The accurate diagnosis of acute graft-versus-host disease following liver transplantation (LTx-aGVHD) has been hampered. Chimerism appears in the majority of recipients after LT and its significance in the diagnosis of LTx-aGVHD has not been clearly established. To demonstrate the significance of chimerism on the diagnosis of LTx-aGVHD, we compared the change of chimerism in syngeneic LT recipients, semiallogeneic LT recipients, and LTx-aGVHD induced recipients. Chimerism in PBMCs following sex-mismatched LT was identified by real-time PCR based on a rat Y-chromosome-specific primer. All recipients in semiallogeneic group grew in a normal pattern. However, when 4 × 108 donor splenocytes were transferred simultaneously during LT, the morbidity of lethal aGVHD was 100%. The chimerism appeared slightly higher in the semiallogeneic group than in the syngeneic LT group, but the difference was not significant. However, when the recipients developed lethal aGVHD after LT, chimerism in the PBMCs increased progressively, and even at an early time, a significant increase in chimerism was observed. In conclusion, high level chimerism correlated well with LTx-aGVHD, and detection of chimerism soon after transplantation may be of value in the diagnosis of LTx-aGVHD prior to the onset of symptoms.
Donor- and third-party-induced proliferation of T-helper (Th) and T-cytotoxic (Tc) cells, and their naïve and memory subsets was evaluated simultaneously in single blood samples from 77 children who received steroid-free liver transplantation (LTx) after induction with rabbit anti-human thymocyte globulin. Proliferation was measured by dilution of the intravital dye carboxy-flourescien-succinimidyl-ester (CFSE) in 3–4 day MLR co-culture. The ratio of donor: third-party-induced proliferated, (CFSElow) T-cells was reported as the immunoreactivity index (IR) for each subset. Rejectors were defined as those who experienced biopsy-proven acute cellular rejection within 60 days of the assay. IR>1 signified increased risk of rejection and IR<1 implied decreased risk.
Demographics for 32 Rejectors and 45 Non-Rejectors were similar. Proliferated CFSElow T-cells and subsets were significantly higher among Rejectors, compared with Non-Rejectors. In 33 of 77 randomly selected children, logistic regression, leave-one-out cross-validation and ROC analyses showed that the IR of Tc associated best with biopsy-proven rejection (sensitivity>75%, specificity>88%). Sensitivity/specificity were replicated in the remaining 44 children, comprising the validation cohort. IR of CFSElow Tc correlated significantly with IR of pro-inflammatory, allospecific CD154+Tc (r=0.664, p=0.0005), and inversely with IR of allospecific, anti-inflammatory, CTLA4+Tc (r=−0.630, p=0.007).
Proliferative alloresponses of T-cytotoxic cells can identify rejection-prone children receiving LTx. (200)
Antigen-specific T-cells, which express CD154 rapidly, but remain untested in alloimmunity, were measured with flow cytometry in 16-hour MLR of 58 identically-immunosuppressed children with liver transplantation (LTx), to identify Rejectors (who had experienced biopsy-proven rejection within 60 days post-transplantation). Thirty one children were sampled once, cross-sectionally. Twenty seven children were sampled longitudinally, pre-LTx, and at 1–60 and 61–200 days after LTx. Results were correlated with proliferative alloresponses measured by CFSE-dye dilution (n=23), and CTLA4, a negative T-cell costimulator, which antagonizes CD154-mediated effects (n=31). In cross-sectional observations, logistic regression and leave-one-out cross-validation identified donor-specific, CD154+T-cytotoxic (Tc)-memory cells as best associated with rejection outcomes. In the longitudinal cohort, 1) the association between CD154+Tc-memory cells and rejection outcomes was replicated with sensitivity/specificity 92.3%/84.6% for observations at 1–60 days, and 2) elevated pre-LTx CD154+Tc-memory cell responses were associated with significantly increased incidence (p=0.02) and hazard (HR=7.355) of rejection in survival/proportional hazard analysis. CD154 expression correlated with proliferative alloresponses (r=0.835, p=7.1e-07), and inversely with CTLA4 expression of allospecific CD154+Tc-memory cells (r=−0.706, p=3.0e-05). Allospecific CD154+T-helper-memory cells, not CD154+Tc-memory, were inhibited by increasing Tacrolimus concentrations (p=0.026). Collectively, allospecific CD154+T-cells provide an estimate of rejection risk in children with LTx.
Bronchiolitis obliterans syndrome (BOS) is a major cause of morbidity and mortality post lung transplantation (LTx). We sought to determine the relationship between alloimmune responses and autoimmunity, and subsequently how autoimmunity leads to chronic rejection.
We analyzed the development of donor specific antibodies (Abs) in LTx by flow PRA and the development of Abs to K-α1 tubulin (K-α1T) and collagen V (ColV) by ELISA. The frequency of K-α1T and ColV specific T cells that secrete IFN-γ, IL-17 and IL-10 in LTx recipients was measured by ELSIPOT.
In a retrospective analysis of 42 LTx recipients, we demonstrated a strong correlation between development of donor specific anti-HLA Abs, Abs to self-antigens, and BOS (p<0.05). To test the hypothesis that alloimmunity is related to an immune response to self-antigens, we analyzed 103 LTx patients prospectively for the development of donor specific Abs (DSA) and Abs to self-antigens. 42.7% of recipients developed DSA and 30.1% developed Abs to K-α1T and ColV. Development of DSA preceded development of Abs to self-antigens. BOS+ patients had higher frequency of T-cells secreting IL-17 (p<0.01) and IFNγ (p<0.05) with decreased IL-10 (p<0.05) compared to BOS- patients.
Based on these results we propose that alloimmune responses to donor HLA can induce autoimmune responses to airway epithelial self-antigens, characterized by activation of the IL-17 pathway. These immune responses to self-antigens along with alloimmunity contribute to the pathogenesis of BOS. Strategies to prevent development of autoimmunity may be important in preventing the development of chronic rejection.
Aiming to improve outcome of lung transplantation (LTx) patients, we reviewed risk factors and treatment practices for the LTx recipients who experienced respiratory infection in the late post-LTx period (>1 month after LTx).
We analyzed the clinical data of 48 recipients and donors from 61 LTx, who experienced late respiratory infections. Late respiratory infections were classified according to the etiology, time of occurrence, and frequency of donor-to-host transmission or colonization of the recipient prior to transplantation.
During the period of observation, 42 episodes of respiratory infections occurred. The organisms most frequently involved were gram (-) bacteria: Acinetobacter baumannii (n=13, 31.0%), Pseudomonas aeruginosa (n=7, 16.7%), and Klebsiella pneumoniae (n=4, 10.0%). Among the 42 episodes recorded, 14 occurred in the late post-LTx period. These were bacterial (n=6, 42.9%), fungal (n=2, 14.3%), viral (n=4, 28.5%), and mycobacterial (n=2, 14.3%) infections. Of 6 bacterial infections, 2 were from multidrug-resistant (MDR) A. baumannii and one from each of MDR P. aeruginosa, extended spectrum β-lactamase (+) K. pneumoniae, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae. Infection-related death occurred in 6 of the 14 episodes (43%).
Although the frequency of respiratory infection decreased sharply in the late post-LTx period, respiratory infection was still a major cause of mortality. Gram (-) MDR bacteria were the agents most commonly identified in these infections.
Lung Transplantation; Respiratory Tract Infections
Strategic exposure to donor antigens prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pre-transplant infusion of donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allo-specificities. To target indirect allo-specific T cells, ECDI-SPs induce up-regulation of negative, but not positive, co-stimulatory molecules on recipient splenic CD11c+ DCs phagocytosing the injected ECDI-SPs. Indirect allo-specific T cells activated by such CD11c+ DCs undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allo-specific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4+CD25+Foxp3+ T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SPs infusions target host allogeneic responses via a multitude of mechanisms including clonal depletion, anergy and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.
ECDI (ethylene carbodiimide); Allogeneic transplantation; Tolerance; Graft rejection; Antigen presenting cells; Dendritic cells; Indirect antigen presentation pathway; Direct antigen presentation pathway; Costimulatory molecules
To report the long-term outcomes of 1218 organs transplanted from donation after cardiac death (DCD) donors from January 1980 through December 2008.
One-thousand two-hundred-eighteen organs were transplanted into 1137 recipients from 577 DCD donors. This includes 1038 kidneys (RTX), 87 livers (LTX), 72 pancreas (PTX), and 21 DCD lungs. The outcomes were compared with 3470 RTX, 1157 LTX, 903 PTX, and 409 lung transplants from donors after brain death (DBD).
Both patient and graft survival is comparable between DBD and DCD transplant recipients for kidney, pancreas, and lung after 1, 3, and 10 years. Our findings reveal a significant difference for patient and graft survival of DCD livers at each of these time points. In contrast to the overall kidney transplant experience, the most recent 16-year period (n = 396 DCD and 1,937 DBD) revealed no difference in patient and graft survival, rejection rates, or surgical complications but delayed graft function was higher (44.7% vs 22.0%; P < .001). In DCD LTX, biliary complications (51% vs 33.4%; P < .01) and retransplantation for ischemic cholangiopathy (13.9% vs 0.2%; P < .01) were increased. PTX recipients had no difference in surgical complications, rejection, and hemoglobin A1c levels. Surgical complications were equivalent between DCD and DBD lung recipients.
This series represents the largest single center experience with more than 1000 DCD transplants and given the critical demand for organs, demonstrates successful kidney, pancreas, liver, and lung allografts from DCD donors. (Surgery 2011;150:692-702.)
Analysis of cell-mediated lympholysis in long-term liver allograft recipients indicated that there was a donor-specific unresponsiveness that could not be reversed by the addition of rIL-2 and/or mixed lymphocyte culture supernatant or by nonspecific stimulation of the cultures with PHA. Stimulation of recipient cells with semisyngeneic cells having both donor and third-party HLA antigens failed to reveal the presence of cytotoxic T cells (CTL) specific to the donor, whereas the CTL response to third-party antigens remained normal. Removal of B lymphocytes from the responding cell population did not influence the responses. Furthermore, limiting dilution analysis showed that the liver transplant recipients did not have detectable levels of CTL precursors (CTLp) reactive to the donor antigens, whereas their CTLp to third-party antigens remained normal. Donor-specific CTLp were present before and during the early post-transplant period; these cells were eliminated from the peripheral circulation by 10 mo after transplantation. Taken together, these results indicate that there is a deletion of CTLp specific to donor MHC antigens in the peripheral circulation of long-term liver allograft recipients that may account in part for the success of liver transplantation across MHC barriers.
To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.
Spontaneous orthotopic liver allograft acceptance associated with microchimerism in mice induces tolerance to subsequent skin or heart transplants from the donor but not third-party animals. Despite in vivo hyporesponsiveness, in vitro MLC and CTL assays showed continuing antidonor reactivity. Cells isolated from recipients’ spleens and grafted livers, when tested in MLC and CTL assays, were antidonor reactive out to 3 months to the same degree as splenocytes obtained from either naive or presensitized (with skin or heart) mice. Nevertheless, passive transfer of splenocytes or liver lymphocytes from liver tolerant mice, but not naive or sensitized donor strain mice, were able to prolong skin graft survival significantly in naive irradiated recipients. By using a strain combination in which the donor but not the recipient expressed the stimulatory endogenous super-Ag (Mlsf), it was possible to determine whether super-Ag-reactive T cells bearing Vβ5 and Vβ11 were deleted or anergic. Phenotypic analysis of cells isolated from recipients’ spleens and grafted livers (up to 90 days after transplant), when compared with naive animals, showed no significant difference in Vβ5 and Vβ11 TCR expression. Additionally, when these isolated spleen cells were tested for antibody-mediated stimulation, both anti-Vβ5 and Vβ11 TCR mAb led to marked proliferation of cells obtained from naive and liver-transplanted recipients, but as expected, proliferation was very low in cells from naive donors. These results suggest that liver transplantation induces donor-specific tolerance in vivo, which may not be reflected in in vitro proliferative and cytotoxicity assays (split tolerance). Furthermore, this tolerance does not seem to be induced by clonal deletion or anergy of minor-lymphocyte-stimulating-antigen-reactive T cells in the recipients.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and congenital or acquired aplastic anemias. In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donor's and recipient's cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donor's and recipient's sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent in situ hybridization (FISH) can be applied.
We compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.
We have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.
All the methods applied enable a rapid and accurate detection of post-HSCT chimerism.
Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF.
We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference.
Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA.
The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.
Emergency ABO-incompatible (ABO-I) liver transplantations (LTx) have been performed increasingly to treat severe liver failure. Herein, we report a case of severe hepatic necrosis after ABO-I LTx. A 53-year-old man with blood group O was diagnosed as having severe hepatitis B and acute-on-chronic liver failure, and underwent an emergency liver transplantation implanting a blood-group-B liver from a cardiac-death donor. A routine anti-rejection, anti-infection and anti-virus therapy was given after operation. On post-operative day (POD) 16, the recipient had fever and erythra. Laboratory and radiographic examinations suggested a severe hepatic necrosis of unknown causes. The patient was managed with a 10-d methylprednisolone pulse therapy. He was discharged on POD 35 with stable condition, and no recurrent disease was found during the follow-up.
Liver transplantations; ABO-incompatible; Hepatic necrosis; Graft rejection; Pulse therapy
Role of non-complement activating antibodies (Abs) to mismatched donor HLA in pathogenesis of chronic lung rejection is not known. We utilized a murine model of obliterative airway disease (OAD) induced by Abs to MHC class I and serum from donor specific Abs (DSA) developed human lung transplantation (LTx) recipients (LTxR) to test the role of non-complement activating Abs in development of OAD and bronchiolitis obliterans syndrome (BOS).
Noncomplement activating anti-MHC (ncAbs) were administered intrabronchially in B.10 mice or in C3 knockout (C3KO) mice. Lungs were analyzed by histopathology. Lymphocytes secreting IL-17, IFN-γ or IL-10 to Collagen V (ColV) and K-alpha 1 tubulin (Kα1T) were enumerated by ELISpot. Serum Abs to ColV and Kα1T determined by ELISA. Cytokine and growth factor expression in lungs was determined by RTPCR. DSA from patients with BOS and control BOS-negative LTxR were analyzed by C1q assay.
Administration of ncAbs in B.10 mice or C3KO resulted in OAD lesions. There were significant increases in IL-17 and IFNγ secreting cells to ColV and Kα1T along with serum Abs to these antigens. There was also augmented expression of MCP-1, IL-6, IL-1β, VEGF, TGFβ, and FGF in ncAbs administered mice by day 3. Among LTxR with BOS only 1/5 had C1q binding DSA.
Complement activation by Abs to MHC class I is not required for development of OAD and human BOS. Therefore, anti-MHC binding to epithelial and endothelial cells can directly activate pro-fibrotic and pro-inflammatory cascades leading to immune response to self-antigens and chronic rejection.
Antibody mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor specific alloantibody (DSA) producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC-mismatched heart allografts were transplanted into three groups of recipients: non-sensitized wild type, alloantigen-sensitized wild type and CCR5−/− mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of anti-donor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody secreting cells and the serum titers of anti-donor IgG alloantibody were equivalent in sensitized and non-sensitized recipients six weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor-reactive ASCs than bone marrow at days 7–21 after transplantation. Furthermore, individual spleen ASCs produced more anti-donor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor-reactive alloAb early after transplantation in both sensitized and non-sensitized recipients.
alloantibody; plasma cells; sensitization; spleen; bone marrow
Patient characteristics are important in the liver transplant (LTX) population because of proven associations between individual and environmental factors, treatment adherence, and health outcomes in general medical and other transplant (txp) populations.
The objective of this report is to determine generalizability of the sample to other LTX populations and to establish reliability of measures used to assess individual and environmental resources.
This is a cross sectional analysis of baseline data in a longitudinal study of adherence and health outcomes.
Ninety first-time adult LTX recipients at the University of Pittsburgh Medical Center completed assessments of socio-demographic, health history, psychosocial and environmental factors shortly after surgery; adherence and health outcomes are tracked throughout the study.
The UPMC cohort is older, less racially diverse, and contains more living donors than the national sample. Our sample is generally comparable to the UPMC cohort on pre-txp socio-demographic and clinical characteristics.
Comparable reliability/internal consistency on psychological measures is demonstrated between our sample and most published norms. The mean scores on all coping scales in our sample are higher than normative. Our subjects indicated a more negative perception of family environment and perceived relationships with their primary caregiver more positively than the normative group.
The generalizability of our sample to the parent population and reliability of individual and environmental measures reported here will enable us to examine relationships and predictive capability of patient and contextual resources on treatment adherence and health outcomes among liver transplant recipients.
liver; transplant; psycho-social; socio-demographic
This is an observational study with the primary objective to measure donor-specific immune responses by pediatric liver transplant (LT) recipients, using cell surface expression of lymphocyte activation markers and cytokine secretion in mixed lymphocyte reactions. The secondary objective was to demonstrate possible mechanism(s) involved in those who demonstrated donor-specific hyporesponsiveness. Study participants included 17 recipients, their respective parental donors, the non-donor parent, as well as unrelated third party individuals. Within the CD4+ population, two distinct patterns of CD69 and CD71 expressions were observed: recipients who had a lower percentage of CD4+CD69+ and CD4+CD71+ cells after donor versus non-donor stimulation (therefore a donor/non-donor ratio <1); and recipients who had a higher percentage of CD4+CD69+ and CD4+CD71+ cells after donor versus non-donor stimulation (therefore a donor/non-donor ratio ≥1). Eight recipients had the above defined ratio of <1, with significantly decreased interferon-γ secretion after donor versus non-donor stimulation. CD4+CD25hi˙CD127– regulatory T cells from these eight recipients suppressed donor and non-donor cell induced proliferation. Suppression of proliferation was partially abrogated by interleukin-2. In conclusion, CD69 and CD71 cell surface expression with interferon-γ secretion can be used to identify two distinct populations in pediatric LT recipients. Both active regulation and anergy underlie donor specific hyporesponsiveness.
Lymphocyte activation; Immune reactivity; Liver transplantation
We have previously shown that CD8 T cells from IFN-γ gene knockout (GKO) donors induces more severe lethal graft-vs.-host disease (GVHD) compared to CD8 T cells from wild type (WT) donors in fully MHC-mismatched strain combinations. In this study, we investigated the mechanisms by which IFN-γ inhibits GVHD in a parent→F1 (B6→;B6D2F1) allogeneic hematopoietic cell transplantation (allo-HCT) model. IFN-γ was strongly protective against GVHD in this parent→;F1 haplotype-mismatched allo-HCT model. Irradiated B6D2F1 mice that received GKO B6 CD4-depleted splenocytes develop lethal GVHD with severe lung and liver injury, whereas those receiving a similar cell population from WT B6 donors survived long-term. Donor CD8 cells showed rapid activation, accelerated cell division and reduced/delayed activation-induced cell death in allogeneic recipients in which donor cells were incapable of producing IFN-γ. Consequently, the numbers of activated/effector (i.e., CD25+, CD62L− and CD44high) donor CD8 T cells in the recipients of GKO allo-HCT significantly exceeded those in mice receiving WT allo-HCT. These data show that IFN-γ negatively regulates the CD8 T cell response by inhibiting cell division and promoting cell death, and suggest that blockade of IFN-γ could augment the severity of GVHD in allo-HCT recipients.
Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 μg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 μg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.
•LtxA has anti-proliferative effects on endothelial cells.•LtxA induces a G2/M phase cell cycle arrest in endothelial cells.•LtxA induces apoptosis in endothelial cells.•LtxA increased expression of ICAM-1 and VCAM-1 in endothelial cells.
Endothelium; Leukotoxin; Aggregatibacter actinomycetemcomitans; Apoptosis; Activation
Although anti-human leukocyte antigen antibodies (anti-HLA Abs) are important factors responsible for graft rejection in solid organ transplantation and play a role in post-transfusion complications, their role in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has not been finally defined. Enormous polymorphism of HLA-genes, their immunogenicity and heterogeneity of antibodies, as well as the growing number of allo-HSCTs from partially HLA-mismatched donors, increase the probability that anti-HLA antibodies could be important factors responsible for the treatment outcomes. We have examined the incidence of anti-HLA antibodies in a group of 30 allo-HSCT recipients from HLA-mismatched unrelated donors. Anti-HLA Abs were identified in sera collected before and after allo-HSCT. We have used automated DynaChip assay utilizing microchips bearing purified class I and II HLA antigens for detection of anti-HLA Abs. We have detected anit-HLA antibodies against HLA-A, B, C, DR, DQ and DP, but no donor or recipient-specific anti-HLA Abs were detected in the studied group. The preliminary results indicate that anti-HLA antibodies are present before and after allo-HSCT in HLA-mismatched recipients.
Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation. 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 co-stimulatory blockade.
minor histocompatibility antigen; sensitization; bone marrow transplantation; humoral immune response
The combination of allogeneic bone marrow transplantation (allo-BMT) and donor lymphocyte infusion (DLI) is a useful method for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, there is a risk of inducing uncontrollable fatal graft-versus-host disease (GVHD). In fact, allo-BMT plus intravenous (IV)-DLI using donor splenocytes induces fatal GVHD in recipient mice. In this study, we examined the effects of the combination of intra-bone marrow (IBM)-BMT and the subcutaneous injection of donor splenocytes (SC-DLI) on the allo-BMT system. Recipient BALB/c mice were conditioned by sublethal irradiation (5 Gy), followed by IBM-BMT plus IV-DLI or SC-DLI in C57BL/6 mice. The IV-DLI group showed better engraftment of donor hemopoietic cells than the control group (without DLI) but showed fatal GVHD. The SC-DLI group, however, showed good reconstitution and mild GVHD. These results suggest that the combination of SC-DLI and IBM-BMT promotes the reconstitution of hemopoiesis and helps reduce the risk of GVHD.
The development of antibodies (Abs) to major histocompatibility (MHC) class I related chain A (MICA) and human leukocyte antigen (HLA) and their role in the immunopathogenesis of chronic rejection (bronchiolitis obliterans syndrome (BOS)) following human lung transplantation (LTx) was analyzed. Sera from 80 LTx recipients were analyzed for anti-MICA and anti-HLA Abs using Luminex and flow PRA (panel reactive assay). Development of Abs either to MICA alone or MICA and HLA together significantly correlated (P<0.01) with development of BOS. Kinetic analysis in the post-LTx period revealed that development of anti-HLA Abs (7.6±4.7 months) preceded the development of anti-MICA Abs (10.0±3.5 months). Abs to MICA alleles (*001 and *009) developed approximately 6 months following LTx and peak titers were present at the time of clinical diagnosis of BOS (16.3±2.7 months). The development of Abs to both MICA and HLA was strongly associated with the development of BOS thereby suggesting a synergistic effect. Furthermore, immune response to mismatched HLA can lead to development of Abs to other MHC related antigens expressed on the airway epithelial cells. Cumulatively, these immune responses contribute to the pathogenesis of chronic rejection following human LTx.
MICA; HLA; antibodies; lung transplantation; BOS; Chronic rejection
Host macrophages protect against graft-versus-host disease in part by engulfing donor T cells and inhibiting their proliferation.
Acute graft-versus-host disease (GVHD) results from the attack of host tissues by donor allogeneic T cells and is the most serious limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Host antigen-presenting cells are thought to control the priming of alloreactive T cells and the induction of acute GVHD after allo-HCT. However, whereas the role of host DC in GVHD has been established, the contribution of host macrophages to GVHD has not been clearly addressed. We show that, in contrast to DC, reducing of the host macrophage pool in recipient mice increased donor T cell expansion and aggravated GVHD mortality after allo-HCT. We also show that host macrophages that persist after allo-HCT engulf donor allogeneic T cells and inhibit their proliferation. Conversely, administration of the cytokine CSF-1 before transplant expanded the host macrophage pool, reduced donor T cell expansion, and improved GVHD morbidity and mortality after allo-HCT. This study establishes the unexpected key role of host macrophages in inhibiting GVHD and identifies CSF-1 as a potential prophylactic therapy to limit acute GVHD after allo-HCT in the clinic.
Though remissions have been observed following allo-HSCT for the treatment of CLL, many CLL patients are ineligible for transplant due to the lack of HLA-compatible donors. The use of umbilical cord blood (UCB) permits transplantation of many patients who lack HLA-compatible donors due to reduced requirements for stringent HLA matching between graft and recipient; however, disease relapse remains a concern with this modality. The generation of CLL-specific CTL from UCB T-cells, primed and expanded against the leukemic clone, might enhance the GVL effect and improve outcomes with UCB transplantation. Here we report the generation of functional, CLL-specific CTL using CD40-ligated CLL cells to prime partially-HLA matched UCB T-cells. Functionality and specificity were demonstrated by immune synapse assay, IFN-γ ELISpot, multi-parametric intracellular cytokine flow cytometry, and 51Cr release assay. The use of patient-specific, non-CLL controls demonstrated the generation of both alloantigen and CLL-specific responses. Subsequently, we developed a clinically-applicable procedure permitting separation of alloreactive CTL from leukemia-specific CTL. Leukemia-specific CTL were able to mediate in vivo killing of CLL in humanized mice without concurrent or subsequent development of xenoGVHD. Our results demonstrate that generation of CLL-specific effectors from UCB is feasible and practical, and the results support further exploration of this strategy as a treatment modality for CLL.