Characterization of Human CD8(+)TCR(-) Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγ(null) Mouse Model.

CD8(+)/TCR(-) facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8(+)/TCR(-)/CD56 negative (CD56(neg)); the remainder are CD8(+)/TCR(-)/CD56 bright (CD56(bright)). The CD56(neg) FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL-2 receptor γ-chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56(neg) FC subpopulation promotes rapid reconstitution of donor HSPCs without graft-versus-host disease (GVHD); recipients of CD56(bright) FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56(neg) or CD56(bright) FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56(bright) FCs with HSPCs upregulates cathelicidin and ß-defensin 2, factors that prime responsiveness of HSPCs to stromal cell-derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

Regulatory B cells: the new "it" cell.

Regulatory B cells (Breg) are a subpopulation of B cells that play a suppressive role in the immune system. The mechanism of how these immune cells perform their effects has been explored by experiments in mice and in humans. Intracellular staining for interleukin 10 continues to be a consistent and reproducible method of identifying Breg in mouse and human studies. The lack of Breg is associated with a worsening of several autoimmune diseases such as collagen-induced arthritis, systemic lupus erythematosus, and experimental autoimmune encephalomyelitis in murine studies. The purpose of this review is to provide a concise summary of the role of Breg in the immune system, including the most recently studied cell surface markers associated with Breg, and to describe the role of Breg in the etiology of several autoimmune diseases, the current understanding of Breg development, their role in the development of autoimmune diseases, and their role in inducing tolerance after transplantation.

Evolving approaches of hematopoietic stem cell-based therapies to induce tolerance to organ transplants: the long road to tolerance.

The immunoregulatory properties of hematopoietic stem cells (HSCs) have been recognized for more than 60 years, beginning in 1945, when Owen reported that genetically disparate freemartin cattle sharing a common placenta were red blood cell chimeras. In 1953, Billingham, Brent, and Medawar demonstrated that murine neonatal chimeras prepared by infusion of donor-derived hematopoietic cells exhibited donor-specific tolerance to skin allografts. Various approaches using HSCs in organ transplantation have gradually brought closer to reality the dream of inducing donor-specific tolerance in organ transplant recipients. Several hurdles needed to be overcome, especially the risk of graft-versus-host disease (GVHD), the toxicity of ablative conditioning, and the need for close donor-recipient matching. For wide acceptance, HSC therapy must be safe and reproducible in mismatched donor-recipient combinations. Discoveries in other disciplines have often unexpectedly and synergistically contributed to progress in this area. This review presents a historic perspective of the quest for tolerance in organ transplantation, highlighting current clinical approaches.

Immunology of vascularized composite allotransplantation: a primer for hand surgeons.

Vascularized composite allotransplantation is a recent innovation in the fields of transplantation surgery, plastic and reconstructive surgery, and orthopedic surgery. The success of hand and face transplantation has been based on extensive experience in solid organ transplantation. Advances in understanding the immunology of transplantation have had a major role in achieving excellent results in this new field. The purpose of this article is to introduce the basics of human immunology (innate and adaptive systems) and the immunological basis of human transplantation (the importance of human leukocyte antigen, direct and indirect pathways of antigen recognition, the 3 signals for T-cell activation, and mechanisms and types of allograft rejection) and focus on the mode of action of immunosuppressive drugs that have evolved as the mechanisms and pathways for rejection have been defined through research. This includes recent studies involving the use of costimulatory blockade, regulatory T cells, and tolerance induction that have resulted from research in understanding the mechanisms of immune recognition and function.

Vascularized composite allotransplantation at a crossroad.

Vascularized composite allotransplantation is a relatively young field that has shown significant growth in the past decade. The subspecialty offers opportunities that are not available with solid organ transplants. However, the field also faces significant hurdles in increasing clinical volumes. The development of innovative immune-reduction strategies will likely determine the pace and direction of growth in the field in the years to come.

Sensitization to minor antigens is a significant barrier in bone marrow transplantation and is prevented by CD154:CD40 blockade.

Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation rejection. The mechanism of sensitization to minor histocompatibility antigens (Mi-HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi-HAg to study the Mi-HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi-HAg disparate B10.BR skin grafts. Adaptive humoral (B-cells) and cellular (T cells) responses to Mi-HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC-sensitized recipients. Sera from Mi-HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi-HAg peptides were broadly shared between mouse strains. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B-cell reactivity to Mi-HAg. Moreover, anti-CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi-HAg. Taken together, Mi-HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 costimulatory blockade.

Composite tissue allotransplantation: current challenges.

Composite tissue allotransplantation (CTA) in the clinic is taking firm root. Success at hand, face, knee, trachea, and laryngeal transplantation has led to widespread interest and increasing application. Despite this, skepticism is common, particularly in the realm of reconstructive surgeons. The risks of immunosuppression remain a barrier to the advancement of the field, as these are perceived by many to be prohibitive. Significant progress in the field require the development of newer immunosuppressive agents with less toxicity and methods to achieve donor specific tolerance. This review focuses on the current state of CTA-both in the clinic and the laboratory. A thorough understanding of the immunology of CTA will allow the widespread application of this promising field.

Composite tissue allotransplantation: past, present and future-the history and expanding applications of CTA as a new frontier in transplantation.

Composite tissue allotransplantation (CTA) transplantation is currently being performed with increasing frequency in the clinic. The feasibility of the procedure has been confirmed in over 40 successful hand transplants, 3 facial reconstructions, and vascularized knee, esophageal, abdominal wall, and tracheal allografts. The toxicity of chronic, nonspecific immunosuppression remains a major limitation to the widespread availability of CTA and is associated with opportunistic infections, nephrotoxicity, end-organ damage, and an increased rate of malignancy. Methods to reduce or eliminate the requirement for immunosuppression would represent a significant step forward in the field. Mixed chimerism induces tolerance to solid organ and tissue allografts, including CTA. This overview focuses on the history and expanding applications of CTA as a new frontier in transplantation, and considers the important hurdles that must be overcome through research to allow widespread clinical application.

The history of human composite tissue allotransplantation.

Restoration of amputations and disfigurement are represented in ancient mythology, but the modern history of composite tissue allotransplantation begins with World War II injuries that generated seminal immunologic experiments by Medawar and co-workers. These studies led to the first successful human allografts in the 1950s by Peacock with composite tissue and Murray and co-workers with solid organs. Pharmacologic immunosuppression brought rapid growth of solid organ transplantation over the next 50 years, but composite tissue transplantation virtually disappeared. This evolution was judged to be a consequence of the greater antigenicity of skin, which that was insurmountable by the available immunosuppression. In the mid-1990s, progress in immunosupression allowed skin-bearing grafts, led by successful hand transplants, which produced a renaissance in composite tissue allotransplantation. Since then, graft types have expanded to over 10, and graft numbers to over 150, with success rates that equal or exceed solid organs. The field has emerged as one of the most exciting in contemporary medicine, although accompanied by substantial challenges and controversy. This paper reviews the origins and progress of this field, assessing its potential for future evolution.

Composite tissue transplantation: a rapidly advancing field.

Composite tissue allotransplantation (CTA) is emerging as a potential treatment for complex tissue defects. It is currently being performed with increasing frequency in the clinic. The feasibility of the procedure has been confirmed through 30 hand transplantation, 3 facial reconstructions, and vascularized knee, esophageal, and tracheal allografts. A major drawback for CTA is the requirement for lifelong immunosuppression. The toxicity of these agents has limited the widespread application of CTA. Methods to reduce or eliminate the requirement for immunosuppression and promote CTA acceptance would represent a significant step forward in the field. Multiple studies suggest that mixed chimerism established by bone marrow transplantation promotes tolerance resulting in allograft acceptance. This overview focuses on the history and the exponentially expanding applications of the new frontier in CTA transplantation: immunology associated with CTA; preclinical animal models of CTA; clinical experience with CTA; and advances in mixed chimerism-induced tolerance in CTA. Additionally, some important hurdles that must be overcome in using bone marrow chimerism to induce tolerance to CTA are also discussed.

Cells enriched in markers of neural tissue-committed stem cells reside in the bone marrow and are mobilized into the peripheral blood following stroke.

The concept that bone marrow (BM)-derived cells participate in neural regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. We recently reported that the BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs), including neural TCSCs. Here, we report that these cells not only express neural lineage markers (beta-III-tubulin, Nestin, NeuN, and GFAP), but more importantly form neurospheres in vitro. These neural TCSCs are present in significant amounts in BM harvested from young mice but their abundance and responsiveness to gradients of motomorphogens, such as SDF-1, HGF, and LIF, decreases with age. FACS analysis, combined with analysis of neural markers at the mRNA and protein levels, revealed that these cells reside in the nonhematopoietic CXCR4+/Sca-1+/lin-/CD45 BM mononuclear cell fraction. Neural TCSCs are mobilized into the peripheral-blood following stroke and chemoattracted to the damaged neural tissue in an SDF-1-CXCR4-, HGF-c-Met-, and LIF-LIF-R-dependent manner. Based on these data, we hypothesize that the postnatal BM harbors a nonhematopoietic population of cells that express markers of neural TCSCs that may account for the beneficial effects of BM-derived cells in neural regeneration.

Impact of bone marrow transplantation on type I diabetes.

Type I diabetes is a systemic autoimmune disease. Evidence is accumulating that autoimmune diseases such as type I diabetes are linked to the bone marrow hematopoietic stem cell (HSC) itself rather than its derivatives. HSC chimerism achieved through bone marrow transplantation (BMT) may affect type I diabetes in two ways: first, to induce tolerance to pancreas and islet cell transplants; and second, to reverse the autoimmune process prior to the development of terminal complications. Transplantation of bone marrow from normal donors into patients with hematologic malignancy and coexistent type I diabetes has reversed the systemic diabetic autoimmune process. Donor HSCs can also be utilized for the induction of donor-specific tolerance to islet cell transplants. Islet or whole pancreas transplantation is the most physiologic approach to treating type I diabetes. Currently, this is limited by the requirement for high-dose chronic nonspecific immunosuppression to prevent rejection. Despite these agents, chronic rejection remains the primary cause for late graft loss. Donor-specific tolerance eliminates the requirement for immunosuppression and prevents the development of chronic rejection. Bone marrow transplantation does have limitations. In particular these limitations include the morbidity associated with lethal conditioning, graft-versus-host disease, and failure of engraftment. Currently the morbidity and mortality associated with lethal conditioning could not be justified for tolerance induction or interruption of the autoimmune state in type I diabetes. The goal of current research is to identify those factors in both recipient and donor that optimize engraftment to reverse the risk/benefit ratio associated with BMT. This article reviews the state of the art for HSC chimerism affecting diabetes.

Bone marrow cell graft engineering: from bench to bedside.

Bone marrow transplantation (BMT) has the potential to treat hemoglobinopathies (sickle cell and thalassemia) autoimmunity (diabetes, lupus, multiple sclerosis, rheumatoid arthritis, Crohn's colitis) and enzyme deficiency states. Graft versus host disease (GVHD) is a major complication and limitation to the therapeutic application of BMT. There have been many clinical trials and experimental animal models that have attempted to control GVHD through the engineering of the donor bone marrow cells (BMC). Historically, several methods have demonstrated effectiveness in controlling GVHD; however they were also associated with a marked increase in the rate of graft failure. Highly purified hematopoietic stem cells (HSC) engraft quite readily in genetically-matched recipients while they do not engraft as easily in MHC-disparate recipients. The numbers of HSC must be increased 100-200 fold in order to overcome the allogeneic barrier. We were the first to phenotypically and to functionally characterize a novel cell in the bone marrow that enables engraftment of highly purified HSC in allogeneic recipients. The discovery of graft facilitating cell populations has resulted in the restoration of the engraftment-potential of purified HSC between genetically-disparate individuals. The addition of facilitating cells (FC) to T cell-depleted BMC grafts results in allogeneic engraftment without GVHD or graft failure. New strategies of BMC engineering that retain FC and HSC but avoid GVHD have allowed successful engraftment in mismatched and older recipients. These techniques have expanded the therapeutic potential of BMT to virtually every candidate as well as to non-malignant diseases in which the morbidity associated with conventional BMT could not be accepted. This article reviews the transition of the FC technology from bench to bedside and discuss the potentially broad-reaching applications of BMT and mixed chimerism.

The role of alphabeta- and gammadelta-T cells in allogenic donor marrow on engraftment, chimerism, and graft-versus-host disease.

BACKGROUND: We previously characterized a facilitating cell (FC) in mouse marrow that enables engraftment of allogeneic hematopoietic stem cells (HSCs) without causing graft-versus-host disease (GVHD). The FC shares some cell surface molecules with T cells (Thy1+, CD3epsilon+, CD8+, CD5+, and CD2+) but is T-cell receptor (TCR) negative. Historically, depletion of CD3+ or CD8+ cells from rat marrow was associated with an increased rate of failure of engraftment. In this study, we evaluated whether depletion of alphabeta- and gammadelta-TCR(+) T cells from donor marrow would retain engraftment potential yet avoid GVHD. METHODS: Wistar-Furth rats were conditioned with 950 cGy of total body irradiation and transplanted with ACI bone marrow processed to remove either alphabeta-TCR(+), gammadelta-TCR(+), or alphabeta- plus gammadelta-TCR(+) T cells. Recipients were typed for chimerism at 28 days and monthly thereafter. RESULTS: Recipients of marrow depleted of alphabeta- (group A), gammadelta- (group B), or alphabeta- and gammadelta-TCR(+) T cells (group C) engrafted and had an average chimerism level of 73.0+/-8.3%, 92.3+/-9.2%, and 46.3+/-32.8%, respectively. Aggressive T-cell depletion did not remove the FC population (CD8+/CD3+/TCR(-)). Group A and group B both developed GVHD, with a higher incidence of GVHD in group B compared to group A. None of the recipients in group C developed GVHD. CONCLUSIONS: These data demonstrate that depletion of T cells from rat marrow does not impair engraftment of HSCs, indirectly supporting the existence of FCs in rat marrow. Moreover, donor alphabeta- and gammadelta-TCR(+) T cells contribute to GVHD in a nonredundant fashion, although alphabeta-TCR(+) T cells are more potent as the effector cells. Finally, the level of donor chimerism is influenced by the composition of the graft, because recipients of marrow that contain alphabeta-TCR(+) T cells exhibited significantly higher donor chimerism compared to recipients of marrow depleted of both alphabeta- and gammadelta-TCR(+) T cells.

Mouse xenoantigens contribute to rat T-cell Vbeta repertoire generation in mixed xenogeneic bone marrow chimeras.

We previously demonstrated that rat bone-marrow-derived cells in mixed xenogeneic chimeras (rat + mouse --> mouse) contribute to peripheral selection of mouse T-cell receptor (TCR) variable betas (Vbetas) repertoire. In this study, we analysed rat T cells that developed in the chimeras to assess the contribution of mouse xenoantigens to the development of rat TCR repertoire. The expression of rat Vbetas was analysed using flow cytometry and a reverse transcription-polymerase chain reaction (RT-PCR) method that allows for both semiquantitative analysis of rat Vbeta gene expression and size heterogeneity of the complementarity determining region 3 (CDR3) domain. Three distinct patterns of Vbeta expression were detected. Partial deletion was observed for Vbeta5, 7, 12, 14, 16, 17 and 20 that exhibited reduced levels of peripheral expression by 3.4-, 1.8-, 8.7-, 2.0-, 7.8-, 9.5- and 1.8-fold, respectively, compared with the levels of Vbetas in naYve rats. Higher levels of peripheral expression were detected for three rat Vbeta genes; Vbeta6 (2.2-fold), Vbeta8.2 (3.2-fold), and Vbeta9 (1.7-fold). The relative expression of the other 10 known rat Vbeta families in chimeras was unchanged as compared with that of normal rats. We did not observe detectable changes in the pattern of CDR3 expression in chimeras, suggesting that the mouse xenogeneic environment exerted its influence on the development of rat T cells via the Vbeta-encoded CDR1/2 domains. Our data demonstrate that the rat T-cell repertoire in chimeras is shaped by both contractions as well as expansions of selected Vbetas and suggest that mouse xenoantigens and/or superantigens of endogenous mouse retroviruses may contribute as ligands for these selection processes

The abrogation of allosensitization following the induction of mixed allogeneic chimerism.

The association of preformed anti-donor Abs with the hyperacute rejection of bone marrow and solid organ allografts and the persistence of the anti-donor immune response secondary to immunologic memory make allosensitization an absolute contraindication to transplantation. Mixed allogeneic (A + B-->A) bone marrow chimerism has been demonstrated to confer donor-specific tolerance in nonsensitized recipients, but has not been evaluated in the setting of allosensitization. The current study documents that despite significant anti-donor sensitization, mixed allogeneic engraftment is possible and provides a marked advantage over fully allogeneic (B-->A) models. Moreover, the acceptance of donor skin grafts and loss of circulating anti-donor Abs suggest that allosensitization can be abrogated with the induction of stable mixed allogeneic chimerism.

Mixed allogeneic chimerism and tolerance to composite tissue allografts.

The development of effective immunosuppressive drugs has made solid organ allotransplantation the preferred approach for treatment of end-organ failure. The benefits of these immunosuppressants outweigh their risks in preventing rejection of lifesaving solid-organ allografts. On the contrary, composite tissue allotransplants are non-lifesaving and whether the risks of immunosuppressants justify their benefits is a subject of debate. Hence, composite tissue allografts (CTA) have not enjoyed widespread clinical application for reconstruction of large tissue defects. Therefore, a method of preventing rejection that would eliminate the need for toxic immunosuppressants is of particular importance in CTA. Bone marrow transplantation (BMT) to establish mixed chimerism induces tolerance to a variety of allografts in animal models. This article reviews mixed chimerism-based tolerance protocols. Their limitations and their relevance to CTA are discussed, highlighting some unique characteristics (high antigenicity and the presence of active bone marrow) that make CTAs different from solid organ allografts.

Establishment of fully xenogeneic (mouse-->rat) bone marrow chimeras: evidence for normal development and clonal deletion of mouse T cells.

BACKGROUND: Xenotransplantation is a potential solution to the critical shortage of transplantable organs. However, conventional immunosuppressive agents do not control the vigorous cellular and humoral rejection across species disparities. The induction of donor specific tolerance via bone marrow chimerism may be a method to avoid xenograft rejection. In xenogeneic chimeras, T cell repertoire selection plays an important role in the induction of tolerance. Until now a model of mouse-->rat multilineage chimerism has not been reported. This study reports the establishment of fully xenogeneic mouse-->rat multilineage chimeras and evaluates the role of T cell development and repertoire selection in tolerance induction in a xenogeneic environment. METHODS: Recipient rats were irradiated at a dose of total body irradiation ranging between 800-1100 cGy and injected with 120-300x10(6) donor mouse bone marrow cells. Chimeras were typed for engraftment at 4 weeks and then monthly thereafter. T cell repertoire was evaluated in chimeras using two-color flow cytometry and monoclonal antibodies directed against the variable portion of the beta chain of the T cell receptor. RESULTS: Fully xenogeneic multilineage bone marrow chimerism was produced in a mouse-->rat model by using ablative radiation and a high dose of donor cells. Mouse T cells develop in a phenotypically normal fashion in chimeric rats and the host rat is capable of deleting T cells that are reactive to the donor mouse strain. CONCLUSION: Long-term multilineage bone marrow chimerism can be produced in a mouse-->rat bone marrow transplant model. Mouse T cells develop in a phenotypically normal fashion and negative selection of specific T cell receptor-Vbeta occurs in a xenogeneic environment in a predictable fashion paralleling that for syngeneic or allogeneic transplantation.