Rejection Of Bone Marrow Transplants Research Articles

Clinical and Economic Outcomes in Patients with Transfusion-Dependent β-Thalassemia and Patients with Sickle Cell Disease with Recurrent Vaso-Occlusive Crises Receiving Hematopoietic Stem Cell Transplants in the United States

Background: Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are inherited blood disorders caused by mutations in the β-globin gene, resulting in abnormal, reduced, or absent expression of hemoglobin. TDT is characterized by ineffective red blood cell production that leads to chronic anemia, need for regular red blood cell transfusions (RBCTs), and organ complications. SCD is characterized by sickle-shaped red blood cells that cause vaso-occlusive crises (VOCs), hemolysis, and end-organ damage. Hematopoietic stem cell transplant (HSCT) is currently the only curative option for patients with TDT and patients with SCD. There is limited research on the frequency and outcomes of HSCT in these two patient populations. This study describes the economic and clinical outcomes for a combined population of patients with TDT and patients with SCD with recurrent VOCs who received HSCTs in the United States. Methods: This retrospective cohort study used administrative claims from the MerativeTM Commercial, Medicare, and Multi-State Medicaid Databases to identify patients with ≥1 inpatient or ≥2 outpatient claims for β-thalassemia or SCD between March 1, 2010, and March 1, 2019. Eligible patients with TDT were required to have ≥8 discrete RBCTs in any 12-month period on or after the date of the earliest qualifying β-thalassemia claim. At least 3 days between service dates of RBCT claims were required to be considered discrete RBCTs. Patients with TDT were required to have 1 year of continuous enrollment after their first transfusion to be included. Eligible patients with SCD with recurrent VOCs were required to have ≥2 diagnosed VOCs per year in any 2 consecutive years after and including the first qualifying SCD claim. A VOC was defined as inpatient or outpatient claim of acute chest syndrome, priapism, SCD crisis, or splenic sequestration; at least 3 days between VOC claims were required to be considered discrete VOCs. Eligible patients with SCD with recurrent VOCs were required to have 24 months of continuous enrollment before the second VOC in the second consecutive year and 12 months of continuous enrollment after. Patients with TDT and patients with SCD who met the above criteria and received an HSCT were included in the analysis set. Patients receiving HSCT were identified utilizing relevant procedure codes. The index date was the date of transplant. Patients were followed from the index date to the earliest of end of enrollment, death, or end of study period (March 1, 2020). Demographics were assessed at the index date, and clinical complications, healthcare resource utilization, and costs were descriptively summarized in the post-index period. Costs were based on paid amounts of adjudicated claims and patient cost-sharing and included costs in the 10 days before the index date to account for pre-transplant healthcare services associated with the transplant. Results: Two hundred thirty-four and 3500 patients met the criteria for TDT and SCD with recurrent VOCs, respectively, for a total of 3734 patients. Of that total, 62 patients (1.66%) underwent a transplant and were therefore included in the analysis cohort. Mean patient age at the index date was 12.6 years, and 46.8% were female. A proportion of transplant patients experienced graft versus host disease (GvHD) (37/62; 59.7%), unspecified HSCT complications (33/62; 53.2%), or bone marrow transplant rejection or failure (6/62; 9.7%) post-transplant. Average length of stay for the index HSCT admission was 42 days; in the first year post-transplant, patients averaged 58.3 days in the hospital. Total costs in the first 100 days and 1 year post-transplant were $442,752 and $623,301, respectively. Inpatient costs accounted for 93.7% and 88.7% of costs at 100 days and 1 year, respectively. Conclusions: This retrospective cohort study suggests that only a small proportion of patients with TDT and patients with SCD with recurrent VOCs receive HSCTs. Many patients who undergo transplants experience clinical complications, including graft failure and GvHD, and substantial economic burden. Given the limited utilization and potential safety risks of HSCT, additional curative treatment approaches are needed for patients with TDT and patients with SCD with recurrent VOCs.

Transfused Stored or Antibody-Coated Red Blood Cells Are Internalized By and Activate Splenic Professional Antigen Presenting Cells

BACKGROUND: Alloimmunization to antigens on transfused red blood cells (RBCs) is a major problem in transfusion medicine. Development of alloantibodies limits the availability of compatible RBC units for chronically transfused patients. In addition, chronic RBC transfusion therapy is an important risk factor for cell-mediated bone marrow transplant rejection. To develop anti-RBC alloantibodies, professional antigen presenting cells (APCs) are required to digest and load RBC antigens into MHC class II for presentation to CD4 helper T cells. Mouse studies suggest that RBC-associated minor histocompatibility antigens can also be cross-presented into recipient MHC class I; thereby contributing to CD8 T-cell mediated immune responses against bone marrow allografts. Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Splenic red pulp macrophages (RPMs) ingest damaged erythrocytes, catabolize heme, and recycle iron but they are weak APCs. Conversely, dendritic cells (DCs) are the most potent of all APCs. A rare and genetically distinct subset of CD8+ DCs can efficiently ingest dead or dying cells and is uniquely capable of activating CD4 and CD8 T cell responses by presenting antigen via MHC class II and by cross-presenting cell-associated antigens via MHC class I. The RBC storage lesion, in addition to promoting phagocytosis by RPMs, may also induce RBC uptake by splenic DCs. Inflammation induced by stored RBC transfusions, or by antibody-mediated RBC clearance, may enhance DC activation and RBC uptake, thereby triggering humoral and cell-mediated immune responses to RBC antigens.AIMS: A mouse model of green fluorescent protein (GFP)+ RBC transfusion and flow cytometry were used to track the uptake of fresh, antibody-coated and stored RBCs into splenic professional APC subsets and the effects of these RBCs on APC activation.METHODS: Cohorts of 8-12 week old C57BL/6 male mice (n=4 per group) were transfused with one mouse-equivalent unit of syngeneic leukoreduced and citrate-phosphate-dextrose-adenine suspended fresh GFP+ RBCs (<24 hours old), stored GFP+ RBCs (11 days old), fresh GFP+ RBCs coated with anti-RBC polyclonal antibodies, or saline. Two hours after transfusion, spleens were dissociated (Liberase™ DL plus DNAse), FcBlocked, and stained with 7-color antibody panels to define CD8+ DCs (CD11c+ MHC-II+ CD8+ DEC205+), CD8- DCs (CD11c+ MHC-II+ CD8-CD11b+), and RPMs (VCAM-1+ CD11blo). Activation markers included MHC class II and CD86. Data were analyzed using FlowJo and Excel.RESULTS: RBC transfusion after prolonged storage markedly increased GFP+ RBC uptake by RPMs (5.8±0.91% fresh vs. 47±2.3% stored) and CD8+ DCs (1.3±0.27% fresh vs. 13±1.0% stored). A smaller increase in RBC uptake was seen in CD8- DCs (0.6±0.068% fresh vs 1.5±0.083% stored). Antibody-coated RBCs showed two-fold increased uptake by CD8+DCs (25±1.5%) compared with stored RBCs and they showed similar uptake into RPMs (50±1.2%) and CD8- DCs (1.1±0.047%). Lack of GFP+ DC staining with the Ter119 RBC marker confirmed that the GFP signal was internalized and not adsorbed onto the DC surface. CD8+ DCs that internalized GFP+ RBCs showed significantly increased CD86 and MHC class II expression consistent with activation.CONCLUSION: Taken together, these results suggest that transfusion of stored or antibody-coated RBCs increases RBC uptake by, and activation of, professional splenic APCs. CD8+ DC uptake of stored or antibody-coated RBCs may contribute to humoral and cellular anti-RBC immune responses via antigen presentation in MHC class II and class I, respectively. Thus, these mechanisms may increase the risk of alloimmunization to RBC antigens. [Display omitted] DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.

Alterations In Specific Immune Cell Subsets In Children With Sickle Cell Disease

IntroductionPatients with sickle cell disease (SCD) are known to have altered immune systems. In the past, immunology studies in SCD patients have focused largely on splenic dysfunction and the increased risk of infection with encapsulated organisms. Increasingly, however, it is now being recognized that SCD patients also have evidence of abnormal immune activation with baseline chronic inflammation as well as high rates of red blood cell (RBC) alloimmunization and hematopoietic stem cell transplant (HSCT) graft rejection. We have previously demonstrated an increased risk of bone marrow transplant rejection with non-myeloablative conditioning in a SCD mouse model, with both increased T cells and natural killer (NK) cells implicated in this rejection. We have also previously shown that at baseline children with SCD have altered immune cell subsets. We now have expanded on this investigation of quantitative immune deviation in pediatric SCD by exploring how chronic transfusion (CT) or hydroxyurea (HU) therapy affects immunophenotype in a larger cohort of patients. MethodsOne hundred and eleven children (68 patients on CT only, 26 patients on HU only, 17 patients on neither CT nor HU) with SCD (SS or Sβ0) and 29 healthy, age-matched African American controls were recruited for this study. Exclusion criteria included recent illness or another disease associated with known abnormalities of the immune system. Flow cytometry was performed on samples from all participants to quantify the following immune cell populations: total white blood cells (WBC), total monocytes, total lymphocytes, T cells (total, CD4+, CD8+, naïve, central memory, effector memory, effector memory RA, and CD4+ T regulatory), B cells (total, naïve, and memory) and NK cells. ResultsSCD patients on CT and patients not receiving CT or HU (no CT/HU) had significantly increased total WBCs, granulocytes, monocytes, lymphocytes, T cells and B cells compared to healthy controls. Conversely, while SCD patients on HU also had mean counts that were elevated, they were not significantly greater than controls for any of these cell populations (Figure 1). Similarly, CD4+ T cells were significantly increased only in the CT and no CT/HU patients compared to controls (Figure 2). CD8+ T cells, however, were increased only in the CT group. CD4+ T cell subset analysis showed the most striking elevations in the CD4+ central memory and CD4+ effector memory (TEM) populations for both CT and no CT/HU patients compared to controls (Figure 3). CD4+ T regulatory cells were also increased in both of these groups. B cell analysis showed increased numbers of naïve but not memory B-cells in CT and no CT/HU patients. In distinct contrast to most populations that were elevated in CT patients, cytotoxic NK cells were uniquely increased only in no CT/HU patients. [Display omitted] [Display omitted] [Display omitted] ConclusionsChildren with SCD have quantitative differences in many different immune cell populations compared to age and race matched healthy controls. HU treatment appears to bring the absolute number of these cell subsets closer to those of healthy controls, while CT does not. It is unclear at this point to what extent CT therapy itself or baseline patient characteristics leading to the need for CT contribute to these findings. Future work is investigating whether certain cell subset abnormalities are associated with particular therapeutic complications such as RBC alloimmunization or HSCT graft rejection. Better understanding the immunology of SCD may allow for targeted therapies to improve SCD transfusion support and HSCT outcomes. Disclosures:Off Label Use: Abstract includes information related to the use of hydroxyurea in children with sickle cell disease.

Transfusion-Induced Bone Marrow Transplant Rejection Due to Minor Histocompatibility Antigens

Traditionally, alloimmunization to transfused blood products has focused exclusively on recipient antibodies recognizing donor alloantigens present on the cell surface. Accordingly, the immunologic sequelae of alloimmunization have been antibody mediated effects (ie, hemolytic transfusion reactions, platelet refractoriness, anti-HLA and anti-HNA effects, etc). However, in addition to the above sequelae, there is also a correlation between the number of antecedent transfusions in humans and the rate of bone marrow transplant (BMT) rejection-under reduced intensity conditioning with HLA-matched or HLA-identical marrow. Bone marrow transplant of this nature is the only existing cure for a series of nonmalignant hematologic diseases (eg, sickle cell disease, thalassemias, etc); however, rejection remains a clinical problem. It has been hypothesized that transfusion induces subsequent BMT rejection through immunization. Studies in animal models have observed the same effect and have demonstrated that transfusion-induced BMT rejection can occur in response to alloimmunization. However, unlike traditional antibody responses, sensitization in this case results in cellular immune effects, involving populations such as T cell or natural killer cells. In this case, rejection occurs in the absence of alloantibodies and would not be detected by existing immune-hematologic methods. We review human and animal studies in light of the hypothesis that, for distinct clinical populations, enhanced rejection of BMT may be an unappreciated adverse consequence of transfusion, which current blood bank methodologies are unable to detect.

Mechanisms of Alloimmunization and Subsequent Bone Marrow Transplantation Rejection Induced by Platelet Transfusion in a Murine Model

For many nonmalignant hematological disorders, HLA-matched bone marrow transplantation (BMT) is curative. However, due to lack of neoplasia, the toxicity of stringent conditioning regimens is difficult to justify, and reduced intensity conditioning is used. Unfortunately, current reduced intensity regimens have high rates of BMT rejection. We have recently reported in a murine model that mHAs on transfused platelet products induce subsequent BMT rejection. Most nonmalignant hematological disorders require transfusion support prior to BMT and the rate of BMT rejection in humans correlates with the number of transfusions given. Herein, we perform a mechanistic analysis of platelet transfusion-induced BMT rejection and report that unlike exposure to alloantigens during transplantation, platelet transfusion primes alloimmunity but does not stimulate full effector function. Subsequent BMT is itself an additional and distinct immunizing event, which does not induce rejection without antecedent priming from transfusion. Both CD4(+) and CD8(+) T cells are required for priming during platelet transfusion, but only CD8(+) T cells are required for BMT rejection. In neither case are antibodies required for rejection to occur.

CTLA4‐Ig prevents alloantibody production and BMT rejection in response to platelet transfusions in mice

Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion. We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody. CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection. These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions.

Co-Stimulatory Blockade Abrogates Alloimmunization to Transfused Platelets in a Mouse Model

Abstract 717 Background:Platelet transfusion therapy can be a life-sustaining treatment for patients with severe thrombocytopenia. However, alloimmunization is a potential sequelae of platelet transfusion with serious consequences for chronically transfused patients. Induction of alloantibodies, typically against HLA and/or human platelet antigens, can lead to poor survival of transfused platelets expressing the offending antigens. Patients with alloantibodies against multiple HLAs can become refractory to subsequent platelet therapy. Although leukoreduction of platelets has significantly decreased humoral alloimmunization rates, anti-HLA antibodies still form in a significant percentage of transfused patients. In addition to alloantibody formation, animal models have shown that platelet-induced T cell alloimmunity can contribute to the rejection of MHC identical bone marrow transplants across minor antigen barriers. Currently, there are no approved therapeutic interventions in humans to mitigate the risk of alloimmunization other than leukoreduction. Targeted blockade of T cell costimulation has shown great promise in inhibiting alloimmunity in the setting of solid organ transplantation; however, this strategy has not yet been explored in the context of platelet transfusion. Therefore, we tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig, which interrupts the CD28-B7.1/2 T cell signaling pathway, would prevent alloreactivity against major and minor alloantigens on transfused platelets. Methods:C57BL/6 (H-2b) mice were transfused with filter leukoreduced platelets from BALB/c (H-2d) donors. Humoral alloimmunization was measured by indirect immunofluoresence using BALB/c splenocytes or platelets as targets, followed by flow cytometry. Platelet specific CD4+ T cell responses were analyzed by adoptive transfer of TCR75 CD4+ T cells (specific for a peptide from donor MHC I (Kd) presented by recipient MHCII (I-Ab)). Some transfusion recipients were subjected to a bone marrow transplant under reduced intensity conditioning (700 rads) using BALB.B mice as donors (MHC identical, minor antigen mismatched). Engraftment was studied by flow cytometry. Experimental animals received a 500 ug dose of CTLA-4 Ig or 500 ug of a control antibody. Results:The combined data from three separate experiments demonstrated that whereas 14/15 (93%) of control animals had anti-H2d antibodies after 4 weekly platelet transfusions, alloantibody formation was abrogated in all animals (n=15) given a single dose of CTLA4-Ig concurrent with the first platelet transfusion. This effect of CTLA4-Ig lasted at least 49 days after treatment. The proliferation of donor-specific CD4+ T cells was completely blocked by CTLA4-Ig, indicating an effect at the CD4+ T cell level. In addition, administration of a single dose of CTLA4-Ig prevented platelet transfusion induced BMT rejection. The combined results of three independent experiments demonstrated that while 14/15 (93%) of recipients transfused with BALB/c leukoreduced platelets rejected a bone marrow transplant in the presence of isotype control antibody, only 1/15 (7%) of CTLA4-Ig treated recipients rejected donor bone marrow. However, the timing of CTLA4-Ig administration was found to be critical. Delaying treatment until after platelet transfusion failed to prevent bone marrow transplant rejection. Conclusion:These findings demonstrate that costimulatory blockade in general, and CTLA4-Ig in particular, have the capacity to prevent both humoral and cellular alloimmunization to transfused platelets. These findings suggest costimulatory blockade may be a viable approach to decrease alloimmunization rates in humans receiving platelet transfusions. Disclosures:Zimring:Immucor Inc,: Research Funding. Off Label Use: We will describe use of CLTA4-Ig in an animal model for what would be an off label indication in humans. However, there are no human studies in this abstract.

Minor Antigens on Transfused RBCs Crossprime CD8 T Cells but Do Not Induce Full Effector Function

HLA-matched bone marrow transplantation (BMT) is a cure for nonmalignant hematological disorders; however, rejection rates are high and correlate with the number of antecedent transfusions. Recently, using murine models, we reported that minor antigens (mHAs) in transfused leukoreduced red blood cell (RBC) or platelet units induce rejection of subsequent BMT. To study RBCs as an immunogen, we utilized transgenic donors that express a model mHA selectively on RBCs (HOD mouse). Transfusion of HOD blood did not induce BMT rejection of marrow that shared mHAs with the HOD RBCs. Similarly, no endogenous anti-HOD CD8(+) T-cell response was detected with antigen-specific tetramer reagents. Adoptively transferred OT-I T cells rapidly expanded after HOD blood transfusion; however, only a semi-effector phenotype was observed (tumor necrosis factor-α and interferon-γ secretion, but essentially no Granzyme B). After initial expansion, OT-I T cells contracted rapidly to very low levels. A similar trend was observed by in vivo CTL assay, with only transient lytic activity. Together, these data indicate that RBCs may not be the component of RBC units that induces BMT rejection, and suggest that contaminating platelets or leukocytes may be responsible.

40‐O ‐[2‐Hydroxyethyl]rapamycin modulates human dendritic cell function during exposure to Aspergillus fumigatus

40-O -[2-Hydroxyethyl]rapamycin (RAD), a novel derivative of the immunosuppressive drug rapamycin, was analyzed for its immunomodulatory influence during the interaction of human monocyte-derived dendritic cells (moDC) with Aspergillus fumigatus. RAD is clinically used to prevent graft-versus -host disease as well as solid organ and bone marrow transplant rejection. However, it may constitute a risk factor for the development of opportunistic infections, such as invasive aspergillosis which is mainly caused by the most common airborne fungal pathogen A. fumigatus. moDC were generated in the presence or absence of RAD. In this setting, RAD had various modulating effects on the immune function of DC. A decrease of pro- and anti-inflammatory cytokines (IL-12, TNF-α, CCL20, IL-10) was observed. Furthermore, RAD reduced the expression of innate immunity receptors (TLR2, TLR4, dectin-1), impaired the maturation capacity of moDC observed through the reduction of costimulatory factors (CD40, CD80, CD83, CD86), and impaired their capacity to phagocytose and damage A. fumigatus. These data demonstrate that RAD influences the differentiation of DC. RAD modulates the cytokine response of DC to A. fumigatus and reduces their ability to kill germ tubes. Thus, RAD treatment might affect the risk of invasive aspergillosis independently of its capacity of blocking T cell activation.

Minor histocompatibility antigens on transfused leukoreduced units of red blood cells induce bone marrow transplant rejection in a mouse model

AbstractWhen successful, human leukocyte antigen (HLA)–matched bone marrow transplantation with reduced-intensity conditioning is a cure for several nonmalignant hematologic disorders that require chronic transfusion, such as sickle cell disease and aplastic anemia. However, there are unusually high bone marrow transplant (BMT) rejection rates in these patients. Rejection correlates with the number of transfusions before bone marrow transplantation, and it has been hypothesized that preimmunization to antigens on transfused blood may prime BMT rejection. Using a novel mouse model of red blood cell (RBC) transfusion and major histocompatibility complex–matched bone marrow transplantation, we report that transfusion of RBC products induced BMT rejection across minor histocompatibility antigen (mHA) barriers. It has been proposed that contaminating leukocytes are responsible for transfusion-induced BMT rejection; however, filter leukoreduction did not prevent rejection in the current studies. Moreover, we generated a novel transgenic mouse with RBC-specific expression of a model mHA and demonstrated that transfusion of RBCs induced a CD8+ T-cell response. Together, these data suggest that mHAs on RBCs themselves are capable of inducing BMT rejection. Cellular immunization to mHAs is neither monitored nor managed by current transfusion medicine practice; however, the current data suggest that mHAs on RBCs may represent an unappreciated and significant consequence of RBC transfusion.

Transfusion of minor histocompatibility antigen–mismatched platelets induces rejection of bone marrow transplants in mice

Bone marrow transplantation (BMT) represents a cure for nonmalignant hematological disorders. However, compared with the stringent conditioning regimens used when performing BMT to treat hematological malignancies, the reduced intensity conditioning regimen used in the context of nonmalignant hematological disorders leads to substantially higher rates of BMT rejection, presumably due to an intact immune system. The relevant patient population typically receives transfusion support, often including platelets, and the frequency of BMT rejection correlates with the frequency of transfusion. Here, we demonstrate that immunity to transfused platelets contributes to subsequent BMT rejection in mice, even when the BMT donor and recipient are MHC matched. We used MHC-matched bone marrow because, although immunity to transfused platelets is best characterized in relation to HLA-specific antibodies, such antibodies are unlikely to play a role in clinical BMT rejection that is HLA matched. However, bone marrow is not matched in the clinic for minor histocompatibility antigens, such as those carried by platelets, and we report that transfusion of minor histocompatibility antigen-mismatched platelets induced subsequent BMT rejection. These findings indicate previously unappreciated sequelae of immunity to platelets in the context of transplantation and suggest that strategies to account for minor histocompatibility mismatching may help to reduce the chance of BMT rejection in human patients.

Innovative Strategies in In Vivo Apoptosis Imaging

Apoptosis (programmed cell death) plays a key role in the pathogenesis of many disorders including cerebral and myocardial ischemia, autoimmune and neurodegenerative diseases, infections, organ and bone marrow transplant rejection, and tumor response to chemotherapy and/or radiotherapy. Apoptosis in itself represents a complex mechanism where numerous (pro-apoptotic and anti-apoptotic) molecules interact in an elaborate manner. Since the original description by Kerr et al. in 1972, clinical assessment of apoptosis has always required biopsies or aspirated material for in vitro investigations. Several well-established methods are available for in vitro tests using tissue specimens. However, a non-invasive detection of apoptosis would be of great benefit for many patients in various situations. Today, non-invasive techniques for direct in vivo detection of apoptotic cells are rare and urgently need improvement. The early in vivo detection of apoptotic cells can provide the physician with important information to develop further therapeutic strategies in chemotherapy or radiotherapy of tumors, in transplantation of organs, or in healing of infarct areas. In some preliminary publications, several authors reported on the in vivo use of caspase-inhibitors and annexin V, labeled with indium-111, technetium-99m, iodine-123, iodine-124 or fluoride-18. In the present paper, we review the current applicability of both techniques for in vivo apoptosis imaging, and discuss the methodical problems.

Radiolabeling of HYNIC–annexin V with technetium-99m for in vivo imaging of apoptosis

Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of the pathophysiology and treatment of these and numerous other human disorders. Here we describe the most used method for labeling annexin V, a protein with a high affinity for apoptotic cells in vitro, with technetium-99m (99mTc) as a radionuclide imaging agent that can localize and non-invasively quantify apoptosis in vivo when coupled with single-photon emission tomography. In this method, annexin V is first attached to the bifunctional chelator molecule hydrazino nicotinate (HYNIC). Once prepared, HYNIC-annexin V can be labeled with 99mTc, a widely available gamma-radiation-emitting radionuclide, for intravenous injection in as little as 30 min without the need for specialized reagents or equipment.

Immunization to minor histocompatibility antigens on transfused RBCs through crosspriming into recipient MHC class I pathways

Increased rates of graft rejection after bone marrow transplantation (BMT) are observed in patients whose illnesses--such as sickle cell disease, thalassemia, and aplastic anemia--necessitate chronic transfusion before BMT. Because BM transplants in these patients are routinely HLA matched, any immunization responsible for increased rejection is likely against minor histocompatibility antigens (mHAs). It has been assumed that contaminating leukocytes in red blood cell (RBC) units are the main sources of immunization to mHAs. However, in this report, we demonstrate that antigens on donor RBCs are presented in the major histocompatibility complex (MHC) class I pathway of recipient antigen-presenting cells, resulting in activation and expansion of recipient CD8+ T cells specific for donor mHAs. Given that human hematopoietic progenitor cells express many of the known mHAs, this observation provides a mechanism by which chronic transfusion of even stringently leukoreduced RBCs may result in sufficient mHA immunization to increase the frequency of BMT rejection.

Transfusion of Leukoreduced RBC Results in Immunization to Minor Histocompatibility Antigens by Crosspriming into Recipient MHC Class I Pathways.

Background: HLA matched related bone marrow transplantation (BMT) following myeloablative treatment with busulfan, cyclophosphamide and ATG is an effective therapy for children with severe sickle cell disease (SCD). However, a five percent risk for transplant related mortality precludes its use in less severely affected children, and a much higher risk in adults prevents its use in older patients. Low dose total body irradiation based conditioning regimens may provide a safer approach to BMT for patients with SCD. However, although these non-myeloablative regimens reliably allow for sustained engraftment in patients with malignant diseases, there is a high incidence of graft rejection in patients with SCD. One distinct variable in the sickle cell population is chronic transfusion with multiple units of red blood cells (RBC), which may lead to immunization to antigens present on donor bone marrow. Because BMTs in this setting are HLA matched, any immunization responsible for increased rejection is likely against minor histocompatibility antigens (mHAs). It has been assumed that contaminating leukocytes in RBC units are the main source of immunization to mHAs. However, a recent analysis of patients undergoing HLA matched BMT for aplastic anemia performed by the International bone marrow transplant registry, indicates that rejection of multiply transfused patients remains high despite the use of leukoreduced blood products. We hypothesized that RBC are alone sufficient to immunize to mHAs through consumption of donor RBC by recipient antigen presenting cells (APC) and crosspresentation of donor peptides in recipient MHC I molecules.Methods: Chicken Ovalbumin (OVA) is a well studied antigen. We converted OVA into a model RBC associated antigen using a maleimide-sulfhydryl based crosslinking strategy to couple OVA to RBC (OVA-RBC). To monitor activation of CD8+ T cells specific for OVA, we utilized mice transgenic for a T cell receptor that recognizes an OVA peptide 257–264 (SIINFEKL) presented by MHC class I H-2Kb (OT-I mice). Leukoreduced (OVA-RBC) from B10.BR (H-2k) donor mice were transfused into C57BL/6 (H-2b) mice that had been adoptively transferred with OT-I T cells. Activation, and expansion of OT-I cells was assessed by flow cytometry using specific tetramer reagents.Results: Although their lifespan is somewhat decreased, OVA-RBC continue to circulate for 10–14 days with stable persistence of OVA on the RBC. In response to transfusion of OVA-RBC, OT-I T cells expanded 49 fold compared to untransfused mice (p value = .0002). This was antigen specific, as no expansion was seen with RBC crosslinked to a third party antigen (hen egg lysozyme). Moreover, the observed T cell expansion required that the antigen be associated with the RBC, as no expansion was observed after injection with soluble OVA. This experimental design precludes direct presentation of OVA by donor cells, as donor cells do not express the MHC (H-2Kb) to which OT-I T cells are restricted.Discussion: Based upon the above data, we conclude that mHAs on transfused RBC crossprime into the MHC class I pathway of recipient APC. Since human hematopoietic progenitor cells express many of the known mHAs, this observation provides a mechanism by which chronic transfusion of even stringently leukoreduced RBC may result in sufficient mHA immunization to increase the frequency of BMT rejection.

Recent Advances in the Imaging of Programmed Cell Death

A deficiency or an excess of programmed cell death (apoptosis) is an integral component of autoimmune disorders, organ and bone marrow transplant rejection, and cancer. A technique to image programmed cell death would be useful in the development of drugs to treat these and others diseases, and to monitor the effectiveness of therapy. The most widely studied agent for the in vivo study of apoptosis is radiolabeled annexin V, an endogenous protein labeled with technectium-99m, now undergoing clinical trials in both Europe and the United States. While annexin V has been studied extensively in humans the precise mechanism(s) of uptake of this agent in vivo is unclear and needs further study. Other agents are also underdevelopment including radiolabeled forms of Z-VAD.fmk, a potent inhibitor of the enzymatic cascade intimately associated with apoptosis. MR imaging techniques and tracers also hold promise as methods to monitor apoptotic cell death. In this article we will review these and other imaging technologies for the non-invasive imaging of cell death. The mechanism(s) and latest data on the conditions in which cellular stress and early apoptosis occur will also be discussed in detail including potential new strategies for the targeting and novel therapeutic interventions of tissues and organs undergoing stress or apoptosis when cell salvage is still possible.

Population frequencies and putative haplotypes of the killer cell immunoglobulin-like receptor sequences and evidence for recombination.

The killer cell immunoglobulin-like receptors (KIR) are a family of receptors expressed on natural killer (NK) cells and some T cells. Class I HLA molecules on target cells are the ligands for the KIR receptors. The number of KIR genes has been reported to vary between individuals, resulting in different KIR haplotypes. There is little published data on the frequency of each KIR gene and the linkage disequilibrium between the genes. Because there is evidence that NK cells may be involved in bone marrow transplant rejection, we have determined the KIR gene frequencies and possible haplotypic arrangements by linkage disequilibrium analysis in an Australian population. Controls, patients with leukemia, and unrelated bone marrow donor-recipient pairs were typed for the presence of 11 KIR genes by polymerase chain reaction-sequence specific priming. Ninety percent of the population was found to have a sufficient number and variety of KIR genes to detect any mismatch of HLA-A, -B, and -C alleles on NK target cells. The 11 KIR genes could be divided into two groups based on linkage disequilibrium between pairs of genes. Evidence for a recombination within the KIR gene complex is presented. Typing for the presence of particular KIR genes may be indicated for bone marrow donor-recipient pairs for whom a class I HLA mismatch is unavoidable.

Murine natural killer cells and marrow graft rejection.

Rejection of bone marrow transplants in lethally irradiated mice differs from rejection of solid tissue grafts in several respects. The genetic laws that govern rejection of solid tissue grafts often fail with hemopoietic grafts. For example, F1 hybrids between two H-2 disparate strains of mice often reject parental bone marrow cells (BMC), and conversely, marrow cells of F1 hybrids (H-2 heterozygous) are usually not rejected by either parent or an unrelated allogeneic recipient. Thus, unlike the classical MHC antigens, the hemopoietic histocompatibility (Hh) antigens relevant in marrow graft rejection are inherited in a recessive pattern. The major Hh (Hh-1) locus maps within the mouse H-2 complex between the H-2S and H-2D regions, and it can therefore be dissociated from the class-I MHC genes. Nevertheless, it is possible that class-I MHC antigens play a role in the formation or expression of Hh-1 antigens. Three models that explain the possible relationship between class-I MHC and Hh-1 genes and the noncodominant pattern of inheritance of Hh antigens are presented. The effector cells responsible for resisting BMC grafts are different from those responsible for rejection of solid tissue grafts. Three cell types, natural killer cells (CD3-, NK1.1+), cytotoxic T cells (CD3+, CD8+), and T cells with natural killer cell markers (CD3+, NK1.1+) have been implicated in the rejection of BMC grafts. Involvement of these cell types is reviewed and the relative roles played by each are discussed. Evidence supporting the existence of Hh-1 specific subsets of NK cells is presented.

T killer cells play a role in allogeneic bone marrow graft rejection but not in hybrid resistance.

Results of recent experiments have provided compelling evidence supporting the hypothesis that the acute rejection of bone marrow transplants by allogeneic and semiallogeneic recipients is principally due to the action of natural killer (NK) cells. The observed specificity of graft rejection is likely induced by target-specific antibody that guides the NK cells in an antibody-dependent cytolytic reaction resulting in the elimination of the graft. The sole involvement of NK cells in marrow graft rejection, however, is contradicted by several observations that point to the environment of specific T cells. Results presented in this paper demonstrate that in allogeneic marrow graft rejection models, T killer cells are capable of causing graft rejection provided a prior sensitization phase is allowed. Thus, mice not able to reject marrow grafts in a primary response via their NK cells will do so in a primed secondary response via their T cells. Rejection is specific in that only marrow grafts H-2 identical to the sensitizing marrow graft are rejected. Sensitization for NK cell independent marrow graft rejection can be accomplished by prior priming with allogeneic tumor cells or by injection of cloned T killer cells. In contrast to bone marrow allograft rejection, the hybrid resistance model in which F1 hybrid mice reject parental marrow grafts does not appear to induce T killer cells in vivo. Neither marrow grafts nor tumor cells prime F1 hybrids for a second-set parental graft rejection. Moreover, F1 hybrid antiparental T killer cells induced in vitro and adoptively transferred in vivo fail to transfer hybrid resistance. Therefore, there appear to be potent mechanisms acting in vivo that suppress the action or induction of F1 hybrid T killer cells specific to parental antigens.

Bone marrow graft rejection as a function of antibody-directed natural killer cells.

There is conclusive evidence that acute bone marrow transplant rejection in lethally irradiated mice is caused by natural killer (NK) cells. The rejection of marrow allografts is exquisitely specific and is controlled by antigenic determinants encoded in or near the H-2 gene complex. The specificity of in vivo marrow graft rejection contrasts with the in vitro specificity pattern of NK cells in cytotoxicity assays. We therefore examined how NK cells cause H-2-specific marrow graft rejection in vivo. Several experimental approaches are presented that suggest that natural antibody, present in responder strains of mice, specifically directs NK cells in an antibody-dependent cytolytic and/or cytostatic reaction, resulting in marrow graft rejection. The following evidence for this mechanism is documented. The ability to reject a marrow graft can be passively transferred by serum from responder to allogeneic nonresponder mice and the specificity of rejection can be mapped within the H-2 region. Serum-induced marrow graft rejection is abrogated following depletion of immunoglobulin, and the serum of responder mice is able to induce a specific antibody-dependent cytotoxic reaction in vitro.