modulation of natural killer cell activity by serum from ...prior treatment of pbl with normal...

(CANCER RESEARCH 48. 2596-2603, May I. 1988)

Modulation of Natural Killer Cell Activity by Serum from Cancer Patients:

Preliminary Results of a Study of Patients with Colorectal Adenocarcinomaor Other Types of Cancer1

Madalina Pislarasu, A. Oproiu, Doina Taranu, R. B. Herberman,2 and A. Sulica

Department of Immunology, Babes Institute [M. P., A. S.J, and Clinic of Gastroenterologe, Fundeni Hospital [A. O., D. T.J, Bucharest, Romania, and Pittsburgh CancerInstitute and Departments of Medicine and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213-2592 [R. B. H.J

ABSTRACT

As previously reported for natural killer (NK) cells of normal individuals, prior incubation of peripheral blood lymphocytes from cancerpatients with human normal serum or monomeric immunoglobulin Greduced their subsequent capacity to kill K562 target cells in a 4-h 5lCr

release assay. The NK activity of such treated effector cells was significantly inhibited only by 58% of sera from patients with colorectaladenocarcinoma (21 of 36 cases) and by 67% of sera from patients withother lymphoid or nonlymphoid solid tumors (22 of 33 cases). Thecytotoxic activity of cells previously incubated with eight noninhibitorysera was even augmented relative to medium-treated peripheral bloodlymphocytes (control). The 26 untreated cancer sera which did not inhibitsignificantly the NK activity (/ ) always developed significant inhibitorycapacity upon heating at 56Â°Cfor 30 min (A*). An additional seven (21%)

patients with colorectal carcinoma and four (27%) patients with othercancers were identified as having type II NK regulation, defined as serawith untreated inhibitory capacity (/*) but with appreciably more inhibition after heating (A*). The sera of the last group of patients with

colorectal adenocarcinoma (14 of 36 cases) defined as having type IIINK regulation were not different from control sera isolated from normalindividuals (/*A~) except that they induced an inhibition greater than

that caused by normal sera.The modulatory characteristics of sera from the first two categories of

patients appear to be cancer associated, since the patterns /A* or /*A +

were observed with sera from only one of 30 patients with benign digestivediseases and two of 100 apparently healthy individuals. Preliminaryresults of longitudinal investigations of patients with colorectal adenocarcinoma revealed also that these patterns disappeared several monthsafter resection of their tumor in all five tested patients, whereas the typeHI NK regulation found in patients with poor prognostic factors wasunchanged after surgery in the other five of six patients. The threedifferent categories of cancer sera identified by the functional assay ofNK regulation indicated differences among our group of patients whichwere not paralleled by differences in levels of cytotoxic reactivity of theirNK cells assayed in vitro in the absence of autologous serum. Theabnormal behavior of sera from 37 (54%) patients with different types ofmalignant disease suggests the presence of a thermolabile serum factorwhich can either stimulate the activity of NK cells isolated from bothcancer patients and normal donors, or protect them against the seruminhibitory factor which, in fact, are in higher amounts or have higherbinding affinities as compared to those in sera of healthy donors. Sincethe type I of NK regulation appears to be correlated with both early stageof malignancy and a better clinical course postoperative!}', the results ofthis study suggest that the in vivoeffect of cancer patients' sera containing

the putative stimulatory or protective factor may contribute to moreefficient function of their circulating NK cells.

Received 4/23/87; revised 1/7/88; accepted 1/20/88.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.

1This work was performed under the US-Romania Agreement on Cancer

Research.2To whom requests for reprints should be addressed.

INTRODUCTION

NK3 cells are a class of nonadherent, nonphagocytic, rapidly

cytotoxic LGL which can efficiently lyse a wide variety of tumorcells, virally infected cells, and immature cell types of normalorigin (1, 2). Besides their cytolytic function, NK cells cansecrete cytokines (3, 4) and appear to participate in the controlof cell differentiation, in particular of hematopoietic precursorsand thymocytes (5, 6). Their contribution to antitumor resistance has been indicated mainly by the considerable evidenceregarding the ability of NK cells to eliminate metastatic cellsand thereby resist tumor spread (7-9). Nevertheless, knowledgeof the potential role of these cells in the control of humanneoplasia is largely limited to observations that somewhat depressed NK activity is usually found in the peripheral blood ofpatients bearing advanced solid tumors (10,11). Such decreasedfunctional NK levels were not attributable to a decreased number of circulating NK cells, and additional regulatory factorsappear to be involved in the determination of the cytotoxicactivity of the cells isolated from cancer patients.

The basis for the physiological regulation of NK activity isalmost unknown in spite of the large number of studies dealingwith the identification and characterization of humoral andcellular factors acting on NK cells and modulating their killingfunction as assessed mainly by in vitro experimentation (12).Such soluble factors regulating positively or negatively the NKcells are interferon (13), interleukin 2 (14), and prostaglandinE2(15).

We have previously demonstrated (16, 17) that mlgG isolatedfrom human serum can act as an additional negative regulatoryfactor of NK activity. Prior treatment of PBL with normalautologous serum induced inhibition of NK activity which couldbe reduced by selective depletion of IgG from the serum. Morerecent data indicated that the mlgG-induced reduction of thekilling capacity of NK cells was caused by the inhibition of apostbinding event, mediated at least partially by cyclic AMP(18) and triggered by a direct interaction between mlgG andLGL (Ref. 19; Footnote 4) through the Cy3 domain of IgGand the Fc receptors of NK cells (20).

In order to evaluate the possible mechanisms underlying thelow NK reactivity in cancer patients, in the present study wehave analyzed the regulatory effects of untreated or heat-treatedserum isolated from tumor-bearing individuals on autologousPBL and on cells harvested from healthy donors. The resultsof this study indicate that either of two patterns of serum-induced regulation of NK activity is present in malignant dis-

3The abbreviations used are: NK, natural killer, LGL, large granular lymphocytes; mlgG, monomeric IgG; PBL. peripheral blood lymphocytes; CEA, carci-noembryonic antigen; TNM, tumor-nodes-metastases; PCS, fetal calf serum;CCM, complete culture medium; LU, lytic units.

4A. Sulica, A. C. Bancu, C. Galatiuc, M. Manciulea, and R. B. Herberman.

Regulation of human natural cytotoxicity by IgG. IV. Association between directbinding or monomeric IgG to the Fc receptors of large granular lymphocytes andinhibition of the cytotoxic function of natural killer cells, manuscript in preparation.

2596

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MODULATION OF NK CELL ACTIVITY BY SERUM FROM CANCER PATIENTS

ease. The modulatory characteristics of serum from 63% of Serum-induced inhibition of NK activity was arbitrarily dotinoli as

cases with colorectal adenocarcinoma appear to be cancer associated, since this pattern has practically not been observedwith serum from normal individuals or patients with benigndigestive diseases and seems to disappear after tumor resection.

MATERIALS AND METHODS

Populations Studied. The patients studied consisted of a first groupof 38 adults with a variety of lymphoreticular or solid malignant tumors(4 Hodgkin's or non-Hodgkin's lymphoma, 4 breast, 2 skin, 1 thyroid,

6 lung, 12 gastrointestinal, and 9 genitourinary carcinomas) and asecond group of 31 cases with colorectal carcinoma. The immunologicalinvestigations (see below) were performed on hospitalized patientsusually as soon as their clinical diagnosis was established and alwaysbefore any antitumor therapy, except one patient with rectal cancerwho was irradiated 2 yr before. In the case of patients with colorectalcarcinoma, all diagnoses were histopathologically confirmed on biopsyspecimens obtained by colonoscopy. Patients in the second group hadroutine physical and laboratory examinations, including scinti- and

echography and determination of serum CEA and ferritin. The Dukesand TNM staging, available only in surgically treated cases, weredetermined at certain periods of time after immunological investigations (3 to 160 days). Before surgical treatment, 9 patients had undergone radiotherapy. Some of the patients with colorectal cancer wereagain clinically examined and immunologically tested at various timesfollowing surgical resection of their tumor. Control subjects consistedof 30 patients with benign digestive diseases (8 chronic hepatitis, 3chronic pancreatitis, 3 Crohn's disease, 3 multiple colon polyposus, 9

rectocolitis, and 4 rectal polyposus). The 100 apparently healthy individuals used as controls in our experiments were blood donors at theHaematology Center, Bucharest, Romania.

Preparation of Lymphocytes. PBL were isolated from heparinizedblood by sedimentation in Sepcel (Comasim, Bucharest, Romania),washed in K '-65 solution (Cantacuzino Institute, Bucharest, Romania),

and resuspend in RPMI-1640 medium (Flow Laboratory, McLean,VA) supplemented with 10% PCS (Biofluids, Inc., Rockville, MD), 20mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid buffer, 1% glu-tamine, and antibiotics used as (CM. For washing the cells, mediumwith only 2% FCS was used.

Treatment of Human Serum. On the day of the experiments, analiquot of the serum from patients with malignant or benign diseasesor from normal donors was inactivated at 56Â°Cfor 30 min. Aliquots ofsome unheated sera from cancer patients were kept frozen at â€”15Â°C

and used after several weeks or months when additional determinationswere performed with PBL from normal donors (see below).

Treatment of PBL. Lymphocytes (2 x 10' cells/ml) were incubatedin Petri dishes for 2 h at 37Â°Cin CCM, in the presence or absence of

20% untreated or heat-inactivated human serum. The medium-treatedcells were used as control. In most of the experiments, aliquots of cellsuspension were similarly exposed to 20% serum isolated from a normaldonor and, in some experiments, the cells were treated with 0.5 mg ofmlgG isolated, as previously reponed, from normal human sera (16).These different types of serum were usually added also to PBL fromthe individual used as the donor of normal serum, and therefore theeffects of autologous and homologous sera on both cell preparationswere compared under the same technical conditions.

Following incubation, the nonadherent lymphocytes were harvested,washed twice in culture medium with 2% FCS, and finally resuspendedin CCM.

NK Assay. A 4-h 51Crrelease assay was performed in round-bottomedmicrotiter plates as described (16). Four 3-fold dilutions of treatedlymphocytes (0.1 ml) were mixed with equal volumes of "Cr labeledK562 targets (3 to 4 x IO3cells). LU at 30% specific "Cr release were

calculated from the curves obtained at different effector/target cellratios. The value of LU/107 cells of nonadherent PBL previouslyincubated at 37"C in medium alone was considered as the control forNK activity of each cell preparation, and the value of different serum-treated effector cells was expressed as the percentage of the control.

levels at least 20% below the control levels. Differences in the range ofâ€”20to +20% between the inhibition determined by each tested serumbefore and after its treatment at 56Â°Cwere considered nonsignificant,

and therefore the heat-induced difference (A) of the regulatory capacity

of a certain serum was considered as significant when it was equal toor higher than 20%. This value of Â±20%was selected as the cut-offsince it is the limit of the mean Â±2SD calculated for all control seraused in our present study.

Statistical Calculation. The data were analyzed by using a paired ttest.

RESULTS

Investigation on Patients with Various Types of Cancer. Addition of 20% normal serum or 0.5 mg of monomeric IgG toPBL harvested from 19 patients with various types of malignantdisease caused in all experiments significant inhibition of NKactivity (at least 20%), whereas prior incubation of the PBL inthe presence of 20% patients' serum produced a significant

reduction of the NK activity in only 10 of the 19 cases (Table1). In addition, it was noted that the cytotoxic activity of cellsincubated with serum of patients with either Hodgkin's disease

(2 cases), breast cancer (1 case), or colon cancer (2 cases) waseven higher than that of medium-treated PBL (control). Theinability of these cancer sera to inhibit NK activity of PBL wasseen when sera were tested at concentrations of 5 to 20% (datanot shown).

The effect of cancer patients' serum on NK activity, expressed

as the percentage of the control (PBL preincubated in mediumalone), of 19 additional cancer patients is summarized in Table2. In addition, it was found that all 6 noninhibitory sera (i.e.,O. V., M. A., D. N., M. S., V. E., M. C.) acquired the capabilityto down-regulate the cytotoxic reactivity of their NK cells uponheating at 56Â°Cfor 30 min. Such heat-induced differences (A)

between the percentage of NK activity of PBL preincubatedwith untreated autologous serum and that obtained with heatedserum, defined as significant when higher than 20%, were

Table 1 NK activity of PBL isolated from cancer patients when incubated in vitrowith autologous or normal homologous serum or with human monomeric IgG

prior to the cytotoxic assayPBL (2 x 10' cells/ml) from patients with various types of cancer were

incubated in Petri dishes for 2 h at 37Â°Cin the presence of 20% autologous or

normal serum or 0.5 mg of human monomeric IgG or medium alone (control).The nonadherent cells were harvested, extensively washed with cold medium, andused as effector cells in the NK assay.

NK activity (LU/107 cells) of PBL when

treated with

Patient Clinical diagnosis

Patient's Normal Monomeric

Medium serum serum IgG

C.D.M.A.M.CS.

E.D.M.C.I.S.

N.V.R.M.D.C.I.Z.V.C.V.C.

R.P.V.A.P.Z.A.T.P.G.H.R.

M.Hodgkin's

diseaseHodgkin'sdiseaseNon-Hodgkin'slymphomaBreast

cancerBreastcancerThyroidcancerBronchialcarcinomaBronchialcarcinomaBronchialcarcinomaBronchialcarcinomaLivercancerLivercancerAbdominaltumorColoncarcinomaColoncarcinomaBladdercancerBladdercancerBladdercancerProstatic

carcinoma100.0142.81818.2153.828.527.3333.3II

lI.I231.5431.067.228.546.020.8116.2166.6250.031.2100.0280.9185.2689.6210.52.87.3185.2775.218.5378.812.011.428.525.0126.7156.2147.026.892.547.2ND909.0ND13.614.8185.2303.02.7138.835.61.420.44.148.0178.5156.20.122.7ND"94.31000.0121.21.42.3170.9476.2111.0376.62.35.74.012.598.8138.9NDND64.0

" ND, not determined.

2597



MODULATION OF NIC CELL ACTIVITY BY SERUM FROM CANCER PATIENTS

Table 2 Effect of inactivation at 56'Cfor 30 min on the capacity of serum from

cancer patients to regulate the NK activity ofPBL isolated from either patients ornormal donors

Donor of

NK activity (% of the control)"

of PBL incubated with patientserum

PatientM.

E.S.

M.0.

V.I.V.D.

F.M.

A.M.I.I.E.D.M.M.S.Tumor

locationNon-Hodgkin's

lymphomaSkinSkinBreastBreastLungLungColonColonRectumPBL

used inNKassayPatientNormalPatient

NormalPatientNormalPatient

NormalPatient

NormalPatientPatientPatientPatient

NormalPatient

NormalUntreated57.7

75.010.0

40.0157.0

125.73.418.017.4

19.2132.664.927.8109.0

100.0117.0

129.0Heat

inactivated26.4

42.81.6

38.063.0

104.711.4

5.48.7

10.072.540.641.64.0

59.047.8

67.8A*31.332.28.4

2.095.0

21.0-8.0

12.68.7

9.260.124.3-13.8105.0

41.061.2

61.2

V.E.T.

E.N.

A.S.

M.O.I.M.

N.M.

C.P.E.P.

M.AbdomenAbdomenLiverLiverKidneyBladderBladderBladderOvaryPatientPatient

NormalPatient

NormalPatient

NormalPatient

NormalPatient

NormalPatient

NormalPatient

NormalPatient113.062.8

40.577.0

75.035.2

72.430.6

14.448.4

5.6114.6

38.575.0

91.061.076.051.0

53.071.4

45.643.5

82.053.5

53.951.0

6.553.5

10.073.5

86.019.337.011.8

-12.55.629.3-8.3

-7.6-22.0

-19.5-2.5

-0.981.2

28.51.5

5.041.7

" In each experiment, the NK activity calculated as LU/107 cells for medium-

treated PBL (control) obtained from either patient or normal donor was considered 100%.

* A, differences between the percentage of inhibition of NK activity of PBL byuntreated serum and that by heat-treated serum.

observed not only in these 6 cases but also with 3 additionalsera in which the inhibitory effects by untreated serum wereaugmented by heat treatment. In 14 experiments, PBL isolatedfrom healthy donors were used in the NK assays in parallel tocells obtained from cancer patients. In all but two of theseparallel experiments, the cancer sera tested as unheated orheated behaved in a similar fashion on both patient and normalPBL.

In contrast to the effects of heating on the inhibitory activityof sera from cancer patients, results of experiments performedwith 31 sera obtained from normal donors and tested on au-tologous PBL and, additionally in 9 experiments, on PBLisolated from cancer patients, indicated that the NK activitywas consistently inhibited by all but three of these untreatednormal sera. Except for one noninhibitory normal serum and

2598

one inhibitory normal serum, treatment at 56Â°Cdidn't signifi

cantly modify the percentage of inhibition caused by normalsera, since the value of the heat-induced difference was in therange of -20 to +20%, even with 2 noninhibitory untreatedsera.

The behavior of some sera from patients with different typesof malignant disease suggested the presence of a thermolabileserum factor which could either stimulate the activity of NKeffector cells isolated from both normal donors and cancerpatients or protect them against the serum inhibitory effects.Thus, it seems that removal by heat inactivation of the stimulator or protective factor(s) which might be present in thesecancer patients, but not in normal individuals, reveals thepresence of the inhibitory factors in cancer sera. In fact, theinhibitory factors in some cancer patients' sera were found to

be in even higher amounts or to have higher binding affinitiesas compared to those in sera of healthy donors. Thus, the meanÂ±SEM of the percentage of inhibition of NK activity in a totalof 85 experiments performed with different normal or cancerPBL, each of them being treated under the same experimentalconditions, was significantly greater (P < 0.001) after preincu-bation with heated serum from cancer patients (72.0 Â±2.3) ascompared to the percentage of inhibition induced by normalserum (53.4 Â±3.0) whose regulatory effect of NK activity wasrather unchanged upon heating.

Investigations on Patients with Colorectal Adenocarcinoma. Inour initial studies, unheated sera from 4 of 5 patients withcolon or rectal tumors had a noninhibitory effect on the NKactivity of PBL (Tables 1 and 2). Therefore, it was of interestto study a large group of untreated (by either surgery, radio-,or chemotherapy) patients with colorectal adenocarcinoma indifferent stages of disease. Some relevant clinical, histolÃ³gica!,and laboratory data on this group of 31 patients are shown inTable 3. The blood specimens for immunological investigationswere obtained as soon as the clinical diagnosis was established,and therefore there are some patients in which the diagnosis ofmaligancy was not followed for some reasons by surgical treatment, and consequently the stage could not be determined bythe criteria shown in Table 3.

The pattern observed in 4 of 5 colorectal cancer patients andin 46% of all cancer sera tested in our initial studies (Tables 1and 2), namely, the failure of unheated serum to inhibit NKactivity and the detection of inhibitory capacity after heattreatment, was looked for in the sera of the patients listed inTable 3. Untreated and heat-treated sera at a final concentrationof 20% were usually incubated in the same or in separateexperiments with PBL from both patient and normal donors,and as above, the NK activities were determined and expressedas the percentage of medium-treated cells.

Table 4 shows that the sera of 11 of the patients (36%) hadthe following characteristics, which have been defined as type INK regulation: (a) failure of untreated serum to reduce significantly the NK activity of at least one effector cell population;(b) inhibitory effects on NK activity after heating at 56Â°C,with

heat-induced differences generally higher than 50%. In addition, the majority of treated sera from this first group of patientsexpressed an inhibitory capacity equal to or greater than thatof a normal serum added to the same effector cell population,either before or after its heat treatment. These results suggestedthat the untreated type I serum contained either a heat-labilefactor interfering with NK inhibitory activity or a rather highamount of an NK stimulatory factor able to counteract or maskalmost completely the effect of inhibitory factors.

A second group of 7 patients (23%) was identified as having




Table 3 Clinical, histological, and laboratory data regarding the group of patients with colorectal adenocarcinoma

Case12345678910111213141516171819202122232425262728293031Age76685151696256505530596666515240584630327660627071416438547375SexMMFMFMMMMFMMMMFFMFMFMFMMMFMFMFFTumorlocationColonColonColonRectumRectumColonColonRectumColonRectumColonRectumColonRectumRectumColonColonRectumRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonColonTumor

stagingAnatomical

siteSigmoidSigmoidConcomitant

hepaticAngle+descendingRectumRectumDescendingDescendingRectumRectosigmoidRectumAscending

+transverseRectumSigmoidRectumRectumConcomitant

descending+sigmoidRectosigmoidRectumRectumSigmoidSigmoidRectosigmoidRectumDescendingRectumRectumRectumRectumSigmoidCecum

+ascendingSigmoidDukesDB,AB,B,BjB,C,DB,B,C,B,DDB,DB,B,C,B,B,B2C,DDTNMIV(T3Nâ€žM,)II

(T,Nâ€žMâ€ž)la(T.NoMo)II(T,N0Mâ€ž)II(TjNâ€žMâ€ž)II(TjNoMo)II(TjNoMo)III(TjN.^Mo)IV(T.N.M,)II

(T,Nâ€žMâ€ž)Ib(TjNoMo)III(TjN.L.M,,)II(T3Nâ€žMâ€ž)IV

(T.N.M,)IV

(TjN.M,)II(TjNoMo)IV(T2N0M,)II(T,Nâ€žMâ€ž)II(T,NoMâ€ž)III(T3Nâ€ž;)Mâ€ž)II

(TjNoMo)II(TjNoMo)II(T,Nâ€žMâ€ž)III(TjN^.Mo)IV(T.N.M,)IV

(T.N.M,)Histological

dataTypeTubularColloidalMixedTubularTubularTubularMixedTubularTubularMixedTubularTubularTubularTubularTubularTubularMixedTubularTubularTubularMixedTubularTubularTubularTubularTubularGradingPDÂ°WDWDMDNDWDNDNDNDNDMDWDWDNDPDWDPDWDNDWDWDPDWDNDPDCEA(ng/ml)<5.0<5.0<5.01.04.5>60.07.01.024.01.035.016.0>60.019.05.5<5.0<5.011.04.510.07.016.08.04.0>600.0>60.0Ferritin

(ng/ml)7.460.024.04.8>

100.056.0>

100.04.7>

100.027.0>

100.016.076.06.43.962.0>

100.0>100.086.025.0<2.537.025.056.0>

100.042.0

' PD, poorly differentiated; WD, well differentiated; MD, medium differentiated; ND, nondifferentiated.

type II NK regulation, defined as sera with untreated inhibitoryactivity but with appreciably more inhibition (>20% difference)after heating, exhibited with at least one of the PBL populationsused as effector cells. This pattern suggested that these seraprobably possess the stimulatory or protective factor in a loweramount than that found in the sera with type I NK regulation.

The sera of a third group of 13 patients, defined as havingtype III NK regulation, had the following pattern: untreatedserum inhibited strongly the NK activity; and this effect wasnot significantly changed by heat treatment (A < 20%). Almostall of these patients had poor prognostic factors, such as distantmÃ©tastasesor tumor invasion into neighbouring organs, youngage, or a personal (previous noncolonic cancer) or familialhistory of cancer.

Tests of the majority of cancer sera on both autologous PBLand cells from a normal donor revealed a good correlationbetween the data provided with each effector cell population inregard to the percentage of the control value of NK activity (r= 0.62) and A value (r = 0.78). Since the particular type ofreactivity of each serum could be defined irrespective of thedonor of cells used in the NK assay, the reproducibility of themodulatory effects of sera from 18 cancer patients on the NKactivity of normal PBL was tested several times after the initialdetermination. Although for about 25% of the investigated serathere were appreciable differences in their ability to modulateat different times the NK activity of 2 to 4 different PBL, theoverall interassay variability was not significant, as the mean Â±SEM of NK activity (percentage of the control) of the firstinvestigation (68.9 Â±10.5) was similar to that obtained whenthe serum-induced regulation of NK activity was tested thesecond (64.7 Â±11.8) or the third time (80.9 Â±35.0). Additionally, Table 5 shows the reproducibility of the A values of 12cancer sera tested 2 to 3 times in separate assays on normalPBL. Although there was a certain degree of variation, the

category of sera was only changed in 2 cases (Patients 17 and18) whose first determined A values were, in fact, near theborderline for categorization.

The type I or II patterns of NK regulations in patients withcolorectal adenocarcinoma (Table 4) were found in only one of30 cases with inflammatory digestive diseases or with benigncolorectal polyposus. The mean Â±SEM of NK activity (percentage of the control) of patients' PBL treated with their

autologous serum (32.1 Â±5.0) was very similar to that obtainedupon exposure to normal serum (32.2 Â±4.6) and, except forone case, heating of these patients' sera didn't induce significant

elevation of their inhibitory capacity.Table 6 shows a summary of the results of our studies on 36

patients with colorectal adenocarcinoma (presented in Tables1, 2, and 4) together with the data of 30 patients with benigndigestive disease and those of 100 normal individuals. As compared to the results obtained with PBL isolated from healthydonors when incubated with untreated or heat-treated autologous serum, sera from the first group of cancer patients (42%of the total cases) were highly significantly (P < 0.001 ) differentfrom normal sera with regard both to their inability to inhibitNK activity and their high A values, whereas the sera from the7 type II cancer patients (19% of cases) were significantly (P <0.001) different only in terms of the heat-induced difference.The sera of cancer patients of the third group (59% of cases)were not different from control sera isolated from healthyindividuals with regard to A value, but a significantly higher (P< 0.005) mean Â±SEM inhibition (67.0 Â±4.1) of NK activityof normal and cancer PBL was found in 24 determinations incomparison to that induced by normal sera (45.8 Â±5.4). Nodifferences were also observed between sera of patients withbenign digestive diseases and those isolated from apparentlyhealthy donors. However, of all of these sera, there were 1 ofthe patients with benign diseases (3% of investigated cases) and

2599




Table 4 Distinct types of NK regulation by serum from patients with colorectal carcinoma

NK activity of PBL incubatedwithCase12345678910111213141516171819202122232425262728293031Tumor

locationColonColonColonRectumColonColonColonRectumColonRectumColonRectumColonRectumRectumColonColonRectumRectumColonColonRectumRectumColonRectumRectumRectumRectumColonColonColonDonor

ofcells used in

N KassayPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalCancer

serum%

ofcontrolNDÂ°90.3100.061.0144.0100.090.0108.089.050.0100.0144.472.0116.0100.093.3101.0113.5145.0ND67.0128.023.068.554.387.746.714.238.360.555.768.8ND34.0ND30.521.4ND30.036.67.622.421.541.323.560.029.593.060.5ND50.00.754.559.785.335.919.532.211.610.929.020.0AND88.653.2ND114.066.772.0ND46.443.862.079.448.081.660.0ND73.273.673.4ND36.5ND21.331.848.029.625.25.423.08.56.924.8ND21.0ND21.020.0ND-14.211.6-9.8ND-4.5ND3.5ND15.3ND0.0ND20.00.04.53.012.82.66.28.0-2.46.319.02.0Normalserum (% of

control)ND28.048.6ND25.746.330.046.340.046.338.065.034.065.029.421.0129.1105.760.0ND73.060.00.846.36.346.352.831.45.770.742.353.7ND43.564.416.5ND67.266.718.210.032.466.764.572.059.0ND82.2ND30.025.0100.0105.774.448.331.064.050.072.060.022.5Typeof NK

regulationIIIIIIIIIIIIInnnnnnHIininminHIininilimHImHIClinical

observationsDukesNDDB/ABBBBCDNDBBCBNDNDDNDDBDBBCNDBBBCDDOther30

yr old, diagnosed&irradiated2 yr be

foreExtendedprocess, ra

diotherapyRadiotherapyRadiotherapyRadiotherapyCancer

previouslyRadiotherapy30

yr old, diffuse colon polyposus, ra

diotherapy32yroldExtended

processRadiotherapyMultiple

colon polyposusRadiotherapyRadiotherapyRadiotherapyDeceasedExtended

process

* ND, not determined.

2 from normal donors (2% of tested individuals) which presented the typical type I or II patterns of NK regulation. Itmust be mentioned that 4 other normal sera were also found inour present investigation to be noninhibitory, but after heatingthey did not acquire the ability to inhibit NK activity, andconsequently they were not considered to belong to type II NKregulation.

Of the 25 colorectal cancer patients who have been treatedsurgically, 11 were subsequently tested at various times afterresection of their tumor (Table 7). Five type I patients (Cases

3, 5, 7, 8, and 11) with improving clinical courses, as assessedby weight, Karnofsky scores, and freedom from detectabletumor, were found to have changed their type of NK regulationto III when determinations several months after surgery wereagain performed. In the case of Patient 11 with an initiallyextended process of malignancy and postoperative residualtumor tissue, the serum characteristics defining the type I NKregulation were again found about 1 yr later, when resection ofadditional tumor was not possible to be performed. Sequentialimmunological determinations of patients with initial type III

2600




Table 5 The value of serum from patients with colorÃ©ela!adenocarcinomaassayed at various time intervals on normal PBL isolated from different donors

A value provided by cancer serumtested at various times on different

PBL of normaldonorsCaseD.

N.6791112131718212226Donor's

serum(tumor

location)ColonColonColonColonColonRectumColonColonRectumColonColonRectum179.4

(If79.4(I)81.6(1)73.6

(I)95.0(I)20.3(II)20.9(II)21.0(11)21.0(11)17.2(111)12.6(111)0.0(111)241.0(1)52.0(1)49.6(1)145.8(1)62.1

(I)31.8(11)29.6

(II)6.7(111)1.3(111)19.4(111)-5.6(111)0.0

(III)3-1.2(111)17.4(111)" Type of NK regulation indicated in parentheses was characterized by taking

into account, besides the A value, the other 2 parameters, namely the regulatoryeffects of untreated cancer serum as compared to medium-treated and to normalserum-treated PBL.

of NK regulation revealed that the sera of 5 of 6 cases hadsimilar inhibitory effects as those found before surgery (Table7). The results with sera from two additional treated patientsnot studied before surgery are also presented in Table 7. Theserum of Case M. T., also in good clinical condition and freeof any detectable tumor, showed a type III pattern of NKregulation. A different pattern was observed with Case A. G.,who had sigmoid colon cancer resected 1 yr before our firstinvestigation when the recurrence was not detected clinicallybut whose serum showed a significant A value. Nine mo later,the patient presented radiologically detectable paracolic lymph-adenopathy which was correlated with the appearance of type INK regulation.

DISCUSSION

Our study provides evidence regarding the ability of serumfrom cancer patients (a) to induce negative /// vitro modulationof NK activity (/, inhibition) expressed as the percentage of theactivity of medium-treated control cells, (b) to acquire or notan appreciable difference (A) in its regulatory effects upon heattreatment, and (c) to inhibit or not the NK function morestrongly relative (R) to normal control serum. These 3 param

eters tested together allowed the identification of 3 distinct NKregulation types characterized, in general, as /~A+/?* (type I),/+A+/T (type II), or r\~R+ (type III). Lack of inhibition of

NK activity by serum from 36% of all our investigated patients(25 of 69 cases) with various lymphoid or nonlymphoid solidmalignant tumors, a percentage similar to that identified in themore homogeneous group of patients with colorectal carcinoma(15 of 36 cases) (Table 6), was found in only 3 of the 100normal donors and 1 of the 30 patients with benign digestivediseases. It should be mentioned that 4 noninhibitory sera ofpatients with type I NK regulation (Table 4) produced even anaugmentation of NK activity relative to medium-treated PBL.The noninhibitory characteristics (/") of these cancer sera,

indicated by a lack of negative modulation of NK activity or byeven positive modulatory effects, were always reversed by theirtesting at 56Â°Cfor 30 min, a treatment which endowed them

with the ability to reduce NK activity. This heat-induced difference in the regulatory capacity was also observed with 10 /*

cancer sera (20% of tested cases), defined as having the type IINK regulation. The A* characteristic of 56% (28 of 50) of the

cancer sera tested was observed with only 3 of the 60 controlsera from either patients with nonmalignant digestive diseases(1 of 23) or apparently healthy donors (2 of 30). The lastpattern of NK regulation (type III) was provided by those cancersera which usually possessed only the characteristic to morestrongly inhibit NK activity as compared to normal sera.

These 3 different categories of cancer sera revealed differences among our group of patients with colorectal cancer whichwere not paralleled by differences in levels of cytotoxic reactivityof their NK cells. Thus, the values for the mean Â±SEM of LU/IO7 cells of medium-treated PBL isolated from patients with

type I, II, or III NK regulation were 124.5 Â±51.8, 94.5 Â±19.1,and 99.3 Â±46.1, respectively, not different among them orfrom the NK activity expressed by PBL of normal individuals(data not shown). Interestingly the reported decrease in relativenumber of circulating HNK-1* cells in colon cancer patients

was associated with an increased cytolytic function comparedto the population of HNK-1* cells purified from healthy subjects (21), accounting perhaps for the "normal" base-line NK

activity expressed by medium-treated PBL of patients investigated in the present study. Additionally, the effector cells ofalmost all cancer patients tested responded normally by inhi-

Table 6 Summary of the results obtained with untreated and heat-treated serum isolated from patients with colorectal adenocarcinoma or with benign digestivediseases

Statistical differences with respect to control values were calculated by Student's ( test for paired data. The control values of NK activity, expressed both aspercentage of the control (41.6 Â±2.3, n = 85) and as heat-induced difference (A) (3.1 Â±2.2, n = 31), are means of the results furnished by all experiments performedin this study with normal PBL incubated with normal serum before or after its heat treatment. Incubation of some of these unheated and heated normal sera withpatient PBL in parallel experiments provided mean Â±SEM values of NK activity very similar to control values, e.g., 41.6 Â±2.5 (n = 100) for the percentage of thecontrol, and -0.7 Â±6.2 (n = 9) for A.

NK activity of PBL treatedwithDonors

ofserumColorectal

cancer (typeI)Colorectal

cancer (typeII)Colorectal

cancer (typeHI)Benign

digestive diseasesNo.

ofcases1571430Donor

of cells usedin NKassayPatientNormalPatient

NormalPatient

NormalPatient

Normal%

ofcontrol104.5Â±6.0Â°'*(14)'

102.8 Â±7.9*(12)43.6

Â±6.0 (5)52.0 Â±9.9(7)33.7

Â±5.7(14)37.5 Â±8.2(11)32.1

Â±5.0(29)28.2 Â±4.3 (30)Patient

serum(A)67.1

Â±6.6*(12)67.0 Â±6.2*(8)24.9

Â±6.611(5)20.3 Â±3.8*(7)4.0

Â±3.2(14)4.8 Â±1.5(7)4.8

Â±1.3(29)6.8 Â±2.6(17)Normal

serum(% ofcontrol)50.3

Â±7.7(14)54.2 Â±6.8(11)21.6

Â±10.8(5)50.9 Â±5.0(7)52.5

Â±6.6 (14)57.3 Â±9.8(10)32.2

Â±4.6 (29)34.7 Â±4.4 (27)

" Mean Â±SEM.'/>< 0.001.' Numbers in parentheses, number of experiments.dP<0.01.

2601




Table 7 Preliminary results of the longitudinal study of serum-induced NK regulatory effects of patients with colorectal adenocarcinoma at various times after tumorresection

NK activity of PBL incubatedwithCase3S7811202122242628A.C.M.

T.Mo

after tumorresection1716815412411153914571645il71221IS1923Donor

ofcells used in

NKassayPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalPatientNormalCancer

serum%of

control0.10.258.018.861.648.341.332.771.510.769.042.83.832.4116.7100.053.887.536.638.733.735.40.127.660.040.015.326.818.085.70.10.117.610.443.31.271.661.654.387.7114.3100.020.09.716.66.6ND35.4A0.00.0-3.2-16.60.68.36.9-6.62.43.23.14.51.5-3.851.195.026.062.611.6NDÂ°10.010.00.09.617.219.412.6-5.64.22.40.00.00.20.4-2.5-1.437.449.348.029.698.360.1-0.5-4.03.03.8ND10.4Normalserum (% of

control)0.150.070.840.467.733.332.646.194.357.787.846.145.226.891.30.121.864.825.066.741.440.51.522.550.931.414.426.820.042.80.10.160.054.357.764.832.080.46.346.3NDND33.357.78.525.0ND40.4Type

of NK regulationBefore

AftersurgerysurgeryI

IIII

IIII

HImi

HIini

iniiHI

inHIinin

inin

inininin

inIII

HIIIIHI

IIIND

IIIND

IIIIIIinHIClinical

observationsTumor

freeTumor

recurrenceTumor

freeTumor

freeTumor

freeTumor

freeResidual

tumortissueExtended

recurrenceExtended

recurrenceTumor

freeTumor

freeHepatic

mÃ©tastases.tumorrecurrenceHepatic

mÃ©tastases.deceased2 molaterResidual

tumortissueHepatic

mÃ©tastasesTumor

recurrenceTumor

freeParacolon

fistulaTumor

recurrenceTumor

freeParacolon

ade-nopathyTumor

freeTumor

freeTumor

freeTumorfree

' ND, not determined.

bition upon incubation in the presence of mlgG or normalserum (Tables 1 and 4). These results raise the possibility thatthe usual measurements of NK activity of cancer patients'

lymphocytes, at least in regard to patients with colorectalcarcinoma, may not be as indicative of clinical course of diseaseor host resistance as the assessment of the patients' reactivity

in the presence of their serum, with the latter more closelyapproximating the in vivo situation.

The distinctive patterns of NK regulation by cancer sera werealso observed when the in v/iro-treated cells were PBL isolated

from cancer patients as well as from normal donors. Thus, thedetection of / and A characteristics of sera obtained from

patients with malignant (Tables 2 and 4) or benign diseases orfrom healthy individuals and tested comparatively in 70 experiments on both patient and normal PBL was concordant in86% and 88% of experiments, respectively. This rather goodreproducibility, in addition to the small interassay variabilityshown in Table 5, strongly supports the conclusion that the /~and/or A+ characteristics of the substantial number of sera

tested were cancer associated. In fact, these characteristics oftype I pattern of 5 of 5 patients disappeared after tumorresection, when the patients' sera were obtained again several

months later (Table 7).An examination of the correlation between the type of serum

2602



MODULATION OF NK CELL ACTIVITY BV SERUM FROM CANCER PATIENTS

regulatory activity and the extent of tumor in a given patientshould take into account that the Dukes and TNM stagingswere determined in some cases at intervals greater than 1 mo,when the surgery was performed, and they were very likelyinaccurately evaluated in those cases treated by radiotherapybefore tumor resection. In spite of these difficulties, it seemsthat the type III NK regulation in tumor-bearing patients canbe correlated with more advanced tumor staging, as Dukes Cor D stages were found in 50% of patients with this pattern (6cases) and in all but one of the other 6 cases in whom radiotherapy was administered or poor prognostic factors were identified (Table 4). Follow-up of 6 patients of this category revealedin 5 cases tumor recurrence and even mÃ©tastases(Table 7).Conversely, the apparent correlation between the early stage ofmalignancy and the type I NK regulation suggests that the 7~A*

serum characteristics of these cancer patients may reflect themore efficient function in vivo of their circulating NK cells,refractory to the negative regulatory factors actually present inthe serum. The mechanism underlying the noninhibitory capacity of these cancer sera may represent either a block in theinhibition of cytophilic IgG or other serum inhibitory factorsor, more likely, a stimulatory factor which just regulates in theopposite direction as mlgG.

It should be noted that the clinical correlations with type ofNK regulation by cancer patients' serum varied considerably

for specimens obtained prior to treatment versus those obtainedafter treatment. In the untreated tumor-bearing patients, typeIII regulation was associated with poor prognosis. In contrast,after tumor removal, the shift from type I or II to type III wasassociated with a tumor-free state, i.e., the development of apattern essentially the same as that seen with normal donors.

The nature of the postulated stimulatory factor in type I sera,which has been identified functionally in this study, is virtuallyunknown except for its thermolability. One possibility is 7-interferon, which can be inactivated at 56Â°C,but recent observations made by us5 and others (22) reveal that this lymphokine

in highly purified form is, in fact, unable to augment within aperiod of 4 h the activity of human NK cells. Another majorcandidate as potent activator of NK activity might be interleu-kin 2, but this molecule is thermostable. Experiments are presently under way to identify the nature of this putative stimulatory factor. Whether its measurement will prove to be valuablein the assessment of a malignant transformation or the courseof disease in a given patient needs to await further longitudinalNK studies and clinical follow-up.

ACKNOWLEDGMENTS

We would like to thank Mariana Feher, Elena /.men, and T. Regaliafor excellent technical assistance. The authors gratefully acknowledgeMariana Pavlovski, Laura Bates, Lori Novak, and Barbara Klewien forsecretarial assistance.

' A. Sulica, M. Gherman, M. Manciulea, and R. B. Herberman. Regulation ofhuman natural cytotoxicity by IgG. III. Ability of i-interferon to restore the NKactivity of cells negatively regulated by cytophilic IgG, manuscript in preparation.

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17. Sulica, A., Gherman, M., Manciulea, M., Galatiuc, C., and Herberman, R.B. Negative regulation of human NK activity by monomeric IgG. In: R. B.Herberman (ed.), NK Cells and Other Natural Effector Cells, pp. 621-628.New York: Academic Press, 1982.

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2603



1988;48:2596-2603. Cancer Res Madalina Pislarasu, A. Oproiu, Doina Taranu, et al. Colorectal Adenocarcinoma or Other Types of CancerPatients: Preliminary Results of a Study of Patients with Modulation of Natural Killer Cell Activity by Serum from Cancer

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