuclear leukocytes from CMV mononucleosis patients. This new subset did not bear either suppressor/cytotoxic (Leu 2a) or helper (Leu 3a) surface markers detectable by immunofluorescence and flow cytometry. These Leu 1+ 2a- 3a- cells comprised approximately one-third of the T cells after preculture during acute CMV mononucleosis. Monoclonal antibody to Leu 1 (8, 15), but not to Leu 4 (16), has been reported to occasionally react with some B cells in normal lymphoid and splenic tissue and immunoglobulin-bearing cells in chronic lymphocytic leukemia. Therefore, it is possible that the Leu 1 + 2a- 3a- cells found after preculture in our studies are B cells. However, our preliminary results with anti-leu 4 support the conclusion that these are T lymphocytes (Table 1). The alteration of Leu 2a+ cells during in vitro culture was apparently not related to direct infection with CMV, as infectious virus was not isolated from mononuclear cells. We have also recently shown that the specific blastogenesis to viral antigens during CMV mononucleosis is suppressed and is not enhanced after preculture (C. R. Rinaldo, Jr. and R. L. DeBiasio, Clin. Immunol. Immunopathol., in press). This would suggest that factors in addition to culture-sensitive, Leu 2a-expressing cells are operative in the immunoregulation of certain lymphocyte functions during CMV infection. Suppressor T cells are thought to consist of subtypes that interact to suppress immune func- INFECT. IMMUN. tion (2). For example, recent evidence has indicated that at least two phenotypically distinct subsets of suppressor cells (Leu 2a+ 8- and 2a+ 8+) are necessary to produce the suppression of Leu 3a-induced immunoglobulin synthesis in normal individuals (11). The increased numbers of suppressor T cells noted during CMV mononucleosis may represent a significant expansion of such a subset of Leu 2a+ cells present in normal donors. These immunoregulatory T cells may interact with culture-sensitive monocytes (3, 25) to suppress blastogenesis to ConA. The interaction of suppressor T cells and monocytes has previously been related to a similar suppression of blastogenesis found during fungal infection (26). Normal subjects have significant circulating NK cell activity, which can be further enhanced by in vitro incubation with interferon (31). Interestingly, the patients with CMV mononucleosis and normal healthy subjects had comparable mean base-line NK cell activity and enhancement after incubation with interferon. Our results confirm those of Lemon et al. (17), who found high levels of NK cytotoxicity against B lymphoblastoid targets in a patient with acute CMV mononucleosis. Similarly, patients with Epstein-Barr virus mononucleosis have relatively normal NK cell responses (28). However, the NK cell activity of mice infected with murine CMV is transiently elevated 2 to 3 days after infection (23), but the enhancement of NK cell activity by in vitro incubation with interferon is suppressed 6 to 9 days after acute infection (20). It is possible that both changes may have been missed in our studies as patients were not seen earlier than 10 days after the onset of symptoms. More important, however, is our finding that circulating NK cell activity persists in the face of the significant immunosuppression that develops during CMV mononucleosis. This suggests a role for the NK cell response in the resolution of this normally self-limiting illness. ACKNOWLEDGMENTS We thank A. McCoy, L. Youngblood, and M. Lassak for technical assistance, J. Fossati for secretarial assistance, and E. Merrall and B. Rabin for aid in the monoclonal antibody analysis. We are grateful to R. Myerowitz, N. Rao, and other physicians for referrals of CMV mononucleosis patients. This work was supported in part by grant AI from the National Institutes of Health and the Pathology Education Research Fund from the Department of Pathology, University of Pittsburgh. LITERATURE CITED 1. Boyum, A Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 4: Cantor, H., and E. A. Boyse Regulation of cellular and humoral immunity by T cell subclasses. Cold Spring Harbor Symp. Quant. Biol. 41: Carney, W. P., and M. S. Hirsch Mechanisms of

6 VOL. 40, 1983 SUPPRESSOR AND NK CELLS IN CMV MONONUCLEOSIS 477 immunosuppression in cytomegalovirus mononucleosis. II. Virus-monocyte interactions. J. Infect. Dis. 144: Carney, W. P., V. lacoviello, and M. S. Hirsch Functional properties of T lymphocytes and their subsets in cytomegalovirus mononucleosis. J. Immunol. 130: Carney, W. P., R. H. Rubin, R. A. Hoffman, W. P. Hansen, K. Healey, and M. S. Hirsch Analysis of T lymphocyte subsets in cytomegalovirus mononucleosis. J. Immunol. 126: Chao, W., and M. M. Yokoyama Determination of B lymphocyte population using antibody-coated polyacrylamide beads. Clin. Chim. Acta 78: Engleman, E. G., C. J. Benike, E. GUckman, and R. L. Evans Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction of man. J. Exp. Med. 153: Engleman, E. G., R. Warnke, R. I. Fox, J. DiLey, C. J. Benike, and R. Levy Studies of a human T lymphocyte antigen recognized by a monoclonal antibody. Proc. Natl. Acad. Sci. U.S.A. 78: Evans, R. L., D. W. Wall, C. D. Platsoucas, F. P. Siegal, S. M. Flkrig, C. M. Testa, and R. A. Good Thymus-dependent membrane antigens in man: inhibition of cell mediated lympholysis by monoclonal antibodies to the TH2 antigen. Proc. Natl. Acad. Sci. U.S.A. 78: Fuks, Z., S. Strober, D. P. King, and H. S. Kaplan Reversal of cell surface abnormalities of T lymphocytes in Hodgkin's disease after in vitro incubation in fetal sera. J. Immunol. 117: Gatenby, P. A., G. S. Kansas, C. Y. Xian, R. L. Evans, and E. G. Engleman Dissection of immunoregulatory subpopulations of T lymphocytes within the helper and suppressor sublineages in man. J. Immunol. 129: Ho, M., L. T. White, H. W. Haverkos, M. C. Brehnig, G. J. Pazin, and J. A. Armstrong Depression of natural killer cell activity by interferon. J. Infect. Dis. 144: Kaplan, M. E., M. Woodson, and C. Clark Detection of human T lymphocytes by rosette formation with AET-treated sheep red blood cells, p In B. R. Bloom and J. R. David (ed.), In vitro methods in cellmediated and tumor immunity. Academic Press, Inc., New York. 14. Klemola, E., and L. KUirinen Cytomegalovirus as a possible cause of a disease resembling infectious mononucleosis. Br. Med. J. 2: Ledbetter, J. A., R. L. Evans, M. Lipinski, C. Cunningham-Rundles, R. A. Good, and L. A. Herzenberg Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/ suppressor subpopulations in mouse and man. J. Exp. Med. 153: Ledbetter, J. A., A. E. Frankel, L. A. Herzenberg, and L. A. Herzenberg Human Leu T cell differentiation antigens: quantitative expression on normal lymphoid cells and cell lines, p In G. J. Hammerling, U. Hammerling, and J. F. Kearney (ed.), Monoclonal antibodies and T cell hybridomas. Elsevier/North-Holland, Publishing Co., New York. 17. Lemon, S. M., L. M. Hutt, and Y. T. Huang Cytotoxicity of circulating leukocytes in cytomegalovirus mononucleosis. Clin. Immunol. Immunopathol. 8: Levin, M. J., C. R. Rinaldo, Jr., P. L. Leary, J. A. Zaia, and M. S. Hirsch Immune response to herpesvirus antigens in adults with cytomegaloviral mononucleosis. J. Infect. Dis. 140: Lozzlo, C. R., and B. B. Lozzlo Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45: Melder, R. J., and M. Ho Modulation of natural killer cell activity in mice after interferon induction: depression of in vitro enhancement by interferon. Infect. Immun. 36: Moghe, M. V., S. H. Advani, and S. G. Gangal Demonstration of inhibitory factors affecting cell-mediated immunity in patients with Hodgkin's disease. Eur. J. Cancer 16: Oill, P. A., M. Flala, J. Schofferman, P. E. Byfield, and L. B. Guze Cytomegalovirus mononucleosis in a healthy adult. Association with hepatitis, secondary Epstein-Barr virus antibody response and immunosuppression. Am. J. Med. 62: Quinnan, G. V., J. E. Manischewitz, and F. A. Ennis Cytotoxic T lymphocyte response to murine cytomegalovirus infection. Nature (London) 273: Rinaldo, C. R., Jr., P. H. Black, and M. S. Hirsch Interaction of cytomegalovirus with leukocytes from patients with mononucleosis due to cytomegalovirus. J. Infect. Dis. 136: Rinaldo, C. R., Jr., W. P. Carney, B. S. Richter, P. H. Black, and M. S. Hirsch Mechanisms of immunosuppression in cytomegaloviral mononucleosis. J. Infect. Dis. 141: Stobo, J. D Immunosuppression in man: suppression by macrophages can be mediated by interactions with regulatory T cells. J. Immunol. 119: Stobo, J. D., S. Paul, R. E. Van Scoy, and P. E. Hermans Suppressor thymus-derived lymphocytes in fungal infection. J. Clin. Invest. 57: Svedmyr, E., and M. Jondal Cytotoxic effector cells specific for B cell lines transformed by Epstein-Barr virus are present in patients with infectious mononucleosis. Proc. NatI. Acad. Sci. U.S.A. 77: Ten Napel, C. H. H., and T. H. The 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: Ten Napel, C. H. H., and T. H. The Acute cytomegalovirus infection and the host immune response. II. Relationship of suppressed in vitro lymphocyte reactivity to bacterial recall antigens and mitogens with the development of cytomegalovirus-induced lymphocyte reactivity. Clin. Exp. Immunol. 39: Trinchieri, G., and D. Santoli Antiviral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis. J. Exp. Med. 147: Wood, G. W., and R. A. Morantz In vitro reversal of depressed T-lymphocyte function in the peripheral blood of brain tumor patients. J. Natl. Cancer Inst. 68:27-33.