Cancer Immunol Immunother (1981) 10:261-263 a ncer mmunologyand mmunotherapy

© 8pringer~Verlag 1981

Lymphocytes from Children and Infants Mediate Natural Cell Mediated Cytotoxicity*, ** (Cytotoxicity)

Eda T. Bloom**

Department of Medicine, University of Southern California, and the USC Cancer Center, Los Angeles, California 90033 Geriatric Research, Education, and Clinical Center, V.A. Wadsworth Medical Center, Los Angeles, CA 90073, USA

Summary. Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRE-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lympho- cytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in deve- lopment is consistent with early mobilization of the policing mechanism.

Introduction

Blood lymphocytes frommost donors are cytotoxic against a variety of normal and malignant target cells in vitro. The phenomenon has been termed natural cell mediated cytotoxicity (NCMC) since it can be measured in normal individuals as well as in a variety of patients. Many investigators believe that NCg~ may function in immunological surveillance against cancer (3). If this is true, NCMC effector cells would be expected to arise early in development. Since human NCMC has been shown to be a polyspecific system (1,8,9), effec- tor cells may develop through early and subse- quent sensitization and response to a variety of antigenic stimuli. Alternatively, nonselective N]< recognition mechanisms (3) would also be expected to develop early if the surveillance theory is correct. In order to determine how early in life the NCMC effector cells develop, NCMC by lymphoc~es from infants and children was compared to that by adults against two lymph- oblastoid cell lines. No correlation between age and NCMC levels could be discerned.

* This work was supported by Grant CA 25765 from the National Cancer Institute

** Partially supported by a Grant from the Concern Founda- tion.

Present Address: Geriatric Research, Education and Clinical Center, 691/11G, V.A. Wadsworth Medical Center, Los Angeles, California 90073 and the Geriatric Center, V.A. Wadsworth Medical Center, Los Angeles, California 90073

Materials and Methods

Effector Cells

The residual blood after routine blood counts from children and infants was obtained from the hematology laboratory in the Pediatric Pavilion of Los Angeles County General Hospital. Blood from children with abnormal total or differential leukocyte counts was excluded, and donors were essentially normal. Usually less than i ml blood was obtained from each donor. The volume of blood available was the limiting factor in the number of tests done with individual donors. Pediatric donors ranged in age from 2 weeks to 17 years. Since this blood was necessarily drawn one day before testing in vaeutainer tubes con- tainingEDTA, blood from normal adult donorswas obtained similarly and tested in parallel for comparison.

Lymphocytes were isolated by centrifugation over Eicoll-Hypaque and purified by treatment with carbonyl iron. Suspensions were prepared in RPMI-1640 containing 15% fetal calf serum. Prep- arations from day-old blood which had been stored refrigerated or at room temperature were usually >90% ly~hocytes and_)97% viable. Although these effector cell suspensions generally contained more contaminating cells than similar preparat- ions from fresh blood, all suspensions used in the present study were of comparable composition.

Cytotoxicity Test

A 4-hour chramium release test was used. Target cells were K562, obtained from a chronic myelocy- tic leukemia (4), and CCRF-CEM (CEM), a T cell lymphoblastoid cell line (7). They were labeled with Na51Cr04 (Amersham~ Arlington Heights, IL), and suspended at 5 x i0 ~ cells/ml in RPMI-1640 with 10% fetal calf serum. One tenth ml was added to 0.1 ml of the lymphocyte suspension in U-bottom Microtiter plates (Dynatech, Alexandria, VA) and the plate was centrifuged at 600 xg for two minutes, incubated for 4 hours at 37°C in 5% C02, and recentrifuged. One tenth ml was collec- ted from each well and counted in a ganma counter.

0340-7004/81/0010/0261/$ 01.00

262 E.T. Bloom: Natural Cell Mediated Cytotoxicity in Children

Effector cell: target cell (EC: TC) ratios were 40:1 and 20:1. Lower ratios resulted in little significant killing. The test was run in quadru- plicate and cytotoxicity calculated as follows:

% Cytotoxicity =

Experimental Release - Spontaneous Release i00 x

Maximum Release - Spontaneous Release

Results and Discussion

Results of cytotoxicity tests against K562 are shown in Figure 1 , and those against CEM in Fig- ure 2. Although NC94C does differ between groups, the differences are not statistically significant by the Student's _t test. Moreover, the variation is not in the same direction when results are compared using the different EC:TC ratios or different target cell lines. For example, the 0-i year age groups had the least NCMC of any group against CEM at an EC:TC ratio of 20:1, in- termediate at 40:1, and the greatest NCMC of any group against CEM at an EC:TC ratio of 40:1. Thus, no consistent difference was observed bet- ween NCMC by donors of different ages. Similarly,

lymphocytes from older individuals have been shown to mediate NC/V~ cc~parable to that by lym- phocytes from young adults (i0).

The lymphocytes obtained from infants and chil- dren effected NCFC comparable to that by adults. The youngest age group was comprised of children aged 2 weeks, 1 month, 5 1/2, 8, 8, 8, 9, 9 1/2, i0, i0, ii, and 12 months. This finding is con- sistent with the report that blood from infants, children, and adults contains comparable percen- tages of B and T cells, and that the juvenile and adult T cells mediate a variety o£ functions at the same levels (2). Lymphocytes from cord blood also mediate NC~3; however, whether such cells are of maternal or newborn origin is uncertain (5). The present data are in contrast to the demonstration that the incidence of cytotoxic activity against noncultured leukemia cells was greater in adults than in older children (6).

That lymphocytes from very young children medi- ate NCMC against cultured susceptible tumor tar- gets as effectively as those from adults and older children suggests that NC~S effector cells may develop extremely early in life and/or during gestation. This implies that NCMC is ontogeneti- cally primitive, which would be predicted by the hypothesis that it functions in i~mundlogical surveillance against cancer.

I00

90

80

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4°

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20

I0

CYTOTOXICITY AGAINST K562

A E:T = 40:]

I . ,,. ,,

±&

I i I I I

0-I I-5 6-10 11-17 22-55

L B E:T:20:I

T T :

0-1 1-5 6-10 11-17 22-55 AGE(YEARS)

Fig. I. Cytotoxicity against K562 at ef-

fector: target ratios of 40:1 (A) and

20:1 (B) is shown for individuals aged

O-1, I-5, 6-10, 11-17, and 22-54. The

means in each group are indicated by the

horizontal line. The standard deviation

is shown by the error bar on the left

of each group ( ) , and the standard

error by the bar on the right ( ) .

For each ratio, no group is significantly

different from any other

CYTOTOXICITY AGAINST CEM

A E:T: 40:1

T ~.0

I

T

i • I0

i" - - L

0-1 I-5

i

i i . l '1

1-. i

, b

± . I 'I

B E:T:20:~

, T I T i

" ; ' I . T T

6-~ 11-17 22-55 0-1 I-5 ~ 11-17 22-~ AGE(YEARS)

Fig. 2. Cytotoxicity against CEM at ef-

fector: target ratios of 40:1 (A) and

20:1 (B) is shown for individuals aged

O-I, I-5, 6-10, 11-17, and 22-54. The

means in each group are indicated by the

horizontal line. The standard deviation

is shown by the error bar on the left of

each group ( ), and the standard error

by the bar on the right ( ). For each

ratio, no group is significantly different

from any other

E. T. Bloom: Natural Cell Mediated Cytotoxicity in Children 263

Acknowledgement. < thank Dr Darleen Powars for her cooperation in providing the pediatric blood samples.

References

I. Callewaert DM, Kaplan J, Johnson DF, Peterson ~ Jr (1979) Spontaneous cytotoxicity of cul- tured human cell lines mediated by normal peripheral blood lymphocytes. II. Specifi- city for target antigens. Cell Immunol 42: 103

2. Handzel ZT, Levin S, Dolphin A, Schlesinger M, Hahn T, Altman Y, Schechter B, Shneyour A, Trainin N (1980) Immune competence of newborn lymphocytes. Pediatrics 65:491

3. Herberman RB, Djeu JY, Kay HD, Ortaldo JR, Riccardi C, Bonnard GD, Holden HT, Fagnani R, Santoni A, Puccetti P (]979) Natural killer cells: Characteristics and regulation of activity. I/nmunological Reviews 44:43

4. Lozzio CB, Lozzio BB (1975) Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45 : 321

5. Pross HF, Baines M3 (1977) Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. Cancer Inmunol Immuno- ther 3:75

6. Rosenberg EB, Herberman RB, Levine PH, Halter- man RH, McCoy JL, Wunderlich JR (1972) Lympho- cyte cytotoxicity reactions to leukemia-asso- ciated antigens in identical twins. Int J Cancer 9 : 648

7. Smith LW, Smith P~{, Hathcock KS et al. (1974) T and B lymphocyte markers in lymphoid cell research and in human disease. Proc. 8th Leukocyte Cdlture Conference. K. Lindahl- Kiessling and D. Osaba (eds) Academic Press, New York, p 127

8. Takasugi M, Akira D, Takasugi J, Nickey MR (1977) Specificities in human cell mediated cytotoxicity. J Natl Cancer Inst 59:69

9. Takasugi M, Hardiwidjaja M, McAllister R, Peer M (1979) Search for specificity in natural cell-mediated cytotoxicity. J Natl Cancer Inst 63:1299

i0. Takasugi M, Mickey MR, Terasaki PI (1973) Reactivity of lymphocytes from normal persons on cultured tumor cells. Cancer Res 33:2898

Received June 11, 1980/

Accepted February 19, 1981