Moloney. MuLV-M to infect and replicate in cultures of. and in thymocytes. Second, we examined the. virus replication and the leukemogenicity in

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1 INFECTION AND IMMUNITY, June 1983, P /83/ $02.00/0 Copyright , American Society for Microbiology Vol. 40, No. 3 T and B Lymphocyte Susceptibility to Murine Leukemia Virus Moloney DALE D. ISAAKl* AND JAN CERNY2 Department of Microbiology and Immunology, Kirksville College of Osteopathic Medicine, Kirksville, Missouri ; and Department of Microbiology, University of Texas Medical Branch, Galveston, Texas Received 7 June 1982/Accepted 4 February 1983 The susceptibility of T and B lymphocytes to productive infection and transformation by murine leukemia virus Moloney was determined by enumeration of cells producing infectious virus after in vitro infection of mitogen-stimulated, isolated cell populations and by in vivo infection of euthymic BALB/c and thymus-deficient (nude) mice. Our in vitro results demonstrated that the majority of splenic T cells and thymocytes are resistant to productive infection in vitro; a specific subpopulation of susceptible nylon-adherent splenic T cells was identified, however. Similarly, surface immunoglobulin-positive B cells also represent susceptible targets in vitro; mature B cells, however, did not represent the principal target for transformation in the in vivo experiments. Infected euthymic mice expressed increasing titers of murine leukemia virus and uniformly developed fatal T-cell lymphomas at 10 to 12 weeks postinfection; nude mice, in contrast, maintained high, stable levels of viremia throughout the 28 weeks of observation. Infected nude mice remained free of malignancy or developed either granulocytic leukemias or, in one case, reticulum cell sarcoma. Collectively, the results indicate that While the majority of T cells are resistant to productive infection, they represent the principle targets for transformation; B cells, however, represent permissive targets for virus replication, but are resistant to transformation. Murine leukemia virus-moloney (MuLV-M) induces a fatal lymphatic leukemia within several months after infection of newborn mice (24). The disease is characterized by enlarged thymus glands, hepatosplenomegaly, and enlargement of both peripheral and mesenteric lymph nodes. The neoplastic process begins in the thymus (11, 32) and cell lines established from leukemic mice express both Thy 1.2 and MuLV-M-specific cellassociated antigens (27, 32), indicating that the virus infects and transforms T cells. However, several studies have provided evidence that MuLV-M also infects antibody-forming cells (i.e., B cells). In mice infected with MuLV-M and then immunized with an unrelated antigen, individual antibody-forming cells were shown to contain viral glycoprotein(s) detectable by immunofluorescence (10), virus particles (26), and virus-specific, cell-associated surface antigen(s) detectable by cytotoxic treatment with MuLV-M-specific antibody plus complement (5). Thus, it appears that MuLV-M infection may offer a model of leukemia virus restriction in which both T and B lymphocyte classes may be infected, but only the T cell is transformed. 977 Since the evidence for B cell permissiveness is indirect, however, and no experiments on transformation of B cells by MuLV-M have been reported, we have examined this issue more thoroughly. First, we determined the ability of MuLV-M to infect and replicate in cultures of mitogen-activated, purified splenic T and B cells and in thymocytes. Second, we examined the virus replication and the leukemogenicity in congenitally thymus-deficient nude mice, which have normal B cells but lack differentiated T cells. The results of these studies indicate a dichotomy between ecotropic MuLV infection and transformation of target cells within some lymphocyte subclasses. MATERIALS AND METHODS Animals. BALB/c as well as nude mice were derived from breeding stock maintained in our animal facilities. BALB/c stock was obtained initially from Charles River Laboratories, Inc., Wilmington, Mass.; nude mice and their phenotypically normal, euthymic littermates (LM) were the progeny of heterozygous breeding stock (derived from mice supplied by N. D. Reed, Montana State University, Bozeman) backcrossed 10 generations on the BALB/c strain.

2 978 ISAAK AND CERNY Virus. The origin of our stock MuLV-M was described previously (9). Virus passages were prepared as 10% (wt/vol) cell-free homogenates from the spleens of infected, leukemic BALB/c mice and were characterized for their content of infectious MuLV by determining the number of focus-forming units (FFU) per milliliter on sarcoma virus-positive, leukemia virus-negative (S+L-) indicator fibroblasts (3), as described previously (6); virus stocks routinely contained approximately 106 FFU of ecotropic MuLV per ml. In addition, the content of mink lung cytopathic focus-forming virus, determined by focus induction on CCL-64 indicator cells as previously described (15), was approximately 4.4 x 103 infectious units per ml. Mitogens. Escherichia coli strain 0127:B8 lipopolysaccharide (LPS) (Difco Laboratories, Detroit, Mich.) was dialyzed against deionized water, lyophilized, solubilized in 0.15 M NaCl, and boiled for 2 h before use; the optimum mitogenic concentration, 10 p.g/ml of culture medium, was used. Concanavalin A (ConA) (grade IV; Sigma Chemical Co., St. Louis, Mo.) was used at its optimum mitogenic concentration, 2,ug/ml of culture medium, or as otherwise indicated. Separation of spleen cells on nylon fiber columns. B cell-enriched and T cell-enriched fractions were obtained from BALB/c spleen cell suspensions by a modification (6) of the procedure of Handwerger and Schwartz (14). The principal modification was that cells were filtered through two consecutive nylon fiber columns. The adherent fraction from the second column was enriched for B cells in that >85% of the recovered cells were surface immunoglobulin-positive (sig+) after membrane immunofluorescence staining with goat anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate, but reproducibly contained a low percentage of nylon wool-adherent T cells. The nonadherent fraction from the second column was judged to be greater than 90%o T cells by sensitivity to lysis with AKR/J anti-c3h/hej Thy 1.2 plus complement treatment; it contained <10% sig+ cells. Separation of sig+ and sig- ceils on immunoadsorbent columns. Immunoadsorbent columns prepared with rabbit anti-mouse F(ab')2-conjugated Sephadex G200 were used to obtain sig+ and sig- cells as previously described (30). Greater than 98% of the recovered adherent cells were sig+, whereas less than 2% of the effluent, nonadherent cells were sig+ after staining with fluoresceinated rabbit anti-mouse (F(ab')2 antiserum. Cytotoxic treatment. sig+ cells were removed from spleen cell suspensions via a two-step cytolytic procedure involving treatment with rabbit polyvalent antimouse immunoglobulin and guinea pig complement as previously described (19). The T cell origin of cells releasing MuLV (infectious centers) was tested by specific cytotoxic treatment with anti-thy 1.2 antibody plus complement. Cell cultures. Single cell suspensions of normal, uninfected lymphocytes were prepared in RPMI 1640 medium supplemented with 2 mm HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, 2 mm glutamine (Microbiological Associates, Bethesda, Md.), 5% fetal calf serum, antibiotics (100 U of penicillin, 100,ug of streptomycin, and 25,ug of fungizone per ml; GIBCO Laboratories, Grand Island, N.Y.) and 2,ug of polybrene (Aldrich Chemical Co., Milwaukee, Wis.) per ml of culture medium. INFECT. IMMUN. For in vitro infection of cells, 107 pelleted cells were suspended in 0.2 ml of a 1:2 dilution of MuLV-M (-105 FFU; multiplicity of infection, 102 FFU of MuLV per nucleated cell); supplemented RPMI medium with 4,ug of polybrene per ml was used to dilute the virus stocks. Cell-virus mixtures were incubated at 4 C for 40 min and then suspended in 4 ml of medium or medium containing LPS or ConA; cultures were incubated at 37 C in humidified air with 5% CO2 until the cells were harvested and assayed for the presence of cells releasing infectious MuLV. Cultures were set in duplicate in 17- by 100-mm plastic culture tubes (Falcon Plastics, Oxnard, Calif.). The mitogen stimulation of lymphocytes was assayed by [3H]thymidine uptake as previously described (9). Data are expressed as the stimulation index, the mean counts per minute in cultures of 106 cells with mitogen divided by the mean counts per minute in unstimulated cultures. Infection of mice. Within 24 h of birth, nude and LM mice were injected intraperitoneally with 1 x 104 to 2 x 104 FFU of MuLV-M. On various days postinfection, peripheral blood was harvested from a small cut of the ventral tail artery by using calibrated glass microcapillary tubes rinsed in complete medium containing 10 U of heparin per ml. The microliter samples were diluted 10-fold in culture medium and assayed fresh or after storage at -70 C. Assay for IC and MuLV. Cell suspensions recovered from in vitro infection cultures were assayed for the presence of MuLV-releasing cells in an infectious center (IC) assay on S+L- indicator fibroblasts as previously described (6). Recovered cells from duplicate cultures were washed three times in supplemented medium, counted, and assayed at a minimum of two dilutions (each in duplicate) on dishes seeded previously with S+L- fibroblasts. Individual centers of fibroblast transformation, reflecting the number of MuLV-releasing cells, were scored on day 5 and are expressed as IC/106 viable lymphocytes plated. The number of IC was proportional to the lymphocyte dilution. Data represent means from dishes with optimal IC numbers (30 to 70 IC/dish). In addition, serial dilutions of peripheral blood recovered from MuLV-M-infected nude and LM mice were assayed on S+L- cells. The foci of transformation were expressed as the number of FFU of MuLV per ml of blood assayed. Since whole blood was assayed, the observed foci were the result of free MuLV and circulating, virus-releasing blood cells. RESULTS The results from representative experiments on the infection of various lymphocyte populations by MuLV-M in vitro are shown in Table 1. The infection of normal spleen cells was enhanced almost 10-fold by the addition of ConA, a T cell-specific mitogen, into the culture medium. The effect seen on day 3 after infection appeared to be transitory, however, since the number of IC in ConA-stimulated cultures on day 6 decreased to the level observed in unstimulated cultures. Stimulation with the B cellspecific mitogen LPS also resulted in enhanced infection, but the effect was not transitory. LPS

3 VOL. 40, 1983 TABLE 1. LEUKEMIA TARGET CELLS 979 Infection of normal mouse lymphocytes with MuLV-M in vitro: effect of lymphocyte classspecific mitogens Cells infected' Mitogenb IC/10' cells' Day 3 Day 6 Splenocytes Unseparated Medium 400 ± ± 150 ConA 3,050 ± ± 100 LPS 6,575 ± 1,610 28,500 ± 5,500 Nylon adherent Medium 1,200 ± 400 3,700 ± 600 ConA 3,200 ± ,500 ± 2,500 LPS 21,000 ± 7,000 43,500 ± 4,500 Nylon nonadherent Medium <50d <100 ConA < LPS <50 1,400 Thymocytes Medium 20 <50 ConA (0.5 1lg) <10 60 ConA (2.0 jig) 15 <50 ConA (10.0 pg) 45 <50 LPS <10 <50 LPS + ConA (0.5 F±g) 25 NTe LPS + ConA (2.0,ug) <10 NT LPS + ConA (10.0,ug) <10 NT a Single cell suspensions prepared from the spleens or thymuses of normal, uninfected BALB/c mice were centrifuged, and the pellets were suspended in MuLV-M (107 cells per 10' FFU of MuLV). After 40 min of incubation at 4 C, cell-virus mixtures were suspended in'culture medium (with or without mitogens) and incubated at 37 C in air with 5% CO2. Splenocytes were infected directly or were separated on nylon wool columns before infection. The nylon-adherent, B cell-rich fraction contained >85% sig+ cells; the nylonnonadherent, T cell-rich fraction was <5% sig', and most cells (80% or more) were susceptible to lysis by anti- Thy 1.2 plus complement. b ConA was added at 2.0 p.g/ml of medium or as specified; E. coli LPS was used at 10,ug/ml. c On days 3 and 6 postinfection, cells from duplicate cultures were pooled, washed, and assayed for the presence of MuLV-releasing cells, which were detected as IC on S+L- indicator fibroblasts. The data represent the average number of IC-producing cells scored on duplicate plates and corrected to 106 viable lymphocytes assayed; cells were assayed at two or more dilutions, each in duplicate. d e No infectious centers scored by plating a given cell number. NT, Not tested. enhanced IC development in both day 3 and day 6 cultures. Interestingly, the cells that became virus permissive upon ConA stimulation, as well as those that were infected in the presence of LPS, segregated with the nylon wool-adherent, B cellenriched fraction of the spleen (Table 1). The nylon-nonadherent fraction, comprised primarily of T cells, was resistant to productive infection with MuLV-M. The low number of IC detected in LPS-stimulated cultures of nonadherent cells on day 6 presumably reflects the small proportion of (sig+) B lymphocytes present (5 to 10% of cells in the nylon-nonadherent fraction). Virtually no MuLV replication was observed in cultures of thymocytes from newborn mice that were infected in vitro and stimulated either with ConA at various concentrations, with LPS, or with both mitogens simultaneously (Table 1). The apparent nonpermissiveness to MuLV-M was not due to a failure of thymocytes to proliferate in vitro. A 10-fold increase in [3H]thymidine uptake after ConA stimulation (2.0,ug/ml) was observed (stimulation index, 10; data not shown). Furthermore, the addition of LPS, itself nonmitogenic for thymocytes, to ConA-stimulated cultures increased the stimulation index to 20, but the synergism between the two mitogens did not render the thymocytes permissive to in vitro infection with MuLV-M. Thymocytes from adult mice were also resistant to in vitro infection (data not shown). These results suggest that splenic B cells, as well as a minor subpopulation of nylon-adherent splenic T cells (-10% of the Thy 1.2+ splenocytes), are target cells for MuLV-M infection and replication in vitro, whereas thymocytes and the major population of splenic T cells are not. However, interpretation of experiments involving partially purified lymphoid cell populations stimulated with mitogens is difficult because cell-free products (interleukins) from mitogen-activated cells may influence infection

4 980 ISAAK AND CERNY TABLE 2. Effect of treatment with rabbit antimouse immunoglobulin plus complement on mitogenenhanced in vitro infection of BALB/c spleen cells with MuLVWMa Treatmentb Mitogenc IC/106 cellsd Untreated Medium 100 ± 0 ConA 1,075 ± 258 LPS 6,300 ± 1,745 Rabbit anti-immuno- Medium 5 ± 5 globulin plus com- ConA 1,500 ± 552 plement LPS 650 ± 150 Normal rabbit serum Medium 250 ± 50 plus complement ConA 950 ± 150 LPS 2,725 ± 858 a Treated or untreated spleen cells from normal, noninfected B3ALB/c donor mice were adjusted to 107 cells per culture, infected in vitro with MuLV-M, and cultured as in Table 1. b Surface immunoglobulin-positive cells were depleted from spleen cell suspensions in a two-step cytolytic treatment with rabbit anti-mouse immunoglobulin plus complement. Cells were resuspended in antiserum appropriately diluted in saline containing 0.01 M sodium azide, incubated at 0 C for 45 min, centrifuged, and suspended in guinea pig complement diluted 1:3. After 45 min at 37 C, the cells were washed and infected. Control cells were treated with normal rabbit serum plus complement or left untreated ċ LPS was used at 10,ug/ml of culture medium, and ConA was used at 2,ug/ml of culture medium. d On day 3 postinfection, recovered cells were washed, and known numbers of cells were assayed for the presence of MuLV-releasing cells. The data represent average numbers of IC scored on duplicate plates, corrected to 106 viable lymphocytes assayed. of other cell types. To confirm that LPS-stimulated B cells were the target cells in question, two approaches were used. First, normal spleen cells were treated with rabbit anti-mouse immunoglobulin antiserum plus complement before infection. Such pretreatment reduced the number of IC seen in LPS-stimulated cultures by 89% compared with untreated spleen cell cultures (650 versus 5,700 IC/106 cells) and by 77% compared with treatment with normal rabbit serum and complement (650 versus 2,725 IC/106) (Table 2). In a second approach, splenic B cells were purified with immunoadsorbent columns prepared with goat anti-mouse F(ab')2-conjugated Sephadex G200. The elution of adherent cells with mouse F(ab')2 resulted in the recovery of highly purified B cells (>98% of the cells were sig+). LPS enhanced the in vitro infection of both unseparated spleen cells and the sig+ B cells (10,000 and 14,538 IC/106 cells, respectively) (Table 3). The enrichment of permissive cells in the sig+ fraction (45%) was less than a twofold increase expected after elimination of INFECT. IMMUN. TABLE 3. LPS-enhanced MuLV-M infection in B cells purified on immunoadsorbent columnsa Cells infectedb Mitogen IC/106 cellsc Unseparated spleen Medium 250 ± 20 LPS 10,000 ± 2,000 Immunoglobulin-positive Medium NDd cells LPS 14,538 ± 1,125 a Unseparated spleen cells or purified splenic B cells isolated on immunoadsorbent columns prepared with goat anti-mouse F(ab')2 conjugated to Sephadex G200 were infected in vitro with MuLV-M and incubated in medium with or without LPS. b Cells adhering to the immunoadsorbent columns were eluted with mouse F(ab')2 and assayed for purity before in vitro infection. Greater than 98% of the cells were positive for membrane immunoglobulin after staining with fluorescinated goat anti-mouse immunoglobulin. I On day 4 postinfection cells from duplicate cultures were pooled, washed, and assayed for the presence of MuLV-releasing cells (detected as infectious centers on S+L- cells). Cells were assayed at two or more dilutions, each in duplicate. d ND, Not done. 50% of spleen cells as sig-. This reflects the existence of sig-, LPS-responsive target cells in the spleen which we observed in our earlier experiments (19). Thus the data show that purified sig+ B cells are a major, but not the only, target for MuLV-M. A specific cytotoxic treatment was used to demonstrate that the splenocytes that became permissive to MuLV-M upon ConA stimulation were indeed T cells (Table 4). Normal spleen cells were infected and cultured in medium with ConA for 3 days. Before the assay on S'Lfibroblasts, the infected lymphocytes were incubated with anti-thy 1.2 antibody plus complement. The treatment reduced the number of detectable IC by 75%, compared with control cells treated with normal mouse serum plus complement (500 versus 2,000 IC/106 cells). Because cells in each group were adjusted to equal numbers of viable cells before assay, this represents a minimum estimate of Thy 1.2+ targets. The observation that cultured B lymphocytes are permissive for MuLV-M (Tables 2 and 3) prompted studies on MuLV-M replication and its leukemogenic potential in vivo with nude mice, which have normal B lymphocytes but lack both thymocytes and mature T cells. Preliminary experiments established that the in vitro infection of LPS-stimulated spleen cells from nude mice by MuLV-M was quantitatively similar to that observed with cells from euthymic mice (unpublished results from our laboratory). Nude mice and LM mice were infected with MuLV-M within 24 h of birth. At various times

5 VOL. 40, 1983 TABLE 4. Expression of Thy 1.2 antigen on spleen cells infected with MuLV-M in vitro and stimulated with mitogena Mitogen Treatmenth IC/106 celis' None None 260 t 20 ConA None 1,900 t 400 NMS + C 2,000 t 300 anti-thy C 500 ± 40 a Normal BALB/c spleen cells were infected with MuLV-M and cultured for 3 days, as in Table 1. b At the end of the culture period, cells were washed in medium and incubated sequentially with mouse anti-thy 1.2 (or with normal mouse serum [NMS] as a control) followed by complement (C). Treatment with antibody plus complement killed approximately 80%o of the cells. c After treatment, cell preparations were adjusted to contain the same number of viable cells before assay for infectious centers. o U. U. postinfection, peripheral blood was collected and assayed for the presence of MuLV and MuLV-releasing cells. During the early course of the infection, the level of viremia in nude mice was equal to or higher than that observed in LM mice (Fig. 1). LM mice, however, all died within 3 months of typical Moloney lymphoma involving thymus, peripheral lymph nodes, and periario7 LEUKEMIA TARGET CELLS 981 teriolar sheaths of the spleen (i.e., T lymphocyte-dependent regions) (the histological evaluation was performed by E. A. Hoover from the Ohio State University, Columbus). Concomitant with the appearance of malignancy was an enhanced MuLV replication in LM mice (weeks 10 to 12 postinfection; Fig. 1). In marked contrast, infected nude mice did not develop this enhanced level of MuLV replication preceding overt lymphoma; rather, MuLV replication was maintained at a stable level for an additional 16 weeks. The life span of infected nude mice did not differ from that of noninfected nude mice in our colony (6 to 8 months). Although splenomegaly frequently developed in nude mice after the fourth month of infection, no evidence for the development of T- cell lymphomas was observed during the 28 weeks of study. Nude mice experienced no malignancy (five of nine animals), or they developed neoplasias classified as either granulocytic cell leukemias (three of nine) or reticulum cell sarcomas (one of nine). Our preliminary analysis of cell-surface markers on splenocytes indicates that the majority of cells from euthymic, lymphomatous mice are Thy 1.2+ (78 to 100% of cells in the spleen), whereas the cells from splenic tumors of nude mice have no detectable Thy 1.2 and there are FIG. 1. Newborn nude mice (hatched columns) and their euthymic littermates (open columns) (six mice per group) were infected with MuLV-M within 24 h of birth, and the level of infectious virus in blood (FFU, assayed as described in the text) was determined at various times postinfection. The results were grouped together so that one column represents a mean from repeated measurements (e.g., 4 to 6 weeks postinfection is the mean value from assays done at 4 and 6 weeks). All euthymic mice succumbed to lymphomas by 12 weeks postinfection.

6 982 ISAAK AND CERNY very few sig+ cells (Isaak, Asjo, and Cerny, experiments in progress). It should be noted that the injection of newborn LM mice with MuLV present in the blood of infected, viremic, but lymphoma-free, nude mice resulted in the development of typical Moloney lymphomas (8). DISCUSSION Our results demonstrate that sig+ lymphocytes (B cells) are permissive to ecotropic MuLV-M. Although the number of cells that became infected after in vitro infection and LPS stimulation varied from one experiment to another, up to 5% of lymphocytes in the B cell-rich fraction released infectious MuLV (Table 1). Because the host environment supports cellvirus interactions better than culture tubes (7), and because cells are continuously exposed to high titers of MuLV, it is reasonable to assume that the proportion of B cells infected in vivo would be even higher. Indeed, when lymphocytes from the spleen and bone marrow of MuLV-M-infected, viremic nude or euthymic mice were assayed on S+L- indicator cells, between 20 and 80% of cells released infectious MuLV (Isaak and Cerny, manuscript in preparation). The replication of infectious MuLV in mice with either phenotype was comparable (Fig. 1); furthermore, the virus from the blood of nude mice readily induced lymphomas in euthymic recipients (8). Yet nude mice did not develop lymphoblastic leukemias involving mature B cells, based on morphological and histological criteria as well as on preliminary screenings for sig. Thus sig+ B cells represent permissive target cells for MuLV-M with respect to virus infection, integration, and replication, but not with respect to transformation. A possible mechanism of this restriction in mature B cells is discussed below. The development of granulocytic leukemias was observed in a proportion of nude mice. In addition, 1 of 9 infected nude mice developed a reticulum cell sarcoma, which is thought to be related to the B cell lineage (22). This indicates that some bone marrow-derived cells may represent alternative targets for transformation by MuLV-M, but the uniform development of T-cell lymphomas in euthymic mice emphasizes the preference of the virus for thymus-derived cells. Ricciardi-Castagnoli et al. (29) reported the development of lymphomas in nude mice infected with radiation leukemia virus (Rad LV), an agent that typically induces thymic lymphomas (13, 21). Cell lines established from these lymphomas expressed Thy 1.2 antigen, suggesting the infection with Rad LV may lead to differentiation of immature T cell precursors in nude mice and their transformation in the absence of thymus. We have not observed the development of INFECT. IMMUN. T-cell lymphomas in MuLV-M-infected nude mice during their life span (8 months), and our preliminary studies failed to demonstrate the presence of Thy 1.2 on cells from the enlarged spleens of MuLV-M-infected nude mice that were evaluated as lymphoma negative by histological criteria. The target cells for transformation by MuLV- M are thought to be lymphocytes of the T-cell lineage (11, 20, 24, 27, 28, 33), in part because transformed thymus cells express a receptor for MuLV-M (2) and produce infectious virus (27). Therefore, we expected a large proportion of thymocytes or splenic T cells to be permissive to the virus after in vitro infection. We found, however, that less than 0.005% of cells in cultures of metabolically active thymocytes (<50 infectious centers per 106 cells; Table 1) were permissive. This corresponds with the finding of Baird et al. (2) that only 2% or less of normal thymocytes have specific binding sites for MuLV-M, whereas >90% of MuLV-M-transformed cells bind the virus. Baird et al. speculated that MuLV-M-induced lymphomas may originate from a rare subpopulation of permissive cells. In addition to thymocyte resistance, the majority of splenic T cells, recovered as nylonnonadherent cells, were also nonpermissive to MuLV-M infection in our primary lymphocyte cultures. Similarly, Horak et al. found that cell lines established from nylon-nonadherent T cells were resistant to infection with various ecotropic MuLV, including MuLV-Moloney (16). The lack of virus replication in T cells may in part be due to a transcriptional block, based on the results of Jaenisch et al. (20), which demonstrated the presence of multiple copies of virus genome in various tissues of mice infected with MuLV-M at the embryonic stage, but high levels of viral RNA were found only in overt thymoma. In contrast to the resistance observed in nylon-nonadherent T cells, our present results demonstrate the existence of a minor quantitatively subpopulation of Thy 1.2+, sive ConA-respon- splenic T cells that are susceptible to productive infection. Subsequent characterization indicated that these susceptible targets segregated in the nylon-adherent fraction, but direct attempts to demonstrate Thy 1.2 on the surface of nylon-adherent, ConA-responsive, susceptible targets by lysis with anti-thy 1.2 plus complement resulted only in a 40% reduction in IC (data not shown). Previous demonstration of the very low density of Thy 1.2 on nylon wooladherent, ConA-responsive splenic T cells by other investigators (23) may in part explain our inability to effectively inhibit IC development by such cytotoxic treatments. Recently it has been pointed out that Thy 1 antigen expression in cultures of mouse hemo-

7 VOL. 40, 1983 poietic cells is not limited to T cells and in fact may be induced, in the presence of mitogenactivated T cell culture supernatants, on cells known not to be T cells (31); in this system, bone marrow cells depleted of T cells and cultured in conditioned medium derived from ConA-activated T-cell cultures subsequently developed Thy 1+ cells related to the granulocytic series. Thus, we must consider the possibility that the susceptible Thy 1.2' cells we have identified are not T cells. In addition to expression of Thy 1.2, however, the targets described herein share a number of other characteristics with T cells; they are ConA-responsive, and they appear to be thymus derived since they are absent in nude mice. Also, we have not been able to enhance infection of nude mouse spleen cells by using conditioned media from ConA-activated T cell cultures. The latter observations suggest that we are not dealing with an unusual, non-t-cell target induced by lymphokines secreted from mitogen-activated T cells. Thus, it seems that the susceptible Thy 1.2+ target cells we have identified represent a unique subpopulation of T cells. It is interesting that we (18) and other investigators (23) have described a similar pattern of susceptibility versus resistance in nylon-adherent versus nonadherent T cells using an unrelated ecotropic MuLV, MuLV Friend. If productive infection is a necessary step in transformation by MuLV, as suggested by Ihle (17), such a virus-permissive subset of T cells may represent the actual target cells for lymphomagenesis as well as for productive infection by MuLV-M. Collectively, the data indicate that although cells from both T and B lineages are permissive to MuLV-M infection, T cells become transformed but B cells do not. An interpretation of the apparent cellular restriction of transformation should take into consideration that MuLV- M belongs to the group of leukemia-inducing retroviruses that are apparently incapable of direct cell transformation. Rather, in the course of prolonged replication in the host, these viruses may recombine with endogenous xenotropic retroviruses to result in transforming agents (12, 15, 25, 34). One can speculate that there is a heterogeneity among the endogenous viruses in different lymphocyte classes and that a given exogenous MuLV may form a recombinant with only some of them. Thus, MuLV-M may form the "appropriate" transforming recombinant with endogenous virus in T cells, but not B cells. We have examined the expression of endogenous, xenotropic virus (X) in various lymphoid tissues of MuLV-M-infected mice. In apparent accordance with the pathogenesis, euthymic mice expressed the highest values of X in the thymus, an observation consistent with that of LEUKEMIA TARGET CELLS 983 Asjo et al. (1), whereas nude mice had the highest level of X in bone marrow (8) (Isaak and Cerny, manuscript in preparation). The fact that some nude mice developed granulocytic leukemias fits the pattern of X expression, but it also shows that the restriction of transformation in MuLV-M-infected mice is relative in that cells other than T lymphocytes may become involved. A phenotypic heterogeneity of leukemias in MuLV-M-infected BALB/c mice was recently reported by Boyer et al. (4), who found the leukemic cells to be Thy 1.2+ in some mice, whereas they appeared to be "null" cells (Thy 1.2-, sig-) in other mice. The two observations on the diversity of leukemogenesis among genetically homogenous mice and the absence of transformation in subsets of lymphocyte that are permissive to the virus infection represent a puzzling aspect of the ecotropic MuLV pathogenesis. ACKNOWLEDGMENTS We thank Carol L. Reinisch for her assistance with the antimouse F(ab')2 immunoadsorbent columns, Edward A. Hoover for his evaluation of the histologic sections prepared from leukemic tissues, and Judy Adam for typing of the manuscript. This work was supported by American Osteopathic Association grant , National Science Foundation grant , Public Health Service grants CA and CA from the National Institutes of Health, and by American Cancer Society grant IM 35. LITERATURE CITED 1. Asjo, B., E. M. Fenyo, J. Spira, and G. Klein Appearance and distribution of virally determined antigens in lymphoid organs of mice during leukemogenesis by Moloney leukemia virus. Leuk. Res. 4: Baird, S., W. Raschke, and I. L. 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