Int. J . Cancer: 18, 197-204 (1976)

INTERACTTONS OF MURINE LEUKEMIA VIRUS (MuLV) WITH ISOLATED LYMPHOCYTES. 111. ALTERATIONS OF SPLENIC B AND T CELLS IN FRIEND VIRUS-INFECTED MICE Jan CERNY l, Myron ESSEX and D. Brian THOMAS Department of Microbiology, Harvard School of Public Health, Boston, Massachiisetts 021 15, U S A ; and National Institute for Medical Research, Mill Hill, London, England

SUMMARY

Lymphoid tissues of mice infected with murine leukemia virus (Friend) (MuL V-F) were examined for the presence of cellular markers of MuLV-F infection. The Friend virus-associated cell membrane antigen ( F V M A ) and the virus group-specific antigen (GSA) were detectable on cells from the spleen and, to a lesser degree, on cells from the bone-marrow. In contrast, neither FVMA nor GSA was found in cells from the thymus. Alterations in the B-cell and T-cell spleen populations of MuL V-F-infected mice were then studied, The proportion of Ig-positive cells declined from the initial 45% (in non-infected controls) to about 10% after 2 weeks of infection. A similar decline of theta-positive cells was noted. However, complement-receptor-bearing cells ( E AC rosettes) declined even more rapidly and became undetectable in the second week after infection, The treatment of spleen cells from MuL V-F-infected mice with anti-FVMA serum plus complement in vitro reduced the number of detectable Ig-positive cells, specifically, whereas the number of theta-positive cells remained unchanged. Furthermore, B and T cells from spleens of infected mice were separated on an afmity column with anti-Ig antibody-coated beads. The initial cell suspension contained about 45% FVMA-positive cells, about 40 % Ig-positive cells and about 40 % theta-positive cells. Ig cells were retained on the column. The theta-positive cell fraction was collected in the eluate and contained very few FVMApositive cells with some " null" cells. Most of the FVMA-positive cells were retained on the column, which strongly suggested that they were B cells. These results confirm the previous experiments which showed the selective infections of purified splenic B cells by MuLV-F in cultures. Previous experiments on the infection of isolated, partially purified splenic B and T cells with Friend leukemia virus (MuLV-F) suggested that B-cells are permissive to infection with this virus. Both virus replication and expression of virus-related cell membrane antigen on B cells infected either in vitro and/or in diffusion chambers implanted in the mouse peritoneal cavity were observed (Cerny et al.,

1976a). In contrast, T cells appeared to be relatively resistant to MuLV. Furthermore, the difference in susceptibility to the virus appeared not only in mature lymphocytes, but also at the level of their precursors: bone-marrow cells cultured in diffusion chambers were readily infected by MuLV-F, whereas thymocytes were not (Cerny et al., 1976a). The latter observation concurs with the earlier work of Thompson (1969), who, however, utilized a n adoptive transfer of bone-marrow cells and thymocytes into irradiated, MuLV-F-infected mice. Another approach to the problem of apparent divergence between T and B cells in respect of their interaction with MuLV-F is to isolate these lymphocytes from spleens of previously infected mice and to determine which one is carrying a marker of the infection. Such experiments are complicated by the pathogenic effect of splenic focus-forming virus (SFFV), which causes rapidly developing splenic tumors dominated by characteristic erythroblastic cells (Friend, 1957; Metcalf et al., 1959). The relative proportion of B and T cells in this enlarged spleen declines rapidly (Cerny et al., 1975; Kateley et al., 1974; and this paper) to the point where their isolation is not feasible. Therefore, studies on separated B and T cells in MuLV-F infection are limited to the week or two following administration of the virus. The results of such studies are presented here. In support of our previous experiments on infection of isolated lymphocytes, they showed a specific permissiveness of B cells to MuLV-F. MATERIAL AND METHODS

Mice BALB/c mice (Charles River Laboratories, Wiimington, Mass., USA) of both sexes were used (6 to 8 weeks of age). Received: April 27, 1976. Abbreviations: MuLV = murine leukemia virus; MuLV-F = Friend virus leukemia virus complex; SFFV = splenic focus-forming virus; LLV = lymphatic leukemia virus ; FVMA = Friend virus-associated cell membrane antigen; GSA = virus group-specific antigen; FlTC = fluorescein isothiocyanate; O = theta antigen; BAO = brain-associated theta antigen; EAC = erythrocyte-antibody-complement ; EA = erythrocyte-antibody.

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CEKNY ET AL.

Virus and infection The source and titration of the Friend virus complex (MuLV-F) were described previously (Cerny et a/., 1975, 1976~). Mice were infected either intravenously (IV) or intraperitoneally (IP) with 2 x lo3 splenic focus-forming units. The inoculum contained approximately 2 to 4 x lo3 focus-forming units of MuLV titrated on S+L- cells according to the method of Bassin et al. (1970). Friend virus-associated cell membrane antigen, F V M A FVMA was detected by membrane fluorescence with an isologous mouse anti-FVMA serum (Cerny and Essex, 1974) followed by FITC-conjugated goat anti-mouse Ig (Hyland, Costa Mesa, Calif., USA). The latter was diluted to detect minute quantities of membrane Ig in control preparations of spleen cells, i.e. cells incubated with either normal mouse serum or phosphate-buffered saline in the first step. Additionally, because the specific immunofluorescence reaction for FVMA was so strong, it was possible to reduce the intensity of fluorescence due to lg by using additional BG12 emission filters together with barrier filter reducing the intensity of green FITC fluorescence (FITC filter). The background was well below 10% in the experiments done in our laboratory previously, as well as in those reported here. For the experiments done with affinity columns the background levels of anti-Ig fluorescence were slightly higher because only the FITC filter was used (see " Results "). Membrane lymphocyte markers Membrane Ig was detected by direct immunofluorescence as described elsewhere (Cerny et al., 19766). Theta antigen ( 0 ) was visualized by direct immunofluorescence with mouse anti-0 serum (AKR anti-C,H/Hej thymocyte serum) prepared as previously described (Cerny et al., 1975) and conjugated to FITC. A 50% ammonium sulfate fraction from the anti4 serum was mixed with powdered FITC (Calbiochem, Los Angeles, Calif., USA) at a ratio of 1OO:l (w/w) following the established procedure (Rabellino et a/., 1971). The unconjugated FITC was removed by filtration through a column of Sephadex (3-50 (Pharmacia, Piscataway, N.J., USA) and the conjugate was concentrated by membrane filtration (Diaflo, Amicon Corporation, Lexington, Mass., USA). However, it should be noted that only one out of three different batches of these conjugates retained its specific anti-0 activity; this preparation was used in all experiments, at a dilution of 1 :2. In the column experiments, T cells were detected by indirect inununofluorescence staining using a rabbit anti-mouse brain serum (anti-brain associated 0, BAe) (Gyongyossy and Playfair, 1973) followed by FITC-conjugated goat anti-rabbit Ig. The anti-BAB

antiserum (generously provided by Dr. J. H. L, Playfair from the Department of Immunology. Middlesex Hospital Medical School, London, England) had been rendered specific for T cells by extensive absorption with bone-marrow cells. Complement .(C ) receptor-hearing spleen cells Cells were enumerated by a rosette formation between spleen cells and erythrocytes coated with antibody and complement (EAC) following the technique described by Bianco et al. (1970). The only deviation from their procedure was in the method of incubation. The mixture of spleen cells and EAC (total volume 1.Oml) was placed in 1.O ml plastic tubes (cut from the bottom of Falcon plastic culture tubes) and sealed with parafilm. The tubes were positioned on a reel with a horizontal axis and rotated (I5 rpm) in a 37" C air incubator, for 2 h. The control erythrocytes (EA) were prepared either by omitting the fresh mouse serum (the source of C) or by adding EDTA ( 1 4 m ~ )to the reaction, as originally described (Bianco et al., 1970). The EA rosette background varied from 0 % to 3 % and the values were subtracted from the EAC counts. Detection of virus group-specific antigen ( G S A ) Indirect fixed cell immunofluorescence was done according to the standard procedure (Hardy et at. 1973; Cerny et al., 1975). Smears were made with the same lymphocyte suspensions as used for the living czll immunofluorescence procedures described above. Rabbit serum against ether-disrupted Rauscher virus was undiluted following in vivo absorption in young rats. Normal rabbit serum absorbed in a similar manner was the control. Fluorescein-labelled goat anti-rabbit gamma globulin (Hyland) was used at a 1 :I0 dilution. Cytotoxic treatment of spleen cells Treatment with mouse anti-serum to Friend virusinduced cell membrane antigen (anti-FVMA) was done by incubating 5 x lo6 cells in 1.0 mi Puck's saline G with 0.1 ml of undiluted anti-FVMA (or with normal mouse serum as a control) for 30 min in a 37" C water bath. Three-tenths ml of undiluted normal guinea-pig serum (BioQuest) absorbed with mouse tissues was then added and incubation proceeded for another 30min. Cells were then washed in cold medium, three times, prior to the membrane immunofluorescence assay. Cell fractionation on immunoabsorbent columns Spleen-cell suspensions were depleted of Ig-bearing lymphocytes by fractionation on immunoadsorbent columns (Wigzell and Anderson, 1969) of Degalan V-26 plastic beads (polymethylmetaacrylic plastic, Degussa Wolfgang AG, Hanau-am-Main, West Germany). Cell suspensions in Eagle's medium (EM)

199

B AND T CELLS IN MULV-INFECTED MICE

were incubated with iron powder (ex: carbonyl iron) to remove the excess of polymorphonuclear cells, washed and resuspended in cold Hanks' balanced salt solution with 2 % bovine serum albumin and 5 mM EDTA (HEB). A total of los cells were applied to Degalan bead columns (10 x 1 cm, containing 12 mg bound, purified rabbit anti-mouse Fab) as 1 ml aliquots (10' cells) followed each time by 3 ml of cold HEB medium. The procedure was carried out at 4"C. RESULTS

Distribution of MuL V-F infection

Spleen, bone-marrow and thymus cell suspensions were obtained from mice infected IV with MuLV-F 2 weeks previously. Cells were stained for both FVMA, as a marker of virus-related cell alteration, and GSA, as a marker of virus replication. Spleen and bone-marrow were consistently positive (Table I) with the number of FVMA+ cells ranging from 76 % to 90 % in the spleen and 30 % to 79 %, i.e. somewhat less, in bone-marrow. Both organs were also positive for GSA (Table I). In contrast, no cells positive for either FVMA or GSA were detectable in thymus (Table I). Changes in splenic B and T cells

Young aduIt BALB/c mice were injected IV with 2 x 1 0 3 splenic FFU MuLV-F, and groups of five were killed on days 4, 8 and 14 after infection. The total number of nucleated cells per spleen was calculated and aliquots from each spleen were stained for FVMA, Ig and 0 and tested for the number of EAC rosettes (Fig. 1). FVMA-positive cells appeared on day 4 and their frequency increased steadily until day 14, when their proportion reached 80 % or more. The total number of nucleated cells (plotted on a log scale) increased in parallel with FVMA+cells. However, the relative

number of lymphocytes progressively decreased. Theta-positive cells decreased from 42 % in the control group to 25 % on day 4, 22 % on day 8 and 19 % on day 14 after infection. Corresponding figures for Ig-positive cells were 48 % (control), 43 %, 30% and 8%. However, cells with complement receptor (EAC rosettes) declined more rapidly so that very few (less than 5 % ) were detectable 8 days after infection and no EAC (i.e. no more than the EA background) were seen on day 14. Thus, at two intervals after infection on days 8 and 14, there were still respectable numbers of Ig-positive cells, but very few, if any, C receptor-bearing cells in the spleen. A total of eight mice were investigated on various days during the second month after the infection (days 35-56, Fig. 1). The accumulated data were similar to those for day 14 (Fig. 1). It is noteworthy that, while the percentage (i.e. relative number) of lymphocytes in the spleen declined, a simple calculation shows that their total number in the enlarged spleen increased. As an example, using the formula: percentage B cells x total nucleated cells Total B cells per spleen 100 one can determine that on the 14th day post infection, when B cells represented only 8 % of the spleen cell population, their total number in the enlarged spleen was about 2.8 x lo8which represents a more than two-fold increase over the average total of 0.96 x lo* B cells in non-infected control spleen. Similarly, an increase in total number of T cells in the spleen was noted. Effect of cytotoxic treatment with anti-FVMA serum plus complement on Ig-positive cells

Spleen cells from MuLV-F infected mice (at day 6 after virus injection) were treated with anti-serum to

TABLE I DISTRIBUTION OF FRIEND VIRUS-ASSOCIATED CELL MEMBRANE ANTIGEN (FVMA) A N D VIRUS GROUP-SPECIFIC ANTIGEN@) ( G S A ) I N LYMPHOID TISSUES OF BALB/c MICE AT 14 DAYS AFTER INFECTION WITH MuLV-F Spleen Exp.

1

Mouse No.

FVMA (%)

Bone-marrow

GSA ( %)

FVMA ( %)

GSA ( %)

Thymus

FVMA

GSA

1 2

3

Control 2

11 12 14 16

Control

+

Positive fluorescence is marked (moderate to strong) orf(weak) and the percentage of stained cells is glven in parentheses. Negative results (-) indicate that number of cells with any trace of staining was lower than or equal to background - Non-infected mice. N D = not done.

200

CERNY ET AL.

100-

z W Y

80W

I

I-

z

FIGURE 1

In

2

0--0: Kinetics of cellular changes in the spleen of MuLV-Finfected mice: total nucleated cells per spleen; 0-0: Friend virus cell membrane antigen(s)-positive cells (FVMAf); n---n :Ig-bearing cells (Ig+); : theta-positive cells (O+); v-.-.- v : complement receptor-bearing cells (C-receptor+) detectable as EAC rosettes. Each point is an average value from four to six mice.

60-

V W

2 E

In

g u 40t-

z

W U

DAYS

AFTER INFECTION

FVMA and complement (see " Material and Methods ") to eliminate the majority of FVMApositive cells. The remaining cells were then stained for Ig and 8. Table I1 shows that this treatment diminished the number of detectable Ig-positive cells by 5%, whereas treatment with normal mouse serum and complement had little effect. In contrast, the proportion of 0-positive cells remained undiminished by anti-FVMA and C. No decrease in anti-Ig staining was seen after similar treatment of spleen cells from non-infected control mice. These results provided an indication that B cells were altered by

the virus. Additional pilot studies using a doublestaining of lymphocytes will be mentioned in the " Discussion ". Separation of lymphocytes from MuL V-F-infected spleens on anti-Ig columns A direct approach to the identification of a virus-altered population of lymphocytes in the spleen was the separation of B cells and T cells on a column with an immunoadsorbent containing antimouse Fab antibody. Results of two independent experiments performed on the 7th day after MuLV-F

TABLE I1

IG-POSITIVE AND 8-POSITIVE CELLS FOLLOWING CYTOTOXIC TREATMENT WITH ANTI-FVMA AND COMPLEMENT Percentage positive cells after stainin p with Cells

Treatment

Mouse anti4 (FITC)

Rabbit anti-mouse

Ig (FITC)

Spleen, non-infected (control) Spleen, MuLV-F-infected (-5 days)

' See text for details.

None Anti-FVMAi complement NMS +complement None Anti-FVMA +complement NMS +complement

36.4 38.6 36.8 29.1 30.8 32.8

45.1 44.1 43.7 46.1 20.1 36.0

201

B AND T CELLS IN MULV-INFECTED MICE TABLE 111 PRESENCE OF FVMA, IG A N D THETA (BAB) ANTIGENS ON ALIQUOTS FROM FV-INFECTED SPLEEN CELL SUSPENSIONS BEFORE A N D AFTER PASSAGE THROUGH ANTI-IG COLUMN ' Cells with membrane marker detected with following sera Exp.

Cell fraction Anti-FVMA

A B

Pre-column Post-column Pre-column Post-column

a

(x)

146/326 (45) 38/344 ( I 1) 56/128 (44) 18/200 ( 9)

Anti-Ig (%)

Anti-BAB (%)

138/363 (38) 2/114 ( 2) 45/105 (43) 3/360 ( 1)

91/245 (37) 200/282 (71) 47/115 (41) 180/225 (80)

NMS

a

(%)

51/445 (12) 7/280 ( 3) 40/280 (14) 2/182 ( 1)

I

Spleen cells were pooled from several BALB/c mice 7 days after infection (2-4 x los FFU MuLV-F, IP.). - Incubation with the serum was followed by staining with a goat anti-mouse Ig fluorescein conjugate diluted 1 2 0 ; NMS = normal mouse serum (BALB/c). - Incubation with anti -BAB serum (rabbit anti-serum against mouse brain-associated theta antigen)followed by incubation with goat anti-rabbit Ig fluorescein conjugate. - 4 Number of cells with positive fluorercence/total number o f cells screened (percentage cells with the marker).

infection are given in Table 111. Cell suspensions pooled from several infected donor spleens contained 38-43 % of Ig-positive cells, 37-41 % of BAB-positive cells and 44-45 % of FVMA-positive cells. Following the passage through the column, most Ig-positive cells and most FVMA-positive cells were retained on the column. Thus, for example, in experiment B (Table 111) less than 1% of cells in the fraction eluted from the column were Ig+, i.e. 98% of Ig+ in the original pre-column cell suspension were eliminated by the passage. Furthermore, only 9 % of the cells in the eluate were FVMA-positive, compared to 44% FVMA-positive in the pre-column fraction, meaning that 80% of the FVMA-positive cells were retained on the anti-Ig column. Cells which passed through the column were from 70% to 80% BAB-positive. By subtracting the sum of Ig+ and BAB+ cells one can calculate that about 16% to 25% cells in the original (pre-column) cell suspension did not have any lymphocyte marker, i.e. they were " null " cells. These cells passed through the column; approximately 20% to 30% null cells appeared in the eluate. Thus, the Ig+, FVMA+ B-cells were retained on the column, whereas T cells and null cells were eluted from the column, together with a few FVMA' cells (which could have been either T or null). It should be emphasized that the proportion of " null " cells would be higher if a similar experiment were performed later after infection when the rapidly expanding population of eythroblastic cells occupies most of the spleen. DISCUSSION

Experiments in this paper provide additional support for previous work in which normal lymphocytes, semi-purified B and T lymphocytes, were infected with MuLV-F in vitro and/or in diffusion chambers (implanted into mouse peritoneal cavity) and replication of MuLV and cell membrane alteration was determined. Infection occurred readily in the suspension of B cells and virus replication was

enhanced in the presence of a B-cell-specific mitogen, bacterial lipopolysaccharide. On the other hand, we found no consistent evidence of MuLV-F infection in a population of T cells (Cerny et al., 19766). In the current study we used the opposite approach. Cells were infected in situ and then disengaged from lymphoid tissues and assayed for cellular marker(s) of virus infection. We showed that the elimination of cells carrying Friend virus-induced cell membrane antigen (FVMA) by treatment with anti-FVMA plus complement markedly decreased the number of detectable Ig-posi:ive (Ig+)cells, whereas the number of theta-positive (B+) cells remained unchanged, suggesting that Igt cells, but not O+ cells, carried FVMA. Additional experiments with an anti-Ig affinity column provided more direct evidence of B-cell infection. All B cells and most of the FVMApositive cells were retained on such columns, whereas FVMA-negative, Of cells appeared in the eluate. Experiments, not included here, have shown double-staining of B and T cells for the lymphocyte marker and the FVMA. To examine T cells, spleen cells from MuLV-F-infected mice were incubated in three steps with: (1) anti-FVMA serum; (2) rabbit anti-mouse Ig-rhodamine conjugate; and (3) mouse anti-8-FITC conjugate. No cells with a yellow fluorescence (i.e. T cells bearing FVMA) were noted. However, when the same spleen cells were incubated with: (1) rabbit anti-mouse Ig-FITC conjugate; (2) anti-FVMA; and (3) rabbit anti-mouse Igrhodamine conjugate, the majority of cells stained yellow, indicating the presence of Is+,FVMA+ cdls. Such double-stained cells w x e not seen in con'rol preparations in which normal mouse serum was used in the second (2) step. Thus, the results obtained by these two experimental approaches are in agreement and demonstrate specific infection of B cells, presumably by the LLV component of the Friend virus complex (Rowson and Parr, 1970; Fieldsteel et al., 1969; Steeves et al., 1971), both in vitro and in vivo. The selectivity of

202

CERNY ET AL.

the virus for lymphocytes of B lineage and the resistance of T cells also appear to exist at the level of the precursor cell population. Bone-marrow cells, but not thymocytes, were infected with MuLV in diffusion chambers (Cerny et al., 1976b) and, similarly, bone-marrow cells collected from MuLV-Finfected mice carried both FVMA and gs antigen, whereas thymocytes from these mice were negative (Table r). This is in agreement with the much earlier work of Thompson (1969) who studied virus replication in mice which were lethally irradiated, reconstituted with cells from various lymphoid tissues and infected with MuLV-F; recipients of bone-marrow, but not recipients of thymocytes, showed virus titers. We did not try to determine whether B-cells infected with MuLV of Friend virus became leukemic. However, others who studied the leukemogenic effects of isolated LLV in newborn mice described the development of lymphatic leukemia, which was distinctly different from the rapidly developing erythroblastic leukemia caused by SFFV (Carter et al., 1970a, b ; Steeves et al., 1971). The LLV-induced leukemia begins in the spleen. It is characterized by hyperplasia of the red pulp with many lymphoblasts and normoblasts (as well as other haematopoietic elements such as erythroblasts) in an early stage (Steeves et al., 1971 ; Carter et al., 1970~).The later stage of the lymphoma begins by expansion of Malphigian bodies from what appear to be germinal centers, and by appearance of reticular cells (Carter et al., 1970b). There is no involvement of either thymus or lymph nodes in this leukemia (Steeves et a[., 1971 ; Carter et a[., 1970~)until the terminal stage when all tissues are being infiltrated (Carter et al., 1970b). We are not aware of any report of lymphocyte markers on cells involved in various stages of this disease. However, its description suggests the involvement of thymus-independent areas (i.e. B cells) in the spleen and no involvement of either central or peripheral thymus-derived cells. Is the apparent process of infection of B cells by LLV, the helper virus, an important step in the pathogenic effect of SFFV and/or does SFFV, too, replicate in B cells? The only suggestion that it may be so comes from the work of Kouttab and Jutila (1 972) who showed that inhibition of IgM-producing cells by neonatal injection of the specific antiserum rendered mice completely resistant to the erythroblastic leukemia induced by SFFV and LLV complex. To our knowledge, this important experiment has not been repeated. Infection with MuLV-F produces rapidly developing suppression of antibody responsiveness, i.e. the B-cell function (Notkins et al., 1970; Dent, 1972). The immune deficiency resides in bone-marrow, whereas thymocytes from such infected mice are

capable of their normal helper function (Bennett and Steeves, 1970). Furthermore, the homograft rejection, another T-cell function, is also unimpaired by MuLV-F (Schneider and Dore, 1969; Bainbridge and Bendinelli, 1972). In the same MuLV-F system, Mortensen et al. (1974) described the suppressive effect of MuLV-F infection on T-cell-dependent cytotoxicity of spleen cells against allogeneic target cells in vitro. This discrepancy can perhaps be understood in the light of the most recent work of several laboratories which has revealed that immunosuppression associated with tumors (both virus-induced and chemically induced) is a multifactorial reaction involving, for example, the appearance of inhibitory soluble factors (Hersh and Drewinko, 1974; Kamo et al., 1975; Hrsak and Marotti, 1975; Fauve et al., 1974;Takada et al., 1974) and non-specific suppressor cells (Gorczynski, 1974; Kilburn et al., 1974; Kirchner et a/., 1975; Glaser et al., 1975; Cerny and Stiller, 1975; Stiller and Cerny, 1976). Very active suppressor cells inhibiting both B and T cells were found in the spleen of MuLV-Moloney-infected mice (Cerny and Stiller, 1975). Therefore, it appears that MuLV may cause suppression of immune cells by an indirect mechanism and that findings of alteration of immune response of B and T cells should not be interpreted solely in terms of virus infection of a given type of lymphocyte. Tropisms for infection and/or transformation of lymphoid cell populations with oncogenic herpesviruses have been demonstrated. The Epstein-Barr virus, for example, only infects B cells, and the Burkitt lymphoma tumors, which are presumably caused by this agent, are always B-cell tumors (Shevach et al., 1972). In contrast, lymphoid tumors induced by Herpesvirus saimiri are of T-cell origin (Wallen et al., 1973). As for other oncornavirus-induced tumors, lymphoma cells from MuLV-M-infected mice bear theta antigen (Proffitt et al., 1975), but the possibility that other lymphocytes might also be infected has not been eliminated. The specific infection of B cells with LLV may provide a model for studying relationships between lymphocyte differentiation and oncornavirus infection. ACKNOWLEDGEMENTS

The authors thank Dr. W. R. Hardy, Jr., SloanKettering Institute for Cancer Research, New York, for performing the test for virus group-specific antigen (GSA) and Dr. J. H. L. Playfair, for the gift of a rabbit anti-mouse brain serum (anti-BAO). This work was supported by Grant No. IM-35C from the American Cancer Society and by US Public Health Service Grants No. CA-14922 and No. CA-15277 from the National Cancer Institute.

B AND T CELLS IN MULV-INFECTED MICE

203

INTERACTIONS DU VIRUS DE LA LEUCEMIE MURINE (MuLV) AVEC DES LYMPHOCYTES ISOLES. 111. ALTERATIONS DES CELLULES SPLENIQUES T ET B CHEZ LES SOURIS INFECTEES PAR LE VIRUS D E FRIEND Les auteurs ont recherche' les marqueurs cellulaires de I'infection par le virus de la leucimie murine souche Friend (MuLV-F) dans des tissus lymphoides de souris infectkes par ce virus. Ils ont de'cele' l'antiglne membranaire induit par le virus de Friend ( F V M A ) et I'antiglne spe'c$que de groupe (gsa) sur les cellules spliniques et, dans une moindre mesure, sur les cellules de motile osseuse. Par contre, ils n'ont observk ni FVMA ni gsa dans les cellules du thymus. Ils ont ensuite ktudie' les alterations des populations de cellules spIPniques B et T des souris infecte'espar Ie MuLV-F. La proportion de cellules Ig-positives est pas& de 45 % au depart (chez les tkmoins non infectis) a environ 10% aprls deux semaines d'infection. Le nombre de cellules thita-positives de'croit de fagon comparable. Toutefois, les cellules quiportent des rkcepteurspour le comple'ment (rosettes EAC) diminuent encore plus rapidement et ne peuvent 2tre de'celies dls la deuxilme semaine aprls l'infection. Le traitement des cellules sple'niques de souris infecte'es par le MuL V-F avec du se'rum anti-FVMA additionne' de compliment in vitro abaisse spe'cifquement ie nombre de cellules Ig-positives de'celables,alors que Ie nombre de cellules th2ta-positives reste inchange'. Les cellules spliniques B et T des souris infecte'es ont e'ti se'pare'es sur une colonne d'afinitk contenant des billes recouvertes d'anticorps anti-lg. La suspension cellulaire initiaie contenait environ 45 % de cellules FVMA-positives, quelque 40 % de cellules Ig-positives, et approximativement 40% de cellules thtta-positives. Les cellules Igf ont Ete' retenues sur la colonne. La fraction de cellules th2tapositives a e'te' recueillie dans I'e'luat ; elle contenait trls peu de cellules FVMA-positives et quelques cellules '' nulles ". La plupart des cellules FVMA-positives ont e'te' retenues sur la colonne, ce qui conduit penser qu'il s'agit de cellules B. Ces re'sultats corroborent les expiriences prickdentes, qui avaient montrk que les cellules splkniques B purifkes sont se'lectivemenf infect& par le MuL V-F dans les cultures.

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