Natural killer activity and lymphocyte subpopulations in patients with primary humoral and cellular immunodeficiencies

Natural

Killer Activity and Lymphocyte Subpopulations Patients with Primary Humoral and Cellular lmmunodeficiencies

in

Natural killer (NK) activity against K-562 and MOLT-4 cell lines and the distribution of T lymphocytic subpopulations were analyzed in 24 patients with primary immunodeficiencies. Theophylline sensitive sheep rosette-forming cells (T-sens) and FcIgG receptor-positive (FcIgG+) lymphocytes were normal in the majority of patients with immunoglobuhns and B-cell abnormalities. NK activity was decreased in two patients with pure T-cell deficiency, One patient with severe combined immunodeficiency (SCID) lacked T-sens, FcIgG+ cells. and NK activity. In this case, TP-5. a synthetic thymopoietin-derived pentapeptide. partially corrected these abnormalities. These data suggest that the maturation of NK function can be induced by thymic hormones. The significant positive correlation between T-sens, FcIgG+ cells, and NK activity suggests a partial identity of the lymphocytic subpopulations detected by these surface markers and that such populations could mediate NK function in humans.

INTRODUCTION

In recent years it has become possible also in humans to characterize functional s&populations of T lymphocytes by surface markers (l-4). T cells can be enumerated on the basis of their ability to bind the Fc portion of either IgG (T,) or IgM (T,) (1). More recently, it has been demonstrated that theophylline inhibits the property of some T lymphocytes to bind sheep red blood cells (SRBC) (5). These T sensitive (T-sens) cells exert in ritvo suppressor activity on T-B (2) and T-T (6) cooperation. On the other hand, T cells whose ability to rosette is resistant (T-res) to theophylline inhibition, exert inducer effects (2). A major overlapping between T-sens and TG, and between T-res and TM cells was also shown (2). Increasing evidence has been produced showing a possible relationship between imbalances of T subpopulations and the pathogenesis of immunodeliciency diseases (7-9). The investigation of natural killer (NK) activity (lo- 12) is also considered to be of great importance in the evaluation of the cellular immune function since it appears to be related to protection from viral infections (13) and tumors (10, 11). However, the relationship between NK cells and T,B, non-T. and non-B subpopulations, is not yet well clarified in humans. Some reports indicate that NK cells have no surface Ig and do not form sheep rosettes, whereas they bear highaffinity receptors for the Fc portion of IgG (10, 11). On the other hand, by using optimal conditions for the formation of sheep rosettes, it has been demonstrated by others that NK cells do have low-affinity SRBC receptors (14). 12 0090.12?9/81/100012-08$01.00/0 Copyright G 1981 by Academtc Press. IK .A11 rights of reproduction in any form re5erved.

NK

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13

Primary immunodeficiencies seem to represent comparatively “clean” deletions of a given subpopulation of lymphocytes and we therefore considered it important to analyze NK activity and T-cell subpopulations in these patients together with other immunological and clinical parameters. MATERIALS

AND METHODS

Normal controls and patients. Twenty normal controls (age/sex matched with the patients) were chosen among laboratory personnel and children admitted to our outpatients department for vaccination. Twenty-four patients with primary immunodeficiencies, classified according to WHO criteria (15), were studied: 5 had common variable hypogammaglobulinemia (CVH), 3 x-linked agammaglobulinemia, 10 selective IgA defect, 2 hyper IgE syndrome, 2 a pure T-cell defect, 1 a severe combined immunodeficiency (SCID) with circulating B cells, and 1 a SCID. Diagnosis was based on history, clinical features, cytomorphological peripheral blood and bone marrow smears, tonsillar and intestinal biopsies. The assessment of both the number and function of T and B lymphocytes was repeated several times. Reponses to T-cell mitogens were undetectable in patients with T-cell defects and SCID; similarly, the same was also found in patients with x-linked agammaglobulinemia and SCID, when the Cowan 1 strain from Staphylococcus aureus was used as selective B-cell mitogen. Some of the patients with CVH and IgA defect were treated by therapy with commercial immunoglobulins. Ig levels were measured as reported by Mancini et al. (16). Following in \,itro tests, patient M.S., SCID without B cells, was the only patient of our series to be treated with TP-5, a synthetic pentapeptide which possesses the biological activity of thymopoietin (17) (kindly provided by Dr. J. Symoens, Janssen Pharmaceutics, Beerse, Belgium). Lymphocyte subpopulations were evaluated at the time of the diagnosis and after therapy that was administered according to the following schedule: 2 mg/day for 3 weeks and then 2 mg twice weekly for 96 days, im. Isolation of PBL. Peripheral blood lymphocytes (PBL) were isolated from heparinized venous samples onto a Ficoll-Isopaque (FIsp) gradient, as described by Aiuti et al. (18). PBL were washed in Hank’s balanced salt solution (HBSS) and then resuspended at a concentration of 5 x 106/ml in medium RPM1 1640 (Gibco, Grand Island, N.Y.). Before performing EA rosettes, monocytes were removed by magnet, after incubation of the cell suspension with carbonyl iron, rotating at 37°C for 40 min. Sheep rosette-forming cells (SRFC) were assayed as described elsewhere (18). Surface membrane Ig(SmIg) were determined as reported (18) using a goat anti-human Ig (Cappel Lab., Cocharaneville, Pa.). Test for T-res and T-sens cells. T-res cells were enumerated as previously described (5, 6). In brief, PBL were incubated with a predetermined concentration of theophylline (Calbiochemical, La Jolla Calif.) for 90 min at 37°C. T-res were then evaluated by performing the SRFC test. T-sens cells were calculated as the difference between total SRFC and T-res rosettes. Tests Jbr the detection of EA rosettes. Ox red blood cells (ORBC) were sensitized by hyperimmune antisera, raised in rabbits. Two different antisera were used: one was a purified IgG anti-ORBC obtained by repeated injections of 1oR

14

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ORBC, while the other serum. prepared according to Mayer (191. was a purified IgM anti-ORBC. Rossettes were performed by pelletting equal volumes of a 2% suspension of ORBC, sensitised with a predetermined amount of antiserum and PBL at a concentration of 2 x lOYm1 of RPM1 1640. PBL were incubated overnight at 37°C in 5% CO, before performing the EAoxIgM rosette (RFC) assay. EAoxIgG RFC were counted as described for SRFC, after an incubation of I5 min on ice. EAoxIgM RFC were incubated for 30 min on ice, before reading rosettes. Fc receptor bearing cells were also evaluated using 0 Rh+(CCDeel human red cells. coated with a subagglutinating dilution of an anti-CD antiserum (Ortho, Raritan, N.J.) (EA human, EAh). Rosettes were then performed as already described. centrifuged, and read immediately. NK assay. Effector PBL. isolated through a FIsp gradient and washed twice in HBSS, were suspended in RPM1 1640, supplemented with 10% FCS and antibiotics (complete RPM1 1640). Monocytes were removed as described above. Target cells were the myelogenous derived cell line K-562 and the MOLT-4 cell line. Targets were labeled with 100 &i of Na,“‘CrO, (Searle, Amersham, England) in 0.1 ml of medium at 37°C for 60 min. The cells were then washed and distributed in round-bottomed plates (Linbrol at a concentration of 5 x 1Wwell in 50 ~1. Effector cells were added to triplicate wells to give effecter/target (E/T) cell ratios of 100: 1, 50: 1, 25: I, and 12. 5: 1. Effecters were also added in a final volume of 50 ~1. The plates were incubated for 18 hr at 37°C in a 5% CO, incubator.’ then 50 ~1 of each supernatant was removed, transferred to glass tubes, and counted in a Beckman gamma spectrophotometer. Percentage of specific lysis was calculated as follows: % of lysis = mean cpm experimental release - mean cpm spontaneous release mean cpm maximum release - mean cpm spontaneous release x loo. Spontaneous release was obtained by adding 50 ~1 of medium alone to the targets and never exceeded 5 to 8%: maximum release was obtained by lysing the same cells with Nonidet P40. Cdculution oj’lytic units. Dose-response curves of specific cytotoxicity were determined by plotting specific JICr release versus the number of effector lymphocytes. The number of lymphocytes necessary to lyse 30% of the target cells was referred to as one lytic unit (LU). Determination of 3% LU was done graphically. RESULTS

Table 1 summarizes clinical and immunological data, including the values of T-sens, EAoxIgM and IgG, and EAh rosettes, in children with primary immunodeficiencies. Lymphocyte subpopulations were generally within normal limits in patients with CVH, x-linked agammaglobulinemia, and selective IgA deficiency, except for cases 8 (x-linked agammaglobulinemia), 17, and 18 (IgA defect), which had decreased levels of T-sens. EAoxIgG, and Ekh rosettes. The percentages of cells with these surface markers were increased in the two children with hyper-IgE syndrome, strongly reduced in patients with T-cell defect and in a I This

incubation

time

was

preferred

in order

to detect

possible

fully

expressed

NK

defects

(14).

Diagnosis

” Not done. b Variable according

to the age.

CVH CVH CVH CVH CVH x-Linked agammaglobulinemia x-Linked agammaglobulinemia x-Linked agammaglobulinemia IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect Hyper-IgE Hyper-IgE T-Cell defect T-Cell defect SCID with B cells SCID

Normal values (mean -t SD)

9. 10. II. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22. 23. 24.

8.

7.

1. 2. 3. 4. 5. 6.

Case

CLINICAL

M M M F F M F F M F F F M M F M

M

M

F M M F M M

Sex

AND

8 10 9 8 16 8 7 6 IS 36 9 9 0.2 6 0.5 1.6

13

9

22 42 22 22 43 7

(years

Age l

IMMUNOLOGICAL

69 27.2

60 70 73 66 60 63 66 61 53 59 61 38 5 47 32 35

64

84

56 89 75 83 61 59

SRFC (% )

DAIS

TABLE

I

17.2 +3

ND” ND ND ND ND ND ND ND 3 5 22 26 I 7 8 0

6

21

14 I9 26 13 10 20

&ens (%)

IN PAI-I~NFS

WIIH

12.3 k 1.7

19 23 I 6 ND 0

I2

I2 11 10 12 II I5 11 II 4

7

22

12 20 23 12 9 19

I&

EAox

PRIMARY

49.7 22.3

52 55 49 51 48 48 49 51 50 51 50 40 4 49 ND 34

50

50

49 40 49 53 50 46

kM

(S)

10 t4

10 11 9 IO 9 11 II 10 IO 6 10 16 5 6 ND 0

IO

10

IO 12 10 11 IO 13

EAh t’;)

IMMUNODEFICI~NCI~S

12.3 23.1

I3 11 12 IO 11 12 10 12 23 I1 8 8 8 10 IS 2

0

0

3 2 12 3 12 0

smlg (S’r)

2 460 265 21 111 0 0

760 1064 3560 415 900 150 22 -11

0 0 0 0 0 0 0 0

0

0

60

30 0

-0

44 60 40 170 100 101 3

122 190 272 216 208 200 148 199

0

4

34

0 5 0 0

M 0 0 0 0

A

Ig tmgidll

1049 910 989 1754 1610 1394 1440 1106

250

190

360 IO 400 130 280 100

G

Serum

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Al-.

case of SCID with B cells, and absent in one patient with SICD. Smlg were decreased in three cases of CVH, as also reported in other similar series of patients ( 15). Table 2 reports the values of NK activity against the K-562 and MOLT-4 cell lines in the same patients. NK function was normal in patients with defects of the B-cell lineage, except for a marked reduction in one patient with IgA defect with hyper-IgE syndrome and in the one with SCID with B cells. It is noteworthy that this latter patient’s NK function was evaluated after fetal liver and thymus transplant. In two cases with T-cell defect and one with SCID. NK activity against both targets was extremely reduced. In these cases the assay was repeated several times with consistent results. A significant positive correlation was found between: EAh rosettes and NK activity (r = 0.87); EAoxIgG rosettes and NK activity (r = 0.76); T-sens activity and NK activity (1. = 0.70): and. finally. ‘I’-sens TABLE NK AC-IWITY.

EXPRESSED

AS

LY

PATIENTS

IK

UNII-S,

WITH

2

AGAINST

PRIMARY

rHt K-562

AND

MOLT-4

CELL

LINU

IN

IMMUNODEFIUENTIIZS

NK activity Case I.

2. 3. 4. 5. 6. 7.

8. 9. IO. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Normal values (mean + SD) I’ Not done.

Diagnosis CVH CVH CVH CVH CVH x-Linked agammaglobulinemia x-Linked agammaglobulinemia x-Linked agammaglobulinemia IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect IgA defect Hyper-IgE Hyper-IgE T-Cell defect T-Cell defect SCID with B cells SCID

Sex

K-562

MOLT-4

6.2 5.2 6.9 7. I 8.3 12.5

4.8 5.5 6.4 6.6 IO N 0”

M

9. I

N II

M

9. I

ND

M M M F F M F F M F F F M M F M

6.2 6.3 5.5 4.3 6.9 8.0 6.6 5.4 6.6 2.0 5. I 23.2 2.0 3.3 19.2 0.3

ND ND ND ND ND ND ND ND ND ND 4.8 25.2 2.1 4.0 ND 0.2

8.4 t 2.9 5.2 t 1.9

8.0 i 2.1 4.9 Ifr 1.8

Males Females

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IMMUNODEFlClENClES

and EAoxIgG rosettes (r = 0.89). One patient with SCID (case 24) was treated with TP-5, since this drug had been shown to induce in vitro the maturation of non-T, non-B PBL into mature T cells (20). The effects of this therapy on the immunological parameters are reported in Table 3. A significant increase of SRFC and of NK activity, together with the appearance of T-sens, EAoxIgG, and EAh rosettes was observed. DISCUSSION

In the present report we analyzed NK activity in a number of patients with various primary immunodeficiencies. The results were compared with those obtained by studying surface markers specific for several lymphocytic subpopulations. Our data indicate that B cells do not play a major role in naturally occurring cellular cytotoxicity, since patients with x-linked agammaglobulinemia, lacking circulating B cells, exerted normal NK activity. This agrees with previous reports (21-23). On the other hand, NK activity positively correlated with EAh rosettes, a marker for human lymphocytes with high-affinity receptors for the Fc portion of IgG (24), and also correlated, even if in a slightly lesser degree, with EAoxIgGpositive and T-sens cells. Reduced numbers of cells with these surface markers and concomitant impairment of NK activity were found in two patients with T-cell defect and in one with SCID. In this latter case the administration of a synthetic thymic hormone, TP-5, induced the appearance of EAh+, EAoxIgG+ , and T-sens cells, as well as an increase of NK activity. The observation that patients with primary T-cell defects had decreased NK activity, as already reported by others (21), gives rise to the question whether or not NK cells belong to the T-cell lineage. This hypothesis has been supported by others (IO). Our data indicate a significant relationship between NK function and the frequency of a subpopulation of sheep rosetting cells. The ability of these cells to rosette is, however, characterized by sensitivity to theophylline (T-sens cells). It has been reported that T-sens cells exert in vitro suppressor activity and correlate with T cells bearing receptors for the Fc portion of IgG (T,;) (2, 6). To our knowledge, no data are available on the relationship between T-sens and NK cells. Our data in patients with primary immunodeficiencies support the concept that NK function is mediated by sheep rosetting cells and confirm a previous report by West et al. (14) that cells mediating NK activity have low-affinity receptors for sheep erythrocytes. Although the sheep rosette marker has long been considered to be specific for T cells (18) it has been suggested recently by the use TABLE EFFECTS

OF TP-5

TREATMENT

ON THE

3

IMMUNOLOGICAL

EAox SRFC (5%) No therapy TP-5 (14 days) TP-5 (95 days) ” Expressed

as lytic

35 73 83 units.

T-sens FCC) 0 5 9

IiS 0 5 7

PARAMETERS

(3i) kM 34 43 47

OF CASE

NK

24 (SCID) activity”

EAh (%‘c)

KS62

MOLT-4

0 4 7

0.3 2.2 3.3

0.2 2.6 3.4

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of monoclonal antibodies that a subpopulation of human peripheral mononuclear cells (FcIgG positive), sharing receptors for sheep erythrocytes and a monocytic specific antigen (251, may exert NK function. On the other hand. de Vries c’t rrl. (26) have shown that PBL obtained after careful depletion of monocytes retain NK activity against the K-562 and MOLT-4 cell lines. but not against nonlymphoid targets. We found normal NK activity in patients with CVH, in agreement with previous reports (21-23), whereas it was discovered to be impaired in 1 out of 10 patients with selective IgA deficiency. The association between NK and IgA deficiencies. difficult to interpret, has also been described by Lipinski et ctl. (21). Intriguing mechanisms are involved in the pathogenesis of IgA deficiency ( I_0 and a lack of specific IgA suppression has been postulated for some of these cases (27). T-sens cells were also depressed in the patient lacking NK function and this observation again supports the close relationship between NK activity and the frequency of theophylline-sensitive lymphocytes. It has been frequently reported that NK activity is markedly impaired in patients with SCID (22, 28). In our series, however, one patient affected by SCID with B cells had significantly higher values than normal controls. In this case NK function was tested only after immunological reconstitution was attempted by fetal liver and thymus transplantation. It cannot therefore be excluded that NK cells were of donor origin, and that the abnormally high NK activity reflected an ongoing graft versus host reaction. In fact the patient had the clinical features of such reaction (blood eosinophilia, skin rash). The other patient with SCID had neither circulating EA+, EAoxIgG+. and T-sens cells nor appreciable levels of NK activity. In this child, therapy with a synthetic derivative of thymopoietin, TP-5 (17). led to a marked improvement of all these parameters. This finding suggests a relationship between thymic hormonal influence and the maturation and/or function of NK cells. Some investigators failed to demonstrate the effect of thymic hormones on NK function (29, 30), while others were successful (3 I). This discrepancy could be explained by the different experimental conditions used and, more likely, by the heterogeneity of the hormones employed by the different groups. REFERENCES 1. Moretta, L., Webb. S. R.. Grossi. C. E., Lydyard. P. M.. and Cooper, M. D..J. Elrp. M&. 146, 184, 1977. 2. Shore, A., Dosch, H. M.. and Gelfand, E. W.. Nofurr @on&m) 274, 586, 1978. 3. Evans, R. L.. Breard, J. M.. Lazarus. H.. Schlossman, S. F., and Chess. L.. J. ELrp. Med. 145. 221, 1977. 4. Reinherz. E. L., Kung. P. C.. Goldstein, G., Levy, R. H., and Schlossman. S. F., Pro<,. !Ytrt. Acad. Sci. USA 77, 1588. 1980. 5. Limatibul, B., Shore, A.. Dosch. H. M., and Gelfand. E. W.. C/in. Exp. Immunol. 33, 503, 1976. 6. Pandolfi, F., Strong, D. M.. Slease. R. B.. Smith, M. L.. Ortaldo. J. R.. and Herberman. R. B.. Blood 56, 653, 1980. 7. Gupta, S., and Good, R. A.. Clin. Immonol. fmmunopathnl. 11, 292. 1978. 8. Waldmann, T. A., Durm, M.. Blackman. M., Blease, R. M., and Strober. W.. Luncet 2.609, 1974. 9. Moretta. L., Mingari, M. C., Webb, S. R.. Pearl, E. R.. Lydyard. P. M., Grossi, C. E.. Lawton. A. R.. and Cooper. M. D., Ercr. J. Immunol. 24, 696. 1977. 10. Kiessling. R., and Wigzell, H., Immunol. Rev. 44, 165, 1979.

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19

Il. Herberman, R. B., Djeu, .I. Y., Kay. H. D., Ortaldo, J. R., Riccardi, C., Bonnard. G. D.. Holden. H. T.. Fagnani. R.. Santoni, A., and Puccetti, P., Im~n~no/. Rrl,. 44, 43. 1979. 12. Jensen. P. J.. and Koren, H. S.. J. Immunol. 124, 395, 1980. 13. Santoli, D.. and Koprowsky, H., Imtwnol. Rev. 44, 125. 1979. 14. West, W. H., Boozer, R. B., and Herberman, R. B., J. Iwmunol. 120. 90. 1978. 15. WHO Bulletin, “Immunodeficiency.” Technical report series 630. Geneva, 1978. 16. Mancini. G., Carbonara, A. 0.. and Heremans. J. F., I,nr,lrrn~~~~/~r,,nistr~ 2, 235. 1965. 17. Goldstein, G.. Scheid. M. P.. Boyse. E. A.. Schlesinger. D. M.. and Van Wauwe. J.. Science 203, 1309.

1979.

18. Aiuti. F., Cerottini, J. C., Coombs. R. R. A., et u/.. Sc,unJ. J. I~n/rt~no/. 3, 521. 1974. 19. Kabat. E. A.. and Mayer, M. M.. “Experimental Immunochemistry,” Thomas, Springfield. III.. 1961. 20. Aiuti, F.. Businco. L., Rossi P.. and Quinti, I.. Ltrrnrr 1. 19, 1980. 21. Lipinski. M., Virelizier. J. L.. Tursz. T., and Griscelli. C.. ENT. J. Immrrno/. IO, 246. 1980. 22. Koren. H. S.. Amos, D. B., and Buckley. R. H.. J. Imm~~70/. 120. 796, 1978. 23. P~OSS. H. F., Gupta, S., Good, R. A., and Baines, M. G.. Cell. I~r)n~(no/. 43. 160. 1979. 24. Froland. S.. and Natvig. J. B.. 7’rrrrrspl~~~~. Rev. 16. I 14. 1973. 25. Reinherz, E. L.. Moretta. L., Roper, M.. Breard. J. M.. Mingari. M. C.. Cooper. M. D.. and Schlossman, S. F.. J. Exp. Med. 151, 969, 1980. 26. De Vries. J. E., Mendelsohn. J.. and Bont. W. S.. Ntrolre (Lor~lo,i) 283, 574, 1980. 27. Atwater, J. S., and Tomasi. T. B., Jr.. Clin. Immunol. Itnmunopothol. 9. 379. 1978. 28. Lopez. C., Soreli, M.. Kirkpatrick. D.. O’Reilly. R.. and Ching. C.. Lcr)~cet 1. 1103. 1979. 29. Axberg. I.. Gidlund. M., Grn. A.. Pattengale. P., Riesenfeld. I., Stern, P.. and Wigzell. H., In “Thymus. Thymic Hormones and T Lymphocytes” (F. Aiuti and H. Wigzell. Eds.). p. 155, Academic Press. London, 1980. 30. Herberman. R. B.. Ortaldo, J. R., Holden. H. T., Djeu. J. Y.. Mattes, J.. Brunda. M.. Riccardi. C.. and Santoni. A., In “Thymus. Thymic Hormones and T Lymphocytes” (F. Aiuti and H. Wigzell. eds.). p. 165. Academic Press. London, 1980. 3 I. Bardos. P.. Carnaud, C.. and Bach. J. F.. C. R. A~?I~. .S(;. (L)) ~~~~;,r289, 1251. 1979. Received December 31. 1980; accepted April 17. 1981