Leukemia Research.Vol. 3. No. 3, pp. 163-169. ~,. Pergamon Press. Ltd. 1979. Printed in Great Britain.
0145-2126/79/0601--0163 $02.00~0
DELAYED MITOGENIC RESPONSES TO PHYTOHEMAGGLUTININ IN ACUTE LYMPHOBLASTIC LEUKEMIA SALLY S. FAIRCHILD an d J. JOHN COHEN University of Colorado Medical School, Denver, CO 80262, U.S.A.
(Received I0 October 1978. Revised 1 December 1978. Accepted 25 January 1979) Abstract--Peripheral blood mononuclear cells from seven children with acute lymphoblastic leukemia (ALL) had.delayed responses to the T cell mitogen phytohemagglutinin (PHA) in vitro. Furthermore, responses were usually lower than those of normal peripheral cells. Neither the magnitude nor the time of peak response to mitogen could be correlated with the peripheral white cell count or the absolute number of T lymphocytes in these patients. The cells which responded to PHA formed rosettes with sheep red blood cells, and could be removed by exploiting this property. Furthermore, they had a normal karyotype in patients whose leukemia cells were aneupioid. Therefore, a small number of residual normal T cells is responsible for the late response to PHA in ALL.
INTRODUCTION ACUTE lymphoblastic leukemia (ALL) of children is a heterogeneous disease. In 20-25 % of patients the leukemia cells form rosettes with sheep red blood cells (SRBC) and therefore appear to be of the T cell lineage. In the other 75-80 % of patients the leukemia cells resemble neither mature T nor mature B lymphocytes; they do not rosette with SRBC and lack surface immunoglobulin (sIg) [2, 4, 6]. Although these "null" ALL cells are lymphoid in appearance, their origin remains controversial. In most cases the cells express surface HLA-D [11, 14], the Ia-like antigen found on normal monocytes and B lymphocytes, and contain the enzyme terminal deoxynucleotidyl transferase which is normally restricted to immature T cells in thymus and bone marrow [16, 20]. These observations have led investigators to suggest that the null form of ALL may derive from a common B and T lymphoid progenitor cell. There have also been reports of cases which seem to involve precursors which have already committed themselves to differentiate along B or T cell pathways [1, 22]. Finally, since the cells from chronic myelocytic leukemia (CML) patients in blast crisis express an antigen found on ALL cells, it has been proposed that ALL and CML originate from a multipotential stem cell [18]. The functional capacities of ALL cells have not been nearly as well defined as surface and enzyme markers and an understanding of them could provide further insight into the origin of ALL cells. Several investigators have reported that peripheral blood cells from most ALL patients respond poorly to the T cell mitogens concanavalin A and phytohemagglutinin (PH A) and that the responses are delayed [10, 17]. However, the cells responding to mitogens have not been identified. The late mitogen response could be due to normal T cells either actively inhibited by the leukemia cells or merely diluted so that multiple rounds of division must occur before the response becomes measurable. Alternatively, the leukemia cells themselves could be responding like normal T cells after mitogen-induced maturation. We felt Correspondence to: Sally S. Fairchild, 224 L. B. Wallace Tumor Institute, University of Alabama Medical School, University Station, Birmingham, AL 35294, U.S.A. Abbreviations: ALL, Acute lymphoblastic leukemia; SRBC, Sheep red blood cells; slg, Cell surface immunoglobulin; CML, Chronic myelocytic leukemia; CLL, Chronic lymphocytic leukemia; PHA, Phytohemagglutinin; ER, Sheep red blood cell (erythrocyte) rosette; FCS, Fetal calf serum. 163
164
SALLYS. FAIRCHILDand J. JOHN COHEN
it was w o r t h distinguishing between these possibilities; the d e m o n s t r a t i o n o f i n d u c e d m a t u r a t i o n to P H A - r e s p o n s i v e cells w o u l d s u p p o r t the hypothesis t h a t c o m m o n A L L is a l e u k e m i a o f p r e - T cells. MATERIALS AND METHODS
Peripheral blood cellpreparations Heparinized blood from untreated children with ALL was kindly provided at the time of diagnosis by Dr. L. Odom of Denver Children's Hospital and Dr. A. Mangalik of Colorado General Hospital. Normal blood was obtained from volunteers in our laboratory. Mononu¢leax cells were isolated by discontinuous density gradient centrifugation on Ficoll-sodium metrizoate (Lymphoprep, Nyegaard & Co., Oslo, Norway) according to standard procedures [5]. To obtain a population free of T lymphoeytes, sheep red blood cell rosettes (ER) were removed from peripheral blood. Two to 2.5x l0 s mononu¢lear cells were suspended in a total volume of 10 ml of RPMI-1640 (Grand Island Biological Co., Grand Island, NY) containing 0.5 ~ SRBC (Colorado Serum, Denver, CO) and 20% heat-inactivated fetal calf serum (FCS, Grand Island, N¥, lot No. 856614) that had previously been absorbed with SRBC. The mixture was incubated for 30 min at 370C, centrifuged at 200 g for 8 rain, and incubated for an additional 2 h at 4°C. After gentle resuspension the cells were layered over Lymphoprep and centrifuged at 400 g for 35 rain. ER sedimented to the bottom of the gradient; nonresetting cells (null and B lymphocytes and monocytes) were isolated from the interface and washed thoroughly. T and B lymphocyte enumeration B lymphocytes were detected by direct immunofluorescence using fluoresceinated goat antiserum to pooled human irnmunoglobulin (Hyland Laboratories, Costa Mesa, CA). T lymphocytes were identified by resetting with SRBC. Briefly, l0 s mononuclear cells were incubated at 37°C for 20 rain in RPMI-1640 containing 2~0 washed SRBC and 20% SRBC-absorbed FCS, centrifuged at 200 g for 5 rain, and incubated overnight at 4°C. After dilution and gentle resuspension, the fraction of cells forming rosettes (ER + cells) was enumerated on a hemacytometer. Mitogen stimulation Mononu¢lear cells were diluted to 8 x l0 s per ml in RPMI-1640 containing 5 ~ FCS and antibiotics (Grand Island). Cultures of 0.5 ml were set up in 12 x 75 mm glass tubes fitted with Morton closures. Purified phytohemagglutinin (PHA HA-16, Burroughs Welleome Co., Research Triangle Park, NC) was added to a final concentration of 0.5/~g/ml, and cultures were incubated at 37°C in humidified 5 % CO2 in air. One to ten days after the initiation of culture tubes were pulsed with 0.5 t~Ci of tritiated thymidine (6.7 Ci/mmol, New England Nuclear, Boston, MA), and 6 h later DNA was precipitated with trichloroacetic acid and counted by scintillation spectroscopy. Data are expressed as At.p.m. (counts per minute in PHA stimulated cultures minus counts per minute in medium controls). Karyotyping Metaphase spreads were prepared by standard procedures from unstimulated blood and bone marrow at the time of diagnosis, and from PHA-stimulated peripheral blood cultures after 4, 5 and 6 days of stimulation. Karyotyping was done by Dr. Helvise Morse. RESULTS
Mitogen response kinetics N o r m a l i m m u n o l o g i c a l p a r a m e t e r s were established using p e r i p h e r a l b l o o d cells f r o m 16 l a b o r a t o r y v o l u n t e e r s with a m e a n age o f 27 years (Table 1). W e used y o u n g a d u l t s as c o n t r o l s because we were u n a b l e to o b t a i n e n o u g h b l o o d f r o m n o r m a l children to c a r r y o u t o u r extensive kinetic studies; M e l i e f et aL [17] a n d Ben-Zwi et aL [3] have r e p o r t e d that mitogen responses o f c h i l d r e n a n d y o u n g a d u l t s are similar. U n d e r o u r e x p e r i m e n t a l conditions, cells f r o m n o r m a l i n d i v i d u a l s gave m a x i m u m p r o l i f e r a t i o n to P H A on d a y 2 (or o c c a s i o n a l l y d a y 3) with an average A response o f 58,600 c.p.m. (range 28,200-104,800); all samples gave very low o r negative ,4 responses o n d a y 5. T h e P H A response p a t t e r n s o f 7 children with u n t r e a t e d A L L were studied in detail. D i a g n o s i s o f A L L was b a s e d on m o r p h o l o g i c a l identification o f l y m p h o b l a s t s in b o n e m a r r o w . I n all patients, p e r i p h e r a l white cell counts were significantly elevated (Table 1)
165
Delayed PHA response in ALL
and at least 70 ~o of the white cells were leukemic blasts. In six of seven patients, the leukemia cells lacked B or T cell surface markers; in most of these patients, especially those with white cell counts of less than 50,000 (patients B.O. and K.S.), there were a few small, presumably normal T and B lymphocytes. The leukemia cells of one patient (D.S.) formed rosettes with SRBC; this patient had a mediastinal mass detected by X-ray. TABLE I. SURFACE MARKERS AND PHA gF.SPONSES OF MONONUCLEAR CELLS FROM NORMAL
INDIVIDUALSAND
PATIENTSWITH ALL
Mononuclear cells
Response to PHA~"
Patient
Age
WBC/mm a
sIg +
ER +
Day 2
Late~:
Normal* B.O. C.M. V.C. D.C. K.S. D.S. E.D.
27 3 6 10 3 3 6 6
7500 47,000 500,000 19,000 341,000 40,000 500,000 60,000
11 1 1 1 1 l 1 1
57 1 1 4 I 7 75 1
Ac.p.m. (S.I.) 58,600 (91.0) --760 (0.9) --4640 (0.6) 1870 (4.9) 620 (1.2) 3580 (2.4) 2870 (2.4) 5100 (3.4)
Ac.p.m. (S.I.) d5: 7700 (1.2) d5:31,200 (2.4) d7:31,400 (33..0) d8:44,100 (32.0) d4: 6260 (3.1) d6:30,700 (5.2) d5:15,200 (10.9) d5:47,400 (6.3)
(%)
(%)
* Average results from 16 volunteers. "~Ae.p.m. = c.p.m, in PHA stimulated cultures minus c.p.m, in medium controls; S.I. = stimulation index = c.p.m, in PHA stimulated cultures divided by c.p.m, in medium controls. :~Time of maximum response (day 5 response of normals given for comparison).
7O
E.D.
5O 30 10 ~x 30 I ~ 0 . ~
~
K.S.
C.M.
10
lO 2
4
6
8
lO
Days of
"~/2 4 6 8 10 Culture
FIG. 1. PHA response profiles. Mononuclear cells from seven ALL patients and one normal individual were cultured with PHA for 1-10 days and mitogenesis measured by incorporation of tritiated thymidine into DNA. Background incorporation in the absence of mitogen has been subtracted.
166
SALLY S. FAIRCHILD a n d J. JOHN COHEN
TABLE2. SURFACE MARKERS Patient
K.S. D.C.
BEFORE AND AFTER DEPLETION OF ER. + CELLS
Cells
Unseparated ER-depleted Unseparated ER-depleted
slg+ (%)
ER+ (%)
Null (%~
1 I 1 1
7 0.5 1 0.5
93 99 98 99
In all seven patients the response to PHA was delayed when compared to that of normal controls (Fig. 1). Response peaks were broad and occurred anywhere from day 4 to day 8. The magnitude of response varied greatly. However, neither the time nor the magnitude of peak response showed any relation to the number of residual T cells or the total white cell count. Patient D.S. is of particular interest because although his leukemia cells rosetted like normal T cells, his PHA response was low and delayed. The spontaneous thymidine incorporation of cells from ALL patients was similar to or lower than that of normal samples at all times tested. DeVaan et aL [10] also observed low turnover of peripheral blood cells from ALL patients. Therefore, an early, normal PHA response was not being masked by a high background response in patient samples.
Identification of PHA°responsive cells in ALL Several studies were carried out to determine which cells in ALL blood respond to PHA; do the residual normal cells respond late and weakly because there are so few of them or do the leukemia cells themselves respond ? Several reports in the literature have shown that the time of peak response to mitogen is related to the concentration of cells cultured [19, 23]. Thus, as normal T cells are diluted, the peak response occurs later and later and eventually becomes undetectable. The minimum number of cells giving a measurable but late response can be as few as ten per culture if appropriate "filler" or "feeder" cells are added [23]. Not all cells can subserve this filler effect. However, if ALL cells could do so, the delayed PHA response might be due to a very small number of residual normal T cells diluted in the ALL mononuelear cell preparations. Three different approaches indicated that this is indeed the case. First, we looked at the surface markers of PHA-induced lymphoblasts at the time of peak response to mitogen. The majority of such blasts from the blood of normal donors form rosettes with SRBC (data not shown) [15]. In four different patients, all with ER-negative leukemia, we found that 75-85 ~ of the PHA-induced blasts formed rosettes with SRBC and 10-15 ~o expressed slg. The most reasonable interpretation of this is that it is the few normal T cells, and possibly a few normal B cells as well [7], that actually respond to PHA. However, since mitogens will induce at least partial maturation of normal T cell precursors [9], it was still possible that PHA was first inducing maturation and then proliferation of the ALL cells themselves. If PHA induces proliferation of normal but not leukemia cells, then it should be possible to decrease the response by removing cells which rosette with SRBC before the initiation of culture. We therefore compared the PHA response of peripheral blood cells from two patients with ER-negative leukemia (K.S. and D.C.) before and after removing ER ÷ cells on Lymphoprep gradients. The surface marker characteristics of the unseparated and ERdepleted populations are shown in Table 2. Rosette-forming cells were not detectable after ER depletion, and proportions of B cells and null cells were not significantly altered. Figure 2 shows the PHA response of unfractionated and ER-depleted cells from patient
Delayed PHA response in ALL
167
K.S. It can be seen that removal of ER + T cells greatly decreased the PHA response. In the second patient, D.C., the P H A response was completely eliminated by removing ER ÷ ]ymphocytes (not shown). We did not obtain enough ER + cells in either experiment to examine directly their response to PHA.
1
30
E 2O e~
0145-2126/79/0601--0163 $02.00~0
DELAYED MITOGENIC RESPONSES TO PHYTOHEMAGGLUTININ IN ACUTE LYMPHOBLASTIC LEUKEMIA SALLY S. FAIRCHILD an d J. JOHN COHEN University of Colorado Medical School, Denver, CO 80262, U.S.A.
(Received I0 October 1978. Revised 1 December 1978. Accepted 25 January 1979) Abstract--Peripheral blood mononuclear cells from seven children with acute lymphoblastic leukemia (ALL) had.delayed responses to the T cell mitogen phytohemagglutinin (PHA) in vitro. Furthermore, responses were usually lower than those of normal peripheral cells. Neither the magnitude nor the time of peak response to mitogen could be correlated with the peripheral white cell count or the absolute number of T lymphocytes in these patients. The cells which responded to PHA formed rosettes with sheep red blood cells, and could be removed by exploiting this property. Furthermore, they had a normal karyotype in patients whose leukemia cells were aneupioid. Therefore, a small number of residual normal T cells is responsible for the late response to PHA in ALL.
INTRODUCTION ACUTE lymphoblastic leukemia (ALL) of children is a heterogeneous disease. In 20-25 % of patients the leukemia cells form rosettes with sheep red blood cells (SRBC) and therefore appear to be of the T cell lineage. In the other 75-80 % of patients the leukemia cells resemble neither mature T nor mature B lymphocytes; they do not rosette with SRBC and lack surface immunoglobulin (sIg) [2, 4, 6]. Although these "null" ALL cells are lymphoid in appearance, their origin remains controversial. In most cases the cells express surface HLA-D [11, 14], the Ia-like antigen found on normal monocytes and B lymphocytes, and contain the enzyme terminal deoxynucleotidyl transferase which is normally restricted to immature T cells in thymus and bone marrow [16, 20]. These observations have led investigators to suggest that the null form of ALL may derive from a common B and T lymphoid progenitor cell. There have also been reports of cases which seem to involve precursors which have already committed themselves to differentiate along B or T cell pathways [1, 22]. Finally, since the cells from chronic myelocytic leukemia (CML) patients in blast crisis express an antigen found on ALL cells, it has been proposed that ALL and CML originate from a multipotential stem cell [18]. The functional capacities of ALL cells have not been nearly as well defined as surface and enzyme markers and an understanding of them could provide further insight into the origin of ALL cells. Several investigators have reported that peripheral blood cells from most ALL patients respond poorly to the T cell mitogens concanavalin A and phytohemagglutinin (PH A) and that the responses are delayed [10, 17]. However, the cells responding to mitogens have not been identified. The late mitogen response could be due to normal T cells either actively inhibited by the leukemia cells or merely diluted so that multiple rounds of division must occur before the response becomes measurable. Alternatively, the leukemia cells themselves could be responding like normal T cells after mitogen-induced maturation. We felt Correspondence to: Sally S. Fairchild, 224 L. B. Wallace Tumor Institute, University of Alabama Medical School, University Station, Birmingham, AL 35294, U.S.A. Abbreviations: ALL, Acute lymphoblastic leukemia; SRBC, Sheep red blood cells; slg, Cell surface immunoglobulin; CML, Chronic myelocytic leukemia; CLL, Chronic lymphocytic leukemia; PHA, Phytohemagglutinin; ER, Sheep red blood cell (erythrocyte) rosette; FCS, Fetal calf serum. 163
164
SALLYS. FAIRCHILDand J. JOHN COHEN
it was w o r t h distinguishing between these possibilities; the d e m o n s t r a t i o n o f i n d u c e d m a t u r a t i o n to P H A - r e s p o n s i v e cells w o u l d s u p p o r t the hypothesis t h a t c o m m o n A L L is a l e u k e m i a o f p r e - T cells. MATERIALS AND METHODS
Peripheral blood cellpreparations Heparinized blood from untreated children with ALL was kindly provided at the time of diagnosis by Dr. L. Odom of Denver Children's Hospital and Dr. A. Mangalik of Colorado General Hospital. Normal blood was obtained from volunteers in our laboratory. Mononu¢leax cells were isolated by discontinuous density gradient centrifugation on Ficoll-sodium metrizoate (Lymphoprep, Nyegaard & Co., Oslo, Norway) according to standard procedures [5]. To obtain a population free of T lymphoeytes, sheep red blood cell rosettes (ER) were removed from peripheral blood. Two to 2.5x l0 s mononu¢lear cells were suspended in a total volume of 10 ml of RPMI-1640 (Grand Island Biological Co., Grand Island, NY) containing 0.5 ~ SRBC (Colorado Serum, Denver, CO) and 20% heat-inactivated fetal calf serum (FCS, Grand Island, N¥, lot No. 856614) that had previously been absorbed with SRBC. The mixture was incubated for 30 min at 370C, centrifuged at 200 g for 8 rain, and incubated for an additional 2 h at 4°C. After gentle resuspension the cells were layered over Lymphoprep and centrifuged at 400 g for 35 rain. ER sedimented to the bottom of the gradient; nonresetting cells (null and B lymphocytes and monocytes) were isolated from the interface and washed thoroughly. T and B lymphocyte enumeration B lymphocytes were detected by direct immunofluorescence using fluoresceinated goat antiserum to pooled human irnmunoglobulin (Hyland Laboratories, Costa Mesa, CA). T lymphocytes were identified by resetting with SRBC. Briefly, l0 s mononuclear cells were incubated at 37°C for 20 rain in RPMI-1640 containing 2~0 washed SRBC and 20% SRBC-absorbed FCS, centrifuged at 200 g for 5 rain, and incubated overnight at 4°C. After dilution and gentle resuspension, the fraction of cells forming rosettes (ER + cells) was enumerated on a hemacytometer. Mitogen stimulation Mononu¢lear cells were diluted to 8 x l0 s per ml in RPMI-1640 containing 5 ~ FCS and antibiotics (Grand Island). Cultures of 0.5 ml were set up in 12 x 75 mm glass tubes fitted with Morton closures. Purified phytohemagglutinin (PHA HA-16, Burroughs Welleome Co., Research Triangle Park, NC) was added to a final concentration of 0.5/~g/ml, and cultures were incubated at 37°C in humidified 5 % CO2 in air. One to ten days after the initiation of culture tubes were pulsed with 0.5 t~Ci of tritiated thymidine (6.7 Ci/mmol, New England Nuclear, Boston, MA), and 6 h later DNA was precipitated with trichloroacetic acid and counted by scintillation spectroscopy. Data are expressed as At.p.m. (counts per minute in PHA stimulated cultures minus counts per minute in medium controls). Karyotyping Metaphase spreads were prepared by standard procedures from unstimulated blood and bone marrow at the time of diagnosis, and from PHA-stimulated peripheral blood cultures after 4, 5 and 6 days of stimulation. Karyotyping was done by Dr. Helvise Morse. RESULTS
Mitogen response kinetics N o r m a l i m m u n o l o g i c a l p a r a m e t e r s were established using p e r i p h e r a l b l o o d cells f r o m 16 l a b o r a t o r y v o l u n t e e r s with a m e a n age o f 27 years (Table 1). W e used y o u n g a d u l t s as c o n t r o l s because we were u n a b l e to o b t a i n e n o u g h b l o o d f r o m n o r m a l children to c a r r y o u t o u r extensive kinetic studies; M e l i e f et aL [17] a n d Ben-Zwi et aL [3] have r e p o r t e d that mitogen responses o f c h i l d r e n a n d y o u n g a d u l t s are similar. U n d e r o u r e x p e r i m e n t a l conditions, cells f r o m n o r m a l i n d i v i d u a l s gave m a x i m u m p r o l i f e r a t i o n to P H A on d a y 2 (or o c c a s i o n a l l y d a y 3) with an average A response o f 58,600 c.p.m. (range 28,200-104,800); all samples gave very low o r negative ,4 responses o n d a y 5. T h e P H A response p a t t e r n s o f 7 children with u n t r e a t e d A L L were studied in detail. D i a g n o s i s o f A L L was b a s e d on m o r p h o l o g i c a l identification o f l y m p h o b l a s t s in b o n e m a r r o w . I n all patients, p e r i p h e r a l white cell counts were significantly elevated (Table 1)
165
Delayed PHA response in ALL
and at least 70 ~o of the white cells were leukemic blasts. In six of seven patients, the leukemia cells lacked B or T cell surface markers; in most of these patients, especially those with white cell counts of less than 50,000 (patients B.O. and K.S.), there were a few small, presumably normal T and B lymphocytes. The leukemia cells of one patient (D.S.) formed rosettes with SRBC; this patient had a mediastinal mass detected by X-ray. TABLE I. SURFACE MARKERS AND PHA gF.SPONSES OF MONONUCLEAR CELLS FROM NORMAL
INDIVIDUALSAND
PATIENTSWITH ALL
Mononuclear cells
Response to PHA~"
Patient
Age
WBC/mm a
sIg +
ER +
Day 2
Late~:
Normal* B.O. C.M. V.C. D.C. K.S. D.S. E.D.
27 3 6 10 3 3 6 6
7500 47,000 500,000 19,000 341,000 40,000 500,000 60,000
11 1 1 1 1 l 1 1
57 1 1 4 I 7 75 1
Ac.p.m. (S.I.) 58,600 (91.0) --760 (0.9) --4640 (0.6) 1870 (4.9) 620 (1.2) 3580 (2.4) 2870 (2.4) 5100 (3.4)
Ac.p.m. (S.I.) d5: 7700 (1.2) d5:31,200 (2.4) d7:31,400 (33..0) d8:44,100 (32.0) d4: 6260 (3.1) d6:30,700 (5.2) d5:15,200 (10.9) d5:47,400 (6.3)
(%)
(%)
* Average results from 16 volunteers. "~Ae.p.m. = c.p.m, in PHA stimulated cultures minus c.p.m, in medium controls; S.I. = stimulation index = c.p.m, in PHA stimulated cultures divided by c.p.m, in medium controls. :~Time of maximum response (day 5 response of normals given for comparison).
7O
E.D.
5O 30 10 ~x 30 I ~ 0 . ~
~
K.S.
C.M.
10
lO 2
4
6
8
lO
Days of
"~/2 4 6 8 10 Culture
FIG. 1. PHA response profiles. Mononuclear cells from seven ALL patients and one normal individual were cultured with PHA for 1-10 days and mitogenesis measured by incorporation of tritiated thymidine into DNA. Background incorporation in the absence of mitogen has been subtracted.
166
SALLY S. FAIRCHILD a n d J. JOHN COHEN
TABLE2. SURFACE MARKERS Patient
K.S. D.C.
BEFORE AND AFTER DEPLETION OF ER. + CELLS
Cells
Unseparated ER-depleted Unseparated ER-depleted
slg+ (%)
ER+ (%)
Null (%~
1 I 1 1
7 0.5 1 0.5
93 99 98 99
In all seven patients the response to PHA was delayed when compared to that of normal controls (Fig. 1). Response peaks were broad and occurred anywhere from day 4 to day 8. The magnitude of response varied greatly. However, neither the time nor the magnitude of peak response showed any relation to the number of residual T cells or the total white cell count. Patient D.S. is of particular interest because although his leukemia cells rosetted like normal T cells, his PHA response was low and delayed. The spontaneous thymidine incorporation of cells from ALL patients was similar to or lower than that of normal samples at all times tested. DeVaan et aL [10] also observed low turnover of peripheral blood cells from ALL patients. Therefore, an early, normal PHA response was not being masked by a high background response in patient samples.
Identification of PHA°responsive cells in ALL Several studies were carried out to determine which cells in ALL blood respond to PHA; do the residual normal cells respond late and weakly because there are so few of them or do the leukemia cells themselves respond ? Several reports in the literature have shown that the time of peak response to mitogen is related to the concentration of cells cultured [19, 23]. Thus, as normal T cells are diluted, the peak response occurs later and later and eventually becomes undetectable. The minimum number of cells giving a measurable but late response can be as few as ten per culture if appropriate "filler" or "feeder" cells are added [23]. Not all cells can subserve this filler effect. However, if ALL cells could do so, the delayed PHA response might be due to a very small number of residual normal T cells diluted in the ALL mononuelear cell preparations. Three different approaches indicated that this is indeed the case. First, we looked at the surface markers of PHA-induced lymphoblasts at the time of peak response to mitogen. The majority of such blasts from the blood of normal donors form rosettes with SRBC (data not shown) [15]. In four different patients, all with ER-negative leukemia, we found that 75-85 ~ of the PHA-induced blasts formed rosettes with SRBC and 10-15 ~o expressed slg. The most reasonable interpretation of this is that it is the few normal T cells, and possibly a few normal B cells as well [7], that actually respond to PHA. However, since mitogens will induce at least partial maturation of normal T cell precursors [9], it was still possible that PHA was first inducing maturation and then proliferation of the ALL cells themselves. If PHA induces proliferation of normal but not leukemia cells, then it should be possible to decrease the response by removing cells which rosette with SRBC before the initiation of culture. We therefore compared the PHA response of peripheral blood cells from two patients with ER-negative leukemia (K.S. and D.C.) before and after removing ER ÷ cells on Lymphoprep gradients. The surface marker characteristics of the unseparated and ERdepleted populations are shown in Table 2. Rosette-forming cells were not detectable after ER depletion, and proportions of B cells and null cells were not significantly altered. Figure 2 shows the PHA response of unfractionated and ER-depleted cells from patient
Delayed PHA response in ALL
167
K.S. It can be seen that removal of ER + T cells greatly decreased the PHA response. In the second patient, D.C., the P H A response was completely eliminated by removing ER ÷ ]ymphocytes (not shown). We did not obtain enough ER + cells in either experiment to examine directly their response to PHA.
1
30
E 2O e~
