cLrNrcAL TMMUNoLOGY AND TMMUNOPATHOLoGY 24, 330-341 (1982)

Heterogeneity of T-CLL Defined by Monoclonal Antibodies inNine Patientsl

FnaNco PANoorrr, GteNpIr,rno SBIusNzaro, Grur-lo Dr Rossr,Doucras M. SrnoNc, IseBnLLA Qutnrr, ANroNro Pezzu'rro,

FneNco MANDeLrr, eNo FBnNeNoo Arurr2,3

Department of Clinical Immunology and Department of Hematology, University of Rome, Rome,and Immunopathology Division, Department of Clinical Medicine II, University of Padua, Padua,

Italy; and Transplantation Research Branch, Naval Medical Research Institute,Bethesda, Maryland

Surface markers were examined on peripheral lymphocytes from 450 patients withchronic lymphocytic leukemia (CLL). In nine (2%), the lymphocytes had the character-istics of thymus-derived cells (T-CLL). These nine patients had a milder clinical coursethan that usually attributed to T-CLL, lack of massive hepato- or splenomegaly, andfewer skin lesions. Lymphocytes from these patients were studied with surface markers,including a panel of monoclonal antibodies (MoAb) and also for in vitro functional activ-ity. Whereas cel1s from two patients appear to represent heterogeneous proliferations, asdetected by MoAb, seven cases gave a clearly recognized phenotype. Evaluation ofimmunoregulatory functions showed that OKTS+, Fc IgG receptor-positive cells (threepatients) were able to suppress either lymphocyte proliferation, or immunoglobulinbiosynthesis, or both. In three patients with a proliferation of OKT4+ cel1s, irz vitro helpwas not demonstrable, but in two of them, high levels of serum IgA were detected. Ourresults clearly show that T-CLL may originate from differeni T-cell subpopulations.Some, if not all of the T-CLL cells, seem to represent the proliferation of well-differentiated T cells, corresponding to the normal T-cell subsets. In some cases thesecells retain functional activity. The expression ofa given phenotype pattern, as detectedby MoAb, represents a useful tool in determining the clonal origin of proliferating cells.

INTRODUCTION

Most cases of chronic lymphocytic leukemia (CLL) derive from the clonalexpansion of B lymphocytes; while the malignant proliferation of T cells (T-CLL)represents a very small number of cases (1-3). T-CLL is a rare disorder in bothEurope and the United States; in contrast it seems to be relatively more commonin Japan, where B-CLL has a much lower incidence (2). Although T-CLL is notyet well characterized, clinically, it has been reported that, in comparison to

1 Supported in part by Grants N. 81.01282.96 from the Consiglio Nazionale delle Ricerche, Italy; Co.N. N0014-77-C-0754 from ONR, and Y01-CP-00502 from the National Cancer Institute. The opinionsand assertions contained herein are the private ones ofthe writers and are not to be construed.asofficial or reflecting the views of the Navy Department or the Naval Service at large.

2 To whom reprint requests should be addressed: Department of Clinical Immunology, Vialedell'Universitd, 37, 00161 Rome, Italy.

3 To whom correspondence should be addressed: Universitir degli Studi di Roma, Cattedra diImmunologia Clinica, Policlinico Umberto I, 00161 Rome, Italy.

0090-1229t 82t 090330- I 2$01.00/0Copyright @ 1982 by Academic Press, Inc.All rights of reproduction ia any fom reserved

330

HETEROGENEITY OF T.CLL 33I

B-CLL, these T-CLL patients have a more aggressive clinical course, massivesplenomegaly, only moderate bone marrow infiltration, increased frequency ofskin lesions, and severe neutropenia (1, 2). Immunologically, it is now acceptedthat T-CLL usually represents the proliferation of fairly differentiated T cells(r,2,4, 5).

Recently, a number of techniques have been developed to characterize T-cellsubsets, both by surface markers and by functional activities. However, due to therarity of this disease, few cases of T-CLL have been characterized by eithersubset surface markers , or functional studies, or both (4-9) , confirming that casesmay express suppressor or helper surface markers and/or activities.

With the recent development of monoclonal antibodies (MoAb) with specificityfor T cells, it has become possible to classify lymphoproliferative disorders ac-cording to reactivity with various MoAb (5, 9, 10).

In this paper we describe clinical and immunological features of nine patientswith T-CLL. Peripheral blood lymphocytes (PBL) from these cases have beenstudied with a panel of I I MoAb recognizing different subsets of human PBL. Inaddition, studies with conventional surface markers were performed together withthe assessment of certain functional studies.

MATERIALS AND METHODS

Monoclonal Antibodies and Other Surface Markers

Isolation of PBL from heparinized blood was performed by Ficoll-Hypaquedensity-gradient centrifugation (11). Eleven MoAb that have been previously de-scribed were used. Details on these MoAb are listed in Table I (12- 18). Todetermine the frequency of positive cells, lymphocytes were incubated with ap-propriate amounts of each MoAb in an indirect immunofluorescence test (19).

The frequency of surface membrane Ig (Smlg)-bearing PBL and of lymphocytesforming rosettes with sheep erythrocytes (SRFC) were determined as previouslydescribed (11). Evaluation of sheep rosetting cells bearing Fc-IgM and Fc-IgGreceptors (FcpR+ and FcyR+) was performed as previously reported (20).

TABLE 1

MoNocLoNer- ANrrnoorr,s Useo rN rnr, PnEseNr Sruoy

MoAb Specificity

oKT312UCHT113ap69l7ltaOKT61'oKT101'oKT1l15oKT412OKT81'34.116

OKMl or M217

oKI118

Pan T cellsPan T cellsPan T cellsThymocytesThymocytes and a few PBLPan T cells (SRBC receptor)Helper T cellsCytotoxic/suppressor T cellsHelper and inducible/suppressor T cellsMyelomonocytesIa-like antigens

332 PANDOLFI ET AL.

Functional Studies

Response of PBL to lectin phytohemagglutinin (PHA) was performed in tripli-cate as previously described (21). PBL were placed in culture (106/ml of RpMI1640 with l07o fetal calf serum) and PHA-P (Wellcome, U.K.) at a concentration of1.0 p.g/ml. After 3 days incubation at 37'C in the CO, milieu, DNA synthesis wasstudied by adding 1.0 pCi of tritiated methylrhymidine (sp. act, 5.0 mCilpg) 6 hrprior to termination. The cells were harvested with a MASH harvester and thesamples were counted in a liquid scintillation counter. Since tests were performedat different times, the results are expressed as percentage of mean thymidineuptake compared to a normal control in each experiment.

suppressor or inducer (helper) ability of r-cLL cells was determined usinglectin-induced responses (LIR) of normal PBL and pokeweed mitogen (pwM)-induced B-cell differentiation (BCD) as responder functions, as reported (4,22).lnbrief, activity on LIR was studied by the effect of l0a T cells on the response of 105normal PBL in vitro. Percentage of inhibition with respect to stimulated cultureswas calculated at Day 3 according to a previously described formula (4).

stimulation index of normal PBL + Tstimulation index of normal PBL x 100.

Control experiments showed that the addition of normal PBL to autologous PBL(1:10) resulted in variations not exceeding 10% of the stimulation index.

In order to evaluate helper or suppressor effects on the in vitro PWM-dependentBCD, T-CLL cells were added to a helper combination of normal T FcTR- ceils (5x 104/well) and normal B cells (10 x 105/well) in the presence of pwM (GrandIsland Biological Co.; 1 pllwellof the 5 ml reconstituted powder), according to themethod of Moretta et al (20). Cultures were performed in flat-bottom microtiterplates (Falcon, 3040, Rockville, Md.). Lymphocytes were cultured in 200 p,l ofRPMI 1640 (Grand Island Biological co., Grand Island, N.y.), supplemented withl0To fetal calf serum and antibiotics . Control cultures were B-cell fractions, fromhealthy subjects, alone (105/well) cultured in the presence of pWM. Cell combina-tions were cultured at 37'C in 5% co2 atmosphere for 7 days, then harvested,counted, and cytocentrifuged. The viability was assessed in this cell suspensionby trypan blue exclusion. The slides were fixed in methanol containing 57o aceticacid, rehydrated in PBS, and stained at room temperature for 20 min with polyva-lent goat anti-human fluorescent immunoglobulin antibodies (Meloy Laboratories,springfield, Ill.; molar F/H ratio 2.6-4.3). Slides were examined with a Leitzorthoplan microscope. The percentage of cells stained brightly and diffusely forcytoplasmic immunoglobulins was evaluated and the absolute number of plasmacells per well determined as a function of the number of viable cells per well.Suppression of helper activity was calculated as percentage of variation betweencultures containing l07o (.or 25%) T-CLL cells in comparison to cultures contain-ing the helper combination alone (i.e., normal B and r FcTR cells). preliminaryexperiments had shown that the addition of 10-25% of normal T-PBL to thehelper combination results in variations not exceeding -r 157c, on plasma cellrecovery.

Percentage /inhibition:100-( u!)-CLL ce

HETEROGENEITY OF T-CLL

Clinical Features of T-CLL Cases

333

Routine surface markers for T and B cells (SRFC and SmIg) were performed on

PBL from 450 patients with cLL. Nine patients (2%) had T-GLL. A summary ofclinical parameters at presentation of these nine cases is listed in Table 2. The

majority of these patients were asymptomatic and diagnosis was made in all but

on" .ut" by routine blood examination. One case (case 7) presented with skin

lesions. The mean age was 60 years, with a male-to-female ratio of almost 2 to l,similar to B-CLL. At the time of diagnosis, in addition to a peripheral

lymphocytosis of 10.2 to 55 x 103/mm3, all patients showed bone marrow in-

volvement with 35-90Vc lymphocytes, thus meeting Rai's criteriafor CLL (23).

Four cases had slight to moderate hepatosplenomegaly and lymphoadenopathywith a diffuse infiltration of small- to medium-sized mature-appearing lympho-

cytes. In only two cases (cases 6 and 7) was an abnormal immunoglobulin level

noted with an increase in IgA in both (362and 570 mg/dl). In one case (7) skin

involvement with erythematous desquamative lesions on the face, arms, and legs

\\'as seen since diagnosis. Patient 6 also developed nodular skin lesions in the legs

afrer 22 months of observation. In general, the majority showed a less aggressive

clinical course than that usually reported for T-CLL. Only three patients were

treated (patients 5, 6, and 7). Two patients died during the period of observation'

One (case 1) died accidentally from a traumatic cranial injury, and case 7 from

bronchopneumonia. In the other patients, the time of observation varied from 13

t case 9) to 46 months (case 3 , a previously reported (4) patient) with a mean of 25 ' 6

months.

-{rra1-r'sls of Immunologic Phenotype by MoAb

The results obtained on T-CLL cells with a panel of MoAb are reported in Table

l. OKT11+ cells were present in all nine cases studied; however, using three

anti-total T MoAb (OKT3, UCHT1, and ap69l71), eight cases (all except case 4)

u'ere positive. This discrepancy may be attributed to the fact that OKT11 recog-

nizes the receptor for sheep erythrocytes, whereas the other three anti-total T

\foAb recogniie different T-cell-specific antigens which are not present on all the

SRFC. OKT6 was negative in all of the cases, thus confirming that, in contrast to

nanl'T-cell-acute lymphoblastic leukemias, T-CLL derives from cells lacking this

earl1: differentiation (i.e., common thymocytic) marker. However, OKT10, which

recognizes an antigen present on 957o of thymocytes and about 5 to 97o of PBL'lia-\ detected on 38 and 2l7o of the cells from patients 2 and 8, respectively'

OKlp- cells were absent or very low in all of the samples tested, indicating that

rhese T-CLL lymphocytes do not present antigens expressed by cells from the

rnl'elomonocytic lineage. oKT4, OKTS, and 3A1 MoAb have been claimed to

i,Jentify functionally active subsets of T-PBL (12, 24) . Inducer cells are contained

ri ith OKT4- and 341+ cell populations although these two MoAb do not recognize

precisel-v the same subsets of T cells; in contrast, OKT8+ cells include suppressor

i, mphocl.tes. In normal donors, the ratio between OKT4+ and OKTS+ cells is 1'73

- t-).j ( 19). and OKT8+ cells represent26.6 -r 4.37o of normal PBL. In our series,

rhxree cases with a marked increase of OKTS+ cells were observed (cases 1-3),

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while OKT4+ cells were concomitantly reduced or absent. On the other hand, twocases were shown to represent a proliferation of OKT4+ cells (cases 5 and 6), withlack of the OKTS subset. Three additional cases were comprised of both OKT4+and OKT8+ cells. One of these (case 7) was under therapy at the time of study.This case came to our observation with skin lesions and was found to have highOKT4+ (66%) and low OKTS+ (14%) cells. Analysis of the skin infiltration byMoAb (Pandolfi, in preparation) revealed that the proliferating lymphocytes wereoKT4+.

In case 4, OKT4+ cells were absent and OKTS+ cells were reduced in compari-son to normal controls. 3A1+ cells were present in four cases (cases 2,3,5,8);whereas, they were undetectable in cases 1,4,6,7, and markedly depressed incase 9. An increased percentage of cells positive for Ia-like antigens (as detectedby reactivity with OKII) was observed in cases 3, 6, and 7. Cases 2 and 4 hadnormal percentages, while the remaining cases had no detectable Ia+ cells.

Characterization by Membrane Markers and Functional Tests

Table 4 lists results obtained using certain conventional surface markers andfunctional tests. All the patients studied had high proportions of SRFC, as ex-pected in T-CLL cases, whereas surface membrane Ig was always very low orabsent. The study of T cells binding the Fc portion of IgG or IgM was of particularinterest, since FcTR+ cells have been reported to provide suppression and FcpR+help in in vitro B-cell differentiation (20). Moreover, the correlation betweenFcTR+ and FcpR+ cells, on one hand, and OKT4+, OKTS+, and 3A1+ cells, on theother, is still unclear under normal and pathological conditions (25,26). A highproportion of FcTR+ cells with an inversion of the normal ratio between FcpcR+

and FcTR+ was observed in five cases (cases 1-4 and 7). Cases 1-3 had concom-itantly high proportions of OKTS+ cells. In case 7, OKT4+ and T8+ cells werenormally represented, and in case 4 these two subsets were reduced or absent.FctrrR+ cells were reduced in the majority of our cases and, more noteworthy,were markedly reduced in case 6, where a high proportion of OKT4+ cells wasobserved.

Functional activity of these T-CLL cells was assessed by several tests. Theblastogenic responses to PHA were shown to be impaired, in all the cases tested.The ability of these cells to provide cooperation with normal T and B lymphocyteswas assessed in two assays measuring inhibition or help in lectin-induced re-sponses of normal T-cell (LIR) or normal B-cell differentiation (BCD). Case 1

(showing the phenotypes OKT3+, T8+, T4-, NIz- ,3Al- , and FcTR+) was suppres-sive on BCD, but not on LIR. Case 2 (OKT3+, T8+, T4-, l;sIz-,3A1+, and FcTR+)was able to suppress in both the assays. Case 3 (showing the same phenotype inaddition to Ia-like antigens) was markedly suppressive on LIR, despite the lack ofactivity on BCD. Suppression on BCD and borderline suppression on LIR wasobserved in case 4 (OKT3 ,T8-,T4 ,M2-,3,{1-, and FcTR+). Despite aprepon-derance of OKT4+ cells, help on BCD was not provided by T-CLL cells of cases 5

and 6 (OKT3+, T8 , T4*).The remaining cases showed no activity for eithersuppression or help.

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we also report three cases (cases 5-7) appearing to be a proliferation of oKT4

cells.TwohadprimarilyoKT4+circulatingcells,whereasincaseT,evidencethattheproliferatingcellsboretheT4antigenwasreachedbyadditionalstudiesonlymphocytes isolated i-*,t" skin lesions (Pandolfi et al., in preparation) and

from electror, *i"ro."opi" u'ufy'i'' Since OKT4 cells have been shown to provide

inducer activity in ritro, itias noteworthy that cells from all these patients were

unable to help in vitrlo'the BcD. A remarkable clinical observation was that'

despite negativity in the BCD assay' hyper-7-globulinemia of the IgA class was

found in both of thes" p.,i"r", .ugg"rt6 a iunctional role for these cells in vivo '

It is possible that the ircp u.ruv *ed was not sensitive enough to demonstrate

*f;1T:":T""i?l:;". 8 and e) both oKr4+ and r8+ cells (as well as FcpR and

FcTR)wereobserv"o.-et.t,o,ghproliferationsofmixedpopulationshavebeenreported in T-CLL b; ffi;;t ilT-q Ytlb (34)' further studies' during both fol-

lowup of the oi."u.r'u.ra ,rsing add-itional MoAb, should give further insight into

the malignant procesJfor sucf, patients. one can not clearly distinguish, in such

cases, between *rrl"i"Jity with dilution by normal residual T cells, or a

polyclonal Proliferation'Inconclusion,studieswithMoAbmaydemonstratethatthemajorityofthese

T_.LL cells repres"rri "tonut

expansions of mature-appearing T-cell subsets'

showingavastt'"t".-og","ityofsurfacephenotypesandfunctionalactivities.ACKNOWLEDGMENTS

WewishtothankDrs.A.S.Fauci,M.Ferrarini,.lvl.Fiorilli,C.E.Grossi,andR.B'Herbermanforexcellent discussion; ,r..^ple.-r*ti, p. Beverly,-uno c. Goldstein for providing MoAb; Mrs' A'

Frielingsdorf ror stimut, tect.-i.;i"rpt Mrs. A. ct".**i"" and Mr. c' Bowen for editorial assis-

tance.

l.)3.

4.

6.

7.

5

8.

q

L., Ortaldo, J. R., and Herberman' R' B''

Immunol. ImmunoPathol' 17'

Van Oers, R. H. J', Silberbusch' J''

P., von dem Bourne, A' E' G' K'' and

t0

11Immunol. Clin. 24,83, 1977

Immunol. 126,2205,

HETEROGENEITY OF T-CLL 34I

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1943,1980.18. Reinherz. E. L.. Kung. P. C.. Pesando, J. M., Ritz, J., Goldstein, G., and Schlossman, S. F.,J.

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usA 77,291, 1980.

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S. F.,J. Exp. Med. 157,969,1980.26. Moretta, L., Moretta, A., Canonica, G. W., Bacigalupo, A., Mingari, M. C., and Cerottini, J. C.,

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Received December 9, 1981; accepted with revisions February 10, 1982