importance of tumor-specific cytotoxic cd8+ t-cells in ... · injected s. c. with 1x10' viable...

(CANCER RESEARCH 50. 7641-7649. December I. 1990|

Importance of Tumor-specific Cytotoxic CD8+ T-Cells in Eradication of a LargeSubcutaneous MOPC-315 Tumor following Low-Dose MclpliaIan Therapy1

Blaine Y. Takesue,2 Joseph M. Pyle, and Margalit B. Mokyr1

Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago 60680 [B. Y. T., M. B. M.], and Department of Pathology, Loyola UniversityMedical Center, Maywood 60153 Â¡J.M. P./, Illinois

ABSTRACT

We have previously demonstrated that depletion of CDS* T-cells by

the use of a monoclonal anti-Lyt-2.2 antibody abolishes the curativeeffectiveness of low-dose melphalan (i.-phenylalanine mustard; L-PAM)therapy for BALB/c mice bearing a large (>20 mm) s.c. MOPC-315tumor and extensive mÃ©tastases (Mokyr et al.. Cancer Res., 49: 4597-4606, 1989). Here we show that as a consequence of low-dose L-PAMtherapy, CDS* T-cells accumulate in the s.c. tumor nodules of MOPC-

315 tumor bearers. Specifically, an 80-fold increase in the number ofCDS* T-cells was seen within 5 days after the chemotherapy. Treatment

of MOPC-315 tumor bearers with low-dose L-PAM in conjunction withmonoclonal anti-Thy-1.2 or anti-Lyt-2.2 antibody, in contrast to treatment with monoclonal anti-L3T4 antibody, prevented the appearance ofthe massive CD8* T-cell infiltrate in the s.c. tumor nodules. Fresh CDS*

T-cells derived from s.c. MOPC-315 tumor nodules that were regressingas a consequence of low-dose L-PAM therapy exhibited a potent directlytic activity against the MOPC-315 plasntacytoma in a short-term invitro assay. The specificity of the lytic activity exhibited by the CDS* T-cells was illustrated not only by the inability of the CDS* T-cells to lyse

two antigenically unrelated thymomas (the WEHI 22.1 and the K.I.-4)and a natural killer-sensitive lymphoma (the YAC-1), but also by theirrelatively weak lytic activity against an antigenically related plasmacy-toma (the MOPC-104E). Thus, CDS* T-cells that infÃltrate the s.c.

tumor nodules of MOPC-315 tumor bearers following low-dose L-PAMtherapy most likely exploit a CTL-type lytic mechanism to eradicate at

least part of the large tumor burden not eliminated by the direct antitumoreffects of the drug.

INTRODUCTION

The effect of anticancer drugs on the immune system is notuniformly suppressive. In fact, many investigators have shownthat under defined conditions anticancer drugs can actuallyenhance a variety of immune responses. Specifically, it wasshown that exposure of lymphoid cells to anticancer drugs priorto their antigenic or mitogenic stimulation leads to the appearance of a greatly enhanced immune response (1-6).

The documentation that anticancer drugs can potentiate immune responses has stimulated several attempts to exploitanticancer drugs to enhance the development of antitumorimmunity in tumor bearers with the hope that the resultantpotent antitumor immunity would in turn aid in tumor eradication (7-14). Indeed, in several tumor systems, anticancerdrugs were found to be successful in shifting the balance fromimmunosuppression to potent antitumor immunity (7-10). Forexample, we have shown that a low dose of either cyclophos-phamide or L-PAM,4 two widely used anticancer drugs, can

lead to the appearance of potent T-cell-dependent antitumor

Received 6/21/90; accepted 8/28/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicale this fact.

' Supported by Research Grant IM-435 from the American Cancer Societyand Research Grant B-8806 from the Banes Estate.

J In partial fulfillment of the requirements for the Doctor of Philosophy degree.3Supported by Career Development Award CA-01350 from the National

Cancer Institute. To whom requests for reprints should be addressed.4The abbreviations used are: L-PAM. L-phenylalanine mustard; NK cells,

natural killer cells; CTL, cytotoxic T-lymphocyte; E/T ratio, effector/target cellratio.

immunity in hitherto immunosuppressed MOPC-315 tumor-bearing mice and this immunity is critical for the curativeeffectiveness of the low-dose chemotherapy for mice bearing alarge (>20 mm) s.c. tumor and extensive mÃ©tastases(7, 8, 15).The magnitude of tumor burden eradicated by the antitumorimmunity which emerges shortly after the low-dose chemotherapy of mice bearing a large MOPC-315 tumor is probably quitelarge since, in the absence of the contribution of antitumorimmunity, the direct antitumor effects of the low dose ofcyclophosphamide or L-PAM are not sufficient even for theeradication of a very small (nonpalpable) tumor (8, 16, 17).

Recently we have initiated studies to identify the T-cell sub-set(s) which is important for the eradication of a large tumorburden in low-dose L-PAM-treated MOPC-315 tumor bearers.Thus far we have shown that depletion of Lyt-2* (i.e., CDS*)T-cells, in contrast to depletion of L3T4* (i.e., CD4*) T-cells,

abrogates the curative effectiveness of the low-dose chemotherapy (18). Histological examination of the s.c. tumor nodules onvarious days following low-dose L-PAM therapy revealed widespread lymphocytic infiltration by Day 5 following the chemotherapy and this infiltration was drastically reduced when thedrug-treated tumor bearers were also subjected to treatmentwith monoclonal anti-Lyt-2.2 antibody, but not with monoclonal anti-L3T4 antibody (18).

In the current study we established that as a consequence oflow-dose L-PAM therapy CD8* T-cells accumulate in the s.c.tumor nodules of MOPC-315 tumor bearers. Subsequently, weevaluated the effect of monoclonal anti-Lyt-2.2 or anti-L3T4antibody treatment on the accumulation of CDS* T-cells in thes.c. tumor nodules of low-dose L-PAM-treated MOPC-315tumor bearers. Finally, since CDS* T-cells classically mediatetheir antitumor activity in vitro through a CTL-type lytic mechanism, studies were undertaken to determine whether freshT-lymphocytes isolated from the s.c. tumor nodules of low-dose L-PAM-treated tumor bearers can exert in vitro a directlytic activity for MOPC-315 target cells in an antigen-specificmanner.

MATERIALS AND METHODS

Tumors. We have used primarily the weakly immunogenic (19)MOPC-315 plasmacytoma that was maintained in vivo as previouslydescribed (20) as a s.c. tumor in female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, MA). Routinely the mice wereinjected s. c. with 1x10' viable MOPC-315 tumor cells, a dose whichis ~300 times the minimal lethal tumor dose, and which kills the micein approximately 16 days. For the specificity experiments, we haveused, in addition to the MOPC-315 plasmacytoma, an antigenicallyrelated syngeneic plasmacytoma (the MOPC-104E) (21), two antigenically unrelated thymomas (the WEHI 22.1 of BALB/c origin and theEL-4 of C57BL/6 origin), and an NK-sensitive lymphoma (the YAC-1of A/As origin). The MOPC-104E was maintained in BALB/c mice byserial weekly passage in an ascitic form while the WEHI 22.1, the EL4,and the YAC-1 were maintained in vitro in tissue culture mediumsupplemented with 10% fetal calf serum (Sigma Chemical Co., St.Louis, MO) (21).

Chemotherapy. A fresh stock solution of 10 mg of L-PAM (Bur-

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MELPHALAN-INDUCED CTL-MEDIATED TUMOR ERADICATION

roughs Wellcome Co., Triangle Park, NC)/ml was prepared as previously described (8, 15), and was further diluted with Dulbecco's phos

phate-buffered saline, pH 7.2 (Grand Island Biological Co., GrandIsland, NY), to the desired concentration. A dose of 1.5-2.5 mg L-PAM/kg body weight (mg/kg) was administered Â¡.p.to mice bearing alarge (>20 mm) MOPC-315 tumor which resulted from the s.c. inoculation of 1 x IO6MOPC-315 tumor cells 10 days earlier. This dose ofL-PAM is curative for approximately 90% of mice bearing a large (>20mm) MOPC-315 tumor if T-cell-dependent antitumor immunity alsoaids in tumor eradication (8, 18). Complete tumor regression occurs insuch mice within 8-10 days after L-PAM administration (8, 18).

Hybridomas. As the source of monoclonal anti-Thy-1.2 antibody weused hybridoma 30-H12 (22) obtained from the American Tissue Culture Collection (Rockville, MD). As the source of monoclonal anti-L3T4 antibody we used hybridoma GK 1.5 (23), and as the source ofmonoclonal anti-Lyt-2.2 antibody we used hybridoma 2.43 (24); bothof these hybridomas were available to us through the generosity of Dr.Frank Fitch at the University of Chicago, Chicago, IL. All threemonoclonal antibodies are rat anti-mouse immunoglobulins of theIgG2b subclass (22-24).

Immunohistochemistry. The presence of T-cells or T-cell subsets wasassessed in tumor sections of untreated mice bearing a large (20-22mm) s.c. MOPC-315 tumor or mice that had been treated with low-dose L-PAM 5 to 7 days earlier when they bore a 20- to 22-mm s.c.MOPC-315 tumor. For this purpose, we have used a modified versionof the method originally described by Hsu et al. (25). Briefly, tissuefrom the s.c. tumor nodules was "snap frozen" in Tissue-Tek O.C.T.

compound (Miles, Inc., Elkhart. IN) and sectioned on a cryostat atâ€”20Â°C.Sections, 4 ^m thick, were fixed in acetone for 7 min and airdried. The sections were then incubated overnight at 4Â°Cwith either

monoclonal anti-Thy-1.2, anti-Lyt-2.2, or anti-L3T4 antibody. Afteran additional 2-h incubation at room temperature, the sections werewashed and incubated with a biotinylated rabbit anti-rat IgG antibody(Vector Laboratories, Burlingame, CA). Endogenous peroxidase activity was quenched by incubating the sections for 30 min in a 0.3%H2O2:methanol solution. After washing, the sections were incubatedfor 30 min in an avidin-horseradish peroxidase solution (ABC Immu-noperoxidase System, Vector Laboratories). The sections were thenwashed, counterstained with Mayer's hematoxylin (Sigma Chemical

Co.), and mounted. Finally, the sections were analyzed in a blind fashionby J. M. P.

In Vivo Depletion of T-Cell Subsets. The in vivo depletion of T-cellsubsets was done as previously described (26), utilizing ascites-contain-ing monoclonal antibody that we generated in outbred nu/nu mice(Charles River Breeding Laboratories) (26). The monoclonal antibodytreatments were initiated on Day 8 following tumor inoculation (i.e., 2days prior to the administration of low-dose L-PAM) and were followedby an additional injection 1 day later and thereafter on alternating daysfor a period of up to 6 days. The effectiveness and selectivity of themonoclonal antibody treatments in depleting the corresponding T-cellsubset were verified as previously described (18, 26) by determining thepercentage of Thy-1.2*, CD4*, and CDS* cells [utilizing indirect im-

munofluorescence staining followed by analysis on the EPICS V (Coulter Electronics Inc., Hialeah, FL)j among splenocytes of mice treatedwith a given monoclonal antibody relative to that among splenocytesof mice not subjected to monoclonal antibody treatment (18, 26). Whilespleens of low-dose L-PAM-treated MOPC-315 tumor bearers contained 8.3 Â±0.9% (SE) and 17.8 Â±1.6% CDS* and CD4* T-cells,

respectively, treatment of low-dose L-PAM-treated MOPC-315 tumorbearers with monoclonal anti-Lyt-2.2 or anti-L3T4 antibody routinelyreduced the percentage of the corresponding splenic T-cell subset bymore than 90%, with no significant reduction in the frequency of T-

cells belonging to the other subset. In addition to evaluating theeffectiveness and the selectivity of the monoclonal antibody treatmentin depleting the corresponding T-cell subset in the spleens of the low-dose L-PAM-treated MOPC-315 tumor bearers, a group of the samebatch of mice was routinely monitored for tumor regression/progression to confirm that the treatment with monoclonal anti-Lyt-2.2 antibody, in contrast to the treatment with monoclonal anti-L3T4 antibody,abrogated the curative effectiveness of low-dose L-PAM therapy.

Isolation of T Cells from s.c. MOPC-315 Tumor Nodules. Single cellsuspensions prepared from the s.c. tumor nodules of MOPC-315 tumorbearers were layered on a discontinuous Ficoll-Hypaque density (1.09g/ml) gradient. Cells at the interface between the Ficoll-Hypaque andthe suspending media were collected and the T-cells were then positivelyselected using the panning method of Mage et al. (27) with slightmodifications. Briefly, cells obtained from the Ficoll-Hypaque densitygradient were incubated on plastic IViri dishes to remove the plastic-adherent cells. Subsequently, the nonadherent cells were added toplastic Petri dishes that had been precoated with monoclonal anti-Thy-1.2 antibody and incubated for 1 hat 37Â°C.At the end of the incubation

period, the nonadherent cells were removed and the adherent cells wererecovered by addition of a 0.1 % lidocaine solution, followed by repeatedvigorous washings. The percentage of T-cells in the final cell preparation, as determined by indirect immunofluorescence staining followedby flow cytometric analysis, exceeded 85% and the viability of the cells,as determined by 0.4% trypan blue dye exclusion, exceeded 95%.

In Vitro Depletion of T-Cell Subsets. Lymphocyte preparations derived from the s.c. tumor nodules of mice treated with low-doseL-PAM 5-7 days earlier were depleted of T-cell subsets as previouslydescribed (18, 26) by treatment with the corresponding monoclonalantibody plus low-toxicity rabbit complement. The effectiveness andselectivity of the in vitro antibody treatments were verified by immunofluorescence staining followed by analysis on the EPICS V. Treatment of lymphocytes with monoclonal anti-Lyt-2.2 antibody plus complement led to a more than 80% reduction in the frequency of CDS*T-cells with no reduction in the frequency of CD4* T-cells. At the same

time, treatment of lymphocytes with monoclonal anti-L3T4 antibodyplus complement led to a more than 95% reduction in the frequency ofCD4* T-cells with no reduction in the frequency of CDS* T-cells.

Antitumor Cytotoxicity Assay. The level of antitumor cytotoxicityexhibited in vitro was determined as previously described, utilizing the3.5-h "Cr release assay (20). Briefly, 2 x IO5 "Cr-labeled target cells(MOPC-315, MOPC-104E, WEHI 22.1, EL-4, or YAC-1) were incubated with various numbers of spleen cells or T-lymphocytes derivedfrom the s.c. tumor nodules of untreated or low-dose L-PAM-treatedMOPC-315 tumor bearers. At the end of the incubation period, thecells were pelleted and both supernatant (Sup) and pellet (Pel) werecounted in an Auto-Gamma scintillation counter. The percentage of"Cr release for each sample was calculated as follows:

of "Cr release = cpm in Sup

cpm in Sup + cpm in Pelx 100

Subsequently, the percentage of specific "Cr release was calculated by

the following formula:

T â€”C% of specific s'Cr release = x 100M â€”C

where T is the percentage of release with test lymphocytes, C is thepercentage of spontaneous release by the target cells alone, whichranged between 17 and 22%, and M is the percentage of maximal s'Crrelease obtained by the addition of 1% Nonidet P-40 detergent (ParticleData Corp., Elmhurst, IL) solution which ranged between 90 and 95%.Each experiment was performed at least twice. The percentage of 51Cr

release presented represents the mean Â±SE of results from all experiments done, and the number of experiments executed is provided inthe footnote of each table.

Statistical Analysis. We determined the statistical significance of ourobservations utilizing analysis of variance. A P value of <0.05 wasconsidered significant.

RESULTS

Use of Indirect Immunoperoxidase Staining to Evaluate s.c.Tumor Nodules from Untreated and Low-Dose L-PAM-treatedMOPC-315 Tumor Bearers for the Presence of T-Cells andTheir Subsets. Since T-cells of the CDS* subset were implicated

by the in vivo depletion studies (18) as required for the eradi-

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cation of the large MOPC-315 tumor burden not eradicated bythe direct antitumor effects of low-dose L-PAM, experimentswere performed to determine whether CDS* T-cells are actually

present in s.c. MOPC-315 tumor nodules that are regressing asa consequence of low-dose L-PAM therapy. For this purpose,frozen tumor sections derived from mice on Day 5 after low-dose L-PAM therapy [at a time after low-dose chemotherapywhen a massive lymphocytic infiltration is evident in the paraffin-embedded histolÃ³gica! sections of the s.c. tumor nodules(18)] were evaluated for the presence of Thy-1.2*, CD4*, orCDS* cells. This was done by determining whether cells thatpossess surface markers that bind monoclonal anti-Thy-1.2,anti-L3T4, or anti-Lyt-2.2 antibody are present within theregressing s.c. tumor nodules. As a reference point, tumornodules from untreated tumor bearers were also evaluated forthe presence of T-cells and T-cell subsets. In agreement withour previous observations (18), tumor nodules from untreatedtumor-bearing mice contained few, if any, mononuclear cellsthat bound to monoclonal anti-Thy-1.2 (Fig. 1), anti-Lyt-2.2,or anti-L3T4 (data not shown) antibody. In contrast, MOPC-

315 tumor nodules that were regressing as a consequence oflow-dose L-PAM therapy contained a massive lymphocyticinfiltrate that bound monoclonal anti-Thy-1.2 (Fig. 2a) or anti-Lyt-2.2 (Fig. 2b) antibody. At the same time, only a few of thecells among the massive lymphocytic infiltrate bound monoclonal anti-L3T4 antibody (Fig. 2e). Occasionally (i.e., <5% of thesections), however, a focus of T-cells that bound anti-L3T4antibody was observed within the regressing s.c. tumor nodule(Fig. 2d). Thus, CDS* T-cells are the major component of themassive lymphocytic infiltrate present in MOPC-315 tumornodules that are regressing as a consequence of low-dose L-PAM therapy, although a small number of CD4* cells are also

present.Effect of In Vivo Treatment with Monoclonal Anti-Lyt-2.2 or

Anti-L3T4 Antibody on the Acquisition of a Massive CDS*T-Cell Infiltrate in s.c. Tumor Nodules of Low-Dose L-PAM-treated MOPC-315 Tumor Bearers. To further study theimportance of the CDS* T-cell infiltrate in the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumorbearers, we evaluated whether treatment with monoclonal anti-Lyt-2.2 antibody, which abrogates the curative effectiveness of

the low-dose chemotherapy (18), also prevents the massiveaccumulation of CDS* T-cells in the s.c. tumor nodules. As

part of this study, we also evaluated the effect of monoclonalanti-L3T4 antibody treatment on the accumulation of CDS* T-

cells in the s.c. tumor nodules of low-dose L-PAM-treatedtumor bearers. In contrast to sections derived from the s.c.tumor nodules of mice treated with low-dose L-PAM alonewhich contained a massive CDS* lymphocytic infiltrate (Fig.

2Â¿i),sections derived from the s.c. tumor nodules of mice treatedwith low-dose L-PAM plus monoclonal anti-Lyt-2.2 antibody(Fig. 3a) contained very few, if any, CDS* T-cells. At the same

time, sections derived from the s.c. tumor nodules of micetreated with low-dose L-PAM plus monoclonal anti-L3T4 antibody (Fig. 3Z>)contained a massive CDS* lymphocytic infil

trate which was comparable to that seen in tumor nodules ofmice treated with low-dose L-PAM alone (Fig. 2b). Thus,treatment of MOPC-315 tumor bearers with monoclonal anti-Lyt-2.2 antibody, which abolishes the curative effectiveness oflow-dose L-PAM, prevents the appearance of a massive CDS*lymphocytic infiltrate in the s.c. tumor nodules of low-dose L-PAM-treated tumor bearers.

Use of Cytofluorometric Analysis to Study the LymphocyticInfiltrate in s.c. Tumor Nodules of Low-Dose L-PAM-treatedMOPC-315 Tumor Bearers for the Presence of CDS* T-Cells.

To obtain some quantitative information regarding the magnitude of increase in the number of CDS* T-cells present in thes.c. tumor nodules of MOPC-315 tumor bearers as a consequence of low-dose L-PAM therapy, we determined the numberof CDS* T-cells that can be recovered per s.c. tumor nodule

from mice treated with low-dose L-PAM 5 days earlier relativeto the number recovered per tumor nodule from untreatedtumor bearers (Table 1). As a control, we also determined thetotal number of Thy-1.2* cells as well as the number of CD4*T-cells that can be recovered from the tumor nodules of untreated and low-dose L-PAM-treated MOPC-315 tumor bearers. As can be seen from Table 1, very few T-cells were recoveredfrom the tumor nodules of untreated MOPC-315 tumor bearers.However, by Day 5 following the low-dose L-PAM therapy,when the tumor nodules had regressed by approximately 50%,the number of T-cells recovered per s.c. tumor nodule increasedmore than 120-fold (i.e., from 0.12 x 10" to 15.2 x IO6). The

Â»̂â€¢Fig. I. Immunohistological section of the

s.c. tumor nodules of untreated mice bearing alarge (20-22 mm) MOPC-315 tumor. The section was stained for the presence of Thy-1.2*

cells. Original magnification, x 520.

f f, '

â€¢ â€¢

- .

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ms*- -

â€¢

c

H

Fig. 2. Immunohistological sections of the s.c. tumor nodules of mice treated 5 days earlier with a low dose of L-PAM when the mice bore a large (20-22 mm)MOPC-315 tumor. The sections were stained for the presence of: (a) Thy-1.2* cells; (b) CDS* cells; or (c and d) CD4* cells. Fig. 2c represents a typical microscopic

field, while Fig. 2d represents a rare microscopic field. Original magnification, x 520.

Table 1 Enumeration of the T-cells and T-cell subsets present within thelymphocytic infiltrate recovered from the s.c. tumor nodules of untreated and low-

dose L-PAM-treated MOPC-315 tumor bearers

Tumor sizeDays at the time

following of excisionchemotherapy (mm)

No. of cells/tumor" (xlO6)

Thy-1.2* CDS* CD4*

21.1 Â±1.2 0.12 + 0.06 0.13 + 0.60 0.00 + 0.0010.5+1.2 IS.25 Â±3.10' 10.91 + 2.2T 4.24 Â±0.86f

* Data for untreated MOPC-315 tumor bearers represents the mean Â±SE for3 separate experiments and the data for L-PAM-treated MOPC-315 tumorbearers represents the mean Â±SE for 6 separate experiments.

'Tumor size at the time of low-dose L-PAM therapy was 20-22 mm in

diameter.c Significantly different (P < 0.01) relative to the number of cells with the same

phenotype in the s.c. tumor nodules of untreated mice (Day 0).

Table 2 Effect of in vivo monoclonal anti-Lyt-2.2 antibody treatment on thenumber of CDS" and CD4* T-cells recovered from the s.c. tumor nodules of low-

dose L-PAM-treated MOPC-315 tumor bearers

Monoclonalantibody

treatmentNone

Anti-Lyt-2.2No.Thy-1.2*15.25Â±3.12.75

Â±3.3*of

cells/tumor"(xlO6)cor10.91

Â±3.10.22 Â±0.1*CD4*4.24

+ 0.862.54Â±0.12C

" Mean Â±SE of 3 separate experiments.* Significantly different (P < 0.01) relative to the number of cells with the

same phenotype in the s.c. tumor nodule of mice treated with a low dose of L-PAM alone.

c Not significantly different relative to the number of CD4* cells in the s.c.tumor nodule of mice treated with a low dose of L-PAM alone.

vast majority of the T-cells recovered from the s.c. tumornodules on Day 5 after low-dose L-PAM therapy belonged tothe CD8+ T-cell subset (i.e., approximately 70%); however, a

substantial percentage of CD4+ T-cells was also present (i.e.,approximately 30%). In fact, the number of CD8+ T-cellsrecovered per s.c. MOPC-315 tumor nodule increased morethan 80-fold within the 5-day period after low-dose L-PAMtherapy, reaching as many as 10.9 x IO6cells/regressing tumor.

Effect of in Vivo Treatment with Monoclonal Anti-Lyt 2.2Antibody on the Number of CDS* T-Lymphocytes That Can BeRecovered from s.c. Tumor Nodules of Low-Dose L-PAM-treated MOPC-315 Tumor Bearers. To gain some quantitativeinformation regarding the magnitude of reduction in the number of CD8+ T-cells present in the s.c. tumor nodule as aconsequence of monoclonal anti-Lyt-2.2 antibody treatment oflow-dose L-PAM-treated MOPC-315 tumor-bearing mice, wedetermined the number of CD8+ T-cells that can be recoveredper s.c. tumor nodule from mice treated with low-dose L-PAMplus monoclonal anti-Lyt-2.2 antibody relative to the numberrecovered per s.c. tumor nodule from mice treated with low-dose L-PAM alone (Table 2). As a control, we also determinedthe total number of Thy 1.2+ cells as well as the number ofCD4+ T-cells that can be recovered per tumor nodule frommice treated with low-dose L-PAM plus monoclonal anti-Lyt-2.2 antibody relative to tumor nodules from mice treated withlow-dose L-PAM alone. As a consequence of monoclonal anti-Lyt-2.2 antibody treatment, the total number of T-cells recovered per s.c. tumor nodule from low-dose L-PAM-treatedtumor bearers was reduced by approximately 80%, and thenumber of CD8+ T-cells recovered was reduced by approximately 98%. In other words, the number of CD8+ T-cellsrecovered per s.c. tumor nodule from mice treated with low-dose L-PAM plus monoclonal anti-Lyt-2.2 antibody was com-

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Â»â€¢â€¢

i

Fig. 3. Immunohistological sections of thes.c. tumor nodules of mice treated 5 days earlier with a low dose of L-PAM plus: (a) monoclonal anti-Lyt-2.2 antibody; or (A) monoclonal anti-L3T4 antibody. The sections werestained for the presence of CDS* cells. Original

magnification, x 520.

\*> . ""'

4 â€¢'â€¢s-

O lC

>"

parable to that recovered per s.c. tumor nodule from untreatedMOPC-315 tumor bearers (Table 1). The monoclonal anti-Lyt-2.2 antibody treatment was selective in depleting the CDS* T-

cell subset, and it did not significantly reduce the number ofCD4+ T-cells recovered per s.c. tumor nodule from low-dose L-PAM-treated tumor bearers. Thus, monoclonal anti-Lyt-2.2antibody treatment, which abrogates the curative effectivenessof low-dose L-PAM therapy for mice bearing a large MOPC-315 tumor, reduces the number of CD8+ T-cells that can berecovered from s.c. tumor nodules of low-dose L-PAM-treatedtumor bearers to the level present in the s.c. tumor nodules ofuntreated tumor bearers.

Assessment of the Ability of Fresh T-Lymphocytes Recoveredfrom s.c. Tumor Nodules of Low-Dose L-PAM-treated MOPC-315 Tumor Bearers to Lyse MOPC-315 Tumor Cells in a Short-Term in Vitro Assay. Experiments were performed to determine

whether the massive lymphocytic infiltrate seen in the s.c.MOPC-315 tumor nodule as a consequence of low-dose L-PAM therapy contains T-cells which can exert direct cytotox-icity for MOPC-315 tumor cells. Specifically, T-lymphocytesrecovered from the s.c. tumor nodules of mice treated with low-dose L-PAM 5 days earlier were assessed for their ability tolyse MOPC-315 tumor cells in a 3.5-h s'Cr-release assay at

effector/target cell ratios ranging from 100/1 to 5/1 (Table 3).As a reference point, T-cells recovered from the s.c. tumornodules of untreated MOPC-315 tumor bearers were also evaluated for their anti-MOPC-315 lytic activity under the sameexperimental conditions. As can be seen from Table 3, T-cellsderived from the s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers exerted potent anti-MOPC-315 lytic

activity which was evident even at an E/T ratio of 5/1. Incontrast, T-cells derived from the s.c. tumor nodules of un-

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Table 3 l^vel ofanti-MOPC-315 cyloloxicity exhibited by T-lymphocytes fromthe s.c. tumor nodules of untreated or low-dose L-PAM-treated MOPC-315 tumor

bearers

SourceofeffectorT-cellsL-PAM

TuB*Untreated TuBrfAntitumor

cytotoxicity" (% of specific "Cr releaseÂ±SE)

at an E/T cell ratioof100/175.6

Â±2.5e-1.6Â± 1.550/173.2

Â±2.2ND'25/159.0

Â±4.6'

0.6 Â±0.75/144.6

Â±10.5'-0.4 Â±2.7

a Mean Â±SE of 10 separate experiments.* T-cells derived from the s.c. tumor nodules of mice treated 5 days earlier

with low-dose L-PAM when the mice bore a 20- to 22-mm s.c. tumor.'Significantly different (P < 0.01) relative to the cytotoxicity exhibited by T-

cells derived from the s.c. tumor nodules of untreated MOPC-315 tumor-bearingmice.

**T-cells derived from the s.c. tumor nodules of untreated mice bearing a 20-to 22-mm tumor.

' ND, not determined.

Table 4 Comparison between the level ofanti-MOPC-315 cytotoxicity exhibitedby lymphocytes derived from the s.c. tumor nodules and spleens of the same

low-dose L-PAM-treated MOPC-3I5 tumor bearers

Antitumor cytotoxicity0 (% of specific "Cr

release Â±SE) at an E/T cell ratio ofSource of effector

lymphocytesNormal

spleensUntreated TuB spleens*L-PAM TuB spleens'L-PAM-treated tumors'â€¢100/13.4

Â±0.51.9 Â±0.59.5 Â±O.f>d

78.1 Â±\.\r25/13.1

Â±0.30.4Â±0.2

2.8 Â±0.359.2 Â±4.1 f5/13.2

Â±0.60.9 Â±0.82.9 Â±0.6

42.7 Â±6.8r

" Mean Â±SE of 3 separate experiments.* Spleen cells from mice bearing a 20- to 22-mm tumor.' Spleen cells from mice that had been treated with low-dose L-PAM 5 days

earlier when they bore a 20- to 22-mm tumor.''Significantly different (P < 0.01) relative to the cytotoxicity exhibited by

spleen cells from normal mice.'T-cells derived from the s.c. tumor nodules of low-dose L-PAM-trcated

MOPC-315 tumor-bearing mice.^Significantly different (P < 0.01) relative to the cytotoxicity exhibited by

spleen cells from the same low-dose L-PAM-treated MOPC-315 tumor-bearingmice.

treated MOPC-315 tumor-bearing mice were unable to lyseMOPC-315 target cells even when assayed at an E/T ratio of100/1. Thus, as a consequence of low-dose L-PAM therapy, T-lymphocytes with potent direct anti-MOPC-315 cytotoxicitycan be recovered from the s.c. tumor nodules of MOPC-315tumor bearers.

Comparison of the Anti-MOPC-315 Lytic Activity of Lymphocytes Derived from either s.c. Tumor Nodules or Spleens ofLow-Dose L-PAM-treated MOPC-315 Tumor Bearers. Experiments were performed to determine whether at the time whenT-lymphocytes with potent direct anti-MOPC-315 cytotoxicitycan be recovered from the s.c. tumor nodules of low-dose L-PAM-treated MOPC-315 tumor bearers, lymphocytes with direct anti-MOPC-315 cytotoxicity are also present at a distantsite (e.g., the spleen). Accordingly, in addition to assessing theanti-MOPC-315 lytic activity of T-cells derived from the s.c.tumor nodules of mice treated with low dose L-PAM 5 daysearlier, we also assessed the anti-MOPC-315 lytic activity ofspleen cells derived from the same mice (Table 4). As expected(18), spleen cells derived from untreated tumor bearers or spleencells derived from normal mice were unable to lyse MOPC-315target cells at any of the E/T ratios used (Table 4). On the otherhand, spleen cells derived from the low-dose L-PAM-treatedMOPC-315 tumor bearers exhibited some direct anti-MOPC-315 cytotoxicity. However, this anti-MOPC-315 lytic activitywas substantially weaker than that exhibited by T-cells derivedfrom the s.c. tumor nodules of the same low-dose L-PAM-treated tumor bearers. In fact, the lytic activity exhibited byspleen cells from low-dose L-PAM-treated MOPC-315 tumorbearers at an E/T ratio of 100/1 was much lower than the lyticactivity exhibited by T-cells derived from the s.c. tumor nodules

at an E/T ratio of 5/1. Thus, on Day 5 after low-dose L-PAMtherapy, when the mice are still engaged in tumor eradication,T-cells derived from the s.c. tumor nodules are superior tospleen cells in their lytic activity for MOPC-315 tumor cells.

Specificity of the in Vitro Lytic Activity Exhibited by T-CellsDerived from s.c. Tumor Nodules of Low-Dose L-PAM-treatedMOPC-315 Tumor Bearers. Experiments were performed todetermine whether T-lymphocytes derived from the s.c. tumornodules of low-dose L-PAM-treated MOPC-315 tumor bearersare capable, in a short-term assay, of lysing in addition to theMOPC-315 plasmacytoma, the antigenically related MOPC-104E plasmacytoma (21) and even some antigenically unrelatedtumors. Specifically, T-cells derived from the s.c. tumor nodulesof low-dose L-PAM-treated MOPC-315 tumor-bearing micewere evaluated for their lytic activity against a panel of tumorcell lines which consisted of the MOPC-315 and MOPC-104Eplasmacytomas, the WEHI 22.1 and EL4 thymomas, as well asthe NK-sensitive YAC-1 lymphoma (Table 5). As can be seenfrom Table 5, T-cells derived from the s.c. tumor nodules oflow-dose L-PAM-treated MOPC-315 tumor bearers exerted acytolytic effect for MOPC-315 as well as MOPC-104E tumorcells. However, these T-cells were less cytotoxic for the MOPC-104E than for the MOPC-315 tumor cells. In fact, the level ofanti-MOPC-104E cytotoxicity exerted at an E/T ratio of 50/1was comparable to the level of anti-MOPC-315 cytotoxicityexerted at an E/T ratio of 3.125/1. This translates to anapproximately 15-fold lower cytotoxicity for the MOPC-104Ethan for the MOPC-315 tumor cells. At the same time, T-cellsderived from the s.c. MOPC-315 tumor nodules of low-dose L-PAM-treated MOPC-315 tumor bearers were unable to lysethe WEHI 22.1 and the EL4 thymomas, nor the YAC-1 lymphoma at any of the E/T ratios used. Thus, the lytic activity ofT-cells from the s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers is specific for MOPC-315-associatedantigens.

Identification of the T-Cell Subset(s) That Exerts the Anti-MOPC-315 Lytic Activity Exhibited by T-Cells Derived froms.c. Tumor Nodules of Low-Dose L-PAM-treated MOPC-315Tumor Bearers. Experiments were performed to determinewhether the CD8+ T-cells are responsible for the potent directanti-MOPC-315 cytolytic activity exhibited by T-lymphocytesderived from the s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers. For this purpose, we evaluated theeffect of depleting CD8+ or CD4* T-cells on the magnitude of

anti-MOPC-315 cytotoxicity exerted by the T-lymphocytes. Ascan be seen from Table 6, depletion of CD8+ cells drasticallyreduced the ability of the T-lymphocytes to lyse MOPC-315tumor cells. In contrast, depletion of CD4+ T-cells did notreduce the anti-MOPC-315 lytic activity of the T-lymphocytes.Thus, CD8+ cells and not CD4* cells, exert the potent directanti-MOPC-315 cytotoxicity exhibited by T-cells derived fromthe s.c. tumor nodules of low-dose L-PAM-treated MOPC-315tumor bearers.

DISCUSSION

We have previously shown that the curative effectiveness oflow-dose L-PAM therapy for mice bearing a large MOPC-315tumor is abrogated when the mice are treated with monoclonalanti-Lyt-2.2 antibody (18). Here we show that as a consequenceof low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor, CD8+ T-cells actually accumulate within the regressing tumor nodules. Moreover, fresh CD8+ T-cells derivedfrom the regressing tumors display in vitro potent direct cyto-

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Table 5 Lerei oflytic activity exerted by T-lymphocytes derived from the s.c. tumor nodules of low-dose L-PAM-treated MOPC-315 tumor hearers against a panel oftumor cell lines

Antitumor cytotoxicity"'* (% of specific "Cr release Â±SE) against

E/T cellratio50/1

25/112.5/16.25/1

3.125/1MOPC-31571.9

Â±1.6'

55.2 Â±i.(f47.9 Â±4.9'32.3 Â±2.6'22.3

Â±4.2Â°MOPC-104E23.4

Â±8.3'

6.8 Â±1.04.6 Â±1.41.8 Â±0.32.0 Â±0.2WEH1

22.12.6

Â±1.04.1 Â±2.53.4 Â±2.32.5 Â±2.31.5 Â±1.3EL41.1

Â±1.04.1 Â±0.30.0 Â±0.01.5 Â±0.80.1 Â±0.8YAC-14.5

Â±1.72.3 Â±0.50.9 + 0.10.3 Â±0.40.2 Â±0.5

" Mean Â±SE of 3 separate experiments.*T-lymphocytes derived from the s.c. tumor nodules of mice treated 5 days earlier with low-dose L-PAM when the mice bore a 20- to 22-mm s.c. tumor.' Significantly different (P < 0.01) relative to the cytotoxicity exhibited against MOPC-104E. WEH I 22.1. EL-4, or YAC-1 tumor cells.d Significantly different (P < 0.01 ) relative to the cytotoxicity exhibited against WEH I 22.1, EL-4, or YAC-1 tumor cells.

Table 6 Effect of in vitro treatment with monoclonal anti-Lyt-2.2 or anti-L3T4antibody plus complement on the level ofanti-MOPC-315 cytotoxicity exhibitedby lymphocytes derived from Â¡hes.c. tumor nodules of low-dose L-PAM-treated

MOPC-315 tumor bearers

Antitumor cytotoxicity' (% of specific 51Cr

release Â±SE) at an E/T cell ratio ofIn vitro treatment of

lymphocytes"Complement

aloneAnti-Lyt-2.2 plus complementAnti-L3T4 plus complement50/177.7

Â±3.116.7 Â±5.3e82.1 Â±2.3"25/178.3

Â±7.520.8 Â±1.4'80.1 Â±0.3''5/166.5

Â±15.75.7 Â±8.7'

70.2 Â±12.4"

" Lymphocytes derived from the s.c. tumor nodules of mice treated 5 daysearlier with low-dose L-PAM when the mice bore a 20- to 22-mm s.c. tumor.

* Mean Â±SE of 2 separate experiments.'Significantly different (P< 0.01) relative to the cytotoxicity exhibited by T-

lymphocytes treated with complement alone.Not significantly different relative to the cytotoxicity exhibited by T-lympho

cytes treated with complement alone.

toxic activity for MOPC-315 tumor cells in an antigen-specificmanner. Thus, it is likely that the CD8+ T-cells that accumulate

in the s.c. tumor nodule as a consequence of low-dose L-PAMtherapy use a CTL-type mechanism to eradicate at least part ofthe tumor burden not eradicated through the direct antitumoreffects of the drug.

The importance of antitumor immunity that emerges shortlyafter the chemotherapy for the cure of tumor bearers is notlimited to the MOPC-315 tumor system nor to the anticancerdrug L-PAM. In fact, several investigators have shown thatantitumor immunity which emerges after the chemotherapy isimportant for the therapeutic outcome of a variety of anticancerdrugs such as L-PAM, cyclophosphamide, l,3-bis(2-chloro-ethyl)-l-nitrosourea, or bleomycin for a variety of tumor modelssuch as the MOPC-104E plasmacytoma, the L1578Y, L522,and LSA lymphomas, or the KMT-17 fibrosarcoma (9-11, 28,29). However, only limited studies have been carried out tocharacterize the tumor-eradicating immunity which emergesafter the chemotherapy. In these studies (9, 10, 29), the spleen,and not the tumor nodule, has been used as the source ofeffector cells. However, the fact that a certain cell type presentin the spleen displays tumor-eradicating activity does not necessarily mean that the same cell type is important in the intacttumor-bearing animal for the eradication of the tumor burdennot eradicated through the direct antitumor effects of the drugs.In this regard it should be pointed out that although asialoGMI+, CD4~/CD8~ cells in the spleens of mice bearing a s.c.

1591-PRO4L tumor acquire LAK-type activity as a consequence of anti-CD3 antibody treatment, such cells are notinvolved in the anti-CD3 antibody-induced rejection of the UV-induced skin tumor (30). Thus, it is apparent that in order toidentify the cell type(s) that actually participates in tumoreradication in the original drug-treated tumor bearers, it isnecessary to determine the effect of in vivo depletion of variouscell types (by the use of a panel of monoclonal antibodies) onthe therapeutic outcome of the chemotherapy. In addition, it is

necessary to examine the tumor nodules of the drug-treatedtumor bearers to establish the presence of the "needed" cell

type in the regressing tumor nodules. Finally, isolation of the"particular" cell type from a regressing tumor nodule and

assessment of the mechanism through which it can bring aboutthe lysis of the autochthonous tumor is essential in order togain some insight into the mechanism through which the particular cell type mediates its tumor eradicating activity in situ.

Examination of histolÃ³gica! sections derived from the tumornodules of low-dose L-PAM-treated MOPC-315 tumor bearersfor the presence of CD8+ and CD4+ T-cells by the use of

immunoperoxidase staining revealed that the massive lympho-cytic infiltrate present on Day 5 after the chemotherapy consistsprimarily of CD8+ T-cells. The fact that CD8+ T-cells com

prised the majority of the lymphocytic infiltration in the s.c.tumor nodules of low-dose L-PAM-treated MOPC-315 tumorbearers was also confirmed by the indirect immunofluorescencestaining of T-cells recovered from the tumor nodules. However,there appears to be a discrepancy regarding the extent to whichCD4+ T-cells are present in the lymphocytic infiltrate of tumor

nodules from low-dose L-PAM-treated MOPC-315 tumor bearers. Specifically, while examination of immunoperoxidase-stained tumor sections only rarely revealed the presence ofCD4+ T-cells, approximately 30% of the T-cells recovered fromthe tumor nodules were CD4+. One possible explanation for

this discrepancy is that the sections examined missed areas ofthe tumor containing the CD4+ T-cells. This possibility is

unlikely, however, considering the numerous tumors that werestudied, and the fact that multiple sections from each tumornodule were examined. A more likely explanation is that someT-cells identified as CD4+ by the immunofluorescence methodwere not identified as CD4+ by the immunoperoxidase method

(e.g., T-cells with lower density of CD4 surface antigens) dueto the lower sensitivity of the immunoperoxidase method. Thepossibility also exists that the method used for the recovery ofT-cells from the tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers favors the recovery of CD4+ overCD8+ T-cells and consequently the ratio of CD4+/CD8+ cellsis offset (31 ). Regardless of the exact frequency of CD4+ cells,it is quite clear that some CD4+ T-cells are indeed present inthe s.c. tumor nodules of low-dose L-PAM-treated MOPC-315tumor bearers. More important, however, is the fact that CD8+T-cells constitute the vast majority of the massive lymphocyticinfiltrate in s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers, since CD8+ T-cells were implicated

in our previous studies utilizing in vivo depletion with monoclonal antibody as the T-cell subset required for the curativeeffectiveness of low-dose L-PAM for mice bearing a largeMOPC-315 tumor (18).

As observed by many other investigators in a variety of tumorsystems (32, 33), in the MOPC-315 tumor system too, T-

7647




lymphocytes isolated from the tumor nodules of untreatedtumor bearers were poorly cytotoxic for the autochthonoustumor cells. However, while T-cells derived from the s.c. tumornodules of untreated MOPC-315 tumor bearers were unable tolyse MOPC-315 tumor cells even at the highest E/T ratio used(i.e., 100/1), T-cells derived from the s.c. tumor nodules of low-dose L-PAM-treated MOPC-315 tumor bearers exerted a potent anti-MOPC-315 lytic activity even at the lowest E/T ratioused (i.e., 5/1). Considering that the anti-MOPC-315 cytotoxicactivity resides in the CDS* T-cell subset and virtually all ofthe T-cells derived from the s.c. tumor nodules of untreatedtumor bearers are CDS*, while only 70% of the T-cells derived

from the s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers are CDS*, this translates to a morethan 25-fold increase in the cytotoxic activity of the CDS* T-

cell population. Moreover, when taking into consideration thatthe number of CDS* T-cells in the s.c. tumor nodule increasedas a consequence of low-dose L-PAM therapy more than 80-fold within a 5-day period, this translates to a more than 2000-fold increase in the lytic activity of the CDS* T-cell population

present within the s.c. tumor nodule.The anti-MOPC-315 cytotoxicity exhibited by T-cells derived

from the s.c. tumor nodules of mice that had been treated withlow-dose L-PAM 5 days earlier is much more potent than thatexhibited by spleen cells from the same mice. Specifically, T-lymphocytes derived from the s.c. tumor nodule exerted a muchstronger anti-MOPC-315 lytic activity at an E/T ratio of 5/1than did the spleen cells at an E/T ratio of 100/1. However,since the anti-MOPC-315 lytic activity resides in the CDS*cells for both spleen cells (18) and T-cells derived from the s.c.tumor nodules of low-dose L-PAM-treated MOPC-315 tumorbearers, we should correct for the number of CDS* T-cellsactually present in the 5'Cr release assay when spleen cellsversus when T-cells from the s.c. tumor nodules are evaluatedfor their lytic activity. Specifically, while 70% of the cells amongthe T-cells derived from the s.c. tumor nodule are CDS* cells,the CDS* cells represent only 8-10% of the nucleated cells in

the spleen (18). Even with this correction for the number ofCDS* T-cells present in the assay system, on Day 5 after low-dose L-PAM therapy of MOPC-315 tumor bearers, CDS* T-

cells derived from the tumor nodules exert in vitro a morepowerful anti-MOPC-315 cytotoxic activity than CDS* T-cellsfrom the spleens (i.e., the actual ratio of splenic CDS* T-cellsto tumor cells in the "Cr release assay at the highest E/T ratioused is 8-10/1, while the ratio of CDS* cells from the tumor

nodule at the lowest E/T ratio used is 3.5/1). This may be dueto the presence of a higher frequency of CTLs with anti-MOPC-315 lytic activity among CDS* T-cells derived from the s.c.tumor nodule than among CDS* T-cells derived from a distant

site such as the spleen (34, 35). In addition (or alternatively), itis possible that on a per cell basis CTLs derived from the s.c.tumor nodules of the low-dose L-PAM-treated MOPC-315tumor bearers are more efficient in lysing MOPC-315 tumor

cells than are CTLs derived from the spleens.The lytic activity exhibited by the CDS* T-cells derived from

the tumor nodules of low-dose L-PAM-treated MOPC-315tumor bearers is specific for MOPC-315-associated antigens.Not only are these MOPC-315 T-cells unable to lyse twoantigenically unrelated thymomas and an NK-sensitive lym-phoma, but they exert only a relatively weak lytic activity evenagainst MOPC-104E tumor cells which share plasmacytoma-associated antigens with the MOPC-315 (21). In fact, the lyticactivity exerted by CDS* T-cells derived from the s.c. tumornodules of low-dose L-PAM-treated MOPC-315 tumor bearers

against the MOPC-104E tumor cells was approximately 15-fold lower than that exerted against the autochthonous tumorcells.

The fact that CDS* T-cells from s.c. tumor nodules of low-dose L-PAM-treated MOPC-315 tumor bearers are capable ofexerting a potent CTL-type lytic activity for MOPC-315 tumorcells does not necessarily mean that this is the only effectormechanism exploited by the CDS* T-cells to bring about the

eradication of the large s.c. tumor burden not eradicated by thedirect antitumor effect of the drug. CDS* T-cells, includingthose which are capable of exerting a CTL-type lytic effect,have been shown to be capable of bringing about tumor celldestruction through a delayed type hypersensitivity mechanism(36, 37), and it is possible that such a mechanism is alsooperating in the s.c. tumor nodules of low-dose L-PAM-treatedMOPC-315 tumor bearers. Our failure to detect antigen nonspecific killing in our in vitro cytotoxicity assay may have beena function of the assay system used. Specifically, the manifestation of nonspecific killing by macrophages may require alonger time period than that used in our studies (38). Still, theresults presented here illustrate that CDS* T-cells capable ofexerting in vitro a potent CTL-type lytic activity for MOPC-315 tumor cells accumulate in the s.c. tumor nodule of low-dose L-PAM-treated MOPC-315 tumor bearers and it is conceivable that the CDS* T-cells exploit in situ too a CTL-type

lytic mechanism to eradicate at least part of the large tumorburden not eradicated by the direct antitumor effects of thedrug.

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1990;50:7641-7649. Cancer Res Blaine Y. Takesue, Joseph M. Pyle and Margalit B. Mokyr following Low-Dose Melphalan TherapyEradication of a Large Subcutaneous MOPC-315 Tumor

T-Cells in+Importance of Tumor-specific Cytotoxic CD8

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importance of tumor-specific cytotoxic cd8+ t-cells in ... · injected s. c. with 1x10' viable...

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