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Blut 39, 17-25 (1979)

9 Springer-Verlag 1979

MLC-Activation by Acute Lymphatic Leukemia Blasts* W. Rella 1, H. Winterleitner 2, and W. Knapp 3 1 Krebsforschungsinstitut der Universit/it Wien, Borschkegasse 8-10, A-1090 Wien, Austria 2 St.-Anna-Kinderhospital des Roten Kreuzes, Kinderspitalgasse 6, A-1090 Wien, Austria 3 Immunologisches Institut der Universitfit, Borschkegasse 8-10, A-1090 Wien, Austria

Aktivierung der MLC durch Blasten akuter lymphatischer Leuk/imien Zusammenfassung. Blasten yon 25 akuten lymphatischen Leukfimien (16 ,,comm o n " ALL, 6 T-ALL, 3 nicht identifiziert) wurden auf ihre F/ihigkeit untersucht, allogene Lymphozyten zu aktivieren. Mitomycin-behandelte Blasten oder bestrahlte Lymphozyten wurden mit heparinisiertem Vollblut verschiedenet gesunder Spender gemischt und kultiviert. Die Aktivierung der MLC dutch die Blasten wurde im prozentuellen Verh/iltnis zur MLC-Aktivierung durch die bestrahlten Lymphozyten ausgedrfickt. Die untersuchten leuk/imischen Blasten zeigten eine unterschiedliche Ffihigkeit die MLC zu aktivieren: 11 der 25 F/ille yon ALL stimulierten in der MLC schwach (2-33 %), 12 F/ille von A L L stimulierten im Normbereich (50-120 %) und 2 F/ille stimulierten eine/ibernormale Reaktion (mehr als 200 %). Vier der 6 FNIe yon T-ALL stimulierten in der MLC ebensogut wie normale Lymphozyten, 2 stimulierten schwach. Die meisten schwach stimulierenden Lymphoblasten 16sten jedoch eine normale Reaktion in der MLC aus, sofern das reagierende Vollblut durch isolierte Lymphozyten ersetzt wurde. Die schwache MLC-aktivierende Eigenschaft mancher leukfimischer Blasten beruhte offenbar auf einer verminderten Freisetzung yon blastogenem Fa/~tor wfihrend der Kultur. Es land sich kein Hinweis ffir eine aktive Suppression. Die Bedeutung der MLC-aktivierenden Eigenschaften leuk/imischer Blasten in Hinblick auf die immunologische Klassifikation lymphatischer Leukfimien und deren Verwendung f/it die Immuntherapie wird diskutiert. Sehliisselwiirter: Akute lymphatische Leuk/imie - gemischte LymphozytenBlasten-Zellkulturen - Mitogen-Faktor

* Supported by Fonds zur F6rderung der wissenschaftlichen Forschung in 0sterreich Offprint requests to: Dr. Heide Winterleitner (address see above)

0006-5242/79/0039/0017/$1.80

18

W. Rella et al. Summary. The MLC-activating potential of 25 ALL blasts (16 "common" ALL, 6 T-ALL, 3 not identified) was investigated. Mitomycin-treated leukemic blasts or X-irradiated lymphocytes were cultured with heparinized whole blood from different healthy donors. MLC activation by blast cells was expressed as percentage of MLC activation by X-irradiated lymphocytes. Leukemic blasts showed a heterogeneous pattern of MLC activation, ranging from 2 % to 245 %. Eleven out of 25 cases of ALL poorly stimulated the MLC (2% to 33% response). Twelve ALL stimulated a normal response (50% to 120%); and 2/25 ALL stimulated a supranormal response (more than 200 %). Four of six cases of T-ALL stimulated the MLC as efficiently as irradiated lymphocytes, 2/6 were among the poor stimulators. Most poor stimulator blasts had, however, normal MLC-activating properties if, instead of whole blood, isolated lymphocytes were used as the responding cells. The poor activation of lymphocytes by some leukemic blasts in whole blood appeared to be associated with impaired release of blastogenic factor (s) during the MLBC. No evidence for active suppressor mechanisms was found. The significance of the MLC-activating properties of leukemic blasts for the classification and immunotherapeutic use of ALL is discussed. ' Mixed lymphocyte-blast cell culKey words: Acute lymphocytic leukemia U tures - Mitogenic factor

Abbreviations: MLC = Mixed lymphocyte culture; ALL = Acute lymphocytic leukemia;

MLBC = Mixed lymphocyte-blast cellculture; HTLA = Human Thymus Lymphocyte Antigen; DR-Ag = D-related Antigen The immunological characterization of leukemic cells has rapidly progressed in recent years and has indeed become an important tool for the diagnosis and treatment of malignant hematological diseases [15]. Leukemic cells have most commonly been characterized by different rosette techniques as well as by immunofluorescence using heteroantisera specific for surface determinants of lymphocytic or leukemic cells [5]. We have used these techniques, as well as mitogen stimulation, for the immunological diagnosis and follow-up of our cases of ALL in the Viennese Leukemia Study Group [17]. Little use has been made so far of the MLC to identify immunological properties of leukemic blasts [8]. MLC-activating determinants (LD antigens) are normally expressed on B cells, monocytes and some non-hemopoetic cells and appear to be mostly products of the major histocompatibility complex. To further characterize the leukemic cells of our children with ALL, we studied their MLC-activating potential in comparison to normal lymphocytes. Tests were carried out with both whole blood and isolated lymphocytes as responding cells. Materials and Methods Patients

Blast cells from 25 children with acute lymphocytic or undifferentiated leukemia were obtained prior to any treatment. Mononuclear cells were isolated from heparinized peripheral blood or bone marrow specimens by Fieoll-Hypaque density gradient centrifugation. Aliquots of washed

MLC-Activation by ALL Blasts

19

cells (107-108/ml) were suspended in medium 199 with 10 % DMSO and 15 % fetal bovine serum, distributed in 2 ml glass vials and frozen in liquid nitrogen using a biological freezer. The percentage of leukemic blasts was estimated on Giemsa-stained cytocentrifuge preparations of frozenthawed cells. Some leukemic blasts were particularly sensitive to the toxic action of DMSO. After thawing, dead cells (3-80 ~ ) were removed after aggregation in low ionic strength medium [16]. Following this treatment, dead cells never exceeded 20 ~.

Phenotyping of Leukemic Cells T Cell Markers: Rosette formation with sheep red blood cells and membrane immunoftuorescence with a T cell specific antiserum were performed as described previously [11 ].

B Cell Markers: S-Ig staining was done as described before [1]. For the demonstration of Ia type antigens a rabbit antiserum prepared essentially as described by Billing et al. [3] was used. This serum did not stain HTLA-positive normal or leukemic cells, and completely inhibited the MLC response when stimulating-unlike responding-cells were incubated with high dilutions of this serum (Knapp et al., in prep.). M L C and M L B C Our mixed leukocytes culture technique using whole blood has been recently described [13]. Briefly, heparinized blood from healthy volunteers was diluted 1 : 20 with culture medium RPMI 1640 and each 0.2 ml were distributed in the wells of round bottomed microtiter II plates (NUNC). DNA-synthesis of stimulating blasts was blocked by incubation with mitomycin C (Kiowa) for 30 min at 37 ~ C (50/~g/ml), followed by three washes in phosphate buffered saline. The cells were then resuspended in culture medium and counted in a hemocytometer using trypan blue as vital stain. Experimental cultures received 20/~1 of the stimulating cell suspension (2, 4 and 8 • 10~ cells/ml) while control cultures received 20 tfl medium. Total and differential white cell counts of donor blood were performed on Giemsa-stained smears. As a control, lymphocytes obtained from normal blood donors and irradiated with 6.000 r (courtesy of Dr. Binder, Dept. of Radiotherapy) were used in parallel as stimulator cells. The experimental set up included, with 4 four replicates in each combination, mixed cultures of responding and stimulating cells, cultures of responding cells alone and of stimulating cells alone. Plates were wrapped in kitchen film and incubated for 7 days at 37 ~ C in a moist atmosphere with 8 ~ C Q . 0.2/~Ci of 3H-thymidine (5 Ci/mmol, Amersham) was added for the last 16 h. Cultures were then lyzed with distilled water and particulate material was collected on glass fiber strips and processed for liquid scintillation counting. Modifications of this technique for detection of suppressive cells or soluble mediators are described in the results. In some experiments isolated lymphocytes or leukocytes were used as responding cells instead of whole blood. In that case, 5 • 104 lymphoid cells were cultured per well in medium RPMI 1640, supplemented with 10% fresh autologous plasma. Results are expressed, if not otherwise stated, as cpm per 5 • 104 responding lymphocytes and are corrected for the background. The counting efficiency was 67 ~.

Results

1. L ymphocytes Activating Properties of A L L Blasts T o d e m o n s t r a t e M L C s t i m u l a t o r y activity, m i t o m y c i n - t r e a t e d blasts w e r e c u l t u r e d w i t h w h o l e b l o o d f r o m at least 2 L D - d i f f e r e n t d o n o r s . I n this way, an a c c i d e n t a l L D - i d e n t i t y c a u s i n g false n e g a t i v e s t i m u l a t i o n c o u l d be detected. A t y p i c a l e x a m p l e o f o n e such test is s h o w n in T a b l e 1. T h e l y m p h o c y t e a c t i v a t i n g c a p a c i t y o f blast cells was c a l c u l a t e d as p e r c e n t a g e M L C a c t i v a t i o n b y X - i r r a d i a t e d l y m phocytes. T a b l e 2 lists all cases o f A L L studied. O u t o f 25 cases, 16 b e l o n g e d to the subg r o u p " c o m m o n " A L L , p o s i t i v e f o r D R - A g a n d n e g a t i v e f o r o t h e r surface m a r k e r s ; six h a d surface c h a r a c t e r i s t i c s o f T cells ( D R - A g - , H T L A +) ; a n d three w e r e

W. Rella et al.

20 Table 1. Demonstration of MLC-activating ability of leukemic blasts MLC No.

Stimulating cells

Responding cells

cpm/105 _+ SE lymphocytes

Stimulation Index

1 2 3 4 5 6 7 8 9 10

(I31 29)m (BI 29)m L- 2x L- 2x L- 3x L- 3x Am Bm L- 2x L- 3x

A B A B A B B A L-3 L-2

47369 20737 67770 39790 39360 11950 38870 40450 60690 17688

49.5 38 67.7 73 40 22 49 42 23 43

• 2770 • 2210 • 4930 • 5020 • 4780 ,+ 2000 • 2600 • 3570 ,+ 4460 • 1110

Percent MLC activation is calculated as follows: (pos 1/Pos 3) • 100 = 73 = Percent activation of "A". (Pos 2/Pos 4) • 100 = 52 = Percent activation of "B". (73 + 52) / 2 = 63; the standard deviation is 15. MLC 5-10 are for control Suffix "m" = mitomycin- treated Suffix "x" = irradiated (6000 r) A and B: Healthy donors L- 2 and L- 3 : Control lymphocytes B1 29: No. 13 in Table 2

n o t identified. A s can be seen f r o m the table, blastogenesis i n d u c e d b y leukemic blasts r a n g e d between 2~o a n d 245 ~ . T h e r e was no significant association o f M L C - s t i m u l a t o r y activity with the other p a r a m e t e r s measured. It is, however, interesting to note t h a t m o s t T cell leukemias were n o t a m o n g the p o o r stimulators.

2. Suppressor Activity of ALL Blasts Two possibilities were considered to account for the p o o r M L C - a c t i v a t i n g potential o f some blasts: 1) L a c k o f relevant surface structures ( L D antigens or others) a n d 2. active suppression. To detect suppressor activity, p o o r s t i m u l a t o r blasts were cocultured with h e a l t h y d o n o r whole b l o o d . The possible b l a s t - i n d u c e d a l t e r a t i o n o f r e s p o n d e r cell reactivity was assessed b y adding, as a second stimulus, X - i r r a d i a t e d l y m p h o cytes f r o m 2 days before until 2 days after the blasts to the m i x e d culture. I n two such experiments, using blasts No. 5 a n d 8, the M L C response was n o t influenced by the a d m i x t u r e o f these p o o r s t i m u l a t o r blasts a n d was no different f r o m the response o b t a i n e d with the use o f well s t i m u l a t o r blasts as a control. Thus, u n t e r these conditions, no suppressor activity o f p o o r s t i m u l a t o r blasts was evident.

3. Comparison of MLBC with Whole Blood and Isolated Leukocytes By chance, we f o u n d t h a t m o s t p o o r s t i m u l a t o r blasts expressed quite n o r m a l M L C - a c t i v a t i n g properties, if, instead o f whole b l o o d , l y m p h o c y t e s or leukocytes were used as the r e s p o n d i n g cells. T h e M L B C - r e s p o n s e of whole b l o o d versus leukocytes is c o m p a r e d in Table 3. N o t e t h a t g o o d s t i m u l a t o r blasts (Nos. 14, 16, 21) a n d X - i r r a d i a t e d l y m p h o c y t e s (L 7x, L 8x) effect a b o u t the same response for

MLC-Activation by ALL Blasts

21

Table 2. Surface markers and MLC-activating properties of leukemic blasts

No.

Code

Clinical Diagnosis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

MF HE a SK HU RA SB PU a B1 23 BR a GA B1 11 NG B129 B1 18 SA RM LU RH FG LC JI SL HJ KA PC

ALL ALL AUL ALL ALL ALL ALL Monoc. ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL AUL ALL ALL ALL LS ALL ALL ALL

ALL AUL LS n.d.

Surface markers ( ~ positive) ~ DR-Ag Surface HTLA Blasts a Ig 99 92 84 90 88 68

99 72 89 82 90 90 76 80 99 85 75 69 90 85 80 83 80 70 65

86 87 n.d. 95 25 70 85 78