IN VITRO FUNCTIONAL STUDIES OF MONONUCLEAR CELLS IN PATIENTS WITH CLL

Evidence for Functionally Normal T Lymphocytes and Monocytes and A bnorrnal B Lymphocytes

TIN HAN, MD, A N D BARBARA DADEY, AAS

ha vitro functional studies of mononuclear cells from 34 patients with B-cell type CLL were investigated and the results of these studies were as follows: I ) The T lymphocytes from patients with CLL were capable of responding normally to PHA or PWM, of inducing allogeneic normal B lymphocytes to respond to these mitogens and of stimulating normally to allogeneic lym- phocytes in “one-way” mixed lymphocyte reaction; 2) The monocytes from these patients were capable of enhancing the T lymphocyte response to mito- gens and of stimulating normally to allogeneic lymphocytes; and 3) The leu- kemic B lymphocytes were incapable of responding to mitogens even in the presence of normal T lymphocytes and their enhancer cell activity on T lym- phocyte response or their stimulating capacity on allogeneic lymphocytes was depressed. These observations suggest that the T lymphocytes and monocytes from patients with CLL are functionally normal while the leukemic B lym- phocytes from these patients are functionally abnormal.

Cancer 43:109-117, 1979.

HYTOMITOCENS SUCH AS phytohemag- P glutinin (PHA), concanavalin-A (Con A) and pokeweed mitogen (PWM) have been routinely used to stimulate human Iympho- cytes to divide. The proliferate response of lymphocytes is usually assayed by the uptake of 3H-thymidine incorporation into DNA. There is no disagreement in the literature that fractionated human T lymphocytes from vari- ous sources are stimulated by these mitogens; on the other hand, there are many conflicting reports in regard to mitogen-induced prolif- erative response of fractionated human B

servations suggest that the contradictory re- sults of B lymphocyte proliferative response to phytomitogens are most likely explained by differences in the techniques used for iso- lation of B lymphocytes and, in some cases, to differences in the purity of the B lympho-

~ymp~oCy~es~6-8 ,10 .11 ,14 ,18 ,24 ,25 ,30 ,31 ,37 These ob-

~

From the Department of Medicine B, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York.

Supported in part by UPHS Grant CA-12318. Address for Reprints: Tin Han, MD, Medicine B,

Roswell Park Memorial Institute, 666 Elm Street, Buf- falo, NY 14263.

T h e authors thank Mrs. Ellen Cray, Mrs. Chris Darlak, Miss Paula Diegelman and Mr. Andrew Blidy for techni- cal assistance.

Accepted for publication March 21, 1978.

cyte preparations. It has been reported that peripheral blood B lymphocytes, isolated by the E-rosetting technique, are not responsive LO PHA, Con A or PWM and that the B lym- phocytes cultured in the presence of irradi- ated autologous or allogeneic T lymphocytes, on the other hand, are stimulated by these

It is now generally agreed that chronic lym- phocytic leukemia (CLL) is a neoplastic dis- ease of B-cell origin in almost all instance^.'"^*^^ Earlier investigators have reported that the lymphocyte response to PHA is signifi- cantly depressed or absent when the unfrac- tionated lymphocytes are cultured for 3 days in patients with CLL. However, later inves- tigators including us, have demonstrated that lymphocytes from CLL patients are capable of transformation into blasts when cultured with PHA for a longer period of i n ~ u b a t i o n . ~ , ’ ~ ~ ~ ~ , ~ ~ It should be recognized that this obser- vation of delayed response to PHA in patients with CLL does not represent delayed initia- tion of DNA synthesis, but rather indicates the typical proliferation curve generated by a small number of normal T cells distributed in a large number of unresponsive leukemic B ~ e l l s . ~ ~ , ~ ~ Wybran et al. 40 reported that frac- tionated T lymphocytes from CLL patients respond very well to PHA or PWM, suggesting

~itogens.7.11.13~15.Z5,37

0008-543X/79/0100/0109 $0.95 0 American Cancer Society

109

110 CANCER Junuuqi 1979 VOl. 43

T A B 1 . t 1. DNA Synthesis of' Unstimulated T and B Lymphocytes from Healthy Subjects and Patients with CLL

3H-Thymidine incorporation (cpm) Mean t su

Lymphocyte donor Type of (no. of subjects) lymphocytes 3-4 Days 6-7 Days 9- 10 Days

Healthy subjects ( 1 5) T 379 2 429 370 ? 219 291 t 184 B 534 2 463* 679 ? 645 751 t 905

Patients with CLL ( 1 1) 1 452 t 279 468 t 557 449 t 676 B 199 t 41* 184 2 90 204 t 168

* p < 0.05.

the presence of a normally reactive T-cell pop- ulation, while fractionated B leukemic cells do not respond to either mitogen. These authors have utilized the E-rosetting technique for cell-purification in their study. However, the leukemic B cell response to those mitogens in the presence of T lymphocytes is not in- vestigated.

The present study was undertaken in an attempt to answer the following questions: 1) Are T lymphocytes from patients with B-cell type CLL functionally normal? 2) Can leu- kemic B lymphocytes respond to mitogens? 3) Are leukemic B lymphocytes and mono- cytes enhancer cells or suppressor cells for T-cell response to mitogens? and 4) Is stim- ulating capacity of T lymphocytes, leukemic B lymphocytes or monocytes from these pa- tients impaired?

MATERIALS A N D METHODS Thirty-four patients with CLL were stud-

ied. There were 15 males and 19 females.

Mean age of these patients was 63 years, with ranges from 44 to 86 years. Two patients were in partial remission and 32 patients had ac- tive disease. Of the 2 patients in partial re- mission, one was on maintenance chlorambu- cil therapy and the other patient was receiv- ing no therapy; of the 32 patients with active disease, 26 were receiving no therapy and 6 were receiving chemotherapy (one on cyclo- phosphamide and 5 on chlorambucil andfor prednisone) at the time of this study. Only 6 of 26 patients, who were receiving no ther- apy, had previously been treated. Twenty pa- tients in this study were untreated for CLL. Healthy subjects used for normal controls in this study were 25-55 years old.

Surface Marker Study for T and B Lymphocytes

Spontaneous rosette formation with sheep erythrocytes (E-rosettes) was used as a T-cell marker as previously des~r ibed '~ . '~ and direct

TABLE 2. Kinetics of Mitogen-Induced Human Peripheral Blood T and B Lymphocyte Blastogenesis (15 Experiments)

T Lymphocyte response? B Lymphocyte responset

3H-Thymidine Stimulation 3H-Thymidine Stimulation Day of Mito- incorporation (cpm) index incorporation (cpm) index culture gen* Mean ? SD Mean ? SD Mean ? SD Mean ? SD

3-4 None PHA PWM

6-7 None PHA PWM

9- 10 None PHA PWM

379 t 429 23,662 t 24,854 3,307 f 4,929

370 t 219 150,648 2 106,178

291 2 184 98,368 t 82,801 11,081 t 11,649

19,191 -C 23,866

- 98.3 -t 120.7 14.4 t 28.8

- 483.7 t 450.1

58.5 t 64.3

- 378.4 t 308.3 36.2 t 19.9

~~~

534 -C 463 564 2 452 496 ? 383

679 t 645 2,182 -t 5,352 1,175 k 1,722

751 2 905 1,766 t 3,665 1,785 T 3,786

~~

- 1.1 ? 0.5 1.0 t 0.3

- 2.1 2 2.5 1.6 t 1.6

- 1.5 ? 1.4 1.8 t 2.7

* PHA 5 pg and PWM 0.01 mliculture were used. t Each culture contained 2 ml of T or B lymphocyte

suspensions (1 X . 105/ml).

No. 1 MONONUCLEAR CELLS I N CLL * Han and Dadey 111

membrane immunofluorescence method, us- ing fluorescence-labeled anti-human immu- noglobulin chain antisera ( K , A, a , 6, y and /.L specific) was used as a B-cell marker as pre- viously described.37

Preparation of Purified T Lymphocytes, B Lymphocytes and Monocytes

Peripheral blood leukocytes were isolated from 30-35 ml of heparinized venous blood by centrifugation over a Ficoll-Hypaque gra- dient. One percent (v/v) sheep erythrocyte suspension was incubated with Vibrio chol- erae neuraminidase (25 unitdml) at 37 C for 30 minutes. Then, the cells were washed twice with RPMI 1640 culture medium containing antibiotics (penicillin and streptomycin). Sam- ples of 0.25 ml of leukocyte suspension (1 x 108/ml), 0.25 ml of fetal calf serum and 0.5 ml of neuraminidase-treated sheep erythro- cytes (1 x 109/ml) were added to plastic test tubes, which were centrifuged for 8 minutes at 200 x g and further incubated at room tem- perature for 60 minutes. The cell pellets of all tubes containing rosetted and nonrosetted cells were pooled, gently resuspended and centrifuged over a Ficoll-Hypaque gradient at 400 x g for 30 minutes. The T lymphocytes, rosetted with sheep erythrocytes formed the pellet and nonrosetted cells remained at the interface between the layers. The T cells were washed twice with culture medium. The inter- face cells were washed once and rerosetted in a similar fashion. The two T cell sus- pensions were then pooled and resuspended

in culture medium. Sheep erythrocytes roset- ting to the T lymphocytes and free sheep erythrocytes in the B-enriched fraction were lysed by treating with ammonium chloride (0.84%) at 4 C for 10 minutes. The cells were then washed twice and resuspended in culture medium.

Both T- and B-enriched preparations were incubated with iron particles (carbonyl iron, GAF Corp.), for 60 minutes at 37 C. Mono- cytes ingested the iron particles and these cells were separated from lymphocytes by centrif- ugation over a Ficoll-Hypaque gradient. Monocytes in the pellets were pooled and re- suspended in culture medium.

Lymphocyte Blastogenic Response to Mitogens

Both purified T and B lymphocytes were suspended in RPMI 1640 culture medium containing 10% pooled human plasma and antibiotics (100 units of penicillin and 50 pg of streptomycin/ml) at 1 x 105/ml concen- tration.

For regular mitogen studies, 2 ml of either T or B cell suspension were transferred to 16 x 125 mm Falcon disposable plastic tubes. Various doses of purified PHA and of recon- stituted PWM were added to each tube. The control culture received no mitogen. The ex- periments were carried out in duplicate.

For helper cell activity studies of T lympho- cytes on B lymphocyte response to PHA or PWM, one ml of B lymphocytes (1 X 105/ml) as responding cells and one ml of irradiated

TABLE 3. Kinetics of Mitogen-Induced Peripheral Blood T and B Lymphocyte Blastogenesis in Patients with CLL (5 Experiments)

T Lymphocyte responset B Lymphocyte responset

3H-Thymidine Stimulation 3H-Thymidine Stimulation Day of Mito- incorporation (cpm) index incorporation (cpm) index culture gen* Mean t SD Mean 2 SD Mean t SD Mean t SD

3-4 None PHA PWM

6-7 None PHA PWM

9- 10 None PHA PWM

574 ? 301 32,129 5 27,673 5,385 2 5,933

223 ? 89 163,162 ? 23,085 26,025 t 22,380

302 2 230 139,850 ? 120,648

14,117 ? 15,493

- 59.6 t 50.1 10.5 t 11.3

- 929.2 t 678.9 138.4k 111.2

- 567.8 k 383.7

59.7 t 78.5

232 t 46 275 t 91 167 t 66

256 rt 112 340 t 156 255 ? 62

302 t 218 263 ? 148 284 rt 217

- 1.2 t 0.5 0.7 t 0.2

- 1.5 t 0.8 1.1 t 0.2

- 1.2 t 0.2 1.3 ? 0.4

* PHA 5 pg and PWM 0.01 mVculture were used. t Each culture contained 2 ml of T or B lymphocyte

suspension (1 x 105/ml).

112 CANCER January 1979 Vol. 43

TABLE 4. T-Lymphocyte Dependence of B-Lymphocyte Blastogenic Response to Phytomitogens (6 Experiments)

Composition of culture*

3H-Thymidine Stimulation

Mean i SD

incorporation (cpm) index Mean ? SD

T lymphocytes T lymphocytes + PHA T lymphocytes + PWM

B lymphocytes B lymphocytes + PHA B lymphocytes + PWM

B lymphocytes + T lymphocytesf B lymphocytes + T lymphocytesf + PHA B lymphocytes + T lymphocytesf + PWM

314 ? 188 77,063 ? 51,215 26,110 r 14,377

792 2 490

1,866 ? 1,830

761 ? 569 19,753 ? 20,469 10,800 ? 8.958

2,426 ? 1,884

- 228.0 ? 163.0

93.3 ? 60.6

3.1 ? 2.11 1.9 ? 1.31

20.3 ? 7.5t 9.8 ? 3.81

-

-

* I : 1 of B:Tf was used. Cultures were incubated for

t P < 0.01. 7 days.

(6,000 rad) T lymphocytes at 1 x lo5, 2 x lo5 or 4 x 105/ml concentration as helper cells were transferred to culture tubes. The mito- gens were then added.

For enhancer cell activity studies of B lym- phocytes and monocytes on T lymphocyte re- sponse to mitogens, one ml of T lymphocytes (1 x 105/ml) as responding cells and one ml of irradiated B lymphocytes or monocytes at 2 x lo4 or 4 x 104/ml concentration as en- hancer cells were transferred to culture tubes. The mitogens were then added.

Culture tubes with loose-fitting caps were incubated at 37 C in a humidified atmosphere of 5% CO, in air for 3-10 days. One micro- curie of 3H-thymidine (specific activity, 2.0 Ci/ mmol) was added to each tube 24 hours prior to harvesting the cells. Incorporation of 3H- thymidine into DNA was measured according to the method previously described (29). The lymphocyte response was expressed as plain counts per minute (cpm) in stimulated culture or as stimulation index (SI), which is a ratio of cpm in stimulated culture and cpm in un- stimulated culture.

Stimulating Capacity of T Lymphocytes, B Lymphocytes and Monocytes in “One-way” Mixed Lymphocyte Reaction

Mixed lymphocyte cultures were set up us- ing a slightly modified whole-blood method.29 Heparinized peripheral blood from healthy subjects was mixed with culture medium at a ratio of 1:lO. Three ml aliquots of cell sus- pension, containing approximately 6 x lo5 lymphocytes were transferred to culture

f Helper cells (T lymphocytes) were irradiated with 6.000 rad.

tubes. Stimulating cells (T, B lymphocytes or monocytes), inactivated by irradiation (6,000 rad) in 0.5 ml aliquots of cell suspension (1.2 x lO6/ml) were added to each tube, making a 1:l ratio of responding lymphocytes and stimulating cells. Each “one-way” mixed lym- phocyte reaction experiment also included single cell cultures containing either respond- ing cells only or stimulating cells only. The experiments were carried out in duplicate. Culture tubes were incubated for 7 days and harvesting was done as already described. “One-way” mixed lymphocyte reaction was expressed as SI which is cpm in mixed cell culture minus cpm in stimulating cell culture divided by cpm in responding cell culture.

RESULTS

All of 34 patients with CLL studied for cell surface marker were classified as B-cell type. In 32 patients, many leukemic lymphocytes exhibited both heavy chain surface immuno- globulin IgM (mean value of 80 with ranges from 56 to 99%) and IgD (mean value of 59 with ranges from 21 to 96%) on their cell membranes. In the other two patients, 72 and 81% of leukemic cells were positive for IgM only, respectively. Of 17 of 34 patients stud- ied for light chain immunoglobulin surface marker, 9 had K type (mean value of 52 with ranges from 34 to 71% positive cells) and 8 had A type (mean value of 68 with ranges from 51 to 80% positive cells).

Total lymphocyte counts in 32 patients with active CLL ranged from 8,000 to 245,000, with a mean value of 49,300/mm3, while total

No. 1 MONONUCLEAR CELLS I N CLL * Hun and Dadey 113

lymphocyte counts in 2 patients in partial re- mission were 2,200 and 6,400/mm3. Total T lymphocytes were elevated in both groups; mean value was 2,650 with ranges from 1,900 to 3,400/mm3 for patients in remission, and was 6,000 with ranges from 1,100 to 22,8001 mm3 for patients with active disease. Total B lymphocyte counts in 2 patients in remission were 300 and 1,400/mm3. Total B lympho- cytes were markedly elevated in those with active disease; mean value was 39,100 with ranges from 5,600 to 2OO,OOO/mrn3.

At the completion of cell isolation proce- dures, each of these enriched fractions con- tained 90 to 95% purity of respective cells, with a yield of approximately 75% for T lym- phocytes, 80% for B lymphocytes and 60% for monocytes. Viability by Trypan blue dye exclusion test of each purified fraction of cells ranged from 95 to 99%.

In a study of the 3H-thymidine incorpora- tion of unstimulated T and B lymphocytes, it was observed that the 3H-thymidine uptake in the normal B lymphocytes was consistently higher than that of normal T lymphocytes in healthy subjects, while the uptake in the leukemic B lymphocytes was consistently lower than that of autologous T lymphocytes in patients with CLL. The 3H-thymidine up- take in T lymphocytes was quite similar in both groups (Table 1).

Table 2 shows the kinetics of mitogen-in-

duced T and B lymphocytes from healthy sub- jects. The T lymphocytes responded very well to PHA or PWM while B lymphocytes were not responsive to both of these mitogens. Maximal 3H-thymidine uptake of T lympho- cyte blastogenesis was seen in cultures incu- bated for 6-7 days in the presence of PHA or PWM. Table 3 shows the kinetics of mito- gen-induced T and B lymphocytes from 5 pa- tients with untreated CLL, who had lympho- cytosis over 50,000/mm3. The degree of re- sponse and peak response of T lymphocyte blastogenesis induced by either PHA or PWM was normal while the leukemic B lymphocyte response to these mitogens was absent in all experiments.

In 3 other experiments, the leukemic B lym- phocytes, with higher doses of PWM (0.1, 0.2 and 0.4 ml/culture) also failed to proliferate (not shown in the table).

When the B lymphocytes from healthy sub- jects were cultured with irradiated autologous T lymphocytes, both PHA and PWM became mitogenic to B lymphocytes (Table 4). How- ever, the leukemic B lymphocytes failed to respond to these mitogens, even in the pres- ence of autologous T lymphocytes from pa- tients with active CLL (Table 5 ) . Allogeneic T lymphocytes from healthy subjects also failed to induce the proliferative response to PHA or PWM of these leukemic B lympho- cytes, while allogeneic T lymphocytes from

T A B L E 5. Lack of B Lymphocyte Response to Mitogen even in the Presence of T Lymphocytes in Patients with CLL* (4 Experiments)

Composition of culture$

3H-Thymidine Stimulation

Mean f SD incorporation (cpm) index

Mean t SD

T lymphocytes (1 x 105/ml) T lymphocytes + PHA T lymphocytes + PWM

B lymphocytes (1 X 105/ml) B lymphocytes + PHA B lymphocytes + PWM

B lymphocytes + T lymphocytes? (1: 1) B lymphocytes + T lymphocytest + PHA B lymphocytes + T lymphocytest + PWM

B lymphocytes + T lymphocytes? (1:2) B lymphocytes + T lymphocytes? + PHA B lymphocytes + T lymphocytes? + PWM

409 ? 69 - 49,875 ? 53,224

6,246 t 3,142 122.0 -t 125.3

18.4 f 12.4

405 f 59 393 t 74 362 f 117

349 t 119 423 t 164 354 f 83

400 t 58 569 f 158 512 t 19

- 1.0 -t 0.1 0.9 ? 0.4

- 1.5 2 1.1 1.1 2 0.4

1.5 -t 0.6 1.3 -t 0.1

-

* Each of these four patients had lymphocytosis over

t T lymphocytes (autologous) were irradiated with

6,000 rad. $ Cultures were incubated for 7 days. 50,000/mm3.

114 CANCER January 1979 Vol. 43

TABLE 6. B Lymphocyte Response to PHA in the Presence of T Lymphocytes in 2 Patients with CLL in Remission

W T h ymidine incorporation (cpm) Stimulation index

Composition of culturet Pt. 1 Pt. 2 Pt. 1 Pt. 2

T lymphocytes T lymphocytes + PHA

B lymphocytes B lymphocytes + PHA

B lymphocytes + T lymphocytes* (1: 1) B lymphocytes + T lymphocytes* + PHA

B lymphocytes + T lymphocyte$* (1 :2) B lymphocytes + T lymphocytes* + PHA

300 62,029

207 530

275 18,469

434 38,533

272 46,757

587 344

345 4,978

218 9,015

-

206.6

- 2.6 -

60.9

- 84.7

- 171.6

- 0.6

- 13.2

- 39.4

* T lymphocytes (autologous) were irradiated with

t Cultures were incubated fur 7 days. 6,000 rad.

patients with CLL were able to induce the proliferative response of B lymphocytes from healthy subjects (not shown in the table). The B lymphocyte response to PHA in the pres- ence of autologous T lymphocytes was ob- served in 2 patients with CLL in partial re- mission (Table 6).

Although leukemic B lymphocytes from pa- tients with CLL were unable to respond to mitogens, their enhancer cell activity on ?’ lymphocyte response to PHA was seen when T and irradiated B cells were co-cultured at a ratio of 10:4. Enhancer cell activity of leu- kemic B lymphocytes was not seen when a ratio of 10:2 was used, while enhancer cell activity of normal B lymphocytes was clearly seen at this ratio. Enharicer cell activity of monocytes on T lymphocyte response to PHA, on the other hand, was higher than that of B lymphocytes. The degree of monocyte en-

hancer cell activity was similar in both healthy subjects and patients with CLL. However, the enhancer cell activity of monocyte in either case was statistically not significant (Table 7).

The results of stimulating capacity of T lym- phocytes, B lymphocytes and monocytes from healthy subject and patients with active CLL in “one-way” mixed lymphocyte reactions are shown in Table 8. The stimulating capacities of T lymphocytes or monocytes between healthy subjects and patients with CLL were quite similar; however, the stimulating capac- ity of leukemic B lymphocytes was signifi- cantly lower than that of counterpart normal B lymphocytes (p < 0.01) on allogeneic lym- phocytes.

DISCUSSION In the present study, leukemic lymphocytes

from 34 patients with CLL had surface im-

TABLE 7. Enhancer Cell Activity of B Lymphocytes arid Monocytes on T Lymphocyte Response to PHA in Healthy Subjects and in Patients ~ i t h CLI, (6 Experiments)

Responding cells

T lymphocytes (1 x 105/ml) T lymphocytes (1 x 105/ml) T lymphocytes (1 x 105/ml) T lymphocytes (1 x 105/ml) T lymphocytes (1 X 105/ml) T lymphocytes ( 1 x 105/ml) T lymphocytes (1 x 105/ml)

Enhancer cells* Ratio of

R: E

~~ ~

3H-Thymidine incorporation (cpm) Mean ? SD

Healthy subjects CLL patients

- T lymphocytes (2 x 104/ml) B lymphocytes (2 x 104/ml) Monocytes (2 x 104/ml) T lymphocytes (4 x IO4/ml) B lymphocytes (4 X 1O4/ml) Monocytes (4 x 104/ml)

- 1:0.2 1:0.2 l:0.2 l:O.4 1 :0.4 1:0.4

58,125 ? 25,432 56,010 ? 58,695 58,743 ? 26,661 57,213 ? 60,698 72,692 ? 18,444 56,286 ? 60,769

101,609 ? 43,875 116,251 ? 131,987 107,393 i_ 81,333 56,053 i_ 60,796 125,175 2 75,157 74,862 ? 68,237 152,429 ? 43,829 115,642 ? 70,810

* Enhancer cells (autologous) were irradiated with 3H-thyrnidine incorporation (100-295 cpm/culture), 6,000 rad. Cultures containing either enhancer cells alone or enhancer cells with PHA exhibiting a low

indicating no proliferative response.

No. 1 MONONUCLEAR CELLS IN CLL . Hun and Dadey 115

muiioglobulins (IgM with or without IgD), in- dicating the B-cell origin of neoplastic disease. These observations are in agreement with published Of interest is the fact that some patients with active CLL exhibit a marked elevation in total T lymphocyte count. A similar finding has previously been dem- o n ~ t r a t e d . ~ , ~ ~

Observations that the 3H-thymidine uptake of T lymphocytes from patients with CLL is slightly higher than that of T lymphocytes from healthy subjects while the 3H-thymidine uptake of leukemic B lymphocytes is signifi- cantly lower than that of normal B lympho- cytes, suggest that the DNA synthesis in the leukemic B lymphocyteg is depressed.

It should be pointed out that in the present study, the peak response of PHA-induced T lymphocyte blastogenesis in both healthy sub- jects and patients with CLL was seen on day 6-7 of culture instead of day 3-4 of culture. This observation simply reflects the fact that we have used a small initial inoculum ( 1 O5 cells/ ml) in cultures, which typically produce a late maximum response. Similar observations of late maximum PHA response have previously been r e p ~ r t e d . ~ ~ , ~ ~

The present study unequivocally demon- strated that the T lymphocyte response to PHA or PWM in patients with active CLL is normal, while the B lymphocytes from these patients fail to respond to these mitogens. Similar findings have been observed.40 In this study, the leukemic B lymphocytes are unable to proliferate when exposed to these mito- gens, even in the presence of autologous or allogeneic T lymphocytes. This observation suggests that the leukemic B lymphocytes may be functionally abnormal. However, the B lymphocyte response to PHA in the presence of autologous T lymphocytes is observed in 2 patients in partial remission, suggesting that there are two populations of B lymphocytes (one normal and the other leukemic) in pa- tients with CLL. It should be emphasized that the T lymphocytes from patients with CLL have a helper cell activity to induce the pro- liferative response of normal B lymphocytes from healthy subjects and that the T lympho- cytes from patients with CLL possess normal stimulating capacity in “one-way” mixed lym- phocyte reaction in this study. In a prelimi- nary study, we also observed that the T lym- phocytes from patients with CLL respond normally to recall antigens and to allogeneic

TABLE 8. Stimulating Capacity of T Lymphocytes, B Lymphocytes and Monocytes from Healthy

Subjects and Patients with CLL in “One-way” Mixed Lymphocyte

Reactions

“One-way” MLR, SI Mean k SD

No. of Stimulating experi- Healthy CLL

cell type ments subjects patients

T Lymphocytes 16 4.0 4 3.5 5.4 5 2.6

Monocytes 3 16.0 ? 9.3 18.3 2 6.1 B Lymphocytes 16 17.6 4 14.0* 7.9 ? 6.5*

* p < 0.01.

cells (unpublished observations). These obser- vations suggest that the T cells from patients with CLL are functionally normal.

The depressed stimulating capacity of leu- kemic B lymphocytes seen in this study sug- gests that the stimulating capacity may have been partially lost or masked during the pro- cess of leukemogenesis. We recently reported that cultured leukemic B lymphoid cell line (BALM-2) exerts only a weak to moderate stimulation, while cultured normal B lymph- oid cell lines exert a strong stimulation on allogeneic normal lymphocytes.1fi*20 It is now generally agreed that the T lymphocyte re- sponse to mitogen is enhanced when T cells and unresponsive non-T cells (B lymphocytes and monocytes treated with irradiation or mi- tomycin C) are cocultivated in feeder layer culture^.'^,^^,^^,^^ Enhancer cell activity of B lymphocytes or monocytes on T lymphocytes obtained from healthy subjects, seen in the present study is in agreement with previously reported observations. Of particular interest is the fact that leukemic B lymphocytes do not suppress the T lymphocyte response to PHA, but instead they enhance the T lympho- cyte response although the enhancer cell ac- tivity of leukemic B lymphocytes was some- what lower than that of counterpart normal B lymphocytes. Another interesting finding is that the monocytes from patients with CLL possess an enhancer cell activity on T lympho- cyte response to mitogens, which is compara- ble to that of monocytes from healthy sub- jects. It has recently been reported that the monocytes and B lymphocytes from patients with solid tumor or Hodgkin’s disease are capable of suppressing the T lymphocyte re- sponse to mitogens.2’21’z2

116 CANCER January 1979 VOl. 43

Fauci et ~ 1 . ~ recently reported that the leu- kemic B lymphocytes from patients with un- treated CLL fail to show antibody production assayed by plaque-forming cell (PFC) assay following exposure to PWM in the presence of helper T cells. These authors also found that leukemic B cells do not possess suppres- sor cell activity on normal B cells in coculture system of PFC assay and that the T lympho- cytes from these patients are capable of help- ing allogeneic normal B cells to produce an- tibody. These observations suggest that the

suppressed in vatro antibody response of leu- kemic B lymphocytes is due not to inadequate T cell help or the presence of suppressor cells, but to an intrinsic defect in the leukemic B lymphocytes. Maino et al. ,26 on the other hand, recently reported that the leukemic B lympho- cytes can, under certain circumstances, be stimulated to synthesize and secrete in- creased amount of immunoglobulin mainly in light chain molecules, suggesting a basic defect in the biosynthetic mechanism of these leukemic cells.

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20. Han, T., Sokal, J. E., and Moore, G. E.: “Antigenic” disparity between cultured lymphoid cells and autologous lymphocytes. Am. J . Med. 53:437-445, 1972.

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