The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of...

[CANCER RESEARCH 58, 5795-5802. December 15. 1998]

The Induction of in Vivo Proliferation of Long-Lived CD44hi CD8+ T Cells after theInjection of Tumor Cells Expressing IFN-aj into Syngeneic Mice1

Filippo Belardelli,2 Maria Ferrantini, Stefano M. Santini, Sara Baccarini, Enrico Proietti, Mario P. Colombo,

Jonathan Sprent, and David F. ToughLaboratory of Virology. Istituto Superiore di SanitÃ . Rome 00161. Italy Â¡F.B.. M. F.. S. M. S.. S. B., E. P.]: Division of Experimental Oncology, National Tumor Institute. Milan20133. Italy Â¡M.P. C.]; Department of Immunologo The Scripps Research Institute. La lolla. California 92037 Â¡J.S.Â¡:and The Edward Jenner Institute for Vaccine Research.Campion RG 207 NN. United Kingdom Â¡D.F. T.]

ABSTRACT

The tumorigenicity of transplantable tumor cells in mice is reduced bytransduction with cytokine genes, including IFN-a and interleukin (IL)

12. Although T cells are considered important in tumor rejection, themechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferationof T-cell subsets in mice transplanted with cytokine-producing syngeneictumor cells was assessed by administering the DNA precursor bromode-oxyuridine. The injection of viable cells producing IFN-a or II,-12 causeda marked proliferation of CDS* T lymphocytes in both the spleen and

lymph nodes. Proliferation was most prominent among memory-pheno-type CD44hl CD8+ T cells. In contrast, proliferation of CD8+ T cells did

not occur in mice injected with control cells or with cells expressing IL-4,granulocyte colony-stimulating factor, or IFN-y. Pulse-chase studies inmice injected with IFN-or-producing cells showed that a proportion ofproliferating ( '1)8 * T cells survived for at least 70 days, suggesting that

long-lived memory cells are induced using such an approach. In summary,

these results, together with previous studies on the host immune reactivitytriggered by the injection of tumor cells expressing IFN-a, represent astrong rationale for considering IFN-a as a powerful T-cell adjuvant for

the generation of more effective cancer vaccines.

INTRODUCTION

The development of a protective immune response depends on theexpansion and long-term persistence of antigen-specific T cells (1,2).The extent of T-cell proliferation and survival in vivo after antigenic

stimulation is influenced by many factors associated with the mannerin which the antigen is presented. One factor likely to play a key roleis the balance of cytokines present in the local microenvironment ofthe responding T cells (3). In this regard, infectious agents are stronginducers of a variety of inflammatory cytokines, which may partiallyaccount for the powerful immune response elicited by infection.Therefore, it is logical to predict that infection-induced cytokines,including type I (a/ÃŸ)IFN and IL-12,3 will also act as adjuvants for

coinjected antigens. Recent studies on the former cytokine havedemonstrated that type I IFN is particularly stimulatory for CD8+ T

cells (4). This stimulation was of two types: (a) injection of type I IFNor inducers of type I IFN into mice resulted in the proliferation ofpreexisting CD44hi CD8+ memory-phenotype T cells in a T-cell

receptor-independent manner. The cells stimulated to divide by type IIFN subsequently reverted to a resting state and persisted long-term as

Received 5/22/98: accepted 10/14/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by grants from the Associazione Italiana Ricerca sul Cancro(Milan), the Italy-USA Special Project on "Therapy of Tumors." and the USPHS. D. F. T.

is the recipient of a Centennial Fellowship from the Medical Research Council of Canada.2 To whom requests for reprints should be addressed, at Laboratory of Virology.

Istituto Superiore di SanitÃ . Viale Regina Elena 299. 00161 Rome. Italy. Phone: 39-6-4990-3290; Fax: 39-6-4938-7184; E.mail: [email protected].

* The abbreviations used are: IL, interleukin; BrdUrd. bromodeoxyuridine; LN. lymph

node; TC. transfection control; G-CSF, granulocyte colony-stimulating factor; FACS.fluorescence-activated cell-sorting; MLTC. mixed lymphocyte tumor cell culture: Th. T

helper.

memory-phenotype cells. This bystander proliferation was quite selective for memory-phenotype CD8+ T cells; neither na'ive-phenotypeCD44'Â°CD8+ T cells nor CD4+ T cells proliferated; and (b) induc

tion of type I IFN was shown to enhance the primary response ofnaÃ¯veCD8+ T cells to a specific antigen; increased proliferation and

survival of antigen-specific cells were observed in response to antigenplus poly (I, C) (a type I IFN-inducer) compared to antigen alone.

Taken together, these results suggested that the induction of type IIFN during virus infections may be important in the generation of along-lived memory response, particularly in CDS* T cells.

The potentiation of antitumor immune responses by a high localconcentration of cytokines has been considered as a possible therapeutic approach to cancer. Experimentally, this has been examined byinjecting cytokines directly into the sites of established tumors or bystudying the in vivo behavior of genetically modified tumor cellsexpressing cytokines (reviewed in Refs. 5-7). The activity of a num

ber of different cytokines thought to participate in the generation ofprotective T-cell responses has been examined. Our own work hasfocused on IFN-a, because of its well known antitumor activity both

in mouse models and in certain malignancies in humans (reviewed inRef. 8). Thus, using three different tumor models, we showed that thetransduction of the mouse IFN-a, gene into metastatic tumor cellsresulted in a clear-cut loss of tumorigenicity after injection of the

genetically modified cells into immunocompetent syngeneic mice(9-12). In some cases, we were able to demonstrate that CD8+ T cells

were the major host cell types responsible for the tumor rejectionobserved in mice transplanted with IFN-a-producing tumor cells (10).

These results, together with recent findings obtained by our group aswell as by others in various experimental models (reviewed in Ref.13), suggested that certain IFN effects on T cells could play importantroles in the generation of a protective immune response against bothvirus infections and tumor growth. Thus, in light of the recentlydescribed stimulatory effects of type I IFN on the in vivo proliferationof T cells (4), it was of interest to study the in vivo proliferation ofdifferent T-cell subsets in mice transplanted with genetically modifiedtumor cells producing IFN-a. For these studies, we chose to use twowell-characterized metastatic mouse tumors transplantable in BALB/c

mice (i.e., TSA mammary carcinoma and C26 colon adenocarcinomacells) because several clones producing various cytokines were available for comparative studies, and we have measured the in vivoturnover of T cells in tumor cell-injected mice by the administration

of BrdUrd.

MATERIALS AND METHODS

Mice. BALB/c mice were purchased from Charles River (Calco. Italy) orthe rodent breeding colony at The Scripps Research Institute and maintainedunder pathogen-free conditions in the animul facilities of the Department of

Virology at the Istituto Superiore di SanitÃ or The Scripps Research Institute,respectively. Mice were treated according to the European Community guidelines. Mice were injected s.c. with various numbers of tumor cells in 0.2 ml ofPBS as indicated in the figure legends. For BrdUrd treatment, the mice weregiven sterile drinking water containing 0.8 mg/ml BrdUrd (Sigma Chemical

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IFN-t>-EXPRESSlNG TUMORS AND T-CELL PROLIFERATION

Co.). BrdUrd-containing water was changed daily. In some experiments, micewere thymectomized at 5-6 weeks of age and left for approximately 40 days

before use.Tumor Cells. C26 is a murine colon adenocarcinoma cell line derived from

BALB/c mice treated with /V-nitroso-yV-methylurethane (14). TSA is a spon

taneous mammary carcinoma transplantable in BALB/c mice (15). The majorcharacteristics of TSA cells producing IL-4, IFN-y. or IFN-a as well as thoseof C26 cells expressing G-CSF and IL-12 have been reported in previous

studies (see the references in Table 1). Genetically modified C26 cells expressing IL-4 have been recently obtained in the laboratory of M. P. Colombs

(Milan, Italy), and their major properties are described in Table 1. Cells werecultivated in DMEM supplemented with 50 Â¿ig/mlgentamicin (BioWhittaker,Walkersville, MD) and 10% PCS (Sebam, Berlin, Germany).

The transduced cell lines were cultured in the same medium containing 0.5mg/ml G418 (calculated to give 100% antibiotic activity; Geneticin; LifeTechnologies, Inc., Grand Island, NY).

Transfection and Isolation of C26 Clones Producing IFN-a,. The plasmi. I form of the retroviral vectors pL-neoL-5 and pLMuIFN-alTneoL (9) wastransfected into C26 parental cells by electroporation in a Gene-Pulser apparatus (Bio-Rad, Richmond. CA). Exponentially growing cells (8 X IO6) were

resuspended in 800 /il of PBS, placed in an electroporation cuvette (0.4-cm

gap), and subjected to 400 V and 960 /iF. After electroporation, the cellsuspension was incubated on ice for 30 min, diluted in 10 ml of DMEMsupplemented with 10% PCS, and incubated at 37Â°Cin a 0.5% CO2 humidified

atmosphere. After 48 h, the cultures were replenished with DMEM supplemented with 10% PCS containing 500 fig/ml G418. G418-resistant cells werecloned by limiting dilution in 96-well microtiter plates in the presence of G418.Single-cell clones were expanded and maintained in selection medium. IFN

was titrated on L929 cells as described previously (16). IFN titers are expressed in International Units (lUs).

For the in vivo experiments to be described, we used the following two C26clones: (a) C26-TC (a TC clone): and (b) C26-28 (an IFN-a-C26 cloneproducing approximately 500 lU/ml). The s.c. injection of IO5 C26-28 cells

into BALB/c mice resulted in a marked delay in tumor growth as compared tothat of mice injected with C26-TC cells. The mean day of death (Â±SD) wasapproximately 28 (Â±12) for mice injected with C26-TC cells and 77 (Â±18) foranimals inoculated with the same number of C26-28 cells. Occasionally, in10-20% of the mice injected with C26-28 cells, there was a complete tumor

rejection, which never occurred in mice transplanted with parental cells orC26-TC cells.

Flow Cytometry. Cell suspensions were prepared from spleens or LNs. Inexperiments in which viable tumor cells were injected, LN results are reportedfor cell suspensions made from the axillary, inguinal, and popliteal nodes fromthe side of the mouse on which the tumor cells were injected. These results didnot differ significantly from those obtained when pooled LN (all popliteal,inguinal, axillary, brachial, cervical, and mesenteric nodes) cells from the samemice were analyzed. For experiments in which irradiated tumor cells wereinjected, LN results refer to pooled LNs. The mAbs used for cell surfacestaining were anti-CD8-PE (Life Technologies, Inc., Gaithersburg, MD), anti-CD4-PE (Collaborative Biomedicai Products, Bedford, MA), anti-Ly-6C-bio-tin (PharMingen, San Diego, CA), anti-B220PE (PharMingen), anti-CD69-biotin (PharMingen), anti-CD44-FITC (PharMingen), and anti-CD44-biotin(IM7.8.1). Biotinylated antibodies were detected with RED670-streptavidin(Life Technologies, Inc.). Staining for BrdUrd with anti-BrdUrd-FITC (Becton

Dickinson, Mountain View, CA) was performed as described previously(17). Stained cells were analyzed on a FACScan flow cytometer (BectonDickinson).

MLTCs and CTL Assays. MLTC lymphocytes from LNs draining thevaccination site (5 x 10s cells/well) were cultured with irradiated C26 cells(5 X \04 cells/well) in 2 ml of lymphocyte medium containing 5 units/ml

recombinant human IL-2 (Chiron-Italia, Milan, Italy) in 24-well culture plates

(Costar, Cambridge, MA). MLTCs from splenic lymphocytes were set with2 X IO7 splenocytes plus 5 x IO5 irradiated C26 cells in a 20-ml volume in

25-ctrr culture flasks (Costar). Cytotoxicity was tested after 5 days of cultureat 37Â°C.The induction of CTL response and the CTL specificity were assessedin standard 4-h "Cr release assays as described previously (18, 19).

RESULTS

In Vivo Proliferation of CD8+ and CD4+ T Cells in Mice

Transplanted with Viable C26 or TSA Tumor Cells ProducingVarious Cytokines. Initially, we carried out a preliminary series ofexperiments to determine whether the injection of viable tumor cellsexpressing IFN-a would alter the in vivo proliferation of T cells. Thus,2 x IO6 IFN-a-secreting or control transfectant tumor cells were

injected s.c. into syngeneic BALB/c mice. The recipients (and non-

injected controls) were given BrdUrd in their drinking water continuously from the time of injection and were sacrificed 7, 14, and 21days after tumor transplantation. At 7 days postinjection, BrdUrdlabeling of CD4+ and CD8+ in spleen or pooled LNs was similar in

tumor-injected and noninjected mice. However, at 14 and 21 daysafter tumor injection, a clear-cut increase in the percentage of BrdUrd-labeled CD8+ cells was observed in both the splenocytes and LN cells

of mice injected with IFN-a-producing TSA or C26 cells as compared

to both noninjected mice and mice injected with control tumor cells(data not shown).

Subsequently, we performed a more detailed study of the effect ofcytokine-expressing tumor cells on in vivo T-cell proliferation by

injecting TSA or C26 tumor cells producing various cytokines andmeasuring the extent of labeling after 14 days of continuous BrdUrdadministration. The major characteristics of the TSA and C26 clonesused are shown in Table 1. Confirming the preliminary results, injecting mice with IFN-a-producing cells resulted in a marked proliferation of CD8+ T cells; this was observed with both TSA- and

C26-derived tumor cells (Table 2). A similar increase in BrdUrdlabeling of CDS* cells was also detected in mice injected with

C26-IL-12 cells, but not in animals transplanted with cells expressing

other cytokines. Notably, these changes in BrdUrd labeling did notcorrelate with any significant variation in the total percentage of eitherCD8+ or CD4+ T cells in the spleen or LNs (data not shown). Ingeneral, there was a small increase in CD4+ cell proliferation in all

groups of mice injected with cells producing cytokines compared tothose injected with control cells that was more marked in the C26model.

Phenotype of CD8+ T Cells Proliferating after the Injection of

Cytokine-producing Tumor Cells. We next investigated the pheno-

type of cells proliferating in response to tumor cell injection. Becausetype I IFN has previously been shown to selectively stimulate theproliferation of CD44hl CD8+ cells (4), we compared the response ofCD44hi and CD441" cells; this was initially done for TSA tumor cells.

Table I Main properties of the cytokine-producing tumor cells used in this study

Cell typeIn vitro cytokine

production (IO6 cells/48 h)Percentage of tumor engraftment

(s.c. injection of 10 cells) References

C26-TC(TC)C26-28C26-IL-4C26-G-CSFC26-IL-12TSA-pc

paremalcellsTSA-A4TSA-IFN-yTSA-IL-4NoneIFN-a

(500lU/ml)IL-4( 18-22ng/ml)G-CSF

(90pg/ml)1L-12(5000pg/ml)NoneIFN-a

(60-120lU/ml)IFN--y(500lU/ml)IL-4

(10-26 ng/ml)10080-90100040100357520This

article("Thisarticle("Thisarticle("202115102223

and 24Materials

andMethods")MaterialsandMethods")Materialsand Methods")

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IFN-a-EXPRESSINO TUMORS AND T-CELL PROLIFERATION

Table 2 Effect of the injection of TSA and C26 clones producing various cylokines onCD8+ and CD4 + cell proliferation in vivo

BALB/c mice were injected s.c. with 2 X IO6 cells as indicated. All mice were given

BrdUrd from the time of tumor cell injection. On day 14. mice were sacrificed, andsplenocytes and cells from pooled LNs were subjected to FACS analysis as described in"Materials and Methods." The data represent the mean values from two or three mice/

group (Â±SD).

BrdUrd* LN cells â€¢BrdUrd+ spleen cells

Cell type CD8 + CD4 + CDS"1" CD4 +

NoneTSA-pcTSA-IFN-aTSA-IFN-yTSA-IL-4C26-TCC26-IFN-aC26-G-CSFC26-IL-4C26-IL-127.7Â±0.111.0Â±0.820.6

Â±0.611.2Â±1.811.2Â±0.66.9Â±2.021.5Â±2.98.5Â±1.510.5Â±2.617.4Â±1.87.4

0.811.30.815.1

1.913.41.112.81.79.4

0.313.91.110.22.214.5

Â±2.013.6Â±1.19.7

0.212.00.917.10.712.83.613.81.27.4

2.521.11.28.2

2.214.0Â±0.519.5Â±2.516.8

Â±2.017.7Â±1.721.4

0.519.60.517.92.811.44.721.41.316.42.518.22.320.5

0.7

As shown in Fig. IA, an obvious increase in the percentage ofBrdUrd-labeled CD8+ CD44hi cells was observed in both LNs and

spleens from mice injected with TSA-IFN-a, but not in the corresponding tissues of animals inoculated with control TSA cells, TSA-IL-4, or TSA-IFN-y. A similar increase in the labeling of CD8+ cells(especially CDS"1"CD44hl) was observed in mice injected with another

TSA clone named A2 (10) that produced IFN-a, (data not shown).The injection of IFN-a-producing TSA cells also resulted in a smallincrease in the BrdUrd labeling of CD44'Â°cells that was consistentlyobserved but was much less marked than the increase seen for CD44hICD8+ cells (Fig. ÃŒA).

Fig. IB shows the results of an equivalent set of experimentsperformed using various C26 clones. The injection of C26-IFN-a cells

resulted in a very similar proliferative response to that observed afterthe injection of TSA-IFN-a cells. Thus, there was a marked increase

in the percentage of BrdUrd-labeled CD44hi CD8+ cells and a smallincrease in the labeling of CD441" CD8+ cells. Of interest, the

injection of IL-12-producing C26 cells also caused a very similar

response. In contrast, no significant change in the proliferation ofCD44hi or CD44'" CDS"1"cells was observed after the injection of

either C26-G-CSF or C26-IL-4 cells.The phenotype of the proliferating CD8+ T cells was further

investigated 14 and 21 days after the transplantation of C26-IFN-acells. As shown in Fig. 2A, increased BrdUrd labeling of total CD8+

T cells was observed at both time points compared to that seen in miceinjected with control tumor cells. Whereas the proliferation was muchmore marked among CD44hl CDS* cells, the percentage of BrdUrd+CD441" CDS"1"cells was also elevated. Significantly, there was an

increased percentage of Ly-6Chl CD8+ cells at both time points (Fig.

2B). The expression of Ly-6C is specifically up-regulated by type I

IFN (25), which implies that the injected tumor cells were producingIFN-a in vivo. In confirmation of this, IFN titers ranging between 40

and 160 lU/ml were generally found in the sera of these mice at thesetime points. Of interest, the injection of control C26 cells consistentlyresulted in a small decrease in the percentage of Ly-6Chl CD8+ cells

in the spleen or LNs as compared to the values observed in thecorresponding cells from untreated mice. Whereas the mechanisminvolved in this down-regulation is unknown, one possibility is that

there is an inhibition of background type I IFN production associatedwith the growth of the tumor. If this is true, this characteristic mightcontribute to the poor immunogenicity and high tumorigenicity of theparental tumor and might have more general non-tumor-specific sup-pressive effects on immune responses in tumor-bearing mice. This

question is currently under investigation.As shown in Table 3, an up-regulation of Ly-6C expression in

CD8+ cells was also observed 2 weeks after injecting mice with TSA

cells producing IFN-a, whereas no increase was found in animals

Fig. 1. Proliferation of CD441" and CD441" CD8"1"T

cells in mice injected with TSA (A) or C26 (ÃŸ)clonesproducing various cytokines. BALB/c mice were injecteds.c. with 2 X IO6 cells as indicated. All mice were given

BrdUrd continuously in their drinking water from the timeof the tumor injection. Mice were sacrificed on day 14.and splenocytes and LN cells were subjected to flowcytometry analysis as described in "Materials and Methods." Data are shown for CD8^ T cells expressing low

(D) or high (â€¢)levels of CD44 in the spleen (upper) orLNs (lower). The data represent the mean values from twoor three mice/group (Â±SD). Similar results were obtainedin two additional experiments using the same clones.

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00

60

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40 -

30

20

10

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60

CD44hlCD44'Â°

â€¢CD44hl

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Li Ã•50

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5797



IFN-a-EXPRESSINO TUMORS AND T-CELL PROLIFERATION

TotalCDS

DAY 14 DAY 21

CD44'Â°

DAY 14 DAY 21

CD44hi B CDS

DAY 14 DAY 21 DAY 14 DAY 21

DAY 14 DAY 21 DAY 14 DAY 21

Fig. 2. Characterization of the phenotype of proliferating CD8+ T cells in mice transplanted with IFN-a-producing C26 cells. BALB/c mice were injected s.c. with 2 X IO6 TC

(B) or IFN-a-producing (â€¢)C26 cells or remained noninjected (D). All mice were given BrdUrd in their drinking water from the time of tumor injection and were sacrificed after14 or 21 days of continuous labeling. Cells from the spleen or LNs were subjected to FACS analyses as described in "Materials and Methods." A, BrdUrd labeling of total CD8+ Tcells (upper left panel), CD44hl CDS* cells (upper right panel), or CD44'Â°cells (lower Â¡eftpanel) and the percentage of CD8+ T cells (lower right panel) in LNs are shown. Verysimilar results were obtained using spleen cells from the same mice (data not shown). B, the percentage of CDS* T cells expressing high levels of Ly-6C in the spleen (top panel)

or LNs (bottom panel). The data represent the mean values from two or three mice/group (Â±SD).

transplanted with tumor cells producing G-CSF (C26), IL-4 (C26 andTSA), or IFN--y (TSA). Notably, some enhancement in the levels ofLy-6C expression on CDS* T cells was observed in both splenocytes

and cells from the LNs of mice transplanted with C26 cells producingIL-12.

On the whole, these data showed a positive correlation between thein vivo production of IFN (as revealed by an up-regulation of Ly-6Cexpression in CDS* cells) and the proliferate response of CDS* T

cells in animals injected with syngeneic tumor cells producing IFN-a.

Evaluation of the in Vivo Proliferative Response of T Cells afterthe Injection of High Numbers of Irradiated Cytokine-producing

Tumor Cells. Although the experiments described above showed aclear induction of CDS* T-cell proliferation after the injection of

Table 3 Expression of Ly-6C antigen in CDS T cells of mice injected with tumorcells expressing various cytokines

The percentage of Ly-6C+ cells was determined 14 days after the s.c. injection of2 X 10 cells, as described in "Materials and Methods." The data represent the mean

values from two or three mice/group (Â±SD).

% Ly-6C" cells

Cell type LNs Spleen

NoneTSA-pcTSA-IFN-oTSA-IFN-yTSA-IL-4C26-TCC26-IFN-0C26-G-CSFC26-IL-4C26-IL-1262.768.194.067.667.447.597.356.44.85.80.66.62.54.50.65.464.3

Â±2.672.6Â±2.854.765.591.566.166.435.795.454.261.14.28.30.33.16.07.50.12.80.271.13.1

viable tumor cells producing IFN-a, the interpretation is complicated

by variables associated with differences in tumor growth. At 2 weekspostinjection, mice injected with IFN-a-producing tumor cells generally exhibited small tumors, which underwent necrosis at 3-4 weeks.

In contrast, control transfectant tumors grew more rapidly and werenot rejected. To dissociate the in vivo proliferation of CDS* T cells

from the extent of initial tumor growth, we performed a set ofexperiments in which we injected high numbers (i.e., 5 X IO7) of

irradiated tumor cells producing the various cytokines.As shown in Fig. 3A, 10 days after the injection of irradiated

C26-IFN-0 cells, there was a marked increase in the BrdUrd labelingof CDS"1"T cells in both the spleen and LNs (especially CD44hi, but

also CD44'Â°)compared to that observed in either noninjected mice or

mice inoculated with the same number of irradiated TC C26 cells. Atthis time, there was also a slight increase in the proliferation of CD4*T cells (both CD44hi and CD44'Â°cell populations) in mice inoculated

with the irradiated cells expressing IFN-a (Fig. 3ÃŸ).

In another set of experiments, we compared the in vivo proliferationof T cells in mice transplanted with irradiated C26-IFN-a, C26-IL-12,or C26-IL-4 cells at 1, 3, 7, and 10 days after injection. At 1,3, and

7 days, no significant changes were observed in any of the groups ofmice injected with cytokine-producing cells as compared to animalsinoculated with control C26-TC cells (data not shown). On day 10, aclear-cut increase in the percentage of BrdUrd* CDS* cells was

observed only in mice injected with C26-IFN-a cells (Table 4). Miceinjected with irradiated IL-12-producing cells showed a slight increase in the percentage of BrdUrd* CDS* cells with respect to the

control values. The changes seemed to be significant only for the totalBrdUrd* CD8*cells (Table 4).

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IFN-0-EXPRESSING TUMORS AND T-CELL PROLIFERATION

Fig. 3. Proliferation of CDS* and CD4* T cells in mice

injected with high numbers of irradiated C26 cells. Five-week-old male BALB/c mice were thymectomized and left for 40 daysbefore use. Mice were either noninjected (Q) or injected s.c.with 0.2 ml of PBS containing 5 X IO7 irradiated (5000 rads)

TC (^) or IFN-a-producing (â€¢)C26 cells. All mice were givenBrdUrd in their drinking water soon after tumor injection. Onday 10, the mice were sacrificed, and splenocytes and cells fromthe LNs were processed for FACS analysis as described in"Materials and Methods." Data show the percentage of total,CD44hl, or CD44'" cells thai were BrdUrd* for CD8 (left panel)

or CD4 (righi panel) cells in the spleen (topi or pooled LNs(bottom). In addition, the percentage of CDS or CD4 cells in therespective cell populations is indicated on the graphs. The datarepresent the mean values from two or three mice/group (Â±SD).

u

LN. LN.

Persistence of BrdUrd-labeled CD8+ T Cells in Mice Injected

with Irradiated IFN-a-producing C26 Cells. Previous work hasshown that CDS* T cells proliferating in the presence of type IIFN

subsequently return to a resting state and survive long term (4).Therefore, it was of interest to examine the survival of T cells thatproliferated after the injection of IFN-a-producing tumor cells. Tomeasure the in vivo persistence of BrdUrd-labeled T cells, we performed a pulse-chase experiment in which mice were injected withhigh numbers of irradiated C26-IFN-a cells. Thus, mice were given

BrdUrd in their drinking water for 10 days after the tumor injection(pulse time) and then transferred to normal water for 30 or 70 days(chase times). Some mice were sacrificed on day 10 to measure theextent of CD8+ T-cell proliferation at time 0 (before the chase times).

As shown in Fig. 4, placing the mice on normal water for 30 daysresulted in only a small decrease in the number of BrdUrd-labeledCDS"1"LN cells. Similar results were observed in the spleen (data not

shown). After a 70-day chase, however, the percentage of BrdUrd-labeled CDS"1"cells had decreased substantially. Nevertheless, 15-20% of the originally labeled cells remained BrdUrd"1" at this later

Table 4 Effect of injection of irradiated cytokine-producing C26 cells on the in vivoproliferation of CDS T cells

Five-week-old male BALB/c mice were thymectomized and left for 40 days beforeuse. Mice were then injected s.c. with 0.2 ml of PBS containing 5 X IO7 irradiated (5000

rads) tumor cells, as indicated. All mice were given BrdUrd in their drinking waterimmediately after tumor injection. On day 10, mice were sacrificed, and splenocytes wereprocessed for FACS analysis as described in "Materials and Methods." The data represent

the mean values from two or three mice/group (Â±SD).

% of BrdUrd*

CelltypeC26-TCC26-IFN-0

C26-IL-12C26-IL-4CD8

+14.0

Â±1.232.4 Â±0.820.5 Â±2.515.6 Â±1.0CDS*

CD44hi21.5

Â±2.443.9 Â±2.225.9 Â±1.825.1 Â±1.4CDS"1"

CD441012.6

Â±2.231.1 Â±2.419.4 Â±4.213.7 Â±2.8

time point. The disappearance of labeled CD4+ T cells in the same

mice followed different kinetics. A relatively large decrease in thepercentage of BrdUrd"1"CD4"1"cells occurred during the first 30 days

after the pulse, whereas there was little or no further decrease observed in the subsequent 40 days. On the whole, the results showedthat a proportion of CD8+ T cells that proliferated after the injection

of irradiated IFN-a-secreting tumor cells could persist long term (at

least 70 days after cell division).

DISCUSSION

Immune responses to infectious agents are associated with intenseproliferation of T cells, and it is likely that the induction of inflammatory cytokines is important in stimulating this response. Thus, typeI IFN, which is rapidly induced upon infection, has been shown toplay two different roles in mediating T-cell proliferation (4). It is bothable to stimulate the proliferation of preexisting CD44hl CDS* T cells

in a T-cell receptor-independent manner (bystander proliferation) and

to act as an adjuvant to enhance the proliferative response of naÃ¯veTcells to a specific antigen. In contrast to viruses and bacteria, synge-neic tumors are generally poorly immunogenic. To improve the im-

munogenicity of tumors with a view to the eventual development ofcancer vaccines, several groups have created genetically modifiedtumor cells that secrete a variety of cytokines. This approach has infact proven successful in generating tumor cells that are more readilyrejected by syngeneic recipients than were the parental tumors (reviewed in Refs. 5-7). We have shown previously that the transductionof tumor cells with the IFN-a gene markedly reduces their tumorige-nicity and induces long-term immunity after injection in immunocom-petent mice (9-12). Therefore, it was of interest to compare the T-cellproliferative response in mice injected with control versus cytokine-

secreting tumor cells.In this study, the use of different tumor cell clones producing

5799



IFN-a-EXPRESSING TUMORS AND T-CELL PROLIFERATION

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Fig. 4. Persislencc of BrdUrd-laheled T cells after in viva T-cell proliferation inducedby the injection of irradiated C26 cells producing IFN-a. Five-week-old male BALB/cmice were thymectomized and left for 40 days under special pathogen-free conditionsbefore use. Mice were then injected s.c. with 5 X IO7 irradiated (5000 rads) IFN-a-

producing C26 cells and were immediately given BrdUrd in their drinking water for 10days. At this time, some mice were sacrificed (D), and the rest of the animals were givennormal water for an additional 30 (Ã‰Ãœ)or 70 (â€¢)days (chase periods). At these time points,the mice were sacrificed, and cells from pooled LNs were analyzed by flow cytometry asdescribed in "Materials and Methods." Data are shown for CDS* (top) and CD4^

(bunion) T cells expressing different levels of CD44 and represent the mean values fromtwo or three mice/group (Â±SD). Very similar results were obtained using splenocytesfrom the same mice (data not shown).

various cytokines (IFN-a, IL-4, G-CSF, IFN-y, and IL-12) allowed us

to perform comparative studies on the effectiveness of these cytokines, which were released by the tumor cells themselves, in enhancing the in vivo proliferation of CD4* or CD8+ T cells. Several

findings are reported: (a) injecting syngeneic mice with viable controltumor cells resulted in little or no increase in T-cell proliferation

above that observed in control untreated animals. This result supportsthe contention that syngeneic tumor cells are poorly stimulatory forthe immune system; (b) in both the TSA and C26 tumor models, theinjection of viable tumor cells producing IFN-a was associated witha marked proliferation of CD8+ T lymphocytes (especially CD44hl

cells), as shown by increased BrdUrd labeling at 14 and 21 days aftertumor transplantation. Because increased proliferation was also observed after the injection of irradiated cells, the formation of a growing tumor was not required for this effect. The proliferative responseof CD8 + T cells was associated with the up-regulation of Ly-6C,

indicating that these cells were exposed to type I IFN in vivo (25).Thus, the secretion of IFN-a clearly increased the ability of the

transplanted tumor cells to stimulate the immune system; (c) tumorcells secreting IL-12 also induced the proliferation of CD44hi CD8+

T cells. The magnitude of the response observed after the injection ofviable IL-12-producing cells and the phenotype of the respondingcells were similar to those observed after the injection of IFN-a-

secreting cells. Although the possibility that these two cytokines arestimulating T-cell proliferation through a common pathway remains

to be investigated (see below), the results are consistent with ourrecent finding that the injection of recombinant IL-12 into miceinduced the proliferation of CD44hi CD8+ T cells;4 (d) injection of

tumor cells secreting IL-4, G-CSF, or IFN-y stimulated little or no

proliferation of T cells. Thus, these cytokines, at least in the contextof a tumor cell inoculum, do not have the same immunostimulatoryeffects on T cells as do IFN-a and IL-12. It should be noted, however,

that tumor cells secreting these cytokines are rejected on transplantation to syngeneic mice (see Table 1), implying that different arms ofthe immune system are activated by these cells: and (e) CDS"1"T cells

that became BrdUrd+ after the injection of IFN-a-secreting tumor

cells persisted for at least 70 days after cell division. Thus, a proportion of the activated T cells were able to return to a resting state andsurvive long term, consistent with the possibility that memory T cellswere generated during the proliferative response.

The relationship between type I IFN and IL-12 is still poorly

understood. There are a number of similarities in the antitumor effectsexhibited by these cytokines. Both type I IFN and IL-12 are very

effective in inhibiting tumor growth in mice injected with metastatictumor cells that are poorly responsive to other cytokines (26-28), and

these antitumor effects are largely mediated by T cells. Moreover,both cytokines are considered to play important roles in the generationof a Th-1 type of immune response. It is well established that IL-12is a major signal for the differentiation of Th-1 cells (29). Recent dataindicate that type I IFN (especially IFN-a) is also a cytokine involvedin the polarization toward the Th-1 pattern of immune response (30).

Although some recent studies with human cells indicate that type IIFN is a powerful inducer of the ÃŸ2chain of the IL-12 receptor on Th

cells (31 ), it is still unclear whether similar mechanisms can operatein mouse cells. In our experiments, we could not find any evidencethat the proliferative response induced by cells expressing IFN-a ismediated by endogenous IL-12. In fact, repeated injections of a mAbto IL-12 neither abolished the proliferative response of CD8+ cells

induced by the injection of IFN-a-producing cells (TSA or C26) norrendered tumor TSA-IFN-a cells more tumorigenic when transplanted

into syngeneic BALB/c mice (data not shown). On the other hand, thefinding that mice injected with IL-12-producing tumor cells exhibitedsome up-regulation of the Ly-6C antigen on CD8+ T cells could

suggest that part of the proliferative effect induced by the injection ofC26-IL-12 cells was mediated by endogenous type I IFN. Conversely,IL-12 could share some ability to up-regulate Ly-6C expression invivo with IFN-a. On this point, it is worth mentioning that we have

been unable to consistently inhibit the proliferative response inducedby IL-12-producing cells by treating the mice with a potent preparation of sheep polyclonal antibodies to mouse IFN-a/ÃŸ (data not

shown). Likewise, we tend to exclude the possibility that the proliferative effect of C26-IL-12 cells on CDS + T cells is simply due to the

induction of IFN-y by IL-12, because TSA cells expressing highlevels of IFN-y did not induce any proliferation of CD8+ T cells.

Thus, we suggest that at least under certain conditions, IL-12 caninduce the in vivo proliferation of CD8+ cells by mechanisms inde

pendent of both type I and type II IFNs.The mechanisms by which IFN-a induces a preferential prolifera

tion of CD8+ CD44hl cells in vivo remain unclear. These effects are

likely to be indirect, because the in vitro exposure of spleen lymphocytes to IFN-a does not result in any in vitro proliferation of CD8+

cells (data not shown). Likewise, only a minimal proliferation of Tcells is observed after the in vitro stimulation of spleen cells withIL-12, suggesting that the in vivo proliferative response of CDS"1"T

cells is likely to be mediated by other factors. Additional in vitro and

4 D. F. Tough and J. Sprent, unpublished observations.

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IFN-a-EXPRESSlNO TUMORS AND T-CELL PROLIFERATION

in vivo studies with other cytokines or cytokine-producing cells are

necessary to characterize these factors.Whereas the injection of IFN-a-secreting tumor cells was associ

ated with both proliferation and long-term survival of T cells, the

specificity of these cells is presently unknown. As described above,type IIFN has been shown both to stimulate bystander proliferation ofmemory-phenotype T cells and to act as an adjuvant for antigen-specific T-cell responses. Thus, in principle, there are three possible

sources for the T cells that proliferated and survived after tumor cellinjection: (a) preexisting memory-phenotype T cells activated in a

bystander manner; (b) T cells responding simultaneously to unrelatedenvironmental antigens, the response to which is enhanced by tumorcell production of IFN-a; and (c) tumor-specific T cells. It seemslikely that all three mechanisms are contributing to the BrdUrd-

labeled cells observed in the present study.Although the specificity of the responding cells has not been

addressed in this work, it is worth mentioning that several studiescarried out with either exogenous type I IFN in tumor-bearing mice

(reviewed in Ref. 13) or genetically modified tumor cells expressingIFN-a (9-12, 32, 33) indicate that this cytokine can act as an adjuvantfor the generation of a T-cell-mediated antitumor response. In sometumor models, a synergistic antitumor effect of IFN and tumor-sensitized lymphocytes has been clearly demonstrated (34-36). In theTSA tumor model, CD8+ T cells were shown to be important fortumor rejection. Thus, the depletion of CD8+ T cells in the recipient

mice rendered TSA-IFN-a (which were rejected in the majority of the

control injected animals) highly tumorigenic (10). Importantly, themajority of nontreated mice that rejected IFN-a-secreting TSA cells

were shown to be resistant to a subsequent challenge with parentaltumor cells, implying that memory T cells were generated uponexposure to the IFN-a-producing cells. However, although we haveobserved the proliferation of CD44hl CD8+ cells in response to

IFN-a-secreting tumor cells in the three different tumor models stud

ied thus far (i.e., TSA, C26, and Friend leukemia cells; data notshown) and therefore assume this to be a general phenomenon, theimportance of CD8+ cells in tumor rejection may differ according to

the characteristics of the tumor model. For example, anti-CD8 treatment only slightly enhanced the tumorigenicity of C26-IFN-a cells,5

implying that other cell types are involved in the rejection of thistumor. In addition, the generation, proliferation, and persistence oftumor-specific CD8+ cells may depend on the balance of immuno-

genic versus immunosuppressive properties of the tumor cells themselves as well as on the correct availability of suitable amounts oftumor antigens needed to induce a protective T-cell-mediated immune

response. In this regard, it is worth mentioning that in both the TSAand C26 tumor models, the injection of irradiated IFN-a-producingcells resulted in a strong induction of tumor-specific CTLs, which are

not detected in animals immunized with the corresponding controlcells.5 Overall, we conclude that the in vivo proliferation of CD44hlCD8+ cells and their long-term persistence observed in mice injected

with cells expressing IFN-a provide a rationale for further consideration of this cytokine as a powerful T-cell adjuvant for the generation

of more effective cancer vaccines.

ACKNOWLEDGMENTS

We thank I. Gresser for polyclonal antibodies to mouse IFN-o/ÃŸ.G. Fornifor TSA-IL-4 cells, and P. L. Lollini for TSA-IFN--y cells. We thank Cinzia

Gasparrini for typing the manuscript.

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1998;58:5795-5802. Cancer Res Filippo Belardelli, Maria Ferrantini, Stefano M. Santini, et al. into Syngeneic Mice

1α T Cells after the Injection of Tumor Cells Expressing IFN-+ CD8hi Proliferation of Long-Lived CD44in VivoThe Induction of

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