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[CANCER RESEARCH 42, 1255-1260, April 1982]0008-5472/82/0042-OOOOS02.00
Depletion of Lymphocyte Subpopulations in Primary and SecondaryLymphoid Organs of Mice by a Transplanted Granulocytosis-inducing Mammary Carcinoma1
Minako Y. Lee2 and Cornelius Rosse
Department of Medicine, Division of Hematology, and Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195
ABSTRACT
Transplanted CE mammary carcinoma causes a markedincrease in the production of neutrophilic granulocytes in miceassociated with the expansion of hemopoietic marrow into theperipheral skeleton. Changes in lymphocyte populations in thefemoral marrow, the expanding peripheral marrow, and thespleen were examined for a period of 3 weeks post-tumor
transplantation using fluoresceinated antisera specific for Eland T-cells. As tumor growth and granulocytic hyperplasiaprogressed, B- and T-cells became reduced in femoral marrow
and the spleen, but lymphocytes of undefined function, devoidof T- and B-cell surface antigens (null cells), transiently in
creased in femoral marrow and in the spleen. In the expandingperipheral marrow, such null cells increased and remained asthe predominant cell type until granulocytic hyperplasia tookplace. These changes suggest shifts in the site of myelogenouslymphocyte production or in the differentiation program oflymphocytes. The thymus invariably showed marked atrophywhich, as shown in adrenalectomized animals, could not beexplained entirely by tumor-induced stress. Thus, the massive
granulocytopoietic effects of CE mammary carcinoma are coupled with marked changes in lymphocyte populations due,most likely, to the tumor's influence on primary lymphoid or
gans.
INTRODUCTION
A number of cells known to be involved in antitumor responses are derived from the bone marrow. These include NK3
cells as well as macrophage precursors, which may participatein antitumor responses in a variety of ways, and B-cells, whicheventually secrete antibody required for antibody-dependentcell-mediated cytotoxicity. The production of these potential
effector cells may be influenced by neoplasms which perturbthe normal pattern of hemopoiesis. CE mammary carcinomahas been found to induce a marked neutrophilic response inthe blood and the bone marrow, and mice transplanted withthis tumor have (a) greatly augmented rates of neutrophilproduction (15), (b) an increased population of granulocyte-macrophage-committed stem cells (17), and (c) fatty marrow in
their peripheral skeleton replaced by hemopoietic marrowwhich initially consists of lymphoid cells and is later replacedby predominant granulocytopoiesis (16).
' This work was supported by Department of Energy Contract 79EV10270and by USPHS Grant CA-28228-01 from NIH.
2 To whom requests for reprints should be addressed, at Division of Hematol
ogy, Department of Medicine RM-10, University of Washington School of Medicine, Seattle, Wash. 98195.
3 The abbreviations used are: NK, natural killer; 1460 MCA, methylcholan-
thr ens-induced fibrosarcoma; CM, caudal-metatarsal bones.Received February 2, 1981 ; accepted January 5, 1982.
The objective of the present study was to gauge the effect ofthe granulocytosis-inducing tumor on the lymphocyte popula
tion of the bone marrow and thymus, which are consideredprimary lymphoid organs, and in the spleen, which is considered a secondary lymphoid organ. Following the transplantation of CE mammary carcinoma, we have quantitated changesin lymphocyte subpopulations in femoral bone marrow, in theexpanding marrow of the metatarsal bones and caudal vertebrae, and in the spleen. B- and T-cells were identified with
fluoresceinated antisera, and changes in null cells were computed from the total lymphocyte count. In addition, we haveexamined the effects of CE mammary carcinoma on the thymusin order to assess the extent to which the splenic changesmight be a reflection of altered lymphocytopoiesis in the 2primary sites of lymphocyte production. To test whether or notsome of the observed changes could be attributed to tumor-
induced stress, we have repeated experiments in adrenalectomized mice. For comparing the stress effect of CE mammarycarcinoma, 1460 MCA was used which has no obvious hemopoietic effects.
MATERIALS AND METHODS
Mice. BALB/c and CE mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). The experiments were performed on 30female CE and 120 male and female BALB/c x CE fi (hereafter calledCCEF,) mice bred in the vivarium, University of Washington. Mice werekilled at the age of 14 to 16 weeks in groups of 3 at various timeintervals after transplantation of the tumor.
Tumor Cells. During the past 2 years, CE 1460 mammary adeno-
carcinoma (7) used in these studies has been passaged in our laboratory through CE, CCEF,, and CBA/Ca strains of mice by s.c. inoculation at 2 to 3 weekly intervals. All of these mice are of the H-2*
haplotype, and we were unable to grow the tumor in several othermouse strains of naplotypes other than H-2*. Dr. I. and Dr. K. E.
Hellström (Fred Hutchinson Cancer Research Center, Seattle, Wash.)kindly donated 1460 MCA. Tumor cell homogenates were prepared asdescribed previously (15), and approximately 1 x 106 viable tumor
cells were injected s.c. in the flank of mice using 0.2 ml of the crudehomogenate. The growth of both tumors was localized without métastases, and the CE 1460 carcinomas reached diameters of approximately 2 cm 3 to 4 weeks after inoculation.
Adrenalectomy. Bilateral adrenalectomy (18) was performed on 14-week-old male CCEF, mice under pentobarbital anesthesia, supported
by preoperative injection of 0.25 mg hydrocortisone acetate (MerckSharp & Dohme, West Point, Pa.). Postoperatively, mice received 1 mlof 0.9% NaCI solution i.p., and their drinking water was replaced by anacidified solution of 0.9% NaCI solution. Age- and sex-matched sham-operated mice received the same pre- and postoperative treatment.
Ten days after the operation, tumor cells were inoculated as described.Mice were killed in groups of 4 at various time intervals, and the wetweight of the thymus and thymic cellularity was recorded.
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M. Y. Lee and C. Rosse
Blood Cell Counts. Just before the mice were killed, leukocyteconcentration in orbital sinus blood was determined by Coulter Counter.Two hundred leukocytes were classified on Wright-Giemsa-stained
smears.Bone Marrow and Spleen Cell Suspension. One femur from each
mouse and all segments of caudal vertebrae as well as 10 metatarsalbones from each mouse (CM) were isolated and placed in a Petri dishcontaining cold Hanks' balanced salt solution. Cells were always suspended in ice-cold medium consisting of Hanks' balanced salt solution,
10% fetal calf serum (Grand Island Biological Co., Grand Island,N. Y.), and 0.1% NaN3 (referred to hereafter as "medium").
Marrow cell suspensions were prepared using a modification of themethod described by Garsten ef al. (3). Bones were cut into smallpieces, were placed in 3 ml of medium in a small porcelain mortar, andwere crushed and ground with a pestle until no intact bone piecesremained. The solution was flushed up and down with a Pasteur pipetseveral times to obtain a single cell suspension. The cell suspensionwas then layered over 3.0 ml of cold Hanks' balanced salt solution
containing 35% fetal calf serum and 0.1% NaN3 in a 15-ml plastic
centrifuge tube. The remaining bone chips were ground similarly twicemore, and a total of 8.5 ml of cell suspension was likewise layered intothe same 15-ml test tube. After the tube was allowed to stand for 60
min in ice, the top 10.0 ml of the suspension were gently transferred toa second tube. This procedure allowed ground bone fragments tosediment to the bottom of the tube while marrow cells remained in thetop layer. Only a negligible number of cells sedimented with the bonefragments. Spleen and thymus cell suspensions were prepared bypressing small pieces of the organ through lens paper in medium asdescribed previously (20). The total number of nucleated cells wasdetermined in femur and CM marrow and spleen cell suspensions usingTurk solution and a hemocytometer.
Antisera. For surface staining of B-cells, goat anti-mouse IgM (Cap-pel Laboratories, Downingtown, Pa.) was used. F(ab')2 fragments were
obtained by pepsin digestion and were conjugated with fluoresceinisothiocyanate (5). T-cells were identified by fluorescein-conjugatedmonoclonal anti-mouse Thy 1.2 (Becton, Dickinson and Co., Sunny
vale, Calif.).Immunofluorescence. RBC were lysed osmotically in spleen and
bone marrow suspensions (20), and in each instance, 2 x 106 cells in
50 /il medium were incubated on ice for 30 min with 25 liters of one ofthe appropriately diluted fluoresceinated antisera. Cells were thenwashed twice, and wet mounts were prepared from a drop of cellsuspension for counting under a Leitz Ortholux II fluorescent microscope with a x 100 oil immersion objective. Cells with circumferentialbright green ring fluorescence were considered positive. Damagedcells were identified as cells with dull and uniform fluorescence. Theremaining cell suspension was spun in the cytocentrifuge and stainedwith Wright-Giemsa for morphological examinations.
Cell Counts. On wet-mount preparations stained with fluoresceinisothiocyanate-conjugated T- or B-cell reagents, 1000 consecutive
cells were scored as fluorescence positive or negative or as damagedcells. On Wright-Giemsa-stained slides, 1000 consecutive cells were
scored as lymphocytes, other hemopoietic cells, or damaged cells. Theabsolute number of lymphocytes and their B- and T-subclasses were
calculated from the total nucleated cell counts, the differential countson Wright-Giemsa-stained slides, and the counts obtained from the
preparations stained with fluoresceinated antisera. The number of"null" or "double negative" lymphocytes was obtained by subtracting
the number of T- and B-lymphocytes from the total number of lymphocytes. The results were expressed in absolute cell numbers of T-, B-,or "null" lymphocytes per femur, CM, or spleen.
RESULTS
Peripheral Blood. As illustrated in Table 1, the blood leukocyte response to the transplanted tumor was in agreement withprevious findings (7, 15), and a remarkable neutrophilia wasobserved in both CE and CCEF, mice as the tumor progressed.The growth rate of the tumor was identical in both strains ofmice. The number of blood lymphocytes remained essentiallyunchanged.
Marrow and Spleen Cellularity. In preliminary experiments,we tested the feasibility of studying marrow cells obtained bygrinding the bones. It was found that the grinding producedapproximately 20% more nucleated cells/femur than did flushing of the medullary cavity with culture medium (17). More than92% of both femur and CM cells were viable immediately aftergrinding. Although in CM it was physically impossible to evaluate completeness of cell extraction from the bones, the groundmarrow suspension from these small bones of tumor-bearing
mice yielded adequate numbers of cells for study, and contaminating bone fragments were as few as in femoral cell suspensions.
The total number of nucleated cells in femoral marrow wasunaffected in both strains by tumor transplantation (Table 2).As shown in Table 1, the splenic weight increased by about50% at 1 week and decreased at 2 weeks after transplantationin both strains. In CE mice, the spleen remained small at 3weeks, but in CCEF! mice, it enlarged again at 3 weeks. Theseweight changes were reflected also in splenic cellularity (Table2).
Before transplantation of the tumor, the peripheral bonemarrow was overwhelmingly fatty. An attempt to obtain marrowcells from CM of normal mice yielded only 6 x 106 cells when
the bones of 10 animals were pooled. Therefore, quantitativecellular studies from these marrows were considered not feasible. By 1 week post-tumor transplantation, however, these
peripheral marrows gave fairly consistent cell yields (Table 2).The pooled CM of a mouse with marked granulocytosis yieldedalmost as many marrow cells as a femur. Tumor cells werenever seen in either the bone marrow or the spleen.
Table 1Tumor-induced changes in blood cell counts and organ weights
MiceCECCEF,Wk
posttumor
transplant01230123Tumordiame
ter(mm)11.3
±1.217.3±0.620.8±1.312.0
±1.316.8±2.322.0±2.8Neutrophils
(x103/nD2.3
±0.9a16.8±
1.969.1±13.271.4±12.41.8±
0.89.8±2.162.2±14.0135.5±19.1Lymphocytes
(X103/fll)6.7
±0.85.3±0.93.0±1.04.8±2.45.6
±0.75.8±0.94.8±1.37.5±1.9Thymus
wt(mg)107.0
±10.088.8±9.013.0±
8.911.5±2.689.1
±18.065.4±8.116.5±3.67.6± 0.8Spleen
wt(mg)70.1
±3.7112.8±10.955.8±15.659.3±9.080.8
±13.9134.4±22.851.4±6.7114.0±58.2
1Mean ±S.D.
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Lymphocyte Subpopulations in Tumor-bearing Mice
Table 2
Cellularity and percentage of granulocyte, erythroblasts, and lymphocytes in femoral or CM marrow and spleen of mice following transplantation of CE mammarycarcinoma
Wk posttumortrans-
Mice OrganplantCE
Femur0123CM
0123Spleen
0123CCEF,
Femur0123CM
0123Spleen
0123No.
ofmice
tested333333333333333333333333Differential
count(%)No.
of nucleatedcells (X10")24.7
±4.7a25.3
±2.522.3±0.624.0±3.010.3
±2.516.4±4.115.2±
3.8105.5±23.1120.6±32.190.6±25.671.0±22.331.2
±4.740.0±4.627.3±3.233.3±3.04.0
±2.311.3±4.622.5
±4.8139.0±27.4178.3±50.845.0±10.0110.3±40.9Neutrophilicgranulocytes56.5
±5.071.3 ±3.681.2 ±2.488.9
±3.230.0
±4.056.5±11.754.8
±17.59.0±2.028.5±9.832.9±6.843.3±8.146.9
±6.162.6±1.6.88.2±2.591
.7 ±4.938.6
±5.059.5±14.274.2±15.59.3±3.219.2±7.838.2±6.160.6±15.0Erythroblasts15.9
±9.710.3±3.34.9±1.42.7±1.20.5
±0.151.5±0.421.6±0.518.8±5.35.4±3.66.8±3.519.1±1.919.0
±2.620.2±1.64.7±2.73.8±0.80.5
±0.21.0±0.50.5±0.16.9±1.24.9±2.33.3±2.116.3±9.1Lymphocytes15.5±
6.710.1±3.15.3±0.25.1±1.745.6
±15.038.0±11.138.6
±12.477.1±5.964.8±5.856.8±8.534.9±3.415.1
±4.312.9±1.03.8
±1.04.5±2.750.2
±14.038.0±11.421.
2 ±8.976.6±6.168.1±6.952.1±7.622.0± 2.3Fluorescent
cell count(%)B-cells7.8
±1.91.9±0.10.5±0.11.9±0.22.8
±0.811.8±3.810.6±3.548.8±7.039.6±5.725.7±4.921.2±5.47.2
±1.62.2±0.60.6±0.11.2±0.83.0
±0.97.6±2.44.3±1.443.4±5.936.7±1.618.8±7.010.9±1.9T-cells1.6
±0.41.1±0.10.5±0.11.0±0.23.8
±1.11.8±0.68.9±2.826.3±6.519.7±4.614.9±8.98.5±0.52.4
±0.20.8±0.10.5±0.10.9±0.14.0
±1.27.0±2.03.6±1.920.4±2.914.3±4.712.7±4.86.5±2.0
" Mean ±S.D.
Granulocytopoiesis and Erythropoiesis. An overwhelmingneutrophilic hyperplasia was observed in the femoral marrowand spleen of both strains, and by the end of the observationperiod, neutrophils predominated also in CM marrow. Erythropoiesis was markedly reduced in femur marrow, and erythroblasts were rarely found in CM marrow; but splenic erythro-
poiesis was maintained (Table 2).Lymphocytes. The changes in the incidence of total lympho
cytes and of their B- and T-subclasses in bone marrow and
spleen are shown in Table 2. Chart 1 shows the effect of thetumor on the absolute numbers of B-, T-, and null or double-
negative lymphocytes per organ.The total number of lymphocytes decreased in femoral mar
row following tumor inoculation, while in the CM of most mice,lymphocytes accounted for approximately 40% of cells overthe 3-week period. However, the percentage of lymphocyteswas also reduced in mice with the most severe neutrophilia.The total splenic lymphocyte population progressively decreased in both strains of mice (Table 2).
Concordant with our previous findings (21 ) in normal femoralmarrow, about 50% of lymphocytes were B-cells. After tumor
transplantation, their percentage and absolute number fell progressively in the femoral marrow, while In CM marrow, aconsiderable number of B-cells appeared as hemopoiesis expanded (Chart 1). The absolute size of the splenic B-cellpopulation fell sharply from over 60 x 106 to less than 50% of
the control value by Week 2 and remained low at 3 weeks.T-cells accounted initially for less than 3% of femoral bone
marrow lymphocytes and by 2 weeks were practically unde-
tectable in femoral marrow in both strains. They appeared inCM marrow, but the incidence was much lower than that of B-
cells, and in the spleen, their population decreased to less than30% of control value (Chart 1).
Prior to tumor inoculation, null lymphocytes accounted for
approximately 40% of the lymphocytes in femoral marrow. Theabsolute number of null lymphocytes in the femoral marrow ofboth strains of mice temporarily increased at 1 week buteventually decreased by the third week. In CM marrow, nullcells were the predominant type of lymphocytes. In both strains,CM contained as many null lymphocytes as did a single normalfemur (Chart 1). In the spleen, null lymphocytes temporarilyincreased at 2 weeks in CE mice and at 1 week in CCEF, mice,but as granulocytes increased, the number of splenic null cellsdeclined in both strains (Chart 1).
Effect on the Thymus. The granulocytosis-inducing CE carcinoma produced pronounced thymic involution in both CE andCCEFi mice (Table 1). Two experiments were performed totest whether or not this thymic atrophy was due to a specificinfluence on the thymus or to nonspecific stress effects in thetumor-bearing animals. Since the hemopoietic effects of CE
mammary carcinoma on CE and CCEF, mice were essentiallyidentical as shown above and the same degree of thymicatrophy was observed in both strains (Table 1), the followingexperiments were performed using only CCEF, mice whichwere more readily available. In one experiment, 1460 MCA,which has no hemopoietic effects demonstrable in the blood,was transplanted into CCEF, hosts, and its effects on theperipheral neutrophil count and the thymus were comparedwith those observed in mice bearing a CE mammary carcinoma.In the other experiment, groups of mice were adrenalectomizedalong with sham-operated controls, and some members of
each group received CE mammary carcinoma.In contrast to the marked neutrophilia produced by the
transplantation of CE mammary carcinoma, there was no suchresponse to the 1460 MCA tumor of comparable size. Themean WBC count of 1460 MCA tumor-bearing mice remainedclose to base-line level [10.5 ± 1.6 (S.D.) x 103//il] and 4weeks after tumor transplantation was 11.7 ±5.0 x 103//tl.
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M. Y. Lee and C. Rosse
CE.F5-,
4-
3-
2-
CE. CM CE. SPL
20-
10-
60
£ (Balb/oCFJF, F (Balb/cxCE)F, CM
B
I
50-
40-
30-
20-
(Balb/c x CE)F, SPL
0123 0123 0123Weeks Post Tumor Transplant
Chart 1. Quantitative changes of marrow lymphocyte subpopulations in thefemoral (F) and CM marrow and the spleen (SPÕ.)of CE and CCEF, mice followingtransplantation of CE mammary carcinoma. •,B-cells; A, T-cells; O, null cells.
Bars, S.D.
The thymus of these mice under the presumed stress of the1460 MCA tumor weighed 79.2 ± 15.3 mg at 4 weeks, notdifferent from the control value (Chart 2), indicating that, unlikeCE mammary carcinoma, the 1460 MCA tumor had no significant effect on granulocytosis or the thymus.
The adrenalectomy experiments revealed that the thymicatrophy seen with the CE mammary carcinoma could not beascribed entirely to stress effects (Chart 3). In normal mice nottransplanted with tumor, there was no change in thymus weightover a 16-day period, and adrenalectomized and sham-oper
ated animals were comparable. Mice transplanted with the CEmammary carcinoma showed a significant fall in thymic weighton Day 14 in both adrenalectomized and sham-operated
groups. In adrenalectomized mice by Day 16, there was anincrease in thymus weight, and this was less evident in sham-
operated mice. Thymic cellularity, for which the data are notshown, paralleled the changes in thymic weight in all groups ofanimals. Adrenalectomy did not alter the granulocytic responseof tumor-bearing mice. The WBC counts of adrenalectomizedtumor-bearing mice reached 47.1 ±12.1 x 103//jl at 14 days
post-tumor transplantation, while those of sham-operated tumor-bearing mice and control mice were 41.8 ±2.8 x 103//il and 9.5 ±1.1 x 103/ftl. respectively.
DISCUSSION
A number of studies have documented the influence of avariety of tumors on hemopoiesis which was reflected in
"leukemoid reactions," granulocytic hyperplasia of the bone
marrow, and redistribution and quantitative changes in hema-
topoietic stem cells (1, 2, 4, 7, 12, 14, 17, 19, 22). A greatlyaugmented marrow granulocyte production to near 10 timesthe normal value in CE carcinoma-bearing mice has been well
documented by our quantitative kinetic measurements (15).The present study was to investigate a possible impact of CEmammary carcinoma and/or the massive granulocyte proliferation on marrow lymphocyte populations. Some cells includedin the marrow lymphoid population may participate in variousantitumor host defense mechanisms, and some may have various stem cell functions (9, 23, 26).
The studies reported here demonstrate that transplanted CEmammary carcinoma causes a marked diminution in the lymphocyte population of bone marrow accompanied by thymicatrophy. Marrow lymphocyte loss was most pronounced in B-
cells, while null cells in the marrow initially and transientlyincreased.
In newly developed marrow of the peripheral skeleton, theincidence of lymphocytes remained higher than in femoralmarrow. Most of these cells were of the null surface phenotype,suggesting a shift in the site of marrow lymphocyte production.Splenomegaly, if present, was mainly due to extramedullaryhemopoiesis, and both B- and T-cells decreased in the spleen.
The decrease in lymphocytes in bone marrow, thymus, andspleen was not reflected in the peripheral blood. All thesechanges in response to the CE mammary carcinoma wereessentially identical in the syngeneic CE strain and in CCEF,hybrid mice.
IOCH
I 50.
Weeks post tumor transplant
Chart 2. Thymic atrophy in tumor-bearing mice. •.observations made fromat least 10 randomly chosen CCEF mice bearing CE mammary carcinoma; O,observations made on 6 CCEF, mice bearing 1460 MCA. Bars, S.D.
IOCH
50
f
o I folieó I toI is I ibl IG I ibI is7 I4 7 M 7 I4 7 M
Days posi tumor transplant
Chart 3. Effect of adrenalectomy and CE mammarycarcinoma on the thymus.Changes in thymus weight of adrenalectomized and sham-operated CCEF, micewere recorded at Days 0, 7, 10, 14, and 16 following transplantation of CEmammary carcinoma and were contrasted with those of adrenalectomized andsham-operated control animals which were not inoculated with tumor. Columns,means; oars, S.D. D, sham-operated controls; 0, adrenalectomized controls; E3,sham-operated tumor bearer; and E3,adrenalectomized tumor bearer.
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The changes observed in the spleen can be attributed atleast partly to the diminished number of lymphocytes in thebone marrow and thymus. Lymphocytes are known to seed tosecondary lymphoid organs from both the thymus and themarrow. Postnatally, in the normal animal, the bone marrow isthe primary site for the origin of B-lymphocytes [reviewed byRosse (23)], and practically all young B-cells in the spleen are
direct immigrants from the marrow. Although the spleen cancontribute significantly to B-cell production when hemopoiesis
in the bone marrow is eliminated (11, 25), the spleen of animalsbearing the CE mammary carcinoma had evidently not readjusted to reverse the decline in the splenic B-cell population,
nor has the activated, vascularized, peripheral bone marrowassumed a major compensatory role in B-cell production. Thetotal number of B-cells recovered from all caudal vertebrae and
10 metatarsal bones represented a mere fraction or, at most,the equivalent of the B-cell population normally present in a
single femur. Thus, it is very likely that not only the splenic butthe total marrow B-cell population was reduced in quantitative
terms.The accumulation of mononuclear cells in activated periph
eral hemopoietic marrow was due chiefly to lymphoid cellsdevoid of both T- and B-cell surface antigens. As we havefound previously, the CE mammary carcinoma exerts its gran-
ulocytopoietic effect in the absence of the spleen (15), andbone marrow lymphocyte changes in splenectomized micewere the same as those in nonsplenectomized mice." It is
unlikely that the mononuclear cells observed in the peripheralmarrow represent circulating blood lymphocytes which consistalmost entirely of differentiated T- and B-cells. The largest
population of null lymphocytes is found normally in hemopoieticmarrow (21), where the majority of them will mature spontaneously into B-cells, while others can be induced in vitro toexpress T-cell membrane markers (23, 24). "Null" cells may
include NK cells as well as a variety of stem cells or progenitorcells. That some of the lymphoid cells in activated fatty marrowhave stem cell or progenitor cell potential is espoused by theobservation that the granulocyte-macrophage colony-forming
units content is increased in the peripheral bones of micetransplanted with CE mammary carcinoma (17). It is not knownwhat proportion of the "null" cells observed in our studies are
hemopoietic progenitors; pre-B-, pre-T-, or NK cells; or NK cell
precursors.Bone marrow T-lymphocytes declined in our tumor-bearing
mice, and there was a significant fall also in the splenic population of T-cells. These changes are consistent with the thymic
atrophy observed in mice bearing CE mammary carcinomawhich could not be attributed to stress. These findings suggestthat CE mammary carcinoma exerts an effect not only on themarrow but also on the thymus.
At present, it is not possible to conclude what primary targetvarious neoplasms influence in hemopoietic differentiation. Theproliferation and differentiation of pluripotent stem cells areinfluenced by various regulatory factors as well as by organ-
dependent microenvironment and cellular interactions (6). CEmammary carcinoma apparently promotes proliferation anddifferentiation of stem cells into an almost entirely granulocyticcell line. The observation that a granulocytosis-promoting factor is responsible for granulocytosis (8) remains to be con-
' Lee and Posse, unpublished observation.
Lymphocyte Subpopulations in Tumor-bearing Mice
firmed. Our findings raise the intriguing possibility of competition between granulocytic and lymphocytic progenitor cells inthe marrow, but how such a competition is mediated awaitsfurther experimentation. In view of initial increase in null lymphocytes, one must also consider the possibility that the differentiation program of newly produced lymphocytes may bealtered under the influence of the tumor.
The marked decrease in the populations of bone marrowlymphocytes and of B-cells in general raises the question of
what impairment the depletion of these cells may lead to in hostdefenses against the tumor. It has been shown that newlygenerated lymphocytes discharged from the marrow home tovarious neoplasms (10, 13), which apparently do not inhibitlymphocyte production. It remains to be determined what relationship such tumor-seeking marrow-derived lymphocytes
may have to the null lymphocytes which can be recovered fromthe marrow and spleen of mice bearing CE mammary carcinoma, and whether or not these cells can contribute to antitu-mor responses of the host.
ACKNOWLEDGMENTS
The technical assistance of Jody Lottsfeldt and Kathy Nielson is acknowledgedand gratefully appreciated.
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1982;42:1255-1260. Cancer Res Minako Y. Lee and Cornelius Rosse Granulocytosis-inducing Mammary CarcinomaSecondary Lymphoid Organs of Mice by a Transplanted Depletion of Lymphocyte Subpopulations in Primary and
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