a lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

Journal of Clinical Immunology, Vol. 3, No. 1, 1983

A Lymphoproliferative Disorder of the Large Granular Lymphocytes with Natural Killer Activity

M A N L I O F E R R A R I N I , I S E R G I O R O M A G N A N I , 2 E L I S A B E T T A M O N T E S O R O , I A N T O N I O ZICCA,1 G I A N F R A N C O D E L PRETE, 2 A R C A N G E L O N O C E R A , I E N R I C O M A G G I , 2 A R N A L D O L E P R I N I , 1 and C A R L O E N R I C O GROSSI 1

Accepted: June 10, 1982

This paper reports the case of a patient with an abnormally expanded population of circulating lymphoid cells displaying the features of the so-called large granular lymphocytes (LGL). These cells were peroxidase negative and nonphagocytic, formed rosettes with sheep erythrocytes, had receptors for IgG, and contained azurophilic (electron-dense) granules. Like normal LGL, the patient cells were positive for two acid hydrolases (acid phosphatase and [3-glucuronidase) but did not stain for c~- naphthyl acetate esterase (ANAE), which is present in normal LGL. Ultrastructural studies revealed that the patient cells were rich in Golgi-derived vesicles, coated vesicles, multivesicular bodies, and immature granules, indicating that, unlike normal LGL, they were engaged in granulogenesis. These features, together with the absence of ANAE activity, are suggestive of some degree of cell immaturity. The patient cells displayed natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities comparable to those of normal peripheral blood mononuclear cells, or even higher, and did not respond to T-cell mitogens or allogeneic cells. Further- more, they were incapable of suppressing normal T-cell proliferation or pokeweed mitogen-induced B-cell differentiation. Analysis of the NK activity at the single-cell level revealed that a large proportion of the patient cells bound to the K562 target cells but could not accomplish the entire lytic process. This finding supports further the possibility that the patient cells were immature LGL. The surface phenotype of the patient cells (as defined by a battery of monoclonal antibodies) was somewhat different from that usually observed in the majority of the normal LGL because, in addition to the HNK-1 marker, the cells were OKT3 +, aLeul +, aLeu4 +, OKT8 +,

'Departments of Immunology and Anatomy, University of Ge- nova, 16131 Genova.

2Department of Clinical Immunology, University of Firenze, Firenze, Italy.

aLeu2a +, and 3Al* but were OKMI- and 4F2-. This phenotype could correspond to that of maturing LGL.

KEY WORDS: Natural killer cells; large granular lymphocytes.

INTRODUCTION

Lymphoprol i fe ra t ive disorders of the T lineage tra- ditionally have been identified through the capaci ty of the malignant cells to fo rm roset tes with sheep ery throcytes (EN) 3 (as reviewed in Ref. 1). More recently, a bet ter definition of the single pathologi- cal entities has been achieved with the use of anti-T- cell monoclonal antibodies (2-4), of cytochemical and ultrastructural methods (5-7), and of functional in vitro studies (8, 9). In this connect ion, the best- known example is represented by the S6zary syndrome, which has been identified as a proliferation of a pos t thymic subset of helper T cells (3, 9). When the above techniques have been applied to the study of T-cell chronic lymphocyt ic leukemias (T-CLL), it has become apparent that, in spite of a certain homogenei ty of the surface phenotype , as defined by convent ional markers [i.e., receptors for EN and for IgG (FcR)], several different disorders coexis ted within this broadly defined group. The cells f rom some of these cases displayed a suppressor function

3Abbreviations used: ADCC, antibody-dependent cellular cytotoxicity; ANAE, ~-naphthyl acetate esterase; AP, acid phosphatase; B-Gluc, [3-glucuronidase; EAC, sheep erythrocytes coated with IgM antibody and human complement; EAG~ ox erythrocytes sensitized with IgG antibody; EAM, ox erythrocytes sensitized with IgM antibody; EN, neuraminidase-treated sheep erythrocytes; FcR, receptors for the Fc portion of IgG; LGL, large granular lymphocytes; Mo, mouse erythrocytes; NK, natural killer; PO, endogenous peroxidase; sIg, surface immunoglobulin; T-CLL, T-cell chronic lymphocytic leukemia; To cells, T cells with receptors for IgG.

30 0271-9142/83/0100~0030503.00/0 ~ 1983 Plenum Publishing Corpm~ation

A LARGE GRANULAR LYMPHOCYTE DISORDER 3]

on a variety of in vitro lymphocyte responses, whereas others had cytotoxic activities (10-14). No uniform morphological characteristics or cytochemical markers have been detected, with the exception of a relatively small number of cases displaying certain common features. The cells from these patients had cytoplasmic azurophilic (electron- opaque) granules and could mediate certain cytotoxic functions, such as antibody-dependent cellular cytotoxicity (ADCC) (13, 15-19). In some studies these cells have been found positive for cytoplasmic acid hydrolases (15-18). These features resemble those of the so-called large granular lymphocytes (LGL) (20, 21). LGL are a subset of nonadherent, nonphagocytic cells with avid receptors for IgG (21-24), which represent the bulk of two major subpopulations of the peripheral blood mononuclear cells, namely, the TG cells (i.e., EN +, FcR + cells) and the third-population cells (i.e., non- T, non-B cells with FcR) (25, 26). LGL are particularly efficient in natural killer (NK) and ADCC activities (21), and because of their NK activity they are sometimes referred to as NK cells (27).

Here we report the case of a patient with an expanded population of peripheral blood mononuclear cells with azurophilic granules. Comparative studies with normal peripheral blood LGL have established that the patient cells were indeed LGL, although immature. The availability of these immature cells has provided some criteria which can be used to define the phenotype of early LGL and to gain information on the debated question of the origin and lineage of these cells.

M A T E R I A L S A N D M E T H O D S

Patient. A 63-year-old man was admitted to the San Giovanni di Dio Hospital in Florence in June 1980 because of asthenia, moderate weight loss, and repeated episodes of fever. On physical examina- tion, the liver was enlarged to 3 cm below the costal margin and the spleen was not palpable. There was no supraclavicular, axillary, or inguinal lymphade- nopathy. The hemoglobin level was 14 g/dl and the white-cell count was 16,000/ram 3, with I4% neutro- phils, 85% lymphocytes, and 1% monocytes. The reticutocyte count was 1%, the platelet count was 220,000/ram 3, and the erythrocyte sedimentation rate was 14 ram/hr. Routine blood chemistries were normal. Serum IgG was 1400 rag% and lgA was 410 rag%, whereas IgM was markedly reduced (15 rag%). A bone marrow biopsy showed hypercellu-

larity with a marked increase in lymphoid cells, the majority of which displayed azurophilic granules. No chromosomal abnormalities were detected, although admittedly a few mitoses could be scored because of the poor response of the cells to mitogens.

The patient remained in relatively good condition, with fluctuations of the total white blood cells from 12,000 to 16,000/ram 3, and was followed in the outpatient clinic. No chemotherapy was considered necessary at the time.

Ten months later the patient was again admitted to the hospital and a chest X-ray revealed a mass with a diameter of 2 cm in the anterior part of the upper lobe of the left lung. Computerized tomogra- phy of the chest and mediastinum confirmed the presence of a solid mass with undefined borders, indicating the presence of a lung cancer. The patient refused bronchoscopy. Because of the progressive- ly deteriorating conditions and invasion of mediasti- hal lymph nodes followed by a left recurrent paraly- sis, the patient could not undergo surgery. Chemo- therapy was subsequently started without any relevant effect and the patient's condition progres- sively deteriorated until he died in November 1981.

Mononuclear-Cetl Suspensions and Surface Markers. Mononuclear cells were obtained from heparinized blood of the patient or healthy donors by centrifugation on Ficoll-Hypaque density gradi- ents. Receptors for neuraminidase-trea~ed sheep red cells (EN), for OX red cells sensitized with IgG antibody (EAt), for mouse red cells (Me,), and for ox red cells sensitized with IgM antibody (EAM) were detected by rosetting techniques previously reported (23, 28, 29). Cells were stained for the presence of surface immunoglobulin (slg) using an F(ab')2 fragment of a goat polyvalent antibody (anti- IgM, tgD, IgG, IgA) conjugated with fluorescein isothiocyanate. Complement receptors were detected by a rosette technique with sheep red cells coated with IgM antibody and complement (EAC) (30). Two antisera directed against DR antigens were used. One was raised in a rabbit and was a gift of Dr. R. Winchester (Rockefeller University, New York) (31), whereas the other was a monoclonal reagent (kindly supplied by Dr. A. Bargellesi, De- partment of Biochemistry, University of Genova). Both antisera reacted with a constant portion of the DR antigen and therefore stained all of the DR- positive cells when used in indirect immunofluorescence. A number of monoclonal reagents detecting antigens specifically expressed by human mononu-

Journal o f Clinical Immunology, Vot. 3, No. t, I983

32 FERRARINI, ROMAGNANI, MONTESORO, ZICCA, DEE PRETE~ NOCERA, MAGGI, LEPRINI, AND GROSSI

clear cell subsets were used. They are listed (together with their specificities and laboratories of origin) in Table Ii. All of them were used in indirect immunofluorescence with a rabbit F(ab')2 anti- mouse polyvalent immunoglobulin conjugated with fluorescein isothiocyanate.

Cytochemistry and Electron Microscopy. The techniques for the cytochemical localization of a- naphthyl acetate esterase (ANAE), acid phosphatase (AP), and [3-glucuronidase (B-Gluc) are detailed elsewhere (21, 25). For the detection of endogenous peroxidase activity (PO) we used the technique of Graham and Karnovsky (32), which allows localization of the enzyme activity at both the light- and the electron-microscopy level. The methods for the electron microscopy preparations (EM) were those described previously (21, 25).

Phagocytosis Assays. Tests for phagocytosis were carried out as previously described (20). Brief- ly, 2 × i0 6 patient cells were incubated with EAG (1:50 ratio) or with 1-1xm latex particles (1:100 ratio) in 1 ml of Hanks balanced salt solution with 20% fetal calf serum (Flow Laboratories, Irvine, Scot- land) and shaken for 2 hr at 37°C. The suspensions were cleared of uningested EA~ or latex particles, incubated for the demonstration of PO activity, and embedded in resin. Semithin sections were stained with toluidine blue and analyzed by light microsco-

pY. Target Cells for Cytotoxic Tests. The human

K562 ceil line was used as the target in the NK assays (33). The murine lymphoma line TLX9 (from the C57BL strain) was the target in the ADCC assays and was kindly provided by Dr. A Manto- vani (Istituto Mario Negri, Milan, Italy) (34). Both cell lines were maintained in vitro using RPMI 1640 (Flow Laboratories) supplemented with 10% fetal calf serum, 100 ~g/ml gentamicin, and 200 mM L- glutamine (RPMI-FCS).

ADCC Assay. First, 2 x 10 6 TLX9 cells were incubated with 200 ixCi 5~Cr (sodium chromate solution; the Radiochemical Centre, Amersham, England) for 2 hr at 37°C in 0.7 ml RPMI-FCS and washed three times in the same medium. Then 2 x 103 cells in 20 txl were seeded in each well of a microtiter plate (Linbro, Flow Laboratories) containing 20 ixl of a 1:1000 dilution of a rabbit anti- TLX9 cell antiserum in RPMI-FCS (kindly donated by Dr. Mantovani) and incubated for 30 rain at 4°C (34). One hundred microliters of an effector-cell suspension adjusted at different cell concentrations to give effector:target ratios of 100:1 and 50:1 was

added. The plate was incubated for 4 hr at 37°C and the radioactivity present in 100 txl of the supernatant of each well was determined in a gamma counter.

The results are expressed as specific chromium release calculated according to the following formu- la:

cpm test release - cpm spontaneous release

cpm maximal release - cpm spontaneous release

x I00,

where the spontaneous release is that of the labeled target cells incubated in medium alone and the maximum release is that of the target cells treated with a 5% saponin solution. The spontaneous release never exceeded 15% of the total incorporated radioactivity, whereas the maximum release was generally about 80%.

NK Assay. First, 2 × 10 6 K562 cells were labeled with 200 IxCi 51Cr for 2 hr at 37°C in 0.7 ml RPMI- FCS and washed three times in the same medium. Then 2 x 103 labeled K562 cells were mixed with various concentrations of effector cells in a 150qxl RPMI-FCS volume in order to give effector:target ratios ranging from 50:1 to 1.5:1. The cytotoxic test was performed in microtiter plates for 4 hr at 37°C in a humidified-air atmosphere with 5% CO2. At the end of the incubation, t00 txl of supernatant was collected from each well and the radioactivity was determined. The results are expressed as lytic units per 10 7 cells. One lytic unit is the number of effector cells necessary to lyse 50% of the target cells. In order to determine 1 lytic unit, the specific release obtained at each effector:tai'get ratio was calculated and then plotted versus the number of effector cells, following a modification of the van Krogh equation (35).

Agarose Single-Cell Binding and Cytotoxicity As- say. The method of Grimm and Bonavida was followed with minor modifications (36). Briefly, equal numbers of K562 and effector cells in RPMI- FCS were incubated for 10 rain at 32°C and pelleted by centrifugation. The supernatant was removed and a convenient volume of a 0.5% agarose solution in RPMI-FCS was added to give a final concentration of 1.2 × 106 cells/ml. The pellet was resus- pended by vigorous pipetting (10 times), and 0.4 ml of this suspension was quickly dispensed in each well of a six-weU tray (Costar, Cambridge, MA) and rapidly spread to form a thin and homogeneous layer. The plate was left at room temperature for a


A LARGE G R A N U L A R LYMPHOCYTE DISORDER 33

few minutes. The semisolid thin layer was covered with 1 ml of RPMI-FCS. Control wells containing target cells alone were prepared in an identical manner. The plates were scored immediately to determine the percentage of conjugated lymphocytes (i.e., the number of single lymphocytes bound to single target cells in a total of I00 lymphocytes). After 4 hr of incubation at 37°C in a humidified atmosphere with 5% CO2, the medium was aspirat- ed from each welt and 2 ml of 0.1% trypan blue was added and allowed to stain for 5 min at room temperature. The wells were destained by a 5-rain incubation with 5 ml of RPMI-FCS and, finally, flooded with 2 ml of medium. The number of lysed conjugated target cells per 100 conjugates was counted and the percentage of cells capable of killing was determined. Spontaneous target-cell death never exceeded 1%.

Ceil Proliferation Assays. Lymphocytes were stimulated in vitro with mitogens or allogeneic cells as detailed elsewhere (37). Briefly, 0.2 × 106 mononuclear cells were cultured for 3 days in 0.2 ml of TC 199 medium (Flow Laboratories) supplemented with 10% FCS in 96-well tissue culture plates with the appropriate concentration of phytohemaggluti- nin (PHA), concanavalin A (Con A) (Difco Labora- tories, Detroit, MI), pokeweed mitogen (PWM) (GIBCO, Grand Islands, NY), or allogeneic cells. Cultures were pulsed with 0.5 fxCi of [3H]Thymi- dine (the Radiochemical Centre) 16 hr before the end of the experiment and harvested by a multiple harvesting machine. [3H]Thymidine uptake was measured in a scintillation counter.

Assay for Immunoglobulin Production in Vitro. The culture method used is detailed elsewhere (38, 39). Briefly, 5 x 10 4 non-T cells were admixed with equal numbers of T-T~ cells in 0.2 ml of RPMI-FCS in a 96-well tissue culture plate and stimulated with PWM (1:100 final dilution). Non-T cells were obtained from peripheral blood mononuclear cells of normal donors by removing T cells (i.e., cells forming rosettes with EN) (25). T cells (EN rosettes) were purified as described elsewhere (25). T cells with receptors for IgG (T~ cells) were identified and removed from T-cell suspensions (T-T6 cells) by rosetting with EAG (25). The possible suppressive effect of various cell populations was tested by adding different numbers of cells (from 1 × 104 to 8 × 104) to the cultures carried out as above. In some experiments, the putative suppressor cells were reacted with EA~ prior to the culture (39). The amount of IgG or IgM released in the culture

supernatants after 7 days was determined by a solid-phase radioimmunoassay in microtiter plates as detailed by Zollinger et al. (40).

RESULTS

Detection o f an Abnormal Proliferation o f Cells with a Homogeneous Surface Phenotype. The surface markers of peripheral blood mononuclear cells from the patient were examined at intervals over a period of approximately 6 months. Tables I and II summarize the results of two different experiments. In both, the presence of an abnormal cell proliferation was apparent from the unusual distribution of the subpopulations and the predominance of a given surface phenotype. Most of the cells formed rosettes with EN and EAG and, therefore, could be considered TG cells. Typical B-cell markers such as sIg and receptors fbr mouse red cells were virtually absent. DR antigens were detected on 30-45% of the cells, indicating that only a fraction of an otherwise homogeneous cell population expressed this marker (Table I). All of the cells stained with anti-pan-T monoclonal antibodies (OKT3, aLeul , aLeu4) but were negative with reagents which recognize surface determinants of immature T cells (OKT6, OKT10). The cells stained with the OKT8 and aLeu2a reagents, which define subsets containing cytotoxic and suppressor cells. Also consistent with the OKT8-aLeu2a positivity was the expression of the antigen detected by the 3A1 monoclonal antibody (41). By contrast, the large majority of the cells was negative for those monoclonals which recognize subsets comprising the helper T cells (OKT4, aLeu3a). A small minority of the cells, most identifiable as monocytes, stained with reagents (OKMI, 4F2) which define predominantly cells of the monocyte-macrophage lineage',. Most of the cells reacted with the NHK-1 reagent, which selectively identifies cells with an NK function (Table II).

Experiment 1 (Tables I and II) demonstrates that the cells with a homogeneous phenotype are more numerous than those detected in experiment 2. These two experiments, canied out at an approxi- mate interval of 4 months, were selected to show the range of variation in the size of the abnormally expanded cell population in different periods.

Morphological and CytochemicaI Studies. Giemsa staining of whole blood smears or of purified mononuclear-cell suspensions demonstrated that the large majority of the patient white cells


34 FERRARINI~ ROMAGNANI, MONTESORO, ZICCA, DEL PRETE, NOCERA, MAGGI, LEPRINI, AND GROSSI

Table I. Surface Markers of Patient Mononuclear Cells

Percentage a

Marker Expt i Expt 2

EN rosettes 97 73 EA~ rosettes 70 76 Surface Ig 2 1 Mo rosettes 1 ND b DR antigens 45 30 EAC rosettes 12 5 EAM rosettes c 14 13

~The two experiments were carried out at a 4-month interval. bNot determined. cMeasured after overnight incubation (29).

(from 70 to 90% at different periods) displayed intracytoplasmic azurophilic granules and the over- all morphology of normal LGL (Fig. 2, inset). Seventy to ninety percent of the patient cells were positive for AP and B-Gluc, with the same pattern of staining as normal LGL, i.e., the cytochemical reaction was localized in the paranuclear area. However, they were negative for ANAE, which gives a strong reaction in normal LGL (Fig. 1).

Similarly to normal LGL, the patient cells were consistently negative for PO and failed to ingest latex particles or opsonized red cells.

At the ultrastructural level, there was a remark-

able morphological uniformity of the cells, which displayed a villous surface and an abundant cyto- plasm with numerous organelles (Fig. 2). In each section every cell showed two to five membrane- bound granules containing a uniform electron- opaque matrix. The number of granules was lower than that generally observed in normal LGL. How- ever, the patient cells displayed many features suggestive of the occurrence of an active process of granulogenesis (Fig. 3a). The cells had a well- developed Gotgi apparatus consisting of several parallel arrays of smooth cisternae surrounded by numerous smooth vesicles (Fig. 3c). Some of these vesicles were seen in the process of fusing with "coated" vesicles to form multivesicular bodies (Fig. 3d). Free coated vesicles were most abundant, particularly in the proximity of the Golgi zone. An electron-dense matrix was sometimes seen within already formed multivesicular bodies (Fig. 3e), indicating that the latter were indeed the precursors of the mature, electron-dense granules (Fig. 3f). The finding that an active process of granulogenesis was taking place in the patient cells also suggested that they might have been less mature than those generally found in the circulation of normal individuals (Fig. 3b). In the latter cells the above-described aspects of granulogenesis are rarely observed (20). Contrary to results reported by others (16, 52), very few cells contained parallel tubular arrays (PTA).

Table II. Surface Phenotype of Patient Mononuclear Ceils as Defined by Monoclonal Antibodies

% positive cells a Monoclonal

reagent Expt 1 Expt 2 Reported cell distribution and reference Comments

HNK-1 b 94 70 OKT3 C 94 80 OKT4 13 15 OKT8 94 70 OKT6 ND 1 OKT10 0 0

OKM1 9 4 3A1 a ND 65

4F2 a ND 2

aLeul e ND 65 aLeu4 ND 85 aLeu2a ND 70 aLeu3a ND 6

Large granular lymphocytes (27) 60% thymocytes, 95% peripheral T (42) 90% thymocytes, 65% peripheral T (42) 90% thymocytes, 30% peripheral T (43) 85% thymocytes (44) Cortical thymocytes (45)

Monocytes and polys (46) 100% thymocytes, 85% peripheral T (47)

Monocytes, 100% thymocytes, 5% peripheral T (48)

95% thymocytes, 95% peripheral T (49) 60-85% thymocytes, 95% peripheral T (50) 85-95% thymocytes, 20-40% peripheral T (51) 95% thymocytes, 40-60% peripheral T (51)

K and NK cells Pan-T reagent Mainly inducer T cells Mainly cytotoxic/suppressor T cells Marker for immature T cells Activated T cells, some B cells and

monocytes Most NK cells Inducer and certain suppressor T

cells Activated T cells

Pan-T reagent Pan-T reagent Mainly cytotoxic/suppressor T cells Inducer T cells

aThe two experiments were carried out at a 4-month interval. bObtained from Drs. T. Abo and C. Balch. ~Reagents of the OK series were purchased from Ortho Pharmaceutical. aObtained from Drs. B. Haynes and A. Fanci. ~Reagents of the aLeu series were obtained from Becton Dickinson.

Journal o f Clinical Immunology, Vot. 3, No. t, 1983

A LARGE GRANULAR LYMPHOCYTE DISORDER 35

Fig. I. Cytochemical localization of acid hydrolases in mononuclear cells from the patient. (a) The majority of the cells is positive for B-Gluc activity, with a paranuclear pattern of staining. (b) The majority of the cells is ANAE negative, as opposed to normal LGL (inset). Arrowheads point to monocytes and to a residual T cell. x 90.0; inset, x 1100.

Fig. 2, Low-power electron micrograph of mononuclear cells from the patient. All of the cells display a remarkable morphological uniformity and contain a relativeIy small number of granules. The inset illustrates the morphological characteristics of a patient cell stained with Giemsa. x 3000; inset, × 1100.

Cytotoxic Activities. The morphology and the predominant surface phenotype of the patient cells suggested that they could be efficient in NK function. The experiments reported in Table III (and two others with identical data) demonstrated that the patient cells had NK activity within the range of

the highest responses of normal peripheral blood mononuclear cells. The ADCC activity was also higher than that generally observed for the most efficient normal mononuclear-cell suspensions (Ta- ble III).

Previous work by Abo and Balch (27) demon-

Journal o f Clinical Immunology, Vol. 3, No. 1, 1983

36 FERRARINI, ROMAGNANI, MONTESORO, ZICCA, DEL PRETE~ NOCERA, MAGGI, LEPRINI, AND GROSSI

Fig. 3. Aspects of granulogenesis in the patient cells. (a, b) A high-power view of a patient cell (a) as compared to a normal LGL (b). Arrowheads in a point to vesicles and multivesicular bodies. (c-f) Details of patient cells showing an expanded Golgi apparatus with numerous vesicles (c), a multivesicular body surrounded by coated vesicles (d), a multivesicular body containing an electron-dense core (e), and a mature granule (f). (a, b) x 12,500; (c-f) x 35,000.

strated that all cells with NK activity are contained in the HNK-l-positive fraction. The HNK-l-positive cells of the patient ranged from 70 to 90%, thus being 5 to 10 times higher than normal. However, a corresponding increase in NK activity was not

observed. In principle, this relative inefficiency could be attributed to a defective binding of the cells to the target, to a defective killing capacity, or to a combination of the two. This problem was investigated further by single-cell binding and cyto-



Table Ill, NK and ADCC Cytotoxic Activities Tested by SlCr Release

Cell NK Cell source (lytic Units/107 cells) source

ADCC(~Cr specific release) at effector:target ratio

100:I 50:1

Patient 137 Control 1 125 Patient Control 2 66 Control 1 Control 3 107 Control 2

74 71 56 42 35 29

toxic assays of K562 target cells. The results are summarized in Table IV. Although the percentage of cells capable of binding was much higher in the patient than in the controls, there was no difference when such values were expressed as the percentage of cells capable of binding relative to the percentage of HNK-l-positive cells (see Table IV). By contrast, the percentage of bound cells capable of killing was much higher in the normal than in the patient. It is noteworthy that the patient cells were not "slow killers" since the maximum number of dead cells in the agarose assay was observed after 6 hr as in the controls. Therefore, the HNK-l-positive cells from the patient were capable of binding to the target but were somewhat defective in their cytolytic capacity. Similar binding experiments could not be carried out for ADCC owing to exces- sive clumping of the cells in the assay system.

The Patient Cells Do Not Have Suppressor Ca- pacities. The predominant surface phenotype of the patient cells suggested that they might exhibit suppressor activities. This hypothesis was investigated by testing the suppressor capacities of the patient cells on both the proliferative response and the production of Ig molecules by normal human lymphocytes. The patient cells failed to suppress the proliferative response of normal lymphocytes to three different mitogens (Table V). Furthermore, they had a profoundly reduced response to all of them (Table V). Likewise, the patient cells failed to respond to allogeneic cells and to suppress an MLC reaction (data not shown).

Graded numbers of the patient cells were added to a mixture of normal non-T and T-TG cells and cultured with PWM for 7 days. In four different experiments no inhibition of the IgG or IgM pro- duced by the cultured cells was detected. These data were obtained independently of whether or not the patient cells were activated by contact with EAc immune complexes. By contrast, in analogy with previous data (39), both autologous and alloge-

neic normal TG cells, when activated by contact with EA~, exerted a significant suppression of Ig production in the same in vitro system (data not shown).

DISCUSSION

The patient described herein presented an abnormal proliferation of cells with receptors for sheep erythrocytes and for the Fc portion of IgG. The cells were positive for the NK cell-specific HNK-1 surface antigen, displayed azurophilic (electron- dense) granules, and stained for AP and B-Gluc with a paranuclear pattern of reaction. Therefore, they could be identified as LGL.

The patient cells displayed a number of ultrastructural features which demonstrated that they were engaged in an active process of granulogenesis. Such a process is usually very limited in normal circulating LGL. Unlike normal LGL, the patient cells were consistently negative for ANAE activity. Granulogenesis generally takes place during the early maturational stages in other heroic cells (53, 54) and ANAE is expressed later than other acid hydrolases, during the maturation of both T and B

Table IV. Agarose Single-Cell Binding and Cytotoxic Assay

Percentage

HNK-1 + Binding a Killingb cells C

Patient 30 (42) 15 70 Controls d 6.1 -+ 1.2 (50) 35.0 - 3.9 12.0 -+ 4.1

~Percentage of lymphocytes forming conjugates with K562 cells. Figures in parentheses correspond to the percentage of cells capable of binding relative to that of HNK-t-positive cells.

bNumber of dead target cells per 100 conjugates scored. The killing assay was scored after 5 hr in vitro, when a plateau was already reached.

cPercentage of HNK-1 + cells determined on the day of the experiment.

~Iean values +- SE of experiments on six different individuals.


38 FERRARINI, ROMAGNANI, MONTESORO, ZICCA, DEL PRETE, NOCERA, MAGGI, LEPRINI, AND GROSSI

Table V. Failure of Patient Cells to Suppress Mitogen-induced Proliferation of Normal Lymphocytes

[3H]Thymidine uptake (cpm) following stimulation with Mononuclear-cell

source PHA a Con A n PWM ~ Control

Patient 10,200 2,900 610 680 Normals ~ 62,300 - 7,000 88,100 --- 10,500 20,200 -+ 2,000 1,650 --+ 300 Normals + patient ~ 64,000 -2_ 8,500 85,000 -+ 9,000 19,300 -+ 3,000 1,710 --- 200

aA titration curve was done for the three mitogens. The figures refer to the highest values detected with 5 ~xg/ml PHA, 5 txg/ml Con A, and a 1:100 dilution of PWM.

bCells from four normal donors. cMononuclear cells from four normal donors (2 x 105) were mixed with graded numbers (from 104 to 5 x 104) of the patient cells. The figures refer to the highest patient-cell concentration.

cells (26, 55). By inference, it is conceivable that the patient cells represented relatively immature LGL. Unlike the response observed for normal LGL, which are weakly positive or negative for DR antigens (56-58), a noticeable proportion of the cells from the patient stained brightly for these antigens. This finding may lend further support to the hypothesis of cell immaturity, since in other hemic lineages (e.g., granulocytes), large quantities of DR antigens are selectively expressed at relatively early stages of maturation (59). In the case of T lineage, cell activation is accompanied by the con- comitant expression of DR antigens (60, 61) and of other markers such as the determinants detected by the OKT10 and 4F2 monoclonal antibodies (48, 62). Interestingly, the patient cells were consistently 4F2 and OKT10 negative and did not stain with the OKT6 monoclonal antibody, which is a marker for immature T cells. Thus, the above findings seem to indicate that the patient cells are not activated T cells. Furthermore, compared to immature T cells, they lack a number of surface markers and express additional surface structures not usually found on immature T cells.

Cytotoxic assays revealed that the patient cells were capable of both ADCC and NK activity. These findings reinforce the view that LGL are the cells which mediate both these cytotoxic functions (26, 63). However, as for the NK activity, there was some inefficiency of the killing, possibly related to the incapacity of a number of cells to accomplish the entire lytic process. These findings seem consistent with the above hypothesis of cell immaturity. Previously, it has been demonstrated that some maturation occurs within the clones of certain lymphoid malignancies (64, 65). The same process could possibly take place within the patient cells, leading to the generation of a relatively small number of mature cells capable of killing. This hypothesis also could be supported by the finding that only

a proportion of cells expressed DR antigens. How- ever, at the ultrastructural level, there were no distinctive features which would suggest that the cells capable of binding and killing the targets were also the most mature (data not shown). Further- more, in the absence of a marker for monoclonality, it is impossible to decide whether or not the abnormally expanded population represented a neoplastic clone. Some of the data (i.e., the homogeneity of the cell phenotype and the infltration of the bone marrow), but not others (i.e., the failure of a proportion of cells to bind to the K562 target), favor a monoclonal proliferation. In analogy with proba- bly similar cases (13, 18), the patient had neutropenia and hypogammaglobulinemia, and the increased number of cytotoxic cells could possibly be second- ary to the immunodeficiency. The fact that the patient later developed a lung cancer complicates the issue further. The relationship among increased LGL number, solid neoplasia and immunodeficiency is unclear, although it may not be fortuitous. Recently, we have observed a marked increase in the proportion of LGL in a child with a transient immunodeficiency, who lately developed a neuro- blastoma.

Previous studies have demonstrated that the majority of cells with NK activity reacts with monoclonal reagents specific for the macrophage/monocyte lineage (66, 67). Observations of purified LGL have confirmed and extended these data by showing that a few cells have the surface phenotype of the suppressor-cytotoxic T cells rather than that of the monocytes (27, 58, 68, 69). Because of their predominant phenotype, the patient cells were likely to derive from this minority of cells with a T cell-like phenotype. These findings raise two questions: (i) Are the LGL with a T cell-like phenotype somewhat related to the T cell lineage? (ii) Is there any close relationship between LGL with different surface phenotypes?

Journal of Clinical Immunology, VoL 3, No. I, 1983


There is evidence, although indirect, that the patient cells may not belong to the T-cell lineage. In addition, in the absence of some T-cell surface markers, they failed to respond to T-cell mitogens and allogeneic cells and were unable to suppress lymphocyte proliferation or Ig synthesis in vitro.

LGL with different surface phenotypes may belong to distinct cell lineages. This would be supported also by studies of murine NK cells showing that they are markedly heterogeneous (70, 71). Alterna- tively, LGL with different phenotypes may represent distinct subsets or even maturational stages of the same lineage. In the case of human LGL, there is no compelling evidence in favor of any of the two hypotheses. Because of the homogeneity of their ultrastructural and cytochemical features, we favor the idea that all of the LGL belong to a unique lineage, possibly distinct from all of the other hemic lineages. The present study shows that cells with a morphologically and enzymatically immature phenotype express the OKT8-aLeu2a surface marker. Interestingly, we have recently found that normal LGL from human bone marrow appear immature and have the OKT8-aLeu2a surface marker, sug- gesting that this may represent the transient surface phenotype of maturing LGL.

A C K N O W L E D G M E N T S

We would like to thank the medical staff of San Giovanni di Dio Hospital (Florence) for permission to study their patient, and Dr. M. Sessarego for the chromosomal analysis of the patient cells.

We are most grateful to Drs. T. Abo, C. M. Balch, A. Bargellesi, A. Fauci, B. Haynes, T. Hoffman, A. Mantovani, and R. Winchester for the generous supply of reagents, to Drs. M. Cooper, T. Hoffman, and A. Mantovani for helpful discussions, and to Miss. M. Dellepiane for secretarial work.

We are indebted to Becton Dickinson (Sunny- vale, CA) for the generous gift of their monoclonat reagents.

This work was supported by grants from the Italian Consiglio Nazionale delle Ricerche (CNR).

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a lymphoproliferative disorder of the large granular lymphocytes with natural killer activity

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