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Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiouronium bromide
CLINICAL
IMMUNOLOGY
AND
IMMUNOI’ATHOLOGY
3,
324-333
(1975)
Enhancement of Sheep Red Blood Cell Human lymphocyte Rosette Formation by the Sulfhydryl Compound 2-Amino Ethylisothiouronium Bromide’ M. A. PELLEGRINO, S. FERRONE,~ M. P. DIERICH,:' AND R. A. REISFELD Department
oj’Experimenta1 Pathology, ScrilqJs Clinic and Research 476 Prospect Street, La Jolla, California 92037 Received
February
Found&ion.
25, 1971
In oitro treatment of sheep red blood cells with the srdthydryl compour~d 2aminoethylisothiouronium bromide (AET) under suitable experimental conditions greatly enhances their ability to form rosettes with human lymphoid cells. The effect of AET depends on the concentration, pH of the solution and time of incnbation. AET treated sheep red blood cells lose their ability to form rosettes after varying time periods depending on the temperature of storage and on the concentration of the AET solution. Treatment of human peripheral lymphocytes with AET abolishes their capability to form rosettes with both normal and AET treated sheep red blood cells. Lymphocytes from some patients with chronic lymphocytic lenkemia react differently with AET-treated sheep red blood cells than with normal sheep red blood cells.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by an intrinsic abnormality of red cell membrane which makes it extremely sensitive to the lytic action of complement. This disease, although rare, has attracted considerable interest because the unique lesion of the membrane confers to red cells a number of peculiar features both in viva and in u&o. The sulfhydryl compound 2-amino ethylisothiouronium bromide (AET) has been extensively utilized to produce the PNH lesion on normal human red cells (for review see Sirchia and Ferrone (1)). That is, under suitable experimental conditions normal human red blood cells treated in vitro with AET resemble PNH erythrocytes in their abnormal susceptibility to the lytic action of complement (2), in their decreased acetylcholinesterase activity (3) and in their reduced survival in oivo (4). The mechanism of action of AET as well as the nature of the lesion in’ This is publication No. SO6 from the Department of Experimental Pathology, Scripps (:iirn(~ and Research Foundation, La Jolla, CA. This work was supported by United States Public Health Service Grant AI 10180 and Grants AI 07007 and 70-615 from the American Heart Association, Inc. and a California Division of the American Cancer Society Senior Fellowship No. D-221. ” S.F. is a Visiting Scientist from the University of Milan, Italy :i M.D. is a Visiting Scientist supported by the Deutsche Forschungsgemeirrsclraft (I’rriyersitk of Mainz), Germany.
Copyright All rights
@> 1975 by Academic Press, Inc of reproduction in any form reserved
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
325
duced in red cell membranes are unknown and are presently under study in our laboratory. In the course of investigations on the effect of AET treatment on red cells from different animal species, it was observed that in U&O incubation of sheep red blood cells with AET solutions markedly enhanced their binding to human peripheral lymphocytes. This phenomenon is described here in view of the numerous reports that sheep red blood cell rosette formation is a specific marker for human T cells (5-8) and that such a test has wide clinical application (8-10). MATERIALS
AND
METHODS
Treatment of cells with AET solutions. AET (Sigma, St. Louis, MO) was dissolved in distilled water and the solution adjusted to pH 8 by addition of 5 N NaOH, unless otherwise stated. Red cells and human peripheral lymphocytes were treated with various concentrations of AET for different times, as previously described (2,ll). Briefly, 4 vol of AET solution were added to 1 vol of packed washed cells and incubated with intermittent mixing at 37°C for the times indicated in Results. After incubation, cells were washed three times with isotonic saline, recounted and then utilized in the rosette formation test. When lysis was still present after three washings, further washings were performed until the supematant was clear and essentially devoid of any visible hemoglobin. Erythrocytes. Human red cells were obtained from heparinized blood of normal adults and one PNH patient; sheep erythrocytes were purchased from Colorado Serum Company Laboratories, Denver, CO, or were obtained from the heparinized blood of animals kept at our institution. Rabbit, goat, guinea pig, rat, and mouse red blood cells were also obtained from animals kept at our institution. Cat erythrocytes were obtained from animals owned by private individuals. Human peripheral lymphocytes. Peripheral blood lymphocytes were isolated from heparinized blood of normal adults and from patients with chronic lymphocytic leukemia (CLL). The supematant from 5 ml of blood sedimented at 37°C for 30 min with 1.25 ml of 3% gelatin (Plasmagel, Laboratories, Roger Bellon, Neuilly-sur-Seine, France) was centrifuged for 10 min at 150g. The pellet was resuspended in 5 ml of HBSS and layered over 3.0 ml of a Ficoll-Hypaque (Lymphoprep, Nyegaard and Company, A/S, Oslo, Norway) gradient and centrifuged for 5 min at 11OOg. The lymphocytes were then washed once with HBSS and adjusted to a concentration of 2 x 10” cells/ml in HBSS. Cultured human Zymphoid cells. The cell lines RPM1 8866, NC-37, RAJI, P3J, and Daudi were grown in Eagle’s minimum essential medium (MEM) (Autopow, Flow Laboratories, Rockville, MD) supplemented with glutamine, nonessential amino acids, pyruvate, fetal calf serum, penicillin and streptomycin, as described by Lemer and Hodge (12). Cell lines RPM1 4098 and RPM1 6410 were perpetuated in medium RPM1 1640 (Associated Biomedic Systems, Buffalo, NY) containing 10% fetal calf serum. All the culture flasks except those containing Daudi’ cells were rotated continuously on a
326
PELLEGHINO
ET
AL.
platform shaker. Daudi cells were grown in stationary culture flasks. Cell viability was assessed by the uptake of trypan blue. Before use, cells were washed once with HBSS and adjusted to a concentration of 2 x 10” cells/ml in HBSS. Preparation of sheep red blood cell-human lymphocyte rosettes. Rosettes were prepared according to the methods described by Jondal et al. (7) and Froland (8) with minor modifications. Briefly, 25 ~1 of 1% sheep red blood cells in HBSS were added to 25 ~1 of lymphocyte suspension (2 x 10” cells/ml) in a Fisher tube and centrifuged at 5OOg for 1 min at room temperature in a Fisher centrifuge. Parellel experiments were performed by incubating a mixture consisting of sheep red cells and human lymphocytes at 37°C for 5 min prior to the centrifugation step. At the end of the centrifugation (referred to as T), the number of rosettes was determined and the mixture was incubated at 4°C for 1 (T,), 6 (T6) or 20 (T,,) hr. Before reading the reaction, the cell pellets were resuspended by very gently tapping the tubes. A minimum of 200 lymphocytes were counted in a hemocytometer and lymphocytes were counted as rosette positive if three or more sheep red cells adhered to them.
Rosette formation human and murine
of human
lymphocytes
with
breakdown
products
of
C3. Rosette formation test was performed as previously described, utilizing sheep erythrocyte-antibody complexes coated with human C1,4,2,3 (EAC 1423hU, a generous gift from Dr. N. R. Cooper) and mouse C1,4,2,3 (EAC 1423m) (13). In vitro response of human lymphocytes to phytohemagglutinin. This response was assessed by incubating normal and AET treated human Iymphocytes with PHA-M (Difco, Detroit, Michigan), utilizing the semimicro culture method of Sengar and Terasaki (14). Treatment of human lymphoid cells with neuraminidase. Cultured human lymphoid cells and lymphocytes from CLL patients (1 x lo’/1 ml of HBSS) were incubated with 50 units of vibrio cholerae neuraminidase (Calbiochem, La Jolla, CA) for 30 min at 37°C. Afier incubation, the cells were washed twice with HBSS, recounted and utilized for rosette formation tests. RESULTS
Effect of AET treatment of sheep erythrocytes on rosette form&ion with normal peripheral blood lymphocytes. Treatment of normal sheep red cells with AET enhanced their reactivity with normal human peripheral lymphocytes. This effect depended on the concentration, the pH of the AET solution, and the time of incubation with sheep red blood cells. As shown in Fig. 1, when sheep red blood cells were treated with a 0.28 M solution of AET for 20 min, the rosettes appeared immediately after incubation with human peripheral lymphocytes in a percentage greater than in the control reactions which had been incubated up to 20 hr. Prolonging the incubation time to 1 and 6 hr, respectively, of AET-treated sheep red blood cells and human peripheral lymphocytes did not result in a further increasing of rosette formation. On the other hand, a slight decrease in the per-
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
327
FIG. I. Reactivity of normal and AET-treated sheep red blood cells incubated for the indicated time periods with human peripheral lymphocytes in the rosette formation test. Normal sheep red blood cells (panel A); sheep red blood cells treated with 0.07 M AET (panel B), with 0.14 M AET (panel C) and with 0.28 M AET (panel D). Treatment of sheep red blood cells with AET solution for 4 min (diagonal shading), for 15 min (open), for 20 min (black) and for 30 min (dots). In panel A the right-hand columns (dots with lines) indicate the percentage of rosettes obtained when prior to centrifugation the mixture of sheep red blood cells and human lymphocytes is incubated at 37°C for 5 min.
centage of rosettes occurred when the incubation time was increased to 20 hr. Treatment of sheep red blood cells with 0.28 M AET solution for periods shorter than 20 min had little effect on their reactivity in the rosette formation test; longer incubation periods did not further enhance the cells’ reactivity. AET was not effective when the pH of the solution was lowered below 8, while treatment at pH 9 did not further increase the rosette formation reactivity. When sheep red blood cells were treated with a 0.14 M solution of AET for 20 or 30 min, the results were similar to those described for sheep red blood cells treated with a 0.28 M AET solution for 20 min; furthermore, there was no marked reduction in the percentage of rosettes when sheep red blood cells and human peripheral lymphocytes were incubated for a total of 20 hr. The effects of a 0.07 M AET solution on the number of rosettes were less marked when read at the end of the centrifugation of the mixture of human peripheral lymphocytes and sheep red blood cells. On the other hand, when this mixture was incubated up to 20 hr, the enhancement of rosette formation was similar to that obtained with 0.14 and 0.28 M AET solutions incubated for 30 min. Concentrations of AET solutions greater than 0.28 M were not utilized because of the large amount of lysis produced by such treatment. AET also caused an increase in the size and stability of the rosettes formed. Some lymphocytes were completely coated with sheep red blood cells, most rosettes had more than six red blood cells attached to the central lymphocyte, and relatively few rosettes contained only three or four red blood cells. The rosettes were also less labile and susceptible to disruption
328
PELLEGRINO
ET AL.
TABLE
- .-..-.--
1
SRBC” (T,)’ :37YY
Subject M. R. M. P. A. C. A. P. L. H. M. D. s. P. E. S. S. F.
Mean .~--61.2 51.5 54.0 52.3 52.3 50.0 50.0 62.0 57.0
AE’I‘XRBC”
Range
Mean
Range
Mean
55-65 48-56 51-59 48-58 44-54 45-57 46-57 53-70 54-60
ND’ 70.0 68.7 70.0 ND 69.5 ND ND 67.0
ND 65-75 60-72 70-75 ND 65-75 ND ND 57-72
79.0 72.0 72.7 73.7 69.5 71 .a 73.4 75.0 74.0
(?‘,,; HLUlgC .-. 7:M35 68-78 70-78 70-78 69-70 70-75 69-76 74-76 70-77
a Sheep red blood cells. b Sheep red blood cells treated with 0.28M AET for 15 min at 37°C ’ Incubation times for the mixtures of sheep red blood cells and human lymphocytes wart 0 (T,,) and 5 (TJ hr, respectively. ” A mixture of sheep red blood cells and human lymphocytes were incubated at 37°C for Ij min prior to centrifugation. * Not done.
due to mechanical manipulations of the tubes. Finally, rabbit natural antibodies which can specifically and completely inhibit the binding of normal sheep red blood cells to peripheral human lymphocytes (15) were far less effective when sheep red blood cells were pretreated with AET. The ranges of values for rosette formation with normal and AET-treated sheep red blood cells are reported in Table 1. Different batches of normal sheep red blood cells from different percentages of rosettes with lymphocytes from a given donor. However, this variability was eliminated when sheep red blood cells from different batches were treated with AET solution (either 0.14 M for 30 min or 0.28 M for 15 mm); lower concentrations of AET solution or shorter periods of incubation were not effective in overcoming this variability. The addition of AET solution in a final concentration of 3 x 10-I to 7 x lop4 M to the mixture of normal sheep red blood cells and human peripheral lymphocytes increased the percentage of rosettes by 100% when the reading of the reaction was performed at the end of the centrifugation (Fig. 2). No effect was detected when the mixture was incubated up to 20 hr.
Reactivity of AET-treated sheep red cells with B type cultured human Zymphoid cells. Cultured human lymphoid cells RPM1 8866, RPM1 4098, RPM1 6410, NC-37, RAJI, P3J, and Daudi are Blymphoid cells; that is, they bear immunoglobulins and/or receptors for the third component of complement and do not form rosettes with normal sheep red bIood cells (13). The treatment of sheep red blood cells with 2.8 x 10-j M AET solution up to 30
SULFHYDRYL
COMPOUND
AND
/’ 0
T-CELL
10-5 10-4 10-j AET Final Concentration
FIG. 2. Effect on rosette formation of the sheep red blood cells and human peripheral (a-@), and 20 hr (A-A).
ROSETTE
10-2 [M]
FORMATION
329
10.’
addition of AET solution lymphocytes. Incubation
to a mixture of normal time: 0 hr (m-m), 1 hr
min did not give these cells the ability to form rosettes with cultured human lymphoid cells. Treatment of these cultured cells with neuraminidase did not change their reactivity with normal or AET-treated sheep red blood cells.
Effect of AET treatment of human peripheral lymphocytes on the rosette formation test. Human peripheral lymphocytes treated in vitro with a 0.07 M solution of AET (pH 8) for 10 min at 37°C appear to be more susceptible to the lytic action of complement and HL-A alloantisera (11). Under these conditions the lymphoid cells are viable as they exclude trypan blue and are able to synthesize DNA when stimulated with PHA. This treatment of human peripheral lymphocytes markedly affects their ability to form rosettes with normal and AET-treated sheep erythrocytes, an effect which was found to be dependent on the concentration of the AET solution utilized (Fig. 3).
Effect of AET treatment on the interaction lymphocytes and red blood cells from different
between human peripheral species. Red cells from one
PNH patient as well as normal human erythrocytes, when incubated up to 9 min at 37°C with an 0.28 M AET solution, did not acquire the ability to form rosettes with human lymphocytes. Similarly, no rosettes were observed when human lymphocytes were incubated with guinea pig, rat, mouse, cat, rabbit or goat erythrocytes pretreated with 0.28 M AET solution for 15 min.
Rosette formation
of CLL lymphocytes
with AET-treated
0
IO’
10-5 10-4 to-3 10.2 AET Final Concentration IM]
sheep red blood
FIG. 3. Effect of AET-treatment on the capacity of human peripheral lymphocytes to form rosettes with normal (0-O) and AET (m-m) treated sheep red blood cells, at Tj and Z’,,, respectively. Sheep red blood cells had been treated with 0.28 M AET for 15 min.
330
PELLEGRINO
ET
AL.
cells. In most of the CLL patients studied, peripheral lymphocytes did not form rosettes with sheep red blood cells (8,9). Neuraminidase treatment enhances the capability of normal human lymphocytes, but not of CLL lymphocytes, to form rosettes with sheep red blood cells (16). When sheep red blood cells treated with 0.28 M AET solution for 15 min were reacted with CLL lymphocytes the following reaction patterns could be distinguished (Table 2) : (1) patients 1 and 4’s lymphocytes formed less than 10% rosettes with normal and AET treated sheep red blood cells; (2) patients 5 and 6 had 20% to 30% rosettes (the percentage could not be increased with AET treated sheep red blood cells); (3) patient 3’s percentage of rosettes was increased by treatment of sheep red blood cells with AET; (4) patient 3’s lymphocytes reacted with normal and AET-treated sheep red blood cells much as peripheral lymphocytes from normal subjects did. This patient ma) be similar to the two cases of CLL with a predominance of T cells described by Bentwich et nl. (9), and by Seligmann ef (11. (17). Treatment of CLL lymphocytes with neuraminidase did not significantly change their reactivity with normal and AET-treated sheep red blood cells.
Loss of reactivity AET-treated sheep red blood cells in the rosette jormution test. At variable times after treatment with AET, sheep red blood cells completely lose their capacity to form rosettes with human lymphocytes, This effect depends on the concentration of AET and the length of treatment with AET as well as on the temperature of storage of the treated cells. .45 shown in Fig. 4, cells treated with either 0.14 or 0.28 M AET solution for 15 min did not form rosettes with human lymphocytes after 24 hr of storage at
1 4 7 5 6 2 3
2 1 5 16 27 26 I1
3 11 22 20 27 63 63
$5 -I 7 I6 27 63 42
4 -I “2 20 34 63 63
2.0 2.8 ND 24.5 2.4 12.5 0.0
ti.6 “2.0 VI) 5.0 10.5 2.. i ll.lJ .___
o Sheep red blood b Sheep erythrocytes ’ Sheep erythrocytes c2, c3. ” Sheep erythrocytes mouse serum. ” Incubation times respectively. ’ Not don?.
cells. treated coated coated
with O.28M with rabbit with
for the mixtures
rabbit SRBC
AET for IS mill at 37,-C. antisheep hemolysin and antisheep and
human
hemolysiu lymphocytes
purified
and Cl-C3 were
-----
.__-
human 1ixxn
(:I.
(:.a,
C5 de!icicut
0 (7-J and 5 (2’;) In,
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
331
FIG. 4. Loss of rosette-forming ability of AET treated sheep red blood cells stored at 37°C (panel A), at 22°C (panel C). Normal sheep red blood cells at T,, (n--n) and at Tzo(A----A); 0.14 M AET-treated sheep red blood cells at To (m-W) and TLO(O----O); 0.28 M AET-treated sheep red blood cells at T,, (0-O) and TgO (O----O).
37°C or 72 hr at 24°C respectively. When the cells were stored at 4°C the time of loss of capacity of AET treated sheep red blood cells to form rosettes depended on the concentration of AET solution. Sheep red blood cells treated with 0.14 M AET solution for 15 and 30 min formed no rosettes with human lymphocytes at To after 8 days of storage and produced only a reduced number of rosettes at TzO after 13 days of storage. The rosette forming capacity of sheep red blood cells treated with 0.28 M AET solution up to 30 min at T, and T,, was lost between the twelvth and thirteenth day of storage. DISCUSSION The present investigation has shown that sheep red blood cells treated in vitro with the sulfhydryl compound AET acquire a markedly enhanced ability to form rosettes with human peripheral lymphocytes as indicated by (1) the increased percentage of rosettes formed, (2) the increased stability of the rosettes, (3) the increase in number of cells bound to each lymphocyte, and (4) the appearance of rosettes immediately after mixing sheep red blood cells with human lymphocytes. Similar findings have been previously mentioned in abstract form by Kaplan and Clark (18). This phenomenon is specific because AET-treated sheep red blood cells do not form rosettes with cultured human lymphoid cells of the B type and with B type peripheral lymphocytes as shown by Kaplan and Clark (18) who utilized a population of lymphocytes reacted with fluoresceinated antiimmunoglobulin reagents. Two possible mechanisms can be envisioned which might explain the enhanced ability of AET-treated sheep red blood cells to bind to human lymphocytes. First, the treatment of sheep red blood cells with the sulfhydryl compound may uncover sites on the cell membrane that are able to react with human T cells. Alternatively, incubation with AET may alter the physicochemical properties of sheep red blood cells surface so as to favor their attachment to the plasma membrane of ractive lymphocytes. The latter explanation is suggested by the effect of AET on the reactivity of human erythrocytes with antibodies and complement. In fact, the AET treated human erythrocytes do not change in their capacity to absorb antibodies (anti-i-antibodies), but are about 20 times more sensitive to complement than untreated red cells (19).
332
PELLEGKINO
ET
AL
Furthermore, it has been reported that in vitro treatment of red cells with AET changes their sulfhydryl content (20). The increased reactivity of AET treated sheep red blood cells in the rosette formation test is not accompanied by an increased susceptibility to lysis by autologous serum (1) and by mouse complement (21). Human and rat erythrocytes, however, display an abnormal sensitivity to autologoua complement after in rjitro treatment with AET (1) but do not acquire the ability to bind to human T lymphoid cells. AET treatment of human lymphocytes completely abolishes their ability to bind normal as well as AET treated sheep red blood cells. This phenomenon does not reflect a selective destruction of T cells by AET, since AET-treated cells maintain, unchanged, their ability to respond to PHA. Therefore, it seems likely that AET treatment causes a destruction of the receptors fog sheep red blood cells. It is of interest that AET has a similar effect on the receptors for C3 breakdown products present on B type lymphoid cells (22). The mechanism of the change in reactivity as well as its reversibility and its effect on the biology of T cells are under investigation. It does seem likely, though, that sulfhydryl groups on lymphocyte membranes play a crucial role in the binding of sheep red blood cells. In this regard, it has been reported that treatment of lymphocytes with iodoacetamide strongly inhibits theii ability to form rosettes because of an effect on the -SH groups of the receptors (8). From a practical standpoint, the use of AET-treated sheep red blood cells may facilitate the clinical application of the rosette formation test because it shortens the incubation time for reading of the rosettes. Also, since such rosettes characteristically have an increased number of sheep red blood cells bound to the central human lymphocytes and are more stable to mechanical manipulation, the reading of the test results is considerably facilitated and the reproducibility of the test is augmented. Additional clinical use of AET treated sheep red blood cells may be worked out which employ the different patterns of reactivity observed with CLL patients, if it can be shown that the patterns correlate with the course of their disease. Studies along this line are in progress in our laboratory. Finally, the homogeneity of the reactivity of AET-treated sheep red blood cells from different batches will help in the standardization of the test which, in turn, will make it more meaningful for the comparison of results from different experiments and various laboratories. ACKNOWLEDGMENT The authors are grateful for their expert technical Hospital, San Diego, CA) Foundation, La Jolla, CA) and leukemic patients.
to Mrs. Anna Pellegrino and Mrs. Sandra Peterson assistance. We also thank Dr. W. Weaver (Naval and Dr. P. Sachs (Scripps Clinic and Research for collecting the blood samples from the PNH
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
333
FORMATION
REFERENCES 1. 2. 3. 4. 5. 6.
SIHCHIA,G.,ANL) FERRONE, S.,
Ser. Haematol.
5,
137,1972.
SIRCHIA,G., FERRONE, S., AND MERCUFUALI, F., Blood 25,502, 1965. SIRCHIA,G., FERRONE, S., MILANI, R., AND MERCURIALI, F., Blood 28,98, 1966. SIRCHIA, G., SPINELLI-RESSI, F., AND FERRONE, S., Bol. Sot. It&. Emat. 13, 225, 1965. WYBRAN,J. H., AND FUDENBERG, H. H., Trans. Ass.Amer. Physicians84,239, 1971. SILVEIRA, N. P., MENDES, N. F., ANDTOLANI,M. A.,J. Immunol. 108, 1456, 1972. 7. JONDAL,RI., HOLhl, G., ANI)WIGZELI.,H.,j. Exp. Med. 136,207, 1972.
8. FH~~LAND, S. S..Scund. I. Immunol. 1,269, 1972. 9. BENTWICH,Z., DOUGLAS,S. D.. SIEGAL,F. P., ANDKUNKEL,G. H., Clin. Immu~aol.Immunopatho/. I, 511, 1973. IO. ANTI, F., CIARLA,M. V., D'ASERO,C., D'AMELIO, R., ANDGAROFALO,J. A., Ifect. Immunot. 8, 110,1973. 11. SIRCHIA,G.,ANDFERRONE, S.,Blood 37,563, 1971. 12. LERNER, R. A., ANDHODCE,L. D.,J. Cell. Physiol.77, 265, 1971. 13. DIEWICH,M. P., PELLEGRINO, M. A., FERRONE, S., ANI) REISFELU,R. A., J. Immunol., 112, 1766,
1974.
14. SENGAR, D. P. S. ANDTERASAKI,P. I., Transplantation 11,260, 1971. 15. PELLECRINO, M. A., FERRONE, S., DIERICH.41. P., PELLECRINO, A. G., ANII REISFELD, R. A.. Clin. Immunol. Immunopathol., 4, 1975. 16. BENTWICH,2.. DOUGLAS,S. D., SKUTELSKY, E., ANDKUNKEL,H. G.,]. Exp. Med. 137, 1532, 17. 18. 19.
1973.
SELIGMANN, M., PREUD'HOMME, J-L., AND BROUET, J-C., Transpkznt. K;APLAN, M. E.,AND CLARK,C.,C~ Res. 21,877,1973. SIRCHIA,G., AND DACIE, J. V., Nature (London) 215, 747, 1967.
Reo.
16, 85,
1973.
20. CANNELLOS, G. P., BRAIN,M. C., AND DAVIE, J. V., Brit. J. Haematol. 18, 269, 1970. 21. MEDHURST, F. A., GLYNN,A. A., ANDDOUMARSHKIN, R. R., Immunology 20,441, 1971. 22. DIERICH,M. P., FERRONE, S., PELLEGRINO, M. A., ANI) REISFELD,R. A., /. Immunol.. 113,940,
1974.
IMMUNOLOGY
AND
IMMUNOI’ATHOLOGY
3,
324-333
(1975)
Enhancement of Sheep Red Blood Cell Human lymphocyte Rosette Formation by the Sulfhydryl Compound 2-Amino Ethylisothiouronium Bromide’ M. A. PELLEGRINO, S. FERRONE,~ M. P. DIERICH,:' AND R. A. REISFELD Department
oj’Experimenta1 Pathology, ScrilqJs Clinic and Research 476 Prospect Street, La Jolla, California 92037 Received
February
Found&ion.
25, 1971
In oitro treatment of sheep red blood cells with the srdthydryl compour~d 2aminoethylisothiouronium bromide (AET) under suitable experimental conditions greatly enhances their ability to form rosettes with human lymphoid cells. The effect of AET depends on the concentration, pH of the solution and time of incnbation. AET treated sheep red blood cells lose their ability to form rosettes after varying time periods depending on the temperature of storage and on the concentration of the AET solution. Treatment of human peripheral lymphocytes with AET abolishes their capability to form rosettes with both normal and AET treated sheep red blood cells. Lymphocytes from some patients with chronic lymphocytic lenkemia react differently with AET-treated sheep red blood cells than with normal sheep red blood cells.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by an intrinsic abnormality of red cell membrane which makes it extremely sensitive to the lytic action of complement. This disease, although rare, has attracted considerable interest because the unique lesion of the membrane confers to red cells a number of peculiar features both in viva and in u&o. The sulfhydryl compound 2-amino ethylisothiouronium bromide (AET) has been extensively utilized to produce the PNH lesion on normal human red cells (for review see Sirchia and Ferrone (1)). That is, under suitable experimental conditions normal human red blood cells treated in vitro with AET resemble PNH erythrocytes in their abnormal susceptibility to the lytic action of complement (2), in their decreased acetylcholinesterase activity (3) and in their reduced survival in oivo (4). The mechanism of action of AET as well as the nature of the lesion in’ This is publication No. SO6 from the Department of Experimental Pathology, Scripps (:iirn(~ and Research Foundation, La Jolla, CA. This work was supported by United States Public Health Service Grant AI 10180 and Grants AI 07007 and 70-615 from the American Heart Association, Inc. and a California Division of the American Cancer Society Senior Fellowship No. D-221. ” S.F. is a Visiting Scientist from the University of Milan, Italy :i M.D. is a Visiting Scientist supported by the Deutsche Forschungsgemeirrsclraft (I’rriyersitk of Mainz), Germany.
Copyright All rights
@> 1975 by Academic Press, Inc of reproduction in any form reserved
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
325
duced in red cell membranes are unknown and are presently under study in our laboratory. In the course of investigations on the effect of AET treatment on red cells from different animal species, it was observed that in U&O incubation of sheep red blood cells with AET solutions markedly enhanced their binding to human peripheral lymphocytes. This phenomenon is described here in view of the numerous reports that sheep red blood cell rosette formation is a specific marker for human T cells (5-8) and that such a test has wide clinical application (8-10). MATERIALS
AND
METHODS
Treatment of cells with AET solutions. AET (Sigma, St. Louis, MO) was dissolved in distilled water and the solution adjusted to pH 8 by addition of 5 N NaOH, unless otherwise stated. Red cells and human peripheral lymphocytes were treated with various concentrations of AET for different times, as previously described (2,ll). Briefly, 4 vol of AET solution were added to 1 vol of packed washed cells and incubated with intermittent mixing at 37°C for the times indicated in Results. After incubation, cells were washed three times with isotonic saline, recounted and then utilized in the rosette formation test. When lysis was still present after three washings, further washings were performed until the supematant was clear and essentially devoid of any visible hemoglobin. Erythrocytes. Human red cells were obtained from heparinized blood of normal adults and one PNH patient; sheep erythrocytes were purchased from Colorado Serum Company Laboratories, Denver, CO, or were obtained from the heparinized blood of animals kept at our institution. Rabbit, goat, guinea pig, rat, and mouse red blood cells were also obtained from animals kept at our institution. Cat erythrocytes were obtained from animals owned by private individuals. Human peripheral lymphocytes. Peripheral blood lymphocytes were isolated from heparinized blood of normal adults and from patients with chronic lymphocytic leukemia (CLL). The supematant from 5 ml of blood sedimented at 37°C for 30 min with 1.25 ml of 3% gelatin (Plasmagel, Laboratories, Roger Bellon, Neuilly-sur-Seine, France) was centrifuged for 10 min at 150g. The pellet was resuspended in 5 ml of HBSS and layered over 3.0 ml of a Ficoll-Hypaque (Lymphoprep, Nyegaard and Company, A/S, Oslo, Norway) gradient and centrifuged for 5 min at 11OOg. The lymphocytes were then washed once with HBSS and adjusted to a concentration of 2 x 10” cells/ml in HBSS. Cultured human Zymphoid cells. The cell lines RPM1 8866, NC-37, RAJI, P3J, and Daudi were grown in Eagle’s minimum essential medium (MEM) (Autopow, Flow Laboratories, Rockville, MD) supplemented with glutamine, nonessential amino acids, pyruvate, fetal calf serum, penicillin and streptomycin, as described by Lemer and Hodge (12). Cell lines RPM1 4098 and RPM1 6410 were perpetuated in medium RPM1 1640 (Associated Biomedic Systems, Buffalo, NY) containing 10% fetal calf serum. All the culture flasks except those containing Daudi’ cells were rotated continuously on a
326
PELLEGHINO
ET
AL.
platform shaker. Daudi cells were grown in stationary culture flasks. Cell viability was assessed by the uptake of trypan blue. Before use, cells were washed once with HBSS and adjusted to a concentration of 2 x 10” cells/ml in HBSS. Preparation of sheep red blood cell-human lymphocyte rosettes. Rosettes were prepared according to the methods described by Jondal et al. (7) and Froland (8) with minor modifications. Briefly, 25 ~1 of 1% sheep red blood cells in HBSS were added to 25 ~1 of lymphocyte suspension (2 x 10” cells/ml) in a Fisher tube and centrifuged at 5OOg for 1 min at room temperature in a Fisher centrifuge. Parellel experiments were performed by incubating a mixture consisting of sheep red cells and human lymphocytes at 37°C for 5 min prior to the centrifugation step. At the end of the centrifugation (referred to as T), the number of rosettes was determined and the mixture was incubated at 4°C for 1 (T,), 6 (T6) or 20 (T,,) hr. Before reading the reaction, the cell pellets were resuspended by very gently tapping the tubes. A minimum of 200 lymphocytes were counted in a hemocytometer and lymphocytes were counted as rosette positive if three or more sheep red cells adhered to them.
Rosette formation human and murine
of human
lymphocytes
with
breakdown
products
of
C3. Rosette formation test was performed as previously described, utilizing sheep erythrocyte-antibody complexes coated with human C1,4,2,3 (EAC 1423hU, a generous gift from Dr. N. R. Cooper) and mouse C1,4,2,3 (EAC 1423m) (13). In vitro response of human lymphocytes to phytohemagglutinin. This response was assessed by incubating normal and AET treated human Iymphocytes with PHA-M (Difco, Detroit, Michigan), utilizing the semimicro culture method of Sengar and Terasaki (14). Treatment of human lymphoid cells with neuraminidase. Cultured human lymphoid cells and lymphocytes from CLL patients (1 x lo’/1 ml of HBSS) were incubated with 50 units of vibrio cholerae neuraminidase (Calbiochem, La Jolla, CA) for 30 min at 37°C. Afier incubation, the cells were washed twice with HBSS, recounted and utilized for rosette formation tests. RESULTS
Effect of AET treatment of sheep erythrocytes on rosette form&ion with normal peripheral blood lymphocytes. Treatment of normal sheep red cells with AET enhanced their reactivity with normal human peripheral lymphocytes. This effect depended on the concentration, the pH of the AET solution, and the time of incubation with sheep red blood cells. As shown in Fig. 1, when sheep red blood cells were treated with a 0.28 M solution of AET for 20 min, the rosettes appeared immediately after incubation with human peripheral lymphocytes in a percentage greater than in the control reactions which had been incubated up to 20 hr. Prolonging the incubation time to 1 and 6 hr, respectively, of AET-treated sheep red blood cells and human peripheral lymphocytes did not result in a further increasing of rosette formation. On the other hand, a slight decrease in the per-
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
327
FIG. I. Reactivity of normal and AET-treated sheep red blood cells incubated for the indicated time periods with human peripheral lymphocytes in the rosette formation test. Normal sheep red blood cells (panel A); sheep red blood cells treated with 0.07 M AET (panel B), with 0.14 M AET (panel C) and with 0.28 M AET (panel D). Treatment of sheep red blood cells with AET solution for 4 min (diagonal shading), for 15 min (open), for 20 min (black) and for 30 min (dots). In panel A the right-hand columns (dots with lines) indicate the percentage of rosettes obtained when prior to centrifugation the mixture of sheep red blood cells and human lymphocytes is incubated at 37°C for 5 min.
centage of rosettes occurred when the incubation time was increased to 20 hr. Treatment of sheep red blood cells with 0.28 M AET solution for periods shorter than 20 min had little effect on their reactivity in the rosette formation test; longer incubation periods did not further enhance the cells’ reactivity. AET was not effective when the pH of the solution was lowered below 8, while treatment at pH 9 did not further increase the rosette formation reactivity. When sheep red blood cells were treated with a 0.14 M solution of AET for 20 or 30 min, the results were similar to those described for sheep red blood cells treated with a 0.28 M AET solution for 20 min; furthermore, there was no marked reduction in the percentage of rosettes when sheep red blood cells and human peripheral lymphocytes were incubated for a total of 20 hr. The effects of a 0.07 M AET solution on the number of rosettes were less marked when read at the end of the centrifugation of the mixture of human peripheral lymphocytes and sheep red blood cells. On the other hand, when this mixture was incubated up to 20 hr, the enhancement of rosette formation was similar to that obtained with 0.14 and 0.28 M AET solutions incubated for 30 min. Concentrations of AET solutions greater than 0.28 M were not utilized because of the large amount of lysis produced by such treatment. AET also caused an increase in the size and stability of the rosettes formed. Some lymphocytes were completely coated with sheep red blood cells, most rosettes had more than six red blood cells attached to the central lymphocyte, and relatively few rosettes contained only three or four red blood cells. The rosettes were also less labile and susceptible to disruption
328
PELLEGRINO
ET AL.
TABLE
- .-..-.--
1
SRBC” (T,)’ :37YY
Subject M. R. M. P. A. C. A. P. L. H. M. D. s. P. E. S. S. F.
Mean .~--61.2 51.5 54.0 52.3 52.3 50.0 50.0 62.0 57.0
AE’I‘XRBC”
Range
Mean
Range
Mean
55-65 48-56 51-59 48-58 44-54 45-57 46-57 53-70 54-60
ND’ 70.0 68.7 70.0 ND 69.5 ND ND 67.0
ND 65-75 60-72 70-75 ND 65-75 ND ND 57-72
79.0 72.0 72.7 73.7 69.5 71 .a 73.4 75.0 74.0
(?‘,,; HLUlgC .-. 7:M35 68-78 70-78 70-78 69-70 70-75 69-76 74-76 70-77
a Sheep red blood cells. b Sheep red blood cells treated with 0.28M AET for 15 min at 37°C ’ Incubation times for the mixtures of sheep red blood cells and human lymphocytes wart 0 (T,,) and 5 (TJ hr, respectively. ” A mixture of sheep red blood cells and human lymphocytes were incubated at 37°C for Ij min prior to centrifugation. * Not done.
due to mechanical manipulations of the tubes. Finally, rabbit natural antibodies which can specifically and completely inhibit the binding of normal sheep red blood cells to peripheral human lymphocytes (15) were far less effective when sheep red blood cells were pretreated with AET. The ranges of values for rosette formation with normal and AET-treated sheep red blood cells are reported in Table 1. Different batches of normal sheep red blood cells from different percentages of rosettes with lymphocytes from a given donor. However, this variability was eliminated when sheep red blood cells from different batches were treated with AET solution (either 0.14 M for 30 min or 0.28 M for 15 mm); lower concentrations of AET solution or shorter periods of incubation were not effective in overcoming this variability. The addition of AET solution in a final concentration of 3 x 10-I to 7 x lop4 M to the mixture of normal sheep red blood cells and human peripheral lymphocytes increased the percentage of rosettes by 100% when the reading of the reaction was performed at the end of the centrifugation (Fig. 2). No effect was detected when the mixture was incubated up to 20 hr.
Reactivity of AET-treated sheep red cells with B type cultured human Zymphoid cells. Cultured human lymphoid cells RPM1 8866, RPM1 4098, RPM1 6410, NC-37, RAJI, P3J, and Daudi are Blymphoid cells; that is, they bear immunoglobulins and/or receptors for the third component of complement and do not form rosettes with normal sheep red bIood cells (13). The treatment of sheep red blood cells with 2.8 x 10-j M AET solution up to 30
SULFHYDRYL
COMPOUND
AND
/’ 0
T-CELL
10-5 10-4 10-j AET Final Concentration
FIG. 2. Effect on rosette formation of the sheep red blood cells and human peripheral (a-@), and 20 hr (A-A).
ROSETTE
10-2 [M]
FORMATION
329
10.’
addition of AET solution lymphocytes. Incubation
to a mixture of normal time: 0 hr (m-m), 1 hr
min did not give these cells the ability to form rosettes with cultured human lymphoid cells. Treatment of these cultured cells with neuraminidase did not change their reactivity with normal or AET-treated sheep red blood cells.
Effect of AET treatment of human peripheral lymphocytes on the rosette formation test. Human peripheral lymphocytes treated in vitro with a 0.07 M solution of AET (pH 8) for 10 min at 37°C appear to be more susceptible to the lytic action of complement and HL-A alloantisera (11). Under these conditions the lymphoid cells are viable as they exclude trypan blue and are able to synthesize DNA when stimulated with PHA. This treatment of human peripheral lymphocytes markedly affects their ability to form rosettes with normal and AET-treated sheep erythrocytes, an effect which was found to be dependent on the concentration of the AET solution utilized (Fig. 3).
Effect of AET treatment on the interaction lymphocytes and red blood cells from different
between human peripheral species. Red cells from one
PNH patient as well as normal human erythrocytes, when incubated up to 9 min at 37°C with an 0.28 M AET solution, did not acquire the ability to form rosettes with human lymphocytes. Similarly, no rosettes were observed when human lymphocytes were incubated with guinea pig, rat, mouse, cat, rabbit or goat erythrocytes pretreated with 0.28 M AET solution for 15 min.
Rosette formation
of CLL lymphocytes
with AET-treated
0
IO’
10-5 10-4 to-3 10.2 AET Final Concentration IM]
sheep red blood
FIG. 3. Effect of AET-treatment on the capacity of human peripheral lymphocytes to form rosettes with normal (0-O) and AET (m-m) treated sheep red blood cells, at Tj and Z’,,, respectively. Sheep red blood cells had been treated with 0.28 M AET for 15 min.
330
PELLEGRINO
ET
AL.
cells. In most of the CLL patients studied, peripheral lymphocytes did not form rosettes with sheep red blood cells (8,9). Neuraminidase treatment enhances the capability of normal human lymphocytes, but not of CLL lymphocytes, to form rosettes with sheep red blood cells (16). When sheep red blood cells treated with 0.28 M AET solution for 15 min were reacted with CLL lymphocytes the following reaction patterns could be distinguished (Table 2) : (1) patients 1 and 4’s lymphocytes formed less than 10% rosettes with normal and AET treated sheep red blood cells; (2) patients 5 and 6 had 20% to 30% rosettes (the percentage could not be increased with AET treated sheep red blood cells); (3) patient 3’s percentage of rosettes was increased by treatment of sheep red blood cells with AET; (4) patient 3’s lymphocytes reacted with normal and AET-treated sheep red blood cells much as peripheral lymphocytes from normal subjects did. This patient ma) be similar to the two cases of CLL with a predominance of T cells described by Bentwich et nl. (9), and by Seligmann ef (11. (17). Treatment of CLL lymphocytes with neuraminidase did not significantly change their reactivity with normal and AET-treated sheep red blood cells.
Loss of reactivity AET-treated sheep red blood cells in the rosette jormution test. At variable times after treatment with AET, sheep red blood cells completely lose their capacity to form rosettes with human lymphocytes, This effect depends on the concentration of AET and the length of treatment with AET as well as on the temperature of storage of the treated cells. .45 shown in Fig. 4, cells treated with either 0.14 or 0.28 M AET solution for 15 min did not form rosettes with human lymphocytes after 24 hr of storage at
1 4 7 5 6 2 3
2 1 5 16 27 26 I1
3 11 22 20 27 63 63
$5 -I 7 I6 27 63 42
4 -I “2 20 34 63 63
2.0 2.8 ND 24.5 2.4 12.5 0.0
ti.6 “2.0 VI) 5.0 10.5 2.. i ll.lJ .___
o Sheep red blood b Sheep erythrocytes ’ Sheep erythrocytes c2, c3. ” Sheep erythrocytes mouse serum. ” Incubation times respectively. ’ Not don?.
cells. treated coated coated
with O.28M with rabbit with
for the mixtures
rabbit SRBC
AET for IS mill at 37,-C. antisheep hemolysin and antisheep and
human
hemolysiu lymphocytes
purified
and Cl-C3 were
-----
.__-
human 1ixxn
(:I.
(:.a,
C5 de!icicut
0 (7-J and 5 (2’;) In,
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
FORMATION
331
FIG. 4. Loss of rosette-forming ability of AET treated sheep red blood cells stored at 37°C (panel A), at 22°C (panel C). Normal sheep red blood cells at T,, (n--n) and at Tzo(A----A); 0.14 M AET-treated sheep red blood cells at To (m-W) and TLO(O----O); 0.28 M AET-treated sheep red blood cells at T,, (0-O) and TgO (O----O).
37°C or 72 hr at 24°C respectively. When the cells were stored at 4°C the time of loss of capacity of AET treated sheep red blood cells to form rosettes depended on the concentration of AET solution. Sheep red blood cells treated with 0.14 M AET solution for 15 and 30 min formed no rosettes with human lymphocytes at To after 8 days of storage and produced only a reduced number of rosettes at TzO after 13 days of storage. The rosette forming capacity of sheep red blood cells treated with 0.28 M AET solution up to 30 min at T, and T,, was lost between the twelvth and thirteenth day of storage. DISCUSSION The present investigation has shown that sheep red blood cells treated in vitro with the sulfhydryl compound AET acquire a markedly enhanced ability to form rosettes with human peripheral lymphocytes as indicated by (1) the increased percentage of rosettes formed, (2) the increased stability of the rosettes, (3) the increase in number of cells bound to each lymphocyte, and (4) the appearance of rosettes immediately after mixing sheep red blood cells with human lymphocytes. Similar findings have been previously mentioned in abstract form by Kaplan and Clark (18). This phenomenon is specific because AET-treated sheep red blood cells do not form rosettes with cultured human lymphoid cells of the B type and with B type peripheral lymphocytes as shown by Kaplan and Clark (18) who utilized a population of lymphocytes reacted with fluoresceinated antiimmunoglobulin reagents. Two possible mechanisms can be envisioned which might explain the enhanced ability of AET-treated sheep red blood cells to bind to human lymphocytes. First, the treatment of sheep red blood cells with the sulfhydryl compound may uncover sites on the cell membrane that are able to react with human T cells. Alternatively, incubation with AET may alter the physicochemical properties of sheep red blood cells surface so as to favor their attachment to the plasma membrane of ractive lymphocytes. The latter explanation is suggested by the effect of AET on the reactivity of human erythrocytes with antibodies and complement. In fact, the AET treated human erythrocytes do not change in their capacity to absorb antibodies (anti-i-antibodies), but are about 20 times more sensitive to complement than untreated red cells (19).
332
PELLEGKINO
ET
AL
Furthermore, it has been reported that in vitro treatment of red cells with AET changes their sulfhydryl content (20). The increased reactivity of AET treated sheep red blood cells in the rosette formation test is not accompanied by an increased susceptibility to lysis by autologous serum (1) and by mouse complement (21). Human and rat erythrocytes, however, display an abnormal sensitivity to autologoua complement after in rjitro treatment with AET (1) but do not acquire the ability to bind to human T lymphoid cells. AET treatment of human lymphocytes completely abolishes their ability to bind normal as well as AET treated sheep red blood cells. This phenomenon does not reflect a selective destruction of T cells by AET, since AET-treated cells maintain, unchanged, their ability to respond to PHA. Therefore, it seems likely that AET treatment causes a destruction of the receptors fog sheep red blood cells. It is of interest that AET has a similar effect on the receptors for C3 breakdown products present on B type lymphoid cells (22). The mechanism of the change in reactivity as well as its reversibility and its effect on the biology of T cells are under investigation. It does seem likely, though, that sulfhydryl groups on lymphocyte membranes play a crucial role in the binding of sheep red blood cells. In this regard, it has been reported that treatment of lymphocytes with iodoacetamide strongly inhibits theii ability to form rosettes because of an effect on the -SH groups of the receptors (8). From a practical standpoint, the use of AET-treated sheep red blood cells may facilitate the clinical application of the rosette formation test because it shortens the incubation time for reading of the rosettes. Also, since such rosettes characteristically have an increased number of sheep red blood cells bound to the central human lymphocytes and are more stable to mechanical manipulation, the reading of the test results is considerably facilitated and the reproducibility of the test is augmented. Additional clinical use of AET treated sheep red blood cells may be worked out which employ the different patterns of reactivity observed with CLL patients, if it can be shown that the patterns correlate with the course of their disease. Studies along this line are in progress in our laboratory. Finally, the homogeneity of the reactivity of AET-treated sheep red blood cells from different batches will help in the standardization of the test which, in turn, will make it more meaningful for the comparison of results from different experiments and various laboratories. ACKNOWLEDGMENT The authors are grateful for their expert technical Hospital, San Diego, CA) Foundation, La Jolla, CA) and leukemic patients.
to Mrs. Anna Pellegrino and Mrs. Sandra Peterson assistance. We also thank Dr. W. Weaver (Naval and Dr. P. Sachs (Scripps Clinic and Research for collecting the blood samples from the PNH
SULFHYDRYL
COMPOUND
AND
T-CELL
ROSETTE
333
FORMATION
REFERENCES 1. 2. 3. 4. 5. 6.
SIHCHIA,G.,ANL) FERRONE, S.,
Ser. Haematol.
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SIRCHIA,G., FERRONE, S., AND MERCUFUALI, F., Blood 25,502, 1965. SIRCHIA,G., FERRONE, S., MILANI, R., AND MERCURIALI, F., Blood 28,98, 1966. SIRCHIA, G., SPINELLI-RESSI, F., AND FERRONE, S., Bol. Sot. It&. Emat. 13, 225, 1965. WYBRAN,J. H., AND FUDENBERG, H. H., Trans. Ass.Amer. Physicians84,239, 1971. SILVEIRA, N. P., MENDES, N. F., ANDTOLANI,M. A.,J. Immunol. 108, 1456, 1972. 7. JONDAL,RI., HOLhl, G., ANI)WIGZELI.,H.,j. Exp. Med. 136,207, 1972.
8. FH~~LAND, S. S..Scund. I. Immunol. 1,269, 1972. 9. BENTWICH,Z., DOUGLAS,S. D.. SIEGAL,F. P., ANDKUNKEL,G. H., Clin. Immu~aol.Immunopatho/. I, 511, 1973. IO. ANTI, F., CIARLA,M. V., D'ASERO,C., D'AMELIO, R., ANDGAROFALO,J. A., Ifect. Immunot. 8, 110,1973. 11. SIRCHIA,G.,ANDFERRONE, S.,Blood 37,563, 1971. 12. LERNER, R. A., ANDHODCE,L. D.,J. Cell. Physiol.77, 265, 1971. 13. DIEWICH,M. P., PELLEGRINO, M. A., FERRONE, S., ANI) REISFELU,R. A., J. Immunol., 112, 1766,
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14. SENGAR, D. P. S. ANDTERASAKI,P. I., Transplantation 11,260, 1971. 15. PELLECRINO, M. A., FERRONE, S., DIERICH.41. P., PELLECRINO, A. G., ANII REISFELD, R. A.. Clin. Immunol. Immunopathol., 4, 1975. 16. BENTWICH,2.. DOUGLAS,S. D., SKUTELSKY, E., ANDKUNKEL,H. G.,]. Exp. Med. 137, 1532, 17. 18. 19.
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SELIGMANN, M., PREUD'HOMME, J-L., AND BROUET, J-C., Transpkznt. K;APLAN, M. E.,AND CLARK,C.,C~ Res. 21,877,1973. SIRCHIA,G., AND DACIE, J. V., Nature (London) 215, 747, 1967.
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16, 85,
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20. CANNELLOS, G. P., BRAIN,M. C., AND DAVIE, J. V., Brit. J. Haematol. 18, 269, 1970. 21. MEDHURST, F. A., GLYNN,A. A., ANDDOUMARSHKIN, R. R., Immunology 20,441, 1971. 22. DIERICH,M. P., FERRONE, S., PELLEGRINO, M. A., ANI) REISFELD,R. A., /. Immunol.. 113,940,
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