interactions of murine leukemia virus (mulv) with isolated lymphocytes. i. virus replication in...

Int. J . Cancer: 18, 176-188 (1976)

INTERACTIONS OF MURINE LEUKEMIA VIRUS (MuLV) WITH ISOLATED LYMPHOCYTES. I. VIRUS REPLICATION IN LYMPHOCYTES INFECTED WITH FRIEND VIRUS AND CULTURED IN DIFFUSION CHAMBERS I N YIVO

Jan CERNY, Samuel H. FISTEL and Patricia A. HENSGEN Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115, USA

SUMMARY

A new technique for infection of mature lymphocytes with murine leukemia virus (Friend) (MuLV-F) is described. Spleen cells from normal, non-infected donors were placed into diffusion chambers (constructed with 0.22 pnpore size Millipore filters) which were then implanted into the peritoneal cavities of normal syngeneic recipient mice. The cells were infected with an injection of MuLV-F into the peritoneal cavity and, in some instances, also by placing virus into the chambers. Cells were recovered by treating the chamber content with elastase and collagenase. The infection was determined in two ways: ( I ) cells with replicating MuLV were enu- merated as infectious centers (IC) on SfL- indicator cells; and ( 2 ) virus-related cell membrane antigen ( M A ) was detected by immunofluorescence. Cells recovered from chambers after 2-3 weeks of’ culture represented about 10% of the original inoculum; viability was approximately 90%. The number of IC in MuLV-F-infected chambers was about I0 times higher than that obtained by infection and cultivation of spleen cells in vitro. The kinetics of ZC and M A in chamber-cultured, MuL V-F-infected spleen cells was similar to that in the spleen of infected mice during the first I0 days after infection. Later on, the process of infection within the chambers slowed down, reaching about 50% MA-positive and about 10% IC-positive cells, whereas the number of both IC- and MA-positive cells in the spleen reached 80 % or more. The infection of splenic lymphocytes in diffusion chambers occurred equally well when chambers were implanted into: ( 1 ) syngeneic, virus susceptible hosts; ( 2 ) syngeneic, lethally irradiated hosts; and ( 3 ) allogeneic, virus- resistant hosts, suggesting that the process within the chamber is independent of MuLV replication in the tissues of the chamber-bearing mouse. The diffusion chamber technique seems to provide an environment in which various types of isolated lymphocytes of different mouse strains can interact with MuLV almost as efficiently as in vivo.

Cellular studies on the interaction of leukemogenic RNA viruses with lymphocytes require a suitable system in which a defined population of isolated

lymphoid target cells can be infected, altered and transformed by the virus. Experiments with murine leukemia virus (MuLV) have been hindered by the lack of such a system. The viruses can be grown in established lines of fibroblastic or epithelial cells (Ginsburg et al., 1961 ; Osato et al., 1964; Peries et al., 1964; Wright et al., 1965) but, apparently, these are not target cells for leukemia in vivo. One report has documented the in vitro transformation of progenitor lymphoid cells from fetal liver with the Abelson virus (Rosenberg et al., 1975). However, an efficient virus infection and transformation of mature lymphocytes in vitro has not been successful. Recently, Sklar et al. (1974) accomplished transformation of spleen cells by a brief exposure of splenic lymphocytes to Abelson virus in a culture followed by their transfer into a syngeneic host; the leukemic cells of donor origin were identified by a chromo- somal marker. Another approach has been the adoptive transfer of lymphocytes into lethally irradiated recipients with subsequent MuLV infection (Thompson, 1969); the major disadvantage of this method is the possible existence of radio- resistant target cells in the host.

Since it appears that in vivo conditions are required for an efficient infection of mature lymphocytes by MuLV, we attempted to develop a system in which isolated, well-defined lymphocytes could be infected with MuLV in an environment which is as close as possible to that of the natural host. For this, we adopted the method of diffusion chambers implanted into the peritoneal cavity of mice. This method has been widely used to study the immune response. It seems clear that the functional potential of both T and B cells is adequately expressed in this culture system (Holub and Riha, 1960; Capalbo et al., 1964;

Received : April 27, 1976. Abbreviations: MuLV = murine leukemia virus;

MuLV-F = Friend leukemia virus complex; SFFV = splenic focus-froming virus ; LLV = lymphatic leukemia virus; S+L- cells = sarcoma-positive, leukemia-negative fibroblasts; FFU = focus-forming units; IC = infectious centers; MA = virus-related cell membrane antigen; RaFV = rabbit antiserum to Friend virus; anti- FVMA = mouse antiserum to Friend virus cell mern- brane antigen.

MULV REPLICATION IN LYMPHOCYTES 177

Dvorak and Waksman, 1962). Borella (1969) used the method of diffusion chambers in studies on oncornavirus-induced immunosuppression.

This paper describes the system in which normal mouse spleen cells were infected with MuLV-F in diffusion chambers. The number of cells with replicating MuLV was almost a hundred times higher than that achieved in vitro. During the first 2 weeks of the culture, the virus replication was comparable to infection of the spleen in situ. Further- more, the appearance of virus-induced cell membrane antigen was noted on cells infected and cultured in chambers. These observations indicate that the method of diffusion chambers provides an efficient culture system for specific studies on MuLV interactions with lymphocytes.

MATERIAL AND METHODS

Animals

Adult BALB/c mice (H-2d) (2-3 months old, from Charles River Laboratories, Wilmington, Mass., USA) and adult DBA/2 mice (H-2d) (either 2-3 months old or retired breeders from Jackson Laboratories, Bar Harbor, Maine, USA) of both sexes were used.

Irradiation

BALB/c mice were X-ray irradiated with 750 rad (180 Kv, 15 mA, 80 rad/min, with 2 mm At and half-value layer mm (p), distance 60 cm).

Viruses

The origin and preparation of an NB-tropic Friend virus complex (MuLV-F) was described elsewhere (Cerny et al., 1975). This virus was used in most experiments. It contained 1-2 x lo5 FFU/ml of the splenic focus-forming virus (SFFV) as detected by the splenic focus assay (Steet-es and Axelrod, 1967) and 1-3 x lo5 FFU/ml of infectious leukemia virus, MuLV, as determined by titration on S+L- cells in vitro (see below). MuLV-F viruses with restricted tropism (N-tropic and B-tropic) were a generous gift from Dr. F. Lilly (Albert Einstein Medical Center, New York, N.Y., USA). These two viruses were passaged once in our laboratory, the B-tropic in BALB/c mice and the N-tropic in DBA/2 mice, and then titrated by the S+L- assay.

Preparation of cells

Cells were obtained by teasing spleens on a stainless steel wire screen in cold Puck's saline G containing antibiotics and antimycotics and 5 %

fetal calf serum inactivated at 56" C for 30 min. The suspension was allowed to stand for 1-2min to remove cell aggregates and the resulting single-cell suspension (viability by trypan blue exclusion higher than 80%) was washed by centrifugation in a refrigerated centrifuge (model PR-J, Damon/IEC Division, Needham, Mass., USA) at 220xg for 10 min at 4" C.

Construction and implantation of diffusion chambers

Diffusion chambers were constructed exactly as described by Nettesheim et al. (1966), from a lucite ring (outer diameter: 20 mm; inner diameter: 10 mm; thickness: 3 mm) sealed with membrane filters (0.22 pm pore size, Millipore) using a synthetic glue (Dekadhese, Tullock and Son, Willmington, Del., USA). Chambers were gas-sterilized 2 days prior to use. Each chamber was filled with 0.7 to 1.5 x lo7 cells, in 0.2 ml, using a 27-gauge needle and the filling hole was sealed with lucite shavings dissolved in chloroform. Filled chambers were maintained in sterile Petri dishes on ice until implantation (5 to 15 min).

Recipient mice were anesthetized with approximately 50 mg per kg body weight of sodium pento- barbital (Nembutal, Abbott Labs, North Chicago, Ill., USA), intravenously. Chambers were inserted into the peritoneal cavity through an incision in the anterior abdominal wall. The peritoneum and abdominal muscles were sutured with silk (4-0, Ethicon, Somerville, N.J., USA) and the skin was closed with clips (Autoclips, 9mm, Clay Adams, Parsippany, N.J., USA). A powdered antibiotic- antimycotic mixture (Gibco, Grand Island, N.Y., USA) was sprinkled on the incision before final closure.

Harvesting of cells from chambers and treatment with enzymes

Mice were killed by cervical dislocation, then the chambers were removed and rinsed in Puck's saline G . The content consisted of a small amount of fluid with free cells and a gel-like clot, apparently composed of connective tissue, in which many more cells were embedded. In some instances, only free cells were collected, washed and resuspended in a concentration appropriate for further assays. In most experiments, the free cells and the clot were treated as follows to increase the cell yield. Chambers rinsed in Puck's saline G were opened by cutting the filter on one side with scissors. The contents were transferred to a sterile culture tube and 0.3 ml of a 1 :I sterile mixture made from 0.3 % elastase and 2 % collagenase (Sigma Chemical Company, St. Louis, Mo., USA) in Puck's saline G was added. After

178 CERNY ET AL.

incubation at 37" C for 60 to 75 min, the fluid content of chambers was collected and the cells washed.

In vitro cultures

A single-cell suspension of lymphoid cells was prepared in RPMI 1640 medium with the addition of 5% fetal calf serum, antibiotics, 2 ,ug/ml of Polybrene (Aldrich Chemical Co., Milwaukee, Wis., USA), 2 m~ HEPES buffer and 1-glutamine (2 mM). On to 2 x 10' cells (either normal or pre-incubated with MuLV-see below) were cultured in 3 to 4 ml of medium in Falcon plastic culture tubes (17 x 100 mm) at 37" C and air/6 % CO, atmosphere.

Infection with murine leukemia virus complex (Friend) ( MuL V-F)

Infection of cells cultured in diffusion chambers was achieved by the injection of 2 to 6x103 FFU MuLV into the peritoneal cavities of chamber- bearing mice within about 1 h after implantation of chambers. In some experiments, virus (lo4 FFU) was also added directly into the chamber, together with the cells.

For infection in vitro, lo7 spleen cells were pelleted by centrifugation and resuspended in 0.2 to 0.4 ml of medium containing 2-6x104 FFU MuLV and Polybrene (2 pg/ml). Infection was allowed to proceeed for 30min at 4" C; 3-4ml of culture medium were then added, and the tubes were cultured as described above. In one experiment, cultures were re-infected with virus at 48 h. The tubes were centrifuged, the medium was removed and the cells were infected by the same procedure as used in the primary infection.

Infectious center assay for virus replication

Replication of MuLV in cultured cells was determined by the enumeration of cells capable of releasing infectious virus in vitro (infectious centers, IC). The indicator cell for the assay was the FG-10 sarcoma-positive, leukemia-negative (S+L-) cell line described by Bassin et al. (1971) and generously provided by Dr. D. Livingstone of the Children's Hospital, Harvard Medical School, Boston, Mass., USA. S+L- cells were seeded at 3-4 x lo4 in 2 ml of medium (McCoy's), with antibiotics and 10% fetal calf serum (Gibco), per 35 mm diameter Petri dish (Falcon Plastics). Two days later, the medium was removed and the cells were treated with 0.5 ml of DEAE-Dextran (mol. wt. 2 x lo6, Sigma, 25 pg/ml PBS) for 30 min at 37" C in air/6 % CO,. The DEAE- Dextran was removed and virus-infected cells harvested either from diffusion chambers or from in vitro cultures were then plated at serial dilutions (in duplicate), 0.5 ml per dish. The dishes were incubated at 37" C, in air/6% COz, for 1-2 h, after which 1.5 ml of fresh medium was added. Cells were incubated and fed with 1.Oml of medium/dish on the third day. After 5 days of incubation, plates were fixed with 10% formaldehyde (30 min) and stained with 0.03 % methylene blue for 4 min. The number of foci of transformation reflected the number of virus-producing cells, i.e. the infectious centers. Two or three serial dilutions of cells were assayed in each experiment. Furthermore, each experiment included a sensitivity control, i.e. dishes infected with a known number of MuLV FFU.

Since the MuLV-infected lymphocytes were left on the indicator S+L- cells for the entire period of incubation (i.e. 5 days) one might have anticipated a development of " secondary " IC due to lympho- cyte mobility and/or spreading of MuLV. This

TABLE I

INFECTIOUS CENTER (IC) ASSAY WITH SPLEEN LYMPHOCYTES FROM MuLV-F INFECTED MlCE '

IC/plate (duplicates) IC x 10d/lOe lymphocytes Spleen

No. Lymphocytes per plate: 10 100 1,000

8, 14 160, 171 TNTC, TNTC 1.1-1.7 ' 15,12 153, 200 TNTC, TNTC 1.3-1.8 * ND 97, 105 TNTC, TNTC 1 .oo ND 108, 98 TNTC, TNTC 1 .oo ND 18,12 154, 144 0.15

ND 104, 76 TNTC, TNTC 0.90

Results from various experiments in which MuLV-infected spleen cells were used as a positive control for the assay (see '' Material and Methods "). - Spleens from individual mice infected with 2-6 x 10' FFU MuLV-F 16-21 days prior to the assay.- a TNTC = too numerous to be counted. - Range obtained by plating log dilutions of cells. - 6 N D = not done.


c

t

T

1 I I I I 1 J 0 4 0 12

I IO) IYI€CTIDl DAYS AFTER INFECTION

FIGURE 1 Kinetics of expression of virus-related cell membrane

antigen(s) detectable with two different antisera in the spleen of MuLV-F-infected mice. Cells were incubated either with a rabbit anti-Friend virus serum (RaFV), followed by staining with a goat anti-rabbit Ig serum conjugated to fluorescein isothiocyanate (0 ) or with an isologous mouse antiserum to Friend virus cell membrane antigen (a-FVMA) followed by staining with a goat anti- mouse Ig fluorescent conjugate (0). Each point represents an average number (percentage) of positively stained cells from six to eight spleens. Vertical bars indicate range within each group (the assay with RaFV on day four was done with cells pooled from four spleens).

would have confounded enumeration of infected lymphocytes; however, our empirical observations suggested that the development of such secondary infection occurred minimally and/or remained undetectable during the time period of the assay. The IC assay was performed repeatedly with spleen cells obtained from mice during the 3rd week after MuLV-F infection. The splenic tumor approaches its maximum size and most, if not all, of the cells carry the membrane marker of infection (Cerny et al., 1975) at this time. With these cells, the number of IC was consistently related to the number of lymphocytes plated and the serial dilutions showed a linear relationship between IC and cells (Table I). It should be emphasized that great care was taken not to move the plates during the culture period.

The S+L- test was also employed for the titration of MuLV in various preparations of MuLV-F. The test was done exactly as described by Bassin et al. (1970, 1971), and the titer was expressed in FFU per ml. The assay detected both N- and B-tropic strains of MuLV-F.

Virus-induced cell membrane antigen (s)

The indirect immunofluorescent assay for cell membrane alteration by MuLV-F-induced antigens was described elsewhere (Cerny et al., 1975). Two different antisera were used: isologous mouse antiserum to Friend virus membrane antigen (anti- FVMA), followed by goat anti-mouse Ig conjugated to fluorescein isothiocyanate (FITC). The preparation and specificity of the serum were previously described (Cerny and Essex, 1974). Rabbit antiserum to MuLV-F antigens (RaFV) was followed by

TABLE I1

RECOVERY OF CELLS FROM DIFFUSION CHAMBERS FOLLOWING VARIOUS LENGTHS OF INCUBATION IN THE PERITONEAL CAVITY OF EITHER NORMAL OR MuLV-F INFECTED MICE

Cells recovered (viable, nucleated) x 10' MuLV-F Treatment (days after implantation and infection)

infection of chamber content 5 7 10 12 16

- 3 ND 6.5 ND 0.50 ' 0.23 + (4-9) (0.2-0.25)

Enzymes 5.3 8.0 16.0 12.0 12.5 (4-7) (6-1 1) (11-25) (6-30) (9-22)

- - 3 5.5 5.0 ND ND ND

Enzymes 5.6 6.0 ND 15.0 9.0 (4-7) (3-7)

(5-6) (3-9) (7-24) (7-12)

1 Cell suspensions from normal, non-infected BALB/c spleens (pooled from several donors) were placed into chambers in numbers varying between experiments from 0.7 x 10' to 1.5 x lo' (viable, nucleated cells). - Recipient mice (BALB/c) received intraperitoneal injections of 2-6 x 10' FFU of MuLV-F (+), immediately after implantation of chambers; non-infected controls (-) received either saline or nothing. - *Only the chamber's fluid content containing free celkCwas collected. - ' The clotted contents of the chambers were digested with a solution containing 0.15% elastase and 1.0% collagenase (see Material and Methods"). - 'ND = not done. - eArithmetic mean from three to seven chambers (range), - 7 Cells from two or three chambers were pooled.

180 CERNY ET AL.

TABLE 111

VIABILITY OF CELLS RECOVERED FROM CHAMBERS ElTHER WlTH OR WITHOUT ENZYME TREATMENT

Percentage viable cells Chamber No. '

No treatment Enzyme treatment

I 72 96 2 50 99 3 81 99 4 67 98

Chambers were implanted for 2 weeks in MuLV-F-infected recipient mice. - * Viability determined by a trypan blue exclusion. - 1 The portion of free cells obtained by aspiration of chamber content with a Pasteur pipette. - Cells obtained after the treatment of the remaining clot with the mixture of collagenase and elastase.

goat anti-rabbit Ig to conjugated fluorescein (Hyland). The RaFV was prepared by the repeated injection of rabbits with spleen cells from MuLV- infected BALB/c mice. The serum obtained after six bi-weekly immunizations (four IM injections of lo7 cells per immunization) was absorbed with

pooled normal mouse spleen cells, repeatedly, until specific fluorescence with MuLV-F infected cells was observed. The specificity of the heterologous RaFV was compared with anti-FVMA in two ways: (1) the kinetics of FVMAt cells in the spleen of MuLV- infected mice was determined with the two antisera and a good correlation was found (Fig. 1). (2) The RaFV serum gave positive membrane fluorescence with cells from mice with progressing MuLV-F- induced leukemia, but not with cells from mice undergoing spontaneous remission, which is comparable with previous results obtained with the specific anti-FVMA (Cerny et al., 1975). Thus, the expression of cellular membrane antigens detectable by RaFV correlates with the development of the MuLV-F-induced tumor.

RESULTS

Recovery and viability of cells from diffusion chambers

Chambers containing spleen cells, bone-marrow cells, or thymocytes from normal donors (0.7 to

TABLE 1V

REPLICATION OF MuLV IN SPLEEN CELLS INFECTED WITH MuLV-F IN DIFFUSION CHAMBERS, I N SITU OR IN TISSUE CULTURE

Infectious centers/lOB viable cells recovered from:

Tissue cultures Day of infection Diffusion chambers Spleen ( in situ) A5 B e

Individual Mean Individual Mean ' Individual Mean ' Individual Mean '

5 6x102 3 7 . 5 ~ 1 0 ~ 2x102 1 1 . 0 ~ 1 0 ~ 0 . 4 ~ 1 0 ~ 0 . 6 ~ 1 0 ~ 1 . 0 ~ 1 0 ~ 1 . 4 ~ 1 0 ~ 12x 102 (0.4%) 2 x lo2 (0.1 %) 0.4 x lo2 (0.006%) 1.0 x lo2 (0.01 %) 24 X lo2 2x102 0.5 x lo2 1 . 4 ~ 1 0 ~ 46 x lo2 2 x 108 0.7 x 102 2.0x 1 0 2

loox 102 4 x lo2 1 .ox 102 15 x loa 1.ox 102 50 x lo2

9 4x103 1 3 . 2 ~ 1 0 ~ 27x103 4 0 . 5 ~ 1 0 ~ 1 . 6 ~ 1 0 ~ 2 . 5 ~ 1 0 ~ N D 8 ND 4x103 (1.3%) 3Ox1O3 (4%) 2.0x103 (0.3%) 8x103 40 x 103 3.4 x 103

16 x lo3 46 x 103 3.5 x 103 22 x 103 50 x 103 25 x 103 50 x 103

15 2x104 5 .3~10 ' 10x104 3 3 . 2 ~ 1 0 ~ ND ND ND ND 6x104 (5%) 25x104 (33%) 8 x 10' 38 x l o 4

40 x 104 53 x 104

19 9 . 0 ~ 1 0 ~ ' 1 . 0 ~ 1 0 ~ 1 . 1 ~ 1 0 ~ ND ND ND ND (9%) 1.2x100 (100%)

~~

Spleen cells were pooled from several normal, non-infected mice and aliquots were either infected and cultured in vitro or placed into diffusion chambers which were implanted into MuLV-F-infected syngeneic recipient mice. - a Cells were recovered by treatment of the chamber contents with elastase plus collagenase. The cell suspension from each chamber was assayed for infectious centers at two or three log dilutions, in duplicate. - Spleen cells obtained by mincing spleens from MuLV-F-infected, chamber-bearing mice. (Assayed as above). - ' Each individual figure represents the number of infectious centers in cells pooled from two culture tubes; each cell pool was assayed at two or three log dilutions, in duplicate. - 5 Cultures were infected with 2-6 x 12: FFU MuLV at the outset as described in " Material and Methods ". - 6 Cultures were infected as in Material and Methods '9. - ' Arithmetic mean; figures in parentheses indicate percentage cells releasing MuLV. -

and then re-infected at 48 h (see ND = not done. - * Cells were pooled from two chamhers.


TABLE V

MuLV REPLICATION IN CHAMBER-CULTURED CELLS IMPLANTED INTO LETHALLY

IRRADIATED RECIPIENTS ‘

Infectious centers/lOB cells Mouse No. MuLV-F

Spleen Cells from chambers

1 + 50 8x10a 2 + < 1 0 2 * 25x103 3 + < 1 0 2 * 1 x 103 4 + < 1 0 2 * 26 x 103 5 - ND3 < 1 0 2 *

Recipient mice were irradiated (750 rad) 20 h prior to theimplanta- tion of chambers with normal spleen cells. MuLV-F (lo4 splenic ffu) was injected IP. - * Calculated per lon viable, nucleated cells recovered from the spleen of an irradiated, chamber-bearing mouse and from the chambers, on day 8. Each cell sample was assayed at two 10-fold dilutions. - a ND = not done. - * No foci seen with lo6 cells plated.

1.5 x 10’ viable, nucleated cells per chamber) were implanted into normal recipients; some of these recipients were subsequently infected with MuLV-F. Chambers were opened at various intervals and cells were collected either without any treatment of the chamber content (i.e. free cells were aspirated with a Pasteur pipette) or following enzyme treatment. Results obtained from several experiments are

summarized in Table 11. The overall pattern shows that without enzyme treatment, progressively fewer cells were recovered as the time of incubation increased.

With chambers bearing spleen cells, about 5 x lo5 cells (ranging from 4 x lo5 to 11 x lo5, in individual chambers) were recovered with or without enzyme treatment during the first week of incubation. However, by days 12 and 16 of the incubation, only about 0.2 to 0.5 x lo5 cells were recovered without enzymes, whereas treatment with collagenase plus elastase increased the yield more than 10-fold, i.e. the number of cells recovered varied from 6 x lo6 to 30 x lo5. This represents 10% or more of the original inoculum.

Infection of host mice with MuLV-F did not appear to have a significant effect on cell recovery (Table II), sugesting that the virus infection of cultured cells (as described below) did not cause an overall increase in the size of the cell population within the chambers.

The enzyme treatment of chamber contents did not diminish the viability of the cells as judged by their ability to exclude trypan blue. Table I11 shows an experiment in which four chambers were opened, fluids and free cells were collected, and the remaining clot was treated with collagenase plus elastase to

TABLE VI

REPLICATION OF MuLV IN VIRUS-SUSCEPTIBLE CELLS CULTURED IN CHAMBERS IMPLANTED INTO VIRUS-RESISTANT, Ha-COMPATIBLE RECIPIENTS

Recipient Cultured cells a Infectious centers/lOa cells ‘ Host Cells fro:

spleen chamber MuLV-F 3;; Group Mouse Strain Strain No. (tropism) (tropism)

BALB/c (B) DBA/2 (N) N-tropic 6 x 10’ 2 x 103

3 x 103 5 x 102 7x103 9 , ,, 9, ,,

1, 1, 7. I,

5 A 1 2 3 5 x lo1

BALB/c (B) B-tropic 7 x 101 10 x 103 t 2 * 2 x 103 6 x loa 1 x 103

BALB/c (B) DBA/2 (N) N-tropic n I xi03 ‘ n <2 *, ** 5 X lo3 ” n 15 x lo3

1, 11 ,, I,

3, 3, ,1 ,, DBA/2 (N) B 7

8 9

8 C 10 11 12

3 , 11 1, 9,

>? 1, 1 3 ,,

DBA/2 (N) BALB/c (B) B-tropic n 4 x lo3 ” n 7x101** 20 x 103 ” n 2x103

I, I, ,9 ,, 1, 11 11 ,,

D 13 14 15

BALB/c (B) BALB/c (B) B-tropic n 6 x 10’ ” n 5x10s** 8 x los ” n 4 x 103

,, 1, 9, ,I

9 , 3, ,I ,, E 16

17 18

Strain of mice into which the chambers were implanted. These recipients were infected with approximately 10‘ FFU MuLV of indicated tropism (see footnote Strain of origin of spleen cells cultured in the diffusion chambers. Cells were obtained from several normal, non-infected donors; MuLV (10’ FFU in 0.1 ml) was added into each chamber prior to its implantation. - MuLV-F, either N-tropic or B-tropic. was employed, as indicated. The same virus was added into chambers and injected into recipients (see footnotes and a). Cells in chambers were susceptible to the virus, whereas the chamber-hearing recipients were resistant. The exception is group C, where both cells in the chamber and the recipient mice had the same tropism, B. - ’ Assay was performed with two or three log dilutions of cells, in duplicate. -

Tropism (B or N) indicates to which virus (B-tropic or N-tropic) the mice are susceptible. (See text for details). - Cells were recovered by treatment with collagenase and elastase. * No infectious centers found in 5 x 10’ cells plated. ** Spleens from three mice were pooled for the assay.

IP. -

182 CERNY ET AL.

TABLE VII

EFFECT OF ENZYME TREATMENT OF CULTURED, MuLV-INFECTED LYMPHOCYTES

ON INFECTIOUS FOCUS FORMATION '

Cells Infectious fnci/lOd cells assayed

Free lymphocytes 13.5 x 103 Clot lymphocytes 37.0 x 103

~~ ~

1 Chambers bearing MuLV-infected spleen cells were opened and fluid with free cells was harvested: the remaining clot was treated with enzymes to recover cells (clot lymphocytes). Cells from four chambers were pooled for theexperiment. - a Arithmetic mean from quadrupli- cate assays.

release the remaining cells. The viability of free cells was, in fact, lower (50% to 80%) than the viability of cells obtained with enzymes (96 % to 99 %).

Replication of MuLV in spleen cells cultured in diffusion chambers in comparison with in vitro cultures

The infectious center (IC) assay described in " Material and Methods " was used to determine

what proportion of spleen cells recovered from chambers implanted into MuLV-infected, syngeneic mice replicated MuLV. A comparison was made between these cells and cells obtained from the spleens of chamber-bearing mice (i.e. spleen cells infected in situ). Furthermore, normal spleen cells pooled from several donors were infected with MuLV-F in vitro and maintained in short-term cultures (see " Material and Methods ") and then tested for IC. Results obtained in several experiments are summarized in Table IV.

The proportion of lymphocytes in diffusion chambers that released infectious MuLV (i.e., infectious centers) increased from 0.4% on the 5th day to 1.3% on the 9th day, 5 % on the 15th day and 9 % on the 19th day after implantation and infection. Corresponding values in the spleens of virus-infected, chamber-bearing mice were 0.1 %, 474, 33% and loo%, respectively. Thus, the rates of infection of spleen cells cultured in chambers and cells infected in situ were comparable during the 1st week. During the course of the 2nd and 3rd weeks,

FIGURE 2 A (Figure 2 B is on facing page)

Immunofluorescent staining of spleen cells recovered from MuLV- F-infected diffusion chambers with a rabbit anti-Friend virus serum and with goat anti-rabbit Ig serum conjugated to FITC. Cells were recovered with elastase and collagenase on day 12 after the infection. A: Cells stained with no further treatment; B: cells incubated for 2 h in a culture medium at 37°C in air/5% CO,, prior to the staining. Photographed on gaf 500 color slide film using a Zeiss fluorescent microscope with FITC filter, a 40 x objective and a lox eyepiece. The total enlargement is approximately 2,000 for both pictures.


the infection within the chambers proceeded at a slower rate than in the spleen (Fig. 3). In comparison, spleen cells infected and maintained in vitro contained only 0.01 % IC, or less, on day 5 and 0.3 % IC on day 9 (Table IV); this is 5-10 times less than the values found in the chambers. It is seen from the assay at day 5 that the appearance of IC in vitro was not increased by repeated infection of the cultured cells with fresh virus (Experiment B, Table 11).

Infection of lymphocytes in diffusion chambers is independent of infection of the host

The membrane filter of the diffusion chambers (0.22,~) is permeable to MuLV. This raises the question of whether the replication of the virus in cells within the chambers occurs in the confined

population independently or whether the virus, in fact, replicates in the tissues of the host, enters the chamber and infects the cells. To demonstrate that the primary infection of cells in the chamber occurs in a host lacking the ability to support virus replication, we employed: (1) lethally irradiated mice; and (2) mice resistant to the strain of MuLV.

BALB/c mice were irradiated with 750 rads 20 h prior to the implantation of chambers containing a suspension of normal spleen cells. Friend virus (2-6 x lo3 FFU) was injected IP immediately after the surgery. Eight days after infection IC both in the chambers and in the spleens of chamber-bearing, irradiated mice were determined. The number of IC in the cell suspension from chambers ranged between 1 and 25 x lo3 per los cells, whereas very few, if any, IC were found in the hosts’ spleens (Table V). The

FIGURE ZB

184 CERNY ET AL.

proportion of IC found in this experiment is comparable to the number of IC in chambers cultured in non-irradiated mice (compare Tables V and IV).

In the next series of experiments, advantage was taken of the genetic restriction of MuLV-F infection regulated by the FV-1 gene (N-, B-tropism) (Lilly and Pincus, 1973). Two strains of mice were employed: BALB/c (H-2d, FV-ld, susceptible to B- tropic MuLV-F and resistant to N-tropic virus) and DBA/2 (H-2d, FV-In, susceptible to N-tropic virus).

Normal BALB/c spleen cells were placed in the chambers which were then implanted into normal DBA/2 mice. B-tropic MuLV-F was both added into the chambers (approx. 1-2 x lo4 FFU) and injected IP into the recipients after implantation (approx. 2-6 x lo3). In the reciprocal experiments, DBA/2 spleen cells were cultured in BALB/c mice and infected with N-tropic MuLV-F. Thus, in each situation, the cell population in the chamber was permissive to the virus, whereas the host was resistant to it. A positive control included BALB/c spleen cells implanted into BALB/c recipients and infected with B-tropic MuLV-F.

Results of infectious focus assays on days 5 and 8 are summarized in Table VI. The number of IC in the chamber (i.e. in the virus-susceptible cells) ranged from 1 to 10 x lo3 per million on day 5, and from 1 to 2 0 ~ 1 0 ~ per million on day 8. There was no apparent difference between chambers containing BALB/c and DBA/2 lymphocytes. In contrast, very few if any IC were found in the spleens of chamber- bearing mice (i.e. in the virus-resistant spleen cell population). Notably, the highest numbers of IC occurred in the spleens of mice bearing chambers with the highest degree of MuLV replication; however, the number of IC in the spleen was 10-100 times lower than in the chambers (mouse No. 2 and mouse No. 7, Table VI).

In the situation when both the cultured cells and the recipients were susceptible to the virus (group E, Table VI) similar numbers of IC were found in the chambers and in the spleen. Furthermore, these numbers were comparable to those of IC in the chambers implanted into virus-resistant recipients (compare IC in group E with IC in the chambers in groups A to D) and these data were also comparable to previous findings (Table IV). It would appear from these results that the replication of MuLV in chamber cultures does not depend on the supply of infectious MuLV from the host.

Enzyme treatment does not inhibit infectious centers in cells recovered from chambers

To determine whether the enzymes interfere with the detection of enzyme-retrieved cultured lymphocytes as infectious centers, the following experiment

was designed. Chambers with cells incubated in MuLV-F-infected hosts for 7 days were opened and two cell fractions were collected: free cells (floating in the liquid portion of the chamber content) and clots. Cells from the clots were recovered with enzymes. The results of the PC assay (Table VII) show that the number of MuLV-replicating cells recovered from the clots was as high, if not higher, than that in free cells.

Virus-induced cell membrane antigen on the surface of chamber-cultured lymphocytes

The process of alteration of lymphocytes by MuLV in diffusion chambers was monitored further with a membrane immunofluorescence assay for virus-induced cellular antigen(s) on viable, non-fixed cells. The expression of this antigen(s) seems to be a correlate of MuLV-F leukemogenesis in that it closely parallels the progression and regression of the splenic tumor (Cerny et al., 1975). At first, the indirect staining of enzyme-recovered cells (using either mouse anti-FVMA serum plus goat anti- mouse Ig fluorescein-conjugated or rabbit anti-FV, RaFV, plus goat anti-rabbit Ig fluorescein conjugate), failed because of non-specific staining. Following enzyme treatment, the cells were enlarged and, upon staining with any fluorescein conjugate, showed a diffuse, yellow-orange cytoplasmic fluorescence with many vacuoles containing the dye (Fig. 2 ~ ) . However, this non-specific staining was greatly diminished by the incubation of enzyme-treated cells in RPMI 1640 medium containing 5 % fetal calf serum for 1-2 h at

TABLE VIII

VIRUS-RELATED CELL MEMBRANE ANTIGEN (MA) ON SPLEEN CELLS CULTURED IN DIFFUSION CHAMBERS '

~

Percentage MA-positive cells a. Cells Virus infection Days after infection :

8-10 14 of host

Chamber-

spleen cells - < l o s < lo Spleen from

carrying host - <lo <lo

cultured + 31 (14-40) 48 (44-55)

chamber- + 38 (18-50) 83 (70-90)

' Spleen cells from normal, non-infected mice were implanted into normal syngeneic recipients which were then infected with MuLV-F. - 2 + = 2 to-6 x los splenic FFU MuLV-F, IP. Non-infected controls (-)were given either saline or no injection. - Cells with membrane fluorescence after incubation with a rabbit anti-Friend virus serum (RaFV) followed by a goat anti-rabbit Ig fluorescent conjugate. -

Arithmetic mean (range is group of six chambers or spleens). - 1 Less than 10% non-infected cells showed a membrane fluorescence with RaFV and GaRlg-FITC; however, the non-specific fluorescence was often less bright (with yellow rather than bright green color). No fluorescence was seen on cells incubated with normal rabbit serum and GaRIg-FITC (data not shown).


Upon such “ regeneration ”, most cells assumed their round appearance while specific, bright-green membrane fluorescence became detectable (Fig. 26). This procedure also permits staining for surface immunoglobulin (Cerny et al., 1976) and was used routinely in subsequent assays.

Table VIII shows the results of an assay of cells cultured in MuLV-F-infected diffusion chambers, at days 10 and 14. About 30% of the cells were positive for virus-induced membrane antigen on day 10 and 45-55 % cells were positive on day 14. Corresponding 37” C in air/CO,, prior to the fluorescent assay. values in the spleen of virus-infected carriers of the

DAYS AFTER INFECTION (non-infactad 1

FIGURE 3 Kinetics of infectious centers (IC, and 0) and cell

membrane antigen (MA, A and A ) in spleen cells infected with MuLV-F either in diffusion chambers (filled symbols, -) or in the spleen in situ (empty symbols, _ _ _ _ - ) see the text for further explanation). Both IC and MA-positive cells were expressed as a percentage of total cells recovered from the chamber or from the spleen. The background of MA staining in non-infected cells was less than 10%; no IC were detectable in these cells. Each point represents a mean from four to eight chambers or spleens; vertical bars indicate range. (The value for IC in chambers on day 20 is a result of assay of cells pooled from two chambers.)

chambers were about 40% and 70-90%, respectively. Less than 10% of non-infected cells were found to have some degree of staining.

A comparison of the data in Tables VIII and I V suggested that in virus-infected lymphocytes the proportion of membrane antigen (MA)-positive cells (Table VIII) is greater than that of infectious centers (Table IV). Data from these Tables, as well as results from additional experiments, are plotted in Figure 3. It is apparent that, during the first week of MuLV-F infection, MA positively exceeds virus replication, both in chamber-cultured cells and in in situ spleen cells. Furthermore, in the spleen, the number of both MA-positive cells and infectious centers increases steadily, reaching 80% or more during the 3rd week of infection. In comparison, the process of infection in spleen cells within chambers appears first to increase and then to level off with about 50% of cells MA-positive and only about 5-10 % of cells releasing infectious MuLV.

DISCUSSION

A new method for infection of normal, mature splenic lymphocytes in suspension, with Friend virus complex (MuLV-F) is described. Cells were cultured in diffusion chambers implanted into the peritoneal cavities of mice and infected with virus added into the chambers and/or injected IP into the recipient mice. The infection was monitored by enumerating the individual lymphocytes with replicating leukemia virus, as infectious centers (IC). The assay of serially diluted single-cell suspensions indicated that the IC test employed here does, in fact, reflect the number of individual lymphocytes releasing MuLV (Table I). Under the conditions described, infection of spleen cells with MuLV in diffusion chambers was about 10 times higher than infection in vitro. In further studies we show that, with purified B-cells, this difference becomes 1,000-fold, highly favoring the diffusion chamber culture system (Cerny et al., 1976). It appears that the environment of diffusion chambers may provide the nutritional and/or hormonal factors which exist in vivo and are required for susceptibility of lymphocytes to oncornavirus infection. In fact, during the 1st week after MuLV administration, the rate of infection of lymphocytes cultivated in the chamber was comparable to that in situ in the spleen, as judged by both the number of cells replicating MuLV (i.e. infectious centers) and the proportion of lymphocytes expressing virus-related membrane antigen (MA) (Fig. 3). Later in its course, the infection in chambers (both MA expression and virus replication) continued at a slower rate compared to the spleen. This might be partially due to a “crowding” of cells in the limited space of the chamber,

186 CERNY ET AL.

Friend virus complex used in our studies contains defective, pathogenic, splenic focus-forming virus (SFFV) and murine lymphatic leukemia helper virus (LLV’) (Rowson and Parr, 1970; Fieldsteel et al., 1969; Sleeves et al., 1971). The sarcoma-positive, leukemia-negative (S’L-) cells used in our assay form foci of cellular transformation upon super- infection with murine leukemia virus of various strains (Bassin et al., 1970, 1971); presumably, the assay detects the MuLV component of the Friend virus complex, i.e. the LLV, as does the XC test (Lilly and Pincus, 1973). However, to determine whether the presence of SFFV in the complex interferes with the LLV assays requires further experiments with isolated LLV.

We have no evidence that the cells infected in chambers actually reached the stage of leukemic transformation. The dissociation between oncornavirus replication and its transforming ability has been demonstrated (Martin, 1970). Nonetheless, the observation of MA on infected cells is significant because previous studies showed that the assay for MA correlated quite well with the progression and regression of MuLV-F-induced splenic tumor, better in fact than the assay for virus group-specific antigen in cytoplasm of lymphocytes (Cerny et al., 1975). The kinetic data from Figure 3 imply that the MA detected here is related to early events in the infection. Experiments to determine whether cells infected with MuLV in diffusion chambers form leukemic colonies upon transfer into a second host are in progress.

The low efficiency of infection in vitro could be an artifact related to the culture conditions and nutritional value of the media. Some of these parameters have been tested in preliminary experiments in our laboratory. Very little, if any, difference in degree of infection was found between lymphocytes infected and maintained in culture tubes (as described in “ Material and Methods ”) and those cultured in Marbrook chambers, even though the latter provides an environment allowing lymphocytes to survive better and to maintain their immune competence (Marbrook, 1967). Sera from various species and/or different species were tested but no consistent improvement of the infection was observed (data not shown).

A significant increase in susceptibility of lymphocytes to MuLV in vitro in the presence of mitogenic or antigenic stimuli has been observed. Sklar et at. (1974) found that murine lymphocytes, stimulated with a mitogen and infected with MuLV in a short- term culture system and then transferred in vivo, underwent leukemic transformation in the host ; the mitogen stimulation was necessary to achieve subsequent transformation (Raschke et al., 1975). Antigenic stimulation, too, may enhance the suscep-

tibility of lymphocytes to MuLV, as shown in our studies on virgin and antigen-sensi‘ized B-cells (Cerny and Waner, 1975). Similarly, Datta et al. (1 975) showed that activation provided by mixing allogeneic lymphocytes in vitro alters the susceptibility of the cells to MuLV infection. The usefulness and limitations of these manipulations for studies of infection with MuLV will be discussed in greater detail in another paper in which we describe the enhancing effect of polyclonal mitogens on MuLV infection of purified B- and T-cells (Cerny et al., 1976).

The apparent superiority of the diffusion chamber culture system over an in vitro culture might parti- cularly apply to models in which the initial multiplicity of infection is low. In the present experiments, the cells were infected at a ratio of to 10-2.4 FFU/cell depending on the MuLV titer in a given virus passage. In a few experiments, we also used the higher-titered preparation of B-tropic MuLV-F from Dr. F. Lilly to infect lymphocytes both in vitro and in diffusion chambers at a multiplicity of infection of 10-l.G FFU/cell; however, the degree of infection obtained (results not shown) was comparable to that described above.

Another advantage of diffusion chambers is that they support differentiation of xenogeneic cells and can be used to study human cells (Barr et a/., 1975). Since our data show that the infection of the cells within the chamber does not require virus replication in the host, the system could be used for studies on xenogeneic cell-virus interaction.

Among the disadvantages of the method, one should mention the development of a clot inside the chambers and the need to recover cells by the enzyme treatment. This has been found previously by other investigators (Capalbo et al., 1964; Borella, 1969). In our hands, the enzyme treatment did not appear to interfere with the assays for cell infection. However, this might be a matter of enzyme concentration which should be empirically determined for each model.

ACKNOWLEDGEMENTS

The authors thank Dr. F. Lilly for a generous gift of N- and B-tropic strains of MuLV-F, Dr. D. Livingstone for the supply of SfL - cells (FG-10) and Dr. Max Essex from the Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts for a critical review of the manuscript. This work was supported by Grant No. IM-35C from the American Cancer Society and in part by US Public Health Service Grants No. CA-14922 and No. CA-15277 from the National Cancer Institute.


INTERACTIONS DU VIRUS DE LA LEUCEMIE MURINE (MuLV) AVEC DES LYMPHOCYTES ISOLES. I. REPLICATION DU VIRUS

DANS LES LYMPHOCYTES INFECTES AVEC LE VIRUS DE FRIEND ET CULTIVES EN CHAMBRES DE DIFFUSION IN VIVO

Les auteurs ont dkcrit une nouvelle technique d’infection des lymphocytes mars avec le virus de la leucimie murine souche Friend (MuL V-F) . Des cellules splkniques de donneurs normaux, non infectis, ont ktk plackes dans des chambres de diflusion (confectionnies avec des firtres Mi[lipDre dont les pores mesurent 0.22 pm) qui ont kt.4 ensuite implantkes dans la caviti piritonkale de souris synginiques normales. Les cellules ont i t& infectkes au moyen d’une injection de MuL V-F dans la cavitk pe‘ritoniale ou, dans certains cas, par introduction directe du virus dans les chambres. Les cellules ont it6 recueillies par traitement du contenu des chambres avec un mklange d’klastase et de collaginase. L’infection a ktk apprkcike de deux facons: 1 ) les cellules dans lesquelles le MuLV se rkplique (centre infectieux ou IC) ont k t t dknombrkes sur des cellules indicatrices S+L-, et 2 ) I’antigtne membranaire ( M A ) lie‘ au virus a ;ti dttectt par immunofluorescence. Les cellules rkcupirkes dans les chambres aprts deux a trois semaines de culture reprksentaient ir peu prts 10% de I’inoculum initial; la viabilite‘ ktait approximativement de 90 %. Le nombre d’lC dans les chambres infectkes par le MuL V-F ktait environ 10 fois plus klevk que celui que I’on a relevk aprts infection et culture des cellules splkniques in vitro. La cinktique des IC et des M A dans les cellules splkniques infectkes pnr le MuLV-F et cultivies en chambres de diffusion est analogue a celle que l’on observe dans la rate des souris infzctkes pendant les dix premiers jours aprts I’infection. Par la suite, le processus infectieux s’est ralenti dans les chambres et l’on a dinombrk environ SO% de cellules MA-positives et yuelque 10% d’lC, tandis que dans la rate, le nombre d’IC et de cellules MA- positives atteignait 80 % ou plus. L’infection des lymphocytes splkniques dans les chambres de diffusion se produit aussi bien lorsque les chambres sont implantkes chez I ) des hdtes syngkniques sensibles au virus, 2) des hdtes synginiques lktalement irradiis et 3 ) des hates allogkniques rksistant au virus, ce qui conduit a penser que le processus est indkpendant de la replication du MuL V dans les tissus de la souris porteuse de la chambre de diffusion. La technique des chambres de diffusion selnble fournir un environnement dans lequel divers types de lymphocytes isolks de diffkrentes souches murines peuvent interagir avec le MuL V presque aussi eficacement qu’in vivo.

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