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Enduring allogeneic marrow engraftment via nonspecific bone-marrow-derived regulating factors (MRF)
CELLULAR
IMMUNOLOGY
57,219-228 (1981)
Enduring Allogeneic Marrow Engraftment via Nonspecific Bone-Marrow-Derived Regulating Factors (MRF) WALTER PIERPAOLI,’ GEORGES JEAN-MARIE MAESTRONI, AND EDGAR SACHE* Institute
of Anatomy, University of Ziirich. Ziirich. *Institute Choay. Paris, France Received February
Switzerland,
27, 1980; accepted April
and
9. 1980
An ultrafiltration fraction of MW > 100,000 separated from the original medium in which bone marrow had been suspended (supernatant) stimulated incorporation of [‘Hlthymidine by marrow in vitro and was designated marrow regulating factor (MRF). The administration of MRF to F, hybrid mice transplanted with parental bone marrow resulted in lasting chimerism of the surviving mice. A few of the hybrids receiving parental marrow but no MRF survived: however, none were chimeric. Administration of MRF after irradiation in C57BL/ 6 mice transplanted with bone marrow from DBA/2 and BALB/c donors resulted in endogenous reconstitution. However, administration of MRF before (preconditioning) and again after irradiation resulted in survival of the majority of mice. These C57BL/6 mice were chimeras of DBA/Z or BALB/c marrow but showed no sign of secondary disease. Thus the use of MRF abrogates resistance to and promotes engraftment of foreign marrow and enduring chimerism when the recipients (F, hybrids) appear to be nonreactive to the donor (parental marrow) and also when alloreactivity is bidirectional (allogeneic combinations).
INTRODUCTION In the course of recent studies on transplantation of allogeneic (homologous) bone marrow (BMT) into lethally irradiated mice, we found that the administration of viable bone marrow cells to recipients just prior to lethal, total-body irradiation (TBI) resulted in protection against radiation injury, facilitation of marrow engraftment, and induction of a persisting xenogenic (rat) and allogeneic chimerism in mice. The operational procedure preceding TBI was designated “preconditioning” since this maneuver profoundly shifted the manner in which the host reacted to the engrafting increment of allogeneic marrow (14). Utilizing the rat to mouse model of marrow transplantation it was established that the critical factors for success included the number of viable bone marrow cells given before and after TBI, the timing of these injections, and the route of inoculation; each of these had to be correctly poised to assure avoidance of secondary disease, the development of chimerism, and long-term survival (2-4). It soon became evident that the overall protection by the marrow given prior to TBI was not at all related to the immune specificity of the donor (24). The fact that the preparatory inoculation of bone marrow was effective when given shortly before TBI and that administration via ’ Present address and requests for reprints: Institute for Integrative Biomedical Research, Lohwisstrasse 50, 8 I23 Ebmatingen, Switzerland. 219 OOOS-8749/81/010219-10$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved
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the systemic route resulted in a more pronounced protection (2) led progressively to the interpretation that the viable bone marrow cells were exerting this markedly favorable effect via synthesis and/or secretion, in the host, of components facilitating engraftment of the transplanted, incompatible marrow after lethal TBI. The implications of such an interpretation prompted further studies on the influence of a pre- and postconditioning regimen in xenogeneic (rat to mouse) and allogeneic BMT where soluble products of bone marrow cells or bone marrow cells suspended in their original suspension medium (supernatant, SN) were inoculated before or after TBI (Maestroni and Pierpaoli, submitted for publication). This report communicates a series of further experiments which led to the finding that such active substances were indeed present in, or elaborated by, marrow and to some of their properties in vitro and in vivo. The preliminary data reported here attest to their essential role in achieving successful engraftment of allogeneic marrow and enduring chimerism in mice. MATERIALS
AND METHODS
Animals The animals used in this investigation were generously provided by the Animal Farm of Hoffman-La Roche & Company, Ftillinsdorf, Switzerland. The mice were bred under specific pathogen-free conditions and then maintained, as adults, under strictly standardized and controlled hygienic conditions in the Central Biological Laboratory of the Cantonal Hospital, Zurich. However, no special precautions were taken to avoid pathogenic bacteria or viruses. No antibiotics were administered via drinking water either before or after irradiation. Donors or recipients of bone marrow were inbred young adult (8-12 weeks old) C57BL/6, DBA/2, BALB/ c, and C57BL/6 X A/J F, hybrid mice. The rabbits used were an outbred Swiss strain, 1.5 to 2 kg body wt. They were used as donors of xenogeneic marrow for preparation of supernatant (SN) from which the marrow regulating factors (MRF) were separated. Preparation of Rabbit Bone Marrow Supernatants (SN) for Separation of Marrow Regulating Factors (MRF) Groups of 10, male or female, rabbits were killed by cervical dislocation. Care was taken to use young adult donors. The long bones were isolated and put into refrigerated TC 199 or saline (NaCl 0.9%). The bones were then cut at the extremities and TC 199 or saline was flushed repeatedly through the bones to expel all the marrow, including fat tissue and connective tissue and all that make up the “milieu interieur” of the bone marrow. The material (clots of marrow, fat, other tissues) was then dissociated by using a large syringe without needle, delicately aspirating and expelling the suspended material which was always kept in melting ice, until no cell aggregates were visible. The number of cells in the suspensions ranged between 50 and 100 X 106/ml of SN. At this stage, the whole initial suspension of material was centrifuged in a refrigerated centrifuge at 5°C for 30 min at 10,OOOgand the fat condensed at the top of the tubes was eliminated by aspiration. The rest of the supernatant was collected in plastic containers and frozen immediately at -30°C. The cells were discarded. All this preparatory work was carried out under sterile conditions. SN is intended to be the original suspension
MARROW
FACTORS
PROMOTE
MARROW
ENGRAFTMENT
221
medium in which the bone marrow cells were dissociated and suspended. The pH of all cell suspensions and of preparations of SN was kept or corrected at 7.2 to 7.5. Preparation
of MRF from Rabbit Bone Marrow
Supernatant
(SN)
After thawing the SN, it was centrifuged for 20 min at 40,000 g in a refrigerated centrifuge to remove all possible particles and precipitate. The SN was then passed over a Diaflo XM 100 filter (MW > 100,000; Amicon Co., Oosterhout, Holland) using an Amicon Model 202 cell. An equal volume of water was used to wash the material which did not pass through the filter. The material remaining on the filter was withdrawn with a syringe and lyophilized. Preparation
of Bone Marrow
Cell Suspensions
Suspensionsof bone marrow cells were freshly prepared 2-3 hr before inoculation. Mice were killed by cervical dislocation; the long bones (humeri, tibias, and femurs) were isolated and cut at the extremities. Ice-cold TC 199 medium was flushed repeatedly through the cavities by syringe with a needle fitting the bone size. The pooled marrow was gently dispersed by a needleless syringe and filtered through gauze. The cells were then washed two to three fold by low-speed centrifugation, using ice-cold TC 199 medium. The final cell suspensions were adjusted to the desired number and volume. Quantities varying from 20 to 40x 1O6cells per donor mouse could be harvested. The cells were administered in a volume of 0.5 ml per mouse, iv. Trypan blue exclusion tests showed that over 95% of the cells were viable just before their inoculation. Irradiation
A dose of 850 to 900 rad total-body irradiation (TBI) was given to the recipients depending on the known strain sensitivity to irradiation (5). This dose led to death of all unprotected mice within 8-15 days. The irradiation apparatus was a Cobalt Gammatron 3 (6000 Ci). Field size was 30x30 cm, main focus distance was 90 cm. No filters were used. Transplantation
of Allogeneic
Marrow
(a) Preconditioning regimen. When this regimen was adopted the mice were injected iv 1.5 to 2.0 hr before irradiation with 1.0, 2.5, or 5 mg of rabbit MRF. The lyophilized material (fraction with MW > 100,000) was dissolved in TC 199. The volume injected was 0.5 ml. Controls were injected with cells or medium without MRF. (b) Postconditioning regimen. The mice were injected 24 hr after TBI with 30 to 37 x lo6 washed bone marrow cells. Depending on the experimental plan the cells were injected alone or suspended in the medium containing the fractions of the MRF. The cells were resuspended in the medium (TC 199, pH 7.4) containing MRF shortly before iv inoculation. Tests for Chimerism
(a) Hemoglobin migration pattern. At monthly intervals after TBI, all mice were individually tested for chimerism of the erythroid cell line. A few drops of blood
222
PIERPAOLI,
MAESTRONI,
AND
SACHE
were taken from the retroorbital plexus, suspended in heparinized saline, and washed repeatedly. The washed blood cells were hemolyzed in 15 cell-water volume. The pattern of hemoglobin migration was examined by cellulose-acetate electrophoresis. The strips were stained with amido black. Engraftment of the donor eythropoietic line corresponded to complete chimerism as evidenced by skin grafting (4). Passively transferred, donor-type red cells were also found in the peripheral blood of nonchimeric mice. They persisted as long as 3 months when donors were BALB/c mice and 2 months when donors were DBA/2 mice. (b) Skin grafts. In the situation of cross-transplantation of bone marrow from or into C57BL/6 and DBA/2 or BALB/c donor-recipient, donor skin was grafted on groups of allogeneic marrow recipients. Grafting was by conventional technique; the corsets were removed after 8-10 days and the viability of the grafts was checked daily. The graft was considered as rejected when the first signs of infiltration, edema, and induration appeared. They were considered as accepted only when none of these signs were discernible and luxuriant hair grew on the transplanted skin. Incorporation of [3H]Thymidine by Bone Marrow Cells in Vitro 6-[3H]Thymidine (27 Ci/mol) was purchased from the Radiochemical Center, Amersham, England. Washed (once) bone marrow cells freshly taken from adult C57BL/6 mice were incubated for 1, 2, and 3 hr in sealed tubes in the presence of 2 PCi of [3H]thymidine. Samples were in triplicate. Each tube contained 2 X lo6 washed bone marrow cells suspended in 1 ml of medium TC 199, or in TC 199 with 200 pg of the ultrafiltration fractions of the original supernatant of rabbit bone marrow cell suspensions (see above preparation of MRF). The pH of the medium was adjusted to 7.5. After 1, 2, and 3 hr of incubation, 0.1 ml of 10% sodium dodecyl sulfate (SDS) was added to each tube. After a vigorous shaking, DNA was precipitated at 4°C for 15 min by adding to each tube 1 ml of 10% trichloroacetic acid (TCA). The precipitate was collected on Whatmann GF/C glass fiber filters and washed with 5% TCA and absolute ethanol. The filters were dried and the activity was measured in a LKB-Wallac 81000 liquid scintillation counter. RESULTS Effect of Ultrafiltration Fractions of Bone Marrow Supernatants on Incorporation of [ 3H]Thymidine by Bone Marrow Cells in Vitro As a preliminary screening test, the effect of different fractions of rabbit bone marrow supernatants obtained by Amicon Diaflo membrane ultrafiltration was tested on the incorporation of [ 3H]thymidine by suspensionsof mouse bone marrow cells in vitro. Table 1 shows the results of one experiment which were fully confirmed by repeated tests. Apparently, only the ultrafiltration fraction with MW > 100,000 stimulated constantly the mitotic activity and/or replication by bone marrow cells. The other fractions (MW < 100,000 and MW > 30,000) did not constantly produce an inhibition or stimulation of the incorporation. The fraction of MW < 30,000 often showed a clear inhibiting action (Table 1). The same fraction of MW > 100,000, when inoculated in vivo, also increased by 50% incorporation of [)H]thymidine at 1 and 2 hr after inoculation (Maestroni and Pierpaoli,
I
62,392 + 5909
32,420 + 4705 35,049 + 5626
31,185 k 4311 18,008 k 1152
2841
35,064 f
23,327 f 2814 20,265 f 4294
1 2
3
4 5
126,631 37,868 98,586 49,918 65,291
f + + 2 + The incubation
7842 4478 2901 6752 8203
3 hr (cpm f SD)
Note. Donors of bone marrow (BM) cells were C57BL/6 mice (2 X lo6 BM cells/ml). was added to each tube. Samples were in triplicate. SD = standard deviation.
73,180 f 6972 19,347 + 2059
1 hr (cpm f SD)
2 hr (cpm k SD)
Incubation
200 200 200 200 IO6 X lo6 BM cells in TC
X lo6 BM cells + X IO* BM cells + X lo6 BM cells + BM cells + X
pg Fraction pg Fraction rg Fraction rg Fraction 199
ml [‘Hlthymidine
> 100,000 < 30,000 > 30,000 < 100,000
Cells
MW MW MW MW
of samples
by Bone Marrow
Composition
Incorporation
medium was TC 199, pH 7.5. 2 &i/O.1
2 2 2 2 2
Fraction of MW > 100,000 from Bone Marrow Supernatant Stimulates [‘H]Thymidine in Vitro. A fraction of MW < 30,000 Inhibits Incorporation
Code
An Ultrafiltration
TABLE
-1
%
2
8
E
%
3
z
2
3 iz
b
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PIERPAOLI,
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AND
SACHE
unpublished). Thus, this fraction of the ultrafiltrates with MW > 100,000 was chosen for testing in experiments in vivo on BMT. It was called marrow regulating factor (MRF). Effect of MRF on Allogeneic
Engraftment
and Chimerism
(a) F, Hybrids injected with parental bone marrow. The in vitro results shown above on activity of MRF and the previous findings on induction of chimerism in C57BL/6 X A/J F, hybrids transplanted with parental marrow suspended in its original SN (Maestroni and Pierpaoli, submitted for publication) suggested the use of this well-known model. C57BL/6 X A/J F, hybrid mice were transplanted with washed bone marrow cells from C57BL/6 mice; the cells were suspended in a solution containing MRF (fraction of MW > 100,000). As can be seen in Table 2, when the hybrid recipients were injected with parental bone marrow cells without MRF, only some of the animals survived thanks to the reconstitution of the endogenous marrow, but no chimerism was achieved. On the contrary, all the surviving hybrids inoculated with parental bone marrow suspended in a medium containing the fraction of MW > 100,000 were permanent chimeras. Apparently, the MRF fraction with MW > 100,000 facilitated or enhanced the engraftment of the parental marrow when injected after TBI (postconditioning) in a fashion identical to that shown previously in the same model by using marrow supernatants (Maestroni and Pierpaoli, submitted for publication). (b) Allogeneic donor-recipient strains. As shown previously, the inoculation of bone marrow cells with or without their supernatants before TBI (preconditioning regimen) facilitated the engraftment of xenogeneic marrow in mice or facilitated the endogenous reconstitution in particularly resistant recipients. However, no chimerism was obtained when the donors and the recipient mice were fully allogeneic (Maestroni and Pierpaoli, submitted for publication). A modified preconditioning regimen has now been adopted using as recipients C57BL/6 mice, which are known to be particularly resistant to allogeneic and xenogeneic engraftment (6). Donors were DBA/2 and BALB/c mice. The timing of administration before TBI was TABLE Marrow
2
Regulating Factor (MRF) Promotes Enduring Chimerism in Hybrid Recipient Inoculated with Parental Bone Marrow after Lethal Irradiation
Mice
Postconditioning
Animals
Group
No.
TBI (rad)
h+
Product
No. BM cells (X 106)
Route
Survival at 6 months
Chimerism
A B
20 40
900 900
24 24
3 mg MRF -
35 35
iv iv
18 (90%) 8 (20%)
18 (100%) 0 (0%)
Note. TBI = total-body irradiation. MRF = marrow regulating factor; Amicon Diaflo membrane ultrafiltration fraction (MW > 100,000) of supernatant of rabbit bone marrow cells. Donors of bone marrow were adult C57BL/6 mice, recipients were F, hybrid, 3- to 4-month-old C57BL/6 X A/J female mice. The parental bone marrow cells were washed once and then suspended in TC 199 containing the MRF immediately before inoculation. The cells were inoculated 24 hr after TBI. Chimerism was assessed by individual hemoglobin typing in each surviving recipient hybrid mouse (4). The results above derive from two identical experiments.
3
iv iv iv iv iv iv
Route 1.5 1.5 1.5 1.5 1.5 1.5 850 850 850 850 850 850
TBI dose bad) 24 24 24 24 24 24
h’ 3 3 3 3 3 3
mg mg mg mg mg mg MRF MRF MRF MRF MRF MRF
Product 37 31 31 31 30 30
No. of BM cells (X 106)
Postconditioning
iv iv iv iv iv iv
Route
6 5 4 3 9 10
(60%) (50%) (40%) (10%) (90%) (50%)
Survival (6 months after TBI)
with Allogeneic
Nore. TBI = total-body irradiation. MRF = marrow regulating factors; Amicon Diaflo membrane ultrafiltration fraction (MW > 100.000) of supernatant bone marrow cells. Donors of bone marrow were adult, 3-month-old DBA/2 and BALB/c female and male mice, recipients were 3- to 4-month-old male mice. MRF or TC 199 was injected 1.5 hr before TBI (h-). The allogeneic bone marrow cells were washed once and then suspended in TC 199 containing immediately before inoculation. The cells were inoculated 24 hr (h’) after TBI. Chimerism was assessed by hemoglobin typing and skin grafting of the surviving recipients (4). For the cause of mortality in groups A, B, C, and F, see Results section. The results above derive from four different experiments and D, two experiments; E and F, two experiments).
mg MRF mg MRF mg MRF 199 199 mg MRF
A B C D E F
5.0 2.5 1.0 TC TC 2.5
DBA/2 DBA/Z DBA/2 DBA/2 BALB/c BALB/c
Group
10 10 10 30 10 20
Product
Donor strain
No. of recipients
Preconditioning
with Marrow Regulating Factors (MRF) Results in Enduring Chimerism of the surviving C57BL/6 Mice Transferred Marrow. MRF Given Only in PostConditioning Results in Endogenous Reconstitution of the Surviving mice.
Animals
Preconditioning
TABLE
(100%) (100%) (100%) (0%) (0%) (100%) of rabbit C57BL/6 the MRF individual (A, B, C,
6 5 4 0 0 IO
Chimerism
Bone
j;r
F 3$
E 22 G 3
5
5 2
2 0
a E
2 2
2
226
PIERPAOLI,
MAESTRONI,
AND
SACHE
chosen on the basis of the stimulatory effects of MRF on BM cells observed in vivo (unpublished) and in vitro (Table 1). As can be seen in Table 3 (groups A, B, C, and F), preconditioning of the recipients with 1.0, 2.5, and 5.0 mg of MRF induced survival and permanent chimerism in a large number of recipients of this extremely alloreactive strain combination, while the lack of it in the preconditioning and its presence in the postconditioning (after TBI) eventually promoted endogenous bone marrow reconstitution but no chimerism of some recipients (Table 3, groups D and E). Chimerism has persisted and no signs of secondary disease have been observed to the present (6 months after marrow transplantation). All the chimeric C57BL/6 mice accepted permanently the skin grafts from DBA/Z or BALB/c donors. Chimerism was specific and immune reconstitution was complete since the chimeric mice rejected, within lo-12 days, skin grafts from strains different at the main H-2 locus (C3H, CBA). The mortality in groups A, B, C, and F could be attributed to several causes such as infection, deficient engraftment, secondary disease, and others. DISCUSSION There have been many attempts to achieve a persisting hematopoietic and immune reconstitution via transplantation of allogeneic bone marrow, but this has proved to be an insurmountable goal despite intensive efforts (7). This report describes an alternative modality for transferring bone marrow between genetically different individuals of the same species. It is based on the use of recently identified marrow regulating factors (MRF) which appear to be intimately linked to marrow function; of primary importance for engraftment and for well-balanced marrow proliferation and maintenance of hematopoiesis. A number of investigators have explored the presence in marrow of inhibitors of marrow proliferation (8-l 0). Such components could certainly play a part in regulating marrow function and possibly account for its rejection. However, to the best of our knowledge components that facilitate marrow proliferation and its engraftment have not previously been reported. It is evident that these substances would be of importance in determining the outcome of bone marrow transplantation. Without in any way implying a lesser relevance of the host-donor immunogenetic compatibility for successful transplantation of bone marrow, it may be useful to analyze and interpret the facts emerging from our own experimental model and the new directions they provide. Apparently, the rapid initiation and progression of a well-balanced hematopoiesis in the irradiated host is a prerequisite for maintenance of the grafted marrow and for enduring chimerism with avoidance of early or late symptoms of GVHR. In fact, the model in which the Fr hybrids were transplanted with marrow from the parental strain demonstrates unequivocally that no reaction of the donor cells against the host ensues when MRF is administered together with the parental marrow (Table 2). Also, in the extreme situation, i.e., the most incompatible model of C57BL/6 mice transplanted with DBA/2 or BALB/c marrow, the apparent enduring eradication of the resistance to engraftment by the preirradiation treatment with MRF changes completely the normal inability of the host to accept this donor marrow. Conversely, the donor marrow rapidly colonizes the host taking care that no “aggression” reaction will initiate (Table 3, compare groups A, B, C, and F to groups D and E). Thus, it seems to
227
MARROW FACTORS PROMOTE MARROW ENGRAFTMENT
Q +@
A)
TEMPORARY
ENDOGENOUS
Cl
OR PIRTIAL
RECONSTITUTION
CHIMERISM;
G~.C57l3+$
MRF
FIG. 1. Marrow regulating factors (MRF) promote engraftment and enduring chimerism in lethally irradiated mice transplanted with allogeneic bone marrow: (A) Allogeneic bone marrow transplantation, as generally performed, involves the inability of the foreign marrow to colonize the host combined with host resistance to engraftment. This combination generates a conflicting situation in which immune reactions also add their negative effects, resulting in deficient or abnormal hematopoiesis. The result is acute or chronic secondary disease, immunodeficiency, runting, infection, and the varied disorders connected with a disturbed hematopoiesis (lymphopenia, agranulocytosis, thrombocytopenia, anemia, etc.). Acute or chronic alterations of liver, gut, and skin also appear variably. (B) Administration of MRF after TBI will lead to either endogenous reconstitution or chimerism, depending on the immunogenetic character of the donor-recipient combination. Endogenous reconstitution takes place when high resistance to engraftment is present (e.g., C57BL/6 mice, see Table 3), whereas chimerism is achieved when no host resistance is offered (e.g., C57BL/6 X A/J F, hybrids transplanted with parental marrow, see Table 2). (C) When MRF is given before as well as after TBI, resistance to engraftment is abrogated and the donor marrow colonizes the host (e.g., C57BL/6 mice transplanted with DBA/2 marrow, see Table 3). This system permits induction of chimerism when alloreactivity is bidirectional.
us evident that a rapid and efficient hematopoietic functionality of the grafted marrow is perhaps the essential requirement for avoidance of GVHR or HVGR. Improvement of the transplantation technique is needed to reduce or eliminate the mortality of the allogeneic chimeras (Table 3). There are indications that some further shifts in dosage and timing of administration of MRF in the pre- and postconditioning will effect a reduction in the death rate of allogeneic chimeras even under these conventional conditions. Our present and necessarily preliminary interpretation of the phenomena connected with the use of MRF in BMT is illustrated in Fig. 1. It is considered possible that pretreatment of the donor with MRF before TBI renders the recipient’s marrow more sensitive to irradiation, thus making for a more complete elimination of any minority or subpopulation of radioresistant marrow cells. In fact, we have repeatedly observed that when MRF is administered after TBI in the highly resistant strains, it facilitates endogenous reconstitution; there then ensues rejection of the foreign marrow. A conflicting situation pertains when neither host nor donor cells prevail; in this situation secondary disease arises. It is at this stage that immunological phenomena and reactions are probably further deviating hematopoiesis and superimposing themselves on the original dysfunction of certain hematopoietic cell lines.
228
PIERPAOLI, MAESTRONI,
AND SACHE
Whatever the mechanism proves to be, the preliminary results given here and the large variety of experiments in course point the way to the reality of allogeneic marrow transplantation with immune and hematopoietic reconstitution. The physiological and hematological investigation of bone marrow factors and their functions clearly hold promise for achieving a safer reconstitution, especially for its farranging potential in therapeutic application. ACKNOWLEDGMENTS We are deeply grateful to Dr. Maurice Landy for his constant interest, advice, and encouragement and for the improvement of the manuscript. We are indebted to Dr. Wolf Weihe for the maintenance of the animals used and to Miss Petra Stijckel for excellent technical assistance. We thank Mrs. Michelle Kunz of Institute Choay for technical help in the preparation of MRF. We are indebted to Professor Gustav0 Cudkowicz for his comments and criticism. This work has been mainly supported with a research grant from Institute Choay, Paris. Further financial support was received from the Swiss National Science Foundation, Bern (Grant 3.251-0.77), from the Cilag-Chemie Foundation for therapeutical research, Schaffhausen, and from the Piombino Foundation for Life Sciences, Populonia, Livorno, Italy.
REFERENCES 1. Pierpaoli, W., and Maestroni, G. J. M., Transplantation 26, 456, 1978. 2. Pierpaoli, W., and Maestroni, G. J. M., Stand. J. Huemotol. 22, 165, 1979. 3. Pierpaoli, W., and Maestroni, G. J. M., J. Lab. Clin. Immunol. 2, 125, 1979. 4. Pierpaoli, W., and Maestroni, G. J. M., Cell. Immunol. 52, 62, 1980. 5. Green, E. L., (Ed.), “Biology of the Laboratory Mouse,” Chap. 22. McGraw-Hill, New York, 1966. 6. Trentin, J. J., Gallagher, M. T., and Lotzova, E., In “Immunobiology of Bone Marrow Transplantation” (B. DuPont and R. A. Good, Eds.), pp. 137-142. Grune & Stratton, New York, 1976. 7. Cudkowicz, G., Landy, M., and Shearer, G. (Eds.), “Natural Resistance Systems against Foreign Cells, Tumors and Microbes.” Academic Press, New York, 1978. 8. Cline, M. J., Herman, S. P., and Golde, D. W., Transplunt. Proc. 10, 99, 1978. 9. Benestad, H. B., Testa, N. G., and Lajtha, L. G., Stand. J. Huematol. 20, 18, 1978. 10. Lajtha, L. G., Nouv. Rev. Fr. Hematol. 21, 59, 1979.
IMMUNOLOGY
57,219-228 (1981)
Enduring Allogeneic Marrow Engraftment via Nonspecific Bone-Marrow-Derived Regulating Factors (MRF) WALTER PIERPAOLI,’ GEORGES JEAN-MARIE MAESTRONI, AND EDGAR SACHE* Institute
of Anatomy, University of Ziirich. Ziirich. *Institute Choay. Paris, France Received February
Switzerland,
27, 1980; accepted April
and
9. 1980
An ultrafiltration fraction of MW > 100,000 separated from the original medium in which bone marrow had been suspended (supernatant) stimulated incorporation of [‘Hlthymidine by marrow in vitro and was designated marrow regulating factor (MRF). The administration of MRF to F, hybrid mice transplanted with parental bone marrow resulted in lasting chimerism of the surviving mice. A few of the hybrids receiving parental marrow but no MRF survived: however, none were chimeric. Administration of MRF after irradiation in C57BL/ 6 mice transplanted with bone marrow from DBA/2 and BALB/c donors resulted in endogenous reconstitution. However, administration of MRF before (preconditioning) and again after irradiation resulted in survival of the majority of mice. These C57BL/6 mice were chimeras of DBA/Z or BALB/c marrow but showed no sign of secondary disease. Thus the use of MRF abrogates resistance to and promotes engraftment of foreign marrow and enduring chimerism when the recipients (F, hybrids) appear to be nonreactive to the donor (parental marrow) and also when alloreactivity is bidirectional (allogeneic combinations).
INTRODUCTION In the course of recent studies on transplantation of allogeneic (homologous) bone marrow (BMT) into lethally irradiated mice, we found that the administration of viable bone marrow cells to recipients just prior to lethal, total-body irradiation (TBI) resulted in protection against radiation injury, facilitation of marrow engraftment, and induction of a persisting xenogenic (rat) and allogeneic chimerism in mice. The operational procedure preceding TBI was designated “preconditioning” since this maneuver profoundly shifted the manner in which the host reacted to the engrafting increment of allogeneic marrow (14). Utilizing the rat to mouse model of marrow transplantation it was established that the critical factors for success included the number of viable bone marrow cells given before and after TBI, the timing of these injections, and the route of inoculation; each of these had to be correctly poised to assure avoidance of secondary disease, the development of chimerism, and long-term survival (2-4). It soon became evident that the overall protection by the marrow given prior to TBI was not at all related to the immune specificity of the donor (24). The fact that the preparatory inoculation of bone marrow was effective when given shortly before TBI and that administration via ’ Present address and requests for reprints: Institute for Integrative Biomedical Research, Lohwisstrasse 50, 8 I23 Ebmatingen, Switzerland. 219 OOOS-8749/81/010219-10$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved
220
PIERPAOLI,
MAESTRONI,
AND
SACHE
the systemic route resulted in a more pronounced protection (2) led progressively to the interpretation that the viable bone marrow cells were exerting this markedly favorable effect via synthesis and/or secretion, in the host, of components facilitating engraftment of the transplanted, incompatible marrow after lethal TBI. The implications of such an interpretation prompted further studies on the influence of a pre- and postconditioning regimen in xenogeneic (rat to mouse) and allogeneic BMT where soluble products of bone marrow cells or bone marrow cells suspended in their original suspension medium (supernatant, SN) were inoculated before or after TBI (Maestroni and Pierpaoli, submitted for publication). This report communicates a series of further experiments which led to the finding that such active substances were indeed present in, or elaborated by, marrow and to some of their properties in vitro and in vivo. The preliminary data reported here attest to their essential role in achieving successful engraftment of allogeneic marrow and enduring chimerism in mice. MATERIALS
AND METHODS
Animals The animals used in this investigation were generously provided by the Animal Farm of Hoffman-La Roche & Company, Ftillinsdorf, Switzerland. The mice were bred under specific pathogen-free conditions and then maintained, as adults, under strictly standardized and controlled hygienic conditions in the Central Biological Laboratory of the Cantonal Hospital, Zurich. However, no special precautions were taken to avoid pathogenic bacteria or viruses. No antibiotics were administered via drinking water either before or after irradiation. Donors or recipients of bone marrow were inbred young adult (8-12 weeks old) C57BL/6, DBA/2, BALB/ c, and C57BL/6 X A/J F, hybrid mice. The rabbits used were an outbred Swiss strain, 1.5 to 2 kg body wt. They were used as donors of xenogeneic marrow for preparation of supernatant (SN) from which the marrow regulating factors (MRF) were separated. Preparation of Rabbit Bone Marrow Supernatants (SN) for Separation of Marrow Regulating Factors (MRF) Groups of 10, male or female, rabbits were killed by cervical dislocation. Care was taken to use young adult donors. The long bones were isolated and put into refrigerated TC 199 or saline (NaCl 0.9%). The bones were then cut at the extremities and TC 199 or saline was flushed repeatedly through the bones to expel all the marrow, including fat tissue and connective tissue and all that make up the “milieu interieur” of the bone marrow. The material (clots of marrow, fat, other tissues) was then dissociated by using a large syringe without needle, delicately aspirating and expelling the suspended material which was always kept in melting ice, until no cell aggregates were visible. The number of cells in the suspensions ranged between 50 and 100 X 106/ml of SN. At this stage, the whole initial suspension of material was centrifuged in a refrigerated centrifuge at 5°C for 30 min at 10,OOOgand the fat condensed at the top of the tubes was eliminated by aspiration. The rest of the supernatant was collected in plastic containers and frozen immediately at -30°C. The cells were discarded. All this preparatory work was carried out under sterile conditions. SN is intended to be the original suspension
MARROW
FACTORS
PROMOTE
MARROW
ENGRAFTMENT
221
medium in which the bone marrow cells were dissociated and suspended. The pH of all cell suspensions and of preparations of SN was kept or corrected at 7.2 to 7.5. Preparation
of MRF from Rabbit Bone Marrow
Supernatant
(SN)
After thawing the SN, it was centrifuged for 20 min at 40,000 g in a refrigerated centrifuge to remove all possible particles and precipitate. The SN was then passed over a Diaflo XM 100 filter (MW > 100,000; Amicon Co., Oosterhout, Holland) using an Amicon Model 202 cell. An equal volume of water was used to wash the material which did not pass through the filter. The material remaining on the filter was withdrawn with a syringe and lyophilized. Preparation
of Bone Marrow
Cell Suspensions
Suspensionsof bone marrow cells were freshly prepared 2-3 hr before inoculation. Mice were killed by cervical dislocation; the long bones (humeri, tibias, and femurs) were isolated and cut at the extremities. Ice-cold TC 199 medium was flushed repeatedly through the cavities by syringe with a needle fitting the bone size. The pooled marrow was gently dispersed by a needleless syringe and filtered through gauze. The cells were then washed two to three fold by low-speed centrifugation, using ice-cold TC 199 medium. The final cell suspensions were adjusted to the desired number and volume. Quantities varying from 20 to 40x 1O6cells per donor mouse could be harvested. The cells were administered in a volume of 0.5 ml per mouse, iv. Trypan blue exclusion tests showed that over 95% of the cells were viable just before their inoculation. Irradiation
A dose of 850 to 900 rad total-body irradiation (TBI) was given to the recipients depending on the known strain sensitivity to irradiation (5). This dose led to death of all unprotected mice within 8-15 days. The irradiation apparatus was a Cobalt Gammatron 3 (6000 Ci). Field size was 30x30 cm, main focus distance was 90 cm. No filters were used. Transplantation
of Allogeneic
Marrow
(a) Preconditioning regimen. When this regimen was adopted the mice were injected iv 1.5 to 2.0 hr before irradiation with 1.0, 2.5, or 5 mg of rabbit MRF. The lyophilized material (fraction with MW > 100,000) was dissolved in TC 199. The volume injected was 0.5 ml. Controls were injected with cells or medium without MRF. (b) Postconditioning regimen. The mice were injected 24 hr after TBI with 30 to 37 x lo6 washed bone marrow cells. Depending on the experimental plan the cells were injected alone or suspended in the medium containing the fractions of the MRF. The cells were resuspended in the medium (TC 199, pH 7.4) containing MRF shortly before iv inoculation. Tests for Chimerism
(a) Hemoglobin migration pattern. At monthly intervals after TBI, all mice were individually tested for chimerism of the erythroid cell line. A few drops of blood
222
PIERPAOLI,
MAESTRONI,
AND
SACHE
were taken from the retroorbital plexus, suspended in heparinized saline, and washed repeatedly. The washed blood cells were hemolyzed in 15 cell-water volume. The pattern of hemoglobin migration was examined by cellulose-acetate electrophoresis. The strips were stained with amido black. Engraftment of the donor eythropoietic line corresponded to complete chimerism as evidenced by skin grafting (4). Passively transferred, donor-type red cells were also found in the peripheral blood of nonchimeric mice. They persisted as long as 3 months when donors were BALB/c mice and 2 months when donors were DBA/2 mice. (b) Skin grafts. In the situation of cross-transplantation of bone marrow from or into C57BL/6 and DBA/2 or BALB/c donor-recipient, donor skin was grafted on groups of allogeneic marrow recipients. Grafting was by conventional technique; the corsets were removed after 8-10 days and the viability of the grafts was checked daily. The graft was considered as rejected when the first signs of infiltration, edema, and induration appeared. They were considered as accepted only when none of these signs were discernible and luxuriant hair grew on the transplanted skin. Incorporation of [3H]Thymidine by Bone Marrow Cells in Vitro 6-[3H]Thymidine (27 Ci/mol) was purchased from the Radiochemical Center, Amersham, England. Washed (once) bone marrow cells freshly taken from adult C57BL/6 mice were incubated for 1, 2, and 3 hr in sealed tubes in the presence of 2 PCi of [3H]thymidine. Samples were in triplicate. Each tube contained 2 X lo6 washed bone marrow cells suspended in 1 ml of medium TC 199, or in TC 199 with 200 pg of the ultrafiltration fractions of the original supernatant of rabbit bone marrow cell suspensions (see above preparation of MRF). The pH of the medium was adjusted to 7.5. After 1, 2, and 3 hr of incubation, 0.1 ml of 10% sodium dodecyl sulfate (SDS) was added to each tube. After a vigorous shaking, DNA was precipitated at 4°C for 15 min by adding to each tube 1 ml of 10% trichloroacetic acid (TCA). The precipitate was collected on Whatmann GF/C glass fiber filters and washed with 5% TCA and absolute ethanol. The filters were dried and the activity was measured in a LKB-Wallac 81000 liquid scintillation counter. RESULTS Effect of Ultrafiltration Fractions of Bone Marrow Supernatants on Incorporation of [ 3H]Thymidine by Bone Marrow Cells in Vitro As a preliminary screening test, the effect of different fractions of rabbit bone marrow supernatants obtained by Amicon Diaflo membrane ultrafiltration was tested on the incorporation of [ 3H]thymidine by suspensionsof mouse bone marrow cells in vitro. Table 1 shows the results of one experiment which were fully confirmed by repeated tests. Apparently, only the ultrafiltration fraction with MW > 100,000 stimulated constantly the mitotic activity and/or replication by bone marrow cells. The other fractions (MW < 100,000 and MW > 30,000) did not constantly produce an inhibition or stimulation of the incorporation. The fraction of MW < 30,000 often showed a clear inhibiting action (Table 1). The same fraction of MW > 100,000, when inoculated in vivo, also increased by 50% incorporation of [)H]thymidine at 1 and 2 hr after inoculation (Maestroni and Pierpaoli,
I
62,392 + 5909
32,420 + 4705 35,049 + 5626
31,185 k 4311 18,008 k 1152
2841
35,064 f
23,327 f 2814 20,265 f 4294
1 2
3
4 5
126,631 37,868 98,586 49,918 65,291
f + + 2 + The incubation
7842 4478 2901 6752 8203
3 hr (cpm f SD)
Note. Donors of bone marrow (BM) cells were C57BL/6 mice (2 X lo6 BM cells/ml). was added to each tube. Samples were in triplicate. SD = standard deviation.
73,180 f 6972 19,347 + 2059
1 hr (cpm f SD)
2 hr (cpm k SD)
Incubation
200 200 200 200 IO6 X lo6 BM cells in TC
X lo6 BM cells + X IO* BM cells + X lo6 BM cells + BM cells + X
pg Fraction pg Fraction rg Fraction rg Fraction 199
ml [‘Hlthymidine
> 100,000 < 30,000 > 30,000 < 100,000
Cells
MW MW MW MW
of samples
by Bone Marrow
Composition
Incorporation
medium was TC 199, pH 7.5. 2 &i/O.1
2 2 2 2 2
Fraction of MW > 100,000 from Bone Marrow Supernatant Stimulates [‘H]Thymidine in Vitro. A fraction of MW < 30,000 Inhibits Incorporation
Code
An Ultrafiltration
TABLE
-1
%
2
8
E
%
3
z
2
3 iz
b
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PIERPAOLI,
MAESTRONI,
AND
SACHE
unpublished). Thus, this fraction of the ultrafiltrates with MW > 100,000 was chosen for testing in experiments in vivo on BMT. It was called marrow regulating factor (MRF). Effect of MRF on Allogeneic
Engraftment
and Chimerism
(a) F, Hybrids injected with parental bone marrow. The in vitro results shown above on activity of MRF and the previous findings on induction of chimerism in C57BL/6 X A/J F, hybrids transplanted with parental marrow suspended in its original SN (Maestroni and Pierpaoli, submitted for publication) suggested the use of this well-known model. C57BL/6 X A/J F, hybrid mice were transplanted with washed bone marrow cells from C57BL/6 mice; the cells were suspended in a solution containing MRF (fraction of MW > 100,000). As can be seen in Table 2, when the hybrid recipients were injected with parental bone marrow cells without MRF, only some of the animals survived thanks to the reconstitution of the endogenous marrow, but no chimerism was achieved. On the contrary, all the surviving hybrids inoculated with parental bone marrow suspended in a medium containing the fraction of MW > 100,000 were permanent chimeras. Apparently, the MRF fraction with MW > 100,000 facilitated or enhanced the engraftment of the parental marrow when injected after TBI (postconditioning) in a fashion identical to that shown previously in the same model by using marrow supernatants (Maestroni and Pierpaoli, submitted for publication). (b) Allogeneic donor-recipient strains. As shown previously, the inoculation of bone marrow cells with or without their supernatants before TBI (preconditioning regimen) facilitated the engraftment of xenogeneic marrow in mice or facilitated the endogenous reconstitution in particularly resistant recipients. However, no chimerism was obtained when the donors and the recipient mice were fully allogeneic (Maestroni and Pierpaoli, submitted for publication). A modified preconditioning regimen has now been adopted using as recipients C57BL/6 mice, which are known to be particularly resistant to allogeneic and xenogeneic engraftment (6). Donors were DBA/2 and BALB/c mice. The timing of administration before TBI was TABLE Marrow
2
Regulating Factor (MRF) Promotes Enduring Chimerism in Hybrid Recipient Inoculated with Parental Bone Marrow after Lethal Irradiation
Mice
Postconditioning
Animals
Group
No.
TBI (rad)
h+
Product
No. BM cells (X 106)
Route
Survival at 6 months
Chimerism
A B
20 40
900 900
24 24
3 mg MRF -
35 35
iv iv
18 (90%) 8 (20%)
18 (100%) 0 (0%)
Note. TBI = total-body irradiation. MRF = marrow regulating factor; Amicon Diaflo membrane ultrafiltration fraction (MW > 100,000) of supernatant of rabbit bone marrow cells. Donors of bone marrow were adult C57BL/6 mice, recipients were F, hybrid, 3- to 4-month-old C57BL/6 X A/J female mice. The parental bone marrow cells were washed once and then suspended in TC 199 containing the MRF immediately before inoculation. The cells were inoculated 24 hr after TBI. Chimerism was assessed by individual hemoglobin typing in each surviving recipient hybrid mouse (4). The results above derive from two identical experiments.
3
iv iv iv iv iv iv
Route 1.5 1.5 1.5 1.5 1.5 1.5 850 850 850 850 850 850
TBI dose bad) 24 24 24 24 24 24
h’ 3 3 3 3 3 3
mg mg mg mg mg mg MRF MRF MRF MRF MRF MRF
Product 37 31 31 31 30 30
No. of BM cells (X 106)
Postconditioning
iv iv iv iv iv iv
Route
6 5 4 3 9 10
(60%) (50%) (40%) (10%) (90%) (50%)
Survival (6 months after TBI)
with Allogeneic
Nore. TBI = total-body irradiation. MRF = marrow regulating factors; Amicon Diaflo membrane ultrafiltration fraction (MW > 100.000) of supernatant bone marrow cells. Donors of bone marrow were adult, 3-month-old DBA/2 and BALB/c female and male mice, recipients were 3- to 4-month-old male mice. MRF or TC 199 was injected 1.5 hr before TBI (h-). The allogeneic bone marrow cells were washed once and then suspended in TC 199 containing immediately before inoculation. The cells were inoculated 24 hr (h’) after TBI. Chimerism was assessed by hemoglobin typing and skin grafting of the surviving recipients (4). For the cause of mortality in groups A, B, C, and F, see Results section. The results above derive from four different experiments and D, two experiments; E and F, two experiments).
mg MRF mg MRF mg MRF 199 199 mg MRF
A B C D E F
5.0 2.5 1.0 TC TC 2.5
DBA/2 DBA/Z DBA/2 DBA/2 BALB/c BALB/c
Group
10 10 10 30 10 20
Product
Donor strain
No. of recipients
Preconditioning
with Marrow Regulating Factors (MRF) Results in Enduring Chimerism of the surviving C57BL/6 Mice Transferred Marrow. MRF Given Only in PostConditioning Results in Endogenous Reconstitution of the Surviving mice.
Animals
Preconditioning
TABLE
(100%) (100%) (100%) (0%) (0%) (100%) of rabbit C57BL/6 the MRF individual (A, B, C,
6 5 4 0 0 IO
Chimerism
Bone
j;r
F 3$
E 22 G 3
5
5 2
2 0
a E
2 2
2
226
PIERPAOLI,
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AND
SACHE
chosen on the basis of the stimulatory effects of MRF on BM cells observed in vivo (unpublished) and in vitro (Table 1). As can be seen in Table 3 (groups A, B, C, and F), preconditioning of the recipients with 1.0, 2.5, and 5.0 mg of MRF induced survival and permanent chimerism in a large number of recipients of this extremely alloreactive strain combination, while the lack of it in the preconditioning and its presence in the postconditioning (after TBI) eventually promoted endogenous bone marrow reconstitution but no chimerism of some recipients (Table 3, groups D and E). Chimerism has persisted and no signs of secondary disease have been observed to the present (6 months after marrow transplantation). All the chimeric C57BL/6 mice accepted permanently the skin grafts from DBA/Z or BALB/c donors. Chimerism was specific and immune reconstitution was complete since the chimeric mice rejected, within lo-12 days, skin grafts from strains different at the main H-2 locus (C3H, CBA). The mortality in groups A, B, C, and F could be attributed to several causes such as infection, deficient engraftment, secondary disease, and others. DISCUSSION There have been many attempts to achieve a persisting hematopoietic and immune reconstitution via transplantation of allogeneic bone marrow, but this has proved to be an insurmountable goal despite intensive efforts (7). This report describes an alternative modality for transferring bone marrow between genetically different individuals of the same species. It is based on the use of recently identified marrow regulating factors (MRF) which appear to be intimately linked to marrow function; of primary importance for engraftment and for well-balanced marrow proliferation and maintenance of hematopoiesis. A number of investigators have explored the presence in marrow of inhibitors of marrow proliferation (8-l 0). Such components could certainly play a part in regulating marrow function and possibly account for its rejection. However, to the best of our knowledge components that facilitate marrow proliferation and its engraftment have not previously been reported. It is evident that these substances would be of importance in determining the outcome of bone marrow transplantation. Without in any way implying a lesser relevance of the host-donor immunogenetic compatibility for successful transplantation of bone marrow, it may be useful to analyze and interpret the facts emerging from our own experimental model and the new directions they provide. Apparently, the rapid initiation and progression of a well-balanced hematopoiesis in the irradiated host is a prerequisite for maintenance of the grafted marrow and for enduring chimerism with avoidance of early or late symptoms of GVHR. In fact, the model in which the Fr hybrids were transplanted with marrow from the parental strain demonstrates unequivocally that no reaction of the donor cells against the host ensues when MRF is administered together with the parental marrow (Table 2). Also, in the extreme situation, i.e., the most incompatible model of C57BL/6 mice transplanted with DBA/2 or BALB/c marrow, the apparent enduring eradication of the resistance to engraftment by the preirradiation treatment with MRF changes completely the normal inability of the host to accept this donor marrow. Conversely, the donor marrow rapidly colonizes the host taking care that no “aggression” reaction will initiate (Table 3, compare groups A, B, C, and F to groups D and E). Thus, it seems to
227
MARROW FACTORS PROMOTE MARROW ENGRAFTMENT
Q +@
A)
TEMPORARY
ENDOGENOUS
Cl
OR PIRTIAL
RECONSTITUTION
CHIMERISM;
G~.C57l3+$
MRF
FIG. 1. Marrow regulating factors (MRF) promote engraftment and enduring chimerism in lethally irradiated mice transplanted with allogeneic bone marrow: (A) Allogeneic bone marrow transplantation, as generally performed, involves the inability of the foreign marrow to colonize the host combined with host resistance to engraftment. This combination generates a conflicting situation in which immune reactions also add their negative effects, resulting in deficient or abnormal hematopoiesis. The result is acute or chronic secondary disease, immunodeficiency, runting, infection, and the varied disorders connected with a disturbed hematopoiesis (lymphopenia, agranulocytosis, thrombocytopenia, anemia, etc.). Acute or chronic alterations of liver, gut, and skin also appear variably. (B) Administration of MRF after TBI will lead to either endogenous reconstitution or chimerism, depending on the immunogenetic character of the donor-recipient combination. Endogenous reconstitution takes place when high resistance to engraftment is present (e.g., C57BL/6 mice, see Table 3), whereas chimerism is achieved when no host resistance is offered (e.g., C57BL/6 X A/J F, hybrids transplanted with parental marrow, see Table 2). (C) When MRF is given before as well as after TBI, resistance to engraftment is abrogated and the donor marrow colonizes the host (e.g., C57BL/6 mice transplanted with DBA/2 marrow, see Table 3). This system permits induction of chimerism when alloreactivity is bidirectional.
us evident that a rapid and efficient hematopoietic functionality of the grafted marrow is perhaps the essential requirement for avoidance of GVHR or HVGR. Improvement of the transplantation technique is needed to reduce or eliminate the mortality of the allogeneic chimeras (Table 3). There are indications that some further shifts in dosage and timing of administration of MRF in the pre- and postconditioning will effect a reduction in the death rate of allogeneic chimeras even under these conventional conditions. Our present and necessarily preliminary interpretation of the phenomena connected with the use of MRF in BMT is illustrated in Fig. 1. It is considered possible that pretreatment of the donor with MRF before TBI renders the recipient’s marrow more sensitive to irradiation, thus making for a more complete elimination of any minority or subpopulation of radioresistant marrow cells. In fact, we have repeatedly observed that when MRF is administered after TBI in the highly resistant strains, it facilitates endogenous reconstitution; there then ensues rejection of the foreign marrow. A conflicting situation pertains when neither host nor donor cells prevail; in this situation secondary disease arises. It is at this stage that immunological phenomena and reactions are probably further deviating hematopoiesis and superimposing themselves on the original dysfunction of certain hematopoietic cell lines.
228
PIERPAOLI, MAESTRONI,
AND SACHE
Whatever the mechanism proves to be, the preliminary results given here and the large variety of experiments in course point the way to the reality of allogeneic marrow transplantation with immune and hematopoietic reconstitution. The physiological and hematological investigation of bone marrow factors and their functions clearly hold promise for achieving a safer reconstitution, especially for its farranging potential in therapeutic application. ACKNOWLEDGMENTS We are deeply grateful to Dr. Maurice Landy for his constant interest, advice, and encouragement and for the improvement of the manuscript. We are indebted to Dr. Wolf Weihe for the maintenance of the animals used and to Miss Petra Stijckel for excellent technical assistance. We thank Mrs. Michelle Kunz of Institute Choay for technical help in the preparation of MRF. We are indebted to Professor Gustav0 Cudkowicz for his comments and criticism. This work has been mainly supported with a research grant from Institute Choay, Paris. Further financial support was received from the Swiss National Science Foundation, Bern (Grant 3.251-0.77), from the Cilag-Chemie Foundation for therapeutical research, Schaffhausen, and from the Piombino Foundation for Life Sciences, Populonia, Livorno, Italy.
REFERENCES 1. Pierpaoli, W., and Maestroni, G. J. M., Transplantation 26, 456, 1978. 2. Pierpaoli, W., and Maestroni, G. J. M., Stand. J. Huemotol. 22, 165, 1979. 3. Pierpaoli, W., and Maestroni, G. J. M., J. Lab. Clin. Immunol. 2, 125, 1979. 4. Pierpaoli, W., and Maestroni, G. J. M., Cell. Immunol. 52, 62, 1980. 5. Green, E. L., (Ed.), “Biology of the Laboratory Mouse,” Chap. 22. McGraw-Hill, New York, 1966. 6. Trentin, J. J., Gallagher, M. T., and Lotzova, E., In “Immunobiology of Bone Marrow Transplantation” (B. DuPont and R. A. Good, Eds.), pp. 137-142. Grune & Stratton, New York, 1976. 7. Cudkowicz, G., Landy, M., and Shearer, G. (Eds.), “Natural Resistance Systems against Foreign Cells, Tumors and Microbes.” Academic Press, New York, 1978. 8. Cline, M. J., Herman, S. P., and Golde, D. W., Transplunt. Proc. 10, 99, 1978. 9. Benestad, H. B., Testa, N. G., and Lajtha, L. G., Stand. J. Huematol. 20, 18, 1978. 10. Lajtha, L. G., Nouv. Rev. Fr. Hematol. 21, 59, 1979.