Dominance and persistence of donor marrow in long-lived allogeneic radiation chimeras obtained with unmanipulated bone marrow

Immunology Letters, 6 (1983) 197-202 Elsevier Biomedical Press

DOMINANCE AND PERSISTENCE OF DONOR MARROW IN LONG-LIVED ALLOGENEIC RADIATION CHIMERAS OBTAINED WITH UNMANIPULATED BONE MARROW Walter PIERPAOLI and Georges J. M. MAESTRONI* lnstitute Jbr Integrative Biomedical Research, Lohwisstrasse 50, 8123 Ebmatingen, Switzerland (Received 17 November 1982) (Modified version received and accepted 7 February 1983)

I. Summary Allogeneic, H-2-incompatible irradiation chimeras (H-2 d -- H-2 b) constructed with normal, unmanipulated bone marrow and with marrow-derived factors live long and do not manifest a GvH disease. Their response to primary immunization is deficient but their alloreactivity is normal. This chimeric allotolerance cannot be passively transferred from chimeric donors to normal irradiated recipients. Passive transfer of both donor- or recipient-type immunocompetent T-cells into the chimeric mice does not lead to syngeneic reconstitution, rejection of the engrafted marrow or GvH disease and the mice maintain permanently their chimerism. This new model demonstrates that chimerism is not eradicable in long-lived chimeras reconstituted with unmanipulated bone marrow, and that the bone marrow itself plays a dominant role in maintenance of chimerism.

2. Introduction The significance of therapeutic approaches for adoptive immunotherapy by allogeneic bone marrow transplantation (BMT) stems from acquisition by radiation chimeras of donor immunocompetence. * Present address: lstituto Cantonale di Patologia, 6604 LocarnoSolduno, Switzerland

Key words: allogeneic chimeras - bone marrow

transplanta-

tion 0165-2478/83/0000-0000/$3.00 © 1983 Elsevier Science Publishers B.V.

Recent studies on allogeneic bone marrow chimeras are suggestive of basic restrictions of cellular interactions and a persisting deficiency in immunological functions in those animals [1~,]. Our methodology for BMT diverges radically from prior work inasmuch as fully allogeneic and clinical graft versus host disease (GVHD)-free chimerism has heretofore been limited to special situations in which the recipients of allogeneic marrow had either been raised and maintained under germfree conditions [5], or the cellular composition of the bone marrow to be transplanted had been manipulated so as to remove or inactivate cytotoxic T-cells [6 19]. From our studies on BMT there has evolved a system which makes for induction of enduring engraftment of fully allogeneic, H-2-incompatible marrow in mice and the establishment of GVHD-free hemopoietic chimeras which survive as long as syngeneically reconstituted mice. This system is based on the concept that the problem of BMT is more hematological than immunogenetical in character and that maintenance of the integrity of cellular composition of donor marrow (including cytotoxic T-cells) and of its internal and still largely unexplored regulatory microenvironmental components is a sine qua non for harmonious engraftment of allogeneic marrow, eradication of resistance in recipients and avoidance of G V H D [20-22]. The sheer continued existence of these chimeric animals brings several issues to the fore; perhaps the most important relate to the functional aspects of the chimeric identity-defence immune system, and the character 197

of the tolerance underlying their hemopoietic chimerism. S o m e of these aspects have been here investigated.

3. Methods and results

3.1. Mice A d u l t (3~,-months-old), inbred C 5 7 B L / 6 , D B A / 2 , B A L B / c and C 3 H / H e male and female mice bred and maintained in our animal r o o m s were used. The mice were kept u n d e r conventional conditions and no antibiotics were given in the course of the experiments.

the m e m b r a n e was washed with an equal v o l u m e of water, collected and lyophilized. The whole procedure of M R F preparation was carried out under sterile conditions and at + 4 ° C. All M R F preparations were checked for e n d o t o x i n c o n t a m i n a t i o n by the Limulus test. Only e n d o t o x i n - f r e e M R F was used, containing less t h a n 1 ng e n d o t o x i n / m g . M R F was dissolved in the same m e d i u m containing the bone m a r r o w cells to be transplanted shortly before injection and both M R F and allogeneic cells were inoculated over 24 h after irradiation by intravenous route t h r o u g h the retro-orbital plexus under ether anesthesia. 3.3. Immune response of long-lived allogeneic chi-

3.2. Construction of allogeneie chimeras The system is illustrated in Table 1 and details of it have been reported [20 22]. The so-called m a r r o w regulating factors ( M R F ) were prepared as follows: bone m a r r o w was extracted f r o m the long bones of young, adult rabbits and suspended in cooled saline. T h e cells were dissociated by gentle shaking and the suspension was then centrifuged at high speed. The cells were discarded and the supernatant was ultrafiltrated t h o u g h A m i c o n Diaflo m e m b r a n e , 100,000 mol. wt. cut-off. The material remaining on

meras A l t h o u g h the allogeneic bone m a r r o w chimeras showed a considerably delayed and reduced primary i m m u n e response to sheep red blood cells ( S R B C ) and two different viruses, their reaction to a second antigenic challenge was significant, a l t h o u g h still less than the reduced response manifested by syngeneically reconstituted mice [4]. O n the other hand, it should be noted that even syngeneically reconstituted mice m o u n t a lesser a n t i b o d y response as c o m pared to n o r m a l animals [4].

Table 1 Marrow regulating factors ( M RF) promote engraftment of allogeneic, H-2-incompatible bone marrow and permanent allogeneic chimerism in lethally irradiated mice Group

No. of mice

A B C D

22 43 44 10

TB1 950 tad

Post-conditioning

Survival

Cbimerism (%)

15 20 × l06 BM cells i.v. -- 2 5 mg MRF/mouse 15 20× 106 BM cells i.v. 15 20× 106 BM cells i.v. + 3 mg kidney extract

0 31 (72%) 4(9%) 0

31 (72%) 4(9%)

Donors of BM were adult, DBA/2 (H-2d) mice. Recipients were adult, C57 BL/6 ( H-2b) mice. BM cells with or without M R F were injected over 24 h after TBI. Enduring and complete allogeneic bone marrow chimerism is achieved in lethally irradiated mice across major histocompatibility loci ( H-2d ~ H-2b). This is accomplished by a new approach involving time administration of'washed" bone marrow cells suspended in a solution of a physiological component of the bone marrow microenvironment, a recently identified bone marrow-derived ultrafiltration fraction designated 'marrow regulating factor', with non-species-specificactivity (M R F) [20-22]. Fully allogeneic chimerism is ascertained starting at 2 months after TBI and BMT by analyzing chimeric spleen cells for susceptibility to cytolysis by specific alloantisera in randomly selected mice, and at monthly intervals in individual animals by allogeneic skin grafting and by electrophoretic evaluation of hemoglobin migration pattern [20 22]. The animals are then maintained in a conventional environment. Their life span approximates that of irradiated and syngeneically reconstituted mice (over 2 years). All the animals used in the tests reported in this communication were perfectly healthy, with no clinical symptoms of chronic graft versus host disease (GV H D). Onset of spontaneous tumors and other anomalies were not encountered. They showed a progressive greying of their fur as a consequence of irradiation; subsequently hair growth was healthy. In contrast, the fur of irradiated mice suffering of subacute or chronic GVHD did not grey and runting animals maintained their original fur colour as long as they survived (4 -6 months after TBI). 198

The same mice utilized for the evaluation of humoral immunity were grafted with allogeneic skin. Without exception, these marrow chimeras (H-2 d marrow in H-2 b mice) all retained, indefinitely, large skin grafts from mice of the same strain as the marrow donors (H-2d), while rejecting within 10 14 days unrelated allogeneic (H-2 k) skin grafts. The rejection time for the allogeneic grafts in the irradiated and syngeneically reconstituted, and in the normal control mice, was the same 10 14 days [4]. The results are in harmony with those of many others [1,14 19] and affirm that bone marrow radiation chimeras constructed with non-manipulated bone marrow display a slowly corrected, deficient or retarded response to primary and secondary sensitization with thymus-dependent antigens. The vigor and magnitude of allograft reactivity in the chimeric animals is, however, fully retained. Chimerism is consistently allospecific and persists [4]. 3.4. Passive transfer of allo-tolerance by B M T from chimeric mice to irradiated normal host The character of the tolerance underlying chimerism is quite unknown. In a study of allogeneic bone marrow chimeras, Ono6 et al. [14] attributed the tolerance of chimeric mice to a clonal deletion mechanism. We have been unable to confirm this report. On the contrary, the marrow in our chimeras seems to possess intact donor character and cell composition, T-cells included. In fact, in our transfer experiments [23] neither bone marrow nor spleen cells from fully allogeneic chimeras (H-2d in H-2 b) passively induced enduring chimerism in lethally irradiated mice syngeneic (H-2 b) to the chimeric host. O n the other hand, the same bone marrow and/or spleen cells from the allogeneic chimeras fully reconstituted lethally irradiated mice syngeneic (H-2d) to the chimeric hemopoietic system. Furthermore, these 'syngeneically' reconstituted mice did not acquire any tolerance to the chimeric host allo-antigens as would have been expected if the chimeric cells had been 'educated' to be specifically unresponsive. In fact, when transplanted with skin from mice of the same strain of the chimeric recipients (C57BL/6) these chimeras-transferred 'syngeneically' reconstituted mice promptly rejected the skin graft [23].

3.5. Passive transfer of immunocompetent cells in long-#ved chimeras We sought to determine whether the so-called recipient-thymus-dependent H-2 restriction of donor T-cells conditions the tolerogenic mechanism. The experimental answer to this question was sought by passive transfer of immunocompetent spleen cells from normal mice of donor, recipient arid a tlaird, unrelated, allogeneic strain, to the allogeneic marrow chimeras. Groups of C57BL/6 mice, hemopoietically chimeric with BALB/c, were inoculated i.v. with a large number of BALB/c, C57BL/6 or C 3 H / H e spleen cells. The experiment is illustrated in Fig. 1. The rationale was that if the thymus-directed H-2 restriction were of relevance for maintenance of the chimeric state, inoculation of donor, mature immunocompetent cells (spleen cells) into the allogeneic chimeras would interfere with the tolerogenic mechanism by inducing a GVH-type reaction or syngeneic reconstitution depending on the H-2 type of the inoculated cells. In fact, however, the contrary result was obtained. No GVH reaction, nor syngeneic reconstitution, nor hemopoietic graft rejection occurred. The chimeric mice remained healthy and maintained their chimerism thus providing unequivocal evidence that the chimeric tolerance is persistent, specific, and is determined by the bone marrow itself (Fig. l). Furthermore, when bone marrow from the mice of group B (Fig. 1), which carry 50% of BALB/c nucleated cells in their spleen, was transferred into lethally irradiated BALB/c and C57BL/6 new hosts, the BALB/c recipients survived and were syngeneically reconstituted while all the C57BL/6 succumbed (Table 2). This result shows that the decreased degree of chimerism of the chimeric C57BL/6 donors was confined to the spleen, but did not involve the bone marrow. Apparently the bone marrow of the C57BL/6 chimeras remained fully donor type (BALB/c) and free of hemopoietic and immunocompetent cells (cytotoxic T-cells) of C57BL/6 H-2 type. Consequently the irradiated BALB/c recipients were protected and fully reconstituted, while the new C57BL/6 recipients died of G V H D (Table 2). Most interesting also is the finding that the impaired or delayed reactivity to primary sensitization persists in these radiation chimeras which had been challenged with a large inoculum of donor- or recip199

Long-lived, GvI-ID-free, bone marrow

BAL.B/c TB I ~

8xlO7BALB/c

]~ M+I:IF

30days

s~3~ells -8.107C57B~

I~ ~

P e r c e n t of BALB/c, I-I-2d-type spleen cells

--

~98~9~° (A) 5o ~0 (B)

8xl O7C31~e

P r i m a r y response to T-dependent antigens I//a.t.±s.e. S R B C (n) BA(n)

S k i n transplantation Donors C3/-//H e

BALB/c

rejected

accepted

A

6+-2 IIO)

13*-4(10)

B

4+-2 (10)

6"-3(10~ rejected

accepted

C

4±3 (10)

rejected

accepted

CO 61±7(15)

40~-9(I0)

groups of healthy, individually checked and established chimeras (10 mice each group) were injected i.v. with 8 0 x 106 nucleated spleen cells from normal BALB/c (H-2d), C57BL/6 (H-2 ~) or C3H/He (H-2 k) mice. Thirty days thereafter, the spleen cells of 4 mice from each group were analyzed for their H-2 chimerism by the capacity of allospecific anti-H-2 sera to lyze their splenic cells. Complete peripheralchimerism with donor-type H-2 lymphoid cells was obtained in the spleens of chimeric mice of group A and C where donors of the spleen cells had been normal BALB/c and C3H/He, whereas 50% or more of the nucleated spleen cells in group B (BALB/c-chimeric C57BL/6 mice) were of donor origin (C57BL/6). The chimeras were injected i.p. with 0.1 ml of a 20% suspension of sheep red blood cells (SRBC) or 0.2 ml of Brucella abortus Antigen (BA, Difco) and the agglutinins (against SRBC) or precipitins (against BA) were measured in serum 5 days later. The antibodies are expressed as the reciprocal of antibody titer+ S.E. The number of mice tested is given in brackets (n). The primary response to SRBC and BA in the 3 groups of chimeric mice which had been challenged with 3 different H-2 types of spleen cells showed that the inoculum of spleen cells, containing many mature and presumably immunocompetent cells, did not normalize the original inability of allo-chimeric mice to respond efficiently to primary immunization [4]. When grafted with skin from C3H/He or BALB/c mice, all 3 groups of mice showed complete maintenance of the original chimeric state. BALB/c skin was permanently accepted whereas skin from the unrelated H-2k donor (C3H/He) was promptly rejected (10 14 days). Bone marrow from these mice was then transferred into irradiated normal BALB/c and C57BL/6 mice (see Table 2).

Fig. I. Persistence of central, marrow chimerism in long-lived allogeneic chimeras challenged with spleen cells from 3 different H2 donors at 7 and 9 months after BMT. Permanent allogeneic chimeras across the H-2 barrier (BALB/c, H-2d to C57BL/6, H2 b) were obtained by the method described in Table 1 [20 22]. Seven to 9 months after BMT and induction of chimerism,

Table 2 Long-lived allogeneic chimeras passively transferred with donor- or recipient-type immunocompetent cells maintain their central marrow chimerism as shown by reconstitution of chimera-syngeneic, irradiated recipients. The chimeric bone marrow (BALB/c) is tolerant to its chimeric host (C57BL/6) but, when transferred to BALB/c mice, shows no tolerance to alloantigens of its original host (skin grafting)* Donors of bone marrow (see Fig. I)

No. of BM cells transplanted (× 106)

Normal irradiated recipients (no.) (950 tad)

Survival (3>3 months)

(A) BALB/c ~ C57BL chimeras transferred with BALB/c spleen cells

15 and 24 from (A)

BALB/c (10) C57BL/6 (10)

5 0

(B) BALB/c -- C57BL chimeras transferred with C57BL spleen cells

15 and 24 from (B)

BALB/c (10) C57BL/6 (10)

8 0

Skin graft* from C57BL/6

rejected (12 days)

The mice of groups (A) and (B) (see Fig. I) were used as donors of bone marrow 3 months after injection of the spleen cells. Results of 2 experiments. 200

ient-type immunocompetent T- and B-cells (Fig. 1). Thus, contrary to what would be expected [1 3] the thymus-dependent H-2 restriction of progenitor T-cells does not appear to be the principal consideration in accounting for the partial immunodeficiency of the allogeneic bone marrow chimeras. Presumably still other mechanisms are likely to be modulating the identity defence system of the chimeric animals.

4. Discussion

The immunocompetence of radiation chimeras has been explored previously [I, 14 19]. However, such allogeneic marrow chimeras h~tve almost invariably been rather short-lived and in some cases were reconstituted and thereafter maintained under germfree conditions [5]. Accordingly, these earlier findings are considered to have but limited applicability to the present situation. In fact, only perfectly healthy and GVHD-free chimeras are equipped to cope successfully with the microbial threats of a conventional environment. It is our belief that only such bone marrow chimeras can be considered as meaningful models for adoptive immunotherapy in man. The results reported here essentially affirm that chimeric mice manifesting no intercurrent infections and remaining healthy must be operationally immunocompetent despite their retarded, diminished primary response to thymus-dependent antigens. As to the nature of the tolerance of these chimeras, passive transfer of their spleen or bone marrow cells to new chimera-syngeneic, irradiated hosts yielded a clear-cut answer: allogenic lymphohemopoietic chimerism across the H-2 barrier is not passively transferable with the presently adopted methods. The recently investigated thymus-dependent H-2 restriction of crucial cellular interactions and immunological functions posed the issue: can such restrictions play a part in the maintenance of the chimeric tolerance? This possibility was tested simply by inoculating allogeneic bone marrow chimeras (BALB/c marrow in C57BL/6 mice) with large numbers of immunocompetent cells of donor (BALB/c), host (C57BL/6) and third party unrelated allogeneic (C3H/He) type (Fig. 1). The answer was unequivocal: the mice maintained their bone

marrow chimerism, including those inoculated with host-type immunocompetent cells. No H-2 restriction processes could account for maintenance of chimerism. On the other hand, the striking continuance of the specific impairment of the primary immune response in the spleen cell-transferred chimeras might well reflect one facet of this tolerance. That is to say, the chimeric state with the continuation of H-2 incompatibility, however harmonious, could well be responsible for this reduced responsiveness to immunization, as a secondary- or as a side-effect. Nonetheless, the secondary responsiveness attests to the maintenance of this capability. Allogeneic interaction in vivo has been shown to produce rapid hormonal changes in peripheral blood [24]. It could well be that an active humoral mechanism based on a changed central and/or peripheral neuroendocrine regulation operates in allogeneic chimerism by maintaining an altered hormonal state, analogous to the situation of the mother-fetus relationship and in an experimental model developed recently in which newborn mice were injected sequentially with different H-2-type spleen cells [25]. In some respects similar to allogeneic chimerism, pregnancy is in fact characterized by striking changes of immune reactions, including T-lymphocyte function and resistance to viruses [26]. However, whether the peculiar immune deficiency of allochimeras is dependent on and secondary to, persistent neuroendocrine changes remains to be determined. In any event neither the clonal deletion nor the more recent evidence of suppressor mechanisms would seem to provide an adequate explanation for this distinctive kind of tolerance. Allogeneic BM chimerism induced with our method is remarkably persistent. It is not transferable and not eradicable. It does not reverse to the original recipient-type character even by infusion of massive amounts of recipient-type, mature lymphohemopoietic cells containing lots of immunocompetent, cytotoxic T-cells (see Fig. I and Table 2). This demonstrates that the BM is central to self-identity and that, while difficult to establish, it will persist in the allogeneic host once hematopoietic reconstitution by the new marrow is complete. Essential to engraftment are the marrow microenvironmental factors (MRF) which, by a still unknown mechanism, influ201

ence hemopoiesis and prevent the initiation, or suppress GVHR. Thus appropriate intervention with intact BM and BM factors can lead to permanent change of the 'immune' identity of an individual and consequently donor marrow 'identity-defence' imprints enduringly the immune character in longlived, H-2-incompatible radiation chimeras [27].

Acknowledgements We thank Miss R. Kellerhals for technical assistance and Mrs. B. Stader for secretarial work. This work has been supported by a grant from Institute Choay, Paris, France, by NIH Grant RO-I CA29992-O2 and by a contribution from the Piombino Foundation for Life Sciences, Populonia, Livorno, Italy.

References [I] Zinkernagel, R. M, Althage, A., Callaghan, G. and Welsh, R. M. (1980)3. lmmunol. 124, 2356 2365. [2] Zinkernagel, R. M. (1978) lmmunol. Rev. 42, 224 270. [3] Zinkernagel, R. M. and Doherty, P. C. (1979) Adv. Immunol. 27, 51 177. [4] Maestroni, G. J. M., Pierpaoli, W. and Zinkernagel, R. M. (1982) Immunology 46, 253-260. [5] Truitt, R. L. and Pollard, M. (1976) Transplantation 21, 12 16. [6] Gengozian, N., Congdon, C. C., Allen, E. A. and Toya, R. E. (1971) Transpl. Proc. 3,434 442. [7] Urso, P. and Gengozian, N. (1974) J. lmmunol. 113, 1770-1779. [8] Nordin, A. A. and Farrar, J. J. (1974) Cell. lmmunol. I0, 218 225. [9] Trier, L. and Rubin, R. (1974) Acta Path. Microb. Scand. 82, 724 730.

202

[10] Gengozian, N. and Urso, P. (1976) Transpl. Proc. 8, 631 635. [11] Mattingly, J. A. and Webb, P. M. (1978) J. Immunol. 120, 1274 1277. [12] Reisner, Y., Itzicovich, L., Meshorer, A. and Sharon, N. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2933-2936. [13] Aizawa, S., Sado, T., Muto, M. and Kubo, E. (1981) J. Immunol. 127, 2426 2431. [14] Ono6, G., Fernandez, G. and Good, R. A. (1980) J. Exp. Med. 151, 115 132. [15] Von Boehmer, H., Hudson, L. and Sprent, J. (1975) J. Exp. Med. 142, 989 997. [16] Erb, P. B., Meier, B., Matsunaga, T. and Feldman, M. (1979) J. Exp. Med. 149, 686 701. [17] Katz, D. H., Katz, L. R., Bogowitz, C. A. and Skidmore, B. J. (1979) J. Exp. Med. 149, 1360 1370. [18] Wagner, H., Roellinghoff, M., Rodt, H. and Thierfelder, S. (1980) Eur. J. lmmunol. 10, 521 525. [19] Krown, S. E., Coico, R., Scheid, M. P., Fernandez, G. and Good, R. A. (1981) Clin. lmmunol. Immunopath. 19, 268 283. [20] Pierpaoli, W., Maestroni, G. J. M. and Sache, E. (1981) Cell. lmmunol. 57, 219 228. [21] Pierpaoli, W. and Maestroni, G. J. M. (1983) in: lmmunoregulation 81, Int. Workshop, Urbino, Italy (Fabris, N. ed.) Plenum Press, New York, in press. [22] Pierpaoli, W. and Maestroni, G. J. M. (1983) in: Organ Transplantation. Present State, Future Goals (Slavin, S. ed.) Elsevier/North-Holland, Amsterdam, in press. [23] Maestroni, G. J. M., Pierpaoli, W. and Zinkernagel, R. M. (1983) lmmunobiology, in press. [24] Pierpaoli, W. and Maestroni, G. J. M. (1977) Cell. lmmunol. 31,355 363. [25] Pierpaoli, W., Kopp, H. G., Mueller, J. and Keller, M. (1977) Cell. lmmunol. 29, 16 27. [26] Gusdon, J. P. (1976) in: Immunology of Human Reproduction (Scott, J. S. and Jones, W. R. eds.) pp. 103 125, Academic Press, London. [27] Pierpaoli, W. (198 I) in: Psychoneuroimmunology (Ader, R. ed.) pp. 575 606, Academic Press, New York.