major histocompatibility complex class i and unique ... · murine tumors that escaped from cd8+...

[CANCER RESEARCH 50. 3851-3858, July I. 1990]

Major Histocompatibility Complex Class I and Unique Antigen Expression byMurine Tumors That Escaped from CD8+ T-Cell-dependent Surveillance1

Patricia L. Ward,2 Hartmut K. Koeppen, Teresa Hurteau, Donald A. Rowley, and Hans Schreiber

Department of Pathology, The University of Chicago, Chicago, Illinois 60637

ABSTRACT

The rejection of murine UV-induced skin cancers by normal mice is astriking example of powerful immune surveillance of the normal hostagainst malignant cells. In this study, we show that UV-induced rÃ©gressertumors regularly grew progressively and killed mice that were depletedof CDS* T-cells. Depletion of CD4* T-cells had no effect, suggestingthat CDS* but not CD4* T-cells were required for this immune surveil

lance. To determine whether change in major histocompatibility complex(MHO class I expression was a frequent event that caused low iiiiinu-nogenicity of tumors or facilitated escape from immune destruction,recently isolated murine tumors of varying degrees of immunogenicity,including highly Â¡mmunogenicUV-induced regressor, less Â¡mmunogenicUV-induced progresser, and poorly immunogenic spontaneous progressertumors, were compared. There was no correlation between the ability ofa tumor to grow progressively in a normal immunocompetent host andthe level of constitutive class I expression or the level of expressioninduced in vitro by 7 Interferon. (Only 1 of more than 20 progressertumors analyzed showed complete loss of a MI K class I molecule.)Some progresser variants showed loss of a unique tumor-specific cyto-toxic T-lymphocyte-defined antigen, consistent with earlier evidence ofantigen loss providing a mechanism for tumor escape. However, most ofthe host-selected progresser variants retained both MHC class I antigensand the unique tumor antigens that we could detect with cytotoxic I -lymphocyte clones, suggesting that mechanisms other than loss of MHCclass I or of the unique target antigen may be involved in escape of sometumors from a highly effective CDS-dependent host surveillance.

INTRODUCTION

T-cell-mediated immunity is critical for the rejection of virallyand chemically induced tumors in immunized mice and for therejection of UV-induced tumors or allogeneic tumors in normalmice (1-4). While the relative importance of different T-cellsubsets has been the subject of many studies and much controversy (5, 6), the CDS* cytolytic T-cell subset is essential for

immunological rejection of at least some experimentally induced tumors by immunized mice. Since CDS* T-cells recognize antigens in the context of MHC' class I molecules (7, 8),

changes in MHC class I antigens may allow tumors to escapeimmune destruction by cytolytic T-cells (9-11); e.g., some virally induced tumors lose expression of MHC class I antigensand escape immune destruction presumably because of this loss(12-17). But other mechanisms may account for escape oftumors of nonviral etiology from host surveillance.4 UV-in

duced tumors appear to be an ideal model for studying this

Received 12/19/89.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 indicate this fact.

' This work was supported by grants from the NIH. ROI CA-37156, POI CA-19266. and R37 CA-22677, and a gift from the Passis family.

1Supported in part by Grant T32 AI-07090. To whom requests for reprints

should be addressed, at Department of Pathology. The University of Chicago.5841 S. Maryland Ave.. Box 414. Chicago. IL 60637.

5Abbreviations used are: MHC. major histocompatibility complex: IFN->,recombinant murine y interferon; FACS. fluorescence-activated cell sorter; CTL,cytotoxic T-lymphocytes; MCA. 3-methylcholanthrene.

4 Immune surveillance was originally conceived as immunological resistanceof the normal host against the development of primary cancers (18-20). In thispaper, the word surveillance is used to describe the immunological resistance ofthe normal host to transplanted cancer cells, an example being the rejection ofUV-induced regressor tumors by normal mice.

question. The advantages of the model are (a) UV-inducedregressor tumors commonly require the CDS* T-cell subset fortumor rejection by normal mice (shown in this paper), (b) CDS*

cytolytic T-cell clones can be generated as reliable probes forthe individually distinct antigens on these tumors (21), and (c)progresser tumors (i.e., tumors that grow progressively in normal mice) can be isolated either as primary tumors or asprogressor variants following immune selection in vivo (22) orin vitro (23). In the present study, we have analyzed MHC classI and unique tumor antigen expression by a large number ofboth UV-induced and spontaneous tumors occurring in inbredmice, and we conclude that loss of expression of MHC class Imolecules is not a frequent mechanism by which these tumorsbecome resistant to CTL and acquire progressive growth potential. Of 24 progressor tumors tested, only 1 appeared to havelost expression of the K-molecule. Two progressor variants lostexpression of a unique tumor antigen but did not lose expression of MHC class I antigens. However, three of four progressorvariants selected in vivo did not show changes either in MHCclass I or in unique tumor antigen expression, a finding suggesting that other yet to be identified heritable changes mayalso be important for some tumors to escape from immunedestruction in vivo.

MATERIALS AND METHODS

Mice. Five- to 10-week-old germ-free-derived, pathogen-free, femaleC3H/HeN mammary tumor virus-negative (MTV~), BALB/cAnN, or

nude NCR/Nu mice were purchased from the National Cancer InstituteFrederick Cancer Research Facility Animal Production (Frederick,MD) and were maintained as previously described (24). The originalstock of nude C3H mice was in its 23rd backcross generation when itwas obtained from a colony at the Biology Division of the Oak RidgeNational Laboratory (Oak Ridge, TN).

Tumor Cell Lines. Derivation of the UV-induced and spontaneoustumors in C3H/HeN and BALB/cAnN mice has been described (21).Regressor tumors, designated RE, are strongly immunogenic in thatthese tumors when transplanted into young syngeneic mice grow duringthe first 10 days and then regress. These tumors grow progressively innude mice and eventually kill these mice by infiltrative growth withoutmacroscopic evidence of distant mÃ©tastases.Progressor tumors, designated PRO, progress to form lethal tumors when tumor fragments aretransplanted into normal mice. All cell lines were cultured in minimumessential medium (410-1100; Gibco Laboratories, Grand Island, NY)supplemented with 10% fetal calf serum.

Derivation of Progressor Variants. To derive progressor variants fromregressor tumors through selection in vivo, normal (24) or UV-irradi-ated (22) mice were each given ten 1-mm3 solid tumor fragments

implanted s.c. in one inguinal region using a trocar. Mice were observedfor at least 8 weeks following tumor challenge, unless the tumor grewout earlier. Mice with such tumors were killed when moribund and thetumor was reisolated for further transplantation studies to confirm theprogressor phenotype. To derive progressor variants through selectionin vitro, regressor tumor cells were exposed to cytolytic T-cells specificfor the parental regressor tumor, as described previously (21, 23). Cellswhich grew out after selection were tested in a "Cr-release assay forresistance to lysis by the regressor-specific CTL.

Cytofluorometric Analysis of MHC Class I Antigens. The specificitiesof the anti-MHC class I monoclonal antibodies 11-4-1. 15-5-5,31-3-4,

3851

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UNIQUE TUMOR ANTIGENS. MHC CLASS 1, AND TUMOR PROGRESSION

28-14-8, 34-2-12, and 16-3-22 have been described (25-28). Hybrido-mas secreting these antibodies were gifts from the Salk Institute (LaJolla, CA), C. Waltenbaugh (Northwestern University, Chicago, IL),and D. Lancki (University of Chicago, Chicago, IL). For quantifyinglevels of normal MHC class I antigen expression on the surface oftumor cells, 2 x 105cells were incubated with a saturating concentrationof the MHC class I-specific antibody for 30 min at 4Â°C.The cells were

then washed twice and resuspended in phosphate-buffered saline containing 10% bovine serum albumin, 0.1% sodium azide, and a 1:10dilution of fluorescein-coupled goat anti-mouse immunoglobulin (Hy-clone E-1081-A; Hyclone Laboratories, Logan, UT). After 30 min, cellswere washed twice again, and IO4cells were analyzed using the FACS

(EPICS C; EPICS Division of the Coulter Corporation, Hialeah, FL).The relative amount of fluorescence was determined as the meanchannel number of fluorescence after staining with both antibodies (A")

minus the mean channel number of fluorescence after staining with thesecond antibody alone ( Y) and was converted to a linear scale using theformula:

(X-Y)

10 85

where 85 channels equal a 10-fold increase in the fluorescence intensityon the scale of the EPICS instrument used for analysis.

Treatment of Tumor Cells with 7 Interferon. For analysis of inductionof MHC class I expression in tumors, 30 units of recombinant murineIFN-7 (lot 4407-47; 1.97 x IO7units/mg; Genentech, Inc., South San

Francisco, CA) were added per ml of tissue culture medium during 48h of culture prior to staining with MHC class I-specific antibodies.This dose of IFN-7 is effective in induction of class I expression oncells which previously had no detectable levels of class I when analyzedby immunofluorescence and cytofluorometry.

Depletion of T-Cell Subsets in yivo. The anti-CD4-producing hybrid-oma GK1.5 (29) and the anti-CD8 (anti-Lyt-2.2)-producing hybridoma2.43 (30) were kind gifts from Dr. F. Fitch (University of Chicago,Chicago, IL). The anti-CD8 (anti-Lyt-2)-producing hybridomaYTS169.4 (31) was a generous gift from Dr. H. Waldmann (Universityof Cambridge, Cambridge, UK). Fluorescein-coupled anti-CD8 (anti-Lyt-2-fluorescein conjugate 1353; Becton Dickinson, Mountain View,CA), anti-CD4 (H129.19; Ref. 32), and mouse anti-rat Â«-chain(MAR-18.5) antibodies (33) were used for analysis of spleen cells after antibodytreatment in vivo. These antibodies were generous gifts from Dr. J.Bluestone (University of Chicago, Chicago, IL). The three antibodiesused for depletion studies (GK1.5, 2.43, and YTS 169.4) were derivedin rats and are IgG2b antibodies. This subclass of antibodies haspreviously been shown to be effective in depletion of the appropriateT-cell subset in vivo (31). The GK1.5, 2.43, and YTS 169.4 hybridomaswere grown in pristane-primed nude (NCR/Nu) mice, and the asciticfluid was used as a source of antibody. On day 0, normal C3H/HeN orBALB/cAnN mice received 0.2 ml ascites fluid i.p. On day 4, the micewere challenged s.c. with 1 x IO7 tissue culture-grown UV-induced

rÃ©gressertumor cells. An additional dose of 0.2 ml ascites was injectedi.p. on day 7. Three weeks following initial treatment, spleen cells oftreated mice were analyzed for the presence of CD8+ and CD4* cells

by staining with fluorescein isothiocyanate-conjugated CD4- or CD8-specific antibodies and FACS analysis. To rule out the possibility thatT-cells were coated with the rat-derived antibodies in vivo rather thanbeing eliminated, spleen cells were also stained with the mouse anti-ratantibody MAR-18.5.

"Cr-Release Assay. Cytotoxicity of CTL was determined in a 4-6-h"Cr-release assay, as previously described (24). The percentage of

specific lysis was calculated by the formula:

% specific lysis =(experimental release â€”spontaneous release)-100

(total release - spontaneous release)

Spontaneous release was <15% of total release.

RESULTSCD8+ T-Cells Are Required for Rejection of UV-induced Re-

gressor Tumors. UV-induced regressor tumors grow in nude

but not normal mice, illustrating the importance of T-cellimmunity for rejection of these tumors (4). The mechanism oftumor escape from this immunity may be influenced by the typeof T-cell subset(s) involved in the rejection. Therefore, we firstexamined whether CDS* T-cells and/or CD4* T-cells were

required for rejection of these highly immunogenic tumors.Mice depleted of CD4+ cells remained capable of rejecting a

challenge dose of eight different regressor tumors (Table 1).However, all of these tumors grew progressively in the majorityof mice depleted of CDS* cells. Analysis of spleen cells 3 weeks

following the initial antibody treatment in vivo showed that thedepletion was selective, since virtually no positive cells of therelevant subclass could be detected by direct immunofluorescence using appropriate antibodies and FACS analysis (Fig. 1).The spleen cells were not coated with the rat-derived anti-CD4and anti-CDS antibodies, since a mouse anti-rat immunoglobulin antibody did not stain these cells (data not shown). Finally,the anti-CD4-treated mice that rejected regressor tumors werenot only completely depleted of CD4+ T-cells detectable by

FACS analysis but were also functionally depleted of this subclass of T-cells, since these mice failed in repeated experimentsto mount an allogeneic mixed leukocyte reaction (data notshown).

Level of MHC Class I Expression Is Not a Reliable Indicatorof the Immunogenicity of a UV-induced or Spontaneous Tumor.As shown above, CDS* cells are required for rejection of UV-induced regressor tumors. Since CDS* T-cells recognize anti

gens in the context of MHC class I molecules, we investigatedwhether tumors that become progressive do so because of lossor reduction in MHC class I antigen expression. We comparedtumors that vary in immunogenicity (i.e., UV-induced regressortumors, UV-induced progresser tumors, and spontaneous tumors) for expression of MHC class I K-, D-, and L-antigens(Figs. 2 and 3). Clearly, there was no consistent correlationbetween the level of expression of antigens and the immunogenicity of the tumors. This lack of correlation was observed intumors of the H-2k as well as the H-2d haplotype. For example,some of the UV-induced regressor tumors expressed very lowamounts of MHC class I antigens, while some poorly immunogenic spontaneous tumors expressed very high levels of classI antigens. When comparing H-2k haplotype with H-2d haplotype tumors, it was apparent that the D-molecule is generallyexpressed in higher amounts in H-2d haplotype tumors, whilethe K-molecule is expressed at higher levels in H-2k haplotype

tumors; this finding is consistent with earlier studies showingdifferences in expression of D-molecules on normal cells between these two strains (34). We have no evidence that thesedifferences affect the immunogenicity of these tumors.

Since multiple characteristics other than the level of MHC

Table 1 CDS* T-cells are required for rejection of UV-induced regressor tumors

TumorUV-5117

UV-5125AUV-6107UV-6139BUV-6132AUV-6130UV-4102UV-6134ARecipient

strainBALB/c

BALB/cC3HC3HC3HC3HC3HC3HTumor

incidence inT-cell subset-

depletedmiceflcor8/8

6/64/43/32/24/43/4

2/4*CD4-0/4

0/30/40/30/20/40/40/4

a Animals received two injections of 200 n\ ascites fluid 1 week apart and werechallenged with 1 x IO7tumor cells s.c. 4 days after the first antibody injection.

Results are pooled from several experiments.h Data from two experiments; Â¡neach case, one of two mice grew tumors.

3852



UNIQUE TUMOR ANTIGENS. MHC CLASS I, AND TUMOR PROGRESSION

NORMAL ANIMAL ANTI-CD4-TREATED ANTI-CD8-TREATED

120

0!

U

oa

AUTO-FLUORESCENCE

ANTI-CD4-STAINED

ANTI-CDS-STAINED

15 20 0 10 15 20 0 10 15 20

Fluorescence Intensity(channel number x 10~1)

Fig. 1. Depletion of CD4* or CDS* T-cells in mice treated with GK1.5 (anti-CD4) or YTS169.4 (anti-CD8) antibodies. Spleen cells from representative mice wereanalyzed by cytofluorometry 3 weeks after the first of two antibody treatments, as described in "Materials and Methods." On the EPICS instrument used for thisanalysis, 85 channels equal a 10-fold increase in the fluorescence intensity. Each histogram represents analysis of 10,000 cells.

CDÃ¼

Â§2SIo >

ifsâ€¢*-â€¢

>Â£~ 05CO *â€”'"Ã–J

DC

Ariti H-2K AntibodyAnti H-2D Antibody

ik .la

<CO

n<o>

oo

IOo

LO 00o *-co coI I

3 D>3

>3

>3

(9< (9

<

UV-lnduced Regressors UV-lnduced Progressors SpontaneousProgressors

H-2k Haplotype TumorsFig. 2. Similar range in level of expression of MHC class I antigens in three groups of H-2k haplotype tumors differing widely in immunogenicity. Tumor cell

lines appear in the rÃ©gresseras well as the progresser category as parental and variant tumors. The relative amount of fluorescence was quantified by cytofluorometryusing monoclonal antibodies specific for K-molecules (11-4-1) and D-molecules (15-5-5), as described in "Materials and Methods."

0>u

, 60<Dcoo ~co'eÂ£ =40hO >,

l|20h

*- 03_03 ^â€”'

O)OC

â€¢ Anti H-2K Antibody

0 Anti H-2D Antibody

D Anti H-2L Antibody

UV-lnducedRegressors

UV-lnducedProgressors

Spon.Progr.

H-2d Haplotype Tumors

Fig. 3. Similar range in level of expression of MHC class 1 antigens in threegroups of H-2" haplotype tumors differing widely in immunogenicity. The relativeamount of fluorescence was quantified by cytofluorometry (K-molecule, antibody31-3-4; D-molecule. antibody 34-2-12; L-molecule. antibody 28-14-8), as described in "Materials and Methods." Spon. Progr., spontaneous progresser.

class I expression can affect the immunogenicity of primarytumors, a better correlation might be observed by comparingthe level of MHC class I antigen expressed by regressor tumorswith that expressed by more closely related progressor variantsderived from selection in vivo or in vitro. The frequency oftumor outgrowth of the progressor variants is shown in Table2. Fig. 4 shows that there is no consistent difference in the levelof MHC class I expression between regressor tumors and theirvariants that have acquired progressive growth behavior. Theonly exception to this general finding was tumor UV-6130 (notshown in Fig. 4), which will be discussed in detail below. Asimilar lack of correlation was observed when clones of anindividual regressor tumor randomly selected for differences inMHC class I expression in vitro were analyzed for growth invivo (Table 3). The level of class I expression of individualclones remained stable, as indicated by cytofluorometric analyses repeated over several weeks. Individual clones were injectedinto nude mice to ensure that these clones could grow in vivo,and fragments of the tumors were then transplanted into normalmice. All of the tumor fragments were equally well rejected,

3853



UNIQUE TUMOR ANTIGENS. MHC CLASS I. AND TUMOR PROGRESSION

Table 2 Growth in vivo of rÃ©gressertumors and their derived progresser variants

Designationoftumor"UV-4102-RE

UV-4102-PROUV-S117-RE

UV-5117-PRO'UV-5125A-RE

UV-5I25A-PROUV-6I05-RE

UV-6105-PROVU-6107-RE

UV-6107-PROUV-6118A-RE

UV-61ISA-PROUV-6132A-RE

UV-6132A-PROUV-6134A-RE

UV-6134A-PROUV-6139B-RE(A*B*C*)

UV-6139B-PRO1(A*B-C*)UV-6 139B-PRO2(A-B*C*)UV-6130-RE(A*B*C*)UV-61

30-VAR3(A*B*C~)UV-6 130- VAR4( A~B*C*)UV-6130-VAR5(A*B-C*)UV-6l30-VARl(A-B-C'yUV-6l30-Var2(A^B^C*)/Derived

byselection^In

vivoIn

vivoIn

vivoIn

vivoIn

vivoIn

vivoIn

vivoIn

vivoIn

vitroInvitroIn

vitroIn vitroIn vitroIn vitroIn vitroTumor

incidencerNormal

mice1/10(10)

9/9(100)0/10(0)

7/7(100)0/6

(0)5/5(100)0/6

(0)3/3(100)0/6

(0)2/3(67)0/6

(0)3/3(100)1/9(11)

9/13(69)0/9

(0)7/9(77)0/6

(0)5/8 (63)7/1 1(64)0/9

(0)0/3 (0)1/6(17)1/8(13)4/8 (50)4/6 (67)Nude

mice1/1

(100)ND*1/1

(100)ND2/2

(100)ND2/2(100)

1/1(100)2/2(100)

1/1(100)2/2(100)

1/1(100)3/3(100)

ND3/3(100)

1/1(100)2/2(100)

NDND4/4

(100)1/1 (100)

NDND

1/1 (100)ND

" RE, rÃ©gresser;PRO, progressor; VAR, variant.* See "Materials and Methods."c Number of mice with progressively growing tumors per number challenged

(percentage in parentheses). Mice were challenged s.c. either with five 1-mm'tumor fragments or with 1 x IO7tissue culture cells.

d ND, not done.' All in vivo selected progressor variants except for UV-5117 were derived by

selection in normal mice (see "Materials and Methods"). The UV-5117 progressorvariant was derived by passage of the rÃ©gresserparental tumor through a UV-irradiated recipient. This method has previously been found to be effective forderivation of progressor variants (22). Progressively growing tumors were reiso-lated from the UV-irradiated mice and transplanted into normal mice to confirmthe progressor phenotype.

^UV-6130-VARl and -VAR2 variants were derived from independent selections using anti-A and anti-B CTL clones.

regardless of whether the tumors were derived from clonesexpressing high or low amounts of MHC class I antigens invitro. Thus, even though the clones were probably closely relatedexcept for expression of MHC class I in vitro, these differencesdid not correlate with regressive or progressive growth behaviorin normal mice.

Level of Expression of K-, D-, or L-Molecules Can Be Equally

Well Stimulated in Regressors, Progressors, and SpontaneousTumors. Although there was no consistent correlation betweenthe level of MHC class I expression and tumor immunogenicity.

a significant correlation might exist between immunogenicityand the level of MHC class I expression induced by IFN-7 (35)or other similarly acting lymphokines secreted by host cellssurrounding or infiltrating a tumor. Therefore, the level ofexpression of MHC class I antigens observed in a growingtumor should be a function not only of the constitutive abilityof the tumor to express a certain level of MHC class I antigensbut also of its ability to attract, activate, and respond to lym-phokine-producmg cells. These different but not mutually exclusive mechanisms cannot be distinguished by analysis in vivo,but we could determine whether poorly immunogenic tumorswere less effective than highly immunogenic tumors in responding to IFN-7 in vitro. Fig. 5 shows that there is no correlationbetween the immunogenicity of a tumor and increased levels ofK-, D-, or L-antigens after stimulation with IFN-7. In fact, twoof the more poorly immunogenic spontaneous tumors, AG-106and AG-104A, were most effectively stimulated to express K-molecules. Furthermore, as shown in Fig. 6, variant tumor cellsthat have acquired progressive growth behavior continue torespond with increased MHC class I expression after stimulation with IFN-7. These results were similar regardless ofwhether the progressor variants were derived by selection invivo or in vitro.

Progressor Variants Can Lose or Retain Sensitivity to Tumor-specific Cytolytic T-Cell Clones. Progressor variants derivedfrom highly immunogenic regressor tumors do not lose theexpression of MHC class I antigens or the ability to respond toIFN-7; however, loss of a tumor-specific antigen rather thanMHC class I antigen expression could be an effective mechanism for escape. This would be manifest by retention of MHCclass I antigen expression despite increased or complete resistance to tumor-specific cytolytic T-cells. Five regressor tumorsand six progressor variants derived by selection in vivo or invitro were tested with CTL clones generated against the regressor tumors. Three of the six progressor variants, UV-5117-PRO (derived by in vivo selection), UV-6193B-PRO1(A-B+C+), and UV-6139-PRO2 (A+B~C+) (both derived by in

vitro selection) were resistant to lysis (Fig. 7, A-C) by therelevant CTL clone, while the remaining three variants, UV-6132A-PRO, UV-4102-PRO, and UV-6134A-PRO (Fig. 7, />-F) (all derived by selection in vivo), were killed by the relevantCTL clones. The CTL clones used in Fig. 7, A, B, C, and E,were specific for the relevant tumors, in that they did not killautologous normal control cells. (Anti-UV-6134A and anti-UV-4102 CTL clones were not tested in this manner.) Theantigen-loss variants obtained by T-cell selection in vitro retained other antigens that were recognized by additional CTLclones (Fig. 7, B and C); however, we were unable to generateCTL responses to the antigen-loss variant UV-5117-PRO derived in vivo (data not shown). All six progressor variantsmaintained significant levels of MHC class I expression (shown

Fig. 4. Comparison of MHC class I antigenexpression by UV-induced regressor tumorcells and progressor tumor variants selected invitro or in vivo. No consistent change in thelevel of expression of these antigens is apparent when tumors acquire the progressive phenotype. H-2k haplotype tumors originatedfrom C3H mice, which do not express the L-antigen. The relative amount of fluorescencewas quantified by cytofiuorometry, as described in "Materials and Methods."

100

lÃ¯60ESÂ«

ilRE PRO RE PRO RE PRO RE PRO RE PRO RE PRO RE PRO RE PRO

H-2k Tumors H-2" Tumors

3854



UNIQUE TUMOR ANTIGENS, MHC CLASS I. AND TUMOR PROGRESSION

Table 3 Clones derived from the same tumor but varying in MHC class iexpression are equally well rejected by the T-cell competent host

Cell lineSublineUV-5117-RE'ParentalClone

234567810UV-4I02-RE"

ParentalClone

1235678Class

Iexpression"7.71.614.01.72.17.810.613.011.07.02.86.515.05.92.04.32.4Tumor

growth*Nude1/11/11/1ND"1/1NDI/I1/1ND1/1N

DND1/1NDNDN

Dl/lNormal0/90/30/3N

D0/3ND0/30/3ND0/10NDN

D0/5NDNDND0/5

" Measured by FACS analysis using the anti-Ld monoclonal antibody 28-14-8(H-2a haplotype) or the anti-K" monoclonal antibody 11-4-1 (H-2k haplotype), asdescribed in "Materials and Methods."

* Animals were challenged with three 1-mm3 solid tumor fragments implanted

s.c. with a trocar and were observed for at least 8 weeks after tumor challenge.e UV-5117 is of BALB/cAnN origin (H-2d haplotype).d ND, not done.' UV-4102 is of C3H/HeN origin (H-2" haplotype).

â€¢Anli H-2K AntibodiesO Ann H-2D AntibodiesD Anti H-2L Antibodies

SPONTANEOUSPROGRESSOR

AG-106 AG-104A

TumorFig. 5. Comparison of the inducibility of MHC class I antigen expression on

tumor cell lines derived from primary UV-induced regressor tumors, primaryUV-induced progressor tumors, and spontaneous tumors following IFN-7 treatment. Tumor cells were either untreated (â€”) or exposed to 30 units/ml IFN-7 for48 h prior to staining (+). The relative amount of fluorescence was quantified bycytofluorometry, as described in "Materials and Methods." (Note difference in

scale used for the spontaneous progressor tumors.)

for five of these progressor tumors in Fig. 4).Certain CTL-selected Progressor Variants of UV-6130 RE

Selectively Lose the K Molecule. The UV-induced regressortumor 6130 represents an exceptional case, compared to theother tumors analyzed. We were unable to derive any progressorvariants of this tumor by selection in vivo; however, afterselection in vitro for loss of CTL-recognized antigens A and B,progressive growth was observed (Table 2). Fig. 8 shows thatvariants could be selected that were completely resistant toeither anti-A CTL or anti-B CTL or both, and all three of thesevariants grew progressively in normal mice. Variants that wereresistant to anti-C CTL, however, retained the regressor phe-notype (Fig. 8, Table 2). We then compared the MHC class I

Ann H-2K Antibody

Anti H-2D Antibody

l .1 11Parental In Vivo

RÃ©gresserSelectedProgresser

Parental In VivoRegressor Selected

ProgresserUV-6152 B

Parental In VitroRegressor Selected

ProgressorUV-6139BUV-6132 A

Fig. 6. Comparison of the inducibility of MHC class I antigen expression ofUV-induced regressor tumor cells and their progressor variants in response toIFN-7 treatment. Tumor cells were either untreated (â€”)or exposed to 30 unitsof IFN-7/ml of culture medium for 48 h prior to staining (+). The relative amountof fluorescence was quantified by cytofluorometry. as described in "Materials andMethods."

LOSS OF ANTIGEN RETENTION OF ANTIGEN

V)

05O)l_

CO

oCO

40

0

40

20

0

40

20

0

ANTI-UV-5117 CLONE

A

.ANTI-UV-6139B CLONE

(Anti-A)

BUV-6139B (A*B*C*).)

Regressor

ANTI-UV-6139B CLONE(Anti-B)

CUV-6139B(A*B*C*)

Regressor

6139_ Autologous

Normst.

UV-6139B -(A'B'C*)

Progressor _

40

20

0

40

20

20

10

ANTI-UV-6134A CLONE

D

ANTI-UV-6132A CLONE

ANTI-UV-4102 CLONE

UV-4I02Regresso

-4102Progressor

UV-6107Regressoroh i-r-

0.6:1 2.5:1 10:1 0.3:1 1.25:1 5:1Effector-to-Target Ratio

Fig. 7. Analysis of progressor variants of UV-induced regressor tumors withCTL clones. Three variants, one derived in viro and two derived in vitro, loseexpression of a unique CTL-defined antigen (A, B. and C) but three other variants,all derived by host selection in vivo, retain the antigen recognized by the CTLclones (D. E. and F). Variants were derived from highly immunogenic regressortumors following selection in vivo (A, D, E. and F) or in vitro (B and C), asdescribed in Table 1 and "Materials and Methods." Lysis of the targets wasanalyzed in a 4.5-h "Cr-release assay, as described in "Materials and Methods."

expression of the CTL-selected variants with that of the parental regressor tumor and found that the K-antigen was selectivelylost in variants that were resistant to both the anti-A and anti-B CTL but not in variants that were sensitive to either one ofthe two CTL clones. (Fig. 9). Furthermore, this A~B~C+ variant

could not be induced to express K-antigens by treatment withIFN-7 (Fig. 9). Variants selected for resistance to anti-C CTL

did not lose K expression (Figs. 8 and 9).3855



UNIQUE TUMOR ANTIGENS. MHC CLASS 1, AND TUMOR PROGRESSION

ANTI-A CTL CLONE ANTI-B CTL CLONE ANTI-C CTL CLONE

606130 and Variants

AÂ»BÂ»CÂ»(PAR>A-B-CÂ»(VAR1>A-B-CÂ»(VAR2>

B+C-(VAR3)A A-BÂ»C*(VAR4)

B-C*(VAR5)

2.5:1 10:1 0.15:1 06:1 2.5:1 10:1 0.15:1 0.6:1 25:1 10:1Effector-to-Target Ratio

Fig. 8. Characterization of the antigenic phenotypes of variants selected withCTL clones from the UV-induced rÃ©gressertumor 6130. Selection pattern clearlyshows the independence of the antigen-loss variants. Growth of these tumorvariants in viro is shown in Table 2. Each panel represents an independentexperiment.

DISCUSSION

In this study, we have investigated whether reduction in MHCclass I expression, loss of tumor antigens, or other unidentifiedmechanisms are commonly involved in tumor escape fromimmune destruction. We analyzed a large number of experimentally induced and spontaneous tumors differing greatly inimmunogenicity. We also compared progressor variants thatwe derived by immunoselection in vivo or in vitro with therelevant parental rÃ©gressertumors. For two different mousestrains, no consistent correlation was found between growth ofthe primary tumor in the normal immunocompetent host andthe level of MHC class I expression by the tumor. In fact, someof the poorly immunogenic spontaneous tumors expressed thehighest levels of MHC class I antigens. Also, no consistent lossor change in the expression of MHC class I antigens occurredwhen progressor variants of UV-induced rÃ©gressertumors escaped immune surveillance. Of the 24 progressor tumors analyzed, loss of MHC class I antigens was observed in only 1tumor. In this tumor, UV-6130, we observed loss of K expression after successive selection in vitro to obtain the 6130 A~B~

variant. We are not certain whether this tumor is exceptionalor whether loss of K also occurs when variants of other tumorsare selected for loss of multiple independent tumor antigens.But it is of interest that a variant of another tumor, UV-6139BRE, which also had lost two independent CTL-recognizedantigens, retained expression of both K- and D-antigens (datanot shown), suggesting that the loss of K expression by the UV-6130 A~B~C* variant may be unusual. Conceivably, progressors

may escape rejection because of failure to up-regulate MHCclass I expression in response to the cytokines released bytumor-infiltrating lymphocytes in situ but quantitation ofchanges in levels of MHC expression is difficult to accomplishin vivo. There were, however, no consistent differences in thelevels to which MHC class I expression could be up-regulatedin vitro by IFN-7.

Loss of expression of MHC class I antigens has previouslybeen reported in a few nonviral tumors that grew progressively

when the original (primary) tumor was transplanted into normalimmunocompetent mice; e.g., sarcoma T10, a methylcholan-threne-induced tumor that lacks expression of Kk- and Kb-

antigens, acquires increased immunogenicity after transfectionand expression of these genes (9). It is possible that (similar toUV-induced tumors) loss of MHC class I molecules is notcommonly observed in MCA-induced tumors, since no consistent changes in MHC class I expression that correlated withtumor immunogenicity were observed in other MCA-inducedsarcomas (36). However, MCA-induced tumors may be a lesspertinent model for studying changes in MHC class I expressionduring tumor escape because (a) there is no evidence that theCD8+ T-cell subset is commonly required for rejection of MCA-

induced tumors or that CTL-recognized antigens are the targetsfor rejection for most of these tumors and (b) the immunogenicity of these tumors is not as strong as that of UV-inducedregressor tumors but is more similar to that of certain UV-induced progressor tumors. MCA-induced tumors, like UV-induced progressor tumors, usually require previous immunization for rejection. Loss of MHC class I antigens has alsobeen observed in some virally induced cancers. Since expressionof certain virally encoded genes can be essential for the maintenance of the malignant phenotype (37), failure to observemalignant cells that lose a viral antigen encoded by such a gene

BACKGROUND ANTI-H-2K

FLUORESCENCE ANTIBODY

ANTI-H-2D

ANTIBODY IFN y

6130 PARA+B+C+

6130VAR4A-B-1-C +

6130VAR5A+B-C+

5 10 15 20

Fluorescence Intensity(channel number x 10"')

Fig. 9. Selective loss of expression of the H-2Kk molecule by heritable tumorvariants of the UV-induced regressor tumor 6130 selected in vitro with CTLclones for loss of the A and B antigen. The loss of H-2 KLexpression cannot beovercome by treatment with IFN-->. while the expression of H-2Dk is increased.The relative amount of fluorescence was quantified by cytofluorometrv usingmonoclonal antibodies for K (16-3-22) and D (15-5-5). as described in "Materialsand Methods." On the EPICS instrument used for this analysis, 85 channelsequal a 10-fold increase in fluorescence intensity. Each histogram represents theanalysis of 10.000 cells.

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UNIQUE TUMOR ANTIGENS, MHC CLASS I, AND TUMOR PROGRESSION

is expected. These virally transformed cells may, therefore, havebeen immunologically selected for reduction of MHC class Iexpression (5).

Resistance to the uniquely specific CTL clone is then bestexplained by loss of the unique CTL-recognized target antigenin all progresser variants in which MHC class I expression wasretained. Thus, for some of the tumors we studied, selection forloss of expression of tumor antigens appears to be an importantmechanism whereby tumors escape immune destruction in thenormal host. Such antigen loss was observed in three variants,UV-5117-PRO, UV-6139B (A~B*C*)-PRO1, and UV-6139B(A*B~C*)-PRO2, the first selected in vivo and the latter two in

vivo. The finding of antigen-loss variants in the present studyagrees with previous results showing that the UV-induced re-gressor tumor 1591 -RE loses certain CTL-defmed antigens (22,23, 38), while retaining the expression of normal MHC class Imolecules, before growing progressively in normal mice (39).Although tumor escape due to loss of a CTL-defined antigenhas been shown previously by us and by other investigators (22,39-41), these earlier studies lacked autologous normal controlcells. Several recent studies by us and others have demonstratedin vitro selection of antigen-loss variants with CTL clones thatfail to kill autologous normal cells, but none of these earlierstudies explored the effect of this antigen loss in vivo. This isimportant because loss of a unique antigen will not necessarilylead to a progresser tumor (this paper and Ref. 23).

For every UV-induced regressor tumor we have analyzed sofar, CDS* T-cells appeared to be required for tumor rejection,i.e., if CDS* T-cell immunity was eliminated, then the regressor

tumors grew progressively. However, the converse may not betrue, i.e., some tumors may grow progressively in the presenceof CDS* T-cell immunity or tumor cells may retain sensitivityto CDS* T-cells but still grow progressively for other reasons.

This is important when considering our finding that three offour escape variants derived from different UV-induced regres

sor tumors and selected in vivo by the normal host retainedCTL-recognized antigens as well as MHC class I antigens butfailed to be rejected by the immunocompetent host. We havenot formally proven that these CTL-recognized antigens arethe targets for rejection in vivo that is mediated by CDS* T-cells, and possibly we have failed to detect the relevant CTL-recognized rejection antigens that determine the regressor phe-notype and are lost during tumor progression. A second possibility is that other changes not directly related to MHC class Ior tumor antigen expression may have allowed these variantsto escape CDS* T-cell-mediated immunity; for example, the

progressor variants may produce immunosuppressive substances and/or fail to stimulate CDS* immunity in vivo (42).Finally, other mechanisms in addition to CDS* cell immunity

may be required for tumor rejection, e.g., tumoricidal macrophages, natural killer cells, or cytokines produced by immunecells. In any event, these host-selected variant progressor tumors that show neither loss of MHC class I nor loss of uniquetumor antigen expression may be helpful for discovering otherimportant mechanisms(s) of tumor surveillance.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Jeffrey Bluestone for criticalreview of the manuscript and Patricia Barnes for secretarial assistance.

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1990;50:3851-3858. Cancer Res Patricia L. Ward, Hartmut K. Koeppen, Teresa Hurteau, et al. T-Cell-dependent Surveillance

+Expression by Murine Tumors That Escaped from CD8Major Histocompatibility Complex Class I and Unique Antigen

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