Allogeneic H-2d→H-2b irradiation bone marrow chimeras: a) failure to transfer chimerism adoptively and b) immune reactivity of immunocompetent lymphocytes adoptively transferred to chimeras

Immunobiol., vol. 164, pp. 417-429 (1983)

Institute for Integrative Biomedical Research, Ebmatingen, Switzerland, and Institute of Pathology, Universitatsspital, Zurich, Switzerland

Allogeneic H-2d~H-2b Irradiation Bone Marrow Chimeras: a) Failure to Transfer Chimerism Adoptively and b) Immune Reactivity of Immunocompetent Lymphocytes Adoptively Transferred to Chimeras G. J. M. MAESTRONI\ W. PIERPAOLI, and R. M. ZINKERNAGEL Received November 18, 1982 . Accepted January 28,1983.

Abstract Long-lived, GVHD-free, H-2 incompatible haemopoietic chimeras (P1--->P2) were constructed transfusing unmanipulated bone marrow cells together with recently identified marrow-regulating factors (MRF) in lethally irradiated recipients. The chimeric tolerance of Pl--->P2 chimeras proved to be adoptively untransferable. Another peculiar property of established Pl--->P2 allochimeras was their ability to «suppress» or reject passively transfused immunocompetent P1 or P2 lymphocytes. Even this «suppression» appeared to be untransferable and to operate in the chimera only in a fashion dependent upon the age of the established chimeras. This chimeric «unidentified suppressive principle» seems not to follow familiar immunologic lines. A relationship with the mechanism of chimeric tolerance is suggested.

Introduction Allogeneic lymphohaemopoietic chimeras induced by irradiating homozygous PI mice and reconstituting them with homozygous H-2incompatible P2 bone marrow cells have been used to study differentiation of T lymphocytes (1-13). Results obtained, and particularly their interpretation, vary greatly. There seems to exist a discrepancy between a primary immune response compared with a secondary one of allogeneic chimeras, in that primary responses signal differences in levels of immunocompetence much better. The reasons for the more or less pronounced defects in primary immune responses of allo-chimeras are not fully understood. Also rather unclear is the process of thymic selection observed in lymphohaemopoietic chimeras (reviewed in 12, 14). Positive selection in the thymus plus postthymic amplification is one possible explanation, but " Present address: Istituto Cantonale di Patologia, Locarno-Solduno, Switzerland Abbreviations: GVHD = graft-versus-host disease; BM = bone marrow: SC = spleen cells; TBl = total body irradiation; BMT = bone marrow transplantation; MRF = marrow regulating factor; SRBC = sheep red blood cells; BA = Brucella abortus antigen; NK = natural killing.

418 . G.

J.

M. MAESTRONI, W. PIERPAOLI, and R. M. ZINKERNAGEL

suppressive mechanisms are still not fully excluded as yet. In allogeneic irradiation bone marrow chimeras, both mechanisms may play a role: Positive selection may be very limited, and postthymic amplification seems not possible because cell-surface H-2 antigen and restriction specificity expressed by T cells should not correspond (3); therefore, primary immune responses measured in vivo are often variable. Nevertheless, positive selection has been demonstrated when thymus cells from allo-chimeras were transferred adoptively (7). Suppression has been searched for, but has not been found so far (15,16), except for a few examples where immunocompetent lymphocytes have been transferred to chimeras in vivo (17, 18). Allochimeras H-2d~H-2b made with untreated bone marrow have been found to lack a measurable degree of primary antiviral T -cell-mediated immune protection (13). In this study, we tested these allochimeras, first, with respect to their lymphohaemopoietic cells' capacity to transfer chimerism to acutely irradiated normal recipients. We found that transfer of chimerism was not possible. Second, several groups of chimeras were transfused with immunocompetent lymphocytes of either parental origin to test whether they can express immunocompetence in this allochimeric environment. In many groups of chimeras, neither donor nor recipient-type spleen cells could express immunocompetence; in other groups of chimeras, donor-type spleen expressed immunocompetence only very early after transfer; in others, recipient-type spleen cells were capable of reversing chimerism, but without causing a graft-versus-host disease (GVHD).

Materials and Methods Animals Inbred male and female C57BL!6, BALB/c and DBA/2 adult mice, bred and maintained under conventional conditions in our animal quarters, were used. They were fed water and food ad libitum, and no vitamins or antibiotics were used in the course of the experiments. Generally, animals between 3 and 6 months of age were used as recipients of bone marrow because of the age-dependent sensitivity of resistance to irradiation.

Irradiation Depending on the known strain-sensitivity to irradiation, a supralethal dose of 900 to 1000 rad total-body irradiation (TBI) was administered. Internal controls were unprotected mice which died within 8-15 days. The irradiation apparatus was a Cobalt Gammatron 3 (6000 Curie). Field size was 30 X 30 cm, the main focus distance was 90 cm (13, 19-21).

Preparation of bone marrow (EM) or spleen cell (SC) suspensions In all experiments, BM or SC were freshly prepared from donor mice killed by cervical dislocation. BM was collected in cooled TC 199 medium from the long bones. The pooled marrow cells were gently dispersed by shaking, and filtered through gauze. Cells were then counted and the supernatant discarded after low-speed centrifugation. Cells were resuspended at the appropriate concentration in TC 199 medium (no serum added). Spleen cell suspensions were washed 3-4 times before use. Trypan blue viability was usually greater that 90 %. Other details are reported elsewhere (13, 19-21).

Allogeneic H-2d---'>H-2h Irradiation Bone Marrow Chimeras . 419

Preparation of marrow-regulating factor (MRF) Rabbit MRF was prepared as already described (20). Several batches were used.

Transplantation of allogeneic BM for induction of allochimerism Enduring and complete allogeneic bone marrow chimerism is achieved in lethally irradiated mice across major histocompatibility loci (H-2 d---,>H-2 b). This is accomplished by a new approach involving timed administration of «washed" BM cells suspended in a solution of physiological component of the BM microenvironment, a recently identified BM-derived ultrafiltration fraction designated MRF, with non-species-specific activity. It is, however, unknown how MRF exterts its action; this is the subject of continuing separate studies. Here in contrast to earlier experiments (20), pre-conditioning of the mice was abandoned before irradiation; also recipient mice were inoculated 24 hours after TBl. Number of BM cellslrecipient mouse was 10 to 20 X 106 . Concentration of MRF was 2 mg/mouse and was added to the BM cells shortly before inoculation. The mice were injected i.v., and the final volume containing both MRF and BM cells was 0.5 m!. Survival of GVHD-free allogeneic chimeras approximated 70 % of the transplanted mice. The animals were maintained in conventional environment and periodically checked for maintenance of chimerism. Their life span approximated that of irradiated and syngeneically reconstituted mice (over 2 years). When control syngeneic ally reconstituted mice were prepared, the syngeneic cells were also suspended in medium containing MRF, but the cells were injected 1-2 hours after TBI. All the animals used in the tests reported in this communication were perfectly healthy, with no clinical symptoms of chronic graft-versus-host disease.

Test for chimerism and immunocompetence of H-z:1~H-:zb radiation chimeras (BALB/C or DBAI2 into C57BLl6) The immune response and durable chimerism of these long-lived allogeneic chimeras reconstituted with MRF and unmanipulated BM has been reported previously (13).

Primary response to SRBC and Brucella abortus antigen (BA) 0.2 ml of a 10 % suspension of SRBC or 0.2 ml of BA (Difco, Detroit, Mich., USA) were injected i.p. Seven days later, blood was collected from the retroorbital plexus and the serum separated. Serial dilutions of individual sera were prepared in microplates, and one drop of 5 % SRBC or BA in methylen blue was added. The plates were left overnight at + 5 0c. The titer was expressed as reciprocal of dilution of last well-showing agglutination ± standard error.

Passive transfer of bone marrow or spleen cells from chimeras into irradiated normal recipients When bone marrow or spleen cells were transfused from chimeras into irradiated chimerasyngeneic or -allogeneic recipients (Table 1), the cells were inoculated 1-2 hours after TBI. No MRF was given.

H-2-typing and mixed lymphocyte culture (MLC) Chimeric lymphocytes were H-2-typed with anti-H-2 antibodies and C' or with MLC as described previously; MLC were prepared according to standard procedures (5).

T-cell-mediated anti-viral cytotoxicity Chimeras or control mice were infected intravenously with about 107 plaque-forming units of WR vaccinia virus. Six days later, mice were killed and spleen cells tested for cytotoxicity on infected, uninfected target cells, or cells susceptible to natural killing (NK). Effector cells were H-2-typed serologically and/or MLC (5, 14).

420 . G.

J.

M. MAESTRONI,

W. PIERPAOLI, and R. M. ZINKERNAGEL

Results

Attempts to transfer chimerism from Pl~P2 allogeneic irradiation bone marrow chimeras to irradiated normal P2 recipients Allogeneic bone marrow chimeras BALB/c~C57BLl6 or DBAI2~ C57BLl6 were established and killed 5 to 8 months later. Their bone marrow or spleen cells were used to reconstitute lethally irradiated BALB/c or C57BLl6 recipients (Table 1). The results clearly demonstrate that chimerism cannot be induced in C57BLl6 recipients with chimeric spleen or bone marrow cells more easily than with bone marrow cells from normal BALB/c donors. Also chimeric bone marrow cells were not able to induce chimerism in original chimeric recipient type C57BLl6 more easily than in unrelated C3H mice. In contrast, chimeric BALB/c~C57BLl6 bone marrow cells reconstituted BALBI c as readily as did normal BALBIc bone marrow cells. BALB/c mice reconstituted with chimeric bone marrow were not tolerant to C57BLl6 skin grafts; (Table 1) nor in mixed lymphocyte cultures against C57BLl6 when tested for cytotoxicity (data not shown). This indicates that tolerance was maintained only in the original chimera, but was not detectable when tested 50 d after adoptive transfer to new irradiated recipients syngeneic with the chimeric bone marrow.

Adoptive transfer of immunocompetent lymphocytes to allogeneic irradiation bone marrow chimeras Since P1~P2 chimeras were not immunocompetent in a primary antivaccinia-virus response, it was interesting to test whether immunocompetent P1 or P2 lymphocytes were capable of expressing their effector function in such chimeras. As shown in Table 2, about 5 X 107 to 108 spleen (or spleen plus lymphnode cells) of P1 or P2 origin were transfused into P1~P2 chimeras. These inoculations of immunocompetent lymphocytes did not provoke any visible sign of GVDH. At various times after this transfer, these chimeras were infected with vaccinia virus, and the effector function of T cells was tested 6 d later. The following results were obtained: BALB/c~C57BLl6 chimeras from several groups of chimeras (Group A, Table 2) (two additional experiments gave similar results) failed to express a substantial immune reactivity to vaccinia virus or SRBC when transfused with either BALB/c or C57BLl6 lymphocytes. Chimeric lymphocytes remained of H-2d type for recipients of BALB/c lymphocytes, and at least partially H-2d in recipients of H-2b lymphocytes. We had considerable problems in H-2-typing many of the H-2d~H-2b chimeras transfused with H-2b lymphocytes, when at least partial chimerism was retained (e.g., group A; 2, 6; B, 1); in these cases, there was less than 50% total anti-H-2+Cmediated cell-lysis. The reasons for this finding are unclear but have been encountered also in some allogeneic chimeras in previous experiments. In 1 out of 3 similar experiments, a H-2 d-restricted cytotoxic T cell response was found when H-2d~H-2b chimeras transfused with H-2d lymphocytes were

BM cells 6 X 106/recipient and spleen cells 12 X 106/recipiem

BM cells 12 X 106/ recipient

BM cells 40 X 106/recipient

BM cells 40 X 106/recipient

C57BLl6 BALB/c

C57BL/6 BALB/c

C57BL/6 DBAI2

C57BL/6 BALB/c CBA/J C57BL/6 BALB/c CBA/J C57BL/6 DBA/2

(H_2 b) (H-2 d)

(H-2 b) (H-2 d )

(H-2h) (H_2 d)

(H-2 b) (H-2 d) (H-2k) (H-2 b) (H-2 d) (H_2k) (H-2 b) (H_2 d )

(4) (4)

(4) (4)

(10) (10)

(12) (12) (8) (8) (8) (8) (10) (10)

Irradiated (950 or 1000 rad) recipients (n)

0 2

0 2

10 0

0 10 0 0 8 0 0 10

Survival at4 months afterBMT

NT

NT d

accepted

accepted b

BALB/c

rejected

accepted

DBAI2

NT

NT

accepted

rejected

rejected

rejected C

C57BL/6

Survival of skin grafts in surviving recIpIents Skin donors

, Spleen and/or bone marrow (BM) cells were inoculated i.v. 1-2 hr. after TBI = The irradiation dose was 950 or 1000 rad. The mice which died showed symptoms of GvHD and runting. Chimeric, syngeneically reconstituted and normal donors were of same age (7-12 mo old). The chimeric mice were used 4 months after BMT and allogeneic marrow reconstitution. Persistence of chimerism was assessed when the chimeras were used as donors of BM or spleen cells. The results shown derive from three separate experiments. Skin graft were performed 60 dafter BM transfer. Accepted as long as observed (120 d). Rejected within 12/14 d. NT: not tested.

Chimeras (6 Mo) BALB/c - C57BLl6 (H-2 d--7H-2 b) chimeras (6 mo)

(H-2d~H-2b)

BALB/c-C57BL/6

(H-2b~H-2b)

Chimeras (6 mol Syngeneically reconstituted C57BL/6

(H-2d~H-2b)

DBAI2 - C57BL/6

Normal BALB/c

chimeras (6 mol

spleen cells 20 or 36 X 106/recipient

spleen cells 20 or 36 X 106/recipient

BALB/c-C57BL/6

(H-2d~H-2b)

Type of cells transferred

Donors of bone marrow or spleen cells (time after reconstitution)

Table 1. Allogeneic bone marrow chimerism across the H-2 barrier is not transferable. Transplantation of spleen and/or bone marrow cells from long-lived chimeras into lethally irradiated, chimera-syngeneic and allogeneic normal recipients results in reconstitution of chimera-syngeneic mice only'

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>90% H-2d

>90% H-2b

>90% H-2d

20%H-2b, 80%H-2d

>90% H-2d

>90% 50% >90% >90%

Time after H-2 type of lymphocytes transfer tested

Number of spleen cells transfused

Chimeras

Group

13 ±4 (10) 6±3 (10) n. d. 40±9 (10)

anti-BA

<1 13 5 2

21 <1 <1 <1 <1 <1 8 <1 0 0

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anti-vaccinia response specific StCr-release from targets H_2b H-2d (D2) (MC57G) nor. vacc. nor. vacc.

6 ±2 (10) 4±2(10) 4±3 (10) 61 ±7 (10)

anti-SRBC lIa.t. ± S. E.

Immune function tested (number of mice tested)

Table 2. Immune functions expressed by lymphocytes from normal H_2d or H_2b donors adoptively transfused into H-2 d__ H-2 b chimeras

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85

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<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

<15

<1 <1 <1 <1 <1 <1

anti-vaccinia response specific 51Cr-release from targets H_2b H-2d (MC57G) (02) vacc. nor. vacc. nor.

anti-SRBC lia.t. ± S. E.

Immune function tested (number of mice tested)

UIJ

Time after H-2 type of lymphocytes transfer tested

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Number of spleen cells transfused

BALB/c (control)

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10

Group

Table 2 - continued

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(120 d)

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(120 d)

DBAI2~C57BLl6

9

8

7

6

Group Chimeras

Table 2 - continued

DBAI2 (8 X 10

7 )

DBA/2 (9 X 107 )

C57BLl6 (5 X 10 7)

Number of spleen cells transfused

60 d

60 d

60 d

>90% H_2d

>90% H-2d

>90% H-2h

Time H-2 type of after lymphocytes transfer tested anti-BA

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anti-vaccinia response MLC-reactivity specific 51Cr-release from targets antiantiH-2d C57BLl6(MC57G) B6 DBA/2 (D2) vacc. nor. vacc. nor.

anti-SRBC lIa.t. ± S. E.

Immune function tested (number of mice tested)

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C57BLl6 (control)

2

3

10

65 d

65 d

>90% H-2d

>90% H-2b

Time H-2 type of after lymphocytes transfer tested anti-BA

CD .,0 21 <1 <1 <1

14 <1 <1 25 <1 <1

vacc.

specific H_2d nor.

<1 <1 <1 15 9 <1 <1 <1 <1 <1 <1 <1

51

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23

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66

35 <1 <1 <1

[]JeD

MLC-reactivity Cr-release from targets antiantiH-2b (D2) (MC57G) B6 DBA/2

anti-vaccinia response

anti-SRBC lIa.t. ± S. E.

Immune function tested (number of mice tested)

". Chimeras were established (see Materials and Methods) and at the times indicated transfused with the reported number of immunocompetent lymphocytes (spleen cells, spleen + lymphnode cells). At different times thereafter chimerism was determined by a haemoglobin-typing.

C1

C57BLl6 (10")

Number of spleen cells transfused

DBA/2 ____ C57BLl6 (180 d)

Group Chimeras

Table 2 - continued

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426 . G.

J.

M. MAESTRONI,

W. PIERPAOLI, and R. M. ZINKERNAGEL

infected early after transfer (i.e., at 5 d, group B, 2, 3). In contrast, chimeras receiving H-2b lymphocytes did not express measurable effector T cells (B, 1). When chimeras of the same group were infected 54 d after transfer, no immunocompetence was detected in recipients of H-2d lymphocytes. However, recipients of H-2b (chimeric recipient type) lymphocytes had lost their H-2d lymphohaemopoietic cells and were not chimeric any longer as determined by both serology and mixed-lymphocyte culture typing. These latter chimeras expressed a measurable H-2 b-restricted anti-vaccinia-virus response (group B6); they were alloreactive against DBAI2 (H2d) stimulator cells. The latter results were found also in group C1, 2, 3. H-2d lymphocytes transfused to H-2d~H-2b chimeras apparently did not transfer immunocompetence to chimeras as measured by anti-viral or allo-reactivity (Group C2). H-2b lymphocytes transfused to the same chimeras apparently rejected the chimeras H-2d lymphohaemopoietic cells, and chimerism disappeared (group C). Therefore, 5-6 months later, these mice responded very well in a H-2 b-restricted fashion (group C1) to vaccinia virus and were alloreactive to H-2d and H-2k. Some chimeras were also injected with Brucella abortus antigen. Whereas BALB/c~C57BLl6 chimeras transfused with C57BLl6 lymphocytes only gave a barely detectable response (group A2), those transfused with BALBIc lymphocytes responded twice as well (group A1); but these chimeras responded 4 to 8 times less than a C57BLl6~C57BLl6 syngeneic control (A4).

Discussion Allogeneic irradiation bone marrow chimeras, long-lived and healthy, have been tested with respect to two parameters: (1) Can P1~P2 bone marrow cells transfer chimerism to new irradiated P2 recipients more readily than bone marrow from normal P1 mice? Transfer of chimerism with bone marrow from chimeras was not possible, suggesting that chimerism was not due to transferable mechanism of active suppression. (2) Does the milieu interieur of the lymphohaemopoietic system of Pl ~ P2 chimeras have an influence on the immunocompetence expressed by immunocompetent lymphocytes from normal adult P1 or from P2 donor mice, when transferred to chimeras? Immunocompetent lymphocytes histocompatible with the bone marrow donors of the chimera were able to transfer some immune reactivity when tested in the first 10 d after transfer; to our surprise, when tested later, immunocompetence was either very weak or in most cases not measurable. It should be noted that no clinical signs of graftversus-host reactions were detected after transfer of immunocompetent lymphocytes to P1~P2 chimeras. Transfer of immunocompetent chimeric

recipient type (C57BLl6) lymphocytes did not lead to recipient-type-

Allogeneic H-2d--->H-2b Irradiation Bone Marrow Chimeras . 427

restricted immune effector functions when tested early after transfer. Chimeras that had been established for more than about 7 months and that were transfused with C57BLl6 lymphocytes did not lose chimerism and failed to express a substantial degree of immunocompetence when tested up to 6 months later. Other chimeras that were transfused with C57BLl6 lymphocytes 3-4 months after induction of chimerism lost chimerism and regained H _2b -restricted immunocompetence when tested 6-12 weeks later. It, therefore, seems that old established chimeras H-2d~H-2b possess a remarkable capacity to resist and/or reject a great number of immunocompetent H-2b lymphocytes. Younger chimeras do seem to be less resistant, and the injected C57BLl6 lymphocytes induced reversal of chimerism. These results may indicate that there is a relationship between length of chimerism and resistance against «reversal of chimerism». It is unfortunate that for technical reasons (not enough C57BLl6~BALB/c chimeras could be prepared until now) the present experiments could not be performed in a symmetrical fashion. Also some chimeras were made with BALB/c, others with DBA12 bone marrow cells. It may be that the time-dependent differences seen in the chimeras with respect to C57BLl6 lymphocytes' capacity to reverse chimerism reflects differences in the bone marrow donors; BALBIc bone marrow cells had been used for the older group (Table 1 A). However, recent experiments using BALB/c bone marrow chimeras transfused with C57BLl6 cells 3 months after induction of chimerism yielded the same results as the ones with DBA12~C57BLl6 chimeras summarized in Table 2. Overall we conclude that no suppression mechanism exists in the bone marrow that allows adoptive transfer of chimerism. In long-term established chimeras Pl~P2, some sort of resistance or «suppression» exists against immunocompetent lymphocytes expressing their immune reactivity (Table 2): PI lymphocytes may express immunocompetence early but not later, whereas P2 lymphocytes are either «suppressed» permanently (group A) or sometimes only (groups B, C) in a fashion dependent on the age of the established chimeras. Since old PI~P2 chimeras (from group A) have failed to mount an anti-P2 response in vivo or in vitro, it seems unlikely that tolerance to P2 is broken in the usual term. Whatever this anti-P2 «suppression» is, it appears to function in the chimera only. No suppression of mixed lymphocyte reactions were observed in vitro when lymphocytes from PI~P2 chimeras were mixed with PI anti-P2 or P2 anti-PI reacting lymphocytes (data not shown). These latter results are compatible with data by SMITH and MILLER who showed that in (PI X P2) FI ~ PI irradiation bone marrow chimeras adoptively transferred FI lymphocytes were suppressed with respect to expression of P2-restricted immunocompetence when tested in a delayed-type hypersensitivity test. They also found that lymphocytes from such chimeras did not suppress reactivity in an in vitro assay.

428 . G.

J.

M. MAESTRONI, W. PIERPAOLI, and R. M. ZINKERNAGEL

The results reported here were difficult to obtain. Also in this model it is not easy to raise enough allogeneic chimeras for doing great numbers of transfer experiments. Therefore, results from relatively small groups have to be compared. This handicap makes their interpretation and generalization difficult. However, the evidence of an untransferable and unidentified suppressive principle operating in GVHD-free P1~P2 allochimeras against immunocompetent P1 or P2lymphocytes seems quite clear, this «suppression» might be related to a central mechanism keeping the chimerism. Acknowledgements We are indebted to Dr. WOLF WEIHE for maintenance and care of some of the animals utilized in this work. We would like to thank Miss R. KELLERHALS for expert technical assistance and Miss T. BACCIARINI for secretarial help. This work was supported with a grant from Institute Choay, Paris, a contribution from the Piombino Foundation for Life Sciences, Populonia, Livorno, Italy, and NIH grant RO-l CA 29992-02 (to G. M. and W. P.), and grants SNF No 3.488-0.79, NIAID-AI-I7285 and the University of Zurich (to R. Z.).

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