Long-term Chimeras Research Articles

Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade.

Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2b) mice received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2 × 107 bone marrow cells (BMC) from either of three donor strains: Balb/c (H2d) (MHC plus multiple minor histocompatibility antigen (mHAg) mismatched), B10.D2 (H2d) or B10.A (H2a) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 of Balb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (p < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (p < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities.

Subclinical GvHD in non-irradiated F1 hybrids: severe lymphoid-tissue GvHD causing prolonged immune dysfunction

GvHD is an important complication of allogeneic hematopoietic SCT. Parent-in-F1 models are frequently used to study GvHD immunobiology; the characteristics of parent-in-F1 GvHD vary between strain combinations and induction protocols. Here, we observed that a high-dose challenge of non-irradiated B6DBA2F1 and B6SJLF1 recipients with C57BL/6 splenocytes left the majority of recipients clinically healthy, while inducing progressive high-grade donor T-cell chimerism. We investigated this previously undescribed pattern of parent-in-F1 T-cell alloreactivity and studied the effect of serial parental splenocyte infusions on epithelial and lymphohematopoietic tissues. The majority of recipients of 4 weekly splenocyte infusions showed long-term survival with gradual establishment of high-grade donor chimerism and without any signs of epithelial-tissue GvHD. A minority of recipients showed BM failure type of GvHD and, respectively, graft rejection. Moreover, long-term F1 chimeras showed protracted pancytopenia, and in peripheral lymphoid tissues severe lymphopenia and near-complete eradication of APCs and dysfunction in antigen-presenting capacity in remaining APC. Hematopoiesis and lymphoid tissue composition recovered only after multilineage donor chimerism had established. In conclusion, we report on a novel type of parent-in-F1 hybrid GvHD, where a cumulative high dose of C57BL/6 parental splenocytes in non-irradiated F1 mice induces subclinical but severe hematolymphoid-tissue GvHD, causing prolonged immuno-incompetence.

Tolerance Versus Immunosuppression: A Perspective

To the Editor: It was with general agreement that we read your editorial and cautionary credo ‘do not try this at home’ (1Halloran PF Bromberg J Kaplan B et al.Tolerance versus immunosuppression: A perspective.Am J Transplant. 2008; 8: 1365-1366Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Tolerance-inducing protocols as routine are ‘not yet ready for prime time’ before more mechanisms are clarified. To attempt to reproduce the landmark neonatal mice tolerance work in 1953 of Billingham, Brent and Medawar in a human with a mature immune system, it seems obvious that an initial immunoablative state that entails some risk must be introduced. Where we disagree with you is in your statement referring to the work of Hofmann et al. ‘that such mice (chimeric from your previous sentence) accept third party heart transplants, that is, are immunosuppressed’. Admittedly, there may be an early spread of a nonspecific regulatory cell effect even in neonatal chimeric tolerance. Indeed, a regulatory effect of recipient suppressor cells was even described by Brent himself in adult mice years after their initial observations (2Kilshaw PJ Brent L Pinto M Suppressor cells in mice made unresponsive to skin allografts.Nature. 1975; 255: 489-491Crossref PubMed Scopus (111) Google Scholar). Certainly regulatory cells by their very nature are (nonspecifically) infectious (bystander inducing) in their local surroundings. However, even the more extensive earlier work by West and Tau of that same group (3West LJ Tao K. Acceptance of thirdparty cardiac but not skin allografts induced by neonatal exposure to semi-allogeneic lymphohematopoietic cells.Am J Transplant. 2002; 2: 733-744Crossref PubMed Scopus (16) Google Scholar) should not be interpreted as being associated with longlasting or even shortterm chimerism. Moreover, even in their skin graft survival data, 30% of the specific (BALB/c) donor (of the bone marrows and spleens) skin grafts were lost in the first 2 weeks and no longterm data after 60 days were reported (3West LJ Tao K. Acceptance of thirdparty cardiac but not skin allografts induced by neonatal exposure to semi-allogeneic lymphohematopoietic cells.Am J Transplant. 2002; 2: 733-744Crossref PubMed Scopus (16) Google Scholar). This may well mean that chimerism was not established, just as it could not be in many of the initial experiments by Medawar’s group between several fully MHC mismatched strains, that is, it did not work in many combinations. The Hofmann animals indeed may well not have been long-term chimeras, that is, keeping their grafts due to tolerance induced by (total) clonal deletion, but more by some active regulatory mechanism. This might have been associated with an element of ‘linked’ or infectious inhibition to nonspecific antigenic stimuli of third party heart grafts due to the ‘proximity’ of (bystanding) regulatory cells, perhaps what you referred to as ‘immunosuppressed’. In addition, there might have been the not inconsiderable positive effect of the evolution toward graft quiescence (absence of inflammation/danger) in the longer term accepted skin grafts (4Matzinger P Tolerance, danger, and the extended family.Annu Rev Immunol. 1994; 12: 991-1045Crossref PubMed Scopus (4013) Google Scholar). Were true long-term mixed chimerism established, implying bi-directional tolerance of these cells residing in the recipient, perhaps one might expect more donor specificity long term (Medawar chimerism model). As such, the earlier nonspecific ‘immunosuppression’ brought on by regulatory cell bystander effects might be minimized long term. This may even have occurred in our previous clinical reports of longer term results of simultaneous donor bone marrow and kidney transplantation in which an early immunosuppressed state appears to have resolved 1 or 2 years after transplantation (5Ciancio G Miller J Garcia-Morales RO et al.Six-year clinical effect of donor bone marrow infusions in renal transplant patients.Transplantation. 2001; 71: 827-835Crossref PubMed Scopus (120) Google Scholar). However, it may or may not have occurred in the groundbreaking tolerance work of the Cosimi, Sykes and Sachs group to which you addressed your editorial and to which they responded in the next (August) issue of the Journal.

Central Tolerance to Myogenic Cell Transplants Does Not Include Muscle Neoantigens

Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.

Rapamycin Promotes Emergence of IL-10-Secreting Donor Lymphocyte Infusion-Derived T Cells Without Compromising Their Graft-Versus-Leukemia Reactivity

There are limited data examining the effects of pharmacological immunosuppression on the in vivo fate of donor lymphocyte infusions (DLI)-derived T cells, their function, and their antitumor efficacy. We addressed this question in a murine model in which DLI is given to stable mixed chimeras resulting in lymphohematopoietic graft-versus-host (LH-GVH) response. In this model, LH-GVH potency can be directly measured as the kinetics of conversion to full donor chimerism and can be correlated with associated graft-versus-leukemia (GVL) reactivity. We found discordance in DLI-mediated LH-GVH reactivity depending on the timing of rapamycin (RAPA) administration. Delayed administration of RAPA in contrast to its early administration at the time of adoptive transfer did not interfere with conversion to full donor chimerism. Moreover, delayed administration of RAPA preserved the GVL reactivity of DLI. Analysis of the long-term chimeras showed that regardless of RAPA administration, adoptively transferred T cells mediating the LH-GVH response contribute minimally to the reconstitution of the peripheral T-cell compartment and exhibit profound hyporesponsiveness and decreased production of interleukin (IL)-2 on restimulation in vitro. However, we observed only in the RAPA-treated chimeras that the remaining hyporesponsive DLI-derived CD4+ T cells secrete large amounts of IL-10, a known immunoregulatory cytokine. We conclude that delayed administration of RAPA after DLI does not interfere with their LH-GVH reactivity but promotes the emergence of IL-10-secreting DLI-derived CD4+ T cells that might contribute to the drug's known ability to promote bilateral donor host tolerance without interfering with GVL reactivity.

Donor CD8+ T Cells Mediate GVL without GVHD in Recipients Conditioned with Anti-CD3 mAb.

Donor CD8+ T cells play a critical role in mediating graft versus leukemia (GVL), but also induce graft versus host disease (GVHD) in recipients conditioned with total body irradiation (TBI). Here, we report that injections of donor C57BL/6 (H-2b) or FVB/N (H-2q) CD8+ T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without GVHD in anti-CD3-conditioned BALB/c (H-2d) recipients. In contrast, the same dose of donor CD8+ T and marrow cells induced lethal GVHD in TBI-conditioned recipients. In addition, the anti-CD3-conditioned long-term complete chimeras without prior exposure to host-type BCL1 cells also eliminated the tumors when being challenged with BCL1 cells 120 days after HCT. This is in contrast to the report that long-term complete chimeras induced with delayed donor lymphocyte infusion lost GVL activity. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8+ T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8+ T cell expansion in anti-CD3-conditioned recipients was confined to lympho-hematological tissues. This confinement was associated with lack of up-regulated expression of α4β7 integrin and chemokine receptors (i.e. CXCR3) on donor CD8+ T cells. In addition, host-reactive donor CD8+ T cells in anti-CD3-conditioned recipients were only partially deleted, and the residual cells were rendered heterogeneous: some unresponsive/anergic, some Tc2, some Foxp3+ suppressive cells, and some effector cells. The whole population of residual donor CD8+ T cells from anti-CD3-conditioned recipients mediated GVL without GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3-conditioning separates GVL from GVHD via confining donor CD8+ T cell expansion to host lympho-hematological tissues as well as tolerization of the residual donor CD8+ T cells, in which the residual host-reactive effector cells mediate persistent GVL, and the regulatory CD8+ T cells prevent them from damaging host tissues.

Graft-versus-Leukemia and Graft-versus-Host Reactions after Donor Lymphocyte Infusion Are Initiated by Host-Type Antigen-Presenting Cells and Regulated by Regulatory T Cells in Early and Long-Term Chimeras

Regulatory T (T reg) cells and host antigen-presenting cells (APCs) have been implicated in graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect after donor lymphocyte infusion (DLI), but their relative contributions remain unclear in early versus long-term complete donor or mixed murine allogeneic hematopoietic stem cell (HSC) chimeras. We have previously demonstrated that donor HSC-derived Thy1 + T reg cells, consisting primarily of CD4 +CD25 + cells, play an important role in the suppression of graft-versus-host (GVH) reactivity when DLI is given to complete donor chimeras 28 days after HSC transplantation. Data presented here demonstrate that protection against GVHD exerted by Thy1 + T reg cells is less evident with time and eventually is not required in long-term complete donor chimeras because of an absence of host-type APCs to activate alloreactive T cells. Lethal GVHD was observed when Thy1 + T reg cells were depleted from complete donor chimeras given by DLI at day 28, 35, or 42; however, T reg cell depletion and DLI at day 70 no longer induced GVHD-associated mortality. Moreover, the failure of DLI to induce GVHD with T reg depletion correlated with a loss of the DLI-induced GVL effect in long-term (day 100) complete donor chimeras. In contrast to the results from complete donor chimeras, GVL reactivity in day 100 mixed chimeras was robust after DLI. Loss of a DLI-induced GVL effect in long-term complete donor chimeras was attributed to the absence of host APCs because the infusion of exogenous host-type dendritic cells partially restored both DLI-induced GVL and GVH reactions in day 100 complete donor chimeras. The GVL and GVH reactions restored by infusion of host dendritic cells in day 100 complete donor chimeras were at least partially regulated by T reg cells because transient depletion of CD25 + cells increased both the GVL effect and the severity of GVHD after DLI. Taken together, these data suggest that T reg cells can regulate DLI-induced GVL and GVH reactions in both early and long-term complete donor chimeras, and a state of mixed chimerism is superior to complete donor chimerism because host-type APCs facilitate a DLI-induced GVL effect without severe GVHD.

Radiation dose determines the degree of myeloid engraftment after nonmyeloablative stem cell transplantation

A multivariate analysis of 121 dogs conditioned with 200, 100, or 50 cGy of total body irradiation (TBI) followed by hematopoietic stem cell transplantation from matched littermates showed that TBI dose was the only factor examined that was statistically significantly associated with the percentage of donor myeloid engraftment in stable long-term chimeras ( P = .008). To understand the direct effects of low-dose irradiation on hematopoietic stem/progenitor cells, nonirradiated and irradiated human CD34 + cells were evaluated for competitive repopulating ability in nonobese diabetic/severe combined immunodeficiency beta2m -/- mice. As expected, the results showed a radiation dose-dependent loss of competitive repopulating ability. Flow cytometric analysis indicated that, within a viable cell gate, there was reduced expression of P-selectin glycoprotein ligand-1 and L selectin on irradiated compared with nonirradiated CD34 + cells; this suggests that irradiated stem/progenitor cells may be compromised in their ability to home to or interact with the marrow microenvironment. However, the CD34 + /P-selectin glycoprotein ligand-1 dim cells also showed activation of caspase-3, indicating that they were destined to die. These results suggest that the TBI dose determines the degree of myeloid engraftment by compromising the resident stem/progenitor cell compartment.

Donor-specific tolerance in fully major histocompatibility major histocompatibility complex-mismatched limb allograft transplants under an anti-alphabeta T-cell receptor monoclonal antibody and cyclosporine A protocol.

Recent studies have demonstrated that treatment with alphabeta-T-cell receptor (TCR) monoclonal antibody and cyclosporine A (CsA) can extend survival in composite tissue allografts (CTA). The purpose of this study was to induce tolerance in fully major histocompatibility complex (MHC)-mismatched rat limb allografts under 7 days of a combined alphabeta-TCR-CsA protocol. The authors performed 30 hind-limb allotransplantations across the MHC barrier between Brown Norway donors (BN; RT1n) and Lewis recipients (LEW; RT1l). Isograft and allograft controls received no treatment. The experimental groups received monotherapy of alphabeta-TCR and CsA or a combination of alphabeta-TCR and CsA for 7 days only. Donor-specific tolerance and immunocompetence were determined by standard skin grafting in vivo and mixed lymphocyte reaction (MLR) in vitro. The efficacy of immunosuppressive therapy and the level of donor-specific chimerism were determined by flow cytometry. Long-term survival (>350 days) was achieved in allograft recipients (n=6) under the 7-day protocol of combined alphabeta-TCR-CsA. Donor-specific tolerance and immunocompetence of long-term chimeras were confirmed by acceptance of skin grafts from the donors and rejection of the third-party alloantigens (AxC Irish). At day 120, MLR demonstrated unresponsiveness to the host and donor antigens but strong reactivity against third-party alloantigens. Flow cytometry confirmed the high efficacy of immunosuppressive treatment and the development of donor-specific chimerism (7.6% of CD4+-RT1n+ cells, 1.3% of CD8+-RT1n+ cells, and 16.5% of CD45RA+-RT1n+ cells) in the periphery of tolerated recipients. Combined therapy of alphabeta-TCR-CsA for 7 days resulted in tolerance induction in fully MHC-mismatched rat hind-limb allografts. Tolerance was directly associated with stable, donor-specific chimerism.

Phenotype and function of hematopoietic stem cells.

Using an in vivo selection technique, we can isolate individual cells that can repopulate the hematopoietic system of a lethally irradiated murine recipient. These cells rapidly acquire a CD34 phenotype in the animal. Progenitors in our long-term chimeras are of donor type. We also have evidence that transplantation of limiting numbers (as low as a single cell) that have this long-term repopulating ability (LTRA) can self-renew. This is demonstrated by serial transplantation of marrow from engrafted recipients 11 months post transplant into new hosts for four additional months.

Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment.

Allogeneic bone marrow transplantation (in immunocompetent adults) has always required cytoreductive treatment of recipients with irradiation or cytotoxic drugs to achieve lasting engraftment at levels detectable by non-PCR-based techniques ('macrochimerism' or 'mixed chimerism'). Only syngeneic marrow engraftment at such levels has been achieved in unconditioned hosts. This requirement for potentially toxic myelosuppressive host pre-conditioning has precluded the clinical use of allogeneic bone marrow transplantation for many indications other than malignancies, including tolerance induction. We demonstrate here that treatment of naive mice with a high dose of fully major histocompatibility complex-mismatched allogeneic bone marrow, followed by one injection each of monoclonal antibody against CD154 and cytotoxic T-lymphocyte antigen 4 immunoglobulin, resulted in multi-lineage hematopoietic macrochimerism (of about 15%) that persisted for up to 34 weeks. Long-term chimeras developed donor-specific tolerance (donor skin graft survival of more than 145 days) and demonstrated ongoing intrathymic deletion of donor-reactive T cells. A protocol of high-dose bone marrow transplantation and co-stimulatory blockade can thus achieve allogeneic bone marrow engraftment without cytoreduction or T-cell depletion of the host, and eliminates a principal barrier to the more widespread use of allogeneic bone marrow transplantation. Although efforts have been made to minimize host pre-treatment for allogeneic bone marrow transplantation for tolerance induction, so far none have succeeded in eliminating pre-treatment completely. Our demonstration that this can be achieved provides the rationale for a safe approach for inducing robust transplantation tolerance in large animals and humans.

Influence of the additional injection of host-type bone marrow on the immune tolerance of minor antigen-mismatched chimeras: possible involvement of double-negative (natural killer) T cells.

It has previously been demonstrated that adding T cell-depleted (TCD) host bone marrow (BM) to an MHC-mismatched BM inoculum allows for induction of long-term stable chimeras without graft-versus-host disease (GVHD) even when non-TCD allogeneic BM was used. The present study was undertaken to investigate immune tolerance mechanisms in minor antigen-mismatched allogeneic BM chimeras when host-type BM was added to the BM inoculum. C3H (H2k, Thy 1.2, Mls 2a) recipients were conditioned with 9.5 gray (Gy) of total body irradiation. To exclude any interference with possible subclinical GVHD, 5x10(6) TCD AKR (H2k, Thy 1.1, Mls 1a) BM cells were injected with (syn + allo) or without (allo) 5x 10(6) TCD C3H BM cells. Chimerism, clonal deletion, and T lymphocyte subsets were scored using FACS and anti-mouse Thy, Vbeta6, Vbeta3, CD3, CD4, or CD8 monoclonal antibodies. The stability of tolerance was studied by investigating mixed lymphocyte reaction and cytotoxic T cell induction in chimeras after immunization with host, donor, or third-party (BALB/c) splenocytes. Breaking of chimerism was attempted by injecting nontolerant 40x10(6) host-type splenocytes 2 months after BM transplantation. Cytokines and Valpha14 mRNA were assayed using real time quantitative reverse transcriptase-polymerase chain reaction at 4 and 48 hr, respectively, after injection of nontolerant host-type splenocytes. Both groups of mice became long-term stable mixed chimeras without any clinical sign of GVHD. Neither group was able to produce antihost nor antidonor cytotoxic T cells, even after immunization. The addition of syngeneic BM to the allogeneic inoculum reduced the overall level of allogeneic chimerism (from approximately 70% or approximately 85% in peripheral blood lymphocytes and spleen, respectively, in allo chimeras versus approximately 35% and approximately 60% in syn + allo chimeras). Moreover, it resulted in complete clonal deletion of both host-reactive (Vbeta3) and donor-reactive (Vbeta6) lymphocytes in syn + allo chimeras in contrast to in allo chimeras, in which only donor-reactive lymphocytes were completely deleted. After nontolerant C3H splenocyte injection, high levels of interleukin 2 mRNA were produced and chimerism decreased in syn + allo chimeras. In contrast, in allo chimeras, this maneuver was followed by the production of higher levels of interleukin 4 and interferon-gamma, and of Valpha14 mRNA, as well as by the proliferation of CD3+CD4-CD8- (double-negative) T cells and by an increase of donor chimerism. The addition of host-type BM to the allogeneic inoculum has an influence on the level of chimerism, the extent of clonal deletion, and the reaction of chimeras after the injection of nontolerant host-type splenocytes. In the latter phenomenon, cytokine production and proliferation of Valpha14+ CD3+CD4-CD8- (double-negative, natural killer T) lymphocytes may be involved.

Anti-CD154 or CTLA4Ig obviates the need for thymic irradiation in a non-myeloablative conditioning regimen for the induction of mixed hematopoietic chimerism and tolerance.

Thymic irradiation (TI) or repeated administration of T cell-depleting monoclonal antibodies (TCD mAbs) is required in a previously described non-myeloablative regimen allowing allogeneic marrow engraftment with stable mixed chimerism and tolerance. As both treatments might be associated with toxicity in the clinical setting, we evaluated whether T-cell costimulatory blockade could be used to replace them. C57BL/6 mice received depleting anti-CD4 and anti-CD8 mAbs on day -5, 3 Gy whole body irradiation (day 0), and 15x10(6) fully MHC-mismatched, B10.A bone marrow cells. In addition, hosts were injected with an anti-CD154 mAb (day 0) and/or CTLA4Ig (day +2). Chimerism in peripheral blood was followed by flow cytometric (FACS) analysis, and tolerance was assessed by skin grafting, and also by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. The frequency of certain Vbeta families was determined by FACS to assess deletion of donor-reactive T cells. Chimerism was transient and tolerance was not present in animals receiving TCD mAbs on day -5 without costimulatory blockade. The addition of anti-CD154 and CTLA4Ig, alone or in combination, reliably permitted induction of high levels of stable (>6 months) multi-lineage chimerism, with specific tolerance to skin grafts and donor antigens by MLR and CML assays. Long-term chimeras showed deletion of donor-reactive CD4+ peripheral blood lymphocytes, splenocytes, and mature thymocytes. Administration of TCD mAbs only 1 day before bone marrow transplantation plus anti-CD154 also allowed induction of permanent chimerism and tolerance. One injection of anti-CD154 or CTLA4Ig overcomes the need for TI or prolonged host TCD in a preclinical model for the induction of mixed chimerism and deletional tolerance and thus further decreases the toxicity of this protocol. Achievement of tolerance with conditioning given over 24 hr suggests applicability to cadaveric organ transplantation.

Role of intrathymic rat class II+ cells in maintaining deletional tolerance in xenogeneic rat-->mouse bone marrow chimeras.

Mixed xenogeneic bone marrow chimerism and tolerance can be induced in mice conditioned with a nonmyeloablative regimen followed by injection of T cell-depleted rat bone marrow cells. We hypothesized that, despite a gradual decline in rat hematopoiesis observed in these chimeras, as long as rat class II+ antigen-presenting cells remain in their thymi, tolerance will persist as a result of deletion of donor-reactive thymocytes. The level of chimerism and of mouse Vbeta5 and Vbeta11 T-cell deletion was followed over time. These results were correlated with the presence of rat class II+ cells in the thymus by immunohistochemistry and the presence of tolerance in long-term chimeras by in vivo and in vitro assays. (1) Proliferation and cytotoxicity assays, as well as skin graft survival, demonstrated the presence of specific tolerance to host and to donor rat, with normal reactivity to third-party rat and mouse stimulators, even as late as 85 weeks after bone marrow transplantation. (2) The absence of mature Vbeta5+ and Vbeta11+ host T cells in the thymus and periphery was always associated with the presence of rat class II+ cells in the thymus, and incomplete deletion of T cells expressing these Vbeta families was observed in thymi in which rat class II+ cells were not detectable. Donor-specific T-cell tolerance is maintained during the period when donor-type reconstitution declines, and is most likely mediated by intrathymic clonal deletion of T cells that recognize antigens expressed on class II+ rat cells.

Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning.

Donor-specific tolerance induced by bone marrow transplantation (BMT) would allow organ allografting without chronic immunosuppressive therapy. However, the toxicity of conditioning regimens used to achieve marrow engraftment has precluded the clinical use of BMT for tolerance induction. We have developed a BMT strategy that achieves alloengraftment without toxic or myelosuppressive host conditioning. B6 mice received depleting anti-CD4 and anti-CD8 monoclonal antibodies, local thymic irradiation, and a high-dose (174 x 10(6)) of major histocompatibility (MHC)-mismatched B10.A bone marrow cells (BMCs) divided over days 0 through 4. High levels of donor cells were observed among white blood cells (WBCs) of all lineages. Permanent, multilineage mixed chimerism; donor-specific skin-graft tolerance; and in vitro tolerance were observed in most animals. Large numbers of donor class II(high) cells were detected in thymuses of long-term chimeras, and their presence was associated with intrathymic deletion of donor-reactive host thymocytes. The treatment was not associated with significant myelosuppression, toxicity, or graft-versus-host disease (GVHD). Thus, high levels of allogeneic stem-cell engraftment can be achieved without myelosuppressive host conditioning. As stem-cell mobilization and in vitro culture techniques have increased the feasibility of administering high doses of hematopoietic cells to humans, this approach brings hematopoietic cell transplantation closer to clinical use for the induction of central deletional T-cell tolerance.

SCID Mouse as a Model for Transplantation Studies

Mice expressing the severe combined immunodeficiency trait (SCID) lack functional T and B lymphocytes and have been widely used for the study of B-cell development and for cancer and HIV research. The purpose of this study was to evaluate the SCID mouse as a potential model for T-cell maturation and transplantation studies. C3H/HEN SCID mice screened by fluorescence-activated cell sorting (FACS) and radial immunodiffusion assay (RID) were verified homozygous recessive if CD3−, CD22−, and serum (IgG) <5 mg/liter. C3H/HEN SCID mice pretreated with 250 R total-body γ irradiation were reconstituted with 2.5 to 4 × 107donor bone marrow cells derived from [syngeneic (syn): male wild-type C3H/HEN, allogeneic (allo): male BALB/c or C57BL/6) mice by intravenous injection. Four weeks post-transplant, engraftment was determined by FACS; repopulation of blood, thymus, and spleen; RID; and histologic evaluation. Immune function against donor, recipient, and third-party antigen was assayedin vitroby mixed lymphocyte response (MLR) andin vivoby full-thickness skin grafting. Greater than 90% of both syngeneic and allogeneic reconstitutions expressed CD3+, CD4+, CD8+, and CD22+cells of donor origin in peripheral blood and spleen. FACS analyses of lymphocyte subpopulations in blood and spleen were not significantly different between reconstituted SCID mice and wild-type C3H/HEN or BALB/c controls, with engraftment stable for >4 months. No evidence of graft-versus-host disease was observed in stable, long-term (>4 months postreconstitution) chimeras. White blood cell, total thymocyte, and total splenocyte counts were significantly elevated (P< 0.05: ANOVA, Student'st) following reconstitution of homozygous SCID mice to levels found in wild-type controls. Serum (IgG) for reconstituted allo- and syn-SCID mice was consistently >150 mg/liter (n= 22), with histologic lymphocyte engraftment of spleen, duodenum, and thymus. Histologic examination of lymphocyte engraftment in spleen, duodenum, and thymus was indistinguishable from normal controls in SCID mice after reconstitution. Prior to reconstitution, scant lymphoid cells were observed at these sites. Allo-SCID splenocyte response against third-party antigen was significantly elevated (P< 0.01: ANOVA, Student'st) when compared with donor and recipient antigen response with a proliferation index (PI) comparable to wild-type controls. Unreconstituted SCID mice were unresponsive.In vivo,allo-SCID mice demonstrated rejection of only third-party skin grafts between postoperative days 9 and 14 (controls: postoperative days 7 and 11). The SCID mouse model demonstratesin vitroandin vivoB- and T-cell immune function comparable to that of wild-type mice and provides a useful model for T-cell maturation and transplantation studies.

Thymic dependence of loss of tolerance in mixed allogeneic bone marrow chimeras after depletion of donor antigen. Peripheral mechanisms do not contribute to maintenance of tolerance.

A nonmyeloablative conditioning regimen has recently been developed that allows allogeneic marrow engraftment with induction of permanent mixed chimerism and donor-specific tolerance across fully MHC-mismatched allogeneic barriers. We recently demonstrated that tolerance can be broken in these chimeras by administration of an anti-donor class I-specific monoclonal antibody that eliminates donor hematopoietic cells. We have now investigated the role of the thymus in the loss of tolerance observed when chimerism is eliminated in this manner. Mixed chimeras were prepared in B10 (H2b) recipients by treatment with depleting anti-CD4 and anti-CD8 mAbs, 3-Gy whole body irradiation, and 7-Gy thymic irradiation, followed by B10.A (H2a) bone marrow transplantation. Chimeras were thymectomized 7 weeks later, and were either untreated or were depleted of donor cells with anti-donor class I (Dd-specific) mAb 34-2-12. Control chimeras that were not thymectomized also received anti-donor monoclonal antibodies or no further treatment. Of the four groups, only euthymic animals that were depleted of donor antigen showed a loss of tolerance, as evidenced by rejection of B10.A skin grafts. In contrast to untreated control and thymectomized, anti-Dd-treated chimeras, these euthymic anti-Dd-treated chimeras showed significant recovery of Vbeta11+ T cells, which can recognize Mtv antigens presented by donor I-E molecules. The requirement for a thymus for loss of tolerance in the absence of donor antigen was verified in an adoptive transfer model, in which chimera (B10.A-->B10) spleen cells were depleted of donor-type cells ex vivo, adoptively transferred into B6 nu/nu mice, and then further depleted of donor-type antigen with monoclonal antibody treatment in vivo. These B6 nu/nu mice maintained donor-specific tolerance to B10.A skin grafts. The absence of active suppression as a potent mechanism of tolerance in long-term mixed chimeras was confirmed by the loss of mixed chimerism and of tolerance that was readily induced by injection of naive host-type spleen cells. Together, our results suggest that in mixed allogeneic chimeras, intrathymic clonal deletion, and not peripheral suppression or anergy, is the major mechanism maintaining donor-specific tolerance.

Lack of GVHD across classical, single minor histocompatibiliTy (miH) locus barriers in mice.

To determine whether a disparity at a single miH genetic loci are sufficient to generate GVHD in mice, we focused on well-known genetic alleleic differences at the miH gene loci, H3 and H4. For H3 congenic GVHD studies, C57BL/10 (H2b) mice were used as recipients of miH-disparate B10.LP-H3b donor cells. For H4 congenic GVHD studies, C57BL/10 were used as recipients for miH-disparate B10.129 (21M)-H4b. To overcome the low frequency of miH-reactive CTLs in naive mice, multiple immunizations of the donor strains with host lymphohematopoietic cells were used. Peripheral blood cells from immunized mice were shown to have potent CTL activity against their respective host-type stimulator cells when analyzed 1 week prior to obtaining donor splenocytes for GVHD induction. Lethally irradiated C57BL/6 recipients of either 50 X 10(6) donor B10.LP-H3b or B10.129 (21M)-H4b splenocytes did not develop acute or chronic GVHD as assessed by monitoring the animals for survival, weight loss, splenic flow cytometry, and histological examination of skin, liver, colon, and lung in long-term survivors. Engraftment was documented in long-term chimeras in both strain combinations by using the post-BMT cells as alloantigen targets for cloned CTL lines specific for donor and not host-type miH antigens (H3b or H4b). On day 6 post-BMT, donor antihost CTL activity could not be detected in the spleen, although third-party responses were intact. These results suggest a rapid downregulation or disappearance of miH antigen-reactive CTL after BMT. These data have implications for the use of in vitro assays to predict GVHD risk in recipients of miH loci-disparate donor grafts.

Oligoclonal expansion of CTLs directed against a restricted number of dominant minor histocompatibility antigens in hemopoietic chimeras.

To understand how T cells respond to allogeneic minor histocompatibility Ags (MiHAs), we studied the fate of Thy-1.1+ lymphocytes, as well as their TCR usage and functional activity, in irradiated LP (Thy-1.2+) recipients transplanted with a mixture of C57BL/6 (Thy-1.2+) hemopoietic progenitors supplemented with either low or high numbers of B6.PL lymphocytes (Thy-1.1+). Mice transplanted with low numbers of T cells experienced a dramatic expansion (> or = 10(5)-fold) of donor Thy-1.1+/CD8+ cells during the first 15 days post-transplant. Flow-cytometric analysis and sequencing of junctional nucleotide sequences showed that the nature of this expansion was oligoclonal and involved primarily one or a few clones using V beta 5.1 or V beta 8.1 TCR elements. Expanded T lymphocyte populations displayed MHC-restricted cytotoxicity for a restricted number of MiHAs, that were found in only two peaks following fractionation of LP MiHAs by reverse-phase HPLC. Expansion of donor T cells was limited to the spleen, and short-lived in these recipients that became healthy long-term chimeras without any signs of graft-vs-host disease (GVHD). In contrast, mice transplanted with high numbers of T cells (GVHD+) showed proliferation of Thy-1.1+ donor cells not only in the spleen, but also in the thymus, and recipients died rapidly of GVHD. These results show that: 1) in the MiHA-incompatible transplantation setting, lack of GVHD cannot be explained simply by the absence of antihost T cell responses, 2) GVHD+ recipients present a massive thymic infiltration by donor mature T lymphocytes, and 3) antihost T cell responses are oligoclonal in nature and targeted to only a few MiHAs. These findings shed new light on the pathogenetic mechanisms involved in GVHD and on the selection of the T cell repertoire involved in response to immunodominant MiHAs.

Split tolerance in spleen chimeras.

Transferring small doses of T cells to heavily irradiated F1 mice expressing isolated MHC class I or class II differences invariably leads to rapid death from graft-vs-host disease (GVHD). Paradoxically, GVHD is mild or absent when irradiated F1 mice are reconstituted with large doses of unseparated parental strain spleen cells. This applies when bulk populations of B6 spleen cells are transferred to irradiated class II-different (B6 x bm12)F1 mice or class I-different (B6 x bm1)F1 mice. In this study, we examined whether the donor T cells in long-term spleen chimeras become tolerant to host MHC Ags. On the basis of skin-allograft rejection and induction of GVHD on adoptive transfer, the results show that the donor T cells display strong tolerance to host antigens; this applies to CD4+ cells in class II-different chimeras and to CD8+ cells in class I-different chimeras. In marked contrast to the profound tolerance seen by these in vivo parameters, little or no tolerance is observed in standard in vitro assays. The results illustrate that typical in vitro tests for alloreactivity are an imprecise guide to physiologic tolerance of T cells in vivo.