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464 NATURE MEDICINE • VOLUME 6 • NUMBER 4 • APRIL 2000

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Allogeneic bone marrow transplantation (in immunocompetentadults) has always required cytoreductive treatment of recipi-ents with irradiation or cytotoxic drugs to achieve lasting en-graftment at levels detectable by non-PCR-based techniques(‘macrochimerism’ or ‘mixed chimerism’)1–11. Only syngeneicmarrow engraftment at such levels has been achieved in uncon-ditioned hosts12,13. This requirement for potentially toxic myelo-suppressive host pre-conditioning has precluded the clinical useof allogeneic bone marrow transplantation for many indicationsother than malignancies, including tolerance induction. Wedemonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatchedallogeneic bone marrow, followed by one injection each ofmonoclonal antibody against CD154 and cytotoxic T-lympho-cyte antigen 4 immunoglobulin, resulted in multi-lineagehematopoietic macrochimerism (of about 15%) that persistedfor up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145days) and demonstrated ongoing intrathymic deletion ofdonor-reactive T cells. A protocol of high-dose bone marrowtransplantation and co-stimulatory blockade can thus achieve al-logeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier tothe more widespread use of allogeneic bone marrow transplan-tation14–18. Although efforts have been made to minimize hostpre-treatment for allogeneic bone marrow transplantation fortolerance induction, so far none have succeeded in eliminatingpre-treatment completely. Our demonstration that this can beachieved provides the rationale for a safe approach for inducingrobust transplantation tolerance in large animals and humans.

To determine whether allogeneic bone marrow could engraftwithout host pre-conditioning and induce tolerance, we gave B6mice a high dose of fully major histocompatibility complex(MHC)-mismatched bone marrow from B10.A donors (2 × 108

unseparated bone marrow cells (BMCs)) followed by a single in-jection of 0.5 mg monoclonal antibody against CD154 (CD40ligand) intraperitoneally immediately after bone marrow trans-plantation (BMT) and 0.5 mg cytotoxic T-lymphocyte antigen 4immunoglobulin (CTLA4Ig) intraperitoneally 2 days after BMT.Recipient mice did not receive any conditioning before the BMT.

We determined donor macrochimerism at multiple time pointsafter BMT by flow cytometry analysis (FCM) of peripheral whiteblood cells. We calculated the percentage of donor cells amongthese lineages by staining with a monoclonal antibody specificfor donor class I compared with various lineage markers.

Two weeks after BMT, there was multi-lineage macrochimerismin almost all recipients (14 of 15 treated mice). In most of thesemice (9 of 14), multi-lineage white blood cell chimerism per-sisted throughout the follow-up period of up to 30 weeks (‘long-term chimeras’) (Fig. 1a). In long-term chimeras, donorrepresentation among peripheral blood T cells remained rela-tively stable at approximately 2–3% throughout follow-up,whereas myeloid chimerism (about 15% immediately after BMT)gradually declined to levels approaching those of T-cellchimerism. The remaining mice (‘transient chimeras’) showed asharp decrease in chimerism, so that multi-lineage chimerismwas lost within four months after BMT (not shown). All BMT re-cipients remained healthy throughout the follow-up, withoutdeveloping signs of graft-versus-host-disease.

At the time mice were killed (31 weeks (first experiment) and34 weeks (second experiment) after BMT), we determined thepercentage of donor MHC class I-positive cells in spleen, thymusand bone marrow by FCM to confirm the presence of multi-lin-eage chimerism. All four long-term chimeras from the first twoexperiments demonstrated distinct donor chimerism amongsplenocytes and BMCs, and three of four showed clearly de-tectable donor class I-positive thymocytes (Table 1). Transientchimeras and a control mouse receiving bone marrow withoutco-stimulatory blockade, in contrast, did not show chimerismdetectable by FCM in these tissues.

The persistence of substantial multi-lineage donorhematopoiesis for 34 weeks after BMT indicates that engraftmentof true donor stem cells or at least very early donor hematopoi-etic progenitor cells occurred19. The level of donor representationachieved in these allogeneic long-term chimeras resembled re-sults obtained in previous studies using an essentially syngeneic(Ly5-congenic) mouse model in which a comparable number ofBMCs were injected into untreated recipients13. It thus seemsthat treatment with high-dose marrow plus antibody againstCD154 and CTLA4Ig is effective in completely overcoming al-loresistance in this BMT model.

Allogeneic bone marrow transplantation with co-stimulatoryblockade induces macrochimerism and tolerance without

cytoreductive host treatment

THOMAS WEKERLE1,3, JOSEF KURTZ1, HIROSHI ITO1, JOSEPH V. RONQUILLO1, VICTOR DONG2,GUILING ZHAO1, JUANITA SHAFFER1, MOHAMED H. SAYEGH2 & MEGAN SYKES1

1BMT Section, Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School,MGH East, Bldg. 149-5102, 13th Street, Boston, Massachusetts 02129, USA

2The Laboratory of Immunogenetics and Transplantation, Department of Medicine, Brigham and Women’sHospital/Harvard Medical School, Boston, Massachusetts 02115, USA

3Current affiliation: Department of Surgery, Vienna General Hospital/University of Vienna, Währingergürtel 18, A-1090 Vienna, Austria

Correspondence should be addressed to M.S.; email: [email protected]

© 2000 Nature America Inc. • http://medicine.nature.com©

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In the least-toxic protocol so far, host pre-treatment with largedoses of depleting antibodies against T cells and thymic irradia-tion was required to allow permanent engraftment of allogeneicmarrow given in high doses5. A relatively high dose of local irra-diation to the thymic area (7 Gy), however, was an indispensablecomponent of the pre-conditioning and the dose of irradiationcould not be successfully decreased in this model (T.W. andM.S., unpublished data). In another model, T-cell-depletedBMCs successfully engrafted in hosts receiving sublethal totalbody irradiation when high doses of marrow were adminis-tered20. Other protocols required cytotoxic drug treatment of therecipient to allow bone marrow engraftment9–11. Thus, host con-ditioning with irradiation or cytotoxic drugs has been an essen-tial part of all published protocols for the induction of lastingallogeneic macrochimerism. Our data (Fig 1a and Talbe 1)demonstrate that easily measured levels of long-lastinghematopoietic mixed chimerism can be achieved across a fullMHC barrier without any pre-conditioning of the recipients, byusing high-dose BMT with co-stimulatory blockade. This proto-col could facilitate the more widespread use of allogeneic BMT inthe treatment of non-malignant diseases, such as autoimmunediseases14–16, certain hematologic conditions17,18, and for trans-plantation tolerance.

Mixed chimerism has been shown in various models to leadto donor-specific tolerance that is measurable by the most strin-gent assays2,7,10. We evaluated tolerance induction with this pro-tocol of high-dose BMT and co-stimulatory blockade by graftingwith donor and third-party skin 4 or 13 weeks after BMT (onelong-term chimera, one transient chimera and one controlmouse died before skin grafting because of a technical error). Alllong-term chimeras accepted donor skin grafts long-term (mon-itored up to 145 days), while promptly rejecting third-partygrafts (Fig. 1b). Donor skin graft survival among transientchimeras was moderately prolonged (to a median of 36 days)compared with that of third-party grafts (median survival time,11 days), but only one (of five such mice) accepted its donorgraft for more than 100 days. Thus, all long-term chimerasshowed donor-specific tolerance according to the stringent test

of skin grafting. Transient chimeras had some evidence for re-duced responsiveness to the donor.

We also evaluated tolerance by in vitro assays at the time micewere killed. Cell-mediated lympholysis assays demonstrated thatlong-term chimeras did not generate cytotoxic T lymphocytesagainst donor antigens, whereas excellent anti-third-party re-sponses were achieved (Fig. 1c). One of three transient chimerasalso showed a specific absence of anti-donor cytotoxic T lym-phocytes, whereas the two other mice in this group responded toboth donor and third-party antigens. Similarly, all long-termchimeras tested showed no reactivity against donor antigens bymixed lymphocyte reaction assays (stimulation index (SI), lessthan 1.1); in contrast, transient chimeras and a control mouse re-ceiving bone marrow alone without co-stimulatory blockadeshowed reactivity (SI, more than 2) against donor antigens(Table 2). Reactivity against stimulators of the selected third-party strain was generally relatively weak in these assays (evenfor naive B6 mice). This reflected poor responsiveness only to thethird-party strain chosen, as there were high responses to an-other third-party strain (B10.RIII; SI, more than 4) in tolerantlong-term chimeric mice in another experiment (not shown).These in vitro data confirm the presence of donor-specific toler-ance in long-term chimeras, and indicate a state of donor-spe-cific hyporesponsiveness in some transient chimeras, consistentwith the somewhat prolonged donor skin graft survival seen inthis group. All chimeras showed an absence of anti-host respon-siveness in both mixed lymphocyte reaction (Table 2) and cell-mediated lympholysis assays (Fig. 1c).

For clinical applicability to cadaveric transplantation, organgrafting and BMT need to be done around the same time. Wetherefore evaluated whether tolerance is still induced with thisprotocol when skin grafting is done at the time of bone marrowinfusion. We treated B6 recipients with 2 × 108 fully allogeneicB10.A BMCs and antibody against CD154 (0.5 mg intraperi-toneally immediately after BMT) plus CTLA4Ig (0.1 or 0.5 mg in-

Table 1 Tissue chimerism after high-dose BMT with co-stimulatory blockade

Percentage donor cells

Spleen Thymus Bone marrow

CD4+ CD8+ B220+

NL B10.A 100.00 99.55 99.92 84.46 93.10NL B6 0.00 0.00 0.24 0.00 0.00control 0.00 0.00 0.10 0.00 0.00

long-term chimeras:# 1 0.39 0.47 4.83 0.08 1.77# 2 8.41 5.45 4.07 2.89 18.55# 3 1.67 1.98 11.10 7.12 23.57# 4 2.08 2.36 10.97 2.67 14.29

transient chimeras:# 5 0.00 0.00 0.00 0.00 0.00# 6 0.08 0.00 0.21 0.02 0.00# 7 0.00 0.00 0.14 0.00 0.00

Percentage of donor MHC class I-positive cells was determined by two-color FCM 31(long-term) and 34 weeks after BMT. Data represent results from the first and sec-ond experiments. Mice were treated with BMCs, antibody against CD154 andCTLA4Ig. NL, normal; control, mouse treated with bone marrow only without co-stimulatory blockade.

Table 2 Mixed lymphocyte reaction results after high-dose BMTwith co-stimulatory blockade

Stimulation Index

anti-host anti-donor anti-3rd party(D) (3rd) D:3rd

NL B6 0.82 5.14 1.82 2.83control 1.02 3.24 1.70 1.91

long-term chimeras:# 1 0.70 0.66 1.22 0.54# 2 0.75 0.94 1.17 0.80# 3 0.88 0.88 1.34 0.66# 4 0.99 1.09 1.74 0.63

transient chimeras:# 5 0.74 2.12 1.68 1.26# 6 0.67 2.33 1.15 2.03# 7 0.84 3.24 1.76 1.85

Mixed lymphocyte reaction results of spleen cells from BMT recipients of the first andsecond experiment were obtained 31 and 34 weeks after BMT. Anti-donor responsewas specifically reduced in long-term chimeras (P < 0.05, SI anti-donor compared withSI anti-third-party) but not in transient chimeras (P > 0.05). Likewise, the ratio of the SIfor the anti-donor response (D) and the SI for the response against third-party (3rd) wassignificantly lower for long-term chimeras than for transient chimeras (P < 0.05). SIswere calculated by dividing mean c.p.m. from responses against host (B6), donor(B10.A) or third-party (A.SW) by mean background c.p.m. (that is, c.p.m. with no stim-ulator population). Mice were treated with BMCs, antibody against CD154 andCTLA4Ig. NL, normal; control, mouse treated with bone marrow only without co-stim-ulatory blockade.


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traperitoneally 2 days after BMT) and grafted them with donorand third-party (B10.RIII) skin within 24 hours of the bone mar-row infusion (n = 9). Most of these mice (seven of nine) showedmulti-lineage hematopoietic macrochimerism by FCM 2 weeksafter BMT (5–14% donor cells among granulocytes). Three ofthese mice retained substantial levels of chimerism 8 weeks afterBMT. Most BMT recipients that showed macrochimerism at twoweeks after BMT (six of seven) accepted donor skin grafts long-term (for more than 100 days) while rapidly rejecting third-partyskin grafts (median survival time, 22 days). Control mice treatedwith co-stimulatory blockade alone without BMT (n = 5) rejecteddonor and third-party skin grafts promptly (median survivaltime, 17 and 16 days, respectively). Thus, high-dose BMT withco-stimulatory blockade rapidly leads to robust donor-specific

tolerance that allows solid tissue grafting at the time of BMT.The induction of robust, permanent tolerance is still an elu-

sive goal in clinical organ transplantation. This new protocolpotentially constitutes a safe and feasible approach to reachingthis goal. T-cell co-stimulatory blockade with antibody againstCD154 and CTLA4Ig has profound immunosuppressive effectsin various transplantation models21–24. Co-stimulatory blockadealone, however, has not induced tolerance for the most strin-gent test: skin grafts across full MHC barriers25. We chose a fullyMHC-mismatched strain combination for our experiments andused a recipient strain (C57BL/6) that is reported to be one ofthe most difficult for achieving graft prolongation with co-stimulatory blockade26. The weak effect of co-stimulatoryblockade alone was demonstrated by the short skin graft sur-

Fig. 1 Macrochimerism and tolerance after high-dose BMT with co-stimulatory blockade. a, Multi-lineage chimerism in peripheral blood for30 weeks after high-dose BMT with co-stimulatory blockade. B6 recipientsreceived BMCs from B10.A donors, antibody against CD154 andCTLA4Ig. Data represent mean percentages of donor hematopoiesisamong various lineages as determined by two-color FCM, in long-termchimeras from three experiments (n = 9). Five additional mice developedtransient multi-lineage chimerism and one mouse did not demonstrateany chimerism (not shown). Control mice receiving bone marrow alonewithout co-stimulatory blockade (n = 3) did not show any chimerism de-tectable by FCM even 1 week after BMT, the earliest time point tested.When bone marrow was administered with antibody against CD154alone (without CTLA4Ig), two of three mice developed transientchimerism, which was lost by week 6 (not shown). b, Donor-specific tol-erance in long-term chimeras, as assessed by primary skin grafting. Long-term chimeras (n = 8) uniformly accept donor grafts long-term (�) whilepromptly rejecting third-party grafts. Donor skin graft survival is moder-ately prolonged in transient chimeras (squares) (n = 5, including the onemouse that did not show any chimerism) to a median survival time of 36d, with only one mouse in this group showing long-term skin graft accep-tance. Third-party grafts (triangles) are rejected by all mice within 14 d(median survival time, 11 d). The recipients of bone marrow with anti-

body against CD154 alone (without CTLA4Ig) rejected both donor andthird-party grafts without delay (survival, less than 13 d) (not shown).Control mice receiving bone marrow alone without co-stimulatory block-ade (n = 2) and controls receiving antibody against CD154 and CTLA4Igalone (without bone marrow; n = 6) all promptly rejected donor andthird-party grafts (survival, less than 15 d). Donor (B10.A) and third-party(A.SW) skin was grafted 13 weeks (in the first and second experiments)and 4 weeks after BMT (in the third experiment). One long-term chimera,one transient chimera and one control mouse were lost to follow-up be-fore skin grafting. c, Long-term chimeras show donor-specific hyporeac-tivity in cell-mediated lympholysis assays. Long-term chimeras (n = 4; #1,#2, #3 and #4) uniformly fail to generate cytotoxic T lymphocytes re-sponses to donor (B10.A; left) antigens, while effectively responding tothird-party antigens (A.SW; middle). One of three transient chimeras (#5,#6 and #7) also show a specific absence of anti-donor cytotoxic T lym-phocytes; the two remaining mice in this group respond to both donorand third-party antigens. All chimeras show no cytotoxic T lymphocyteresponses against host (B6; right) antigens. A control mouse receivingbone marrow alone without co-stimulatory blockade responded to donorand third-party antigens effectively. Cell-mediated lympholysis assayswere done 31 and 34 weeks after BMT. control, mouse treated with bonemarrow only without co-stimulatory blockade; NL, normal controls.

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vival (median survival time, 17 and 16 days) in B6 controls re-ceiving co-stimulatory blockade alone without BMT. Thus, it isunlikely that the induction of macrochimerism and toleranceby high-dose BMT with co-stimulatory blockade is a strain-spe-cific phenomenon, although we cannot rule out this possibilityat present. Both reagents used in our model (antibody againstCD154 and CTLA4Ig) have recently been successfully tested ina phase I clinical trial27, and in nonhuman primate studies22,and it can be anticipated that they will become available clini-cally in the near future. Our results argue that these reagentswill be most effective if used in combination with high-dosedonor stem cell transplantation.

To study the involvement of clonal deletion in tolerance in-duction, we analyzed peripheral blood lymphocytes, splenocytesand mature thymocytes for the presence of certain Vβ subunitson their T-cell receptors. The donor strain B10.A expresses MHCclass II I-E, which is required to present superantigens derivedfrom endogenous retroviruses encoded in the B6/B10 back-ground genome. Developing thymocytes whose T-cell receptorscontain Vβ11 or Vβ5.1/2, which bind to these superantigens, aredeleted in I-E-positive B10.A mice28,29, but not in B6 mice, be-cause they do not express I-E (refs. 30, 31).

One week after BMT, mice that received 2 × 108 BMCs plus an-tibody against CD154 and CTLA4Ig had a highly significant re-duction in the percentages of Vβ11+ and Vβ5+ cells among CD4+

peripheral blood lymphocytes, compared with that of controlmice receiving 2 × 108 BMCs alone (0.98% ± 0.35 compared with3.26% ± 0.42 for Vβ11 (P = 0.005); and 0.57% ± 0.22 comparedwith 1.57% ± 0.4 for Vβ5 (P < 0.05); combined results from threesimilar experiments). There was no reduction in the percentageof irrelevant Vβ (Vβ 8.1/2). Deletion of donor-reactive T cells inrecipients of BMT with co-stimulatory blockade and immediateskin grafting occurred to a degree similar to that in the experi-ments with delayed skin grafting (2.32% ± 1.11 compared with5.38% ± 0.10 for Vβ11+CD4+ peripheral blood lymphocytes, and

0.86% ± 0.46 compared with 3.07% ± 0.32 forVβ5+CD4+ peripheral blood lymphocytes at 1week in BMT recipients and controls receivingco-stimulatory blockade without BMT, respec-tively; P < 0.00001 for Vβ11 and Vβ5). The dele-tion of CD4+ peripheral blood lymphocytesbecame even more profound in long-termchimeras in the ensuing weeks and persistedthroughout follow-up. There was a substantialdecrease in the percentages of Vβ11+ andVβ5+CD4+ splenocytes in long-term chimeras atthe time mice were killed (0.77% ± 0.52 for Vβ11and 0.71% ± 0.54 for Vβ5, 31–34 weeks afterBMT) both in experiments with day 1 skin graft-ing and in those with delayed skin grafting.

At the time mice were killed, long-termchimeras also had a decrease in the percentagesof Vβ11+ and Vβ5+ (but not Vβ8+) single-positiveCD4 and single-positive CD8 thymocytes to alevel resembling that in naive B10.A controls(Table 3). Neither transient chimeras nor a con-trol mouse receiving bone marrow alone (no co-stimulatory blockade) showed similar reductionsin these Vβs. MHC class II-positive macrophagesand dendritic cells are considered to be effectivemediators of intrathymic deletion32. We there-fore looked for donor MHC class II-positive cells

in the thymus by immunohistochemical staining. Three of four long-term chimeras showed evidence of donor class II-positive cells intrathymically (one chimera could not be evalu-ated unequivocally because of high background staining) (Table 3). Transient chimeras were negative for donor class II-positive cells.

Our data indicate that in long-term chimeras, intrathymicclonal deletion of donor-reactive T cells is an ongoing mecha-nism of tolerance, even as late as 34 weeks after BMT. Clonaldeletion is a robust mechanism of tolerance, and thus would bedesirable for clinical application33. Intrathymic clonal deletionhas been determined to be the main mechanism for long-termtolerance in mixed chimeras produced with low-dose total-bodyirradiation plus host T-cell depletion or co-stimulatory block-ade7,34,35. In addition, extrathymic clonal deletion of donor-reac-tive T cells has been found to occur shortly after BMT withco-stimulatory blockade in recipients of low-dose total-body ir-radiation7. We have demonstrated similar mechanisms here.Thus, peripheral and central clonal deletion seem to be chiefmechanisms of tolerance in this model, although it remains tobe determined whether additional mechanisms participate inthe initial peripheral tolerance induction phase. Although wedid not directly measure deletion in the graft-versus-host direc-tion, we hypothesize that similar mechanisms are involved inthe tolerization of graft-versus-host-reactive donor T cells, thusaccounting for the absence of acute or chronic graft-versus-hostdisease in the mice.

Thus, we induced allogeneic bone marrow engraftment androbust donor- and host-specific tolerance without irradiation,cytotoxic drugs or T-cell depletion using high-dose BMT with co-stimulatory blockade. Peripheral and central clonal deletionwere involved in the induction and maintenance of tolerance,respectively. In future, these studies might lead to clinically ap-plicable treatment protocols for the induction of robust transplantation tolerance.

Table 3 Intrathymic deletion of donor-reactive T cells after high- dose BMT with co-stimulatory blockade

CD4 SP CD8 SP DonorMHC

Vβ8 Vβ5 Vβ11 Vβ8 Vβ5 Vβ11 class II+

NL B10.A 13.95 0.83 1.42 9.95 0.78 0.93 +NL B10.A 15.32 0.31 0.27 13.28 0.40 0.40 +NL B6 14.54 3.09 3.53 10.12 7.04 2.93 –NL B6 11.58 3.50 2.70 7.38 5.97 2.00 –control 14.75 2.09 2.14 11.37 3.17 1.75 ND

long-term chimeras:# 1 14.03 0.96 0.57 10.11 1.39 0.89 ±# 2 18.01 0.73 0.26 12.97 1.35 0.89 +# 3 17.19 0.62 0.32 9.37 1.75 0.62 +# 4 15.21 0.87 0.73 13.15 1.26 0.85 +

transient chimeras:# 5 16.08 1.87 2.43 12.52 3.87 2.08 –# 6 15.81 2.26 2.30 10.24 4.78 2.23 –# 7 16.81 2.53 2.18 11.71 4.43 2.81 –

The percentage of Vβ8+, Vβ5+ and Vβ11+ cells among host MHC class Ihigh, CD8– (CD4 SP) and CD4– (CD8 SP)thymocytes was determined by three-color FCM 31 or 34 weeks after BMT. The presence (+) or absence (–)of donor MHC class II-positive cells in the thymus was determined by immunohistochemical staining. Datarepresent results from the first and second experiment. Mice were treated with BMCs, antibody againstCD154 and CTLA4Ig. NL, normal; control, mouse treated with bone marrow only without co-stimulatoryblockade. SP, single-positive; ND, not determined. ±: Because of high background the presence of donorMHC class II-positive cells could not be determined with certainty.


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MethodsBone marrow transplantation protocol. Age-matched (6- to 12-week-old)female C57BL/6 (B6: H-2b) mice were injected intravenously with a dose ofapproximately 2 × 108 (suspended in 1 ml) unseparated BMCs from the tib-iae and femora of fully MHC-mismatched B10.A (H-2a) donors (7–11 weeksold). In addition, recipients were treated with a hamster monoclonal anti-body against mouse CD154 (MR1; 0.5 mg injected intraperitoneally on theday of BMT) and with mouse CTLA4Ig (0.1 mg or 0.5 mg injected intraperi-toneally 2 d after BMT). The MR1 hybridoma was provided by R.J. Noelle(Dartmouth Medical School, Lebanon, New Hampshire), and CTLA4Ig wasprovided by Bristol-Myers, Squibb Pharmaceuticals (Seattle, Washington) orproduced in our laboratory from a transfected cell line provided by T. Strom.In the last three experiments, recipients were treated with heparin before thebone marrow injection. All mice were maintained in a specific-pathogen-freemicroisolator environment.

Monoclonal antibodies for FCM. Nonspecific Fcγ receptor binding wasblocked by the monoclonal antibody against mouse Fcγ receptor, 2.4G2.Fuorescein isothiocyanate (FITC)-conjugated monoclonal antibodies in-cluded antibodies against CD4, CD8 and B220 (all from PharMingen, SanDiego, California) and MAC1 (Caltag, San Francisco, California), as well asantibodies against Vβ5.1/2, Vβ11 and Vβ8.1/2 (PharMingen, San Diego,California). Negative control FITC-conjugated monoclonal antibodyHOPC1, with no reactivity to mouse cells, was prepared in our laboratory.Biotinylated 34-2-12 (against H-2Dd, prepared in our laboratory), KH95(against Db; PharMingen, San Diego, California) and control monoclonalantibody HOPC1 were developed with phycoerythrin–streptavidin (PEA;PharMingen, San Diego, California) for two-color FCM or withCyChrome–streptavidin (PharMingen, San Diego, California) for three-color FCM. Phycoerythrin (PE)-conjugated antibodies included antibodiesagainst CD4 and CD8 and a nonspecific rat IgG2a (PharMingen, San Diego,California). Propidium iodide staining was used to exclude dead cells intwo-color FCM.

Analysis of multi-lineage chimerism. Allogeneic reconstitution of variouslineages was evaluated by FCM analysis of white blood cells at multiple timepoints. At the time mice were killed, spleen, bone marrow and thymus cellsuspensions were prepared. Forward-angle and 90-degree light-scatterproperties were used to distinguish lymphocytes, monocytes and granulo-cytes in peripheral white blood cells, as described34. Two-color FCM wasused to distinguish donor and host cells of particular lineages, and the per-centage of donor cells was calculated as described34 by subtracting controlstaining from quadrants containing donor and host cells expressing a par-ticular lineage marker, and by dividing the net percentage of donor cells bythe total net percentage of donor plus host cells of that lineage.

Analysis of T-cell receptor Vβ families. Peripheral blood lymphocyteswere stained at multiple time points with specific FITC-conjugated mono-clonal antibodies against Vβ or HOPC1 versus PE-conjugated antibodyagainst CD4. FCM analysis used gated CD4+ cells, and staining with controlmonoclonal antibody was subtracted to determine the percentage of cellsexpressing each Vβ in the gated population. Splenocytes were stained forthree-color analysis at the time mice were killed, with specific FITC-conju-gated monoclonal antibodies against Vβ (or control monoclonal antibody)versus PE-conjugated antibody against CD4, and also versus 34-2-12-biotinplus CyChrome–streptavidin. Gated 34-2-12–CD4+ cells were analyzed, andpercentages of cells expressing each Vβ were determined as describedabove. For B10.A controls, CD4+ cells were analyzed. Thymocytes werestained for three-color analysis with FITC-conjugated antibody againstVβ5.1/2, Vβ11 or Vβ8.1/2 or HOPC1 versus PE-conjugated antibody againstCD4 or CD8, and also versus KH95-biotin. Gated class I (KH95)high CD4–

(gating mainly on CD8+ (single-positive) thymocytes) or CD8– (gatingmainly on CD4+ (single-positive) thymocytes) cells were analyzed. ForB10.A controls, gated 34-2-12high cells were analyzed in a similar way.Background staining was subtracted from the percentage of gated cellsstaining with each monoclonal antibody against Vβ.

Skin grafting. Skin grafting was done 4 or 13 weeks after BMT, or within 24h of BMT. Full-thickness tail skin from B10.A mice (donor-specific) and fullyMHC-mismatched A.SW mice (H-2s; third-party) or B10.RIII mice (H-2r;

third-party) was grafted onto the lateral thoracic wall, secured with 5-0 silksutures and adhesive bandages; grafts were monitored by visual and tactileinspections daily from day 8 onward for three weeks, then at least everyweek thereafter. Grafts were considered to be rejected when less than 10%of the graft remained viable.

Mixed lymphocyte reaction. Splenocytes were cultured at 37 °C in 5% CO2

for 3–4 d in triplicate wells containing 4 × 105 responders with 4 × 105 stimu-lators (irradiated with 30 Gy) in RPMI 1640 medium (Life Technologies) sup-plemented with 15% (volume/volume) controlled processed serumreplacement (CPSR-2; Sigma), 4% nutrient mixture (7.3 mg/ml L-glutamine,4× nonessential amino acids (Life Technologies), 2.75 mg/ml sodium pyru-vate, 250 U/ml penicillin and 250 mg/ml streptomycin), 1% HEPES buffer,and 10 mM β-mercaptoethanol. Then, cells were pulsed with 3H-thymidineand collected approximately 18 h later. Stimulation indices (SIs) were calcu-lated by dividing mean counts per minute (c.p.m.) from responses againsthost (self), donor or third-party by mean background c.p.m. (that is, c.p.m.with no stimulator cell population).

Cell-mediated lympholysis assay. Splenocytes from controls, BMT recipi-ents and normal mice were suspended in RPMI 1640 (Mediatech, Herndon,Virginia) containing 10% fetal bovine serum (Sigma), 0.09 mM non-essen-tial amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mlpenicillin, 0.2 µg/ml streptomycin, 0.025 mM β-mercaptoethanol and 0.01M HEPES buffer. Responder and stimulator cells (irradiated with 30 Gy)were diluted to a concentration of 8 × 106 cells/ml, and 100 µl respondercells were co-cultured with 100 µl stimulator cells per well. Cultures wereset up in two rows of three replicates each, and after 5 days of incubation in5% CO2 at 37 °C, twofold serial dilutions were prepared from the secondrow of triplicates, so that cytolytic capacity could be examined at five differ-ent responder-to-target ratios. Lymphoblasts (8 × 103, 51Cr-labeled, two-dayconcanavalin-A-stimulated) were added to each well and incubated for 4 hin 5% CO2 at 37 °C before they were collected. Percent specific lysis was cal-culated with the formula: % specific lysis = [(experimental release–sponta-neous release)/(maximum release–spontaneous release)] × 100%.

Immunohistochemical staining. Frozen thymus sections (4 µm in thick-ness) were prepared and stained as described34. Sections were stained withISCR-3 (mouse IgG2b antibody against MHC class II I-E; 1:200 dilution ofascites) and 25-9-17 (mouse IgG2a antibody against MHC class II I-Ab; 1:50dilution). Ascites containing monoclonal antibody HOPC-1 (non-reactivemouse IgG2a antibody; 1:100 dilution) or 74-11-10 (mouse IgG2b anti-body against pig MHC class I;1:200 dilution) were used as negative controlstains. Biotinylated rat monoclonal antibodies against mouse IgG2a orIgG2b were used as secondary reagents. Stained sections were analyzed byan observer ‘blinded’ to sample identity.

Statistical analysis. Statistical significance was determined with a two-tailed Student’s t-test for comparison of means with unequal variances. A Pvalue of less than 0.05 was considered statistically significant.

AcknowledgmentsWe thank H. Auchincloss Jr., H. Winn and L.A. Turka for review of themanuscript; D.H. Sachs for support and advice; and D. Plemenos for secretarialassistance. This study was supported by National Institutes of Health GrantR01 HL49915 and in part by a sponsored research agreement betweenMassachusetts General Hospital and BioTransplant. M.H.S. is a recipient of theNational Kidney Foundation Clinician Scientist Award. T.W. was supported byfellowships from the Max Kade Foundation and the Austrian Science Fund(Fonds zur Foerderung der wissenschaftlichen Forschung).

RECEIVED 12 JANUARY; ACCEPTED 23 FEBRUARY 2000

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