CD40L-stimulated B cells for ex-vivo expansion of polyspecific non-human primate regulatory T cells for translational studies.

The therapeutic applications of regulatory T cells (Tregs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific Tregs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded Tregs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of Treg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific Tregs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.

Sublimation in bright spots on (1) Ceres.

The dwarf planet (1) Ceres, the largest object in the main asteroid belt with a mean diameter of about 950 kilometres, is located at a mean distance from the Sun of about 2.8 astronomical units (one astronomical unit is the Earth-Sun distance). Thermal evolution models suggest that it is a differentiated body with potential geological activity. Unlike on the icy satellites of Jupiter and Saturn, where tidal forces are responsible for spewing briny water into space, no tidal forces are acting on Ceres. In the absence of such forces, most objects in the main asteroid belt are expected to be geologically inert. The recent discovery of water vapour absorption near Ceres and previous detection of bound water and OH near and on Ceres (refs 5-7) have raised interest in the possible presence of surface ice. Here we report the presence of localized bright areas on Ceres from an orbiting imager. These unusual areas are consistent with hydrated magnesium sulfates mixed with dark background material, although other compositions are possible. Of particular interest is a bright pit on the floor of crater Occator that exhibits probable sublimation of water ice, producing haze clouds inside the crater that appear and disappear with a diurnal rhythm. Slow-moving condensed-ice or dust particles may explain this haze. We conclude that Ceres must have accreted material from beyond the 'snow line', which is the distance from the Sun at which water molecules condense.

Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model.

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-γ) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Macrochimerism in Intestinal Transplantation: Association With Lower Rejection Rates and Multivisceral Transplants, Without GVHD.

Blood chimerism has been reported sporadically among visceral transplant recipients, mostly in association with graft-vs-host disease (GVHD). We hypothesized that a higher degree of mixed chimerism would be observed in multivisceral (MVTx) than in isolated intestinal (iITx) and isolated liver transplant (iLTx) recipients, regardless of GVHD. We performed a longitudinal prospective study investigating multilineage blood chimerism with flow cytometry in 5 iITx and 4 MVTx recipients up to one year posttransplant. Although only one iITx patient experienced GVHD, T cell mixed chimerism was detected in 8 out of 9 iITx/MVTx recipients. Chimerism was significantly lower in the four subjects who displayed early moderate to severe rejection. Pre-formed high-titer donor-specific antibodies, bound in vivo to the circulating donor cells, were associated with an accelerated decline in chimerism. Blood chimerism was also studied in 10 iLTx controls. Among nonsensitized patients, MVTx recipients exhibited greater T and B cell chimerism than either iITx or iLTx recipients. Myeloid lineage chimerism was present exclusively among iLTx and MVTx (6/13) recipients, suggesting that its presence required the hepatic allograft. Our study demonstrates, for the first time, frequent T cell chimerism without GVHD following visceral transplantation and a possible relationship with reduced rejection rate in MVTx recipients.

Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Allospecific rejection of MHC class I-deficient bone marrow by CD8 T cells.

Avoidance of long-term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low-toxicity, nonimmunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC-mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti-CD154 and T cell-depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of preexisting alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC Class I-deficient and Class I/Class II-deficient marrow in a CD8 T cell-dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC Class I-deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC Class I-deficient donor cells in association with rejection. These data implicate a novel CD8 T cell-dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a T cell receptor-independent manner, of Class I-deficient donor cells.

Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression.

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5-11.4 years, while three required reinstitution of IS after 5-8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

B-cell-dependent memory T cells impede nonmyeloablative mixed chimerism induction in presensitized mice.

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor-specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B-cell-deficient µMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long-term (LT) multilineage chimerism and tolerance to donor-type skin. To apply these regimens in wild-type (WT) animals while avoiding antibody-mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T-cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized µMT B6 mice had diminished memory T-cell responses compared to WT B6 mice. These data implicate T-cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.

Mechanisms of donor-specific tolerance in recipients of haploidentical combined bone marrow/kidney transplantation.

We recently reported long-term organ allograft survival without ongoing immunosuppression in four of five patients receiving combined kidney and bone marrow transplantation from haploidentical donors following nonmyeloablative conditioning. In vitro assays up to 18 months revealed donor-specific unresponsiveness. We now demonstrate that T cell recovery is gradual and is characterized by memory-type cell predominance and an increased proportion of CD4⁺ CD25⁺ CD127⁻ FOXP3⁺ Treg during the lymphopenic period. Complete donor-specific unresponsiveness in proliferative and cytotoxic assays, and in limiting dilution analyses of IL-2-producing and cytotoxic cells, developed and persisted for the 3-year follow-up in all patients, and extended to donor renal tubular epithelial cells. Assays in two of four patients were consistent with a role for a suppressive tolerance mechanism at 6 months to 1 year, but later (≥ 18 months) studies on all four patients provided no evidence for a suppressive mechanism. Our studies demonstrate, for the first time, long-term, systemic donor-specific unresponsiveness in patients with HLA-mismatched allograft tolerance. While regulatory cells may play an early role, long-term tolerance appears to be maintained by a deletion or anergy mechanism.

Acute renal endothelial injury during marrow recovery in a cohort of combined kidney and bone marrow allografts.

An idiopathic capillary leak syndrome ('engraftment syndrome') often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here, we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10-16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68(+) MPO(+) mononuclear cells and CD3(+) CD8(+) T cells, the latter with a high proliferative index (Ki67(+) ). B cells (CD20(+) ) and CD4(+) T cells were not detectable, and NK cells were rare. XY FISH showed that CD45(+) cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti-rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2-4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery.

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.

Skin graft tolerance across a discordant xenogeneic barrier.

Specific T-cell tolerance may be essential for successful xenotransplantation in humans. Grafting of thymectomized, T cell-depleted normal mice with xenogeneic fetal pig thymus and liver (FP THY/LIV) tissue results in the recovery of functional CD4 antigen-positive cells. We have tested T-cell tolerance by skin grafting. Donor-matched pig skin survived permanently (> 200 days), whereas allogeneic mouse skin was rapidly rejected. Nontolerant control mice rejected pig skin within 26 days. Both porcine and murine histocompatibility class IIhigh cells were detected in long-term thymus grafts, and T-cell repertoire analyses suggested that tolerance to both donors and recipients developed, at least in part, by intragraft clonal deletion. This study demonstrates the principle that tolerance, measured by the stringent criterion of skin grafting, can be induced across a widely disparate species barrier.

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.

Differentiation of the asteroid Ceres as revealed by its shape.

The accretion of bodies in the asteroid belt was halted nearly 4.6 billion years ago by the gravitational influence of the newly formed giant planet Jupiter. The asteroid belt therefore preserves a record of both this earliest epoch of Solar System formation and variation of conditions within the solar nebula. Spectral features in reflected sunlight indicate that some asteroids have experienced sufficient thermal evolution to differentiate into layered structures. The second most massive asteroid--4 Vesta--has differentiated to a crust, mantle and core. 1 Ceres, the largest and most massive asteroid, has in contrast been presumed to be homogeneous, in part because of its low density, low albedo and relatively featureless visible reflectance spectrum, similar to carbonaceous meteorites that have suffered minimal thermal processing. Here we show that Ceres has a shape and smoothness indicative of a gravitationally relaxed object. Its shape is significantly less flattened than that expected for a homogeneous object, but is consistent with a central mass concentration indicative of differentiation. Possible interior configurations include water-ice-rich mantles over a rocky core.

Effects of T cell depletion in radiation bone marrow chimeras. II. Requirement for allogeneic T cells in the reconstituting bone marrow inoculum for subsequent resistance to breaking of tolerance.

The ability of normal recipient-type lymphocytes to break tolerance in long-term allogenic radiation chimeras has been investigated. Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic T cell-depleted (TCD) bone marrow (BM) has previously been shown to lead to mixed chimerism and permanent, specific tolerance to donor and host alloantigen (3-5). If allogeneic T cells are not depleted from the reconstituting inoculum, complete allogeneic chimerism results; however, no clinical evidence for GVHD is observed, presumably due to the protective effect provided by syngeneic TCD BM. This model has now been used to study the effects of allogenic T cells administered in reconstituting BM inocula on stability of long-term tolerance. We have attempted to break tolerance in long-term chimeras originally reconstituted with TCD or non-TCD BM by challenging them with inocula containing normal, nontolerant recipient strain lymphocytes. tolerance was broken with remarkable ease in recipients of mixed marrow inocula in which both original BM components were TCD. In contrast, tolerance in chimeras originally reconstituted with non-TCD allogeneic BM was not affected by such inocula. Susceptibility to loss of chimerism and tolerance was not related to initial levels of chimerism per se, but rather to T cell depletion of allogeneic BM, since chimeras reconstituted with TCD allogeneic BM alone (mean level of allogeneic chimerism 98%) were as susceptible as mixed chimeras to the tolerance-breaking effects of such inocula. The possible contribution of GVH reactivity to this resistance was investigated using an F1 into parent strain combination. In these animals, the use of non-TCD F1 BM inocula for reconstitution did not lead to resistance to the tolerance-breaking effects of recipient strain splenocytes. These results suggest that the ability of T cells in allogeneic BM inocula to confer resistance to late graft failure may be related to their graft-vs.-host reactivity, even in situations in which they do not cause clinical GVHD. These findings may have relevance to the mechanism whereby T cell depletion of allogeneic BM leads to an increased incidence of late graft failure in clinical BM transplantation situations.

Graft-versus-host-related immunosuppression is induced in mixed chimeras by alloresponses against either host or donor lymphohematopoietic cells.

Graft-vs.-host (GVH)-related immunosuppression has previously been demonstrated in F1 rodent recipients of parental lymphoid cells, and has been thought to result from an immunologic attack of the donor against the host. Since all cells of such F1 recipients could potentially bear target class I MHC alloantigens, it has not previously been possible to determine precisely the target tissues responsible for development of GVH-related effects. In the present studies we have used mixed allogeneic chimeras as recipients of host or donor-strain lymphocyte inocula, and have made the surprising observation that "GVH-induced" immune unresponsiveness does not require GVH reactivity, per se, but develops in the presence of a one-way alloresponse against lymphohematopoietic cells in either the GVH or the host-versus-graft direction.

In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment.

Previous work from this laboratory has demonstrated that T cell-depleted (TCD) syngeneic marrow can delay, but not prevent, the mortality from acute graft-vs.-host disease (GVHD) caused by MHC-mismatched lymphoid cells administered to lethally irradiated mice. We demonstrate here that a protective effect against GVHD is also observed after in vivo treatment with IL-2. Administration of 10,000-50,000 U of IL-2 twice daily for the first 5 d after bone marrow transplantation markedly reduced the mortality from both acute and chronic GVHD induced across complete MHC barriers in lethally irradiated mice, and frequently led to long-term survival. Complete allogeneic reconstitution was demonstrated in all long-term survivors of this treatment regimen. While either IL-2 or TCD syngeneic marrow administered alone was protective in some experiments, the maximal protective effect was observed after administration of both IL-2 and TCD syngeneic marrow, especially when the effects of IL-2 were suboptimal. The timing of IL-2 administration was critical to this beneficial effect, since a delay of 7 d in commencing IL-2 treatment was associated with accelerated GVHD mortality. This new approach to the prevention of GVHD permits the administration of allogeneic T cells, and may therefore avoid the increased incidence of graft failure and loss of antileukemic effects associated with the T cell depletion of allogeneic marrow, which is otherwise required for the prevention of GVHD.

Specific tolerance induction across a xenogeneic barrier: production of mixed rat/mouse lymphohematopoietic chimeras using a nonlethal preparative regimen.

The development of safe methods for inducing donor-specific tolerance across xenogeneic barriers could potentially relieve the critical shortage of allograft donors that currently limits the applicability of organ transplantation. We report here that such tolerance can be induced in a xenogeneic combination (rat----mouse) using a nonmyeloablative and nonlethal preparative regimen. Successful induction of chimerism and donor-specific transplantation tolerance required pretreatment of recipients with monoclonal antibodies (mAbs) against NK1.1, Thy-1.2, CD4 and CD8, followed by administration of 3 Gy whole body radiation (WBI), 7 Gy thymic irradiation, and infusion of T cell-depleted rat bone marrow cells (BMC). Rat cells appeared among peripheral blood lymphocytes (PBL) of such recipients by 2-3 wk, and rat T cells by 2-5 wk following bone marrow transplantation (BMT). Donor-type rat skin grafts placed 4 mo after BMT were accepted, while simultaneously placed non-donor-type rat skin grafts were promptly rejected. In addition to its clinical potential, the ability to induce donor-specific tolerance across xenogeneic barriers using such a nonlethal preparative regimen provides a valuable model for the study of mechanisms of xenogeneic transplantation tolerance.

Hematopoietic cells and radioresistant host elements influence natural killer cell differentiation.

Radioresistant host elements mediate positive selection of developing thymocytes, whereas bone marrow-derived cells induce clonal deletion of T cells with receptors that are strongly autoreactive. In contrast to T cell development, little is known about the elements governing the natural killer (NK) cell repertoire, which, similar to the T cell repertoire, differs between individuals bearing different major histocompatibility complex (MHC) phenotypes. We have used murine bone marrow transplantation models to analyze the influence of donor and host MHC on an NK cell subset. We examined the expression of Ly-49, which is strongly expressed on a subpopulation of NK cells of H-2b mice, but not by NK cells of H-2a mice, probably because of a negative effect induced by the interaction of Ly-49 with Dd. To evaluate the effect of hematopoietic cell H-2a expression on Ly-49 expression of H-2b NK cells, we prepared mixed allogeneic chimeras by administering T cell-depleted allogeneic (B10.A, H-2a) and host-type (B10, H-2b) marrow to lethally irradiated B10 mice, or by administering B10. A marrow to B10 recipients conditioned by a nonmyeloablative regimen. Expression of H-2a on bone marrow-derived cells was sufficient to downregulate Ly-49 expression on both H-2a and H-2b NK cells. This downregulation was thymus independent. To examine the effect of H-2a expressed only on radioresistant host elements, we prepared fully allogeneic chimeras by administering B10 bone marrow to lethally irradiated B10.A recipients. B10 NK cells of these fully allogeneic chimeras also showed downregulation of Ly-49 expression. The lower level of H-2a expressed on H-2b x H-2a F1 cells induced more marked downregulation of Ly-49 expression on B10 NK cells when presented on donor marrow in mixed chimeras than when expressed only on radioresistant host cells. Our studies show that differentiation of NK cells is determined by interactions with MHC molecules expressed on bone marrow-derived cells and, to a lesser extent, by MHC antigens expressed on radioresistant host elements.