Characterization of target cells for MCF viruses in AKR mice

Cell, Vol. 32, 217-225.

January

1983,

Copyright

0 1983 by MIT

Characterization of Target Cells for MCF Viruses in AKR Mice Miles W. Cloyd National Institute of Allergy and Infectious Diseases Laboratory of Persistent Viral Rocky Mountain Laboratories Hamilton, Montana 59840

Diseases

Summary The recombinant (MCF) class of murine leukemia virus appears to play an important role in lymphomagenesis in AKR and other mice. Although much effort has been extended in characterizing MCF viruses, relatively little is known about the cells they infect. I examined what cells were targets in AKR mice for both lymphomagenic and nonlymphomagenie MCF viruses. Lymphomagenic MCF viruses of thymic origin (AKR-247 and C58Ll) were found to infect and replicate selectively in immature lymphocytes only present in thymic cortex, whereas nonlymphomagenic MCF viruses of splenic origin (C58v-1 -C77 and C58v-2-C45) selectively infected and replicated in cells that appeared to B lymphocytes. Virus-binding studies suggested that neither T- nor B-lymphocyte tropisms were determined by selective attachment of virus to the respective cells. These findings demonstrate that in contrast with ecotropic viruses, which can infect many types of cells in the mouse, specific cellular tropisms can exist for MCF viruses, and that MCF infection, and therefore oncogenicity, is closely linked to cellular differentiation. Introduction Spontaneous lymphomagenesis in AKR mice has been shown to be a complex, autogenous disease process involving the interplay of various host genes, including endogenous retroviruses (murine leukemia viruses), as well as nongenetic elements (Gross, 1970; Steeves and Lilly, 1977; Rowe, 1978). Of the endogenous murine leukemia viruses (MuLV) present in AKR mice, at least two classes (MCF, ecotropic) appear to be directly involved in genesis of lymphomas. One of these, MCF virus, is a virus derived from recombination of ecotropic virus with endogenous xenotropicor MCF-like proviral gene sequences (Fischinger et al., 1975; Hartley et al., 1977; Rommelaere et al., 1978; Chattopadhyay et al., 1981; Chattopadhyay et al., 1982). Isolates of this type of virus have been shown to be quite heterogeneous, and certain ones have been implicated as the proximal transforming agent in lymphomagenesis in AKR and other mice (Hartley et al., 1977; Cloyd et al., 1980; Green et al., 1980; O’Donnell et al., 1981; Bedigan et al., 1981). The other virus, ecotropic virus, is inherited in AKR mice as two or more chromosomal loci (Rowe, 1972;

Chattopadhyay et al., 1980; Steffen et al., 1980; Quint et al., 1981; Yoshimura and Breda, 1981) and serves during lymphomagenesis not only to generate MCF viruses but also to facilitate MCF infection of the host (Cloyd et al., 1981). Although knowledge concerning the details of the viral involvement in AKR lymphomagenesis is increasing rapidly, the system is quite complex and mechanisms still remain obscure. It is well known that endogenous MuLVs, in general, are not able to transform many kinds of cells that they can infect (fibroblasts in vitro, for example). Usually, oncogenicity is only revealed by the induction of transformation of a certain type of cell in the mouse after latencies of months and only certain viruses appear to have this property. The reasons for selective oncogenicity based on cell and virus type are not entirely clear. Although relatively little has been done with the cells involved, a great deal of study has been focused on characterization of the viruses. It has been shown that the MCF viruses that are lymphomagenic efficiently infect and replicate in vivo and have ecotropiclike gene sequences in the far 3’ region of their genome (Lung et al., 1980; Cloyd et al., 1980; O’Donnell et al., 1981 ; Chattopadhyay et al., 1981). Nonlymphomagenic MCF viruses do not have the same 3’ sequences and do not replicate as well, if at all, in vivo, although they can replicate in fibroblast cultures in vitro. It also has been demonstrated that MCF viruses that do replicate in vivo often are restricted to certain organs (O’Donnell et al., 1980; Cloyd et al., 1980). This apparent fastidiousness of MCF viruses in mice probably reflects important aspects of virus-cell interaction, and may be an important property that could, in part, be responsible for expression or absence of oncogenicity. Since MuLV oncogenicity is so closely associated with cell type, it seems warranted that studies looking at oncogenicity of MuLVs also should examine the cells involved. The study reported herein examined the target cells for two types of MCF viruses. One type was the lymphomagenic MCFs of thymic origin from AKR or C58 mice, and the other was the nonlymphomagenic MCF viruses isolated from leukemic spleens of NFS mice congenic for C58v-7 or C58v-2. After injection into neonatal AKR mice, the lymphomagenic isolates were shown to be recoverable primarily from thymus but not spleen (Cloyd et al., 1980) while the MCF viruses from C58v congenic mice displayed the opposite pattern, that is, replication in spleen but not thymus (unpublished results). In each case, only a small percentage of cells registered as infected by infectious center assays, which invoked further questions about which cells within a given organ were being infected. Using cell selection or depletion procedures, I found that the lymphomagenic MCF viruses selectively replicated in cortical (immature) T-lymphocytes in the thymus of AKR mice and not exclusively in thymic

Cell 218

epithelial or stromal cells. The nonlymphomagenic MCFs from C58v congenic mice likewise preferred hematopoietic cells, but these were probably B-lymphocytes. Furthermore, it appeared that differential binding of the viruses to the cell surfaces could not account for the difference in cell tropisms. These results demonstrated the existence of differentiationassociated cellular mechanisms involved in the biology of MCF viruses.

Infectious ecotropic virus, endogenous to AKR mice, was detected in all of these organs (data not shown). The age of the animal at the time of inoculation was found to be important for recovery of the MCF viruses. Neither of the C58v congenic MCF viruses could be recovered by infectious center assays if the animal was greater than 2 weeks of age at the time of inoculation. In contrast, AKR-247 or C58Ll MCF viruses replicated efficiently in mice injected at 4 weeks of age (Table 1). It was evident that these two subclasses of MCF virus selectively replicated in different organs, with age-dependent susceptibility to infection.

Results Organ Tropism of Two Subclasses of MCF Virus Cloned MCF viruses, two lymphomagenic and two nonlymphomagenic, were individually injected intraperitoneally or in the region of the thymus in 2-5-dayold AKR mice, and various organs were tested for replication of MCF virus 6-8 weeks later (Table 1). The two lymphomagenic isolates (AK&247, C58Ll), originally derived from prelymphomatous or lymphomatous thymus, were recovered only from thymus of these inoculated mice, and not from spleen, bone marrow, mesenteric lymph node, brain or pancreas. In contrast, the nonlymphomagenic MCF isolates from leukemic spleens of NFS.C58v-7 and NFS.C58v-2 mice were found in spleen, bone marrow and lymph node, but not in thymus or the nonlymphoid organs tested. This pattern of virus recovery did not change with alteration of route of inoculation; both intraperitoneal or parathymic routes gave the same results. Table

1. Organ

Tropisms

of Four MCF Viruses Quantity

MCF Virus Inoculateda

Age at Inoculation

No. Mice Testedb

AKR-247

2-5

C58Ll

C58v-2-C45

C58v-l-C77

Target Cells for the MCF Viruses The organ tropisms of these MCF viruses may be due to preferential infection of cells unique to each organ. Specific cell depletion or selection procedures were used to examine what these target cells might be (Table 2). Anti-Thy-l and complement (C’) treatment of thymocytes from mice injected with AKR-247 or C58Ll MCF viruses eliminated greater than 99% of the MCFpositive cells. Treatment with anti-Thy-l alone or C’ alone had no significant effect. The target cells for these two viruses therefore expressed the Thy-l Tlymphocyte antigen. Filtration of dispersed thymus cells through nylon wool or their incubation on plastic, which largely removes B-lymphocytes and stromal (fibroblastic) or macrophage cells (Julius et al., 1973;

of MCF Virus

Recovered

from

ThymusC

Spleen”

Lymph Node’

Bone Marrow”

Braind

Pancreas?

7

6.000-14,000” (10.000)

0 trace’ (0)

0

0

0

0

4 weeks

5

5,000-l (9.000)

2,000 0

0

0

0

0

2-5

5

7,000-l (11,000)

5,000 0

0

0

0

0

4 weeks

5

5.000-12.000 (9.000)

0

0

0

0

0

2-5

6

0 trace (0)

34-560 (220)

79-520

(200)

52-600 (250)

0

0

4 weeks

5

0

0 trace (0)

0

0

0

0

2-5

5

0 trace (0) 0

68-450

29-370 (98) 0

35-580 (240) 0

0 0

0 0

days

days

days

days

4 weeks

5

(210) 0

’ AKR mice were inoculated with 103.‘-104.’ mink cell focus-forming units (FFW of designated viruses intraperitoneally. b Mice were killed 5-6 weeks postinoculation. Control (uninoculated) mice express no detectable endogenous ’ Mink cell FFU per 10’ cells. ’ Mink cell FFU per 0.2 ml of 20% tissue extract. ’ Range (median value). ’ Trace amounts of MCF virus were revealed only after passage of the mink cells.

in the

region

of the

MCF virus at these

thymus

ages.

or

MCF Target 219

Cells

Table 2. Characterization

of Target

Cells for MCF Viruses

by Specific

Cell Depletion

or Selection

Virus Inoculated AKR-247 Organ

Tested

(No.)

Thymus

C58Ll (5)

Thymus

C58v-1

-C77

C58w2-C45

(3)

Spleen

(4)

Spleen

(4)

Treatment None

11,700

Anti-Thy-l

a

Anti-Thy-l

* 1,700

11,000

‘- 580

225

f 56

296 f

74

f

1 ,100

10,300

2 870

185 f 55

286 f

79

14

123 f 33

222 * 70

7,400

f

780

7,300

-c 330

150 t 35

240 + 67

8,700

f

1 ,180

9,000

-c 580

8.680 + C’”

106%

C’a Filtration

Through

Adherence

Nylon Wool (Nonadherent

to Plastic

(Nonadherent

Cells)

Cells)

30

9,300

f

1,460

755

10.000

f

580

11 f4

22 c 9

180 f 47

256 f

68

Panningb Lyt-l+

Cells

7,700

f

660

7,600

-c 230

nd

nd

Lyt-2+

Cells

6.900

k

840

7,000

F 290

nd

nd

kg+ Cells

nd

nd

413

f 90

376 f

lg- Cells

nd

nd

152 f 52

112t30

nd

nd

Steroid

(Lysis

In Viva)

52 +

30

631t

18

79

Data are number of MCF infectious centers (mean ? SE) per 10’ cells after treatments. Following treatments, serial dilutions of cells were overlayed on mink lung indicator cells, with foci characteristic of MCF viruses scored 5-7 days later. a 10’ cells were treated and all cells that remained were tested for MCF virus. ’ 90%-98% of cells not attached to dish during panning procedure was negative for the selection antigen, and 96%-l 00% of attached cells was antigen-positive by membrane immunofluorescence. nd = not determined; no Ig+ cells were recoverable from thymus, and exclusive selection of Lyt-1 + or Lyt-2+ cells from spleen suspensions was not possible, since the panning method used also selected lg’ cells.

see Unanue, 1972) also did not significantly reduce the number of MCF-positive cells. These data therefore indicated that these two MCF viruses predominantly replicated from T-lymphocytes in the thymus, and not exclusively from thymic stromal or epithelial cells. Thymus cells from 247 or C58Ll MCF-inoculated mice were further fractionated based on Lyt-1 or Lyt2 phenotype or on glucocorticoid sensitivity (cortical cells lysed, medullary cells resistant). The MCF target cells appeared to express either or both Lyt-I and Lyt-2 antigenic markers as determined by the panning procedure, because cells selected for each marker replicated MCF virus (Table 2). Thus there was no mutually exclusive Lyt-7 or Lyt-2 antigenic profile characteristic of these target cells. However, administration of the glucocorticoid, dexamethasone, eliminated nearly all the MCF-positive cells from thymuses of these mice, showing that the targets for 247 or C58Ll MCF viruses were cortical lymphocytes rather than medullary cells (Table 2). Cortical depletion and medullary sparing was confirmed by histological examination (data not shown). In contrast to those results with lymphomagenic MCF viruses, anti-Thy-l + C’ treatment had little effect on the frequency of MCF-positive cells from spleens of mice inoculated with either of the nonlymphomagenic MCF viruses from C58v congenic mice, whereas filtration through nylon wool did (Table 2). These target cells also did not adhere to plastic, so

they were not likely stromal or macrophage cells. The obvious candidates were B-lymphocytes, which lack Thy-l, do not adhere to plastic, but do adhere to nylon wool. Furthermore, since mature B-lymphocytes express immunoglobulin (lg) on their surfaces, spleen cells from inoculated mice were fractionated into cells lacking or expressing surface lg by the panning procedure and each population was tested for replicating MCF virus (Table 2). A greater percentage of MCFpositive cells were found in the population of cells expressing lg on their surface than in the same number of unfractioned spleen cells. Nevertheless, some MCF-positive cells also were found in the cell population lacking surface lg. If B-lymphocytes are the targets for these MCF viruses, perhaps some are in the pre-B stage when they lack surface lg. Specific Stimulation of MCF Target Cells by Mitogens Mitogens (Con A, LPS) that specifically stimulate certain populations of lymphocytes were used to further define the target cells for the two types of MCF viruses. Representative examples of these tests are illustrated in Figure 1, but at least 2 mice inoculated with each MCF (AKR-247, C58L1, C58v-l-C77, C58v-2-C45) were tested. Only results of testing spleen cells are reported, because thymus cells were, at most, poorly responsive to the mitogens as judged by incorporation of 3H-thymidine. Therefore, to test mice inoculated with the thymotropic MCF viruses

Cdl 220

LPS stimulation significantly increased the amount of MCF virus recovered from spleen cells of mice inoculated with either C58v MCF virus (Figure 1 B), but Con A did not. These data support the cell depletion or selection data above, showing that target cells for these two types of virus appear to be T- and B-lymphocytes, respectively.

-220

-180

-140 r -100

3 : Jj

-60

...kJ.

,,,,,,,,,,,,,,,,,,,,,

-20 8

3000

-

'

m 0

; 0

.

-300

5

2 -260

6 k 0

.220

Y

.180

Y 6 zj : +

0

1

5 MITOGEN

IO

20 CONCENTRATION

30

40 ( ug/ml

)

Figure 1. Effects of Mitogen Stimulation on Number of MCF tious Centers from Spleens of MCF Virus-Inoculated Mice

Infec-

(A) Spleen cells taken from an AKR mouse inoculated with AKR-247 MCF virus 3 months previously were cultured for 46 hr in various concentrations of mitogens Concanavalin A (Con A) or lipopolysaccharide (LPS). Stimulation of cell division was monitored in a 10 pl sample of cells by pulsing 6 hr with 3H-thymidine (O---O), and the number of MCF-positive cells (infectious centers; U) per 10’ cells sampled was quantitated on mink indicator cells. (B) Number of MCF infectious centers per 10’ cells and relative 3H-thymidine incorporation in mitogen-stimulated spleen cells from a 6-week-old AKR mouse inoculated with C5&-7X77 MCF virus as a neonate.

(AKR-247 or C58Ll), individuals were chosen which had not yet developed obvious lymphoma at 3-4 months postinoculation, because occasionally small numbers of MCF-positive cells can be detected in their spleens. These splenic MCF-positive cells were probably infected cells that had migrated from the thymus. In vitro stimulation of spleen cells from these older 247 or C58Ll MCF-inoculated mice markedly increased the amount of MCF virus recovered when a T-lymphocyte mitogen, Concanavalin A (Con A), was used. Stimulation by LPS, a B-lymphocyte mitogen, however, did not increase MCF virus recovery (Figure 1 A). 3H-thymidine incorporation showed that both mitogens stimulated spleen cells, although LPS stimulation was always less than that of Con A. In contrast,

Phenotype of AKR Lymphomas Induced by AKR247 MCF Virus The cells comprising lymphomas induced in AKR mice by AK&247 MCF virus were examined to compare them to the early target cells of the virus (Table 3). Similar to the cells infected early, lymphoma cells were almost exclusively T-lymphocytes, expressing large quantities of Thy-l. The percent of cells expressing Lyt-7 and/or Lyf-2 was quite variable from mouse to mouse, and sometimes from organ to organ within a mouse (A84-2, Al 14-5). This result also is compatible with the cell selection data described previously that showed early target cells did not preferentially express either Lyt-7 or Lyt-2. A large percentage of the cells of most lymphomas expressed MCF antigens, but this also varied somewhat between mice and even among different organs of individual mice (A73-5, A84-3). These antigens, however, stained very weakly, and some positive cells were likely missed. In general, many more cells were infected with MCF, as determined by MCF antigen expression, than were indicated by infectious center assays (Table 3). It was likely that most, if not all, of the cells in a tumor were infected. Even though only a few markers were compared, it appeared that the phenotype of lymphomas induced by MCF virus resembled the phenotype of early target cells for the virus. MCF Tropisms Appear Not to be Due to Differential Binding to Cells The specific cellular tropisms of the two types of MCF virus could be due to either or both of two mechanisms: a particular virus attaches to and infects only cells that have the corresponding receptors for that virus, and/or the viruses can infect many cell types but only certain cells support their replication. To distinguish if one or the other mechanism might be involved, I used virus binding assays to examine possible virus-cell attachment specificities. The basic assay was to mix infectious virus with dispersed thymus or spleen cells, allow the virus to attach to the cells, rinse off excess virus, and then overlay the cells on the mink lung indicator cells. Virus transferred with the cells was scored on the mink cells as MCF foci. To corroborate the results of this assay, I also used a similar assay with virus labeled with 3Huridine (see Experimental Procedures). To ensure that virus binding and not productive infection was being measured in both assays, I performed the binding and

MCF Target 221

Cells

Table 3. Phenotype

of AKR Lymphomas

Induced Percent

by AK!+247

of Cells Staining

MCF Virus for These

Antigensa MCF

Organ Tested

Thy-l

A73#3

Thymoma

100

56

95

60

80

0.08

A73#5

Thymoma

100

85

98

55

60

0.03

Mes Ln

100

85

100


40

0.01

Thymoma

100

100

46

82

68

0.06

Mes Ln

100

92

61

60

90

0.04

Thymoma

100

8

25

92

100

0.11


Mouse Number

A84#2

A84#3

Lyt-1

Lyt-2

Hy-40

Rab

Spleen

100

10

95

A84#1

Thymoma

100

95

100

61

87

A114#5

Thymoma

100

45

93

nt

15

0.07

Mes Ln

100

27

30

nt

90

0.04

Thymoma

100

nt

nt

43

50

A19


Percent fectious

a Indirect live-cell membrane immunofluorescence in which 100-200 cells were scored for surface staining. Monoclonal 1 and Lyt-2 are described in Experimental Procedures. MCF-specific monoclonal antibody Hy-40 and rabbit antiserum described elsewhere (Cloyd et al., 1979; 1982). Mes Ln = mesenteric lymph node: nt = not tested. b Percentage of cells registering infectious MCF virus in the mink cell focus assay.

rinsing reactions at 4°C and a control included elimination of the virus detected on the cells by short treatments with trypsin (Pincus et al., 1975; Figure 2). The latter treatment eliminated nearly all the detectable virus attached to cells, but did not diminish significantly virus detectable from productive infection (that is, endogenous ecotropic virus replicating from these AKR cells; data not shown). Both assays gave similar results (Figure 2); there was no apparent thymus or spleen cell specificity in binding of either type of MCF virus. Both AKR-247 and C58v-2-C45 MCF viruses bound similarly to thymus as well as to spleen cells from 1 O-l 4-day-old AKR mice, although thymus cells seemed consistently to bind slightly more virus than did spleen cells (Figures 2A, 28, 2C). Spleen cells from older mice (4 weeks of age) did not bind either virus significantly, whereas thymus cells did (Figure 2D). It thus seemed that obvious virus-cell attachment could not explain the cellular specificities of these viruses in mice. Discussion This study revealed that, unlike endogenous ecotropic MuLVs, which can widely infect tissues of the mouse, MCF viruses can have specific tropisms for different lymphocyte subpopulations in vivo, and these tropisms appear to be determined by postattachment mechanisms and not by virus-cell binding specificity. Such tropisms are obviously determined by properties of both virus and cell, but while considerable effort has been extended in defining the properties and structure of MCF viruses, little is known about their target cells.

MCF InCente&

0.05 0.09

0.10 antibodies to Thy-l, Lyt(Rab, diluted 1:80) are

This study showed that the major target cells for two lymphomagenic MCF viruses appeared to be a small population of lymphocytes present in the thymic cortex, which is reported to harbor mostly immature and dying lymphocytes (Bryant, 1972). Infectious center and membrane immunofluorescence tests of thymus cells from 247 MCF-inoculated mice have indicated that a very small number of cells were infected by 2-3 weeks after inoculation (-O.OOl%-0.01% of thymus cells; unpublished results), and that the number of infected cells eventually expanded to comprise nearly all the cells of tumors (Table 3). Similar findings have been reported for thymotropic RadLV, which appears to infect selectively the small population of primitive lymphoblasts in the thymic cortex with eventual spread to most of the cortical lymphocytes (Boniver et al., 1981). The possibility that minor cellular components of the thymus, such as stromal or epithelial cells, may be the only MCF target cells, with lymphocytes being transformed indirectly or infected after transformation, appears not to be the case. The thymic cells infected early by these lymphomagenic MCF viruses may be the cells eventually transformed, because their phenotype is not unlike that of the cells of overt lymphomas. Both early target cells and lymphoma cells induced by AKR-247 were not restricted to expression of either Lyt-1 or Lyt-2 phenotypes, which similarly has been shown for spontaneous AKR lymphomas (Mathieson et al., 1978). The nonlymphomagenic MCF viruses from spleens of C58v congenic mice infect different types of cells in the same mouse strain. These cells are not in the thymus, but are in spleen, lymph node and bone marrow. In the spleen, they lack Thy-l, adhere to

Cell 222

/

THYMUS

THYMUS+247

THYMUs+Cs&-l-c77

5

3.0

4.0

3.5

FFU OF AKR-247 IN REACTION

4.5

FFU OF MCF VfRUS N REACTION

MCF VIRUS (LOG,,)

Figure Mice

B

THYMUS

2zL5 SPLEEN

.

TsHplFZZ

2.5

3.5

3.0

FFU OF C58v-l-C77 IN REACTION

4.0

STV ‘REATMENT

4.5

MCF VIRUS (LOG,, 1

2. Binding

of MCF Virus to Thymus

(LOG,,) or Spleen Cells from AKR

(A) Binding of AKR-247 MCF virus to thymus or spleen cells from 12day-old AKR mice. 10’ ‘-1 O4 4 focus-forming units (FFU) of MCF virus were incubated with 10” dispersed thymus or spleen cells for 3 hr at 4°C. The cells were then rinsed and overlayed onto mink lung indicator cells with MCF virus-induced foci scored 5-7 days later. Treatment of the thymus or spleen cells for 10 min with saline containing 0.25% trypsin and 0.02% versene (STV) before overlaying onto mink cells showed that virus scored was virus attached to the thymus or spleen cells, and not virus replicating from the cells due to productive infection (the latter unaffected by trypsin treatments; unpublished results). (6) Binding of C58v-7-C77 MCF virus to thymus or spleen cells from lo-day-old mice. (0 Extent of binding of ‘Huridine-labeled AKR-247 MCF virus to thymus or spleen cells from 1 a-day-old AKR mice. Indicated number of cells was incubated with 1036 FFU of virus (40.000 cpm) in 0.25 ml volume for 3 hr at 4°C as described above. The cpm remaining with rinsed cells minus the cpm remaining with controls (no cells) is plotted against the number of cells in reaction. (D) Binding of AKR-247 or C58v-I-C77 MCF viruses to thymus or spleen cells from 4-week-old AKR mice.

SPLEEN THYMUS

SPLEEN THYMUS 4

;

6

NUMBER OF CELLS IN REACTION

STV I TREATMENT

7 (LOG,,)

nylon wool but not plastic and are stimulated by LPS but not Con A in vitro. These are characteristics of Blymphocytes. Not all of these cells appear to express surface lg, which could indicate that if they are all Blymphocytes, some may be in early stages of differentiation. In the bone marrow, pre-B cells are especially prevalent, being as frequent as mature B-lymphocytes (Ryser and Vassalli, 1974). The finding that

only mice younger than 2 weeks of age can be infected with these viruses may be explained by the finding that spleen cells from weanling mice did not bind MCF virus well. Itis of additional interest that the predominant type of neoplasm occurring naturally in the NFS mice congenic for various murine ecotropic viral loci are B-cell lymphomas (T. Frederickson and W. P. Rowe, personal communication). It seems likely, then, that the target cells for both types of MCF virus are probably cells in early or intermediate stages of differentiation and ontogeny. In fact, end stages of differentiation may, in the case of 247 targets, suppress expression of virus, since organs (spleen, lymph node) seeded from thymus but possessing mature T-lymphocytes do not express much, if any, detectable MCF virus. Of course, an alternative explanation would be that the 247 target cells may not even leave the thymus until after transformation. Virus suppression upon cellular maturation, however, appears’not to occur with the target cells for the B-cell tropic MCFs, because virus was expressed from mature (Ig-bearing) cells, and was expressed long after the time when they can be infected by inoculation. It may be further suggested that early stages of differentiation are required for MCF infection

MCF Target

Cells

223

rather than just cell proliferation, because it has been shown that MCFs do not infect mitogen-stimulated proliferating mature T-lymphocytes (Horak et al., 1981). The virus binding studies showed that either type of MCF virus could attach equally well to thymus or spleen lymphocytes from suckling mice, and that the specific cell tropisms apparently cannot be accounted for by differential virus-cell binding. Similar nonspecific cell-binding properties have been shown for the avian retroviruses (Piraino, 1967). It appears that retroviruses can largely bind to cells in a nonspecific fashion, but penetration or perhaps postpenetrational mechanisms may be specific. It is not known at this time whether specific penetration or postpenetrational mechanisms are determining the lymphocyte tropisms of the two MCF types studied here, but work is in progress to elucidate the mechanism. In several cases examined, lymphomagenesis does not occur or is diminished when the virus replicates suboptimally (Cloyd et al., 1980). Recovery of the nonlymphomagenic B-lymphocyte-tropic MCFs from mice is much less efficient than the recovery of lymphomagenic MCFs, and it also could be that the degree of virus production is controlled by the differentiation pathway of the cell. If so, it would indicate that the cellular mechanisms involved in MCF replication and associated with the differentiated state of the cell may also be important in MCF transformation. That the tropisms demonstrated for these viruses may be important determinants of pathology (oncogenicity) appears quite likely, because any mechanism(s) involved in infection and replication of the virus should be important. These results demonstrate that MCF viruses follow other examples of differentiation-associated retrovirus oncogenicity, such as the effects exerted by a truncated MCF virus, Friend SFFV, on the proliferative properties of cells of the erythrocytic lineage (Evans et al., 1980; Hankins and Troxler, 1980; MacDonald et al., 19811, or the oncogenic specificity of avian acute transforming viruses on various hematopoietic cell lineages (Graf and Beug, 1978). It also is possible that the MCF viruses generated in mice during expression or infection by endogenous or exogenous ecotropic MuLVs (for example, AKR or C58 mice for the former; inoculated Friend, Rauscher, Moloney, Gross MuLVs for the latter) are determining the resulting specificity of neoplasms observed, especially because the ecotropic parental viruses show very little tissue or cell tropisms and widely infect the mouse (Rowe and Pincus, 1972; Nagao et al., 1978, unpublished results). Experimental

Procedures

Viruses and Mice The viruses originated from the following mice: AKR-247 MCF virus from the thymus of a g-month AKR mouse (Hartley et al., 1977);

C58Ll

MCF

from a spontaneous thymoma of a C58/Lw mouse; MCF from a spontaneous splenic neoplasm of a NFS.C58v-1 mouse; and C58v-2-C45 from a spontaneous splenic neoplasm of NFS.C58v-2. All viruses have been cloned by three cellfree passages at limiting dilution; most were gifts from J. Hartley. The lymphomagenic properties of the viruses have been described previously (Cloyd et al., 1980). AKR/J mice (Jackson Laboratory. Bar Harbor. Maine) were bred and maintained at Rocky Mountain Laboratories. Mice were injected with approximately 1 O3 ‘-” FFU of MCF virus either intrathoracically, in the region of the thymus. or intraperitoneally. C58v-l-C77

Virological Assays Infectious MCF virus in tissues was quantitated by plating serial dilutions of dispersed cells (from hematopoietic organs) or 0.2 ml of a 20% homogenate (pancreas, brain) onto cultures of SC-l (Hartley and Rowe, 1975) and mink lung (ATCC-CCl64) cells that were plated at 2 x 10’ cells per 60 mm dish the previous day in Dulbecco-Vogt modification of Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The culture medium containing 8 pg/ml polybrene was changed the following day with medium without polybrene, and 2 days later the SC-l cells were killed by ultraviolet irradiation and overlayed with 2 X 1 O5 mink cells as described by lshimoto et al. (1977). This “UV-mink” procedure is able to score, in addition to complete MCF viruses. MCF viruses that are phenotypically mixed with ecotropic virus envelope host-range determinants. The mink or UV-mink cultures were scored under a dissecting microscope for foci characteristic of MCF virus when confluent 5 and 7 days after infection. Specific Cell Selection or Depletion Procedures Anti-Thy-l Plus Complement Treatment Thymus or nucleated spleen cells (20 X 1 06) were incubated for 1 hr at 4°C in 2 ml of anti-Thy-l monoclonal antibody 31-11 (McGrath et al., 1980). provided by I. Weissman. This amount of antibody was determined by membrane immunofluorescence to be more than enough to saturate the cell surfaces. The cells were rinsed in RPM1 1640 medium containing 5% FBS, and were then incubated for 1 hr at 37’C in 1 ml of rabbit or guinea pig complement (C’) (optimum dilution previously determined by titration in trypan blue exclusion assays). The cells were subsequently centrifuged, rinsed and then overlayed onto mink or SC-1 indicator cells as described above. Controls included treatments of cells with either anti-Thy-l or C’ alone. Cell Fractionation on Nylon Woof A 10 ml column containing 1 g of sterile packed nylon wool was equilibrated for 1 hr at 37’C with 60 ml of phosphate-buffered balanced salt solution (PBBS) containing 10% FBS (Julius et al., 1973). Cells dispersed from lymphoid organs of inoculated mice were passed slowly through the column over a period of 1 hr, and nonadherent cells were then eluted by infusing -15 ml of fluid dropwise through the column. Of this nonadherent cell population, 95%-98% was comprised of cells lacking surface immunoglobulin (lg) as detected by live-cell immunofluorescence. Plastic Adherence Cells (macrophages. fibroblasts) capable of adherence to plastic surfaces were removed from cell populations by incubation for 2 hr at 37°C in 60 mm tissue culture dishes containing RPM1 1640 supplemented with 10% FBS. The nonadherent cells were rinsed from the dish and collected. Panning The cell selection procedure of Wysocki and Sato (1978) was used to select for lymphoid subpopulations expressing surface lg (mature B-lymphocytes) or Lyt-1 or Lyt-2 surface antigens (T-lymphocyte subpopulations). Fischer polystyrene petri dishes (100 mm) were coated overnight at 4°C with affinity-purified rabbit anti-mouse lg (50 gg/ml; provided by J. Portis), rinsed extensively and then further coated with protein (PBBS containing 2% FBS) to bind any nonblocked plastic sites. Dispersed whole spleen or thymus cells (20 x lo? were allowed to settle and adhere to the antibody-coated dishes

Cell 224

for 90 min at 4°C. The Lyt-1 or Lyt-2 antigen-expressing cells were isolated from thymus by first incubating the dispersed thymocytes with the corresponding mouse monoclonal antibody (53-7.313 and 53-6.72 provided by L. A. Herzenberg; Ledbetter and Herzenberg, 1979) for 1 hr and then rinsed twice before incubation on dishes. Nonadherent cells were gently rinsed off with cold medium, collected and counted. Adherent cells were removed by additional incubations for 30 min at 37°C in RPMI-10% FBS, and then squirting them off with a Pasteur pipette. Monitoring purity of cell fractions was determined by membrane immunofluorescence. Gkrcocorficoid Treatment For lysing the steroid-sensitive lymphocytes in the thymic cortex, MCF-inoculated mice were given a single i.p. injection of 3 mg of dexamethasone 36-46 hr before virological assays were to be performed on their tissues. Thymic cortical depletion and medullary sparing was confirmed by histological examinations.

Acknowledgments I am grateful to J. Hartley (NIH), I. Weissman (Stanford University), L. Herzenberg (Stanford University), E. Ribi (Hamilton). W. Hadlow, J. Portis, Dr. Richard Race and Dr. William Britt (all from RML) for providing many reagents and materials. I am also grateful to Dr. Bruce Chesebro for helpful discussions, and to Margie Thompson for excellent technical assistance. I wish to thank Helen Blahnik for expert preparation of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

August

13, 1982;

revised

October

7. 1982

References Stimulation of Lymphocyte Subpopulations with Mitogens Dispersed spleen cells (5 X 106) from MCF-inoculated mice were cultured in wells of TC-24 plates (Linbro) containing 2 ml of RPM1 1640, 10% FBS and various concentrations of either Concanavalin A (Con A; Calbiochem-Behring Corp.) or LPS (phenol-water whole cell extract of S. typhimurium Re mutant strain G30/C21, provided by E. Ribi). Forty-eight hr later a 0.1 ml aliquot of cells from each well was transferred to wells of a 96 well microtiter plate, and were pulselabeled with 1 &i of 3H-thymidine (ICN) for 6 hr at 37°C. These cells were then washed and collected on filter paper with a MASH unit, and were dried and counted by liquid scintillation. The remaining cells in the TC-24 wells were washed, counted and overlaid on mink or SC-1 cells to score for MCF virus as described above.

lmmunofluorescent Assay Monitoring the results of panning or cell depletion procedures and quantitating the proportions of cells expressing certain surface antigens (Lyt-1 , Lyt-2, Thy-l, MCF antigens) was accomplished by live cell immunofluorescence as described by Cloyd and Bigner (1977). The assay entailed incubating 1 X IO5 cells with antibody in wells of a microtiter plate for 30 min. washing the cells, and then treating them with fluorescein-isothiocyanate-conjugated goat anti-mouse lg (heavy and light chain) antisera (Cappel Laboratory). After the cells were washed and mounted under coverslips. they were scored for surface fluorescence under a Leitz Orthopan incident-light fluorescence microscope.

Virus Binding Assays Binding of virus to thymus or spleen cells was examined in two ways. One procedure entailed mixing various dilutions of infectious MCF virus with freshly dispersed thymus or spleen cells in medium containing 10% FCS and 8 pg/ml of polybrene (total volume 0.25 ml), allowing binding to occur for varying time intervals and at varying temperatures, and then centrifuging and rinsing the cells with medium twice before overlaying them on mink indicator cells. MCF virus transferred with the cells was thus scored as mink cell foci. The second type of assay used MCF virus metabolically labeled with 3H-uridine. The virus, grown in mink cells in DMEM supplemented with 2% FBS and 20 &i/ml of 3H-uridine (ICN), was harvested every 24 hrs and stored at 4°C. Approximately 200 ml of virus suspension was centrifuged for 20 min at 2500 rpm to eliminate cell debris, and was filtered at 4°C through an Amicon Diaflo AM1 0 ultrafilter under 40 Ibs psi nitrogen pressure. This retained the virus and any proteins greater than 10,000 daltons. Fresh medium without serum was continually added to the virus suspension until the filtrate lacked any radioactivity. The virus, thus rinsed free of unincorporated label, was concentrated approximately tenfold, aliquoted and stored frozen at -70°C. This virus was incubated with thymus or spleen cells as described above. The radioactivity remaining with rinsed cells was determined by liquid scintillation.

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a