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Lymphocytotoxic T lymphocytes in a patient with B-chronic lymphocytic leukemia and pure red cell aplasia
Leu£'emia tl~¢,ar~'h Vol. 9, No. 9, pp. 1189-1194, 1985. Printed in (;real Ilrilain.
0145-2126/8553.00 ~ .(X) (~ 1985 Pergamon Press Lid.
LYMPHOCYTOTOXIC PATIENT
WITH
LEUKEMIA
AND
'F
I_YMPHOCYTES
B-CHRONIC PURE
RED
IN
A
LYMPHOCYTIC CELL
APLASIA
Nt!II [~. KAY, MARl'IN M. ()KIN, .l~}.x(} A',cf N'-.;',,(t:.tltd MANt;I:I. E. KAPI.AN Veterans Administration Mcdic~d (.'Clllt_'t-.Minneapolis, Mim~csola and the Lluiversity of Minnesota Medical School. \linneapolis, Minnesota, U.S.A. (Received 19 Jn(v 1984..,Ict.t7~ted 29 .hmuat3' 1985)
Abstract--The peripheral blood T cells of a hyperlranq'uscd patient with B-chronic lymphocytic leukemia and pure red cell aplasic were found to exhibit tmusual spontaneous cytotoxic activity in vitro. The patient's E-rosette positive cells wcrc cytotoxic for K562 (cultured human crythroleukemia cells) and allogeneic B and T lymphocytes freshly isolated from the peripheral blood of normal and CLL donors. They failed to kill autologous B cells, erythroid progenitors present in allogencic bone marrow, and a number of cultured human tumor cells (Maline, CAKI) even after prolonged (36 h) co-culture. Peripheral blood T cells isolated from normal controls, other CLL patients, and hypertransfused individuals (tl = 13) did not exhibit spontaneous lymphoc.vtotoxic activity. Circulating cytotoxic T cells having the ability to kill freshly isolated allogeneic lymphocytes have, heretofore, not been reported in humans. Our findings suggest that among this patient's peripheral blood T cells, there exists a subpopulation of lymphocytotoxic cells that closely resemble cytotoxic T cells generated in vitro after priming with allogeneic target cells. Although the lymphocytotoxic cells could ha~e been induced in this patient by previous HLA-mismatched transfusions, it is possible they may have arisen spontaneously and underly the ~atient's erythroblastopenic state. Key word~: Lymphocytotoxic, T cells, CLL.
INTRODUCTION FRESHLY isolated human T cells are known to contain several cytotoxic subpopulations [2, 10, 13]. These subpopulations which have been defined and distinguished from each other by a number o f criteria are designated as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and antibody-dependent cytotoxic cells (ADCC). Recently we and others have demonstrated a number of peripheral blood T cell abnormalities in patients with typical B-ceU chronic lymphocytic leukemia (CLL) including: (I) increased absolute numbers of T cells [14]; (2) qualitative and quantitative increases in T suppressor cells, with decrease in T helper cells [5, 14, 15]; (3) deficient T-cell colony formation in vitro [8] and (4) depressed NK function [3, 22, 26]. We recently had the opportunity to study a B - C L L patient who presented
Abbreviations: CLL, chronic lymphocytic leukemia; CTL, cytotoxic T lymphocytes; NK, natural killer cells; ADCC, antibody-dependent cytotoxieity; AET, 2-aminoethylisothiouronium bromide hydrobromide; SRBC, sheep erythrocyte red blood cells; Slg, surface immunoglobulin; BFU-E, CFU-E, erythroid precursors; CFU-GM, myeloid precursors. Correspondence and reprint requests to: Nell E. Kay, M.D., Hematology-Oncology Section (I 1IE), V. A. Medical Center, 54th Street and 48th Avenue South, Minneapolis, MN 55417, U.S.A.
with pure red cell aplasia. His peripheral blood T lymphocytes exhibited significant spontaneous cytotoxicity against K562 (a human erythroleukemic cell line) and rapidly killed allogeneic B and T lymphocytes freshly isolated from the peripheral blood of normal donors and other CLL patients. We report here the cytotoxic properties and preliminary characterization of this tmusual, perhaps unique, cytotoxic T cell.
MATERIALS
AND METHODS
Isolation and characterization of peripheral blood B and T lymphoqvtes
Heparinized venous peripheral blood was obtained after informed consent, and the mononuclear cells isolated by Ficoll-Hypaque centrifugation. Monocytes were removed by adherence to plastic. B cells were detected by immunofluorescent staining of surface immunoglobulin (Slg) with rhodamineconjugated rabbit anti-human lgG-(Fab')2 (Cappel Labs, Cochranville, PA) and T cells were assayed by rosetting with 2-aminoethyl isothiouronium bromide hydrobromide (AET) (Aldrich Chemical Co., Milwaukee, WI) treated sheep erythrocyte red blood cells (SRBC) [1, 12]. Rosettes were defined as lymphocytes with three or more adherent erythrocytcs. Purified T-lymphocytes were isolated by rosetting twice with SRBC-AET followed by FicolI-Hypaque centrifugation. T cells were harvested by brief hypotonic lysis (i.e. 30 s exposure with 5000.1 of sterile H20) of the SRBC-AET lym-
1189
1190
Nil, E. KA~et ul.
phoeyte pellet. Non-T cells `.~'ere collected from the interfaces. Isolated T cells `,`.ere > 9 8 % viable and > 9 5 % re-rosetted `.`.ith SRBC-AET. The patient's non-rosetting cells ,.`.ere 92-96% B cells by Slg staining, while control SIg% varied from 78-80% positive. T-lymphocyte subsets were detected using monoclonal antibodies as follows: twice rosetled cells were incubated at 4°C tbr 1 h with various monoclonal antibodies (MAb) (Orthomune, Ortho Pharm Corp., Raritan, NJ): OKT3 (1:100), OKT4{I:I0J, O K T 8 ( I : I 0 ) O K M I ( I : I 0 ) o r O K T I I ( l : 1 0 ) . T h e cells were then washed and incubated for 30 min `.`,ith 0.1ml of a 1:50 dilution of fluorochrome-conjugated F(ab') 2 goatantimouse lgG (Cappel Labs). After thorough washing, the cells were examined for m e m b r a n e fhmrescence with a Zeiss photomicroscope. The specificities of these MAB are: OKT3, peripheral T lymphocytes; OKT4, T helper cells: OKT8, T suppressor ceils; O K M I , h u m a n monocyte, null cells and PMN leukocytes and OKTI IA, the sheep erythrocyte receptor [4.9, 16, 231. Cytotoxic assays All experiments were performed with purified (twice rosetted peripheral blood) T effector cells, whenever possible. Control donors were sex- and age-matched. Cultured K562 cells ser`,ed as the target for measurement of NK activity [7]. Autologous or allogeneic non-T and T cells, isolated as previously described, served as targets in some studies. 2 x 10' target cells suspended in lml RPMI 1640 (Gibco, Inc.) supplemented with 10% heat-inactivated fetal calf serum (FXS), gentamycin {100p.g/ml) and glutamine (60gg/ml) were labelled with 100p.Ci sodium " c h r o m a t e solution ( N a / ' C r O , ) (New England Nuclear, Boston, MA) for Ih at 22°C. The ceils were washed three times and diluted to a concentration of 5 x 10'/ml. 0. I ml aliquots of these cell suspensions were pipetted into 96 `,`,ell kinbro plates (Linbro Chemical C o m p a n y , Hamden, CT). 0.1m[ effector cells, prepared in various concentrations, were added to triplicate wells resulting in defined effector:target ratios. Plates were spun at 100g for 5 rain and incubated for 4-16 h at 37°C in 5% CO2. Thereafter, each supernatant was carefully aspirated, centrifuged to remove intact cells, and 100pl counted for 2 min in a g a m m a spectrometer (Beckman Instruments, Inc., Fuller,on, CA). Triplicate control wells containing only 5'Cr-tabelled target cells in medium or in 0.1% Triton X-100 were included to assess the extent of spontaneous and total cytotoxicity respectively. The degree of cytotoxicity was calculated by the formula: % c-ytotoxicity
=
experimental release - spontaneous release ×
t00.
total release - spontaneous release
resuspended cell suspensions were cytocentrifuged on micro.~opc slides, fixed with methanol and stained for 8-10 rain with 10% Giemsa IFisher Scientific C o m p a n y , Fairlawn, N J). The number of lymhocytes binding to tile target cells was then enumerated by oil immersion microscopy counting at least 200 target cells. When studying the cytoadherence of effector -l cells to allogencic lymphocyte targets, the effector cells were stained with 0.1% toluidine blue prior to rosetting. Hematopoietie elonal assays Erythroid (BFU-E, CFU-E) and myeloid precursors (CFUGM) were assayed as previously described [24]. Briefly, bone marrow cells were separated on a Fico[I-Hypaque gradient ( d = 1.077) and used as (a) unfractionated ,narrow; {b) depleted of monocytes by adherence to plastic in the presence of fetal calf serum; (c) depleted of lymphocytes by rosetting with SRBC; and (d) mixtures of depleted marrow and autologous or allogeneic peripheral blood lymphocytes were cultured for 7-14 days in plasma clots, were fixed, stained with benzidinc, and bursts or colonies (greater than 8 benzidine positive cells) counted. To enumerate C F U - G M , colonies (greater than 40 cells) forming in agar were counted under a dissection microscope. Statistical attalysis o f data Data was analyzed by Student's t test. CASE
REPORT
A 62-yr-old male with the diagnosis o f asymptomatic C L L since 1973 presented in 1978, with anemia and thrombocytopenia, at another institution and was treated with splenic irradiation. In 1979 he was referred to the Minneapolis V.A. Medical Center because of severe red cell aplasia (RCA). This diagnosis was determined because of profound reticulocytopenia and absent marrow erythroid precursors. He was initially treated with corticosteroids but developed intractable complications o f diabetes requiring discontinuation of this therapy. Thereafter, recurrent episodes of RBC have necessitated use o f alkylating agents and intensive plasmapheresis. During remissions,, apparently induced by these various treatments, both a reticulocytosis and reappearance of marrow erythroid precursors occurred. In 1979 immune hemolytic anemia occurred, the direct antiglobulin test revealing both IgG and Cj. In August, 1981, he underwent splenectomy because of a rapidly enlarging spleen and severe thrombocytopenia. His total white blood cell counts have varied from 15 to 100 × 10/I during the clinical course known to us. His percentage of non-T cells has varied from 81 to 880/0 ~`,ith T cells ranging from 5 to 16°70. Our patient had required 2-3 units of red cells every 2-3 weeks during his bouts of RCA. The in vitro studies herein reported were initiated 4 weeks after splenectomy. They have been repeated on numerous occasions thereafter Cxhen the patient was receiving no chemotherapy.
Cytoadherenee r4l e./.7t'ctor cells to target cells To measure thc ability of different cffector cell populations to bind to various target cells, I x 10' SRBC-AET rosette positive lymphocytcs were incubated with I x 10 ~ target cells (K562 or allogcneic t:I or T cells) in I ml of medium for l0 rain at 37°C. Cell suspensions ,.,.ere then gently ccmrifuged (130.ti for 5 mini. alnw, st all lilt" stff,ernatant f h f d remo;ed, and the pelleted ceils carcl'ully rcsuspcnded ,.~ith a Pasteur pipette. [hc
RESULTS SuUace markers l h c p a t i e n t ' s isolated n o n - T cells were f o u n d to be monoclonal IgM ( I g M × ) . His S R B C - A E T rosett-
B and T cell killing by CLL T lymphocytes ing cells reacted with OKT3 (91°/0), O K T I I A (96%), O K M I (15o70), O K T I 0 (15O70), OKT4 (23o70) and OKT8 (58°/0). These cell markers represent one study but they remained relatively constant during the entire study period. Cytotoxicit.v studies Since the patient exhibited pure red cell aplasia, we hypothesized that his lymphocytes might kill red cell progenitors. As expected, the patient's marrow conlaincd significantly reduced numbers o f hematopoietic progenitors. Removal of monocytes (by adherence to plastic) a n d / o r T-cells (by rosetting) or by exposure to high concentrations of OKT3 and complement did not improve colony formation in vitro by the patient's bone marrow. Co-culture of patient serum of lymphocytes failed to affect the development of erythroid or myeloid colonies by normal allogeneic marrows (data not shown). However, we were surprised to observe that the patient's T cells killed freshly isolated allogeneic B and T l y m p h o c y t e s (n = 4), while control T cells (as expected) were noncytotoxic for these targets (Fig. 1.). Malignant (i.e. C L L ' B cells) and normal B and T cells from allogeneic donors were killed equally well (Fig. 2), although in some instances neoplastic B cell targets appeared slightly more vulnerable than normal B cells (54 -+- 3.5°7o vs 45.5 __+ 2.8o70). In no instance did our patient's T cells lyse autologous 5'Cr-labelled B or T cells. Since the patient had been repeatedly transfused prior to these studies, we examined the cytotoxic activity of T lymphocytes isolated by similar techniques from
1191
the peripheral blood of 13 hypertransfused control donors, in no instance did they kill allogeneic B or T cells derived from normal controls or from CLL patients, confirming the previously noted marked resistance of these cells to cell-mediated cytotoxicity in vitro I211. The patient's T cells also exhibited significant cytoloxic activity against K562 (Figs. 3, 4). While no spontaneous cytotoxicity was noted against two human tlllnor cell lines, Mahne and CAK I (melanoma and bladder tumors), cylotoxicity toward K562 activity was significantly less than that induced by normal T cells (p <().01L and it was consistently higher than that observed with other CI_L patients ( n = 2 0 ) studied (p <0.001) (Fig. 3). We studied the rate of killing over time (Fig. 4). Patient T cells killed K562 somewhat more rapidly than normal allogeneic lymphocytes (Fig. 4). The pattern of our patient's cytotoxicity toward both K562 and allogeneic lymphocytes has remained constant from May 1981 to January 1982. C vtoudherence studies Patient and control T cells were found to adhere equally well to K562 (39 -t- 4 vs 36 -+" 2) (Table I). in contrast, only the patient's T cells rosetted significantly with allogeneic B and T lymphocytes (27 _ 3 and 31 q4). When patient T cells were added to a mixture of K562 and allogeneic non-T lymphocytes, no mixed rosettes, i.e. T cells binding to non-T and K562 simultaneously, were observed. There was no binding of patient non-T cells to K562 or to allogeneic lymphocytes.
Pf. T Cells Lyse AIIocjeneic B or T Cells 60
50
~Pt.T Effectors
'°[ 0
,', 2:1
~5:l
I,~:l
B or T Cells~ Normal T .1 { "P ~ Effectors ,50:1 IO0:l
Effecfor to Torget Rotio FI(~. I. NK activity of normal and patient T cells vs ~'Crlabelled allogeneic B or T cells. The pooled mean -4- S.E.M. % cytotoxicity for allogeneic B and T cells (n=4) is shown separately while pooled mean -I.-S.E.M. is shown for normal T effectors.
Ni u E. KAY etal.
1192
Lymphocyte end K56z Cytotoxicity by Patient T Cells Kinetic A n a l y s i s ~/a, Patient Lymphocyte Cytotoxicity against B end T Cells
50
/~
40 70
K562
,, ~ / ~ " - "
/
o 8 Cell
~
'
T
Cell
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3o
'~
2o
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.~
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5O
~D
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t0
40
.~, o o
i
0
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i ~' 3 4 5 6 7 8 9 I0 tl 12 13 14 15 t6 HOURS
20
Fla. 4. NK activity of patients T cells towards allogeneie B ((-' C)), T ( O - O), or K562 (A A) targets with an effector to target ratio of 50:1. The data is plotted as % cytotoxicity observed over 1-16 h incubation period.
IO I
Pt
I
N
CLL
PI
B Cells
CLL
N
T Cells Targets
T.\BI.E I. ROSETTING OF K562 AND at- AI.I.O(;ENEIC L'YMPItOC'~TFSBY T CEI,I S F~G. 2. NK activity (% cytotoxicity at 6 h, mean __. S.E.M.) of patient's T cells vs different sources of B or T cell targets. All data was derived with an effector to target ratio of 50:1. Results represent pooled data (mean + S.E.M.) of four different sources of normal (N) or CLL B cells.
% Rosettes
Patient T Control T
B cells
T cells
K562
27 +~ 3 0.8 + 0.2
31 _+_ 4 1.5 + 0.5
36 __+ 2 39 + 4
Pt. T Cells Spontaneously Lyse K56a Comparisonto Normalend Other CLL T Cells 90 80 Normal
70 (9 "6
60 50
o
40 :30 20 tO 0
#" " 2:1
+ 5:1
4' 16:1
"" 50:1
Effector to Target Ratio
Fit. 3. NK aeti,.ity (mean -+- %.E.M.) of norlnal. CI.I. and patient T ceils '.s "Cr-hlbclled K562 folh',willg a 6 h incubation. Nolnl:.tl f cell\ (@ .,D), ( 1 . 1 -I cells {,A . . . . . a.). patienl 1" c,.'lls i. -1.
DISCUSSION These studies demonstrate that the peripheral blood o f this p a t i e n t c o n t a i n s S R B C - A E T r o s e t t i n g cells t h a t s p o n t a n e o u l s y kill K562 a n d freshly isolated a l l o g e n e i c B a n d T cells w i t h o u t a p p a r e n t m e d i a t i o n o f a n t i b o d y . O u r c y t o a d h e r e n c e s t u d i e s ( T a b l e I) s u g g e s t t h a t different T cell s u b p o p u l a t i o n s kill K562 a n d a l l o g e n e i c l y m p h o c y t e s . H o w e v e r , o u r initial e f f o r t s to s e p a r a t e t h e s e e f f e c t o r cells by c y t o a d h e r e n c e d e p l e t i o n h a v e been u n s u c c e s s f u l ; a d d i t i o n a l s t u d i e s in p r o g r e s s a r e d e s i g n e d to a c h i e v e this goal. T h e l y m p h o c y t o t o x i c p r o pcriies o b s e r v e d are clearly d i f f e r e n t f r o m t h o s e o f o t h e r p r e x i o u s l y r e p o r t e d h u m a n c y t o t o x i c T l y m p h o c y l e s [2, 10. 13]. It is u n l i k e a n a t u r a l killer in thai it h a s a n u n u s u a l t a r g e t f r e s h l y isolated l y m p h o c y l e a n d it d i f f e r s f r o m t h e classically d e f i n e d c y t o t o x i c T cell in b e i n g active as a f r e s h l y isolated cell. Recent s t u d i e s by v a r i o u s l a b o r a t o r i e s e m p l o y i n g i h e s a t n e m o n o c l o n a l a n t i b o d i e s s u g g e s t that N K cells d o not cc, n t a i n t h e O K T 3 rn,.'mbrane a n t i g e n but are reactive xxith O K M I n m n o c l o n a l a n t i b o d y that c o n t b i n e s With a n a n t i g e n f)rescilt on myeh'fid cells an(.I n l o r t o c y t e s
B and T cell killing by CLL T lymphocytes [4]. T h e m e m b r a n e p h e n o t y p i n g o f our p a t i c n t ' s T cells did detect cells reactive, in part, for putative NK related antigens, i.e. O K M I , O K T I 1 , a n d O K T I 0 [20, 251. It is possible that positive sorting by a fluorescence activated cell sorter using one o f these antibodies will permit us 1o further define this lymphocytotoxic cell. In a d d i t i o n , wc detected a m a j o r i m b a l a n c e in this p a t i e n t ' s T cell O K T 4 (helper) OKT8 (suppressor) s u b g r o u p s with a revcrsal of the n o r m a l ratio. This observation is consistent with a pattern we have noted in other B - C L L patients (unpublished) a n d recently reported by others [181. T h e relationship, if any, o f the unique lymphocytotoxic cell detected in o u r patient to his unttsual clinical presentation, i.e. pure red cell aplasia, is uncertain. Since we failed to observe T cell mediated lymphocytotoxic activity in o t h e r hypertransfused patients ( C L L a n d others), we are tmwilling to a t t r i b u t e it to allosensitization. Recently M a n g a n et al. [17] have described erythroid aplasia ( P R C A ) in two patients with B-cell CLL; patient T cells exhibiting Fc receptors for IgG were f o u n d to inhibit in vitro colony f o r m a t i o n by n o r m a l marrow. In addition, there have been a n u m b e r o f patients [I I, 19] with T cell C L L in w h o m the neoplastic cells inhibited colony f o r m a t i o n by n o r m a l erythroid precursors. However, in this patient we were unable to d e m o n s t r a t e any inhibition o f n o r m a l erythroid colony growth in vitro by the p a t i e n t ' s serum or circulating lymphocytes. T h e lymphocytotoxic cells present in this patient are distinctly unusual a n d most closely resemble cytotoxic T lymphocyte t h a t arise after priming in vitro [6]. In m u r i n e systems rapidly cytotoxic (i.e. within 1--4 h) T cells are induced in vitro after n o r m a l m u r i n e lymphocytes are i n c u b a t e d with irradiated s t i m u l a t o r y allogeneic l y m p h o i d cells for 2--4 days. These activated cells are then capable o f killing freshly o b t a i n e d target cells genetically identical to the priming cells. W h e t h e r o u r p a t i e n t ' s lymphocytotoxic T cells represent a n in vivo c o u n t e r p a r t to these preprimed cytotoxic T cells remains to be elucidated.
6.
7.
8.
9. 10. I I.
12.
13.
14.
15.
16. work was supported in part by research funds from the Veterans Administration, Acknowledgement--This
17. REFERENCES 1. Aisenberg A. C. & Block K. J. (1972) lmmunoglobulins on the surface of neoplastic lymphocytes. N e w Engl. J. Med. 287, 272. 2. Alvarez J. M., de Landazuri M. O., Bonnard G. D. & Herberman R. B. (1978) Cytotoxic activities of normal cultured human T cells. J. hnmun. 121,270. 3. Behelak Y., Bamerjei D. & Richter M. (1976) Immunocompetent cells in patients with malignant disease: the lack of naturally occurring killer cell activity in the unfractionated circulating lymphocytes from patients with chronic lymphocytic leukemia. Cancer 38, 2274. 4. Breard J., Reinherz E. L., Kung P. C., Goldstein G. & Schlossman S. F. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. I m m u n . 124, 1943. 5. Catovsky D., Lauria F., Matutes E., Foa R., Mantovani
18.
19.
20.
21.
22.
1193
V., Tura S. & Gallon D. A. G. (1981) Increase in Ty lymphocytes in B-cell chronic lymphocytic leukemia: correlation with clinical stage and findings in B-prolymphocytic leukemia. Br. J. Haemat. 47, 539. Cerottini J.-C. & Brunner N. T. (1974) Cell mediated cylotoxicity, allograft rejection and tumor immunity. In Advances in Immunology, Vol 18 (Dixon F. J. & Kinkel H. G. Eds.), p. 67. Academic Press, New York. Dokhclar M.-C., Testa U., Vainchenkcr W., Finale Y., Tetaud C., Salem P. & Tursz T. (1982) NK sensitivity of the leukemic K562 cells: effect of sodium butyrate and heroin induction..L hnmun. 128, 211. Foa R., Catovsky D., Lauria F., Gahon D. A. G. (1981) Reduced T-colony fornfing capacity by T-lymptlocyles from B-chronic lymphocytic leukemia. Br. J. Haemat. 56, 623. Haynes B. F. (1981) Human T lymphocyte antigens as defined by monoclonal antibodies. Immun. Rev. 57, 127. Herberman R. B. & Holden H. T. (1978) Natural cell mediated immunity. Adv. Cancer Res. 27,305. Hoffman R., Kopel S., Hsu D. et al. (1978) T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro. Blood 52, 255. Kaplan M. E. & Clark C. (1974) An improved rosetting assay for detection of human T lymphocytes. J. l m m u n . Meth. 5, 131. Kay H. D., Bonnard G. D., West W. H. & Herberman R. B. (1977) A functional comparison of human Fcreceptor-bearing lymphocytes active in natural cytotoxicity and antibody dependent cellular cytotoxicity. J. l m m u n . !18, 2058. Kay N. E., Johnson J. D., Stanek R. & Douglas S. D. (1979) T cell supopulations in chronic lymphocytic leukemia: abnormalities in distribution and in in vitro receptor maturation. Blood 54, 540. Kay N. E. (1981) Abnormal T-cell subpopulation function in CLL: excessive suppressor (TT) and deficient helper (Tla) activity with respect to B cell proliferation. Blood 47, 418. Kung P. C., Goldstein G., Reinherz E. L. & Schlossman J. F. (1979) Monoclonal antibodies defining distinctive human T-cell surface antigens. Science 206, 347. Mangan K., Chikkappa G. & Farley P. C. (1978) T gamma (T',/) cells suppress erylhropoiesis in vitro. Blood 52, 255. Matutes E., Wechsler A., Gomez R., Cherchi M. & Catovsky D. (1981) Unusual T-cell phenotype in advanced B-chronic lymphocytic leukemia. Br. J. Haemat. 49, 635. Nagasawa T., Abe T. & Nakagawa T. Pure red cell aplasia and hypogamma-globulinemia associated with T-cell chronic lymphocytic leukemia. Blood 27, 1025. Ortaldo J. R., Sharrow S. O., Timonu T. & Herberman R. B. (1981) Determination of surface antigens on highly purified human NK cells by flow cytometry with monoclonal antibodies. J. h n m u n . 127. 240. Pattengale P. K., Gidlund M., Nilsson K., Sundstrom C., Sallstrom J., Simonsson B. & Wigzell H. (1982) Lysis of fresh human B lymphocyte-derived leukemia cells by interferon-activated natural killer (NK) cells. Int. J. Cancer 29, 1. Platsoucas C. D., Fernandes G., Gupta J. L., Kempin S., Clarkson B., Good R. A. & Gupta S. (1980) Defective
1194
NJll E. KA',' et al.
spontaneous and antibody-dependent cytotoxicity mediated by E-rosette positive and E-rosette negati',e cells in untreated patients with chronic lymphocytic leukemia: augmentation ill vitro treatment with inlcrl'eron..J, hltllnltt. 125, 2116. 23. Reinherz E. L. & Schlossman J. F. (1980) The differentiation and function of human T lymphocytes. Cell 19, 821. 24. Roodman G. D., Ascensao J. L., Banisadre M. et aL
(1980) At, loimmune pancylopenia: lymphocyte inhibition of aulologous but not allogeneic bone marrow growth in vitro. A m . J. M e d . 69, 325. 25. Zarling J. M., Cl6usc K. A., Biddison W. E. & Kung P. C. ( 1981 ) Phenotypes of human natural killer cell populations detected with monoclonal antibodies. J. I m m u n . 127,257. 26. Zicgler H.-U., Kay N. E., Zarling J. M. (1981) Deficiency of natural killer cell activity in patients with chronic lymphocytic leukemia. Int. J. Cancer 27, 321.
0145-2126/8553.00 ~ .(X) (~ 1985 Pergamon Press Lid.
LYMPHOCYTOTOXIC PATIENT
WITH
LEUKEMIA
AND
'F
I_YMPHOCYTES
B-CHRONIC PURE
RED
IN
A
LYMPHOCYTIC CELL
APLASIA
Nt!II [~. KAY, MARl'IN M. ()KIN, .l~}.x(} A',cf N'-.;',,(t:.tltd MANt;I:I. E. KAPI.AN Veterans Administration Mcdic~d (.'Clllt_'t-.Minneapolis, Mim~csola and the Lluiversity of Minnesota Medical School. \linneapolis, Minnesota, U.S.A. (Received 19 Jn(v 1984..,Ict.t7~ted 29 .hmuat3' 1985)
Abstract--The peripheral blood T cells of a hyperlranq'uscd patient with B-chronic lymphocytic leukemia and pure red cell aplasic were found to exhibit tmusual spontaneous cytotoxic activity in vitro. The patient's E-rosette positive cells wcrc cytotoxic for K562 (cultured human crythroleukemia cells) and allogeneic B and T lymphocytes freshly isolated from the peripheral blood of normal and CLL donors. They failed to kill autologous B cells, erythroid progenitors present in allogencic bone marrow, and a number of cultured human tumor cells (Maline, CAKI) even after prolonged (36 h) co-culture. Peripheral blood T cells isolated from normal controls, other CLL patients, and hypertransfused individuals (tl = 13) did not exhibit spontaneous lymphoc.vtotoxic activity. Circulating cytotoxic T cells having the ability to kill freshly isolated allogeneic lymphocytes have, heretofore, not been reported in humans. Our findings suggest that among this patient's peripheral blood T cells, there exists a subpopulation of lymphocytotoxic cells that closely resemble cytotoxic T cells generated in vitro after priming with allogeneic target cells. Although the lymphocytotoxic cells could ha~e been induced in this patient by previous HLA-mismatched transfusions, it is possible they may have arisen spontaneously and underly the ~atient's erythroblastopenic state. Key word~: Lymphocytotoxic, T cells, CLL.
INTRODUCTION FRESHLY isolated human T cells are known to contain several cytotoxic subpopulations [2, 10, 13]. These subpopulations which have been defined and distinguished from each other by a number o f criteria are designated as cytotoxic T lymphocytes (CTL), natural killer cells (NK) and antibody-dependent cytotoxic cells (ADCC). Recently we and others have demonstrated a number of peripheral blood T cell abnormalities in patients with typical B-ceU chronic lymphocytic leukemia (CLL) including: (I) increased absolute numbers of T cells [14]; (2) qualitative and quantitative increases in T suppressor cells, with decrease in T helper cells [5, 14, 15]; (3) deficient T-cell colony formation in vitro [8] and (4) depressed NK function [3, 22, 26]. We recently had the opportunity to study a B - C L L patient who presented
Abbreviations: CLL, chronic lymphocytic leukemia; CTL, cytotoxic T lymphocytes; NK, natural killer cells; ADCC, antibody-dependent cytotoxieity; AET, 2-aminoethylisothiouronium bromide hydrobromide; SRBC, sheep erythrocyte red blood cells; Slg, surface immunoglobulin; BFU-E, CFU-E, erythroid precursors; CFU-GM, myeloid precursors. Correspondence and reprint requests to: Nell E. Kay, M.D., Hematology-Oncology Section (I 1IE), V. A. Medical Center, 54th Street and 48th Avenue South, Minneapolis, MN 55417, U.S.A.
with pure red cell aplasia. His peripheral blood T lymphocytes exhibited significant spontaneous cytotoxicity against K562 (a human erythroleukemic cell line) and rapidly killed allogeneic B and T lymphocytes freshly isolated from the peripheral blood of normal donors and other CLL patients. We report here the cytotoxic properties and preliminary characterization of this tmusual, perhaps unique, cytotoxic T cell.
MATERIALS
AND METHODS
Isolation and characterization of peripheral blood B and T lymphoqvtes
Heparinized venous peripheral blood was obtained after informed consent, and the mononuclear cells isolated by Ficoll-Hypaque centrifugation. Monocytes were removed by adherence to plastic. B cells were detected by immunofluorescent staining of surface immunoglobulin (Slg) with rhodamineconjugated rabbit anti-human lgG-(Fab')2 (Cappel Labs, Cochranville, PA) and T cells were assayed by rosetting with 2-aminoethyl isothiouronium bromide hydrobromide (AET) (Aldrich Chemical Co., Milwaukee, WI) treated sheep erythrocyte red blood cells (SRBC) [1, 12]. Rosettes were defined as lymphocytes with three or more adherent erythrocytcs. Purified T-lymphocytes were isolated by rosetting twice with SRBC-AET followed by FicolI-Hypaque centrifugation. T cells were harvested by brief hypotonic lysis (i.e. 30 s exposure with 5000.1 of sterile H20) of the SRBC-AET lym-
1189
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phoeyte pellet. Non-T cells `.~'ere collected from the interfaces. Isolated T cells `,`.ere > 9 8 % viable and > 9 5 % re-rosetted `.`.ith SRBC-AET. The patient's non-rosetting cells ,.`.ere 92-96% B cells by Slg staining, while control SIg% varied from 78-80% positive. T-lymphocyte subsets were detected using monoclonal antibodies as follows: twice rosetled cells were incubated at 4°C tbr 1 h with various monoclonal antibodies (MAb) (Orthomune, Ortho Pharm Corp., Raritan, NJ): OKT3 (1:100), OKT4{I:I0J, O K T 8 ( I : I 0 ) O K M I ( I : I 0 ) o r O K T I I ( l : 1 0 ) . T h e cells were then washed and incubated for 30 min `.`,ith 0.1ml of a 1:50 dilution of fluorochrome-conjugated F(ab') 2 goatantimouse lgG (Cappel Labs). After thorough washing, the cells were examined for m e m b r a n e fhmrescence with a Zeiss photomicroscope. The specificities of these MAB are: OKT3, peripheral T lymphocytes; OKT4, T helper cells: OKT8, T suppressor ceils; O K M I , h u m a n monocyte, null cells and PMN leukocytes and OKTI IA, the sheep erythrocyte receptor [4.9, 16, 231. Cytotoxic assays All experiments were performed with purified (twice rosetted peripheral blood) T effector cells, whenever possible. Control donors were sex- and age-matched. Cultured K562 cells ser`,ed as the target for measurement of NK activity [7]. Autologous or allogeneic non-T and T cells, isolated as previously described, served as targets in some studies. 2 x 10' target cells suspended in lml RPMI 1640 (Gibco, Inc.) supplemented with 10% heat-inactivated fetal calf serum (FXS), gentamycin {100p.g/ml) and glutamine (60gg/ml) were labelled with 100p.Ci sodium " c h r o m a t e solution ( N a / ' C r O , ) (New England Nuclear, Boston, MA) for Ih at 22°C. The ceils were washed three times and diluted to a concentration of 5 x 10'/ml. 0. I ml aliquots of these cell suspensions were pipetted into 96 `,`,ell kinbro plates (Linbro Chemical C o m p a n y , Hamden, CT). 0.1m[ effector cells, prepared in various concentrations, were added to triplicate wells resulting in defined effector:target ratios. Plates were spun at 100g for 5 rain and incubated for 4-16 h at 37°C in 5% CO2. Thereafter, each supernatant was carefully aspirated, centrifuged to remove intact cells, and 100pl counted for 2 min in a g a m m a spectrometer (Beckman Instruments, Inc., Fuller,on, CA). Triplicate control wells containing only 5'Cr-tabelled target cells in medium or in 0.1% Triton X-100 were included to assess the extent of spontaneous and total cytotoxicity respectively. The degree of cytotoxicity was calculated by the formula: % c-ytotoxicity
=
experimental release - spontaneous release ×
t00.
total release - spontaneous release
resuspended cell suspensions were cytocentrifuged on micro.~opc slides, fixed with methanol and stained for 8-10 rain with 10% Giemsa IFisher Scientific C o m p a n y , Fairlawn, N J). The number of lymhocytes binding to tile target cells was then enumerated by oil immersion microscopy counting at least 200 target cells. When studying the cytoadherence of effector -l cells to allogencic lymphocyte targets, the effector cells were stained with 0.1% toluidine blue prior to rosetting. Hematopoietie elonal assays Erythroid (BFU-E, CFU-E) and myeloid precursors (CFUGM) were assayed as previously described [24]. Briefly, bone marrow cells were separated on a Fico[I-Hypaque gradient ( d = 1.077) and used as (a) unfractionated ,narrow; {b) depleted of monocytes by adherence to plastic in the presence of fetal calf serum; (c) depleted of lymphocytes by rosetting with SRBC; and (d) mixtures of depleted marrow and autologous or allogeneic peripheral blood lymphocytes were cultured for 7-14 days in plasma clots, were fixed, stained with benzidinc, and bursts or colonies (greater than 8 benzidine positive cells) counted. To enumerate C F U - G M , colonies (greater than 40 cells) forming in agar were counted under a dissection microscope. Statistical attalysis o f data Data was analyzed by Student's t test. CASE
REPORT
A 62-yr-old male with the diagnosis o f asymptomatic C L L since 1973 presented in 1978, with anemia and thrombocytopenia, at another institution and was treated with splenic irradiation. In 1979 he was referred to the Minneapolis V.A. Medical Center because of severe red cell aplasia (RCA). This diagnosis was determined because of profound reticulocytopenia and absent marrow erythroid precursors. He was initially treated with corticosteroids but developed intractable complications o f diabetes requiring discontinuation of this therapy. Thereafter, recurrent episodes of RBC have necessitated use o f alkylating agents and intensive plasmapheresis. During remissions,, apparently induced by these various treatments, both a reticulocytosis and reappearance of marrow erythroid precursors occurred. In 1979 immune hemolytic anemia occurred, the direct antiglobulin test revealing both IgG and Cj. In August, 1981, he underwent splenectomy because of a rapidly enlarging spleen and severe thrombocytopenia. His total white blood cell counts have varied from 15 to 100 × 10/I during the clinical course known to us. His percentage of non-T cells has varied from 81 to 880/0 ~`,ith T cells ranging from 5 to 16°70. Our patient had required 2-3 units of red cells every 2-3 weeks during his bouts of RCA. The in vitro studies herein reported were initiated 4 weeks after splenectomy. They have been repeated on numerous occasions thereafter Cxhen the patient was receiving no chemotherapy.
Cytoadherenee r4l e./.7t'ctor cells to target cells To measure thc ability of different cffector cell populations to bind to various target cells, I x 10' SRBC-AET rosette positive lymphocytcs were incubated with I x 10 ~ target cells (K562 or allogcneic t:I or T cells) in I ml of medium for l0 rain at 37°C. Cell suspensions ,.,.ere then gently ccmrifuged (130.ti for 5 mini. alnw, st all lilt" stff,ernatant f h f d remo;ed, and the pelleted ceils carcl'ully rcsuspcnded ,.~ith a Pasteur pipette. [hc
RESULTS SuUace markers l h c p a t i e n t ' s isolated n o n - T cells were f o u n d to be monoclonal IgM ( I g M × ) . His S R B C - A E T rosett-
B and T cell killing by CLL T lymphocytes ing cells reacted with OKT3 (91°/0), O K T I I A (96%), O K M I (15o70), O K T I 0 (15O70), OKT4 (23o70) and OKT8 (58°/0). These cell markers represent one study but they remained relatively constant during the entire study period. Cytotoxicit.v studies Since the patient exhibited pure red cell aplasia, we hypothesized that his lymphocytes might kill red cell progenitors. As expected, the patient's marrow conlaincd significantly reduced numbers o f hematopoietic progenitors. Removal of monocytes (by adherence to plastic) a n d / o r T-cells (by rosetting) or by exposure to high concentrations of OKT3 and complement did not improve colony formation in vitro by the patient's bone marrow. Co-culture of patient serum of lymphocytes failed to affect the development of erythroid or myeloid colonies by normal allogeneic marrows (data not shown). However, we were surprised to observe that the patient's T cells killed freshly isolated allogeneic B and T l y m p h o c y t e s (n = 4), while control T cells (as expected) were noncytotoxic for these targets (Fig. 1.). Malignant (i.e. C L L ' B cells) and normal B and T cells from allogeneic donors were killed equally well (Fig. 2), although in some instances neoplastic B cell targets appeared slightly more vulnerable than normal B cells (54 -+- 3.5°7o vs 45.5 __+ 2.8o70). In no instance did our patient's T cells lyse autologous 5'Cr-labelled B or T cells. Since the patient had been repeatedly transfused prior to these studies, we examined the cytotoxic activity of T lymphocytes isolated by similar techniques from
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the peripheral blood of 13 hypertransfused control donors, in no instance did they kill allogeneic B or T cells derived from normal controls or from CLL patients, confirming the previously noted marked resistance of these cells to cell-mediated cytotoxicity in vitro I211. The patient's T cells also exhibited significant cytoloxic activity against K562 (Figs. 3, 4). While no spontaneous cytotoxicity was noted against two human tlllnor cell lines, Mahne and CAK I (melanoma and bladder tumors), cylotoxicity toward K562 activity was significantly less than that induced by normal T cells (p <().01L and it was consistently higher than that observed with other CI_L patients ( n = 2 0 ) studied (p <0.001) (Fig. 3). We studied the rate of killing over time (Fig. 4). Patient T cells killed K562 somewhat more rapidly than normal allogeneic lymphocytes (Fig. 4). The pattern of our patient's cytotoxicity toward both K562 and allogeneic lymphocytes has remained constant from May 1981 to January 1982. C vtoudherence studies Patient and control T cells were found to adhere equally well to K562 (39 -t- 4 vs 36 -+" 2) (Table I). in contrast, only the patient's T cells rosetted significantly with allogeneic B and T lymphocytes (27 _ 3 and 31 q4). When patient T cells were added to a mixture of K562 and allogeneic non-T lymphocytes, no mixed rosettes, i.e. T cells binding to non-T and K562 simultaneously, were observed. There was no binding of patient non-T cells to K562 or to allogeneic lymphocytes.
Pf. T Cells Lyse AIIocjeneic B or T Cells 60
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Effecfor to Torget Rotio FI(~. I. NK activity of normal and patient T cells vs ~'Crlabelled allogeneic B or T cells. The pooled mean -4- S.E.M. % cytotoxicity for allogeneic B and T cells (n=4) is shown separately while pooled mean -I.-S.E.M. is shown for normal T effectors.
Ni u E. KAY etal.
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Lymphocyte end K56z Cytotoxicity by Patient T Cells Kinetic A n a l y s i s ~/a, Patient Lymphocyte Cytotoxicity against B end T Cells
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Fla. 4. NK activity of patients T cells towards allogeneie B ((-' C)), T ( O - O), or K562 (A A) targets with an effector to target ratio of 50:1. The data is plotted as % cytotoxicity observed over 1-16 h incubation period.
IO I
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T.\BI.E I. ROSETTING OF K562 AND at- AI.I.O(;ENEIC L'YMPItOC'~TFSBY T CEI,I S F~G. 2. NK activity (% cytotoxicity at 6 h, mean __. S.E.M.) of patient's T cells vs different sources of B or T cell targets. All data was derived with an effector to target ratio of 50:1. Results represent pooled data (mean + S.E.M.) of four different sources of normal (N) or CLL B cells.
% Rosettes
Patient T Control T
B cells
T cells
K562
27 +~ 3 0.8 + 0.2
31 _+_ 4 1.5 + 0.5
36 __+ 2 39 + 4
Pt. T Cells Spontaneously Lyse K56a Comparisonto Normalend Other CLL T Cells 90 80 Normal
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Fit. 3. NK aeti,.ity (mean -+- %.E.M.) of norlnal. CI.I. and patient T ceils '.s "Cr-hlbclled K562 folh',willg a 6 h incubation. Nolnl:.tl f cell\ (@ .,D), ( 1 . 1 -I cells {,A . . . . . a.). patienl 1" c,.'lls i. -1.
DISCUSSION These studies demonstrate that the peripheral blood o f this p a t i e n t c o n t a i n s S R B C - A E T r o s e t t i n g cells t h a t s p o n t a n e o u l s y kill K562 a n d freshly isolated a l l o g e n e i c B a n d T cells w i t h o u t a p p a r e n t m e d i a t i o n o f a n t i b o d y . O u r c y t o a d h e r e n c e s t u d i e s ( T a b l e I) s u g g e s t t h a t different T cell s u b p o p u l a t i o n s kill K562 a n d a l l o g e n e i c l y m p h o c y t e s . H o w e v e r , o u r initial e f f o r t s to s e p a r a t e t h e s e e f f e c t o r cells by c y t o a d h e r e n c e d e p l e t i o n h a v e been u n s u c c e s s f u l ; a d d i t i o n a l s t u d i e s in p r o g r e s s a r e d e s i g n e d to a c h i e v e this goal. T h e l y m p h o c y t o t o x i c p r o pcriies o b s e r v e d are clearly d i f f e r e n t f r o m t h o s e o f o t h e r p r e x i o u s l y r e p o r t e d h u m a n c y t o t o x i c T l y m p h o c y l e s [2, 10. 13]. It is u n l i k e a n a t u r a l killer in thai it h a s a n u n u s u a l t a r g e t f r e s h l y isolated l y m p h o c y l e a n d it d i f f e r s f r o m t h e classically d e f i n e d c y t o t o x i c T cell in b e i n g active as a f r e s h l y isolated cell. Recent s t u d i e s by v a r i o u s l a b o r a t o r i e s e m p l o y i n g i h e s a t n e m o n o c l o n a l a n t i b o d i e s s u g g e s t that N K cells d o not cc, n t a i n t h e O K T 3 rn,.'mbrane a n t i g e n but are reactive xxith O K M I n m n o c l o n a l a n t i b o d y that c o n t b i n e s With a n a n t i g e n f)rescilt on myeh'fid cells an(.I n l o r t o c y t e s
B and T cell killing by CLL T lymphocytes [4]. T h e m e m b r a n e p h e n o t y p i n g o f our p a t i c n t ' s T cells did detect cells reactive, in part, for putative NK related antigens, i.e. O K M I , O K T I 1 , a n d O K T I 0 [20, 251. It is possible that positive sorting by a fluorescence activated cell sorter using one o f these antibodies will permit us 1o further define this lymphocytotoxic cell. In a d d i t i o n , wc detected a m a j o r i m b a l a n c e in this p a t i e n t ' s T cell O K T 4 (helper) OKT8 (suppressor) s u b g r o u p s with a revcrsal of the n o r m a l ratio. This observation is consistent with a pattern we have noted in other B - C L L patients (unpublished) a n d recently reported by others [181. T h e relationship, if any, o f the unique lymphocytotoxic cell detected in o u r patient to his unttsual clinical presentation, i.e. pure red cell aplasia, is uncertain. Since we failed to observe T cell mediated lymphocytotoxic activity in o t h e r hypertransfused patients ( C L L a n d others), we are tmwilling to a t t r i b u t e it to allosensitization. Recently M a n g a n et al. [17] have described erythroid aplasia ( P R C A ) in two patients with B-cell CLL; patient T cells exhibiting Fc receptors for IgG were f o u n d to inhibit in vitro colony f o r m a t i o n by n o r m a l marrow. In addition, there have been a n u m b e r o f patients [I I, 19] with T cell C L L in w h o m the neoplastic cells inhibited colony f o r m a t i o n by n o r m a l erythroid precursors. However, in this patient we were unable to d e m o n s t r a t e any inhibition o f n o r m a l erythroid colony growth in vitro by the p a t i e n t ' s serum or circulating lymphocytes. T h e lymphocytotoxic cells present in this patient are distinctly unusual a n d most closely resemble cytotoxic T lymphocyte t h a t arise after priming in vitro [6]. In m u r i n e systems rapidly cytotoxic (i.e. within 1--4 h) T cells are induced in vitro after n o r m a l m u r i n e lymphocytes are i n c u b a t e d with irradiated s t i m u l a t o r y allogeneic l y m p h o i d cells for 2--4 days. These activated cells are then capable o f killing freshly o b t a i n e d target cells genetically identical to the priming cells. W h e t h e r o u r p a t i e n t ' s lymphocytotoxic T cells represent a n in vivo c o u n t e r p a r t to these preprimed cytotoxic T cells remains to be elucidated.
6.
7.
8.
9. 10. I I.
12.
13.
14.
15.
16. work was supported in part by research funds from the Veterans Administration, Acknowledgement--This
17. REFERENCES 1. Aisenberg A. C. & Block K. J. (1972) lmmunoglobulins on the surface of neoplastic lymphocytes. N e w Engl. J. Med. 287, 272. 2. Alvarez J. M., de Landazuri M. O., Bonnard G. D. & Herberman R. B. (1978) Cytotoxic activities of normal cultured human T cells. J. hnmun. 121,270. 3. Behelak Y., Bamerjei D. & Richter M. (1976) Immunocompetent cells in patients with malignant disease: the lack of naturally occurring killer cell activity in the unfractionated circulating lymphocytes from patients with chronic lymphocytic leukemia. Cancer 38, 2274. 4. Breard J., Reinherz E. L., Kung P. C., Goldstein G. & Schlossman S. F. (1980) A monoclonal antibody reactive with human peripheral blood monocytes. J. I m m u n . 124, 1943. 5. Catovsky D., Lauria F., Matutes E., Foa R., Mantovani
18.
19.
20.
21.
22.
1193
V., Tura S. & Gallon D. A. G. (1981) Increase in Ty lymphocytes in B-cell chronic lymphocytic leukemia: correlation with clinical stage and findings in B-prolymphocytic leukemia. Br. J. Haemat. 47, 539. Cerottini J.-C. & Brunner N. T. (1974) Cell mediated cylotoxicity, allograft rejection and tumor immunity. In Advances in Immunology, Vol 18 (Dixon F. J. & Kinkel H. G. Eds.), p. 67. Academic Press, New York. Dokhclar M.-C., Testa U., Vainchenkcr W., Finale Y., Tetaud C., Salem P. & Tursz T. (1982) NK sensitivity of the leukemic K562 cells: effect of sodium butyrate and heroin induction..L hnmun. 128, 211. Foa R., Catovsky D., Lauria F., Gahon D. A. G. (1981) Reduced T-colony fornfing capacity by T-lymptlocyles from B-chronic lymphocytic leukemia. Br. J. Haemat. 56, 623. Haynes B. F. (1981) Human T lymphocyte antigens as defined by monoclonal antibodies. Immun. Rev. 57, 127. Herberman R. B. & Holden H. T. (1978) Natural cell mediated immunity. Adv. Cancer Res. 27,305. Hoffman R., Kopel S., Hsu D. et al. (1978) T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro. Blood 52, 255. Kaplan M. E. & Clark C. (1974) An improved rosetting assay for detection of human T lymphocytes. J. l m m u n . Meth. 5, 131. Kay H. D., Bonnard G. D., West W. H. & Herberman R. B. (1977) A functional comparison of human Fcreceptor-bearing lymphocytes active in natural cytotoxicity and antibody dependent cellular cytotoxicity. J. l m m u n . !18, 2058. Kay N. E., Johnson J. D., Stanek R. & Douglas S. D. (1979) T cell supopulations in chronic lymphocytic leukemia: abnormalities in distribution and in in vitro receptor maturation. Blood 54, 540. Kay N. E. (1981) Abnormal T-cell subpopulation function in CLL: excessive suppressor (TT) and deficient helper (Tla) activity with respect to B cell proliferation. Blood 47, 418. Kung P. C., Goldstein G., Reinherz E. L. & Schlossman J. F. (1979) Monoclonal antibodies defining distinctive human T-cell surface antigens. Science 206, 347. Mangan K., Chikkappa G. & Farley P. C. (1978) T gamma (T',/) cells suppress erylhropoiesis in vitro. Blood 52, 255. Matutes E., Wechsler A., Gomez R., Cherchi M. & Catovsky D. (1981) Unusual T-cell phenotype in advanced B-chronic lymphocytic leukemia. Br. J. Haemat. 49, 635. Nagasawa T., Abe T. & Nakagawa T. Pure red cell aplasia and hypogamma-globulinemia associated with T-cell chronic lymphocytic leukemia. Blood 27, 1025. Ortaldo J. R., Sharrow S. O., Timonu T. & Herberman R. B. (1981) Determination of surface antigens on highly purified human NK cells by flow cytometry with monoclonal antibodies. J. h n m u n . 127. 240. Pattengale P. K., Gidlund M., Nilsson K., Sundstrom C., Sallstrom J., Simonsson B. & Wigzell H. (1982) Lysis of fresh human B lymphocyte-derived leukemia cells by interferon-activated natural killer (NK) cells. Int. J. Cancer 29, 1. Platsoucas C. D., Fernandes G., Gupta J. L., Kempin S., Clarkson B., Good R. A. & Gupta S. (1980) Defective
1194
NJll E. KA',' et al.
spontaneous and antibody-dependent cytotoxicity mediated by E-rosette positive and E-rosette negati',e cells in untreated patients with chronic lymphocytic leukemia: augmentation ill vitro treatment with inlcrl'eron..J, hltllnltt. 125, 2116. 23. Reinherz E. L. & Schlossman J. F. (1980) The differentiation and function of human T lymphocytes. Cell 19, 821. 24. Roodman G. D., Ascensao J. L., Banisadre M. et aL
(1980) At, loimmune pancylopenia: lymphocyte inhibition of aulologous but not allogeneic bone marrow growth in vitro. A m . J. M e d . 69, 325. 25. Zarling J. M., Cl6usc K. A., Biddison W. E. & Kung P. C. ( 1981 ) Phenotypes of human natural killer cell populations detected with monoclonal antibodies. J. I m m u n . 127,257. 26. Zicgler H.-U., Kay N. E., Zarling J. M. (1981) Deficiency of natural killer cell activity in patients with chronic lymphocytic leukemia. Int. J. Cancer 27, 321.