Divisione di Ematologia dell'Ospedale Generale Regionale di Reggio Calabria

(Primario: Prof. A. NE~I)

FUNCTIONS OF T CELLS AND RECRUITMENT OF CELLULAR RESPONSE AFTER I N V I T R O MITOGENIC STIMULATION OF

LYMPHOCYTES tN CHRONIC LYMPHOCYTIC LEUKAEMIA

ALBERTO NERI PAOLO ALBERTO PACIUCCI MAURA BRUGIATELLI

LIONELLO CAMBA MARGHERITA COMIS PASQUALE IACOPINO FRANCESCO NOBILE VINCENZO TRAPANI LOMBARDO

Many important aspects of the in vitro stimulation of lymphocytes by mitogens and specific antigens in chronic lymphocytic leukaemia (CLL) are now well estab- lished ~' 12-15, 17

Some problems, however, have yet to be clarified, especially the behaviour of normal thymus-derived (T) and 'leukaemic' bone marrow-derived (B) lympho- cytes in the proliferative response.

In this paper we will concentrate on these problems, since in our previous report 9 we explored the mitogenic response of whole CLL lymphocyte populations from different sources under various experimental conditions. The present study thus attempted to define the role that T and B cells can play in the proliferative response; another aim was to detect any recruitment of CLL lymphocytes by normal T cells in a co-culture system after stimulation in vitro with phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (ConA).

MATERIALS AND METHODS

Cell source - Ten C L L patients, whose clinical and haematological findings are summarized in tab. 1, and also healthy donors were used as sources for leukaemic and normal lymphocytes.

Key-words: Chronic lymphocytic leukaemia (CLL); Concanavalin A (ConA); In vitro co-cultures; In vitro cultures; Lymphocyte stimulation; Phytohaemagglutinin (PHA); Pokeweed mitogen (PWM).

Accepted for publication on November 24, 1978. La Ricerca Clin. Lab. 9, 67, 1979.

67

T CELLS AND LYMPHOCYTE STIMULATION IN CLL

age sex period without therapy E rosettes SIg

stage (months) (/%) (%)

P.G. 70 M I I I

A.G. 61 M II I

C.F. 68 F IV

~.M. 57 F II

G.D. 71 M I

L.G. 65 M II

M.D. 65 M II

B.R. 71 M II

2.P. 75 F I I I

F.C. 8i F IV

Tab. i - Main clinical and laboratory data to the RAI et al ." classification.

2.5

5

2

3

3

untreated

untreated

untreated

untreated

untreated

15

12

32

3

3

2

4

11

34

9

80

82.5

62

89,5

79

91

90

77

49

78.5

of the patients investigated. The stage given is according

Enumeration of T and B cells - Lymphocytes, isolated from heparinized sterile blood by gradient sedimentation on Ficoll-hypaque, were enumerated by the formation of E rosettes with sheep red blood cells (SRBC), using fluorescein-conjugated anti- immunoglobulin goat antisera as described previously 9

T cell purification - A modification of the method of WYBRAN et al. 2o was employed. In short, 1 x 108 to 1 x 109 lymphocytes were incubated with a suspension of fresh, sterile SRBC at a ratio of 1:10. The mixture was incubated for 20 min at 37"C and then pelleted at 260 x g for 5 min, and kept in an ice bath for 2-3 h. The pellet was then gently suspended and layered over a Ficoll-hypaque gradient and spun at 400 x g for 30 min. The SRBC of the pellet were eliminated by lysis with distilled water followed by an adequate amount of phosphate buffered solution 10x. The viability of cells after hypotonic treatment was evaluated with 1% eosin. To ascertain the degree of purification, the cells recovered from the pellet and at the interface were subsequently tested for their ability to form E rosettes.

Culture medium - RPMI 1640 supplemented with 4 mM glutamine, 100 U/ml penicillin, 100 ,ug/ml streptomycin and 15% pooled decomplemented human serum were employed.

Culture conditions - Basically the same as previously described 9. Flat bottom microtitre plates were used. The culture volume was 0.25 ml in each well, containing 1 x 10 s cells (0.4 x 106/ml). The ceils and the various stimulants were added with a Hamilton syringe adjusted to act as a repeating dispenser. Macrocultures were performed in 17 x 100 mm Falcon tubes, and the culture volume was 2 ml, contain- ing 1 x 106 lymphocytes. Each culture, in plates or in tubes, was performed in triplicate or quadruplicate. The vials and plates, covered with lids, were placed in a humidified incubator with 5% CO2 at 37 "C for 72 and 120 h.

Mitogens . Phytohaemagglutinin (PHA), purified pokeweed mitogen (PWM) and concanavalin A (ConA) were used at different doses, and the values observed at the optimal doses (1 and 10 ~tg/ml for PHA and PWM; 10 and 100 btg/ml for ConA) are given.

68

A. NERI e t al.

D N A synthesis was evaluated after 72 and 120 h. Before harvesting, each culture was labelled with 2 ~tCi 3H-thymidine (specific activity 30 mCi/ml), and after 12 h the cells were harvested, and the isotope incorporation was assessed by scintillation counting as described previously.

Co-cultures were carried out by adding to the CLL cell suspensions in each well or tube a cellular suspension of Iymphocytes obtained from healthy donors. The ratio normal/CLL cells was 1:10 and was kept constant in each culture system.

Microcytotoxic i ty test - The technique introduced by ZARLIN6 et al. 21, using round bottom microtitre plates, was followed. As a stimulant, a pool of allogeneic lymphocytes, obtained from. 10 normal donors and preserved at -80"C in 10% dimethyl-sulphoxide, was used. The results of the chromium (SlCr) release assay were expressed, at the various killer/target ratios, as the % of radioactivity found in the supernatant, collected with Skatron harvesting frames after incubating the plates for 8 h at 37 "C.

E rosettes a/ter incubation with P H A - SRBC rosette formation was determined in some experiments after incubation of lymphocytes with PHA 10 ~tg/ml) for 30 min.

RESULTS

The isolation of T cells, after rosetting with SRBC and separation on a Ficoll- hypaque gradient, enabled us to obtain T ceil-enriched fractions with an amount of rosette forming ceils (RFC) varying from 33 to 80% in the various subjects (tab. 2).

The comparison between the in vitro reactivity of the unseparated peripheral blood lymphocytes (PBL) and that of the T-enriched fractions with PHA (tab. 3) suggests that the mitogenic response is increased in the T cell-enriched fractions of

% RFC % RFC in T ceil- % RFC in T cell- in PBL enriched population depleted fractions

;LL 1

2

3

4

5

6

7

8

9

10

1

2

15

12

32

8

3

2

2

11

34

9

81

73

80

73

74

56

72

61

33

55

58

52

95

90

2

2

1.2

1.5

3

1.5

3

2.4

i

2

0.5

1

Tab. 2 - Degree of purification obtained after T cell separation in CLL and normal subjects (NS).

69

T CELLS AND LYMPHOCYTE STIMULATION IN CLL

PHA (1 ttg/ml)

PBL T cell-enriched fractions

3 days 5 days

ormal

:LL 1

2

3

4

5

6

7

8

9

10

3 days 5 days

121,731 • 7,701 111,158 • 2,245

3,000 4- 53 33,395 • 1,170

150,220 • 11,158

15,050 4- 1,004

105,160 • 3,015

27,796 4- 1,091

1,009 -4- 46

3,950 -• 905

2,258 -• 341

3,218 • 158

29,100 ~ 480

8,000 • 132

12,800 4- 728

nd

nd

1,116 • 76

7,508 • 523

3,190 • 90

12,156 • 463

36,506 • 360

I1,006• 452

5,936 • 359

nd

nd

i6,098 4- 1,736

1,480 4- 211

2,032 --+ 162

2,128 • 166

nd

nd

nd

7,563 • 96

16,556• 1,231

25,837 4- 2,357

3,078 • 41

1,768 • 28

13,421 4- 491

nd

nd

nd

nd

13,883 • 1,089

PHA (10 ~g/ml)

normal

CLL l

2

3

4

5

6

7

8

9

I0

208,233 4- 7,600

14,101 ~ 2,215

19,433 4- 4,330

16,208 • 23

4,426 4- 70

22,260 • 1,438

31,923 • 315

6,541 4- 113

11,240--- 231

nd

nd

127,173 ~- 2,048

6,634 • 875

10.348 • 941

32,896 • 483

1,638 • 26

88,513 • 40l

60,850 • 80

23,976 4- 734

25,308 • 697

nd

nd

162,702 • 4,185

52,236 • 1,926

157,489 • 6,425

23,491 • 208

17,938 • 538

58,951 • 758

nd

nd

nd

17,433-~ 218

20,483 • 57i

123,042 -~ 11,922

212,106 4- 4,868

118,666 ~ 7,116

55,043 --- 848

85,801 -4- 216

200 ,89l • 1,189

nd

nd

nd

nd

18,137 4- 593

Tab. 3 - P H A st imulat ion of unseparated blood lymphocytes and T cell-enriched fractions, in CLL patients and normal controls. Values are expressed as cpm ~ sd; nd = not determined.

CLL lymphocytes, and that it becomes comparable to that of the normal T cell- enriched fractions after five days of culture, thus showing the same delay in proliferation as whole CLL PBL.

In addition, stimulation with PHA in the T cell-enriched cultures has a mito- genic dose-dependent effect that cannot be detected in the T cell-en'riched cultures of control subjects or in the PBL cultures of both CLL and control lymphocytes (tab. 4).

Table 5 and fig. 1 give the results of the experiments carried out to evaluate the alloantigen recognition of CLL lymphocytes and the cloning of effector T cells. 3H-thymidine incorporation in mixed leukocyte cultures (MLC) and the peak of proliferative activity were lower and more delayed than in the control MLC, but

70

A. NERI et al.

the cloning of cytotoxic T cells, as measured by the S'Cr release assay, demonstrated a preservation of the T cell effector mechanism.

The evaluation of these preliminary results led us to raise the question whether the hyporesponsiveness of CLL lymphocytes could be ascribed, at least in part, to some alteration in T-B cell cooperation in vitro during the mitogenic response 8. Co*cultures were thus performed in which whole CLL PBL were supplemented with 10% of normally reactive lymphocytes obtained from normal donors and then stimulated with PHA, PWM and ConA.

The results of these co-cultures are shown in fig. 2 after five days of culture at one mitogen concentration. A higher reactivity was obtained from CLL PBL under these culture conditions. Furthermore, it can be seen that the same pattern of response is found in co-cultures as in whole and separated CLL populations. The increase in the mitogenic response, although statistically significant; is not as striking as on the third day of culture.

The increase in DNA synthesis in the co-cultures is due to an amplification of the response of the leukaemic lymphocytes, and this is supported by the following experiment: 10 4 normal lymphocytes cultured with l0 s mitomycin-treated autologous or leukaemic lymphocytes, thus reproducing the microenvironment of the co-cultures, failed to yield the activation values that had been observed in the co-cultures in which the CLL ceils were allowed to respond (tab. 6).

NS

CLL

PBL enriched T cells

0.84

1.14

0.20

9.18

4.38

0.51

7.11

0.55

1.10

1.17

7.63

4.58

17.88

48.52

14.96

1.30

Tab. 4 - P H A dose-dependent activation of CLL lymphocytes expressed as tee ratio between the cpm of cultures stimulated with 10 ,t~g/ml P H A and the cpm of cultures stimulated with 1 ~tg/ml PHA. Evaluation after culture for 120 h.

cultures cpm • sd

CLL + pool,~ 4,095 • 1,718

NS + pool,~ 24,098 • 3,212

CLL 4- NS + pool,,, 36,077 • 2,410

Tab. 5 * Mixed leukocyte cultures from I CLL case and a pool of allogeneic lymphocytes obtained from 10 different donors. The concomitant culture of CLL and normal (NS) lymphocytes did not depress 3H-thymidine incorporation. Subscript m indicates that the cells were rendered unrespon- sive by treatment with mitomycin.

71

100

80"

20-

60,

4o-

de, __ *A, 1~

1:1 5:1 25:1

killer/target ratios

T C E L L S AND L Y M P H O C Y T E S T I M U L A T I O N I N C L L

Fig. 1 - Microcytotoxicity test. Targets are: unrelated normal subject (o), and CLL lymphocytes (o, g); autologous normal (A) and CLL (n) lymphocytes.

%

>r

E o_ o

100-

50-

PHA (10}Jglml) PWM (l~Jg/ml) ConA (lO0pglml)

Fig. 2 - PI-tA, PWM and ConA stimulation of CLL PBL cultures (o) and CLL 105 + normal 104 PBL co-cultures (o), Evaluation after five days of culture.

72

A. NERI e t al.

co-culture system cpm -4- sd

N + Plm

N + P2,,,

N + N S m

Pt + N

P1 + NS.,

P1 + P1m

P 2 + N

P2 + NS,,,

P2 + P2.,

P2

18,628 -+ 642

15,828 • 951

9,776 • 636

94,456 • 1,36t

8,338 -4- 475

10,796 • 2.116

44,175 -+ 1,236

3,998 -4- 238

1,481 -4- 184

2,632 • 447

Tab. 6 - Co-culture experiments with 10' normal (N) and l0 s CLL (P) lymphocytes or 10 ~ autologous normal cells (NS) in the presence of PHA 10 ,ttg/ml. Subscript m indicates that the cells were rendered unresponsive by treatment with mitomycin. Evaluation after culture for 120 h.

60

40 �84 %

•

E {3.

20

PHA (10 }Jglml) PWM (1 Hg/ml) ConA (100 ~JglmD

Fig. 3 - Co-culture experiments, as in fig. 2: | = CLL RFC-depleted fractions, o = CLL RFC- depleted fractions + 10% normal PBL. Evaluation after five days of culture.

The same 'synergic ' effect was present when T cell-enriched fractions from normal PBL were used in the co-cultures; however , when CLL T-enriched populat ions were added, they were unable to elicit such an effect (data not shown). Also in the co-cultures performed with CLL RFC-deple ted fractions, the addit io, l of 10~ normal lymphocytes greatly amplified the mitogenic response (fig. 3).

73

T CELLS AND LYMPHOCYTE STIMULATION IN CLL

In conclusion, a synergic effect was achieved in all co-culture systems, regardless of the number of ceils employed, from 10 s to 106, under the different culture conditions (microtitre plates or tubes): fig. 4 shows the increase in RFC in co- cultures in tubes, starting from 106 CLL PBL plus l0 s control lymphocytes.

DISCUSSION

Our study aimed at answering two of the many problems concerning the interpretation of the defects of in vitro lymphocyte stimulation in CLL: the function of residual T ceils, and the possible involvement of the leukaemic clone in the proliferative processes after mitogenic stimulation.

As regards the first question, it is recognized that T cell function in vitro in CLL is controversial: in fact in some reports 2,3. ~0, ts they are described as severely impaired, whereas in others they appear to possess a normal reactivity ,,20

Our experiments indicate that CLL T ceils react in a different fashion from normal T lymphocytes. They show in fact a good reactivity to mitogens, but require a stronger mitogenic stimulus (about ten times greater than normal) in order to be triggered to an optimal response, and even then still display a retarded DNA synthesis peak.

The delayed activation peak of T cells could be explained by the fact that, with our separation procedure, we were not able to obtain highly purified T cell fractions; however, this factor is probably not of great importance when we consider that almost in every case the enrichment that was produced yielded a % of RFC that was near to the usual T cell % of normal PBL preparations. Another possible explanation could be that the enriched T cell fractions of our CLL patients were composed of two different cell populations. In fact, in some experiments in which the RFC recovered from the pellet after the first gradient separation were mechanically dissociated from SRBC and then layered over another Ficoll-hypaque

40

30

20

10

%

•

m 1

0 3 5

days of c u l t u r e Fig. 4 - Increasing number of RFC in three co<ulture experiments.

74

A. NERI et al.

before after

CLL

NS

28

11

8

5

29.5

21

63

63

46.5

86

28.5

27

41

34

70

77

Tab~ 7 - Number of E rosetting CLL and normal lymphocytes before and after incubation with PHA at 10 t~g/m~ for 30 rain,

gradient, the weakly rosetting lymphocytes from the second interface were unre- sponsive. Stimulation of cultures lacking this latter cell population, however, gave activation results not dissimilar from those obtained in the cultures performed with lymphocytes recovered from the first gradient (data not shown).

Finally, the dose-dependent PHA reactivity of CLL T cells could be explained on the basis of a recent observation ~6 that PHA can reveal a T antigenic substance on the lymphocytes of CLL after exposure of the cells to the mitogen for a brief period; it also increases the number of RFC in normal and CLL cultures 6.7 (tab. 7). The disclosure of T antigenic sites in CLL could thus account for the augmented response to higher mitogenic doses. It does not explain, however, why the pattern of this response remains delayed, like that of unseparated CLL cell populations.

We feel, therefore, that these findings suggest that CLL RFC may be responsible, at least to some extent, for the impaired reactivity of CLL lymphocytes.

On the other hand, it is reasonable to argue that the most important part in this impaired proliferative response should be attributed to the large number of leu- kaemic cells, defined as 'B' by their inability to form rosettes.

Taking all these considerations into account, we investigated the possibility of a recruitment of these 'leukaemic' cells by normal T lymphocytes in the mitogenic response.

Analysis of the co-culture experiments indicates that, in fact, a great number of leukaemic cells are recruited. We would like to point out that this recruitment was more evident in the co-cultures in which PHA and ConA were employed as stimulants, that is a mainly T cell type response was obtained. Furthermore, it should be noted that the same type of response is also evoked in co-cultures with CLL RFC-depleted cell populations. In other words, it seems that such a system favours the cloning of CLL lymphocytes, regardless of their characterization by immunological markers.

We can say little about the interpretation of these co-cuhure results. In these circumstances the involvement of previously unresponsive cells may be due either to some sort of cell-to-ceil interaction or to the presence of soluble mediators produced by the small number of normal activated cells. Evidence of such in vitro interaction

75

T CELLS AND LYMPHOCYTE STIMULATION IN CLL

mechan isms dur ing mi togenic s t imula t ion of n o r m a l lymphocytes has been repor ted recent ly 5.

Anyway , wha teve r the exp lana t ion of these events is, we bel ieve that our ex- pe r imen t s suppor t the con t en t i on 19 that to define CLL as a c lon ing of un respons ive B lymphocytes may be an overs implif icat ion of the complex biological i m p a i r m e n t of cells in this lymphopro l i fe ra t ive disorder.

SUM2vLARY

In vitro stimulation of peripheral blood Iymphocytes from ten patients with chronic lympho- cytic leukaemia (CLL) was investigated under various culture conditions. It was confirmed that the mitogenic reactivity of whole cell populations (PBL) was delayed and depressed. When CLL rosette-forming cells (RFC) were stimulated, their 3H-thymidine uptake was increased, but the pattern of the response was similar to that of whole PBL, thus suggesting some impairment of these cells. Furthermore, in order to evaluate the possible recruitment of B cells, generally thought to be unresponsive, some co-culture experiments were performed in which 104 normal lymphocytes and 10 s CLL whole PBL or RFC-depleted ceil populations were stimulated with mitogens. An amplified response of the CLL lymphocytes was obtained in all co-cultures, and this effect was more evident when specific T cell stimulants were used; autologous CLL T lymphocytes, on the contrary, failed to display such a 'synergic' effect. These results indicate that normal lymphocytes are able to recruit a large number of CLL lymphocytes in the mitogenic response; furthermore, the fact that in co-cultures of CLL T-depleted fractions a better response was obtained with T cell mitogens suggests that the definition of CLL as a clonal expansion of unresponsive 'B' lympho- cytes may be inadequate.

REFERENCES

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2. EPSTEIN L.B., CLINE M. J.: Chronic lymphocytic leukemia: studies on mitogen-stimulated lymphocyte interferon as a new technique for assessing T lymphocyte effector function - Clin. exp. Immunol. 16, 553, 1974.

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4. JAKSIC B., PAGLIARm G. L., PEGORARO L.: Kinetics of phytohaemagglutinin,stimulated lympho- cytes in chronic lymphocytic leukaemia - Ceil. Tiss. Kinet. 9, 419, 1976.

5. KONDRACK* E., MILGROM F.: Direct and cooperative mechanism of lymphocyte triggering in liquid and solid cultures - J. Immunol. I18, 381, 1977.

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7. MANCONI P. E., DEL Glacco G. S., TOGNELLA S., MANTOVANI G., FADDA M. F., CORNAGLIA" P., ZICCA A., ZACCHEO D., GRIFONI V.: Ulteriori osservazioni concernenti l'azione della fitoemo- agglutinina (PHA) sulla formazione di rosette E da parte di linfociti periferici umani - In: Atti del XXV Congr. naz. Soc. itai. Ematol., Milano, 1975.

8. MORETTA L., \VEBB S.R., GROSSI C.E., LYDYARD P., COOPER M.: Functional analysis of human T-cell subpopulations: help and suppression of B-ceil response by T-cells bearing receptors for IgM or IgG - J. exp. Med. 146, 184, 1977.

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10. PEGORARO L., GAVOSTO F.: Phytohaemagglutinin responsive lymphocytes in chronic lymphocytic leukaemia - Lancet i, 1508, 1973.

11. RAI K.R., SAWITSKY A., CRONKITE E.P., CHANANA A.D., LEVY R.N., PASTERNACK g.s . : Clinical staging of chronic lymphocytic leukaemia - Blood 46, 219, 1975.

12. RUBIN A.D., Davis S.: The role of the lymphocyte in producing the clinical manifestations of chronic lymphocytic leukaemia - Med. Clin. N. Amer. 37, 463, 1973.

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A. NERI et ah

13. SCHULTZ E.F., DAvis S., RUBIN A.D.: Further characterization of the circulating cell in chronic lymphocytic leukaemia - Blood 48, 223, 1976.

14. SMITH J. L., BROWNE E., SLUNGAARD A.: The impaired responsiveness of chronic lymphocytic leukaemia lymphocytes to allogenic lymphocytes - Blood 41,505, 1973.

15. SMITH J. L., COWLING D. C., BARKER C.R.: Response of lymphocytes in chronic lymphocytic leukaemia to plant mitogens - Lancet i, 229, t972.

16. TmEL E., RODT H., HUHN D., THIER~ELDER S.: Decreased and altered distribution of human T antigen on chronic lymphatic leukaemia cells of T type suggesting a clonal origin - Blood 47, 723, 1976.

17. TURSI. A.: Deficit of cell-mediated immunity in chronic lymphatic leukaemia - Folia allerg. Immunol. clin. 22, 186, 1975.

18. UTZINGE~ P.D.: Lymphoid T cell transformation in chronic lymphocytic leukaemia: demon- stration of a blastogenesis inhibitory factor - Blood 46, 883, 1975.

19. WHITESIDE T.L., WIN~(ELSTEIN A., RABIN B.S.: Immunologic characterization of chronic lymphocytic leukaemia cells - Cancer 39, !109, 1977.

20. WYBRAN J., CHANTLER S., FUDENBE~G H.H.: Isolation of normal T cells in chronic lymphocytic leukaemia - Lancet i, 126, 1973.

21. ZA~LING J.M., McKEouGH M., BACH F.H.: A sensitive micromethod for generating and assaying allogenically induced cytotoxic human lymphocytes - Transplantation 21, 468, 1976.

Requests jor reprints should be addressed to:

ALBERTO NERI

Divisione di Ematologia dell'Ospedale Generale Regionale 89100 Reggio Calabria - Italia

77