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Reduced T-colony forming capacity in B-chronic lymphocytic leukaemia—II. Correlation with clinical stage and findings in B-prolymphocytic leukaemia
Leukemia Research Vol. 6, No. 3, pp. 329-333, 1982. Primed in Greal Brilain.
0145-2126/82/030329-05503.00/0 © 1982 Pergamon Press Ltd.
REDUCED T-COLONY FORMING CAPACITY IN B-CHRONIC LYMPHOCYTIC LEUKAEMIA--II. CORRELATION WITH CLINICAL STAGE AND FINDINGS IN B-PROLYMPHOCYTIC LEUKAEMIA ROBER'I FOA,* FRANCESCO LAURIA,'I" DANIEL CATOVSKY+ and DAVID A. G. GALTON + *lstituto di Medicina lnterna, I Cattedra di Patologia Speciale Medica, University of Torino, Italy: tlstituto di Ematologia 'Lorenzo e Ariosto Ser:~gnoli', University of Bologna, Italy: ~MRC keukaemia Unit, Royal Postgraduate Medical School and Hammersmith Hospital, London. U.K. (Received 29 September 1981. Revised 21 December 1981. Accepted 5 January 1982)
Ahstrael--The T-colony forming capacity of T-lymphocytes from 33 cases of B-chronic lymphocytic leukaemia (B-CLL) and five of B-prolymphocytic leukaemia (B-PLL) was studied. An absent or reduced (less than 50) colony growth was observed in 21 of the 33 B-CLL and in four of the five B-PLL studied. Seven of the nine stage 0 patients (77.7?.) (according to Rai's clinical staging) gave rise to more than 50 colonies, compared with five out of 24 (20.9°~i,) in stages I-IV patients. Furthermore, the mean number of colonies was significantly (p < 0.01) higher in stage 0 patients (57 + 33.6), compared with more advanced stages (22 + 29.9). Since in normal peripheral blood. T-colony formation appears to be a property of Tp lymphocytes, and T7 cells are significantly increased in B-PLL and B-CLL, mainly in advanced disease, the T-colony growth was correlated with the percentage of T7 cells. Despite a negative trend, a statistical correlation was not observed. Our findings are suggestive of a functional defect of the T-cell population in the majority of cases of B-CLL. with a partial sparing in stage 0 patients. This abnormality, apparently unrelated to the Tp:T7 ratio, is probably due to an intrinsic defect of the Tp cell population. Key words: Chronic lymphocytic leukaemia, chronic prolymphocytic leukaemia, T-lymphocyte colonies, T-lymphocyte subsets.
INTRODUCTION THE IMBALANCEDdistribution of T-lymphocyte subsets recently reported in B-cell chronic lymphocytic leukaemia (B-CLL) has focused the attention on the role played by T-lymphocytes in this disease [8, 9-1. A reduced response to phytohaemagglutinin (PHA) by purified T-lymphocytes has also been reported in many cases of B-CLL ['13, 7], suggesting a possible functional impairment of the T-cell population. Using a double layer technique which enables the in vitro formation of T-lymphocyte colonies, we have recently described in a preliminary study that purified T-cells from B-CLL often show a reduced T-colony forming capacity [5-1. We now report our findings in a larger series of patients, in which the colony growth has been correlated with Rai's clinical staging [12] and with the proportion of circulating T7 cells. Furthermore, the T-colony formation of T-lymphocytes from B-cell prolymphocytic leukaemia (B-PLL) is described. Ahh~'el'iatiolT.~: B-CLL. B-cell chronic lymphocytic leukaemia: B-PLL. B-cell chronic prolymphocytic leukaemia: FCS. fetal calf serum: PHA. phytohaemagglutinin: Tp cells. T-lymphocytes with helper phenotype: T~ ~el[.~, T-lymphocytes with suppressor phenotype. Corre,spomh'Jwc and reprinl rcql~e.st.s to: Dr. R. Foa. lstituto di Medicina lnterna. I Cattedra di Patologiza Speciale Medica. Corso Polonitl 14. 10126 Torino. Italy.
329
ROBERT FOA et al.
330
MATERIALS AND METHODS Samples. Heparinised peripheral blood samples were collected from 33 patients with B-CLL and five with B-PLL. Controls from normal individuals were always collected at the same time. The B-cell nature of the leukaemic cells was documented by techniques reported elsewhere [1]. All patients were untreated or had been off therapy for at least five months. According to Rai's clinical staging [12] the B-CLL patients were: nine in stage 0, eight in stage I, 14 in stage II and two in stages Ill-IV. Cell separation. The mononuclear cells were obtained by "'Lymphoprep" (Nyegaard) separation and washed twice with TC199 (Wellcome). The percentage of T-lymphocytes was assessed by rosette formation with sheep RBC (E-rosettes). T-lymphocytes were purified by E-rosette formation followed by further Lymphoprep separation. This procedure was always repeated at least twice. After the last separation, the sheep RBC were lysed with distilled water and the lymphocytes washed twice with TCl99. The degree of T-lymphocyte purification was checked by E-rosette formation. T-colony assay. The double layer technique has been described in detail elsewhere [4]. Briefly, it consists of an underlayer containing 1 ml modified McCoy's 5A (Gibco) supplemented with 10°,o fetal calf serum (FCSt and 10~ horse serum (HS), agar at a final concentration of 0.50/0 and 2 × 106 unseparated peripheral blood leucocytes irradiated with 2000 rad. The overlayer contains 0.4 ml modified McCoy's 5A (supplemented with 10~o FCS and 10% HS), 0.01 ml PHA (WellcomeJ/disb and 1 x 105 test cells. Cultures are placed at 37 C in an incubator with 5Vo CO2 in humidified air. Colonies of more than 50 cells are scored in duplicate or triplicate after 4-6 days' incubation. The T-colony forming capacity of T-cell enriched fractions from B-CLL, B-PLL and from normal controls was tested. T-lymphocyte subsets. The percentage of T~ and T': cells of the purified T-cell fractions was assessed in the majority of the cases studied, by rosette formation with ox RBC coated with rabbit IgM or IgG. as described elsewhere [9] according to the technique of Moretta et al. [11]. The number of colonies was correlated with the percentage of T7 cells.
RESULTS On the unseparated lymphocytes the mean percentage of E-rosettes was 17~o + 9.6 S.D. in B-CLL and 6Vo + 2.8 in B-PLU compared with 70% + 7.9 in normal controls. After purification, the T-cell fractions contained 85% + 10.9 E-rosettes in B-CLL, 78~o + 13.4 in B-PLL and 94~o + 3.3 in the controls (25 samples). The overall proportion of T7 cells was significantly (p < 0.01, Student's t-test) increased in B-CLL and B-PLL compared with normal controls (Table 1). Conversely, the proportion of T/~ cells was significantly (p < 0.01) reduced in both diseases (Table 1). Both in B-CLL and B-PLL the absolute number of T/~ cells was either normal or increased, while that of T;' cells was always increased, up to 30-fold the normal values. TABLE 1. PERCENTAGEOF T/.4 AND T)' LYMPHOCYTESUBSETS IN B-CLL AND B-PLL Samples B-CLL B.PLL Normal controls
YoT~ + S.D.
~ T',, + S.D.
28 + 12.4 22 + 10.9 49 + 4.2
39 + 11.7 43 _+ 10.8 20 + 6.5
In B-CLL the mean number ofT-colonies was 31 + 32.5, significantly lower (p < 0.01) than with normal T-lymphocytes (mean 121 +_ 36.1). Figure 1 shows that T-colony forming capacity was related to the clinical stage of the disease. Overall, 12 of the 33 cases studied gave rise to more than 50 colonies. Of the nine stage 0 cases, seven originated more than 50 colonies (77.7~o), while only five of the 24 stages I-IV patients (20.90:/0) fell within this group. Furthermore, the mean number of colonies in stage 0 patients was 57 + 33.6 compared with 22 + 29.9 in stages I-IV patients. This difference was statistically significant (p < 0.01). In four of the five B-PLL tested, the T-colony growth of the purified T-lymphocytes was markedly reduced (Fig. 1).
T-colonies in B-CLL and B-PLL
331
200
1SO i "E
V
o ,
Ioo
..D £
$0
8
0
Irlorma controls
uaiaAI I--IV
0
B'PLL
(cl. stage) I
IB- CF.L
FIG. l. T-colony growth in 33 patients with B-CLL, divided according to Rai's clinical staging, and five with B-PLL.
100
..e.
80
_o
D
o 6o ~, 40 ~0
o
= 20-
0
o~
0
10
20
30
,
40
t.
50
60
FIG. 2. Correlation between T-colony numbers and percentage of T i' cells in 26 cases of B-CLL and B-PLL.
The T-colony numbers and the percentage of T7 cells were correlated in 26 cases of B-CLL and B-PLL. Although a negative trend could be observed (Fig. 2), this failed to reach significance ( r = 0.147). DISCUSSION We have shown that in B-CLL and B-PLL there is a significant decrease in the T-colony forming capacity of purified T-lymphocytes. It appears, however, that in B - C L L this decrease is not random. The T-colony forming capacity seems correlated to disease progression (Fig. i). Of the 12 cases with the highest T-colony formation, seven were in stage 0 and the overall mean number of colonies was significantly higher in stage 0 than in stages l-IV patients. Furthermore. almost 80°,0 of stage 0 cases were capable of giving rise to more than 50 colonies, while this occurred only in about 20°0 of the patients in more advanced stages. These data indicate that the T-lymphocyte function in B-CLL, although generally defective, is partly spared in the initial phases of the disease. This abnormality tends to worsen following the natural course of the disease. Our findings confirm and extend earlier observations of Chandra et al. [3]. who suggested a better i~7 t'itro transformation with PHA in stage 0 patients.
332
ROBERT FOA era/.
We have recently demonstrated that in normal peripheral blood the capacity to produce T-colonies is a property mainly of T-lymphocytes bearing Fc receptors for IgM (T~L) [6]. The overall reduced growth observed in B-CLL and in B-PLL may thus reflect the imbalanced distribution of T-lymphocyte subsets and mainly the significant increase in T7 cells recently reported [8, 9]. This hypothesis appeared sustained by the observation in B-CLL of an increase in T7 cells correlated with disease progression, while the overall decreased proportion of T# cells seems unrelated to the clinical state of the disease [2]. However, although a negative trend could be observed, we were unable to demonstrate a statistically significant correlation between proportion of T7 cells and T-colony numbers (Fig. 2). This suggests that the reduced T-colony forming capacity observed in the majority of patients with B-CLL may not be due only to the increase in T? cells and reduction in T# cells, but possibly to an intrinsic defect of the T~ cell population. This is further confirmed by findings in cases of hairy-cell leukaemia with a T#:T7 imbalance similar to that observed in B-CLL and a normal T-colony growth [10]. Results from our laboratories, showing that, at least in some cases of B-CLL, purified T# cells are still incapable of giving rise to T-colonies and of promoting the differentiation of normal B-lymphocytes to plasma cells in a pokeweed mitogen stimulated assay, seem to be in accordance with this suggestion (in preparation). Data from two patients (one B-CLL, one B-PLL) indicate that the T-colony formation may be improved following continuous treatment with chlorambucil (6-8 mg daily) (Table 2). It is possible that the changes in growth may be due to a selective effect on a lymphocyte subset(s) incapable of giving rise to T-colonies or to an improvement of the T#:T7 ratio. The latter has already been suggested in B-CLL with splenic irradiation which was capable of reversing to normal the T~:T7 ratio and of improving serum levels of immunoglobulins [2]. Whether the same can occur with chemotherapy is still uncertain. It is of interest, however, that in both our cases the proportion of T;, cells decreased following treatment (Table 2). TABLE 2. T-COLONY GROWTH OF T-LYMPHOCYTES AND PERCENTAGE OF T7 CELLS IN TWO PATIENTS (ONE B-CLL. ONE B-PLL) BEFORE AND AFTER TREATMENT WITH CHLORAMBUCIL Before treatment T-colony ~,~ T 7 Patients numbers (Abs. No.) B-CLL
52
B-PLL
20
35 (1260) 32 (1760)
After treatment T-colony ~, T7 n u m b e r s (Abs. No.) 103 80
9 (211) 30 (315)
In conclusion, our data confirm, on a large series, that in the majority of cases of B-CLL and B-PLL, the T-lymphocyte population shows a defective T-colony forming capacity. This impairment, partly spared in stage 0 patients, increases with disease progression. The lack of correlation between percentage of T7 cells and T-colony numbers is suggestive of a functional defect of the T# cells in many cases of B-CLL. Finally, treatment may improve the functional state of T-lymphocytes in B-CLL. Repeated evaluations, before and after therapy, of the functional behaviour of T-lymphocytes in B-CLL and B-PLL, together with the distribution of T-lymphocyte subsets, may play a part in the management of this disease, and in the pathogenesis of one of the most frequent complications of B-CLL, hypogammaglobulinaemia, which is known to depend on the functional behaviour of T-lymphocytes. Acknowledctemen!'s--This work was partly supported by C N R P F C C N , Rome.
T-colonies in B-CLL and B-PLL
333
REFERENCES I. CATOVSKYD.. CHERCHI M., GALTON D. A. G , HOFFBRANDA. V. & GANESHAGURU K. (1978l Cell differentiation in B- and T-lymphoproliferative disorders. In Differentiation Of Normal and Neoplastic hematopoietic Cells (MARKS P A.. TILL J. E. & CLARKSOY B.. Eds.). p. 811. Cold Spring Harbor Laboratory. 2. CATOVSKYD., LAURIA F, MATUTESE., FOA R.. MANTOVANIV., TURA S. & GALTON D. A. G. (19813 Increase in T;' lymphocytes in B-cell chronic lymphocytic leukaemia. 11. Correlation with clinical stage and findings in B-prolymphocytic teukaemia. Br. J. Haemat. 47, 537. 3. CHANDRA P., CHANANA A. D., CHIKAPPA G. & CRONKITE E. P. (19773 Chronic lymphocytic leukemia: concepts and observations. Blood Cells 3, 637. 4. FOA R. & CATOVSKYD. (19793 T-lymphocyte colonies in normal blood, bone marrow and lymphoproliferative disorders. Clin. exp. hnmun. 36, 488. 5. FOA R.. CATOVSKY D., LAURIA F. & GALTON D. A. G. (1980) Reduced T-colony forming capacity of T-lymphocytes from B-chronic lymphocytic leukaemia. Br. J. Haemat. 46, 623. 6. FOA R., LAURJAF. & CATOVSKY D. (19803 Evidence that T-colony formation is a property of TI~ (helper) lymphocytes. Clin. exp. Immun. 42, 152. 7. FOA R,. CATOVSKYD.. LAURIA F., ZAEAR M. N. & GALTO~ D. A. G. (19813 T-lymphocytes in B-cell chronic lymphocytic leukaemia. Haematoloqica 66, 105. 8. KAY N. E., JOHNSON J. D., STANEK R. & DOUGLAS S. D. (19793 T-cell subsets in chronic lymphocytic leukemia: abnormalities in distribution and in vitro receptor maturation. Blood 54, 540. 9. LAURIA F.. FOA R. & CATOVSKY D. (19803 Increase in T 7 lymphocytes in B-cell chronic lymphocytic leukaemia. Scand. J. Haemat. 24, 187. 10. LAURIAF., FOA R., MANTOVANIV., GOBBI M., MATUTES E., CATOVSKYD. & TURA S. (19813 T-lymphocyte studies in hairy-cell leukaemia (HCL). In Hemolymphopoiesis: Normal and Pathological Cell Differentiation (GAVOSTO F., BAGNARAG. P., BRUNELLI M. A & CASTALDINI C., Eds.), p. 107. Esculapio, Bologna. 11. MORETTAL., FERRARINI M., DURANTE M. L. & MINGARI M. C. (19751 Expression of a receptor for IgM by human T-cells in vitro. Eur. J. Immun. 5, 565. 12. RA! K. R.. SAWITSKYA., CRONKITE E. P, CHANANAA. D., LEVY R. N. & PASTERNAK B. S. (1975) Clinical staging of chronic lymphocytic leukemia. Blood 46, 219. 13. SCHULTZ E. F., DAVIS S. 8g RUSIN A. D. (1976) Further characterization of the circulating cell in chronic lymphocytic leukemia. Blood 48, 223.
0145-2126/82/030329-05503.00/0 © 1982 Pergamon Press Ltd.
REDUCED T-COLONY FORMING CAPACITY IN B-CHRONIC LYMPHOCYTIC LEUKAEMIA--II. CORRELATION WITH CLINICAL STAGE AND FINDINGS IN B-PROLYMPHOCYTIC LEUKAEMIA ROBER'I FOA,* FRANCESCO LAURIA,'I" DANIEL CATOVSKY+ and DAVID A. G. GALTON + *lstituto di Medicina lnterna, I Cattedra di Patologia Speciale Medica, University of Torino, Italy: tlstituto di Ematologia 'Lorenzo e Ariosto Ser:~gnoli', University of Bologna, Italy: ~MRC keukaemia Unit, Royal Postgraduate Medical School and Hammersmith Hospital, London. U.K. (Received 29 September 1981. Revised 21 December 1981. Accepted 5 January 1982)
Ahstrael--The T-colony forming capacity of T-lymphocytes from 33 cases of B-chronic lymphocytic leukaemia (B-CLL) and five of B-prolymphocytic leukaemia (B-PLL) was studied. An absent or reduced (less than 50) colony growth was observed in 21 of the 33 B-CLL and in four of the five B-PLL studied. Seven of the nine stage 0 patients (77.7?.) (according to Rai's clinical staging) gave rise to more than 50 colonies, compared with five out of 24 (20.9°~i,) in stages I-IV patients. Furthermore, the mean number of colonies was significantly (p < 0.01) higher in stage 0 patients (57 + 33.6), compared with more advanced stages (22 + 29.9). Since in normal peripheral blood. T-colony formation appears to be a property of Tp lymphocytes, and T7 cells are significantly increased in B-PLL and B-CLL, mainly in advanced disease, the T-colony growth was correlated with the percentage of T7 cells. Despite a negative trend, a statistical correlation was not observed. Our findings are suggestive of a functional defect of the T-cell population in the majority of cases of B-CLL. with a partial sparing in stage 0 patients. This abnormality, apparently unrelated to the Tp:T7 ratio, is probably due to an intrinsic defect of the Tp cell population. Key words: Chronic lymphocytic leukaemia, chronic prolymphocytic leukaemia, T-lymphocyte colonies, T-lymphocyte subsets.
INTRODUCTION THE IMBALANCEDdistribution of T-lymphocyte subsets recently reported in B-cell chronic lymphocytic leukaemia (B-CLL) has focused the attention on the role played by T-lymphocytes in this disease [8, 9-1. A reduced response to phytohaemagglutinin (PHA) by purified T-lymphocytes has also been reported in many cases of B-CLL ['13, 7], suggesting a possible functional impairment of the T-cell population. Using a double layer technique which enables the in vitro formation of T-lymphocyte colonies, we have recently described in a preliminary study that purified T-cells from B-CLL often show a reduced T-colony forming capacity [5-1. We now report our findings in a larger series of patients, in which the colony growth has been correlated with Rai's clinical staging [12] and with the proportion of circulating T7 cells. Furthermore, the T-colony formation of T-lymphocytes from B-cell prolymphocytic leukaemia (B-PLL) is described. Ahh~'el'iatiolT.~: B-CLL. B-cell chronic lymphocytic leukaemia: B-PLL. B-cell chronic prolymphocytic leukaemia: FCS. fetal calf serum: PHA. phytohaemagglutinin: Tp cells. T-lymphocytes with helper phenotype: T~ ~el[.~, T-lymphocytes with suppressor phenotype. Corre,spomh'Jwc and reprinl rcql~e.st.s to: Dr. R. Foa. lstituto di Medicina lnterna. I Cattedra di Patologiza Speciale Medica. Corso Polonitl 14. 10126 Torino. Italy.
329
ROBERT FOA et al.
330
MATERIALS AND METHODS Samples. Heparinised peripheral blood samples were collected from 33 patients with B-CLL and five with B-PLL. Controls from normal individuals were always collected at the same time. The B-cell nature of the leukaemic cells was documented by techniques reported elsewhere [1]. All patients were untreated or had been off therapy for at least five months. According to Rai's clinical staging [12] the B-CLL patients were: nine in stage 0, eight in stage I, 14 in stage II and two in stages Ill-IV. Cell separation. The mononuclear cells were obtained by "'Lymphoprep" (Nyegaard) separation and washed twice with TC199 (Wellcome). The percentage of T-lymphocytes was assessed by rosette formation with sheep RBC (E-rosettes). T-lymphocytes were purified by E-rosette formation followed by further Lymphoprep separation. This procedure was always repeated at least twice. After the last separation, the sheep RBC were lysed with distilled water and the lymphocytes washed twice with TCl99. The degree of T-lymphocyte purification was checked by E-rosette formation. T-colony assay. The double layer technique has been described in detail elsewhere [4]. Briefly, it consists of an underlayer containing 1 ml modified McCoy's 5A (Gibco) supplemented with 10°,o fetal calf serum (FCSt and 10~ horse serum (HS), agar at a final concentration of 0.50/0 and 2 × 106 unseparated peripheral blood leucocytes irradiated with 2000 rad. The overlayer contains 0.4 ml modified McCoy's 5A (supplemented with 10~o FCS and 10% HS), 0.01 ml PHA (WellcomeJ/disb and 1 x 105 test cells. Cultures are placed at 37 C in an incubator with 5Vo CO2 in humidified air. Colonies of more than 50 cells are scored in duplicate or triplicate after 4-6 days' incubation. The T-colony forming capacity of T-cell enriched fractions from B-CLL, B-PLL and from normal controls was tested. T-lymphocyte subsets. The percentage of T~ and T': cells of the purified T-cell fractions was assessed in the majority of the cases studied, by rosette formation with ox RBC coated with rabbit IgM or IgG. as described elsewhere [9] according to the technique of Moretta et al. [11]. The number of colonies was correlated with the percentage of T7 cells.
RESULTS On the unseparated lymphocytes the mean percentage of E-rosettes was 17~o + 9.6 S.D. in B-CLL and 6Vo + 2.8 in B-PLU compared with 70% + 7.9 in normal controls. After purification, the T-cell fractions contained 85% + 10.9 E-rosettes in B-CLL, 78~o + 13.4 in B-PLL and 94~o + 3.3 in the controls (25 samples). The overall proportion of T7 cells was significantly (p < 0.01, Student's t-test) increased in B-CLL and B-PLL compared with normal controls (Table 1). Conversely, the proportion of T/~ cells was significantly (p < 0.01) reduced in both diseases (Table 1). Both in B-CLL and B-PLL the absolute number of T/~ cells was either normal or increased, while that of T;' cells was always increased, up to 30-fold the normal values. TABLE 1. PERCENTAGEOF T/.4 AND T)' LYMPHOCYTESUBSETS IN B-CLL AND B-PLL Samples B-CLL B.PLL Normal controls
YoT~ + S.D.
~ T',, + S.D.
28 + 12.4 22 + 10.9 49 + 4.2
39 + 11.7 43 _+ 10.8 20 + 6.5
In B-CLL the mean number ofT-colonies was 31 + 32.5, significantly lower (p < 0.01) than with normal T-lymphocytes (mean 121 +_ 36.1). Figure 1 shows that T-colony forming capacity was related to the clinical stage of the disease. Overall, 12 of the 33 cases studied gave rise to more than 50 colonies. Of the nine stage 0 cases, seven originated more than 50 colonies (77.7~o), while only five of the 24 stages I-IV patients (20.90:/0) fell within this group. Furthermore, the mean number of colonies in stage 0 patients was 57 + 33.6 compared with 22 + 29.9 in stages I-IV patients. This difference was statistically significant (p < 0.01). In four of the five B-PLL tested, the T-colony growth of the purified T-lymphocytes was markedly reduced (Fig. 1).
T-colonies in B-CLL and B-PLL
331
200
1SO i "E
V
o ,
Ioo
..D £
$0
8
0
Irlorma controls
uaiaAI I--IV
0
B'PLL
(cl. stage) I
IB- CF.L
FIG. l. T-colony growth in 33 patients with B-CLL, divided according to Rai's clinical staging, and five with B-PLL.
100
..e.
80
_o
D
o 6o ~, 40 ~0
o
= 20-
0
o~
0
10
20
30
,
40
t.
50
60
FIG. 2. Correlation between T-colony numbers and percentage of T i' cells in 26 cases of B-CLL and B-PLL.
The T-colony numbers and the percentage of T7 cells were correlated in 26 cases of B-CLL and B-PLL. Although a negative trend could be observed (Fig. 2), this failed to reach significance ( r = 0.147). DISCUSSION We have shown that in B-CLL and B-PLL there is a significant decrease in the T-colony forming capacity of purified T-lymphocytes. It appears, however, that in B - C L L this decrease is not random. The T-colony forming capacity seems correlated to disease progression (Fig. i). Of the 12 cases with the highest T-colony formation, seven were in stage 0 and the overall mean number of colonies was significantly higher in stage 0 than in stages l-IV patients. Furthermore. almost 80°,0 of stage 0 cases were capable of giving rise to more than 50 colonies, while this occurred only in about 20°0 of the patients in more advanced stages. These data indicate that the T-lymphocyte function in B-CLL, although generally defective, is partly spared in the initial phases of the disease. This abnormality tends to worsen following the natural course of the disease. Our findings confirm and extend earlier observations of Chandra et al. [3]. who suggested a better i~7 t'itro transformation with PHA in stage 0 patients.
332
ROBERT FOA era/.
We have recently demonstrated that in normal peripheral blood the capacity to produce T-colonies is a property mainly of T-lymphocytes bearing Fc receptors for IgM (T~L) [6]. The overall reduced growth observed in B-CLL and in B-PLL may thus reflect the imbalanced distribution of T-lymphocyte subsets and mainly the significant increase in T7 cells recently reported [8, 9]. This hypothesis appeared sustained by the observation in B-CLL of an increase in T7 cells correlated with disease progression, while the overall decreased proportion of T# cells seems unrelated to the clinical state of the disease [2]. However, although a negative trend could be observed, we were unable to demonstrate a statistically significant correlation between proportion of T7 cells and T-colony numbers (Fig. 2). This suggests that the reduced T-colony forming capacity observed in the majority of patients with B-CLL may not be due only to the increase in T? cells and reduction in T# cells, but possibly to an intrinsic defect of the T~ cell population. This is further confirmed by findings in cases of hairy-cell leukaemia with a T#:T7 imbalance similar to that observed in B-CLL and a normal T-colony growth [10]. Results from our laboratories, showing that, at least in some cases of B-CLL, purified T# cells are still incapable of giving rise to T-colonies and of promoting the differentiation of normal B-lymphocytes to plasma cells in a pokeweed mitogen stimulated assay, seem to be in accordance with this suggestion (in preparation). Data from two patients (one B-CLL, one B-PLL) indicate that the T-colony formation may be improved following continuous treatment with chlorambucil (6-8 mg daily) (Table 2). It is possible that the changes in growth may be due to a selective effect on a lymphocyte subset(s) incapable of giving rise to T-colonies or to an improvement of the T#:T7 ratio. The latter has already been suggested in B-CLL with splenic irradiation which was capable of reversing to normal the T~:T7 ratio and of improving serum levels of immunoglobulins [2]. Whether the same can occur with chemotherapy is still uncertain. It is of interest, however, that in both our cases the proportion of T;, cells decreased following treatment (Table 2). TABLE 2. T-COLONY GROWTH OF T-LYMPHOCYTES AND PERCENTAGE OF T7 CELLS IN TWO PATIENTS (ONE B-CLL. ONE B-PLL) BEFORE AND AFTER TREATMENT WITH CHLORAMBUCIL Before treatment T-colony ~,~ T 7 Patients numbers (Abs. No.) B-CLL
52
B-PLL
20
35 (1260) 32 (1760)
After treatment T-colony ~, T7 n u m b e r s (Abs. No.) 103 80
9 (211) 30 (315)
In conclusion, our data confirm, on a large series, that in the majority of cases of B-CLL and B-PLL, the T-lymphocyte population shows a defective T-colony forming capacity. This impairment, partly spared in stage 0 patients, increases with disease progression. The lack of correlation between percentage of T7 cells and T-colony numbers is suggestive of a functional defect of the T# cells in many cases of B-CLL. Finally, treatment may improve the functional state of T-lymphocytes in B-CLL. Repeated evaluations, before and after therapy, of the functional behaviour of T-lymphocytes in B-CLL and B-PLL, together with the distribution of T-lymphocyte subsets, may play a part in the management of this disease, and in the pathogenesis of one of the most frequent complications of B-CLL, hypogammaglobulinaemia, which is known to depend on the functional behaviour of T-lymphocytes. Acknowledctemen!'s--This work was partly supported by C N R P F C C N , Rome.
T-colonies in B-CLL and B-PLL
333
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