use of adoptive transfer and winn assay procedures in the ... · ported by the finding that...

[CANCER RESEARCH 44, 1489-1498, April 1984]

Use of Adoptive Transfer and Winn Assay Procedures in the Further

Analysis of Antiviral Acquired Immunity in Mice Protected againstFriend Leukemia Virus-induced Disease by Passive SerumTherapy1

Eugene V. Genovesi,2'3 Carolyn L. Pettey,2 and Jeffrey J. Collins4

Departments of Surgery [E. V. G., C. L. P., J. J. C.] and Microbiology and Immunology [J. J. C.], Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT

Previous studies have demonstrated that spleen cells fromDBA/2 mice protected against challenge with a leukemogenicdose of Friend leukemia virus (FLV) by passive administration ofxenogeneic antiviral or anti-FLV M, 71,000 viral envelope glyco-

protein antisera can adoptively transfer antiviral resistance tounimmunized irradiated syngeneic recipients. In addition, elimination of T-cells by treatment with anti-Thy 1.2 antibodies plus

complement had no effect on the ability of spleen cells fromserum-protected mice to adoptively transfer antiviral resistance.We now show that similar depletion of B-cells with rabbit anti-

mouse immunoglobulin G plus complement or macrophages byadherence to Sephadex G-10 columns also leaves intact theprotective capacity of spleen cells from serum-protected mice.

That these results reflect the ability of more than one spleen cellpopulation to transfer antiviral resistance rather than the activityof a non-T, non-B, nonmacrophage cell compartment is supported by the finding that purified splenic T- or B-cells alone fromserum-protected DBA/2 mice can adoptively transfer antiviral

resistance. Given the previously reported effects of sublethalirradiation on FLV leukemogenesis which could potentially complicate the interpretation of adoptive transfer experiments carriedout in this system, analogous studies were performed using aWinn-type assay in which putative effector cells were preincu-

bated with virus before inoculation of the mixture in unirradiatedmice. These Winn assay experiments yielded identical results inthat serum-protected spleen cells again prevented viral leukemogenesis, and the separate elimination of T-cells, B-cells, or

macrophages had no effect on their protective activity. In addition, mixed transfer of serum-protected and normal spleen cells

also protected irradiated mice against FLV challenge, providingfurther evidence that this adoptive protection truly reflects thepresence of virus-specific effector cells in the spleens of serum-

protected mice and not an inability of these spleen cells to replaceradiation-sensitive viral target cells in recipient animals, since

these should be supplied by the normal spleen cells in thetransferred mixture.

'This work was supported by Grants IM-120D from the American CancerSociety and PO1-CA-25863 from NIH. This is Paper 10 in a series on immunother-apy of murine leukemia. See Refs. 3 to 6,12 to 14, and 26 for previous papers inthis series.

2 Supported by NIH Viral Oncology Postdoctoral Training Grant 5T32-CA-09111.3 Present address: United States Department of Agriculture-ARS, Plum Island

Animal Disease Center, P. 0. Box 848, Greenport, NY 11944.4 Scholar of the Leukemia Society of America. To whom requests for reprints

should be addressed, at the Department of Surgery, Box 2926, Duke UniversityMedical Center, Durham, NC 27710.

Received October 12,1983; accepted January 6,1984.

INTRODUCTION

We have reported previously that DBA/2 mice protectedagainst challenge with a leukemogenic dose of FLV5 by passive

administration of xenogeneic antisera raised against disruptedvirus or the purified major viral envelope glycoprotein, gp71,develop antiviral acquired immunity which can be demonstratedin 2 ways: (a) the ability of spleen and bone marrow cells fromserum-protected mice to adoptively transfer antiviral resistance

to unimmunized irradiated syngeneic recipients (4, 14); and (b)the long-term resistance of serum-protected mice to rechallengewith FLV or FLV-infected leukemic spleen cells (12, 14). In

addition, preliminary cell fractionation studies demonstrated thatelimination of T-cells by treatment with anti-Thy 1.2 antibodies

plus complement had no effect on the ability of spleen cells fromserum-protected mice to adoptively transfer antiviral resistance

(4), although subsequent studies using other approaches havesuggested the involvement of an as yet unidentified T-cell sub-

population in the generation of acquired antiviral immunity inserum-protected mice (14).

The adoptive transfer approach is extremely useful not onlyfor demonstrating the development of antiviral immune responses in serum-protected mice, but also, when used in con

junction with cell fractionation techniques, for potentially identifying the cell population(s) responsible. Nevertheless, interpretation of the results obtained using this approach is complicatedby the effect of sublethal irradiation on FLV leukemogenesis.Sublethal irradiation of DBA/2 mice prevents the development ofFLV-induced splenomegaly (which reflects the activity of the

defective SFFV component) without affecting the replication ofthe helper nondefective lymphatic leukemia virus, an effect whichcan be reversed by reconstituting irradiated mice with spleen orbone marrow cells from normal or virus-infected syngeneic ani

mals (4). This observation thus raised the possibility, albeit anunlikely one, that the adoptive transfer results reported previously actually reflected the absence of a cell population, presumably radiation-sensitive SFFV target cells, in the spleens ofserum-protected mice which is necessary for reconstituting sub-

lethally irradiated recipient animals for their ability to support thedevelopment of FLV-induced splenomegaly, rather than the activity of virus-specific immune cells.

In the present paper, studies are presented which rule out any

5The abbreviations used are: FLV, Friend leukemia virus; CpaFLV, chimpanzeeanti-disrupted Friend leukemia virus; CTL, cytotoxic T-lymphocytes; FCS, fetal calfserum; MSV, Moloney sarcoma virus; PBS, phosphate-buffered saline (0.15 UMNaCI-0.1 Ã•<Mphosphate, pH 7.2); PFU, plaque-forming unit; RIA, radioimmunoassay;SFFV, spleen focus-forming virus; gp71, major viral envelope glycoprotein (M,71,000).

APRIL 1984 1489

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E. V. Genovesi et al.

involvement of an irradiation "side-effect" in the analysis of the

acquired antiviral immunity which develops in serum-protectedmice, (a) Mixtures of serum-protected and normal spleen cells

are still effective in transferring antiviral resistance to irradiatedrecipients, which provides convincing evidence that this adoptiveprotection truly reflects the presence of virus-specific effectorcells in the spleens of serum-protected mice and not an inabilityto replace radiation-sensitive SFFV target cells in recipient animals, since these should be supplied by the normal spleen cells.(b) Similar resistance against virus challenge is provided byspleen cells from serum-protected mice in a Winn-type assay in

which unirradiated recipient animals are used, thus obviouslyeliminating any role for irradiation effects. The present studiesalso extend the adoptive transfer approach using fractionatedspleen cells and indicate that serum-protected splenocytes depleted of B-cells or macrophages are still capable of transferringantiviral resistance. That this reflects the ability of more than onecell population to transfer antiviral resistance rather than theactivity of a non-T, non-B, nonmacrophage cell population is

supported by the use of positive selection techniques (enrichment) which indicate that either splenic T- or B-cells alone fromserum-protected mice can adoptively transfer antiviral resistance

to irradiated syngeneic recipients.

MATERIALS AND METHODS

Mice. Female DBA/2 mice (6 to 8 weeks old) were obtained fromHarlan-Sprague Dawley, Inc. (Indianapolis, IN) or The Jackson Laboratory

(Bar Harbor, ME) for use in the serum therapy studies.Virus. Preparation of stock FLV was as described (5). Briefly, the

spleens of FLV-infected DBA/2 mice were collected when they became

grossly palpable (usually 2 to 3 weeks after infection, at which time theyweighed >900 mg each) and used to prepare a 20% (w/v) preparationin RPM11640 medium by homogenization for 30 sec and clarification bycentrifugation at 2000 x g for 15 min. The supernatant was removedand filtered successively through 1.2- and 0.8-^m pore-size Milliporefilters, and the cell-free filtrate was stored in small aliquota at -70Â°.

Antisera. CpaFLV antiserum was prepared as described previously(5). Rabbit anti-mouse Immunoglobulin G antiserum (heavy- and light-chain-specific; Cappel Laboratories, Cochranville, PA) was used at a1:10 dilution for depletion of B-cells (see below), while mouse anti-Thy

1.2 monoclonal antibody, obtained commercially as a hybridoma culturesupernatant preparation (New England Nuclear, Boston, MA), was usedat a 1:100 dilution for depletion of T-cells. Guinea pig complement (Pel

Freeze, Rogers, AR) was used at a final dilution of 1:10.Passive Serum Therapy. The basic serum therapy protocol has been

described in detail elsewhere (5). Briefly, 6- to 8-week-old female DBA/2 mice were inoculated i.p. with ~2 x 103 PFU of FLV [as measured by

the FG-10 sarcoma-positive, leukemia-negative cell plaque assay (1, 5)]

in 0.2 ml of PBS on Day 0. Mice were then given 0.2 ml of the appropriateserum (normal chimpanzee serum or CpaFLV serum) i.p. on Days 3, 6,9, and 12 after virus inoculation. Animals were killed at indicated times,blood was collected, and various lymphoid tissues, including spleen,peritoneal exÃºdate, bone marrow, and lymph node cells (axillary, inguinal,and mesenteric), were removed aseptically for processing as describedpreviously (4, 5, 13). All blood samples were allowed to clot for 30 minat room temperature and then overnight at 4Â°;sera were collected andstored at -70Â°.

Tumor Cell Immunization. DBA/2 mice were immunized with thesyngeneic FLV erythroleukemia FLC-745 cells (10) by 6 weekly i.p.inoculations of 5 x 106 lethally irradiated (6000 R) cells as described

previously (4, 15). Tumor-immune spleen and lymph node cells were

collected 10 to 14 days after the last immunization and prepared asdescribed above.

Adoptive Transfer of Antiviral Resistance. Spleen cells from miceof the indicated experimental groups [after saving a portion of the spleensfor analysis of infectious virus content by the FG-10 assay (see below)]

were examined for ability to adoptively transfer antiviral resistance tosublethally irradiated syngeneic recipients using the procedure describedpreviously (4,14). Recipient mice received 550 R of whole-body radiationat a dose rate of 135 R/min using a 137Cssource 7-irradiator (Gamma

Cell 40; Atomic Energy of Canada, Ltd., Ottawa, Ontario, Canada) justprior to the adoptive transfer of spleen cells inoculated i.p. in 0.5 ml ofmedium. When unfractionated or B-cell- or macrophage-depleted (seebelow) spleen cell populations were used, recipients received 5 x 107cells/mouse; however, only 2.5 x 107 of the T- or B-cell-enriched spleen

cells were transferred per mouse. In addition, mice adoptively transferredwith mixed cell populations received 5 x 107 cells of each donor type fora total of 1 x 10e spleen cells/recipient. Virus challenge was carried out2 days after cell transfer with 2 x 103 PFU of FLV injected i.p. in 0.2 ml

of PBS. All recipient mice were killed at indicated times, blood wascollected, and spleens were processed as in the serum therapy procedure.

Winn-type Procedure. The ability of various lymphoid cell populations

from the indicated experimental groups to transfer antiviral resistance tosyngeneic recipients was also examined using a Winn-type assay (30),

which differed from the adoptive transfer protocol in that the test cellswere mixed and preincubated with virus in vitro before inoculation intorecipient mice, which themselves were not sublethally irradiated. Lymphoid cells were prepared as described previously (4, 5, 13) and weremixed with virus and incubated for 1 hr at 37Â°in amounts adjusted soas to yield 2 x 103 PFU of FLV and the desired number of cells in a 0.5-

ml inoculum (injected i.p.). Cell fractionations were earned out as described below and, in those cases where the preincubated virus wasinoculated in the absence of cells, the latter were removed by centrifugation at 8000 x g for 5 min after completion of the 1-hr incubation.

Spleen Cell Depletion and Enrichment Procedures. Splenic T- andB-lymphocytes were eliminated in certain experiments by prior treatmentof cells with mouse anti-Thy 1.2 monoclonal antibody (1:100) or rabbitanti-mouse IgG antiserum (1:10), respectively, in the presence of guinea

pig complement as described elsewhere (4, 13). Briefly, splenocytes at4 x 107 cells/ml in RPM11640 medium were incubated at 4Â°for 45 min

with the indicated dilution of the appropriate antibody (or medium ascontrol), washed once with PBS, and then incubated for 1 hr afterresuspension in RPMI 1640 medium containing guinea pig complement(1:10). The cells were then washed again in PBS and resuspended tothe desired concentration of viable cells. The efficacy of this procedurein eliminating T- and B-cells was monitored by determining the blasto-genic response of the treated cells to the T-cell and B-cell mitogensconcanavalin A and lipopolysaccharide, respectively, using a [3H]thymi-

dine incorporation assay described in detail elsewhere (13).For depletion of adherent macrophage-like cells, a previously de

scribed (13) modification of the Sephadex G-10 column filtration procedure of Ly and Mishell (22) was used. Briefly, 5x10Â° spleen cells in 5

ml of PBS containing 5% FCS were layered over a 50-ml bed volumeSephadex G-10 column (Pharmacia, Piscataway, NJ). The cells were

eluted with warm RPMI 1640 medium containing 5% FCS, and thenonadherent cells present in the first 50 ml of effluent were pelleted,washed once with PBS, and resuspended to the desired concentration.

Enrichment of splenic T- and B-lymphocytes was earned out usingSephadex G-10 nonadherent cells obtained as described above. The G-

10 nonadherent cells were first fractionated on nylon wool columns by amodification of the procedure of Garaci ef al. (11). Briefly, 3 g of nylonwool (Leuko-Pak Leukocyte Filter; Fenwal Laboratories, Deerfield, IL)were packed in a 50-ml syringe, and 5 x 108 G-10 nonadherent spleen

cells in 25 ml of RPMI 1640, 5% FCS, were loaded on to each columnand were incubated at 37Â° for 45 min. Nylon-nonadherent cells were

eluted by rinsing the column with warm RPMI 1640 medium containing5% FCS, and adherent cells were collected by expelling the medium inthe column with the syringe plunger. The nylon wool was then resatur-

ated with additional medium, and the procedure repeated in order to

1490 CANCER RESEARCH VOL. 44



obtain any remaining adherent cells. The nylon wool-adherent and non-

adherent spleen cell populations were then pelleted and resuspended inRPM11640 medium before the next fractionation step. To further enrichfor T-cells, the nylon wool-nonadherent cells were subjected to depletionof B-cells by treatment with rabbit anti-mouse Immunoglobulin G serumplus complement as described above, while an enriched B-cell populationwas similarly obtained by treating the nylon wool-adherent cells with anti-

Thy 1.2 mouse monoclonal antibodies plus complement. The treatedcells were washed once with PBS and resuspended to the desiredconcentration of viable cells.

Serological Assays. The double-antibody RIA procedure using purified FLV gp71 labeled with 125Iand the [14C]nicotinamide release micro-

cytotoxicity assay using the FEM line of FLV-producing STU mouse

embryo cells (3, 28) have been described in detail previously (4,5).Virus Infectivity Assay. FLV infectivity in serial dilutions of test

material, which consisted of 20% extracts (w/v) of spleen tissue fromexperimental mice (see text for details), was assayed in vitro on the FG-10 clone of sarcoma-positive, leukemia-negative mouse cells as de

scribed elsewhere (1, 5).

RESULTS

Adoptive Transfer of Anti-FLV Resistance to Irradiated

Recipients by Mixed Populations of Spleen Cells from Normaland Serum-protected Donor Mice. If previous results demonstrating the ability of spleen cells from serum-protected mice to

transfer antiviral resistance to irradiated recipients (4, 14) didreflect the absence of a cell population(s) needed to replaceradiation-sensitive SFFV target cells rather than the presence ofvirus-specific immune cells, it would be expected that the transferof a mixed population of normal and serum-protected spleen

cells would not protect recipients against FLV challenge, sincethe former cells would be able to provide the necessary targetcells. However, as indicated by the data presented in Table 1,

FLV Serum Therapy and Antiviral Acquired Immunity

the mixed cell population was as effective in transferring antiviralresistance as serum-protected spleen cells alone (compare

Groups 5 and 7). Note that, despite the presence of low levelsof infectious virus in the spleens of serum-protected donor mice(Group 2), there was no evidence of the previously described (4)"virus carryover" effect with either these cells alone or when

mixed with normal spleen cells, as demonstrated by the absenceof splenomegaly and viremia development in the correspondingrecipients not subsequently challenged with exogenous FLV(Groups 6 and 8). As expected (4), FLV-challenged sublethallyirradiated mice not reconstituted with spleen cells producednormal levels of the nondefective helper lymphatic leukemia virus(measured by the FG-10 assay) while failing to develop spleno

megaly (Group 9).Adoptive Transfer of Anti-FLV Resistance by B-cell and

Macrophage-depleted Spleen Cells from Serum-protected

Mice. Given the above confirmation that the adoptive transferapproach is a valid means for demonstrating the generation ofvirus-specific immune cells in serum-protected mice, we have

extended our earlier attempts at identifying the cell population(s)involved by specific depletion of cells before transfer. Analogousto our previous results which demonstrated that elimination ofT-cells with anti-Thy 1.2 antibodies plus complement did notaffect the ability of spleen cells from serum-protected mice to

adoptively transfer antiviral resistance (4), comparable depletionsof B-cells (anti-immunoglobulin serum plus complement) or macrophages (Sephadex G-10 fractionation) failed to significantlyaffect the protective capability of serum-protected spleen cells

in the adoptive transfer protocol (Table 2; compare Groups 8and 10-12). Note that the "virus carryover" and irradiated non-

reconstituted controls in Table 2 (Groups 9 and 13, respectively)yielded the expected results similar to those described above for

Table 1Ability of mixed populations of spleen cells from normal and serum-protectedDBA/2 mice to adoptively transfer anti-FLVresistance to syngeneic recipients

Donors8

Group1

234

5678910Serum

treatmentCpaFLVSpleen

cellstransferredNormal

FLV + CpaFLVFLV + CpaFLVNormal and FLV +

CpaFLV (1:1)Normal and FLV +

CpaFLV (1:1)FLV

Day ofchallenge'' sacrifice34

+ 34+34FLV

Irradiation Day ofchallenge (550 R)sacrifice+

+ 42+ + 42

+42++42+

42+

+ 42+ - 42Av.

spleen wt(mg)112

Â± 3e

207 Â± 28904 Â±197Recipients'Av.

spleenwt674

Â± 58109Â± 1197 Â± 5

158 Â±5696

Â±5126

Â± 8983 Â±301Splenomegaly/total0/40

2/457/8Splenomegaly/total15/17

1/190/71/130/70/45/5Virus

titer"Small<25

1.15 X102<25Virus

titerSmall<25

<25<25<25<25

1.00x10Â«Large4.25

x 10"2.38x10Â«Large1.36x10Â»

2.31 x10Â»1.66x10Â»

4.27 x 10"a Donor DBA/2 mice were normal (Group 1)or serum-protected(Group 2, FLV-infected,treated with CpaFLV serum).Spleencells were prepared for adoptive transfer

as described in 'Materials and Methods" using only spleens weighing <250 mg.Virus challengeconsisted of ~2 x 103PFUof FLV inoculated i.p. on Day 0.

c Spleenweight >250 mg is considered to represent significant splenomegaly.''FLV titer was determined by the FG-10 sarcoma-positive, leukemia-negativecell plaque assay described in 'Materials and Methods" and expressed as PFU/g.

Spleentissues from mice of the samegroup with either small (<250 mg)or large (>250 mg) spleenswere pooled before preparationof the tissue extracts for assessmentof infectious virus content.

8 Mean Â±S.E.All recipient DBA/2 mice except those of Group 6 received 550 R of whole-body irradiation2 days before virus infection. Mice of Groups 4-6, and 7 and 8 received

5 x 107and 1 x 10* of the indicated spleen cells, respectively, inoculated i.p. in 0.5 ml of medium within 2 hr of irradiation. Virus challengeof recipients (Groups 4, 5, 7,9, and 10) was carried out 2 days after irradiation (i.e.. Day 0) using the same dose as for donor mice. Recipientanimals were killed on Day 42 by exsanguination,andspleenswere weighed and processed for infectious virus content as described in 'Materials and Methods."

APRIL 1984 1491




Table 2Adoptive transfer of anti-FLV resistance to syngeneic recipients with spleen cells from serum-protected DBA/2 mice depleted of B-cells or macrophages

Donors

VirustiterGroup123Serum

treatmentCpaFLVFLVchal

lenge Day ofsacrifice29-35+

29-35+29-35Av.

spleen wt(mg)121

Â±2225Â±151261Â±112Splenomegalyiltotal0/10619/15325/27Small<253.59

x102<25Large1.27x10"4.17x10"Recipients8Group4567891011121314Spleen

cellstransferredNormalNormalNormalNormalFLV

+CpaFLVFLV+CpaFLVFLV-1-CpaFLVFLV+CpaFLVFLV

+ CpaFLVTreatment

of FLVchal-transferredcellslenge+Complement

only+a-immunogtobulin++

complementSephadexG-10+fractionation+â€”Complement

only+a-immunoglobulin++

complementSephadexG-10+fractionation++Irradia

tion Dayof(550R)sacrifice+

30-42+30-38+30-42+

31+

30-42+42+38-42+35-42+

30-35+

38-4727-42Av.

spleenwt(mg)695

Â±37711Â±56664Â±61677

Â±12131

Â±1698Â±4125Â±13190Â±59159

Â±18150

Â±311225+ 140Splenomeg

aly/total57/6222/2222/243/31/320/90/152/91/181/1120/21VirustiterSmall1.60

X103<25<25<251.50X102<25<253.14

x10s<25Large2.93x10"7.62

x10"4.48x10"2.92

x10Â»3.75

x10Â»1.20x10"1.38x10Â»1.14x10Â«4.49

x 10*

* All recipient DBA/2 mice except those of Group 14 received 550 R whole-body irradiation 2 days before virus infection. Mice of Groups 4 to 12 received 5 x 10? of

the indicated spleen cells inoculated i.p. in 0.5 ml of medium within 2 hr of irradiation. Treatment of donor spleen cells before adoptive transfer with rabbit anti-mouseIgG antiserum plus complement (or complement alone) or removal of Sephadex G-10-adherent cells was as described in "Materials and Methods.' Virus challenge of

recipients (Groups 4 to 8 and 9 to 14) was carried out 2 days after irradiation (i.e., Day 0) using the same dose as for donor mice. Recipient mice were killed at theindicated times by exsanguination, and spleens were weighed and processed for infectious virus content as described in "Materials and Methods." For further details,

see footnotes to Table 1.

the analogous control groups in the experiments presented inTable 1. It should also be pointed out that the lower level ofsplenomegaly induction in normal spleen cell recipients (e.g.,Table 1, Group 4; Table 2, Groups 4 to 7) is frequently seen inFLV-challenged irradiated reconstituted mice, which only occa

sionally manifest spleen enlargement comparable to that seen incontrol FLV-infected animals (e.g., Table 1, Groups 3 and 10;

Table 2, Groups 3 and 14).Adoptive Transfer of Anti-FLV Resistance by Purified T-

cells or B-cells from Spleen Cells of Serum-protected Mice.

When the results presented in Table 2 are considered in conjunction with those obtained previously with T-cell-depletedspleen cells from serum-protected mice (4), the possibility arisesthat the virus-specific cell population(s) being measured by theadoptive transfer protocol is distinct from T-cells, B-cells, ormacrophages. While other evidence does support the nonin-

volvement of these cell populations in serum therapy itself (4, 6,14) and, in some cases, in the development of acquired antiviralimmunity in serum-protected mice as well (12,14), an alternativeexplanation is that more than one population of serum-protectedspleen cells can independently transfer antiviral resistance toirradiated recipients. To examine this possibility directly, Sephadex G-10-nonadherent spleen cells from normal and serum-

protected mice were fractionated over nylon wool and thensubjected to specific antibody treatment, as described in "Materials and Methods," in order to obtain separate populations of

T- and B-cells. The efficacy of these treatments was confirmedby analysis of the blastogenic responses to the T-cell and B-cell

mitogens concanavalin A and lipopolysaccharide, respectively(Table 3). The adoptive transfer results obtained with theseenriched cell populations are presented in Table 4 and indicate

that either splenic T- or B-cells alone from serum-protected donormice can transfer anti-FLV resistance, raising the possibility that

macrophages may also have this capability, although we havenot been able to examine this directly because of practicalproblems which prevent recovery of sufficient numbers of thesecells from murine spleens. It should be noted that both T- andB-cells alone from normal control mice can reverse the irradiation

effect in recipient mice (Groups 4 and 5), obviously necessary ifthe results of these experiments are to be meaningful, but alsoindicating that both of these enriched cell populations containcells which serve as SFFV targets. Given the fact that serum-protected B-cells alone can transfer antiviral resistance, as wellas the well-established close correlation between serum protec

tion and the development of a host antiviral humoral immuneresponse (4-7,12, 26, 27), it remains surprising that adoptivelyprotected mice receiving unfractionated or B-cell-containingspleen cells from serum-protected donors (e.g., Table 1, Groups

5, 7, and 8; Table 2, Groups 8, 10, and 12; Table 4, Group 6)characteristically fail to demonstrate the presence of antiviralantibodies in their sera (data not shown for present studies; seealso Ref. 4).

Ability of Spleen Cells from Tumor-immune and Serum-protected Mice to Protect Syngeneic Unirradiated Animalsagainst FLV Challenge in a Winn-type Assay. Although the

results obtained in the adoptive transfer studies using mixturesof normal and serum-protected spleen cells (Table 1) appear to

eliminate previous concerns as to the complicating factor of theeffect of sublethal irradiation on FLV leukemogenesis (4), it wasnevertheless decided to attempt to circumvent this considerationby an even more direct means, namely, the ability of spleen cellsfrom serum-protected mice to protect unirradiated recipients





Table 3Enriched populations of splenic T- and B-cells used in adoptive transfer experiments

Source ofspleencells8NormalFLV

+ CpaFLVEnrichment

procedures8Nylon

woolnonadherentanda-immunoglobulin+

complementNylonwool adherentanda-Thy

1.2 + complementNylon

woolnonadherentanda-immunoglobulin-(â€¢complementNylon

wool adherentanda-Thy1.2 + comple

mentPutative

identityNATBNATBBlastogenic

response0Mitogen"Con

ALPSConALPSConALPSConALPSConALPSConALPScpm2,18044,18319,2971,25868,5452,6697482,74916,5933,02551,89513,4561,37155,8612,5304031,0515,833Sl"20.38.8554.52.123.7422.217.24.4540.71.852.6114.5Acpm43,00317,11767,2871,4112,04715,85448,87010,43154,4901,1596485,430

a See 'Materials and Methods" for details of the passive serum therapy protocol and the procedures used to obtain

enriched populations of splenic T- and B-cells and Table 4 for the DBA/2 donor mice used as the source of spleen cellsfor these adoptive transfer experiments.

'' Concanavalin A and lipopolysaccharide were used at 2.5 and 50 fig/ml, respectively.'' Blastogenic responses were measured using the [3H]thymidine (0.5 /iCi/well) incorporation assay described previously

(13)."SI, mean cpm incorporated with mitogen divided by mean cpm incorporated without mitogen; Acpm, mean cpm

incorporated with mitogen minus mean cpm incorporated without mitogen; NA, not applicable; Con A, concanavalin A;LPS, lipopolysaccharide; T, T-cell; B, B-cell.

Table 4Adoptive transfer of anti-FLV resistance to syngeneic recipients with enriched populations of splenic T- or B-cells from serum-protected DS4/2 mice

DonorsGroup

Serumtreatment1

2 CpaFLV3FLV

challenge Day ofsacrifice30

+ 30+ 30Av.

spleen wt(mg)109

Â± 15*

205 Â±168788 Â±351Splenomegaly/

total0/65

8/6413/14Small<25

<25<25Virus

titerLarge9.53

x 10s4.74 x10Â«Recipients"Group

Spleen cellstransferred4

Normal B-cells5 Normal T-cells6 FLV + CpaFLV B-cells7 FLV -l- CpaFLV T-cells

8FLV

Irradiation Day ofchallenge (550 R)sacrifice+

+ 40+ + 40+ + 40+ + 40+ - 40Av.

spleen wt(mg)657

+ 199*

747 Â±273131 Â± 63164 + 106738 Â±176Splenomegaly/

total8/8

9/91/134/266/6Small<25

<25Virus

WerLarge5.47

x 10Â«5.48 x 10Â«2.76x10Â«1.34x10Â«5.51 x 10Â«

4 Mean Â±S.E." All recipient DBA/2 mice except those of Group 8 received 550 R whole-body irradiation 2 days before virus infection. Mice of Groups 4 to 7 received 2.5 x 107 of

the indicated spleen cells inoculated i.p. in 0.5 ml of medium within 2 hr of irradiation. The identification of the enriched populations of splenic T- and B-cells transferred(prepared as described in 'Materials and Methods") is presented in Table 3. Virus challenge of recipients (Groups 4 to 8) was carried out 2 days after irradiation (i.e.. Day

0) using the same dose as for donor mice. Recipient mice were killed at the indicated time by exsanguinatkxi, and spleens were weighed and processed for infectiousvirus content as described in "Materials and Methods." For further details, see footnotes to Table 1.

against FLV challenge in a Winn-type assay. The Winn assay(30) involves premixing of putative effector cells with the tumor-

igenic agent and subsequent inoculation of the mixture, a procedure which has been used widely in tumor immunobiologyover the past 20 years.

For our purposes, it was first necessary to determine whetherantiviral resistance could be demonstrated using a Winn-type

assay in the FLV system and, if so, to optimize the experimentalconditions. Toward this end, we used as a positive controlsystem the immunization of DBA/2 mice with the FLC-745syngeneic FLV-induced erythroleukemia cell line which has pre

viously been shown to not only result in resistance to direct viruschallenge (12), but also lead to the generation of virus-specific

cytotoxic effector cells (15). The results of these studies (Table

5) indicated that i.p. inoculation of a mixture of 2 x 103 PFU ofFLV and ss1 x 107 tumor-immune spleen cells/mouse after priorincubation in vitro for 1 hr at 37Â°resulted in protection against

the development of FLV-induced disease. Prior incubation ofvirus with medium alone (Group I) or normal spleen or lymphnode cells (groups 2A to 2F) had no effect on its leukemogenicactivity subsequent to i.p. inoculation. Of considerable interestwas the observation that removal of tumor-immune spleen cells

by centrifugation after the In vitro incubation, but prior to i.p.inoculation, resulted in undiminished viral infectivity (compareGroups 3B and 3E), demonstrating the necessity for the transferof the virus-specific immune cells and thereby eliminating thepossibility that virus neutralization occurs during the in vitroincubation with these effectors.

APRIL 1984 1493




Tabte5Protection of unirradiated syngeneic recipients against FLVinfection by lymphoid cells from tumor-immuneDBA/2 mice in a Winn-typeassay

Group12A2B2C2D2E2F3A3BX3D3E3FCellsincubated with

Treatment of donor mice*FLVFLC-745-immunizedFLC-745-immunizedFLC-745-immunizedFLC-745-immunizedFLC-745-immunizedFLC-745-immunizedSpleenSpleenSpleenSpleenSpleenLymph

nodeSpleenSpleenSpleenSpleenSpleenLymph

nodeNo.

of cells inoculated"3.0x10'2.0

x1071.0x1073.3

x10Â«2.0

xIO73.0x1

072.0x1071.0X1073.3x10Â«2.0

x 107Day

of sacrifice Av. spleen wt(mg)32-353435343435353435343435351488Â±181C1364Â±1381309

Â±2671551Â±1291401Â±1241128

Â±258955Â±8699Â±

6118Â±7280

Â±1211327Â±287907

Â±176109Â± 9Spleno

megaly/total7/74/44/44/44/44/43/30/40/41/44/45/50/3Virus

Â«terSmall<25<25<25<25Large4.66x10"7.50

x10"6.00x10Â«4.65x10"5.64

x10"9.28X1057.05

x10"1.75

x10"4.71x10Â«1.67x10"

'' See "Materials and Methods" for the protocol for immunizing mice with the syngeneic FLV-induced erythroleukemia cell line FLC-745. For further details, see

footnotes to Table 1.fi Appropriate numbers of spleen cells or lymph node cells were incubated for 1 hr at 37Â°with an appropriate amount of FLV so that inoculation of 0.5 ml of this

mixture per unirradiated recipient contained 2 x 103 PFU of FLV and the indicated numbers of cells. In the case of Groups 2-E and 3-E, spleen cells (2 x 10') wereremoved after incubationof the cell-virus mixture but, before inoculation,by centrifugation at 8000 x g for 5 min.

c Mean Â±S.E.

Based on the results of the studies using tumor-immuneeffector cells, it was decided to use a standard dose of 2 x 107

cells/recipient mouse in the Winn assay analyzing putative immune cell populations from serum-protected DBA/2 donor mice.

As demonstrated by the results in Table 6, although lymph nodecells from serum-protected mice at this dose did not protect

unirradiated recipients against FLV challenge (Group 10A),spleen cells from the same donors did provide resistance to FLVleukemogenesis. Furthermore, in complete agreement with theresults of adoptive transfer studies (Table 2; Ref. 4), specificdepletion of T-cells, B-cells, or macrophages did not eliminatethe antiviral activity of the remaining spleen cells from serum-

protected mice (Groups 10D to 10F). It should also be notedthat serum-protected spleen cells incubated in vitro without

added FLV did not induce splenomegaly or viremia upon i.p.inoculation (Group 10G), demonstrating the absence of a "viruscarryover" effect (4).

The Winn assay procedure was also used to determinewhether lymphoid cells from mice treated with CpaFLV serumonly (i.e., no FLV challenge) could manifest protection againstFLV infection, since a serum prophylaxis effect has been demonstrated when virus challenge is carried out within 20 days afterthe last inoculation of immune serum (12). Various lymphoid cellpopulations, including spleen, peritoneal exÃºdate, bone marrow,and lymph node cells, were collected from serum-treated micewithin the "prophylaxis window," incubated with virus for 1 hr at37Â°, and inoculated at 2 x 107 cells/recipient. Regardless of

whether the cells had been obtained from mice treated withnormal chimpanzee serum or CpaFLV serum, in no case couldeffective protection against FLV leukemogenesis be demonstrated in the Winn assay (Groups 8A to 8D and 9A to 9D).These results parallel our inability thus far to demonstrate thespecific in vivo arming of these same lymphoid cell populationsin uninfected mice treated only with CpaFLV serum as measuredin in vitro cytotoxicity assays with the syngeneic FLC-745 targetcells (12).

When the antiviral humoral response of mice protected in theWinn assay against FLV challenge by spleen or lymph node cellsfrom tumor-immune or serum-protected donors was analyzed,

CYTOTOXICITY

>â€”â€¢â€¢â€¢â€¢â€¢â€¢Â»â€¢â€¢***â€¢â€¢â€¢â€¢â€¢â€¢oo

RIA

â€”ooooooooooo

- ::::88""Â«"~

No Splenomegaly Splenomegaly No Splenomegaly Splenomegaly

Chart 1. Relationship between the protection of unirradiated DBA/2 miceagainst the development of FLV-induced splenomegaly by syngeneic spleen orlymph node cells in the Winn assay and the appearance of a mouse antiviralhumoral immune response, as determined by serum cytotoxicity (complement-dependent)on FLV-infectedFEM mouse cells and RIA versus 125I-FLVgp71. â€¢,O,

individual serum sample collected between 30 and 35 days after inoculation ofvirus-cellmixtures with sera included from all recipient groups presented in Tables5 and 6 except non-virus-challenged mice of Group 10-G (Table 6). Sera aredistinguished between mice receiving spleen or lymph node cells (plus FLV) fromserum-protected (â€¢)or tumor-immune (O) donor mice, respectively. Serum cyto-toxic reactivity expressed on a scale of + to ++++: +, 20 to 40%; ++, 40 to 60%;+++, 60 to 80%; ++++, >80% specific release based upon complement control(1,4). Serum RIA reactivity is expressed as degree of precipitation of 12SI-FLVgp71by serum at a 1:5 dilution on a scale of + to ++++: +, 10 to 15% precipitation ofinput label; ++, 15 to 20%; +++, 20 to 25%; ++++, >25% (note that mouse seraare much weaker than hyperimmune anti-FLV or anti-FLV gp71 sera and rarelygive 50% precipitation of input label). Spleen weight of Â»250mg is considered torepresent significant splenomegaly

some interesting observations were noted (Chart 1). While thefailure of mice developing virus-induced splenomegaly to gener

ate antiviral antibodies is identical to that seen with both serumtherapy itself and adoptive transfer studies using irradiated recipients (4-7, 12, 26, 27), quite unexpected is the fact that nearlyone-half (17 of 36) of the sera from mice protected in the Winnassay by spleen cells from serum-protected donors containantiviral antibodies measurable in RIA versus 125I-FLV gp71 or





Tablee

ce//s,B-cells, or macrophages in a Winn-typeassay

Group12345Group67A7B7C8A8B8C8D9A969C9010A10B10C10010E10F10GSerumtreatmentNCSCCpaFLVCpaFLVTreatment

ofdonormiceNCS

onlyNCSonlyNCSonlyNCSonlyCpaFLV

onlyCpaFLVonlyCpaFLVonlyCpaFLVonlyFLV

+CpaFLVFLV+CpaFLVFLV+CpaFLVFLV+CpaFLVFLV

+CpaFLVFLV

+CpaFLVFLV

+ CpaFLVFLV

infected-â€”â€”++Cells

incubatedwithFLV'SpleenSpleenLNCPECBone

marrowLNCSpleenPECBone

marrowLNCSpleenLNCSpleenSpleenSpleenSpleenSpleenSpleen

(no virus)Day

ofsacrifice34-36303034-3634-36Donors"Av.spleen wt(mg)115Â±4b159

Â±8176Â±121123

Â±188227Â±23Recipients"Treatment

ofeffectorcete'Complement

onlyComplement

onlya-immunoglobulin+

complementa-Thy1.2 + com

plementSephadexG-10fractionationAv.

spleen wt(mg)1283Â±157"1366

Â±1681283Â±2381783Â±2641606

Â±1091681Â±4481572

Â±1061839Â±1391553

Â±2951155Â±5931638Â±1471608Â±1781876

Â±278"449

Â±211310Â±164112Â±7310

Â±164126

Â±6150

Â± 14Splenomeg

aly/total0/320/110/1611/1111/55Splenomeg

aly/total8/811/115/54/43/33/33/33/33/32/36/68/85/52/92/90/101/50/70/3Virus

titerSmall<25<25<25<25Large2.82x10Â«2.21

x10*Virus

titerSmall3.25

x102<25<25<25<25<25<25Large1.80x10"3.07

x10*3.31x10"1.56x10Â»2.70

x10Â«2.35x10"7.45x10Â«1.29x10Â»1.32x10"1.46X

10"4.66x10Â«1.71

X10"1.12x10Â«3.37

x10Â«7.55x10Â»2.09x10"

'' Donor DBA/2 mice were normal (Group 1), serum-protected (Group 4, FLV-infected, treated with CpaFLV serum), or uninfected mice treated only with normalchimpanzeeserum (Group 2) or CpaFLV serum (Group 3). Lymphoid cells were prepared for the Winn assay as described in "Materials and Methods' using only mice

with spleens weighing <250 mg. See footnotes to Tables 1 and 4 for further details."MeanÂ±S.E.c NCS, normal chimpanzeeserum; LNC, lymph node cell; PEC, peritonealexÃºdatecell.rfAll recipient mice were killed 30 days after inoculationof cell-virus mixtures by exsanguination,and spleenswere weighed and processed for infectious virus content

as described in "Materials and Methods."8Appropriate numbers of lymphoid cells were used for the incubation with FLV so that all recipient mice received 2 x 10?cells when 0.5 ml of this mixture was

inoculated. Note that mice of Group 10G received serum-protected spleen cells that had been incubatedwith medium instead of virus.Treatment of donor spleen cells before incubation with FLV with mouse anti-Thy 1.2 monoclonalantibodies plus complement or rabbit anti-mouse IgG antiserum

plus complement (or complement alone)or removal of Sephadex G-10-adherentcells was as described in "Materials and Methods."

by cytotoxic activity on FLV-producing FEM cells in the [14C]-

nicotinamide release assay. This is in marked contrast to thefailure of irradiated recipients protected against FLV leukemo-genesis by the adoptive transfer of serum-protected spleen cells

to demonstrate a comparable antiviral humoral response (datanot shown for present studies; see also Ref. 4). Nevertheless, itshould be noted that, analogous to findings in the adoptivetransfer system (4), mice protected in the Winn assay by tumor-

immune lymphoid cells manifest a much more intense antiviralantibody response, both as reflected by a higher proportion ofreactive sera (13 of 15) and by a generally stronger response,than those receiving serum-protected cells (Chart 1). This is inaccord with the observation that tumor-immune donor mice

generally produce a stronger antiviral humoral response thanthat seen in serum-protected animals (data not shown), as wellas the previous finding that occasional irradiated recipients oftumor-immune spleen cells do demonstrate the presence of

antiviral antibodies (4), in contrast to the consistent negativity of

mice adoptively protected against FLV challenge by spleen cellsfrom serum-protected donors (4,14).

DISCUSSION

The present results confirm and extend previous observationsthat spleen cells from serum-protected DBA/2 mice can adoptively transfer antiviral resistance to unimmunized irradiated syn-geneic recipients and that elimination of T-cells has no effect on

the protective activity of such splenocytes (4, 14). Analogousdepletion techniques have now demonstrated that the transferof resistance to FLV challenge by spleen cells from serum-

protected donor mice is similarly unaffected by the separatedepletion of B-cells or macrophages (Table 2). These results,

when considered in conjunction with those obtained previouslyconcerning the role of T-cells in the transfer of antiviral immunity

(4), raise the possibility that the responsible effectors reside in apopulation of non-T, non-B, nonmacrophage cells. However, the

APRIL 1984 1495




data obtained using specific enrichment techniques suggest thateither splenic T- or B-cells alone from serum-protected mice are

protective against FLV challenge (Table 4), thus implying thatthe cell depletion results reflect the ability of multiple cell populations to transfer antiviral resistance.

The present results are also instrumental in establishing thatadoptive transfer of anti-FLV resistance to irradiated recipientsby spleen cells from serum-protected mice actually reflects theactivity of virus-specific effector cells rather than the absence ofthe previously described (4) radiation-sensitive SFFV target cellneeded for development of virus-induced splenomegaly. Given

the fact that these SFFV target cells can be replaced in irradiatedmice by normal syngeneic spleen (or bone marrow) cells (4),adoptive transfer experiments were carried out with mixed populations of normal and serum-protected spleen cells to determine

whether protection against FLV challenge could still be achieved.The ability to transfer antiviral resistance under these conditions(Table 1) strongly suggests that serum-protected spleen cellsare effective because of the presence of virus-specific immunecells and not because of an inability to replace the radiation-sensitive target cells, cells which are supplied by the cotrans-

ferred normal spleen cells.That the adoptive transfer results are not influenced by com-1

plications associated with the effects of sublethal irradiation onFLV leukemogenesis has been demonstrated even more directlyby the antiviral protective activity of spleen cells from serum-protected mice in a Winn-type assay using unirradiated recipients

(Table 6). Regardless of whether spleen cells are obtained fromserum-protected or FLV erythroleukemia-immunized donor mice,

and despite the fact that they are preincubated in vitro withchallenge virus before inoculation into recipient animals, the cellsmust be injected along with FLV for effective transfer of resistance in the Winn assay (Table 5); thus, neutralization of virusinfectivity does not occur prior to in vivo administration of thecell-virus mixture. It should be noted that results obtained using

the Winn assay (Table 6) parallel observations made in adoptivetransfer experiments (Refs. 4, 14, and this paper, Table 2) in anumber of regards, including the lack of effect of depleting T-cells, B-cells, or macrophages on the antiviral activity of serum-

protected spleen cells, as well as the failure of spleen cellsobtained within the "serum prophylaxis" window (12) from mice

treated with CpaFLV serum only to mediate resistance to FLVchallenge. Thus, it appears that the Winn assay using unirradiated recipients measures the same activity in spleen cells fromserum-protected mice as the adoptive transfer protocol using

sublethally irradiated recipients, in both cases this being thetransfer of anti-FLV resistance by virus-specific immune cells.

The observation that either splenic T- or B-cells alone fromserum-protected mice can adoptively transfer antiviral resistance

to irradiated syngeneic recipients (Table 4), reflecting the use ofpositive selection techniques to yield specifically enriched cellpopulations, is unusual in that adoptive transfer studies in avariety of other systems have indicated that only T-cells areinvolved (9, 16-19, 21, 23, 25, 29). For example, whereasenriched splenic T-cells from mice hyperimmunized against asyngeneic idiotype-secreting hybridoma can adoptively transferantihybridoma immunity, enriched splenic B-cells from the samedonor mice are ineffectual (19).

Recent studies have further analyzed the T-cells involved inthe adoptive transfer of resistance to tumorigenic or infectiouschallenges, both viral and cellular, with both helper and cytotoxic

T-cell subsets being implicated in different systems. For example,using both positive and negative selection techniques the Lyt 1+

helper T-cell subpopulation from tumor-immune mice has been

demonstrated to be responsible for the adoptive transfer ofresistance to the FLV-induced leukemia FBL-3 in C57BL/6 mice

(16, 17), possibly reflecting mechanisms in which these cellsfunction to induce and/or amplify other effector cell populationsin the recipient animals. Analogous transfer of antitumor immunity has been demonstrated with the monoclonal antibody-defined rat helper-containing T-cell subset and syngeneic sarcomas

induced by either MSV or methylcholanthrene (9). In contrast tothe above studies in murine and rat tumor systems which indicatethe apparent noninvolvement of CTL in the adoptive transfer ofantitumor resistance, adoptive protection of syngeneic miceagainst infection with herpes simplex virus, type 1 has beenshown to be mediated by cloned virus-immune CTL subjected

to secondary stimulation in vitro (29). However, it was alsoclaimed that Lyt 1+ or Lyt 2,3+ T-cell subpopulations isolated

from virus-specific T-cell cultures could individually transfer anti-

herpes simplex virus, type 1, protection, thus indicating thathelper T-cells can function in this system as well. The complexity

of this situation is underscored by the adoptive transfer studiesof Leclerc and Cantor (21) demonstrating that, whereas Lyt 1+

helper T-cells from the spleens of MSV-inoculated donor C57BL/

6 rÃ©gressermice are responsible for the transfer of resistanceagainst the induction of sarcomas by MSV in syngeneic animals,they are ineffectual in transferring protection against the growthof Moloney leukemia virus-induced lymphomas, the latter adoptive protection being mediated by the transfer of Lyt 2,3+ CTL,

which in turn has relatively little influence on MSV-induced tumor

formation.In contrast to the above cell transfer studies which have

implicated T-cells as the essential mediators of adoptive immu

nity, Calvelli ef a/. (2) have claimed that macrophages are capableof transferring resistance to challenge with B16 melanoma cells.However, while in vitro studies clearly demonstrate that B-cells

can participate in the immune lysis of tumor cells (20, 29, 31),an activity which is directly related to antibody production andsubsequent antibody-dependent cellular cytotoxicity reactions(31) or complement-dependent cell lysis (8), previous attemptsto adoptively transfer antitumor immunity with B-cells in several

systems have been unsuccessful (2, 18, 19).At the present time, it is not clear whether specific subpopu

lations are responsible for the adoptive transfer of anti-FLVresistance by splenic T-cells from serum-protected mice, but

studies to answer this question are in progress. While our resultsdo suggest that B-cells from serum-protected donors can transfer protection against FLV-induced disease (Table 4), it is clearthat, while considerably enriched, the B-cells used in the presentstudies are not completely devoid of T-cells (Table 3). Thus, itremains possible that a minor contaminating fraction of T-cells isresponsible for the adoptive protection seen with the serum-protected B-cells. Arguing against this possibility, however, isthe fact that ~5 x 107 unfractionated serum-protected spleen

cells are required/recipient mouse for effective adoptive therapyagainst FLV challenge,6 making it unlikely that a sufficient number

of the active effector cells would be present as a minor contaminant in the 2.5 x 107 enriched B-cells which were administered

to each recipient animal. Nevertheless, the possible involvementof T-cells, perhaps providing accessory functions, in the adoptiveprotection mediated by the serum-protected splenic B-cells used





in the present studies cannot be rigorously excluded at this time.In addition, the possible contribution of immunoglobulin~ Thy 1~

splenocytes can also not be ruled out, since positive selectionbased on these surface markers was not used.

In any event, at the present time, it is still not possible toconclude definitively that a population of non-T, non-B, non-macrophage cells is not involved in the adoptive transfer of anti-FLV resistance by spleen cells from serum-protected mice. Even

if the contamination of the enriched cell populations used is onlyto a minor degree, the effects of such contamination could bemarkedly amplified by replication of these cells during the -40-

day period used to determine the adoptive protection of miceagainst FLV challenge. This could be especially significant if thecontaminating cells include stem cells capable of differentiatingin the cell-recipient animals during this extended assay period.

Further studies are under way to characterize in greater detailthe identity of the cell population(s) present in the spleens ofserum-protected mice which are responsible for the adoptive

transfer of antiviral resistance, including the possible involvementof cells with natural killing activity.

Of considerable interest is the absence of a detectable hostantiviral humoral response in mice adoptively protected againstFLV-induced disease, a response which is closely associatedwith serum protection itself (4-7, 12, 26, 27). Previous results

(4) have demonstrated that mice protected against FLV challengeby the adoptive transfer of unfractionated spleen cells from eitherserum-protected or FLV erythroleukemia-immunized donors fail

to consistently demonstrate antiviral antibody, arguing againstthe notion that this adoptively transferred protection reflects theactivity of mature antibody-producing cells or of primed T-helper

cells capable of supporting antibody synthesis in the irradiatedrecipients. While analysis of sera from mice adoptively protectedagainst FLV challenge by serum-protected spleen cells subjected

to positive or negative selection techniques (data not shown)was in accord with the earlier results, sera from unirradiatedmice protected in the Winn assay by spleen cells from serum-protected or tumor-immune donors unexpectedly demonstrated

the presence of antiviral antibodies in 17 of 36 and 13 of 15cases, respectively (Chart 1). This raises the possibility that thefailure to detect mouse antiviral antibodies in adoptively protected animals reflects the inactivation by sublethal irradiation ofhost accessory cells needed for antibody production, althoughat the present time we cannot rule out irradiation effects whichinfluence the seeding of donor splenocytes.

It should also be noted that the failure to detect antiviralantibody, even with a sensitive RIA technique, could reflect anexcess of viral antigen in the cell recipient FLV-challenged mice

precluding measurement of free circulating antibody, or, alternatively, that antibody may be present at other times aftertransfer of lymphoid cells than those examined. Nevertheless,regardless of the factors involved which may influence the detection of a host antiviral humoral response in sublethally irradiated adoptively protected mice, it is clear that, if such a responsewere to be detected in any mice in this study, it would presumablybe with recipients of the B-cell-enriched serum-protected spleencells. However, with one exception, even sera from B-cell-pro-

tected recipients were negative in RIA and the cytotoxicity assayfor antiviral antibodies (data not shown). Given the fact that theseB-cells were obtained from serum-protected donor mice positive

6E.V. Genovesiand J. J. Collins, unpublishedobservations.

for antiviral antibody at the time of cell transfer, it is possible thatrecipient mice generate antiidiotypic responses which could neutralize the activity of antiviral antibodies produced by donorspleen cells. If this is the case, it could reflect the involvement ofamplifying mechanisms associated with the idiotypic network(24) in the adoptive therapy of FLV-induced disease using spleencells from serum-protected donors. Attempts are under way to

determine whether sera from adoptive transfer recipients arecapable of inhibiting the antiviral antibodies present in the seraof the corresponding serum-protected donor mice as a means

of investigating whether such antiidiotypic humoral responsesare induced in sublethally irradiated mice adoptively protectedagainst FLV-induced disease.

ACKNOWLEDGMENTS

We thank Deborah Sackie and Gail Johnson for excellent technical assistance,Dr. Jo Michaelsonfor critical reading of the manuscript, and Deborah Eubanksforpreparationof the manuscript.

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