- Email: [email protected]
Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients
Cell, Vol. 19, 171-l
79, January
1980.
Copyright
0 1980 by MIT
Phenotypic Alteration by Leukemia-Resistant Leukemia-Susceptible
in Retroviral Gene Expression Thymocytes Differentiating in Recipients
Syamal K. Datta, Samuel D. Waksal and Robert S. Schwartz Departments of Medicine and Pathology and Cancer Research Center Tufts University School of Medicine Boston, Massachusetts 02111
Summary (AKR x NZB)F, mice possess the dominant genes, Akv-7 , Akv-2, Nzv-la and Nzv-2”, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F, thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F, bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F, thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system. Introduction In the leukemia-susceptible AKR mouse a marked amplification in the expression of murine leukemia virus (MuLV) antigens by thymocytes occurs around the age of 6 months. This change develops before the appearance of thymic leukemia (Kawashima et al., 1976a, 1976b) and coincides with the production of xenotropic viruses by the preleukemic thymus. At the same time there is the emergence of recombinant viruses from the N-ecotropic virus that is expressed by AKR mice from birth and the thymic xenotropic precursor viruses (Elder et al., 1977; Hartley et al., 1977; Rommelaere, Faller and Hopkins, 1978). These polytropic viruses accelerate the development of leukemia when injected into AKR mice (Hartley et al., 1977). Other kinds of leukemogenic viruses, which may arise by recombination, have also been isolated from thymuses of old AKR mice (Nowinski and Hays, 1978). These various recombinants may be the direct leukemogenic agents in this strain. In previous experiments we studied the expression of ecotropic and xenotropic viruses, as well as the
incidence of leukemia, in the F, progeny of crosses between AKR and NZB mice (Datta and Schwartz, 1978). These (AKR x NZB)F, hybrids (hereinafter ANF,) inherit four autosomal dominant loci relevant to high grade expression of the two viruses: Akv-1 and Akv-2, from the AKR parent, determine the expression of ecotropic virus (Rowe, 1972; Chattopadhyay et al., 1975); and Nzv-1” and Nzv-2’, from the NZB parent, determine expression of xenotropic virus (Datta and Schwartz, 1976, 1977). Despite the presence of these four loci, the incidence of leukemia in the ANF, mouse is very low and markedly delayed (Holmes and Burnet, 1966; Datta and Schwartz, 1978). Moreover, highgrade expression of both ecotropic and xenotropic viruses occurs in all ANF, lymphoid cells except in thymocytes and peripheral T lymphocytes. This thymus-specific restriction of virus expression in ANF, mice is not found in (AKR x DBA/P)F, (AKD/2) mice and, therefore, seems related to a genetic mechanism derived from the NZB parent. We postulated that the thymus-specific restriction of virus expression in the ANF, mouse accounts for its unexpected resistance to leukemia. The present experiments were designed to determine whether the mechanism that restricts retrovirus expression in T lymphocytes of the ANF, mouse resides in the radiation-resistant thymic stroma (thymic reticuloepithelial elements) or in radiation-sensitive prethymic or thymic lymphoid cells (von Boehmer and Sprent, 1976; Sprent, 1978; Fink and Bevan, 1978; Zinkernagel et al., 1978). We show that there is a specific, high-grade augmentation in the expression of MuLV antigens and of ecotropic and xenotropic viruses in ANF, thymic lymphocytes when they were allowed to differentiate in irradiated, leukemia-prone AKR mice. By contrast, these phenotypic changes did not occur when ANF, thymocytes differentiated in irradiated ANF, and C57BR hosts, which have a very low incidence of spontaneous leukemia (Rowe, 1972; Chattopadhyay et al., 1975; Datta and Schwartz, 1978). The results indicate that the thymic microenvironment exerts a major influence on the expression of retroviral genes by thymocytes. Results Expression of MuLV Antigen In the following experiments chimeras were prepared by repopulating lethally irradiated 2-3 month old mice with anti-8 and complement-treated bone marrow cells from 3-3% month old donors. The recipients were killed 2 months later and tested to ensure that their lymphoid cells were of donor origin (see Experimental Procedures for results of tests for chimerism). Suspensions of thymocytes from proven chimeras were examined microscopically after staining with fluorescein-conjugated antisera for the presence of MuLV
Cell 172
antigens. Parallel tests were done with thymocytes from intact ANF, and AKR mice. A remarkable result was found in the thymuses of ANF, + AKR chimeras. In these animals 80-100% of the thymocytes expressed MuLV antigen, as detected by specific reactions with the antisera (Figures 1 and 2a, Table 2). By contrast, less than 1% of thymocytes from intact ANF, mice (4-9 months old) or intact 3-4% month old AKR mice expressed MuLV antigen. Thus virtually all ANF, thymocytes differentiating in the AKR environment express an MuLV-related antigenic determinant which is barely detectable in either intact ANF, mice or agematched intact AKR mice. In this respect, the chimera’s thymocytes resemble those of 6 month old AKR mice (Table 1 and Kawashima et al., 1976a). The augmented expression of MuLV antigen by thymocytes of the ANF, -+ AKR chimera had both donor and recipient specificity. The former is demonstrated by the low numbers of thymocytes (
; p
BO70.
.s
60 -
$
50-
.
2 40. .F s 30 L
20.
3
io-
Figure 1. lmmunofluorescence cytes of the Chimeric Animals
Expression of Xenotropic Virus Thymocytes and spleen cells from the chimeras shown in Figure 1 were tested by infectious center assays for the presence of xenotropic and ecotropic viruses. Results are given in Table 2 and Figure 3. In intact ANF, mice, the expression of xenotropic virus by thymocytes is severely restricted (Datta and Schwartz, 1978). The same organ-specific restriction was found in syngeneic ANF, + ANF, chimeras. In these mice the thymuses had very low or undetectable levels of xenotropic virus, whereas their spleens had
‘.“.’ . . ..
a90 qo-
f5
was not found (Figure 4, Table 2). This result suggests the existence of an age-dependent factor in the AKR recipients, and that the phenotypic alteration of the donor thymocyte population occurs relatively soon after they reseed the host.
.
Tests for MuLV Antigens (Donor + Recipient)
Figure 2. Examples Specific Anti-MuLV
on Thymo-
Each dot represents an individual mouse. ANF, = (AKR X NZB)F,: AKD/P = (AKR X DBA/P)F,. The recipient mice were 8-l 2 weeks old when reconstituted and 16-l 9 weeks old when tested.
of Thymocytes Antisera
from
the Chimeras
Stained
with
(a) Thymocytes from an ANF, + AKR chimera stained with fluorescein-conjugated goat anti-Moloney MuLV serum. A similar result was obtained on staining with the goat antigp70 Rauscher MuLV serum (X 400). (b) Thymocytes from an ANF, + C57BR chimera stained for MuLV antigen. No fluorescence-positive cells are detectable. The light staining is due to cells taking up the dull orange rhodamine counterstain (X 400).
Retrovirus 173
Expression
in F, -
Parent
Chimeras
high levels. In the thymuses and spleens of AKR --* AKR chimeras, xenotropic virus was either not found or detected in very low amounts, as is the case in intact AKR animals of similar age (Kawashima et al., 1976b; Datta and Schwartz, 1978). In thymuses of all ANF, + AKR chimeras, by contrast, there were numerous xenotropic virus-producing infectious centers. On average, the thymuses of these chimeras contained IO4 times more infectious centers than those of either of the syngeneic chimeras. The augmented expression of xenotropic virus by thymocytes of the ANF, + AKR chimeras was donorand recipient-specific. Donor specificity is shown by low or undetectable levels of the virus in AKD/P + AKR and AKR + AKR chimeras. Recipient specificity is demonstrated by the ANF, -+ C57BR, AKR --, ANF, and ANF, + ANF, chimeras, the thymocytes of which expressed relatively low or undetectable levels of xenotropic virus (Table 2 and Figure 3). Augmented expression of xenotropic virus by thymocytes of ANF, + AKR chimeras was, like the expression of MuLV Table
1. Expression
of MuLV-Antigen
in Thymocytes
of intact
Mice
% MuLV Antigen-Positive Thymocytes” Mice
Age (Months)
Number Tested
ANF,
4-9
6
o-o.001
AKR
3-4%
6
0.02-l
AKR
6-6’h
5
54.0-70.0
Range
Mean
it SD.
0.0005 .o
0.25 60.8
+_ 0.0003 + 0.37
f 8.67
* Expression of MuLV antigen in thymocytes of intact mice. One MuLV antigen positive thymocyte, when present in an entire cytocentrifuge smear containing approximately 2 X lo5 cells, is expressed as 0.0005% in this Table and in Table 2. “0” means no positive cells were detected in the smear.
Table 2. Detailed 1.3and4
Results
of MuLV Antigen
Expression
and Retrovirus
antigen, also related 4, Table 2).
Range
Chimera ANF, +
AKR (10)
ANFI +
ANF,
AKR+
ANF,
ANF,
-+ C570R
80.0-l
(8) (13) (8)
AKD/P
-
C57BR
AKD/P
+
AKR (7)
(6)
ANF, +
AKRGwk
Mean
Production
by Lymphoid
00.0
(7)
93.6
f S.D. f
8.2
Infectious
Cells from the Chimeric
Centers
(Mean
Thymus
f SD.)
Mice Shown
in Figures
per 10’ Cells
Spleen
Xenotropic
Ecotropic
Xenotropic
Ecotropic
4.63
+ 0.26
4.24
f 0.59
3.2
f 0.35
3.89
+ 0.21
0.29
+ 0.26
0.07
+ 0.14
2.8
k 0.48
3.0
+ 0.34
0.0005-0.001
0.0007
0.001-3.8
0.4 zk 0.98
0.55
* 0.43
4.19
* 0.11
0.76
f 0.5
3.48
f 0 28
0.44
f 0.47
0.04
Tk 0.11
2.89
f 0.21
2.9
f 0.55
3.52
2 0.2
0.02
+ 0.025
0.05
+ 0.12
3.11
+ 0.18
0.14
f 0.22
3.6
f 0.06
0.38
f
0.13
f 0.24
3.19
f 0.22
0.15
f 0.28
3.88
+ 0 17
0.11
f 0.17
3.28
2 0.46
0.21
f 0.26
3.71
f 0.27
0.04
+ 0.11
3.04
k 0.23
2.96
f 0.11
3.49
+ 0.19
0.001-l
.o
0.001-0.06 0.001-l
AKR -+ AKR (10)
Thymocytes
(Figure
Expression of Ecotropic Virus Levels of ecotropic virus, which are barely detectable in thymocytes of intact ANF, mice (Datta and Schwartz, 1978) were greatly augmented in thymocytes of ANF, + AKR chimeras (Table 2, Figure 3). This effect was not due to nonspecific factors which might, for example, arise after total body irradiation, because the ANF, + ANF, chimeras maintained the thymocyte-specific restriction of intact ANF, mice. Part of the increased titers of ecotropic virus in the ANF, -+ AKR chimera may have been due to crossinfection of the donor ANF, cells by virus produced in the AKR recipient, but this does not explain the specific localization of the increase in the thymus; the mean number of ecotropic virus-producing infectious centers in the spleens of these animals was not different from that in ANF, + ANF, mice (P > 0.1). Moreover, on average, 10 times more infectious centers were detected in ANF, --, AKR thymocytes than in AKD/2 + AKR or AKR + AKR thymocytes. These latter two chimeras expressed amounts of ecotropic virus which we have found in intact AKD/2 and AKR mice (Datta and Schwartz, 1978) and in the AKD/2 + C57BR chimera (Table 2). Further evidence against cross-infection as the explanation for the increased levels of ecotropic virus production by thymocytes of ANF, + AKR chimeras is that the levels of virus were approximately one tenth lower when younger (6 week old) AKR recipients were used (Table 2, Figure 4). The enhanced expression of ecotropic virus by thymocytes of ANF, + C57BR chimeras (Table 2, Figure 3) is of interest. In this case we are not dealing with cross-infection because cells of the C57BR strain
LogI % MuLV-Antigen-Positive
to the age of the recipient
.6
0.02-40.0
a
0.005-0.1
0.065
f
0.0003
0.56
+ 0.04
Detailed results of MuLV antigen expression and retrovirus production by lymphoid cells from the chimeric mice shown Numbers in parentheses represent numbers of animals tested. B Thymocytes from two animals in the AKR + AKR group of chimeras expressed high levels (23-40%) of MuLV antigen. positive cells in the other eight animals of this group was 0.02-0.3% (mean 0.08 f 0.09 SD.).
in Figures
1, 3 and 4.
The range
of antigen-
Cell 174
%ymus --
Spleen 8 -
. b
Thymus .--
Splen c
.
Thymus Spleen -A
Thvmus
2% 3 *
tA i
t :s -50 k2
-60 .A. ANFt -
ANFI
AKR -ANF
‘I
ANF, -
C57BR
-40
5
-30 5
I
-20 3 -IO -
0
OJ p0n.D ANF, ,AKD/ 2 -AKR
AKR -AKR
Figure 3. Xenotropic (0) and Ecotropic mus and Spleen Cells from the Chimeric
ANF, -AKR (A) Virus Production by ThyMice Shown in Figure 1
ANF, = (AKR x NZB)F,: AKD/2 = (AKR x DBA/P)F,. Open circles and triangles in the AKR + AKR chimeras (E) represent xenotropic (0) and ecotropic (A) virus production by the two mice in this group that had high MuLV antigen expression in their thymocytes (Figure 1).
rarely express ecotropic virus, either spontaneously or after attempts at in vitro induction (Rowe, 1972; Chattopadhyay et al., 1975). This result indicates that the C57BR environment permits expression of ecotropic viral genes that are repressed by ANF, thymocytes in the ANF, environment. Moreover, the effect is virus-specific, because enhanced expression of xenotropic virus or MuLV antigens by thymocytes of the ANF, + C57BR did not occur. There was an increase in MuLV antigen expression in the thymocytes of ANF, + C57BR chimeras as compared to ANF, + ANF, and AKD/2 ---, C57BR chimeras, but the levels in the former were still 80-100,000 fold less than the ANF, + AKR chimeric thymocytes. AKR + ANF, is another noteworthy chimera. The thymocytes of these mice had the virological phenotype of the donor AKR strain and were similar to those of the AKR ---* AKR chimera. The unchecked expression of ecotropic virus by AKR thymocytes in the ANF, host (Table 2, Figure 3) shows that the ANF, environment does not actively restrict ecotropic virus production and spread by thymocytes that differentiate from bone marrow cells of young adult AKR mice. The aug-
s o\”
AKR
Figure 4. Xenotropic (0) and Ecotropic (A) Virus Production by Thymus and Spleen Cells (A) and MuLV Antigen Expression by Thymocytes (B) from (AKR x NZB)F, + AKR Chimeras Where the Recipients Were 6 Weeks Old When Irradiated and Reconstituted These chimeras other chimeras
were rested for 3 months and tested along with the when the recipients were 18-l 9 weeks old.
mented expression of ecotropic virus by the thymocytes of ANF, + C57BR chimera, therefore, in contrast to the ANF, + ANF, chimera, is not simply because the C57BR host provides a permissive environment for spread of virus produced by the ANF, donor thymocytes. The intact ANF, mouse restricts expression of ecotropic virus by both thymocytes and peripheral T cells of lymph node8 and spleen (Datta and Schwartz, 1978). The latter phenomenon was also found in ANF, + ANF, chimeras. Numbers of ecotropic virus-producing infectious centers in nylon wool-purified T lymphocyte-enriched spleen cell fractions were 30270 fold lower than in unfractionated spleen cells. By contrast, the levels in T lymphocyte-enriched fractions of spleens from ANF, -+ AKR chimeras were only 210 fold lower in unfractionated spleen cells, as was found in the case of AKR --* AKR, AKR + ANF, and ANF, + C57BR chimeras (data not shown). Isolation of Polytropic Viruses Although spleens of intact ANF, mice express relatively high levels of both ecotropic and xenotropic viruses, no evidence indicative of polytropic viruses can be detected (Datta and Schwartz, 1978). In light of the expression of both ecotropic and xenotropic
Retrovirus 175
Expression
in F, +
Parent
Chimeras
Table 3. Isolation of Viruses with Polytropic Activity from ANF, + AKR Thymuses following Limiting Dilution Purification in Mink Cell Lines Fluorescent Focus Forming Units (Log,,) per 0.2 ml of Purified Culture Supernatant’ Number Mice
of
Mink (Coverslip)
NIH/3T3 (Coverslip)
XC Test (NIH/3T3)
1
3.62
3.15
Negative
2
4.02
3.75
Negative
3
4.35
3.93
Negative
4
5.24
0.95
Negative
Isolation of viruses with polytropic activity from ANF, -+ AKR thymuses following limiting dilution purification in mink cell lines. (See Experimental Procedures for details of co-cultivation and purification of culture supernatants.) Attempts were made to isolate polytropic virus using similar protocols from thymocytes of three AKR -+ ANF,. three AKD/P -+ AKR and three AKR + AKR chimeras. Four of these yielded only xenotropic virus after the passage in mink cells. The rest were negative on both mink and NIH/3T3 cell assays after the mink passage. “Negative” = no XC-plaques could be detected on inoculation of NIH/3T3 indicator cells with 0.5 ml of purified culture supernatants. ’ See Experimental Procedures.
viruses by ANF, thymocytes in ANF, + AKR chimeras, therefore, it was of interest to seek polytropic viruses in these cells. Of four supernatants tested after passage in tissue culture (see Experimental Procedures), three yielded XC-negative viruses with polytropic activity, with approximately equal and high titers in mink and NIH/3T3 cells (Table 3). No such viruses could be isolated from AKR + AKR, AKD/2 + AKR or AKR + ANF, thymocytes. Further purification of the viruses with polytropic activity for biochemical studies (Elder et al., 1977; Rommelaere et al., 1978) is in progress. Discussion Our experiments demonstrate that virtually all of the thymocytes of ANF, mice express MuLV antigen when they differentiate in the environment of AKR mice. Moreover, such thymocytes also produce high levels of both ecotropic and xenotropic viruses. These manifestations are in striking contrast to those exhibited by thymocytes of intact ANF, mice, which fail to express MuLV antigen and produce very low or undetectable amounts of ecotropic and xenotropic viruses (Datta and Schwartz, 1978). Thymocytes and peripheral T cells in the spleen and lymph nodes of ANF, mice have these characteristics despite the presence of the ecotropic viral genes, Akv-1 and Akv-2, and the xenotropic virus-inducing genes, Nzv-1’ and Nzv-2” (Rowe, 1972; Datta and Schwartz, 1978). Some mechanism that restricts the expression of these genes specificially in the cells of T lymphocyte lineage must therefore exist in ANF, mice. The present ex-
periments also show that the marked augmentation of retrovirus and MuLV antigen expression occurs specifically in the thymocytes of ANF, mice when they differentiate in the AKR host. Since the thymic reticuloepithelial environment is solely responsible for the differentiation and maturation of bone marrow precursor cells to thymocytes in lethally irradiated, bone marrow-restored chimeras, we assume that the phenotypic alteration of ANF, thymocytes is brought about by the radiation-resistant thymic reticuloepithelium of the AKR recipient (von Boehmer and Sprent, 1976; Sprent, 1978; Fink and Bevan, 1978; Zinkernagel et al., 1978). The effects we observed were specific. We can exclude nonspecific effects of irradiation because changes in expression of MuLV antigen or infectious viruses did not occur in the thymuses of syngeneic chimeras (ANF, + ANF, and AKR + AKR). There were also no changes in MuLV antigen expression or production of xenotropic virus in AKD/2 + AKR or ANF, + C57BR and AKR ---* ANF, chimeras. In the former case, the AKD/2 donor mice lack NZB genes; in the latter cases, both C57BR and ANF, recipients are low leukemia strains (Rowe, 1972; Datta and Schwartz, 1978). Therefore, the remarkable changes in the thymuses of the ANF, -+ AKR chimeras are brought about by a specific interaction between ANF, thymocytes and the AKR environment in which they differentiate. An alternative explanation for the augmented retrovirus production we found could be that the thymocytes in this chimeric combination are more susceptible to reinfection and spread of the viruses. For example, in the ANF, + AKR chimera, ANF, cells may produce ecotropic virus which can replicate in AKR cells generating some type of polytropic recombinant virus, which then replicates in the ANF, cells. The evidence against this possibility as the sole cause for the pronounced retrovirus production lies in the specificity of the results. AKR or AKD/2 cells that were allowed to differentiate in the AKR host did not generate either polytropic or xenotropic viruses, although they did produce ecotropic viruses. In those chimeras the donor cells possess and express ecotropic viral genes, and they are not known to restrict the replication and spread of polytropic recombinant viruses (Hartley et al., 1977); yet augmented expression of xenotropic and ecotropic viruses and generation of polytropic viruses occurred on/y in the ANF, -+ AKR chimeras. The same argument applies to MuLV antigen: its expression was markedly amplified only in the thymocytes of the thymuses of the ANF, -+ AKR chimeras. Neither are these specific results explained by a peculiarity in the ANF, donor cells that permits increased virus spread, since thymuses of ANF, + C57BR and ANF, + ANF, chimeras lacked the virologic changes shown by the ANF, + AKR chimeras. Moreover, when the direction of chimeric combination was reversed, as in the AKR + ANF,
Cell 176
chimera, so that AKR thymocytes were allowed to differentiate in ANF, host environment, the virologic changes seen in ANF, + AKR chimeras did not occur. This result also indicates that the ANF, host does not restrict spread of retroviral infection, because the donor AKR thymocytes produce ecotropic viruses in this host, as it does in the AKR + AKR chimeras or the intact AKR animal. Thus the lack of virus production in thymuses of ANF, + ANF,, in contrast to the ANF, + AKR chimeras, is not due to a restriction in virus spread. The evidence therefore favors the interpretation that the thymic reticuloepithelium of the high leukemia strain AKR has the specific ability to alter the phenotype of thymocytes from low leukemia ANF, mice. Kawashima et al. (1976b3) have observed the amplification of MuLV antigen by the thymocytes of syngeneic AKR + AKR chimeras. In their experiments this phenomenon was not found in 2 month old recipients, but only in 6 month old preleukemic recipients, whose thymuses express xenotropic virus in high titers, as well as polytropic (MCF) particles. In our system, the “amplification signal” was detectable when AKR recipients were as young as 2 months. At this age the AKR thymus expresses only ecotropic virus. One reason for the difference between ANF, and AKR donor cells may be that ANF, mice possess NZB genes, including Nzv-7’ and Nzv-2’ (Datta and Schwartz, 1976, 1978). These genes may be highly susceptible to activation (or increased expression) by a signal from AKR thymic reticuloepithelial cells. In the intact ANF, animal, this signal is apparently never produced, perhaps because of restrictive or nonpermissive NZB genes. Our results show that, given a sufficiently sensitive system (the ANF, thymocyte), virus- and viral antigen-amplifying “signals” can be detected in AKR mice many months before they can be observed in the intact animal. We propose that the amplifying “signals” of AKR thymic microenvironment increase with time, so that by the age of 6 months the phenomenon can be detected in AKR thymocytes (Kawashima et al., 1976a, 1976b). It seems improbable, in view of our data, that the amplifying signal(s) is either a xenotropic or polytropic virus. In two AKR + AKR chimeras, 23% and 40% of the thymocytes stained for the presence of MuLV antigen (Figure l), yet the xenotropic virus titers in the thymuses of these mice were low (Figure 3, Table 2). This finding implies that augmented expression of MuLV antigen may not be due entirely to the production of infectious xenotropic virus. The very large difference, in ANF, + AKR chimeras, between the number of thymocytes that stain for MuLV antigen (80-100%) and the number that produce infectious virus, either ecotropic or xenotropic (-1%. as determined by infectious center assays), shows that MuLV antigen is expressed independently of infectious virus. Furthermore, the augmented numbers of ecotropic
virus-producing thymocytes in the face of very low levels of xenotropic virus and MuLV antigen-positive thymocytes in the ANF, + C57BR chimera also point to discordant expression of infectious viruses and MuLV antigens in these chimeras. The restricted expression of ecotropic virus by thymocytes of ANF, mice and ANF, --* ANF, chimeras appears to be prethymic, because AKR prothymocytes, which already produce ecotropic virus before reaching the thymus, expressed the virus unchecked in thymuses of AKR + ANF, chimeras. This prethymic restriction was removed when ANF, precursor cells differentiated in either C57BR or AKR hosts. It is improbable that this result was due to cross-infection by virus produced within the irradiated hosts. C57BR mice do not usually express ecotropic virus spontaneously, or even after attempts at in vitro induction (Rowe, 1972; Chattopadhyay et al., 1975). Furthermore, spleen cells of intact ANF, mice and ANF, --, ANF, chimeras produce ecotropic virus, yet the T lymphocytes within these spleens are not cross-infected (Datta and Schwartz, 1978; see also Results). All of the above indicates that the prethymic restriction in intact ANF, mice is maintained throughout the T lymphocyte lineage. The results in the low leukemia C57BR hosts, in which ANF, thymocytes expressed only ecotropic virus, contrast with those in the high-leukemic AKR hosts, where ANF, thymocytes expressed both ecotropic and xenotropic viruses and high levels of MuLV antigen. This difference is noteworthy because the expression of both viruses occurs during the development of thymic leukemia (Kawashima et al., 1976b; Hartley et al., 1977). Moreover, the thymocytes of ANF, + AKR chimeras also produced a polytropic virus, which we assume arises by recombination between ecotropic and xenotropic viruses (Elder et al., 1977; Hartley et al., 1977; Rommelaere et al., 1978). It will therefore be of interest to compare the incidences of thymic leukemia in these two chimeras, an experiment which is in progress. The amplified expression of proviral genes by thymocytes exposed to the AKR thymic environment may provide the viruses that recombine with the N-tropic virus prevalent in all tissues of AKR mice from birth. The presence of polytropic virus, a presumed leukemogenic agent, in ANF, + AKR chimeras raises the question of whether the ANF, thymocytes of the chimera undergo malignant transformation. None of the animals we tested had evidence of tumor. The transfer of MuLV antigen-positive thymocytes from ten ANF, + AKR chimeras individually into young ANF, mice (50-80 X lo6 cells per recipient), however, has resulted in the appearance of leukemia involving thymus and spleen in three recipients within 3 months. Transfer of thymocytes from the ANF, + C57BR, AKR + AKR, ANF, ---, ANF, and AKR -P ANF, chimeras have failed to produce tumors, even up to 5 months after
Retrovirus 177
Expression
in F, +
Parent
Chimeras
inoculation (S. K. Datta, unpublished observations). Kawashima et al. (1976a) found that the transfer of thymocytes from 6 month old AKR mice into young syngeneic recipients did not cause leukemia, even up to 100 days. By contrast, only a few leukemic cells from AKR thymomas produce evident tumor in syngeneic recipients within 3 weeks. This indicates that the “preleukemic” thymocytes with amplified MuLV antigen and xenotropic virus expression require further residence in the aged AKR environment before leukemic transformation becomes established. Events subsequent to those concerned with the virological changes in the preleukemic AKR thymus may therefore be required for leukemia to develop. In the ANF, + AKR chimera these events may have been accelerated. We may conclude from these experiments that, in the mice we studied, the functional status of the thymic reticuloepithelium is of particular importance in determining the expression of integrated retroviral genes by thymocytes. The evidence in these animals favors a specific genetic mechanism that controls the ability of thymic reticulopeithelium to render possible the expression of these viral genes. This property is lacking in thymic reticuloepithelium of ANF, mice, whose T lymphocytes fail to express their viral genes. Based on our findings in ANF, + AKR and ANF, + C57BR chimeras, the thymic reticuloepithelium augmenting mechanism has three properties relevant to increased expression of MuLV antigen, ecotropic virus and xenotropic virus. The AKR thymus possesses all three capabilities, whereas that of the C57BR strain augments expression of only ecotropic viral genes. The ANF, thymus lacks all of these properties. Attempts to isolate and identify these inductive mechanisms are in progress. Experimental
Procedures
Mice NZB, AKR, C57BR and (AKR x DBA/2)F1 mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). (NZB x AKR)F1 and (AKR x NZB)F, hybrids were bred in our laboratory. Since results were similar in the reciprocal crosses. these were designated simply as ANF,. Radiation Chimeras Chimeras were prepared by irradiating recipient mice with 950-l 000 rads from a I” Cs source and repopulating them with bone marrow ceils in a manner described previously &on Boehmer and Sprent. 1976; Sprent. 1978; Fink and Bevan. 1978; Zinkernagel et al., 1978). 4 hr after irradiation the recipients were injected intravenously with 8-15 x 10’ viable bone marrow cells that had been treated twice with rabbit anti-mouse brain serum (Waksal et al.. 1974) and normal rabbit serum as a source of complement to deplete any T lymphocytes in the inoculum. This step obviates any graft versus host disease in the AKR ---f ANFl combinations. All the other combinations were matched at the major histocompatibility loci. The reconstituted mice received Polymyxin and Neomycin in their drinking water for 2 weeks. They were analyzed 7-9 weeks after reconstitution. The donor mice were 12-14 weeks old, and the age of recipient mice was 8-12 weeks. In one set of ANFl + AKR chimeras the recipients were 6 weeks old (see Results). Donors and recipients were the same sex,
except for ANF, + ANF, and AKR -+ AKR chimeras, where male donor cells were injected into female recipients. The mortality (6080%) was high because of the lethal to supralethal irradiation. All irradiated mice that were not reconstituted with bone marrow died within 2 weeks. Due to the high mortality, the above chimeric combinations were made 3 times. Results were similar in the three sets of experiments and are presented together. Tests for Chimerism Treatment with Anti-K2” Serum In all combinations except ANF, + ANFl and AKR + AKR. lymphoid cell chimerism was assessed by a two-stage, complementdependent cytotoxicity test using specific anti-H-2d sera. (Erb et al., 1979). AntiD-4 was provided by T. Hansen (National Institutes of Health, Bethesda, Maryland) and anti-D-31 was from H. Winn (Massachusetts General Hospital, Boston, Massachusetts). Baby rabbit serum screened for low background cytotoxicity against mouse lymphocytes sewed as a source of complement. Cytotoxicity was determined by the trypan blue dye exclusion test and calculated as % of the control in which cells were incubated with normal BALE/c mouse serum and complement. Cells from ANF, mice were also used as control to test the activity of the anti-H-2” sera. 80-90% of the thymus and spleen cells from all combinations except AKR + ANF, were of the H-2d type, using both anti-H-2 sera. Less than 10% of the lymphoid cells of the AKR + ANF, chimera were killed by this treatment. Chromosome Preparation Thymus and spleen cells from ANF, 6 + ANF, P and AKR 6 - AKR P chimeras were stimulated with Con A (2 pg/ml)(Pharmacia, Upsala, Sweden) or phytohemagglutinin (PHA-P, 5 pg/ml) (Wellcome Reagents, Ltd., Beckenham. England) for 72 hr. The lymphoblasts in 5 ml suspension were incubated with 2 drops of 0.1% solution of colchicine (Calbiochem. La Jolla, California) for 2 hr. harvested. swelled with hypotonic 0.075 M KCL and fixed with 3 parts methanol/ 1 part glacial acetic acid, and air-dried smears were prepared (Wang and Federoff, 1972). The modified Seabright banding technique was used to stain the smears with Giemsa after brief trypsin digestion (Seabright. 1971). Cells from intact male and female mice served as controls. In magnified photographs of the metaphase spreads from cells of the chimeras, Y chromosomes were consistently identified as the unpaired short chromosome lacking heavily stained centromeres (Wiener et al., 1978). All chimeras were tested in this manner at the time of retrovirus assays. Retrovirus Assays Infectious center assays with aliquots of lymphoid cell suspensions from thymuses and spleens of the chimeric mice were carried out as previously described (Datta and Schwartz, 1978; Datta et al., 1978). In some chimeras nylon wool column-purified T lymphocyte fractions of spleen were also obtained, as described (Julius. Simpson and Herzenberg. 1973; Datta and Schwartz, 1978). and tested for virus production. Xenotropic Virus Fluorescent antibody focus assays to detect infectious centers were done on mink lung cells (ATCC CCL64) growing on glass cover slips in Petri dishes, as previously described (Kawashima et al., 1976b; Datta and Schwartz, 1978). Ecotropic Virus Since none of the mice tested are known to produce B-tropic virus, and all were homozygous for Fv-1” (Rowe, 1972; Chattopadhyay et al.. 1975; Datta and Schwartz, 1978). production of N-ecotropic viruses were determined by a fluorescent antibody focus assay with NIH/3T3 indicator cells growing on glass cover slips placed in tissue culture dishes (Kawashima et al., 1976b: Datta and Schwartz, 1978; Datta et al., 1978). This assay detects the expression of both XCpositive and XC-negative N-tropic viruses (Nowinski et al., 1977; Datta and Schwartz. 1978). Titers are expressed as logl,, of the number of foci produced on each cover slip per 10’ lymphoid cells inoculated in the tissue culture dish, although the area of the cover
Cell 178
slips on which culture dishes.
foci were
counted
is one fourth
the area of the tissue
Polytropic Virus The method developed by Hartley et al. (1977) to detect recombinant viruses with dual host range was used. This has been described in detail in previous reports from our laboratory (Hiai et al., 1977; Datta and Schwartz, 1978). Lymphoid cells were co-cultivated with mink cells. After 5-6 passages, the supernatants from these cultures were filtered through 0.22 mp Millipore filters and subjected to limiting dilution purification by reinfection on a fresh line of mink cells. These cells were subsequently split and passaged weekly. Afler 5-6 passages, culture supernatants were tested on mink and NIH/3T3 cells by the cover slip fluorescent focus assay and XC tested with NIH/ 3T3 indicator cells (Rowe, 1972; Datta and Schwartz, 1978). Only xenotropic and polytropic viruses would replicate in this protocol. lmmunofluorescence for Murine Leukemia Virus Antigens Thymocytes from the chimeric animals were adjusted to a concentration of 5 x IO5 cells per ml. 0.45 ml of the cells were pelleted on slides in a cytocentrifuge (Manny, Datta and Schwartz, 1979) and fixed for 10 min in cold methanol. Air-dried smears were stained with fluoresceinated-specific anti-MuLV sera at 1:40 dilution (diluted in a rhodamine counter-stain solution) at 37% for 30 min, washed and examined with a fluorescence microscope with Ploem epiillumination and appropriate filters (Datta et al.. 1978). The entire smear (approximately 2 X lo5 cells) was scanned in those specimens which had very low numbers of positive cells. In some cases, membrane immunofluorescence was sought by incubation of viable thymocytes at 4’C for 30 min with antiserum (Kawashima et al., 1976a). These assays gave results similar to those for fixed cells. There were two anti-MuLV sera used in these tests. The first was fluorescein-conjugated goat antiserum prepared against Tween-ether-disrupted Moloney MuLV (supplied by J. Gruber. National Cancer Institute, Bethesda, Maryland). This anti-serum contains antibodies to the broad spectrum of MuLV-related antigens (Hartley and Rowe, 1976; Kawashima et al., 1978b). The second was a fluoresceinated goat anti-gp69/71 MuLVRauscher (Lerner et al., 1978) provided by R. Lerner (Scripps Clinic, La Jolla, California). Results were similar with both antisera. At a 1: 40 dilution they specifically stained cells replicating and expressing xenotropic. ecotropic and polytropic MuLV (Datta et al., 1978). Acknowledgments We acknowledge the expert technical assistance of Isaac Robert Mendelson. Ann Marie Champ, Norma Robert and Rhonda Sinoff and wish to thank Marrian Gleason and Elaine McGlame for typing the manuscript. This work was supported by two National Cancer Institute grantstoS.K.D.;onetoS.D.W.;and,twotoR.S.S.S.K.D.isaFaculty Research Awardee of the American Cancer Society. S.D.W. is a recipient of a Scholar Award from the Leukemia Society of America. 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
July 23. 1979;
revised
October
10. 1979
References Chattopadhyay. S. K., Lowy. D. R., Teich, N. M., Levine, A. S. and Rowe, W. P. (1975). Qualitative and quantitative studies of AKR-type murine leukemia virus sequences in mouse DNA. Cold Spring Harbor Symp. Quant. Biol. 39, 1085-l 101. Datta, S. K. and Schwartz, R. S. (1976). Genetics of expression of xenotropic virus and autoimmunity in NZB mice. Nature 263, 412415. Datta. S. K. and Schwartz, loci controlling xenotropic 83, 449-452.
R. S. (1977). virus production
Mendelian segregation of in NZB crosses. Virology
Datta, S. K. and Schwartz, R. S. (1978). Restricted expression of ecotropic virus by thymocytes of leukemia-resistant (AKR x NZB)FI mice. J. Exp. Med. 148, 329-334. Datta, S. K.. Manny, N., Andrzejewski, C.. Andre-Schwartz, J. and Schwartz, R. S. (1978). Genetic studies of autoimmunity and retrovirus expression in crosses of New Zealand Black mice. I. Xenotropic virus. J. Exp. Med. 147, 854-871. Elder, J. H.. Gautsch. J. W., Jenson, F. C., Lerner. R. A., Hartley, J. W. and Rowe, W. P. (1977). Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants. Proc. Nat. Acad. Sci. USA 74, 4676-4680. Erb, P., Meier. B.. Matsunaga. T.. Feldman, M. (1979). Nature of T cell macrophage interaction in helper-cell induction in vitro. II. Two stages of T-helper cell differentiation analyzed in irradiation and allophenic chimeras. J. Exp. Med. 749, 686-701. Fink, P. J. and Bevan. M. J. (1978). H-2 antigens of the thymus determine lymphocyte specificity. J. Exp. Med. 148, 766-775. Hartley, J. W. and Rowe, W. P. (1976). Naturally occurring murine leukemia viruses in wild mice: characterization of new “Amphotropic” class. J. Viral. 19, 19-25. Hartley, J. W., Wolford. N. K.. Old, L. J. and Rowe, W. P. (1977). A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Nat. Acad. Sci. USA 74, 789-792. Hiai. H., Morrissey. P., Khiroya. Selective expression of xenotropic mice. Nature 270, 247-249.
R. and Schwartz, R. S. (1977). virus in congenic HRS/J (hairless)
Holmes, M. C. and Burnet, F. M. (1966). The characteristics of F, and backcross hybrids between “high leukemia” (AKR) and “autoimmune” NZB mouse strains. Aust. J. Exp. Biol. Med. Sci. 44, 235249. Julius, M. H.. Simpson, E. and Herzenberg. L. A. (1973). method of the isolation of functional thymus-derived murine cytes. Eur. J. Immunol. 3, 645-649.
A rapid lympho-
Kawashima. K.. Ikeda. H.. Stockert. E., Takahashi. T. and Old, L. J. (1976a). Age-related changes in cell surface antigens of preleukemic AKR thymocytes. J. Exp. Med. 144, 193-208. Kawashima. K.. Ikeda, H.. Hartley, J. W.. Stockert. E., Rowe, W. P. and Old, L. J. (1976b). Changes in expression of murine leukemia virus antigens and production of xenotropic virus in the late preleukemic period in AKR mice. Proc. Nat. Acad. Sci. USA 73, 46804684. Lerner. R. A., Wilson, C. B., Del Villano, B. C.. McConahey, P. J. and Dixon, F. J. (1976). Endogenous oncornaviral gene expression in adult and fetal mice: quantitative, histologic and physiologic studies of the major viral glycoprotein gp70. J. Exp. Med. 743, 151-l 66. Manny, N.. Datta, S. K. and Schwartz, R. S. (1979). by cells of NZB and SWR mice and their crosses. 1220-l 227.
Synthesis of IgM J. Immunol. 122,
Nowinski, R. C. and Hays, E. F. (1978). Oncogenicity enous leukemia viruses. J. Viral. 27, 13-18. Nowinski, R. C.. Hays, E. F.. Doyle, T., Linkhart. Pickering, R. (1977). Oncorna virus produced cells in culture. Virology 87, 363-370. Rommelaere. J.. tion and mapping the genomes of Acad. Sci. USA
of AKR endog-
S., Medeiros, E. and by murine leukemia
Failer. D. V. and Hopkins, N. (1978). Characterizaof RNase Tl -resistant oligonucleotides derived from Akv and MCF murine leukemia viruses. Proc. Nat. 75, 495-499.
Rowe, W. P. (1972). Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-1” strains of mice. J. Exp. Med. 136, 1272-l 285. Seabright, M. (1971). A rapid banding somes. Lancet 2, 971-972. Sprent. J. (1978). Restricted marrow chimeras controlled 747, 1838-1842.
technique
for human chromo-
helper function of F, + parent bone by K-end of H-2 complex. J. Exp. Med.
von Boehmer. H. and Sprent. J. (1976). marrow chimeras: absence of host-reactive
T cell function in bone T cells and cooperation
Retrovirus 179
of helper 23.
Expression
T cells
across
in F1 +
Parent
allogeneic
Chimeras
barriers.
Transplant.
Rev. 29, 3-
Waksal. S. D.. St. Pierre, R. L.. Hostetler. J. Ft. and Folk, R. M. (1974). Brain associated 19antiserum: differential effects on lymphocyte subpopulations. Cell Immunol. 12, 66-73. Wang, H. C. and Federoff, S. (1972). Banding in human chromosomes treated with trypsin. Nature New Biol. 235, 52-54. Wiener, F.. Ohno, S.. Spira, J., Haran-Ghera. N. and Klein, G. (1978). Chromosome changes (trisomes #I 5 and 17) associated with tumor progression in leukemias induced by radiation leukemia virus. J. Nat. Cancer Inst. 61, 227-237. Zinkernagel. R. M.. Callahan, G. N., Althage, A., Cooper, S.. Klein, P. A. and Klein, J. (1978). On the thymus in the differentiation of “H-2 self-recognition” by T cells. Evidence for dual recognition? J. Exp. Med. 147, 882-896.
79, January
1980.
Copyright
0 1980 by MIT
Phenotypic Alteration by Leukemia-Resistant Leukemia-Susceptible
in Retroviral Gene Expression Thymocytes Differentiating in Recipients
Syamal K. Datta, Samuel D. Waksal and Robert S. Schwartz Departments of Medicine and Pathology and Cancer Research Center Tufts University School of Medicine Boston, Massachusetts 02111
Summary (AKR x NZB)F, mice possess the dominant genes, Akv-7 , Akv-2, Nzv-la and Nzv-2”, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F, thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F, bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F, thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system. Introduction In the leukemia-susceptible AKR mouse a marked amplification in the expression of murine leukemia virus (MuLV) antigens by thymocytes occurs around the age of 6 months. This change develops before the appearance of thymic leukemia (Kawashima et al., 1976a, 1976b) and coincides with the production of xenotropic viruses by the preleukemic thymus. At the same time there is the emergence of recombinant viruses from the N-ecotropic virus that is expressed by AKR mice from birth and the thymic xenotropic precursor viruses (Elder et al., 1977; Hartley et al., 1977; Rommelaere, Faller and Hopkins, 1978). These polytropic viruses accelerate the development of leukemia when injected into AKR mice (Hartley et al., 1977). Other kinds of leukemogenic viruses, which may arise by recombination, have also been isolated from thymuses of old AKR mice (Nowinski and Hays, 1978). These various recombinants may be the direct leukemogenic agents in this strain. In previous experiments we studied the expression of ecotropic and xenotropic viruses, as well as the
incidence of leukemia, in the F, progeny of crosses between AKR and NZB mice (Datta and Schwartz, 1978). These (AKR x NZB)F, hybrids (hereinafter ANF,) inherit four autosomal dominant loci relevant to high grade expression of the two viruses: Akv-1 and Akv-2, from the AKR parent, determine the expression of ecotropic virus (Rowe, 1972; Chattopadhyay et al., 1975); and Nzv-1” and Nzv-2’, from the NZB parent, determine expression of xenotropic virus (Datta and Schwartz, 1976, 1977). Despite the presence of these four loci, the incidence of leukemia in the ANF, mouse is very low and markedly delayed (Holmes and Burnet, 1966; Datta and Schwartz, 1978). Moreover, highgrade expression of both ecotropic and xenotropic viruses occurs in all ANF, lymphoid cells except in thymocytes and peripheral T lymphocytes. This thymus-specific restriction of virus expression in ANF, mice is not found in (AKR x DBA/P)F, (AKD/2) mice and, therefore, seems related to a genetic mechanism derived from the NZB parent. We postulated that the thymus-specific restriction of virus expression in the ANF, mouse accounts for its unexpected resistance to leukemia. The present experiments were designed to determine whether the mechanism that restricts retrovirus expression in T lymphocytes of the ANF, mouse resides in the radiation-resistant thymic stroma (thymic reticuloepithelial elements) or in radiation-sensitive prethymic or thymic lymphoid cells (von Boehmer and Sprent, 1976; Sprent, 1978; Fink and Bevan, 1978; Zinkernagel et al., 1978). We show that there is a specific, high-grade augmentation in the expression of MuLV antigens and of ecotropic and xenotropic viruses in ANF, thymic lymphocytes when they were allowed to differentiate in irradiated, leukemia-prone AKR mice. By contrast, these phenotypic changes did not occur when ANF, thymocytes differentiated in irradiated ANF, and C57BR hosts, which have a very low incidence of spontaneous leukemia (Rowe, 1972; Chattopadhyay et al., 1975; Datta and Schwartz, 1978). The results indicate that the thymic microenvironment exerts a major influence on the expression of retroviral genes by thymocytes. Results Expression of MuLV Antigen In the following experiments chimeras were prepared by repopulating lethally irradiated 2-3 month old mice with anti-8 and complement-treated bone marrow cells from 3-3% month old donors. The recipients were killed 2 months later and tested to ensure that their lymphoid cells were of donor origin (see Experimental Procedures for results of tests for chimerism). Suspensions of thymocytes from proven chimeras were examined microscopically after staining with fluorescein-conjugated antisera for the presence of MuLV
Cell 172
antigens. Parallel tests were done with thymocytes from intact ANF, and AKR mice. A remarkable result was found in the thymuses of ANF, + AKR chimeras. In these animals 80-100% of the thymocytes expressed MuLV antigen, as detected by specific reactions with the antisera (Figures 1 and 2a, Table 2). By contrast, less than 1% of thymocytes from intact ANF, mice (4-9 months old) or intact 3-4% month old AKR mice expressed MuLV antigen. Thus virtually all ANF, thymocytes differentiating in the AKR environment express an MuLV-related antigenic determinant which is barely detectable in either intact ANF, mice or agematched intact AKR mice. In this respect, the chimera’s thymocytes resemble those of 6 month old AKR mice (Table 1 and Kawashima et al., 1976a). The augmented expression of MuLV antigen by thymocytes of the ANF, -+ AKR chimera had both donor and recipient specificity. The former is demonstrated by the low numbers of thymocytes (
; p
BO70.
.s
60 -
$
50-
.
2 40. .F s 30 L
20.
3
io-
Figure 1. lmmunofluorescence cytes of the Chimeric Animals
Expression of Xenotropic Virus Thymocytes and spleen cells from the chimeras shown in Figure 1 were tested by infectious center assays for the presence of xenotropic and ecotropic viruses. Results are given in Table 2 and Figure 3. In intact ANF, mice, the expression of xenotropic virus by thymocytes is severely restricted (Datta and Schwartz, 1978). The same organ-specific restriction was found in syngeneic ANF, + ANF, chimeras. In these mice the thymuses had very low or undetectable levels of xenotropic virus, whereas their spleens had
‘.“.’ . . ..
a90 qo-
f5
was not found (Figure 4, Table 2). This result suggests the existence of an age-dependent factor in the AKR recipients, and that the phenotypic alteration of the donor thymocyte population occurs relatively soon after they reseed the host.
.
Tests for MuLV Antigens (Donor + Recipient)
Figure 2. Examples Specific Anti-MuLV
on Thymo-
Each dot represents an individual mouse. ANF, = (AKR X NZB)F,: AKD/P = (AKR X DBA/P)F,. The recipient mice were 8-l 2 weeks old when reconstituted and 16-l 9 weeks old when tested.
of Thymocytes Antisera
from
the Chimeras
Stained
with
(a) Thymocytes from an ANF, + AKR chimera stained with fluorescein-conjugated goat anti-Moloney MuLV serum. A similar result was obtained on staining with the goat antigp70 Rauscher MuLV serum (X 400). (b) Thymocytes from an ANF, + C57BR chimera stained for MuLV antigen. No fluorescence-positive cells are detectable. The light staining is due to cells taking up the dull orange rhodamine counterstain (X 400).
Retrovirus 173
Expression
in F, -
Parent
Chimeras
high levels. In the thymuses and spleens of AKR --* AKR chimeras, xenotropic virus was either not found or detected in very low amounts, as is the case in intact AKR animals of similar age (Kawashima et al., 1976b; Datta and Schwartz, 1978). In thymuses of all ANF, + AKR chimeras, by contrast, there were numerous xenotropic virus-producing infectious centers. On average, the thymuses of these chimeras contained IO4 times more infectious centers than those of either of the syngeneic chimeras. The augmented expression of xenotropic virus by thymocytes of the ANF, + AKR chimeras was donorand recipient-specific. Donor specificity is shown by low or undetectable levels of the virus in AKD/P + AKR and AKR + AKR chimeras. Recipient specificity is demonstrated by the ANF, -+ C57BR, AKR --, ANF, and ANF, + ANF, chimeras, the thymocytes of which expressed relatively low or undetectable levels of xenotropic virus (Table 2 and Figure 3). Augmented expression of xenotropic virus by thymocytes of ANF, + AKR chimeras was, like the expression of MuLV Table
1. Expression
of MuLV-Antigen
in Thymocytes
of intact
Mice
% MuLV Antigen-Positive Thymocytes” Mice
Age (Months)
Number Tested
ANF,
4-9
6
o-o.001
AKR
3-4%
6
0.02-l
AKR
6-6’h
5
54.0-70.0
Range
Mean
it SD.
0.0005 .o
0.25 60.8
+_ 0.0003 + 0.37
f 8.67
* Expression of MuLV antigen in thymocytes of intact mice. One MuLV antigen positive thymocyte, when present in an entire cytocentrifuge smear containing approximately 2 X lo5 cells, is expressed as 0.0005% in this Table and in Table 2. “0” means no positive cells were detected in the smear.
Table 2. Detailed 1.3and4
Results
of MuLV Antigen
Expression
and Retrovirus
antigen, also related 4, Table 2).
Range
Chimera ANF, +
AKR (10)
ANFI +
ANF,
AKR+
ANF,
ANF,
-+ C570R
80.0-l
(8) (13) (8)
AKD/P
-
C57BR
AKD/P
+
AKR (7)
(6)
ANF, +
AKRGwk
Mean
Production
by Lymphoid
00.0
(7)
93.6
f S.D. f
8.2
Infectious
Cells from the Chimeric
Centers
(Mean
Thymus
f SD.)
Mice Shown
in Figures
per 10’ Cells
Spleen
Xenotropic
Ecotropic
Xenotropic
Ecotropic
4.63
+ 0.26
4.24
f 0.59
3.2
f 0.35
3.89
+ 0.21
0.29
+ 0.26
0.07
+ 0.14
2.8
k 0.48
3.0
+ 0.34
0.0005-0.001
0.0007
0.001-3.8
0.4 zk 0.98
0.55
* 0.43
4.19
* 0.11
0.76
f 0.5
3.48
f 0 28
0.44
f 0.47
0.04
Tk 0.11
2.89
f 0.21
2.9
f 0.55
3.52
2 0.2
0.02
+ 0.025
0.05
+ 0.12
3.11
+ 0.18
0.14
f 0.22
3.6
f 0.06
0.38
f
0.13
f 0.24
3.19
f 0.22
0.15
f 0.28
3.88
+ 0 17
0.11
f 0.17
3.28
2 0.46
0.21
f 0.26
3.71
f 0.27
0.04
+ 0.11
3.04
k 0.23
2.96
f 0.11
3.49
+ 0.19
0.001-l
.o
0.001-0.06 0.001-l
AKR -+ AKR (10)
Thymocytes
(Figure
Expression of Ecotropic Virus Levels of ecotropic virus, which are barely detectable in thymocytes of intact ANF, mice (Datta and Schwartz, 1978) were greatly augmented in thymocytes of ANF, + AKR chimeras (Table 2, Figure 3). This effect was not due to nonspecific factors which might, for example, arise after total body irradiation, because the ANF, + ANF, chimeras maintained the thymocyte-specific restriction of intact ANF, mice. Part of the increased titers of ecotropic virus in the ANF, -+ AKR chimera may have been due to crossinfection of the donor ANF, cells by virus produced in the AKR recipient, but this does not explain the specific localization of the increase in the thymus; the mean number of ecotropic virus-producing infectious centers in the spleens of these animals was not different from that in ANF, + ANF, mice (P > 0.1). Moreover, on average, 10 times more infectious centers were detected in ANF, --, AKR thymocytes than in AKD/2 + AKR or AKR + AKR thymocytes. These latter two chimeras expressed amounts of ecotropic virus which we have found in intact AKD/2 and AKR mice (Datta and Schwartz, 1978) and in the AKD/2 + C57BR chimera (Table 2). Further evidence against cross-infection as the explanation for the increased levels of ecotropic virus production by thymocytes of ANF, + AKR chimeras is that the levels of virus were approximately one tenth lower when younger (6 week old) AKR recipients were used (Table 2, Figure 4). The enhanced expression of ecotropic virus by thymocytes of ANF, + C57BR chimeras (Table 2, Figure 3) is of interest. In this case we are not dealing with cross-infection because cells of the C57BR strain
LogI % MuLV-Antigen-Positive
to the age of the recipient
.6
0.02-40.0
a
0.005-0.1
0.065
f
0.0003
0.56
+ 0.04
Detailed results of MuLV antigen expression and retrovirus production by lymphoid cells from the chimeric mice shown Numbers in parentheses represent numbers of animals tested. B Thymocytes from two animals in the AKR + AKR group of chimeras expressed high levels (23-40%) of MuLV antigen. positive cells in the other eight animals of this group was 0.02-0.3% (mean 0.08 f 0.09 SD.).
in Figures
1, 3 and 4.
The range
of antigen-
Cell 174
%ymus --
Spleen 8 -
. b
Thymus .--
Splen c
.
Thymus Spleen -A
Thvmus
2% 3 *
tA i
t :s -50 k2
-60 .A. ANFt -
ANFI
AKR -ANF
‘I
ANF, -
C57BR
-40
5
-30 5
I
-20 3 -IO -
0
OJ p0n.D ANF, ,AKD/ 2 -AKR
AKR -AKR
Figure 3. Xenotropic (0) and Ecotropic mus and Spleen Cells from the Chimeric
ANF, -AKR (A) Virus Production by ThyMice Shown in Figure 1
ANF, = (AKR x NZB)F,: AKD/2 = (AKR x DBA/P)F,. Open circles and triangles in the AKR + AKR chimeras (E) represent xenotropic (0) and ecotropic (A) virus production by the two mice in this group that had high MuLV antigen expression in their thymocytes (Figure 1).
rarely express ecotropic virus, either spontaneously or after attempts at in vitro induction (Rowe, 1972; Chattopadhyay et al., 1975). This result indicates that the C57BR environment permits expression of ecotropic viral genes that are repressed by ANF, thymocytes in the ANF, environment. Moreover, the effect is virus-specific, because enhanced expression of xenotropic virus or MuLV antigens by thymocytes of the ANF, + C57BR did not occur. There was an increase in MuLV antigen expression in the thymocytes of ANF, + C57BR chimeras as compared to ANF, + ANF, and AKD/2 ---, C57BR chimeras, but the levels in the former were still 80-100,000 fold less than the ANF, + AKR chimeric thymocytes. AKR + ANF, is another noteworthy chimera. The thymocytes of these mice had the virological phenotype of the donor AKR strain and were similar to those of the AKR ---* AKR chimera. The unchecked expression of ecotropic virus by AKR thymocytes in the ANF, host (Table 2, Figure 3) shows that the ANF, environment does not actively restrict ecotropic virus production and spread by thymocytes that differentiate from bone marrow cells of young adult AKR mice. The aug-
s o\”
AKR
Figure 4. Xenotropic (0) and Ecotropic (A) Virus Production by Thymus and Spleen Cells (A) and MuLV Antigen Expression by Thymocytes (B) from (AKR x NZB)F, + AKR Chimeras Where the Recipients Were 6 Weeks Old When Irradiated and Reconstituted These chimeras other chimeras
were rested for 3 months and tested along with the when the recipients were 18-l 9 weeks old.
mented expression of ecotropic virus by the thymocytes of ANF, + C57BR chimera, therefore, in contrast to the ANF, + ANF, chimera, is not simply because the C57BR host provides a permissive environment for spread of virus produced by the ANF, donor thymocytes. The intact ANF, mouse restricts expression of ecotropic virus by both thymocytes and peripheral T cells of lymph node8 and spleen (Datta and Schwartz, 1978). The latter phenomenon was also found in ANF, + ANF, chimeras. Numbers of ecotropic virus-producing infectious centers in nylon wool-purified T lymphocyte-enriched spleen cell fractions were 30270 fold lower than in unfractionated spleen cells. By contrast, the levels in T lymphocyte-enriched fractions of spleens from ANF, -+ AKR chimeras were only 210 fold lower in unfractionated spleen cells, as was found in the case of AKR --* AKR, AKR + ANF, and ANF, + C57BR chimeras (data not shown). Isolation of Polytropic Viruses Although spleens of intact ANF, mice express relatively high levels of both ecotropic and xenotropic viruses, no evidence indicative of polytropic viruses can be detected (Datta and Schwartz, 1978). In light of the expression of both ecotropic and xenotropic
Retrovirus 175
Expression
in F, +
Parent
Chimeras
Table 3. Isolation of Viruses with Polytropic Activity from ANF, + AKR Thymuses following Limiting Dilution Purification in Mink Cell Lines Fluorescent Focus Forming Units (Log,,) per 0.2 ml of Purified Culture Supernatant’ Number Mice
of
Mink (Coverslip)
NIH/3T3 (Coverslip)
XC Test (NIH/3T3)
1
3.62
3.15
Negative
2
4.02
3.75
Negative
3
4.35
3.93
Negative
4
5.24
0.95
Negative
Isolation of viruses with polytropic activity from ANF, -+ AKR thymuses following limiting dilution purification in mink cell lines. (See Experimental Procedures for details of co-cultivation and purification of culture supernatants.) Attempts were made to isolate polytropic virus using similar protocols from thymocytes of three AKR -+ ANF,. three AKD/P -+ AKR and three AKR + AKR chimeras. Four of these yielded only xenotropic virus after the passage in mink cells. The rest were negative on both mink and NIH/3T3 cell assays after the mink passage. “Negative” = no XC-plaques could be detected on inoculation of NIH/3T3 indicator cells with 0.5 ml of purified culture supernatants. ’ See Experimental Procedures.
viruses by ANF, thymocytes in ANF, + AKR chimeras, therefore, it was of interest to seek polytropic viruses in these cells. Of four supernatants tested after passage in tissue culture (see Experimental Procedures), three yielded XC-negative viruses with polytropic activity, with approximately equal and high titers in mink and NIH/3T3 cells (Table 3). No such viruses could be isolated from AKR + AKR, AKD/2 + AKR or AKR + ANF, thymocytes. Further purification of the viruses with polytropic activity for biochemical studies (Elder et al., 1977; Rommelaere et al., 1978) is in progress. Discussion Our experiments demonstrate that virtually all of the thymocytes of ANF, mice express MuLV antigen when they differentiate in the environment of AKR mice. Moreover, such thymocytes also produce high levels of both ecotropic and xenotropic viruses. These manifestations are in striking contrast to those exhibited by thymocytes of intact ANF, mice, which fail to express MuLV antigen and produce very low or undetectable amounts of ecotropic and xenotropic viruses (Datta and Schwartz, 1978). Thymocytes and peripheral T cells in the spleen and lymph nodes of ANF, mice have these characteristics despite the presence of the ecotropic viral genes, Akv-1 and Akv-2, and the xenotropic virus-inducing genes, Nzv-1’ and Nzv-2” (Rowe, 1972; Datta and Schwartz, 1978). Some mechanism that restricts the expression of these genes specificially in the cells of T lymphocyte lineage must therefore exist in ANF, mice. The present ex-
periments also show that the marked augmentation of retrovirus and MuLV antigen expression occurs specifically in the thymocytes of ANF, mice when they differentiate in the AKR host. Since the thymic reticuloepithelial environment is solely responsible for the differentiation and maturation of bone marrow precursor cells to thymocytes in lethally irradiated, bone marrow-restored chimeras, we assume that the phenotypic alteration of ANF, thymocytes is brought about by the radiation-resistant thymic reticuloepithelium of the AKR recipient (von Boehmer and Sprent, 1976; Sprent, 1978; Fink and Bevan, 1978; Zinkernagel et al., 1978). The effects we observed were specific. We can exclude nonspecific effects of irradiation because changes in expression of MuLV antigen or infectious viruses did not occur in the thymuses of syngeneic chimeras (ANF, + ANF, and AKR + AKR). There were also no changes in MuLV antigen expression or production of xenotropic virus in AKD/2 + AKR or ANF, + C57BR and AKR ---* ANF, chimeras. In the former case, the AKD/2 donor mice lack NZB genes; in the latter cases, both C57BR and ANF, recipients are low leukemia strains (Rowe, 1972; Datta and Schwartz, 1978). Therefore, the remarkable changes in the thymuses of the ANF, -+ AKR chimeras are brought about by a specific interaction between ANF, thymocytes and the AKR environment in which they differentiate. An alternative explanation for the augmented retrovirus production we found could be that the thymocytes in this chimeric combination are more susceptible to reinfection and spread of the viruses. For example, in the ANF, + AKR chimera, ANF, cells may produce ecotropic virus which can replicate in AKR cells generating some type of polytropic recombinant virus, which then replicates in the ANF, cells. The evidence against this possibility as the sole cause for the pronounced retrovirus production lies in the specificity of the results. AKR or AKD/2 cells that were allowed to differentiate in the AKR host did not generate either polytropic or xenotropic viruses, although they did produce ecotropic viruses. In those chimeras the donor cells possess and express ecotropic viral genes, and they are not known to restrict the replication and spread of polytropic recombinant viruses (Hartley et al., 1977); yet augmented expression of xenotropic and ecotropic viruses and generation of polytropic viruses occurred on/y in the ANF, -+ AKR chimeras. The same argument applies to MuLV antigen: its expression was markedly amplified only in the thymocytes of the thymuses of the ANF, -+ AKR chimeras. Neither are these specific results explained by a peculiarity in the ANF, donor cells that permits increased virus spread, since thymuses of ANF, + C57BR and ANF, + ANF, chimeras lacked the virologic changes shown by the ANF, + AKR chimeras. Moreover, when the direction of chimeric combination was reversed, as in the AKR + ANF,
Cell 176
chimera, so that AKR thymocytes were allowed to differentiate in ANF, host environment, the virologic changes seen in ANF, + AKR chimeras did not occur. This result also indicates that the ANF, host does not restrict spread of retroviral infection, because the donor AKR thymocytes produce ecotropic viruses in this host, as it does in the AKR + AKR chimeras or the intact AKR animal. Thus the lack of virus production in thymuses of ANF, + ANF,, in contrast to the ANF, + AKR chimeras, is not due to a restriction in virus spread. The evidence therefore favors the interpretation that the thymic reticuloepithelium of the high leukemia strain AKR has the specific ability to alter the phenotype of thymocytes from low leukemia ANF, mice. Kawashima et al. (1976b3) have observed the amplification of MuLV antigen by the thymocytes of syngeneic AKR + AKR chimeras. In their experiments this phenomenon was not found in 2 month old recipients, but only in 6 month old preleukemic recipients, whose thymuses express xenotropic virus in high titers, as well as polytropic (MCF) particles. In our system, the “amplification signal” was detectable when AKR recipients were as young as 2 months. At this age the AKR thymus expresses only ecotropic virus. One reason for the difference between ANF, and AKR donor cells may be that ANF, mice possess NZB genes, including Nzv-7’ and Nzv-2’ (Datta and Schwartz, 1976, 1978). These genes may be highly susceptible to activation (or increased expression) by a signal from AKR thymic reticuloepithelial cells. In the intact ANF, animal, this signal is apparently never produced, perhaps because of restrictive or nonpermissive NZB genes. Our results show that, given a sufficiently sensitive system (the ANF, thymocyte), virus- and viral antigen-amplifying “signals” can be detected in AKR mice many months before they can be observed in the intact animal. We propose that the amplifying “signals” of AKR thymic microenvironment increase with time, so that by the age of 6 months the phenomenon can be detected in AKR thymocytes (Kawashima et al., 1976a, 1976b). It seems improbable, in view of our data, that the amplifying signal(s) is either a xenotropic or polytropic virus. In two AKR + AKR chimeras, 23% and 40% of the thymocytes stained for the presence of MuLV antigen (Figure l), yet the xenotropic virus titers in the thymuses of these mice were low (Figure 3, Table 2). This finding implies that augmented expression of MuLV antigen may not be due entirely to the production of infectious xenotropic virus. The very large difference, in ANF, + AKR chimeras, between the number of thymocytes that stain for MuLV antigen (80-100%) and the number that produce infectious virus, either ecotropic or xenotropic (-1%. as determined by infectious center assays), shows that MuLV antigen is expressed independently of infectious virus. Furthermore, the augmented numbers of ecotropic
virus-producing thymocytes in the face of very low levels of xenotropic virus and MuLV antigen-positive thymocytes in the ANF, + C57BR chimera also point to discordant expression of infectious viruses and MuLV antigens in these chimeras. The restricted expression of ecotropic virus by thymocytes of ANF, mice and ANF, --* ANF, chimeras appears to be prethymic, because AKR prothymocytes, which already produce ecotropic virus before reaching the thymus, expressed the virus unchecked in thymuses of AKR + ANF, chimeras. This prethymic restriction was removed when ANF, precursor cells differentiated in either C57BR or AKR hosts. It is improbable that this result was due to cross-infection by virus produced within the irradiated hosts. C57BR mice do not usually express ecotropic virus spontaneously, or even after attempts at in vitro induction (Rowe, 1972; Chattopadhyay et al., 1975). Furthermore, spleen cells of intact ANF, mice and ANF, --, ANF, chimeras produce ecotropic virus, yet the T lymphocytes within these spleens are not cross-infected (Datta and Schwartz, 1978; see also Results). All of the above indicates that the prethymic restriction in intact ANF, mice is maintained throughout the T lymphocyte lineage. The results in the low leukemia C57BR hosts, in which ANF, thymocytes expressed only ecotropic virus, contrast with those in the high-leukemic AKR hosts, where ANF, thymocytes expressed both ecotropic and xenotropic viruses and high levels of MuLV antigen. This difference is noteworthy because the expression of both viruses occurs during the development of thymic leukemia (Kawashima et al., 1976b; Hartley et al., 1977). Moreover, the thymocytes of ANF, + AKR chimeras also produced a polytropic virus, which we assume arises by recombination between ecotropic and xenotropic viruses (Elder et al., 1977; Hartley et al., 1977; Rommelaere et al., 1978). It will therefore be of interest to compare the incidences of thymic leukemia in these two chimeras, an experiment which is in progress. The amplified expression of proviral genes by thymocytes exposed to the AKR thymic environment may provide the viruses that recombine with the N-tropic virus prevalent in all tissues of AKR mice from birth. The presence of polytropic virus, a presumed leukemogenic agent, in ANF, + AKR chimeras raises the question of whether the ANF, thymocytes of the chimera undergo malignant transformation. None of the animals we tested had evidence of tumor. The transfer of MuLV antigen-positive thymocytes from ten ANF, + AKR chimeras individually into young ANF, mice (50-80 X lo6 cells per recipient), however, has resulted in the appearance of leukemia involving thymus and spleen in three recipients within 3 months. Transfer of thymocytes from the ANF, + C57BR, AKR + AKR, ANF, ---, ANF, and AKR -P ANF, chimeras have failed to produce tumors, even up to 5 months after
Retrovirus 177
Expression
in F, +
Parent
Chimeras
inoculation (S. K. Datta, unpublished observations). Kawashima et al. (1976a) found that the transfer of thymocytes from 6 month old AKR mice into young syngeneic recipients did not cause leukemia, even up to 100 days. By contrast, only a few leukemic cells from AKR thymomas produce evident tumor in syngeneic recipients within 3 weeks. This indicates that the “preleukemic” thymocytes with amplified MuLV antigen and xenotropic virus expression require further residence in the aged AKR environment before leukemic transformation becomes established. Events subsequent to those concerned with the virological changes in the preleukemic AKR thymus may therefore be required for leukemia to develop. In the ANF, + AKR chimera these events may have been accelerated. We may conclude from these experiments that, in the mice we studied, the functional status of the thymic reticuloepithelium is of particular importance in determining the expression of integrated retroviral genes by thymocytes. The evidence in these animals favors a specific genetic mechanism that controls the ability of thymic reticulopeithelium to render possible the expression of these viral genes. This property is lacking in thymic reticuloepithelium of ANF, mice, whose T lymphocytes fail to express their viral genes. Based on our findings in ANF, + AKR and ANF, + C57BR chimeras, the thymic reticuloepithelium augmenting mechanism has three properties relevant to increased expression of MuLV antigen, ecotropic virus and xenotropic virus. The AKR thymus possesses all three capabilities, whereas that of the C57BR strain augments expression of only ecotropic viral genes. The ANF, thymus lacks all of these properties. Attempts to isolate and identify these inductive mechanisms are in progress. Experimental
Procedures
Mice NZB, AKR, C57BR and (AKR x DBA/2)F1 mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). (NZB x AKR)F1 and (AKR x NZB)F, hybrids were bred in our laboratory. Since results were similar in the reciprocal crosses. these were designated simply as ANF,. Radiation Chimeras Chimeras were prepared by irradiating recipient mice with 950-l 000 rads from a I” Cs source and repopulating them with bone marrow ceils in a manner described previously &on Boehmer and Sprent. 1976; Sprent. 1978; Fink and Bevan. 1978; Zinkernagel et al., 1978). 4 hr after irradiation the recipients were injected intravenously with 8-15 x 10’ viable bone marrow cells that had been treated twice with rabbit anti-mouse brain serum (Waksal et al.. 1974) and normal rabbit serum as a source of complement to deplete any T lymphocytes in the inoculum. This step obviates any graft versus host disease in the AKR ---f ANFl combinations. All the other combinations were matched at the major histocompatibility loci. The reconstituted mice received Polymyxin and Neomycin in their drinking water for 2 weeks. They were analyzed 7-9 weeks after reconstitution. The donor mice were 12-14 weeks old, and the age of recipient mice was 8-12 weeks. In one set of ANFl + AKR chimeras the recipients were 6 weeks old (see Results). Donors and recipients were the same sex,
except for ANF, + ANF, and AKR -+ AKR chimeras, where male donor cells were injected into female recipients. The mortality (6080%) was high because of the lethal to supralethal irradiation. All irradiated mice that were not reconstituted with bone marrow died within 2 weeks. Due to the high mortality, the above chimeric combinations were made 3 times. Results were similar in the three sets of experiments and are presented together. Tests for Chimerism Treatment with Anti-K2” Serum In all combinations except ANF, + ANFl and AKR + AKR. lymphoid cell chimerism was assessed by a two-stage, complementdependent cytotoxicity test using specific anti-H-2d sera. (Erb et al., 1979). AntiD-4 was provided by T. Hansen (National Institutes of Health, Bethesda, Maryland) and anti-D-31 was from H. Winn (Massachusetts General Hospital, Boston, Massachusetts). Baby rabbit serum screened for low background cytotoxicity against mouse lymphocytes sewed as a source of complement. Cytotoxicity was determined by the trypan blue dye exclusion test and calculated as % of the control in which cells were incubated with normal BALE/c mouse serum and complement. Cells from ANF, mice were also used as control to test the activity of the anti-H-2” sera. 80-90% of the thymus and spleen cells from all combinations except AKR + ANF, were of the H-2d type, using both anti-H-2 sera. Less than 10% of the lymphoid cells of the AKR + ANF, chimera were killed by this treatment. Chromosome Preparation Thymus and spleen cells from ANF, 6 + ANF, P and AKR 6 - AKR P chimeras were stimulated with Con A (2 pg/ml)(Pharmacia, Upsala, Sweden) or phytohemagglutinin (PHA-P, 5 pg/ml) (Wellcome Reagents, Ltd., Beckenham. England) for 72 hr. The lymphoblasts in 5 ml suspension were incubated with 2 drops of 0.1% solution of colchicine (Calbiochem. La Jolla, California) for 2 hr. harvested. swelled with hypotonic 0.075 M KCL and fixed with 3 parts methanol/ 1 part glacial acetic acid, and air-dried smears were prepared (Wang and Federoff, 1972). The modified Seabright banding technique was used to stain the smears with Giemsa after brief trypsin digestion (Seabright. 1971). Cells from intact male and female mice served as controls. In magnified photographs of the metaphase spreads from cells of the chimeras, Y chromosomes were consistently identified as the unpaired short chromosome lacking heavily stained centromeres (Wiener et al., 1978). All chimeras were tested in this manner at the time of retrovirus assays. Retrovirus Assays Infectious center assays with aliquots of lymphoid cell suspensions from thymuses and spleens of the chimeric mice were carried out as previously described (Datta and Schwartz, 1978; Datta et al., 1978). In some chimeras nylon wool column-purified T lymphocyte fractions of spleen were also obtained, as described (Julius. Simpson and Herzenberg. 1973; Datta and Schwartz, 1978). and tested for virus production. Xenotropic Virus Fluorescent antibody focus assays to detect infectious centers were done on mink lung cells (ATCC CCL64) growing on glass cover slips in Petri dishes, as previously described (Kawashima et al., 1976b; Datta and Schwartz, 1978). Ecotropic Virus Since none of the mice tested are known to produce B-tropic virus, and all were homozygous for Fv-1” (Rowe, 1972; Chattopadhyay et al.. 1975; Datta and Schwartz, 1978). production of N-ecotropic viruses were determined by a fluorescent antibody focus assay with NIH/3T3 indicator cells growing on glass cover slips placed in tissue culture dishes (Kawashima et al., 1976b: Datta and Schwartz, 1978; Datta et al., 1978). This assay detects the expression of both XCpositive and XC-negative N-tropic viruses (Nowinski et al., 1977; Datta and Schwartz. 1978). Titers are expressed as logl,, of the number of foci produced on each cover slip per 10’ lymphoid cells inoculated in the tissue culture dish, although the area of the cover
Cell 178
slips on which culture dishes.
foci were
counted
is one fourth
the area of the tissue
Polytropic Virus The method developed by Hartley et al. (1977) to detect recombinant viruses with dual host range was used. This has been described in detail in previous reports from our laboratory (Hiai et al., 1977; Datta and Schwartz, 1978). Lymphoid cells were co-cultivated with mink cells. After 5-6 passages, the supernatants from these cultures were filtered through 0.22 mp Millipore filters and subjected to limiting dilution purification by reinfection on a fresh line of mink cells. These cells were subsequently split and passaged weekly. Afler 5-6 passages, culture supernatants were tested on mink and NIH/3T3 cells by the cover slip fluorescent focus assay and XC tested with NIH/ 3T3 indicator cells (Rowe, 1972; Datta and Schwartz, 1978). Only xenotropic and polytropic viruses would replicate in this protocol. lmmunofluorescence for Murine Leukemia Virus Antigens Thymocytes from the chimeric animals were adjusted to a concentration of 5 x IO5 cells per ml. 0.45 ml of the cells were pelleted on slides in a cytocentrifuge (Manny, Datta and Schwartz, 1979) and fixed for 10 min in cold methanol. Air-dried smears were stained with fluoresceinated-specific anti-MuLV sera at 1:40 dilution (diluted in a rhodamine counter-stain solution) at 37% for 30 min, washed and examined with a fluorescence microscope with Ploem epiillumination and appropriate filters (Datta et al.. 1978). The entire smear (approximately 2 X lo5 cells) was scanned in those specimens which had very low numbers of positive cells. In some cases, membrane immunofluorescence was sought by incubation of viable thymocytes at 4’C for 30 min with antiserum (Kawashima et al., 1976a). These assays gave results similar to those for fixed cells. There were two anti-MuLV sera used in these tests. The first was fluorescein-conjugated goat antiserum prepared against Tween-ether-disrupted Moloney MuLV (supplied by J. Gruber. National Cancer Institute, Bethesda, Maryland). This anti-serum contains antibodies to the broad spectrum of MuLV-related antigens (Hartley and Rowe, 1976; Kawashima et al., 1978b). The second was a fluoresceinated goat anti-gp69/71 MuLVRauscher (Lerner et al., 1978) provided by R. Lerner (Scripps Clinic, La Jolla, California). Results were similar with both antisera. At a 1: 40 dilution they specifically stained cells replicating and expressing xenotropic. ecotropic and polytropic MuLV (Datta et al., 1978). Acknowledgments We acknowledge the expert technical assistance of Isaac Robert Mendelson. Ann Marie Champ, Norma Robert and Rhonda Sinoff and wish to thank Marrian Gleason and Elaine McGlame for typing the manuscript. This work was supported by two National Cancer Institute grantstoS.K.D.;onetoS.D.W.;and,twotoR.S.S.S.K.D.isaFaculty Research Awardee of the American Cancer Society. S.D.W. is a recipient of a Scholar Award from the Leukemia Society of America. 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
July 23. 1979;
revised
October
10. 1979
References Chattopadhyay. S. K., Lowy. D. R., Teich, N. M., Levine, A. S. and Rowe, W. P. (1975). Qualitative and quantitative studies of AKR-type murine leukemia virus sequences in mouse DNA. Cold Spring Harbor Symp. Quant. Biol. 39, 1085-l 101. Datta, S. K. and Schwartz, R. S. (1976). Genetics of expression of xenotropic virus and autoimmunity in NZB mice. Nature 263, 412415. Datta. S. K. and Schwartz, loci controlling xenotropic 83, 449-452.
R. S. (1977). virus production
Mendelian segregation of in NZB crosses. Virology
Datta, S. K. and Schwartz, R. S. (1978). Restricted expression of ecotropic virus by thymocytes of leukemia-resistant (AKR x NZB)FI mice. J. Exp. Med. 148, 329-334. Datta, S. K.. Manny, N., Andrzejewski, C.. Andre-Schwartz, J. and Schwartz, R. S. (1978). Genetic studies of autoimmunity and retrovirus expression in crosses of New Zealand Black mice. I. Xenotropic virus. J. Exp. Med. 147, 854-871. Elder, J. H.. Gautsch. J. W., Jenson, F. C., Lerner. R. A., Hartley, J. W. and Rowe, W. P. (1977). Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants. Proc. Nat. Acad. Sci. USA 74, 4676-4680. Erb, P., Meier. B.. Matsunaga. T.. Feldman, M. (1979). Nature of T cell macrophage interaction in helper-cell induction in vitro. II. Two stages of T-helper cell differentiation analyzed in irradiation and allophenic chimeras. J. Exp. Med. 749, 686-701. Fink, P. J. and Bevan. M. J. (1978). H-2 antigens of the thymus determine lymphocyte specificity. J. Exp. Med. 148, 766-775. Hartley, J. W. and Rowe, W. P. (1976). Naturally occurring murine leukemia viruses in wild mice: characterization of new “Amphotropic” class. J. Viral. 19, 19-25. Hartley, J. W., Wolford. N. K.. Old, L. J. and Rowe, W. P. (1977). A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Nat. Acad. Sci. USA 74, 789-792. Hiai. H., Morrissey. P., Khiroya. Selective expression of xenotropic mice. Nature 270, 247-249.
R. and Schwartz, R. S. (1977). virus in congenic HRS/J (hairless)
Holmes, M. C. and Burnet, F. M. (1966). The characteristics of F, and backcross hybrids between “high leukemia” (AKR) and “autoimmune” NZB mouse strains. Aust. J. Exp. Biol. Med. Sci. 44, 235249. Julius, M. H.. Simpson, E. and Herzenberg. L. A. (1973). method of the isolation of functional thymus-derived murine cytes. Eur. J. Immunol. 3, 645-649.
A rapid lympho-
Kawashima. K.. Ikeda. H.. Stockert. E., Takahashi. T. and Old, L. J. (1976a). Age-related changes in cell surface antigens of preleukemic AKR thymocytes. J. Exp. Med. 144, 193-208. Kawashima. K.. Ikeda, H.. Hartley, J. W.. Stockert. E., Rowe, W. P. and Old, L. J. (1976b). Changes in expression of murine leukemia virus antigens and production of xenotropic virus in the late preleukemic period in AKR mice. Proc. Nat. Acad. Sci. USA 73, 46804684. Lerner. R. A., Wilson, C. B., Del Villano, B. C.. McConahey, P. J. and Dixon, F. J. (1976). Endogenous oncornaviral gene expression in adult and fetal mice: quantitative, histologic and physiologic studies of the major viral glycoprotein gp70. J. Exp. Med. 743, 151-l 66. Manny, N.. Datta, S. K. and Schwartz, R. S. (1979). by cells of NZB and SWR mice and their crosses. 1220-l 227.
Synthesis of IgM J. Immunol. 122,
Nowinski, R. C. and Hays, E. F. (1978). Oncogenicity enous leukemia viruses. J. Viral. 27, 13-18. Nowinski, R. C.. Hays, E. F.. Doyle, T., Linkhart. Pickering, R. (1977). Oncorna virus produced cells in culture. Virology 87, 363-370. Rommelaere. J.. tion and mapping the genomes of Acad. Sci. USA
of AKR endog-
S., Medeiros, E. and by murine leukemia
Failer. D. V. and Hopkins, N. (1978). Characterizaof RNase Tl -resistant oligonucleotides derived from Akv and MCF murine leukemia viruses. Proc. Nat. 75, 495-499.
Rowe, W. P. (1972). Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-1” strains of mice. J. Exp. Med. 136, 1272-l 285. Seabright, M. (1971). A rapid banding somes. Lancet 2, 971-972. Sprent. J. (1978). Restricted marrow chimeras controlled 747, 1838-1842.
technique
for human chromo-
helper function of F, + parent bone by K-end of H-2 complex. J. Exp. Med.
von Boehmer. H. and Sprent. J. (1976). marrow chimeras: absence of host-reactive
T cell function in bone T cells and cooperation
Retrovirus 179
of helper 23.
Expression
T cells
across
in F1 +
Parent
allogeneic
Chimeras
barriers.
Transplant.
Rev. 29, 3-
Waksal. S. D.. St. Pierre, R. L.. Hostetler. J. Ft. and Folk, R. M. (1974). Brain associated 19antiserum: differential effects on lymphocyte subpopulations. Cell Immunol. 12, 66-73. Wang, H. C. and Federoff, S. (1972). Banding in human chromosomes treated with trypsin. Nature New Biol. 235, 52-54. Wiener, F.. Ohno, S.. Spira, J., Haran-Ghera. N. and Klein, G. (1978). Chromosome changes (trisomes #I 5 and 17) associated with tumor progression in leukemias induced by radiation leukemia virus. J. Nat. Cancer Inst. 61, 227-237. Zinkernagel. R. M.. Callahan, G. N., Althage, A., Cooper, S.. Klein, P. A. and Klein, J. (1978). On the thymus in the differentiation of “H-2 self-recognition” by T cells. Evidence for dual recognition? J. Exp. Med. 147, 882-896.