Vol. 19, No. 2

INFECrION AND IMMUNrrY, Feb. 1978, p. 486-492 0019-9567/78/0019-0486$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Friend Virus-Induced Immunodepression: Effect of Neuraminidase Treatment on Thy-1.2 Antigen Expression and Cytotoxic Potential of Splenocytes from Virus-Infected Mice AFEWORK A. MASCIOt AND WALTER S. CEGLOWSKI*

Department ofMicrobiology and Cell Biology, Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 26 July 1977

Infection of susceptible strains of mice with Friend leukemia virus (FLV) results in a profound depression of cell-mediated immunity as assessed by lymphocyte-mediated cytotoxicity. This depression occurs early in the disease, before the onset of splenomegaly, and is associated with a decline in the susceptibility of splenocytes from FLV-infected mice to lysis by anti-Thy-1.2 serum and complement. Treatment of splenocytes from FLV-infected mice with neuraminidase restores, in large part, their susceptibility to anti-Thy-1.2 serum as well as their cytolytic capacity. These studies suggest that one early immunosuppressive consequence of infection with FLV involves alteration of the effector T-lymphocyte cell surface.

In recent years experimental evidence has been presented that demonstrates that infection of susceptible strains of mice with murine leukemia viruses (MuLV's) depresses both their humoral and cellular (4, 10, 11) immune responses. Although the mechanism whereby this depression is mediated has not yet been elucidated, several aspects of the depression of humoral immune responses have been investigated, and reports of such studies suggest that Friend leukemia virus (FLV) interferes with the maturation of antibody-forming precursor cells (5), namely, derived bone marrow or B cells (2; M. J. Siegel, W. S. Ceglowski, and H. Friedman, RES J. Reticuloendothel. Soc., p. 10a, 1974). In contrast, less is known about virus-induced depression of cell-mediated immunity. Cell-mediated immunity is a function of the thymusdependent cells (T cells) and may have a role in an "immunological surveillance" mechanism directed against neoplastic cells (3). Recent studies in our laboratory have been concerned with the assessment of the effect of infection with one of the MuLV's, FLV, on cellular immune responses. One assay system, lymphocyte-mediated cytotoxicity as described by Cerottini and Brunner (9), has been widely used as a sensitive in vitro correlate of allograft rejection. In our present study, we have used this technique to extend our studies concerning the effect of MuLV infection on cytolytic activity directed against alloantigen-bearing target cells. t Present address: Department of Zoology, Drew University, Madison, NJ 07940.

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(This work was submitted by A.A.M. in partial flfillment of the requirements for the Ph.D. degree at Pennsylvania State University.) MATERLS AND METHODS Experimental animals. Inbred female adult C57B1/6 and DBA mice were obtained from Jackson Laboratories, Bar Harbor, Maine. Female BALB/c mice were purchased from either Flow Laboratories, Inc., Dublin, Va.; Cumberland View Farms, Inc., Clinton, Tenn.; or Laboratory Supply, Inc., Indianapolis, Ind. The animals were 6 to 10 weeks old at the start of an experiment. They were maintained in groups of five in plastic mouse cages and fed commercial mouse pellets and water ad libitum. FLV. FLV preparations were prepared as described previously (22). The stock virus was freed (25) of lactic dehydrogenase virus by a single passage in newborn Sprague-Dawley rats. For the present experiments, portions of stock virus were maintained at -70°C until used. This stock virus contained both the spleen focusforming virus (1) and lymphatic leukemia virus (30) components of the complex. According to the recent proposal of Steeves (29), the complete abbreviation to adequately describe this preparation would be FSFFV (MuLV-F). As a consequence of forced passage in BALB/c mice, this virus is NB tropic when assayed by the X-C method of Rowe et al. (26). The stock virus contained 4 x 102 focus-forming units per ml and 6 x lO4 plaque-forming units per ml. Target and spleen celi preparations. Mouse lymphoma cells, EL-4 from C57B1/6 (H-2b) strain mice obtained from C. S. Henney, were used throughout this study as both the sensitizing alloantigen and target cell. These cells were maintained in the ascites form in adult C57BI/6 mice and were propagated by weekly serial, intraperitoneal passage of 2 x 107 cells. Target

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cells for the in vitro assay were prepared as described by Henney (14). In brief, cells were labeled with sodium 5"chromate (New England Nuclear Corp., Boston, Mass.) at a concentration of 50 ,uCi/107 cells. The cells were then washed three times with Eagle minimal essential medium (MEM) and adjusted to 106 cells per ml in MEM containing 10% heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.). Effector spleen cell suspensions were prepared from BALB/c (H-2d) mice that had been sensitized with alloantigen-bearing EL-4 cells 11 days previously. The spleen cells were washed three times with Eagle MEM and then adjusted to 107 cells per ml in MEM containing fetal calf serum, as described in a previous report (22). The viability of these cells as determined by trypan blue exclusion was 90 to 95% (19). Lymphocyte-mediated cytotoxicity test. Labeled target cells were added to various effector spleen cell concentrations to obtain the desired ratio of effector to target cell. Tests were usually performed with 30:1 and 50:1 lymphocyte/target ratios unless stated otherwise. The total volume was adjusted to 1.0 ml, and the tubes were incubated at 37°C in a 10% CO2 atmosphere for 5 h. All tests were performed in duplicate. The tubes were centrifuged at 2,000 rpm for 5 min, and a 0.5-mi portion of the supernatant was then counted for 51Cr content on a gamma-ray spectrometer. This test is based on the assumption that the amount of 5"Cr released is proportional to target cell lysis (14). The percentage of specific cytolysis was determined as described in more detail in a previous report (22). Briefly, the results are expressed according to the following formula: observed percent specific lysis = [cpm in test - cpm spontaneous (5 h)]/[total cpm - cpm spontaneous (5 h)] x 100, where cpm = counts per minute. The spontaneous release of chromium was in the range of 5 to 10% for the 5-h incubation period. In FLV-infected animals, splenomegaly was usually observed only 8 or more days after infection. In an attempt to account for the effect of dilution of effector cells with nonfunctional leukemic cells in assays performed in animals infected for time periods greater than 8 days, the observed specific lysis was multiplied by the ratio of the number of leukocytes per spleen in the appropriate FLV-infected group to that of the noninfected controls. Preparation of anti-Thy-1.2 serum and determination of susceptibility to lysis. The anti-Thy1.2 serum used in these studies was prepared by the method of Reif and Allen (24). The antiserum was absorbed with C3H erythrocytes and then titered, using 51Cr-labeled BALB/c thymus cells as target cells (22). For use in the present studies, 0.2 ml of antiThy-1.2 serum was added to 0.2 ml of 6'Cr-labeled spleen or thymus cell suspension from either control or FLV-infected animals. The mixture was adjusted to 1.0 ml with MEM and incubated for 30 min at 37°C. A 0.1-mi amount of absorbed guinea pig complement was added, and the mixture was incubated for another 30 min. The cell suspension was centrifuged at 2,000 rpm for 5 min, and 0.5 ml of the supernatant was then counted for 51Cr content in a gamma-ray spectrometer. The percent specific lysis was calculated by subtracting the 51Cr released in the absence of antiThy-1.2 serum and complement from that released in

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the presence of anti-Thy-1.2 serum and complement and dividing the total 5'Cr minus that spontaneously released. Preparation and utilization of anti-FLV serum. Antiserum to FLV was prepared by immunizing BALB/c mice with five weekly inoculations of a Formal-treated preparation of spleen cells obtained from BALB/c mice infected with FLV 25 days previously. Seven days after the last injection, the animals were bled, and sera were separated, heat inactivated, and stored at -20°C. These vaccinated mice were immune to FLV infection because a subsequent challenge with the stock viable virus did not induce overt symptoms of leukemia over an observation period of 27 days. In addition, 0.2 ml of sera at a 1:5 dilution neutralized the infectivity of an equal volume of FLV stock virus. For use in the present study, 0.2 ml of spleen cell suspension from either control or FLV-infected mice was incubated with an equal volume of either antiFLV serum or normal mouse serum for 30 min at 370C. A 0.1-ml amount of absorbed, undiluted guinea pig complement was added, and each mixture was again incubated for 30 min. After three washes in MEM, the spleen cells were assayed for cytotoxic

potential. Neuraminidase treatment of splenocytes. Spleen cells from either normal or FLV-infected BALB/c mice were treated with Vibrio chokra neuraminidase (Calbiochem, La Jolla, Calif.). Fifty units of neuraminidase in 0.5 ml of MEM was added to 2 ml of spleen cell suspension, and the mixture was incubated for 15 min at 370C. The cells were then washed three times with MEM before determining their susceptibility to anti-Thy-1.2 serum or their cytolytic capacity.

RESULTS Effect of treatment with anti-FLV serum on effector cell funiction. Table 1 shows that, TABLE 1. Effect of anti-FL V serum on lymphocytemediated cytotoxicitya Target cell cytotoxicity (%) after treatment of splenocytes with:

Spleen cell source

Normal serum +

comple-

Anti-FLV + complement

ment

55 ± 3.2 50 ± 3.1 Control 44 ± 3.9 FLV infected 2 days 49 ± 2.9 26 ± 2.3 22 ± 2.2 FLV infected 5 days 12 ± 2.1 2.9 ± 1.3b FLV infected 10 days a Alloantigen-sensitized spleen cells from either control or FLV-infected BALB/c mice were exposed to either normal or FLV-immune BALB/c serum and complement before assessing their cytolytic potential against alloantigen-bearing EL-4 target cells. All assays were performned at 11 days post-sensitization. In the experimental group, duration of infection of 2, 5, and 10 days corresponds to infection with FLV at 9, 6, and 1 days post-sensitization, respectively. b Significant P < 0.05.

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anticipated, treatment of control-sensitized spleen cells with anti-FLV serum and complement did not reduce their cytolytic potential. Similar treatment of sensitized spleen cells from mice that had been infected either 2 or 5 days before assay also induced no reduction in cytolytic activity. This was observed even in mice infected 5 days before assay that exhibited levels of specific lysis only one-half that of the noninfected controls. Only in the case of animals infected 10 days before assay was a significant reduction in cytolytic potential observed after treatment with anti-FLV serum and complement. The experimental design employed does not permit discrimination between viral synthesis in the effector cell population and passive adsorption of the virus to the effector cell surface at 10 days postinfection. Expression of Thy-1.2 antigen on spleen and thymus celis from FLV-infected and control mice. The Thy-1.2 antigen is the most convenient cell surface marker that identified the effector cell population. To assess possible changes in the display of this marker as a consequence of infection, the susceptibility of splenic and thymic lymphocytes to lysis by antiThy-1.2 serum and complement was determined. For this purpose, appropriate cell suspensions from control and FLV-infected mice were labeled with 5'Cr, treated with antiserum and complement and assayed in the 5"Cr-release assay. The values of percent lysis that were observed after assessment of chromium release are shown in Table 2. It is clear that the susceptibility of spleen cells to lysis by anti-Thy-1.2 serum decreased as the time interval after infection with FLV increased. The data are presented as the actual percent lysis observed and the percent lysis calculated to compensate for the dilution effect due to non-Thy-1.2-bearing neoplastic cells. In contrast to our observations of spleen as

cells, no decrease in susceptibility to anti-Thy1.2 serum was observed with thymus cells from

mice infected with FLV for either 5 or 10 days. However, a decrease in susceptibility of thymus cells to anti-Thy-1.2 serum was noted when cells from animals in which 18 days had elapsed between infection and assay were studied. Effect of neuraminidase treatment on susceptibility of splenocytes to anti-Thy1.2 serum. The decrease in the susceptibility to lysis by anti-Thy-1.2 serum observed in the FLV-infected mice may have reflected a decline in the absolute number of Thy-1.2-bearing cells in the spleen of an FLV-infected animal. An alternative proposal would be that the Thy-1.2 antigen site is altered, masked, or distorted such that an effective interaction with the Thy-1.2specific antibody cannot occur. Studies in a number of laboratories (12, 13, 27, 28, 32) have demonstrated that appropriate treatment of cells with the enzyme neuraminidase can enhance the antigen display and immunogenicity of such cells as well as their immunological responsiveness. To determine if neuramindase treatment would alter the display of Thy-1.2 antigen, spleen cells from control and FLV-infected mice were assessed for susceptibility to anti-Thy-1.2 serum both before and after treatment with neuraminidase. The data shown in Table 3 demonstrate that neuraminidase treatment restores in large measure the susceptibility of spleen cells from FLV-infected mice to lysis by anti-Thy-1.2 serum (Table 3). For example, the percent lysis of control spleen cells increased from 31 to 39% after neuraminidase treatment, whereas similar treatment of spleen cells from FLV-infected mice resulted in an increase in lysis from 10 to 29%. Comparison of these values for anti-Thy-1.2-induced lysis after neuraminidase treatment of the FLV-infected group reveals a level of susceptibility to anti-

TABLE 2. Susceptibility of spleen and thymus cells to anti-Thy-1.2 serum and complementa Spleen cells

Expt group

% Lysis ± SEb

Thymus cells

% of Control

Observed

Calculated

% Lysis ± SEb

% of Control observed

Control 49 ± 3.3 100 100 78 ± 2.3 100 FLV infected 5 days 33 ± 3.1 60 67 75 ± 1.9 96 FLV infected 10 days 32 ± 2.9 17 65 73 ± 2.1 94 FLV infected 18 days 20 ± 3.1 7 41 59 ± 1.7 76 a 51Cr-labeled spleen or thymus cells (2 x 106) were added to 0.2 ml of (1:6 dilution) anti-Thy-1.2 serum. Undiluted guinea pig complement was then added and percent specific lysis was computed based on the release of 5'Cr. To compensate for the FLV-induced splenomegaly, the "calculated" values were obtained by multiplying the observed percent lysis by the ratio of the average number of splenic leukocytes (FLV-infected group)/average number of splenic leukocytes (control group). Because changes in total thymocyte counts were not observed in the FLV-infected groups, no calculations were required. b SE, Standard error.

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Thy-1.2 serum approximating 75% of the control values (29 versus 39%). In an extension of these studies, spleen cells have been treated with anti-Thy-1.2 serum and complement before exposure to neuraminidase. The generation of additional cells susceptible to anti-Thy-1.2 serum was then assessed by reexposing these cells to anti-Thy-1.2 serum and complement. Table 3 shows that treatment of spleen cells with neuraminidase before reexposure to anti-Thy-1.2 serum and complement generates additional cells expressing the Thy-1.2 antigen. This increase is considerably greater in the case of the FLV-infected spleen cells than in the controls (17 versus 7%). Effect of neuramiidase treatment on subsequent lymphocyte-mediated cytolysis. Because neuraminidase treatment appeared to increase the susceptibility of splenocytes to

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antiserum against the identifying effector cell marker (Thy-1.2), additional studies were performed to assess the effect of neuraminidase treatment on subsequent cytotoxic capacity. Table 4 presents the results of these studies. Untreated alloantigen-sensitized spleen cells from FLV-infected mice exhibit less than half the cytotoxic potential of their appropriate noninfected controls (24 versus 59%). As expected, treatment of each of these cell preparations with anti-Thy-1.2 serum and complement resulted in an abolition of cytotoxic activity. Treatment of the residual cells with neuraminidase in the case of the controls resulted in a slight increase (from 3 up to 6%) in their cytotoxic capacity, whereas similar treatment of splenocytes from FLV-infected mice resulted in a substantial increase (from 3 up to 22%) in cytotoxic capacity. This increase in the cytotoxic potential of spleen cells

TABLE 3. Effect of neuraminidase treatment of spleen cells on subsequent susceptibility to anti-Thy-1.2 serum % Lysis Treatmenta Spleen cell source 31 ± 2.3 Anti-Thy-1.2 serum + complement Control 7 ± 1.5 Anti-Thy-1.2 serum + complement, followed by neuraminiControl dase treatment, followed by anti-Thy-1.2 serum + complement 39 ± 1.9 Neuraminidase treatment followed by anti-Thy-1.2 serum + Control complement 10 ± 1.5 Anti-Thy-1.2 serum + complement FLV infected 5 days 17 ± 1.8 Anti-Thy-1.2 serum + complement, followed by neuraminiFLV infected 5 days dase treatment, followed by anti-Thy-1.2 serum + complement 29 ± 2.1 Neuraminidase treatment, followed by anti-Thy-1.2 serum FLV infected 5 days + complement

aSpleen cells from control and FLV-infected mice were labeled with 61Cr and subsequently exposed to antiThy-1.2 serum and complement. After assay for 5'Cr release, the residual unlysed cells were treated with neuraminidase before reexposure to anti-Thy-1.2 serum and complement. The additional 5"Cr released into the supernatant was then assessed. In addition, 61Cr-labeled spleen cells from control and FLV-infected mice were treated with neuraminidase before exposure to anti-Thy-1.2 serum and complement. The total 61Cr released into the supernatant was determined and the percent lysis was calculated. TABLE 4. Effect of neuraminidase treatment of spleen cells on subsequent lymphocyte-mediated cytotoxicity Treatmenta

Spleen cell source

Control

None

Control Control

Anti-Thy-1.2 serum + complement Anti-Thy-1.2 serum + complement, followed by neuramini-

Control

dase treatment Neuraminidase treatment

FLV infected 5 days FLV infected 5 days FLV infected 5 days

None

Anti-Thy 1.2 serum + complement Anti-Thy-1.2 serum + complement, followed by neuramini-

% Specific target 59 ± 3.5

3 ± 1.5 6 ± 2.1 70 ± 2.9

24 ± 2.5 3 ± 1.3 22 ± 2.5

dase treatment 52 ± 3.1 Neuraminidase treatment FLV infected 5 days a Spleen cells from alloantigen-sensitized control and FLV-infected mice were assayed for their cytotoxic potential against alloantigen-bearing target cells. Portions of the cell preparations were treated with anti-Thy1.2 serum and complement and assayed immediately or after neuraminidase treatment. Additional spleen cells were assayed for their cytotoxic capacity after exposure to neuraminidase.

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from FLV-infected mice can also be demonstrated by a single treatment of the cell suspension with neuraminidase. In this case the cytotoxic potential is increased from 24 up to 52%. This enhanced degree (52%) of lysis approaches that of both the untreated (59%) and neuraminidase-treated controls (70%). In contrast, treatment of the target cell population with neuraminidase did not appreciably alter the level of cytotoxic activity of spleen cells from either control or FLV-infected animals (data not shown). Treatment of spleen cells from FLV-infected animals with trypsin or lipase did not increase either susceptibility to anti-Thy-1.2 serum or cytotoxic potential (data not shown). DISCUSSION

These studies confirm and extend our previous observations concerning the effect of infection with FLV on several parameters of cellmediated immunity (4, 22). It is clear that infection of susceptible strains of mice with FLV leads to a marked depression in cell-mediated immune competence early after infection. This depression of cell-mediated immune function appears not to be mediated by a direct MuLV-effector cell interaction. This lack of a direct interaction contrasts with studies reported for humoral immunity, in which case it is clear that MuLV can interact with antibody-forming cells. For example, Koo et al. (17) examined individual plaque-forming cells obtained from FLV-infected, sheep erythrocyte-immunized mice and observed budding C-type virus particles in 55% of such cells. In addition, Oshiro et al. (23) examined lymphoid cells obtained from rats immunized with apoferritin after infection with Moloney leukemia virus and observed individual cells producing both Moloney leukemia virus and anti-apoferritin antibodies. In a related study, Celada and Asjo (6) demonstrated the presence of viral antigens on hemolysin-producing cells by the treatment of spleen cells from MuLV-M-infected mice with anti-MuLV-M serum and complement. If one can apply and extend this approach to the study of cells involved in the lymphocyte-mediated cytotoxicity reaction, we interpret our observations with FLV-antiserum-treated spleen cells as indicating no direct association of FLV with the specific effector cell population (early after infection). It is acknowledged that this interpretation assumes the presence of antibodies in our antiserum to the entire range of viral antigens. Appropriate tests suggest the presence of antibodies to the gp7O envelope antigen and the P60 component. Unequivocal evidence for the presence of antibody activity directed against other viral

INFECT. IMMUN.

antigens would require additional appropriate serological tests. The observation of a reduction in the susceptibility of spleen cells to lysis by anti-Thy-1.2 serum and complement during development of disease parallels studies (8) using immunofluorescent techniques. In an additional related study, Kateley et al. (16) demonstrated an altered expression of immunoglobulin receptors and capping patterns in spleen cells as a consequence of infection with FLV. These studies, although demonstrating generalized alterations in splenocyte cell surface activity or display, do not specifically demonstrate an alteration in a particular subpopulation that constitutes the effector cells. In the present study, the decline in Thy-1.2antigen expression of spleen cells appeared early after infection and continued with increasing time after infection. In contrast to the observation with splenic lymphocytes, thymic lymphocytes remained susceptible to anti-Thy-1.2 serum treatment for at least 10 days after infection, at which time cell-mediated immunity, as measured by lymphocyte-mediated cytotoxicity, was significantly compromised. However, thymus cells from mice infected 18 days previously exhibited a decline in the Thy-1.2 marker. This is consistent with the observations of a previous (Siegel et al., RES J. Reticuloendothel. Soc., p. 10a, 1974) study in which T-helper cell activity was indeed compromised late (18 days) after infection but not early after infection (2 days). However, in that study, at no time was virus isolated from thymus cells of such infected mice (6). This latter observation is consistent with that of Cerny et al. (7) regarding a lack of Friend virus membrane antigen in thymus cells of FLVinfected mice. The sequence of events between the initial effector-target interaction and the lysis of the target cell has been the subject of considerable study (15, 20, 21, 31). Recent studies suggest that adhesions are formed between the effector cell and target cell and that a short but finite period of contact is required in which the target cell becomes programmed for lysis. This programming event is followed by the lytic event, which does not require continued effector-target cell contact. Although leukemia virus-induced inhibition of cell-mediated lysis could occur at any of the steps described above, the observations of altered reactivity to anti-Thy-1.2 serum suggested a possible alteration in the configuration of the effector cell surface which might impede or block "effective" contact with the target cell. In addition, Kuppers and Henney (18) have recently demonstrated that there is a direct link-

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age between antigen recognition and lytic expression in the effector cell population. Any alteration that changed either the configuration of the antigen receptor on the effector cell surface or masked the expression of the antigen receptor would prevent effector-target cell interaction with subsequent, profound effects on cytolytic activitv. Treatment of cells with neuraminidase has led to the enhancement of tumor cell imrmunogenicity (28), alteration of serological reactivity of normal and transformed lymphoid cells (27, 32), as well as the enhancement of the responsiveness of lymphoid cells to stimulation by mitogens (13). The removal of sialic acid by neuramninidase is believed to be responsible for the enhanced tumor cell immunogenicity (28) and in our studies may be responsible for both the observed enhanced susceptibility to lysis by antiThy-1.2 serum and the partial restoration of cytolytic capacity. An alternate explanation one might invoke is that neuraminidase-treated cells are more efficient "killers" than are normal sensitized cells. Although it is not possible to determine precisely the absolute number of effector cells in the infected and control neuraminidasetreated cell populations, it has been conventional practice to consider that differing levels of cytolysis reflect different concentrations of effector cells. Therefore we would interpret our data as reflecting a restoration of activity in a portion of the splenocyte population and not enhanced efficiency. The present study demonstrates that one early consequence of infection with FLV is an alteration in the splenic effector cell surface. Further studies will be required to elucidate both the nature of the alteration and the mechanisms by which these cell surface alterations are mediated. ACKNOWJLEDGMENT This investigation was supported by Public Health Service grant CA15643 from the National Cancer Institute. LITERATURE CrrED 1. Axelrad, A. A., and R. A. Steeves 1964. Assay for Friend leukemia virus: rapid quantitative method based on enumeration of macroscopic spleen foci in mice. Virology 24:513-518. 2. Bennett, ML, and R. A. Steeves. 1970. Immunotompetent cell functions in mice infected with Friend leukemia virus. J. Natl. Cancer Inst. 44:1107-1119. 3. Burnet, M. F. 1971. Immunologic surveillance in neoplasia. Transplant. Rev. 7:3-25. 4. Ceglowski, W. S., B. P. Campbell, R. F. Mortensen, and H. Friedman. 1974. Humoral and cellular immune responses in susceptible and resistant strains of mice infected with Friend leukemia virus. Proc. Soc. Exp. Biol. Med. 146:619-624. 5. Ceglowski, W. S., and H. Friedman. 1970. Immunosuppression by leukemia viruses. IV. Effect of Friend leukemia virus on antibody-precursors as aed by cell transfer studies. J. Immunol. 105:1406-1415.

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6. Celada, F., and B. Asjo. 1973. Studies on Moloney leukemia virus infection of antibody forming cells, p. 263-270. In W. S. Ceglowski and H. Friedman (ed.), Virus tumorigenesis and immunogenesis. Academic Press Inc., New York. 7. Cerny, J., M. Essx, M. A. Rich, and W. D. Hardy, Jr. 1975. Expresion of virus-asociated antigens and immune cell functions during spontaneous regression of the Friend viral murine leukemia. Int. J. Cancer

15:351-365.

8. Cerny, J., M. Essex, and D. B. Thomas. 1976. Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. III. Alterations of splenic B and T cells in Friend virus-infected mice. Int. J. Cancer 18:197-204. 9. Cerottini, J. C., and K. T. Brunner. 1971. In vitro assay of target cell lysis by sensitized lymphocytes, p. 369-372. In B. R. Bloom and P. R. Glade (ed.), In vitro methods in cell-mediated immunity. Academic Press Inc., New York. 10. Dent, P. B. 1975. Immunodepression by oncogenic viruses: mechanisms and relevance to oncogenesis, p. 95-107. In The immune system and infectious diseases. Karger, Basel. 11. Friedman, H., and W. S. Ceglowski. 1971. Immunosuppression by tumor viruses: effects of leukemia virus infection on the immune response, p. 815-829. In B. Amos (ed.), Progress in immunology. Academic Press Inc., New York. 12. Grothaus, E. A., M. W. Flye, E. Yunis, and D. B. Amos. 1971. Human lymphocyte antigen reactivity modified by neuraminidase. Science 173:542-544. 13. Han, T. 1975. Specific effect of neuraminidase on blastogenic response of sensitized lymphocytes. Immunology 28:283-286. 14. Henney, C. S. 1971. Quantitation of the cell-mediated immune response. I. The number of cytolytically active mouse lymphoid cells induced by immunization with allogeneic mastocytoma cells. J. Immunol. 107:1558-1566. 15. Henney, C. S., and J. E. Bubbers. 1973. Studies on the mechanism of lymphocyte-mediated cytolysis. I. The role of divalent cations in cytolysis by T lymphocytes. J. Immunol. 110:63-72. 16. Kateley, J. R., J. Holderbach, and H. Friedman. 1974. Leukemia virus-induced alteration of lymphocyte Ig surface receptors and the "capping" response of mouse spleen and lymph node cells. J. Natl. Cancer Inst.

53:1135-1140.

17. Koo, G. C., W. S. Ceglowski, IL Higgins, and H. Friedman. 1971. Immunosuppression by leukemia viruses. VI. Ultrastructure of individual antibody-forming cells in the spleens of Friend leukemia virus-infected mice. J. Immunol. 106:815-830. 18. Kuppers, R. C., and C. S. Henney. 1976. Evidence for direct linkage between antigen recognition and lytic expression in effector T cells. J. Exp. Med. 143:684-89. 19. McLimans, W. R., E. V. Davis, F. L Glover, and G. W. Rake. 1957. The submerged culture of mammalian cells: the spinner culture. J. Immunol. 79:428-433. 20. Martz, E. 1975. Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. I. Resolution and characterization. J. Immunol. 115:261-267. 21. Martz, E., and B. Benacerraf. 1975. T-lymphocyte mediated cytolysis: temperature dependence of killer cell dependent and independent phases and lack of recovery from the lethal hit at low temperatures. Cell. Immunol. 20:81-91. 22. Mortensen, R. F., W. S. Ceglowski, and H. Friedman. 1974. Leukemia virus-induced immunosuppression. X. Depression of T cell-mediated cytotoxicity after infection of mice with Friend leukemia virus. J. Immunol. 112:2077-2086. 23. Oshiro, L S., N. E. Cremer, D. 0. N. Taylor, and E. H. Lennette. 1969. Electron microscopic studies on

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localization of antibodies in rat lymph node cells producing Moloney virus. J. Natl. Cancer Inst. 43:1109-1118. Reif, A. E., and J. M. V. Allen. 1964. The AKR thymic antigen and its distribution in leukemias and nervous tissues. J. Exp. Med. 120:413-433. Riley, V. 1968. Lactate dehydrogenase in the normal and malignant state in mice and the influence of a benign enzyme elevating virus, p. 493. In H. Busch (ed.), Methods in cancer research, vol. 4. Academic Press Inc., New York. Rowe, W. P., W. E. Pugh, and J. W. Hartley. 1970. Plaque assay techniques for murine leukemia viruses. Virology 42:1136-1139. Schlesinger, M., and B. D. Amos. 1971. Effect of neuraminidase on serological properties of murine lymphoid cells. Transplant. Proc. 3:895-897. Simmons, R. L, and A. Rios. 1971. Immunotherapy of

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cancer: immunospecific rejection of tumors in recipients of neuraminidase-treated tumor cells plus BCG. Science 174:591-593. Steeves, R. A. 1975. Spleen focus-forming virus in Friend and Rauscher leukemia virus preparations. J. Natl. Cancer Inst. 54:289-297. Steeves, R. A., R. J. Eckner, M. Bennett, E. A. Mirand, and P. J. Trudel. 1971. Isolation and characterization of a lymphatic leukemia virus in the Friend virus complex. J. Natl. Cancer Inst. 46:1209-1217. Wagner, H., and M. Bollinghoff. 1974. T cell-mediated cytotoxicity, discrimination between antigen recognition, lethal hit and cytolysis phase. Eur. J. Immunol. 11:745-750. Yu, A., W. Liang, and E. P. Cohen. 1975. Detection of a TL (+) murine leukemia cell line that resists the cytotoxic effects of guinea pig complement and specific antiserum. J. Natl. Cancer Inst. 55:299-308.

Friend virus-induced immunodepression: effect of neuraminidase treatment on Thy-1.2 antigen expression and cytotoxic potential of splenocytes from virus-infected mice.

Vol. 19, No. 2 INFECrION AND IMMUNrrY, Feb. 1978, p. 486-492 0019-9567/78/0019-0486$02.00/0 Copyright © 1978 American Society for Microbiology Print...
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