Cancer Immunol Immunother (1981) 11 : 139-145 Sancer mmunol9gyand mmunotherapy

© Springer-Verlag 1981

Human Natural Cell-mediated Cytotoxicity I. Levels in Peripheral Blood, Cord Blood, and Thoracic Duct Lymphocytes

James T. Forbes 1, G. D. Niblack 2, R. Fuchs 1, R. E. Richie 3, H. K. Johnson 3, and Robert K. Oldham 1

a Division of Oncology, Department of Medicine 2 Department of Pathology, School of Medicine, Vanderbilt University 3 Veterans Administration Hospital, Nashville, TN 37232, USA

Summary. Natural cell-mediated cytotoxicity has been shown to be age-dependent in both rats and mice. The present study was undertaken to study age-related levels of natural cytotoxicity in humans. Mononuclear peripheral blood cells from over 200 normal volun- teers were co-incubated with 51Cr-labeled K-562 for 4 h and the supernatants assayed for released isotope. No striking age-related variations in natural cytotox- icity were observed. Cord blood was shown to exhibit both high and low levels of natural cell-mediated cytotoxicity. There was no significant difference in the levels of natural cytotoxicity between males and females. Thoracic duct lymphocytes (TDL) from prospective kidney graft recipients were tested for natural cytotoxicity at intervals during drainage. Natural cytotoxic activity was undetectable in samples from initial drainage but increased as the drainage progressed. This increased activity was correlated with decreased in vitro responsiveness to PHA and decreased levels of E-rosettes in these TDL.

Introduction

In vitro cell-mediated cytotoxicity (CMC) to tumor cells has become a major experimental assay of tumor immunity [4, 5]. The demonstration that lymphocytes from normal individuals could lyse tumor cells to which they have never been exposed has made interpretation of CMC assay results difficult [22, 23]. This spontaneous CMC or natural cell-mediated immunity (NCMC) has been demonstrated in both rodents and humans [3, 7-10, 12, 14, 18, 28]. The target antigen(s) of this cytotoxicity have not yet been clearly identified [7]. The in vivo function of this system is not firmly established but seems consistent with the existence of a surveillance mechanism against tumor growth. However, it is obvious that any

Reprint requests should be addressed to: J. T. Forbes

future use of CMC for the evaluation of in vivo tumor immunity must rely on a thorough understanding of natural cell-mediated immunity.

The effector cell of NCMC has been found in most of the lymphoreticular organs, except the thymus, with the highest activity in the spleen and peripheral blood and the lowest in the lymph nodes and tonsils [10, 16]. The cell active in NCMC has been termed the natural killer (NK) cell, and its exact lineage is unknown [7]. Some human NK cells have been distinguished by the presence of a Fc receptor [27, 31] and a low-affinity receptor for sheep erythrocytes [32]. The T-cell lineage of NK cells is unclear. There is some evidence to suggest that they are immature T cells. NK cells form spontaneous E-rosettes at 4°C but not at 29°C [17]. Evidence from experiments in mice suggests that NK cells are sensitive to anti-Thy 1 [11]. Recently [15, 26] it has been reported that human NK cells are killed by a T-cell antiserum.

Limited studies to characterize NCMC in man have suggested that males are more reactive than females and that there appears to be a genetic association between decreased reactivity and the HLA-A3, B7 haplotype, while increased reactivity was associated with the HLA-B12 haplotype [30]. Other workers have reported little association between age and level of natural cytotoxicity [23]. This is in contrast to rodents, in which no cytotoxicity is seen early in life and high levels are found in young animals followed by decreased activity with advanc- ing age [7].

In this study, lymphocyte preparations from the peripheral blood (PBL) of normal human donors of all ages were tested for cytotoxicity against the human cell line K-562 in a 4-h 51Cr release assay. The results were correlated with age, gender, and parity. Cells from chronic thoracic duct drainage and from human cord blood, which might reasonably be expected to be

0340-7004/81/0011/0139/$ 01.40

140

en r i ched in i m m a t u r e T cells were also t e s t ed for N K activi ty.

J. T. Forbes et al.: Dynamics of Natural Cytotoxicity

elsewhere [25]. Data were compared for statistical significance by Student's t-test. Data were considered significant if P < 0.05.

Materials and Methods

Panel of Blood Donors. The lymphocytes used were obtained from volunteer healthy donors of all ages. Donors receiving medication or with a history of viral infection less than 2 weeks prior to donation, or with a personal history of neoplastic or other serious illness, were excluded. Cord blood samples were taken from healthy full-term infants at birth. All blood was anticoagulated with preservative-free heparin (Fellows Medical Manufacturing Company, Oak Park, Michigan USA).

Thoracic Duct Lymphocytes. Human TDL were collected from the chronic drainage of prospective kidney graft recipients and were processed as described previously [13]. These patients were receiving hemodialysis treatment for control of their uremia.

Separation of Lymphocytes. Effector cells were obbtained from heparinized blood by one-step centrifugation on a Ficoll-Hypaque gradient [2]. These cells were washed twice with balanced salt solution and enumerated, and their viability was determined by trypan blue exclusion. They were resuspended to the desired concentration in RPMI 1640 (Grand Island Biological Company, Grand Island, New York, NY, USA), supplemented with 10% fetal calf serum (GIBCO) and penicillin (100 units/ml). This culture medium will be referred to hereafter as complete medium.

Proliferation Assay. Lymphocyte proliferation assays with ConA, PHA, PWM, and allogeneic cells were performed as previously reported [21]. Generation of CMC in mixed-lymphocyte culture was performed as previously described [21].

SlCr Release Assay. The chromium release assay was performed as previously described [29]. Briefly, K-562, a human cell line derived from the pleural effusion of a patient with acute myelogenous leukemia [20] was incubated with 150 gCi Na 51CRO4 (Amersham, Arlington Heights, Illinois) at a concentration of 5-7 x 106/ml for 45 min at 37 ° C. The cells were washed twice in complete medium and adjusted to 2 x 105/ml. A total of 104 SaCr-labeled K-562 cells were added to effector cell concentrations of 105, 5 x 105 or 106 per ml, which resulted in a final effector-to-target ratio of 10 : 1, 50 : 1, or 100 : 1. The reaction mixtures were carried out in 12 x 75 mm polystyrene tubes, which were incubated in 5% CO2 for 4 h at 37 ° C on a Bellco rocker, cycling at 6 per min. After incubation the tubes were centrifuged at 100 g for 10 rain and the supernatants decanted for determination of unbound isotope. All reactions were performed in quadruplicate. Total labeling was determined by counting an aliquot of 104 5aCr-labeled K-562 cells. Autologous controls (unlabeled target cells added to labeled targets) were used as baseline release values in all experiments. Any experiment in which this baseline value exceeded 10% of the incorporated label was rejected as technically unsound. The percentage isotope release was calculated from the following formula:

Percent release =

CPM released from cells during incubation x 100. Total CPM incorporated into cells

All NK data expressed in this report are mean percent release at an effector-to-target ratio of 50 : 1 unless otherwise indicated. Lytic units are expressed as a function of the number of cells capable of lysing 15% of the target cells and are calculated as described

Results

Reproducibility of NCMC

Cen t ra l to any discuss ion of the p o p u l a t i o n dynamics of N C M C is the r ep roduc ib i l i t y of resul ts in the m e a s u r e m e n t of this p h e n o m e n o n . Tha t N C M C is ser ia l ly r e p r o d u c i b l e in hea l thy n o r m a l dono r s has been d e m o n s t r a t e d p rev ious ly [19, 22, 23], and con f i rmed and e x t e n d e d in the p re sen t s tudy. Tab le 1 d e m o n s t r a t e s the high deg ree of r ep roduc ib i l i t y of this assay with mul t ip le m e a s u r e m e n t s f rom the same donors ove r a 9 -month in terval . The da t a show on ly m i n o r va r i a t ion and suggest tha t the va lues for N C M C col lec ted at var ious t imes m a y be d i rec t ly c o m p a r e d . F u r t h e r ev idence for the r ep roduc ib i l i t y of N C M C is found in Tab le 2, which tests the poss ib i l i ty tha t t a rge t cells m a y r ep re sen t a source of va r iab i l i ty in this assay. K-562 s eeded at 1 x 105 cel l /ml and ha rves t ed at 24-h in terva ls were tes ted as 51Cr-labeled t a rge t cells for N C M C by c r y o p r e s e r v e d ef fec tor cells f rom a single d o n o r [24]. The da t a d e m o n s t r a t e tha t changes in cul ture kinet ics dur ing this p e r i o d a re no t a m a j o r source of va r i a t ion in N C M C . These da t a d e m o n s t r a t e the r ep roduc ib i l i t y of this assay.

Population Distribution

T h e resul ts o f a s tudy of na tu ra l C M C in 200 n o r m a l n o n - p r e g n a n t indiv iduals is shown in Fig. 1. The va lues a re g r o u p e d by decades and iden t i f i ed as to sex. T h e r e is no c lear assoc ia t ion of levels o f na tu ra l cy to toxic i ty wi th age, bu t severa l f ea tu res mer i t c o m m e n t . Leve ls of s p o n t a n e o u s na tu ra l C M C a p p e a r to be h ighes t at the e x t r e m e s of age: in the young and the e lder ly . The lowes t va lues a re n o t e d in young adul ts . This is cer ta in ly in con t ras t to the age associa t ions n o t e d in roden t s , which d e m o n s t r a t e an absence of reac t iv i ty in ea r ly life fo l lowed by inc reased act ivi ty to a p l a t eau , which dec reases g radua l ly wi th age. T h e da t a p r e s e n t e d in Fig. 1 also suggest tha t t he re a re bo th high reac to r s and low reac to r s , wi th a spec t rum of va lues in b e t w e e n these two ex t remes . I t should be n o t e d tha t on r e p e a t e d tes t ing high reac to r s were cons is ten t ly high and low reac to r s were cons is ten t ly low (Tab le 1). A s no exogenous p ressures were n o t e d to be pecu l i a r to the high- or low-reac t ing indiv iduals it is poss ib le tha t s p o n t a n e o u s na tu ra l C M C in the h u m a n is u n d e r gene t ic con t ro l in much the s ame m a n n e r as it is in the mouse .

J T. Forbes et al.: Dynamics of Natural Cytotoxicity

Table 1. Serial NCMC values by healthy donors on K-562

Donor Percent NCMC on 2 - 6 occasions over 9 months

T .S . 9 ± 0,4 a 14 ± 1 12 ± 0.6 15 ± 1.7 11 ± 0.7 16 ± 1.0 F . J . 20 ± 0,7 19 + 1.1 18 ± 1.8 19 ± 1.0 H . C . 25 + 2.6 21 ± 2.5 20 + 1.3 L . W . 31 ± 1,1 33 ± 1.4 34 + 1.9 C . D . 37 + 1.9 33 ± 2.0 J . J . 25 + 2.3 31 + 1.1 C . K . 19 ± 0.9 18 ± 0.8

a Mean percent NCMC ± SE

141

Table 2. Effect of K-562 culture time on NCMC

Days in culture

1 2 3 4

K-562 in culture (x 105/ml) ~ Spontaneous release of SlCr (:~ ± SE) NCMC by cry•preserved effector b

2.5 5.0 6.8 7.3 1 0 ± 0 . 4 8 ± 0 . 3 6 ± 0 . 5 7 ± 0 . 2 4 0 ± 2 . 1 3 5 ± 1 . 6 3 5 ± 1 . 1 4 0 ± 1 . 8

Initial cultures seeded at 1 x 105/ml b Effector ceils from single donor cry•preserved and tested at effect•r: target ratio o f 50 : 1

5 0 "

4 0 '

5 0 '

~ 2 0 ¸

1 0 ¸

Table 3. Effect of age and gender on NCMC

o • "a , Age group Gender o

8 o • o

g, • o - •

• 4 • o "L • "

o ~ ~ - - . . . . .

eOD N O

dx,:o •

.~ ~ " : o o , o

coo o • ~ o

o • o MALE

• FEMALE

<-.10 1 1 - 2 0 21-50 3 1 - 4 0 4 1 - 5 0 5 1 - 6 0 61 -70 > 7 0

A G E G R O U P

Fig. 1. Population distribution of natural cytotoxicity of K-562 by human peripheral blood mononuclear cells at an effect•r-to-target cell ratio of 50 : 1. The results are grouped in decades by the age of the peripheral blood

Influence of Gender on Levels of Natural CMC

The effect of gender on levels of natural CMC to the K-562 cell line is shown in Table 3. There are no

Male Female

Number % NCMC a Number % NCMC a tested tested

11-20 19 32.5 + 2.0 10 29.6 + 2.9 b 21-30 34 20.3 + 1.5 55 21.6 + 1.5 31-40 14 24.7 + 3.1 21 25.1 + 2.7 41-50 5 19.8 + 1.9 33 23.7 + 1.7 51-60 10 27.7 + 3.9 13 22.9 + 2.2 > 60 17 0.8 + 2.5 32 25.1 + 2.2

a Mean percent ± SE b Comparisons male/female NCMC all nonsignificant (P > 0.05). Male groups 11-20 (0.01 > P > 0.001) and 51-60 and > 60 (P < 0.05) are significantly more reactive than those between 21 and 5O

significant differences in cytotoxicity associated with gender (P > 0.05). In the older age groups, males are slightly more reactive than females at all ages in natural CMC [30]. When considered alone, young males (ages 1 1 - 2 0 ) are more reactive than males between the ages of 21 and 50 (0.01 > P > 0.001). Males above 50 are also more reactive than those between 21 and 50 ( P < 0 . 0 5 ) . There were no significant age-related differences in the females tested.

Age group Nulliparous

J. T. Forbes et al.: Dynamics of Natural Cytotoxicity

Number - % N C M C a tested

2 1 - 3 0 34 21.3 + 2.6 3 1 - 4 0 7 30.6 _ 5.9 4 1 - 5 0 7 29.8 _ 1.6 5 1 - 6 0 5 20.3 ___ 2.0 > 60 6 17.0 + 4.2

Multiparous Pregnant P-value

Number % N C M C a tested

Number % N C M C a tested

a Mean percent + SE

21 19.0 + 2.7 14 22.6 + 2.7 26 21.8 _+ 2.2

8 24.7 _+ 3.3 26 27.7 _+ 2.3

9 15.7 + 0.2 > 0.05 0.05 0.05 > P > 0.02

> 0.05 > 0.05

, 4 0 ¸

Table 5. Natural cytotoxicity of human cord blood lymphocytes

Patient Gender % Net cytotoxicity

1 F 6 2 F 32 3 F 3 4 F 15 5 F 12 6 F 13 7 F 8 8 M 18 9 M 6

10 M 5 11 M 26 12 F 4 13 M 0

35-

TIME OF DRAINAGE

[3 I DAY • 05 WK z~ 1.0 WK • 2.0WK

0 2 .5 WK I I 3.0WK 30

-- 25

k-

0 i -

ao

15'

10'

5'

142

Table 4. Effect of pregnancy and parity on NC M C

,; go ,;o RATIO TDL/K-562

Fig, 2. Natural cytotoxicity of K-562 by thoracic duct lymphocytes at various times after cannulation

8 ePt. I I tPt.2 /

~-g .a3 /

2 g, J

I 2 3 Time (Wks.) of Drainage

Fig. 3. Natural cytotoxicity in three individuals with chronic drainage. Data expressed as 15% lyric units/106 T D L

Effect of Pregnancy on Natural CMC

To examine the hypothesis that natural CMC is due in part to sensitization of maternal lymphocytes by fetal antigens, PBL from multiparous or pregnant females were compared with age-group-matched lymphocytes from nulliparous females for their ability to lyse K-562 target cells. The results of tests with lympho- cytes from pregnant females are shown in Table 4. When these data are compared with the cytotoxicity mediated by lymphocytes from age-group-matched nongravid females (Table 3) it can readily be seen that the levels for pregnant females are lower than the levels for non-pregnant females (15.7 + 2.2 versus 21.6 + 1.5), but this difference is not significant (P > 0.05). The NCMC of this group of pregnant females is also not significantly different (P > 0.05) from the NCMC of age-matched nulliparous or multiparous females. These data would not be compatible with natural cytotoxicity being the result of fetal antigen sensitization of maternal lympho- cytes.

J. T. Forbes et al.: Dynamics of Natural Cytotoxicity

t O 0 '

7 5

5 0

tm I*

110,

.= I~" I 0

e <4

I O - o

I x

x t O -

L I 0 - o IX

%_ 10- x = *1 • t O -

o IK

T D L " 4 " C E R O S E T T E S

D

T D L - P H A

T D L - C O N &

T O L - P W M

°

T O L - C M L

~ 7 5 "

o

x o

goo.,,,_~

~ = s -

Q [3

T I M E O F O R A I N A G E ( W K $ )

Fig. 4. Spontaneous sheep erythrocyte (E) rosettes, incorporation of tritiated thymidine, in response to stimulation by PHA, ConA, or PWM, incorporation of tritiated thymidine (MLC) in response to incubation with pooled mitomycin C-blocked allogeneic cells and the generation of cytotoxic effector cells to 51Cr-labeled allogeneic cells (CML) by TDL taken at weekly intervals during thoracic duct drainage

The levels of cytotoxicity against K-562 by lymphocytes from multiparous nongravid females are shown in Table 4 and are compared with those from age-group-matched nulliparous females. The levels of cytotoxicity mediated by lymphocytes from multipa- rous females are lower than those mediated by lymphocytes from nulliparous females 21-50 years of age. The opposite is true for women above 50 years of

143

age; lymphocytes from multiparous females over 50 are more active in NCMC than those from their nulliparous counterparts. However, the only statis- tically significant difference between these two groups is to be found in the 41-50 age group (e < 0.05).

Natural CMC Mediated by Lymphocytes from Human Cord Blood

The natural CMC mediated by lymphocytes from 13 samples of human cord blood are shown in Table 5. Most of these were cytotoxic for the K-562 cell line, though several had cytotoxicity of a very low order. These data demonstrate the existence of natural CMC prior to any influence of the extrauterine environment. As in the adult population there appear to be both high- and low-responder populations. This provides more evidence for a genetic basis of NCMC.

Natural CMC Mediated by Lymphocytes from Chronic Human Thoracic Duct Drainage

The results, as percent cytotoxicity, of a represen- tative experiment to demonstrate the levels of NCMC for human TDL are shown in Fig. 2. These TDLs demonstrate higher levels of activity as the duration of drainage increases. This is even more easily seen in Fig. 3, where the results are expressed as a function of the number of cells capable of lysing 15% of the target cells (lytic units): When the data are expressed in terms of lytic units, the increase with duration of drainage becomes exponential rather than linear. The results are shown for three individuals tested during chronic drainage.

During the same period in which there is an exponential increase in the NK activity, mature T cells, as defined by the ability to form low-affinity (4 ° C) E-rosettes, are decreased in both relative and absolute number (Fig. 4). Mature T-cell function, defined by the incorporation of tritiated thymidine in response to PHA and ConA, also falls in this period. It is interesting to note that the incorporation of thymidine in response to pokeweed mitogen (PWM), to which both T and B cells respond, remains unaffected by chronic drainage. The response of T cells to specific alloantigens is also markedly decreased by chronic thoracic duct drainage (Fig. 4). This decrease is obvious not only as a reduction in the proliferation of T cells in response to alloantigen stimulation, but also as a reduction in the generation of cytotoxic effector cells.

144

Discussion

NK activity has been defined in a number of different ways. Since our original observations that human PBL can lyse a variety of tissue culture lines (tumor-derived and other) without a known previous exposure to the antigens involved, we have used the 4-h 51Cr release assay and the K-562 cell line as a model system to further explore NK activity. Others have used different targets or longer incubations, or defined NK activity in a variety of systems. However, K-562 is the current standard target for human NK [1]. Although the acitvity of normal lymphocytes in cytotoxicity is clearly a reproducible phenomenon, the choice of the systems with which to investigate this activity is arbitrary and may yield different results.

In this report, the levels of cytotoxicity did not vary greatly with age but were highest in the younger and older age segments of the population tested. This is in agreement with our preliminary experiments [23], but is in marked contrast with rodents, which express little natural CMC early in life and demon- strate declining reactivity with increased age after a peak during young adulthood.

Examination of the levels of cytotoxicity elicited by lymphocytes from a large panel of donors revealed the existence of both high responders and low responders. These subpopulations were not clearly bounded as there was a continuum of responses between them. Individuals who had high or low responses reproducibly had high or low responses on repeated testing. An examination of previous data concerning the levels of natural CMC in various strains of mice shows that there are both high- and low-responder strains of mice, with some strains whose responses lie between the two subpopulations [7]. These data suggest that the level of natural CMC expressed by an individual's lymphocytes is under some genetic control. An association between reac- tivity and HLA haplotype has been reported [30]. Male donors with the HLA-A3 B7 haplotype had decreased reactivity, but this was not true of females with the same haplotype. Increased reactivity was associated with the HLA-B12 haplotype. We had previously failed to find any difference in the levels of reactivity related to the race of the donors, while other workers have reported that black donors are significantly more reactive than white donors [6] against certain tumor-derived cell lines. The exper- iments reported in this paper fail to show any significant difference in the levels of reactivity between males and females. This is in contrast to earlier reports that males were more reactive than females [30].

J. T. Forbes et al.: Dynamics of Natural Cytotoxicity

To test the possible significance of fetal antigen sensitization of maternal lymphocytes contributing to the NCMC, lymphocytes from pregnant or non-preg- nant females were compared with lymphocytes from nulliparous females. Since there was little difference in reactivity between these groups during the child-bearing years, it must be concluded that sensitization to fetal antigens is not a major contri- butor to NCMC on the K-562 cell line. However, multiparous women of child-bearing age have lower NCMC values than their nulliparous counterparts. In addition, the observation that pregnant females have lower NK activity than age-matched non-gravid women suggests some suppression of this activity during pregnancy.

Human cord blood lymphocytes were found to be reactive in NCMC suggesting that humans are born with this activity, unlike rodents, in which NCMC is absent at birth. Furthermore, the cytotoxicity exhib- ited by human cord blood lymphocytes is also separable into high and low categories, and thereby reflects early partition into the subpopulations seen in adults.

Human TDL were found to have increased levels of NK activity as the duration of drainage increased. Mature T-cell levels, as indicated by the formation of spontaneous E-rosettes, response to the T-cell mito- gens PHA and ConA, and the proliferation and generation of cytotoxic effector cells in response to alloantigens, were decreasing during this same period of drainage. Thus, NK activity could be shown to be increased during a period of chronic depletion of the mature T-cell subpopulation and to decline with T-cell maturation. Our results are consistent with this observation. An accessory cell type such as the monocyte could also be simultaneously depleted, contributing to the decrease in T-cell function (response to PHA and ConA). However, the con- centration of monocytes in human thoracic duct drainage products was so low that this could not be adequately quantitated.

The data reported here illustrate that although the subpopulation of cells responsible for NK activity in man and rodents seems similar (Fc receptor-pos- itive, low-affinity sheep erythrocyte receptor-nega- tive, non-adherent, non-phagocytic, and complement receptor-negative) there are definite population dif- ferences between the species. In addition, it is clearly apparent that one must account for NK activity before specific cytotoxicity in tumor-associated sys- tems can be reliably assessed.

Acknowledgements. This study was supported in part by Contract NCI-NO1-CB-74166 and grant CA 23477 from the National Cancer Institute.

J. T. Forbes et al.: Dynamics of Natural Cytotoxicity 145

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Received August 18, 1980/Accepted February 10, 1981