Mitogenic stimulation of human tumor-infiltrating lymphocytes by secreted factor(s) from human tumor cell lines

Transcription

1 Proc. Nti. Acd. Sci. USA Vol. 87, pp , June 199 Immunology Mitogenic stimultion of humn tumor-infiltrting lymphocytes y secreted fctor(s) from humn tumor cell lines (cytokines/immunotherpy) BVRLY S. PACKARD Division of Cytokine Biology, Center for Biologics' vlution nd Reserch, Food nd Drug Administrtion, 88 Rockville Pike, Bethesd, MD 2892 Communicted y Christin B. Anfinsen, Mrch 14, 199 ABSTRACT Tumor-infiltrting lymphocytes (TILs) hve shown in vivo ntitumor efficcy in oth niml nd humn studies. Functions thought necessry for ntitumor ctivity include cytolysis, homing, nd prolifertion t tumor sites. TILs, which re T lymphocytes grown ex vivo directly from tumors, er interleukin 2 (IL-2) receptors cple of trnsducing the IL-2 mitogenic signl. However, IL-2 is not normlly synthesized y solid tumor cells. This study ws imed t exploring the possile presence of T-cell mitogens of tumor origin. To this end four TIL lines derived from four melnom ptients were studied for their ility to use the environments of cultured tumor cell lines s mitogenic sources. The presence of four irrdited cultured humn tumor cell lines, three of which were derived from the sme melnom ptients s the TILs, were found to stimulte prolifertion of humn TILs in the sence of IL-2. Further investigtion showed tht the oserved prolifertive stimultion y the fourth tumor line ws due to secreted fctor(s) s mitogenic ctivity ws present in the serum-free tumor cell superntnt. Both immunologic nlyses of this medium nd prolifertive ssys in which TILs were stimulted with recominnt lymphokine stndrds suggest the presence of yet unchrcterized T-cell mitogen. The ide tht the tumor environment is intrinsiclly immunogenic nd, therefore, contins lymphocytes with the potentil to induce tumor regression hs led to the use of tumor-infiltrting lymphocytes (TILs) in clinicl trils (1-14). In this procedure lymphocytes re grown ex vivo directly from tumors in the presence of the lymphokine interleukin 2 (IL-2); ptients re reinfused with their TILs fter the ltter hve een expnded in culture for severl weeks. Although 6% of metsttic melnom ptients who hd not previously received ny immunotherpy showed ojective responses in the lrgest clinicl TIL tril to dte (15), the chrcteristics of the tumor environment tht produces TILs in vivo remin uncler. Two clsses of stimuli-one derived from cell-cell contct etween tumor cells nd immunocytes nd the other from solule fctors-my influence T-cell homing nd prolifertion. IL-2 is potent mitogen for T cells, the cell type of TILs used (Tle 1); however, its iosynthesis ppers to e confined to immunocytes (16). This fct is consistent with the current elief tht immunologiclly competent cells such s lymphocytes nd monocytes represent the mjor sources of secreted mitogenic stimultion for lymphocytes. In this study the suject of solule fctors secreted y tumor cell lines s sources of stimuli for lymphocyte prolifertion ws ddressed. As strting point for this work, the issue of whether tumor cell line estlished from melnom tumor could stimulte the growth of TILs derived from melnom msses ws exmined. After confirming the mplifiction potentil of The puliction costs of this rticle were defryed in prt y pge chrge pyment. This rticle must therefore e herey mrked "dvertisement" in ccordnce with 18 U.S.C solely to indicte this fct. 458 three melnom cell lines, nonmelnom line ws tested for the sme ioctivity nd found to e t lest s potent mitogenic source; tumor-derived mitogenic ctivity for TILs could e scried to secreted fctor(s) s mitogenic ctivity for TILs ws found in its serum-free superntnt. Immunologic nd prolifertive ssys indicte nonidentity of t lest one secreted fctor with ny previously chrcterized lymphokine. MATRIALS AND MTHODS Mterils. Dulecco's modified gle's medium (DMM) (4.5 g of glucose per liter) nd Hm's F-12 medium were purchsed from Flow Lortories; AIM-V nd RPMI 164 medi nd fetl clf serum (FCS) were from GIBCO; insulin/ trnsferrin/sodium selenite medium supplement ws from Sigm; humn lumin (Alutein) ws from Alph Therpeutic (Los Angeles); nd [3H]thymidine (6.7 Ci/mmol; 1 Ci = 37 GBq) ws from New nglnd Nucler. Recominnt humn interleukins 4 nd 6 (IL-4 nd IL-6) nd tumor necrosis fctor (TNF-) s well s neutrlizing murine monoclonl ntiody ginst humn IL-6 (ctlog 428) were from Genzyme; rit nti-humn IL-2 IgG ntiody (ctlog 412) ws from Collortive Reserch; recominnt humn trnsforming growth fctor f (TGF-13) ws from R nd D Systems; nd IL-2 cme from Cetus. Fluoresceinnd phycoerythrin-leled ntiodies (nti-cd3, -CD4, nd -CD8) were from Becton Dickinson. The clone of A-431 cells used in this study ws from J.. DeLrco (Monsnto), dog smooth muscle cells were from T. Innerrity (Gldstone Foundtion, University of Cliforni, Sn Frncisco), Mdin-Dry cnine kidney (MDBK) cells were from the Americn Type Culture Collection, nd the 618, 677, nd 66 tumor lines were estlished from humn melnom tumors y culturing in RPMI 164/1% humn serum. Four T-lymphocyte lines, TILs 618, 641, 66, nd 677, were estlished from four humn melnom tumors s descried (17). A iorector with crtridge contining hollow fiers hving moleculr-weight cutoff of 1 kd [Cellco Advnced Biorectors (Kensington, MD)] ws used for the lrge-scle culture of A-431 cells. LISA kits for IL-2, IL4, nd IL-6 were from Genzyme; second LISA kit for IL-2 ws from Collortive Reserch. MTHODS Culturing. All tumor lines were crried in DMM 1% FCS, nd the four TIL lines were in AIM-V supplemented with IL-2 t 1 units/ml. Arevitions: TIL, tumor infiltrting lymphocyte; IL-2, -4, nd -6, interleukins 2, 4, nd 6, respectively; TGF-,3, trnsforming growth fctor P; TNF-, tumor necrosis fctor ; DMM, Dulecco's modified gle's medium; FCS, fetl clf serum; CSM, extrcpillry spce medium; SFM, serum-free medium.

2 Immunology: Pckrd Tle 1. Phenotypes of TILs mesured y flow cytometry TIL CD3+, % CD8+, % CD4+, % * <3 *Nineteen percent of 641 TILs were doule-positive for oth CD8 nd CD4; none ws singly positive for CD8. The serum-free medium (SFM) used for prolifertion ssys ws modifiction of serum-free medium used for melnom cells (18) with DMM/Hm's F-12 t 1:1 rtio sustituting for DMM lone. The iorector crtridge ws seeded with -5 x 19 A-431 cells in DMM/1% FCS. After 3 weeks, the medium in the extrcpillry spce (CSM) of the crtridge ws switched to the SFM nd the FCS concentrtion in the reservoir medium ws lowered from 1% to 2%. The CSM ws drined dily from the crtridge, which ws then filled with fresh SFM. The conditioned medium (CSM) ws then centrifuged, filtered (.22 gm), nd stored t 4C. Phenotyping. Stining ws done t 4C for 3-6 min in Hnks' lnced slt solution/1% FCS/.2% NN3. Flow microfluorometric nlyses were performed y using Coulter PICS flow cytometer. Cellulr Prolifertion Assys. Tumor cells t 5 x 14 cells per well were plted in 96-well flt-ottomed pltes. Confluent cultures of tumor cells were irrdited (3 rds) (1 rd =.1 Gy)just efore cocultivtion with TILs. Lymphocytes were removed from IL-2-contining AIM-V nd plced in the pproprite medium for iossy 48 hr efore commencement of prolifertion ssys. They were resuspended in SFM or DMM/1%o FCS t 1.25 x 15 lymphocytes per ml for cocultivtion experiments nd t 6 x 15 lymphocytes per ml for experiments in which solule fctors were eing ssyed; 2 gl of cell suspension ws dded per well. Cellulr prolifertion ws determined in the presence of conditioned medi nd cytokines t the indicted concentrtions. For experiments in which neutrliztion y n ntiody ws eing mesured, conditioned medi were preincuted with the ntiody t concentrtions up to 1-fold the leled neutrliztion cpcity for 2.5 hr t 37 C efore the ddition of cells. After 24- or 48-hr stimultion, the level of lymphocyte prolifertion ws ssyed y dding.5,ci of [3H]thymidine to ech well of 96-well plte for 4 or 18 hr. A Sktron hrvester ws used to hrvest the cells, nd the rdioctivity ws counted y using n LKB -plte reder. ch mesurement ws done in sextuplicte; ech experiment ws repeted t lest three times. RSULTS Does the Presence of Tumor Cells Alter the Growth Chrcteristics of TILs? In Fig. l TIL line 618 cells re shown growing in lrge clusters in suspension. In contrst, in Fig. l these TILs in the presence of monolyer of cell line 618 tumor cells re seen growing in tight pposition to the tumor cells; the sence of lrge clusters of lymphocytes suggests the predominnce of cellulr heterophili over homophili. Thus, tumor cells induced striking chnge in the morphology of TIL-TIL interctions. The question of whether this morphologic ltertion ws due to n interchnge of iologic signls etween the two different cell types ws ddressed. Do Tumor Cells Potentite TIL Prolifertion? To determine whether tumor cell line cn stimulte growth of TILs the cellulr prolifertion rtes of TIL lines derived from four (618, 677, 641, nd 66) ptients were determined with nd without n irrdited tumor cell line estlished from one of the ptients (618). Becuse ll four TIL lines hd culture Proc. Ntl. Acd. Sci. USA 87 (199) 459 FIG. 1. TILs growing in the sence () nd presence () of 618 tumor cells. Tumor cells were plted t 5 x 14 per.2 cm2 in DMM/1% FCS. After 1 dy the medium ws chnged to SFM nd left for t lest 48 hr. TILs t 1 x 16 cells per ml were plted without nd with confluent monolyer of 618 tumor cells. Twenty-four hours lter the cultures were exmined nd photogrphed. In the former, lymphocytes grow s lrge clusters, wheres in the ltter cellulr heterophili-i.e., dhesion etween lymphocytes nd tumor cells-predomintes over cellulr homophili-i.e., dhesion mong lymphocytes. Although TILs 618, 66, nd 677 could lyse utologous tumor cells tht hd een frozen ut never cultured, they showed virtully no lytic cpility ginst ny of the cultured tumor lines in the configurtion used in this study-i.e., s dherent monolyers. This shrply contrsts with lymphokine-ctivted killer cells, which effectively lysed ll tumor lines used in this work. (X 11.) history of IL-2 dependence, the initil set of experiments ws done in the presence of IL-2 (1 nd 2 units/ml) s well s in its sence. As seen in Fig. 2 with IL-2 present t either 1 or 2 units/ml, 618 tumor cells did not enhnce y >2 SD [3H]thymidine incorportion into DNA of ny of the four TIL lines except for 677 t 2 units of IL-2 per ml; the ltter ws rely significnt ecuse the oserved enhncement ws y just 2 SD. In contrst, Fig. 2 shows tht without IL-2, prolifertion of oth 618 nd 66 TIL lines ws drmticlly incresed when irrdited 618 tumor cells were present. The response of the 677 TIL line ws gin mrginlly significnt. Presence of tumor cells hd no effect on the prolifertion of the 641 TILs, cell line with phenotype of 1% CD4+. The three other TIL lines (618, 66, nd 677) re composed of, t lest, 8%o CD8+ cells. Hence, lymphocyte prolifertion ssys suggested the prolifertive potentition of tumor cells for CD8+ lymphocytes in the sence of IL-2. Is Serum ssentil to the Tumor Potentition? To determine whether tumor stimultion in the sence of IL-2 ws inde-

3 46 Immunology: Pckrd Proc. Ntl. Acd. Sci. USA 87 (199) ~. C:._ 2Ī AC,, Tlil - IL-2, units./ml ILS FIG. 2. ffect of irrdited 618 tumor cells on prolifertion of four TIL lines t three IL-2 concentrtions: 1, 2, nd units/ml (). () xpnsion of right third of Fig. 2: prolifertion in the sence of IL-2. pendent of serum fctors, prolifertion ssys were done in prllel in SFM nd DMM/1% FCS. Fig. 3 indictes tht the prolifertion levels of oth 618 nd 66 TILs were significntly incresed y 618 tumor cells whether or not serum ws present. The prolifertion of 677 TILs ws mrginlly ffected-i.e., y not >2 SD in either medium. 641 TILs showed sttisticlly insignificnt chnges upon ddition oftumor cells. Hence, the results suggested tht the enhncement of prolifertion of TILs y 618 tumor cells ws independent of serum-derived fctors. Do Tumor Lines stlished from Other Ptients Also nhnce TIL Prolifertion? Prolifertion of TIL 66, the est responder to tumor cell-enhnced prolifertion, ws mesured in the presence of two dditionl melnom lines, 66 nd 677, oth estlished from the respective TIL ptients. Dt listed in Tle 2 show similr 3- to 4-fold increse in the prolifertive response of 66 TILs to the presence of irrdited 66 or 677 tumor lines, seen with the irrdited 618 tumor line. Is Tumor Cell-Induced Potentition Specific to Cells of Melnom Origin? Becuse melnoms derive from the neurl 12 3 md +618 tumor +A431 tumor B3 -tumor crest, cells of line not derived from this emryologic origini.e., the A-431 cell line, which ws originlly derived from n epiderml crcinom, were tested for ility to potentite TIL prolifertion in oth serum-contining nd serum-free medi. Fig. 3 shows tht irrdited A-431 cells potentited prolifertion of ll four TIL lines in serum (Fig. 3) nd showed effects similr to 618 tumor cells in SFM (Fig. 3). Is Potentition Specific to Trnsformed Cell Lines? In contrst to the melnom nd A-431 cell lines tested, irrdited dog smooth muscle cells nd MDBK cells, two untrnsformed cell lines, did not potentite TIL prolifertion (dt not shown). Although this fct, long with stimultion y the murine melnom B16 line, supports the concept of TIL stimultion y trnsformed nd not norml cells, it would e premture to mke this generliztion, s ll cell lines y virtue of their ility to survive ex vivo for extended times hve cquired some trits of trnsformed phenotype. Vlidtion of tumor specificity wits determintion of the presence or sence of purified fctor in fresh uncultured cells. Is Prolifertive Activity Due to Secreted Fctors? A scnning electron microgrph shows A-431 cells in the hollow fier +618 tumor +A431 tumor -tumor 8 2- CL TILs FIG. 3. ffect of serum on tumorpotentited TIL prolifertion. Comprison of enhnced TIL prolifertion due to irrdited 618 tumor cells with tht from irrdited A431 cells in oth serum-contining () nd serum-free () medi. A higher level of rdioresistnce of the A431 cells resulted in higher ckground for the ssys in TILs SFM.

4 Immunology: Pckrd Tle 2. Prolifertion of 66 TILs in SFM in the sence nd presence of irrdited melnom cell lines Tumor [3H]Thymidine, cell line cpm None 355 ± ± ± ± 154 Bckground counts for tumor lone hve een sutrcted. Proc. Ntl. Acd. Sci. USA 87 (199) 461 crtridge in cross section (Fig. 4). Tumor cells were only seen in the spce outside the hollow fiers. The memrne lining these fiers hd moleculr mss cutoff of 1 kd; hence, ny fctors secreted y the tumor cells with moleculr mss >1 kd were retined in the CSM. The medium tht circulted inside the hollow fiers ws composed of DMM/ 2% FCS nd ws designted s the reservoir. The CSM ws drined dily nd ssyed for ioctivity. Rtes of [3H]thymidine incorportion into oth 618 nd 66 TILs s function of incresing concentrtions of CSM re plotted in Fig. S. There is dose-dependent increse in prolifertive ctivity up to the ssy composition of 5% CSM for oth 618 nd 66 TILs. Aove this level oth curves fll off. These declines my e due to the sence of nutrients in the spent CSM or the presence of inhiitory fctor(s). Additionlly, these curves my lso represent ehvior similr to tht of other known mitogens, for which t high concentrtions prolifertive levels re sumximl. In support of the ltter hypothesis-when prtilly purified mteril ws used s the stimulus, the dose-response curve ws U-shped with the mximum oserved t -1 mg of protein per ml (dt not shown). L C I % CSM TlLs A 66 TILs 2 FIG. 4. Scnning electron microgrph of A-431 cells grown in hollow fier iorector. Drk insides of hollow fiers tht contin reservoir medium-i.e., DMM/2% FCS-re seprted from extrcpillry spce contining A-431 cells y memrne with 1-kD cutoff. (x6.) 2o 4o % Reservoir FIG. 5. Prolifertion ssys with serum-free superntnts from iorector CSM secreted y A-431 cells () nd serum-contining medium ofreservoir-i.e., drk insides of hollow fiers shown in Fig. 4 (). In Fig. S rtes of [3H]thymidine incorportion into oth 618 nd 66 TILs s function of incresing concentrtions of reservoir medium re plotted. No stimultory ctivity ws detectle. Thus, moleculr mss of the mitogenic fctor(s) must e, t lest, 1 kd s no mitogenic ctivity ws ssocited with fctor(s) secreted into the reservoir. Is the Secreted Activity Due to Known T-CelI Mitogens? To determine whether the mitogenic ctivity present in CSM ws due to fctors tht hve previously een shown to hve direct mitogenic ctivity for T cells, three CSM smples collected t 2-week intervls were tested for IL-2, IL-4, nd IL-6 y LISA. The presence of neither IL-2 (using kits from the two indicted compnies) nor IL-4 ws mesurle t or ove detection levels for these two lymphokines-i.e.,.4 Cetus unit per ml nd 9 pg/ml, respectively. At these two respective concentrtion levels no significnt prolifertive ctivity ove ckground ws detected. However, IL-6 ws found to e present t 2 ng/ml; the presence of IL-6 in A-431 cell superntnts hs een reported (19).

5 462 Immunology: Pckrd To scertin whether IL-6 could e mitogen for TILs under the conditions used in this study (3H]thyMridine incorportion ws mesured fter culturing 66 TILs for 24 hr in the presence of IL-6 t concentrtions rnging from.1 to 2 ng/ml; no [3H]thymidine incorportion ove ckground ws mesured. Furthermore, the presence of neutrlizing monoclonl ntiody ginst IL-6 hd no effect on the stimultion of [3H]thymidine incorportion induced y CSM, wheres mitogenic stimultion y IL-2 stndrds up to 1 units/ml ws completely inhiited y the nti-il-2 ntiody used. Lck of mitogenic ctivity' for 66 TILs ws lso seen for TGF-pS (1 x 1-7,-8,-9,-1,-11, nd 12 M) nd TNF- (1, 1, nd 1 ng/ml). In ll experiments IL-2 nd CSM served s positive controls. DISCUSSION The ide tht the tumor environment contins lymphocytes tht my exhiit ntitumor ctivity ws the sis for using lymphocytes expnded'in culture s' therpeutic gents (1-14). Much work supports the'concept tht n immune response is initited ginst tumors vi direct tumor celllymphocyte contct, followed y the secretion of fctors mitogenic for lymphocytes from severl immunocyte sources. However, the possiility of tumor cells providing mitogenic stimulus for lymphocytes in the form of secreted fctors hs not een well explored. Thus, the ojective of this work ws to determine whether tumor cells could secrete fctor(s) mitogenic for lymphocytes. Fig. l, which shows TILs growing in the presence of IL-2, is typicl of cultures of ctivted lymphocytes-i.e., clusters of cells in suspension. In contrst, in the presence of monolyers of tumor cells TILs hve spred ppernce (Fig. l). To determine whether the oserved pposition etween TILs nd tumor cells ws prt of n immunostimultory environment, prolifertion of four TIL lines ws compred in the presence nd sence of irrdited tumor cells. In the presence of IL-2 ddition of irrdited culture- 618 tumor cells did not significntly ffect mitosis of the four TIL lines tested (Fig. 2). However, in the sence of IL-2 (Fig. 2) these tumor cells drmticlly incresed the prolifertion rtes of two TIL lines, 618 nd 66, nd incresed less significntly the rte of third, 677. Thus, cultured tumor cells cn support T-cell mitogenesis independently' of IL-2. This fct my e essentil in understnding'the oserved disprity in phenotypic profiles of TIL cultures t the stge of reinfusion into ptients with those of the strting popultions (15, 2). Tht is, ecuse receptors for IL-2 re present on virtully ll ctivted T cells (21), the T-cell popultion tht will grow ex vivo in the constnt presence of this lymphokine is strongly ised towrd those cells cple of trnsducing nd using the IL-2 signl most efficiently. Hence, fctors from the tumor environment, which my e essentil for mintennce of lymphocytes with ntitumor ctivity, my not e retined in long-term cultures in which ll tumor cells hve died nd the only exogenous cytokine is the pn-t-cell mitogen IL-2. In the present study the oserved mitogenic stimultion of TILs y irrdited tumor cells ws shown to e of cellulr origin nd not from the serum, s indicted y Fig. 3. In ddition to the tumor line estlished from ptient 618, dt in Tle 1 show- tht tumor lines from two other melnom ptients, 677 nd 66, were similr in their ility to enhnce prolifertion of 66 TILs in SFM. Thus, the presently reported mitogenic T-cell stimultion y tumor cell lines, ll of which were derived from'unrelted ptients, is clerly not mjor histocomptiility complex-restricted. To determine whether tumor cell-induced lymphocyte mitogenicity is chrcteristic of tumors of specific emryonic Proc. Ntl. Acd. Sci. USA 87 (199) origin, such s the neurl crest, which includes melnoms s well s neurolstoms, cell line derived from tumor of epiderml crcinom origin, the A-431 line ws used s source of potentition of TIL prolifertion. Dt indicting tht A-431 cells cn serve s superior mitogenic source for TILs in serum (Fig. 3) nd equivlently to the 618 melnom line in SFM (Fig. 3) suggest tht this type of immunocyte stimultion my e rodly prevlent. Proof tht ctivity mitogenic for TILs is secreted y A-431 cells ws confirmed y the ctivity in the CSM (Fig. S). These dose-response curves indicte the presence of cytokines tht exhiit ehvior similr to tht of other known mitogens, where sumximl prolifertive levels hve een mesured t high concentrtions. The sence of IL-2 nd IL-4 y LISA nd the lck of prolifertive stimuli from IL-6, TNF-, nd TGF-,f under the experimentl conditions used suggest the presence of direct T-cell mitogen not previously chrcterized. 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