Antigen Disparity Research Articles

Recognition of β-linked self glycolipids mediated by natural killer T cell antigen receptors

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens.

Predicting antigenic sites on the foot-and-mouth disease virus capsid of the South African Territories types using virus neutralization data

Foot-and-mouth disease virus (FMDV) outer capsid proteins 1B, 1C and 1D contribute to the virus serotype distribution and antigenic variants that exist within each of the seven serotypes. This study presents phylogenetic, genetic and antigenic analyses of South African Territories (SAT) serotypes prevalent in sub-Saharan Africa. Here, we show that the high levels of genetic diversity in the P1-coding region within the SAT serotypes are reflected in the antigenic properties of these viruses and therefore have implications for the selection of vaccine strains that would provide the best vaccine match against emerging viruses. Interestingly, although SAT1 and SAT2 viruses displayed similar genetic variation within each serotype (32 % variable amino acids), antigenic disparity, as measured by r(1)-values, was less pronounced for SAT1 viruses compared with SAT2 viruses within our dataset, emphasizing the high antigenic variation within the SAT2 serotype. Furthermore, we combined amino acid variation and the r(1)-values with crystallographic structural data and were able to predict areas on the surface of the FMD virion as antigenically relevant. These sites were mostly consistent with antigenic sites previously determined for types A, O and C using mAbs and escape mutant studies. Our methodology offers a quick alternative to determine antigenic relevant sites for FMDV field strains.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth

The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal-fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.

Concurrent Allorecognition Has a Limited Impact on Posttransplant Vaccination

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.

A Common Biomarker Signature for Tolerated Allografts and Self Tissues

GENERAL COMMENTARY article Front. Immunol., 04 May 2011Sec. Immunological Tolerance and Regulation volume 2 - 2011 | https://doi.org/10.3389/fimmu.2011.00011

Peripheral Blood Stem Cell (PBSC) Haploidentical Transplantation Using a Reduced Intensity Conditioning (RIC), T Cell Replete Approach Proves Both Efficacious and Well Tolerated In a Range of Haematological Malignancies.

AbstractAbstract 4584Haploidentical transplantation is an option for patients who do not have a timely identifiable sibling or volunteer unrelated donor (VUD).Benefits of this stem cell source include donor availability, highly motivated donors and the ability to select the best donor from several relatives taking: age/fitness, cytomegalovirus (CMV) status, ABO group and natural killer cell alloreactivity into account.Historically the high level of human leukocyte antigen (HLA) disparity led to increased graft failure and high rates of acute and chronic graft versus host disease (GVHD). Luznik et al (Blood 2001) demonstrated that the use of post stem cell return cyclophosphamide in RIC haploidentical transplantation (using bone marrow as a stem cell source) reduced acute and chronic GVHD to acceptable levels, but at the expense of higher relapse rates in their cohort.We postulated that the use of PBSC's with their inherently higher T cell complement would reduce relapse rates compared to bone marrow, whilst post cell return cyclophosphamide would reduce acute and chronic GVHD.We present 5 patients treated at our centre using a RIC T cell replete haploidentical transplant protocol utilising PBSC's and post cell return cyclophosphamide.The patients, (median age 51; range 44–58), were treated for: relapsed follicular non Hodgkin's lymphoma (NHL), secondary acute myeloid leukaemia, Mycosis Fungoides and Adult T-Cell Leukaemia/Lymphoma (ATLL).Four patients had received 1st line chemotherapy only and remained chemotherapy sensitive, 3 of whom were in complete remission, one in a partial response. None had undergone a previous transplant. The NHL patient was chemotherapy insensitive following 4 previous lines of chemotherapy, a splenectomy and 2 rejected sibling allografts.Three patients were a major ABO mismatch, the remaining 2 fully matched.Four patients were CMV +/+ and 1 mismatched.HLA disparity ranged from 2–5 alleles (2 and 3 patients respectively).Median CD34+ cell dose returned was 6.98×106 cells/kg (range 4.81–8.00), with a median CD3+ cell dose of 2.36×108 cells/kg (range 1.19–2.97).The conditioning regime used was that of Luznik et al's (Blood 2001) phase I trial:Fludarabine 30mg/m2 day -6 to -2, cyclophosphamide 14.5mg/kg day -6 to -5, total body irradiation 2 Gray day -1, post stem cell cyclophosphamide 50mg/kg day +3 to +4, tacrolimus 1mg IV day +5 onwards, mycophenolate mofetil 15mg/kg TDS day +5 to +35. Outcomes:Four of 5 (80%) patients were fully donor chimeric by day 28 however graft failure with autologous reconstitution due to previously undetected HLA antibodies occurred in 1 patient. This patient reconstituted autologous neutrophils and platelets at 15 and 26 days respectively.Median time to neutrophil and platelet engraftment was 16.5 days (range 14–17) and 12 days (range 11–14) respectively.All 5 patients reactivated CMV (the latest at day 112). With pre-emptive treatment however none developed CMV disease. The incidence of acute GVHD grade II – IV and grade III - IV by day 100 was 40% and 20% respectively. Limited chronic GVHD was seen in 3 patients. 2 were assessed as grade I-II and 1 patient grade III. All cases of acute and chronic GVHD were steroid responsive.In both ATLL patients a sustained suppression of human T-lymphotropic virus (HTLV) viral loads was observed post transplant.One patient subsequently died of sepsis at day 113, the patient who had rejected their graft went on to relapse.The remaining 3 patients continue in CR, performance status 0, currently at day 245, 280 and 438.This data shows that RIC T cell replete haploidentical transplantation using PBSC's is well tolerated and enables both early engraftment and full donor chimerism. The rates of acute and chronic GVHD (40 and 60%) are comparable to sibling and fully matched unrelated donors.All of which has resulted in 60% of patients remaining in CR, including both ATLL patients who have gone on to fully suppress their HTLV viral loads. Disclosures:No relevant conflicts of interest to declare.

Comparison of RH Genotypes In Rh Alloimmunized and Non-Alloimmunized Patients with Sickle Cell Disease.

AbstractAbstract 1122Red cell alloimmunization remains a frequent and potentially serious complication of transfusion for patients with Sickle Cell Disease (SCD). RBC antigen disparities between African American patients and European American blood donors, particularly in the Rh system, are thought to contribute to the elevated frequency of alloimmunization in SCD. Extended RBC antigen-matching has been shown in single institutional and prospective multi-center experiences to reduce the incidence of alloantibody production in SCD. Some transfusion services also supply RBCs from African American donors when possible, but its effect on alloimmunization is not well-studied. Despite a program of providing D, C, E, and K-matched RBCs from primarily African-American donors, we have continued to observe Rh alloimmunization. Many patients produced anti-D, -C and –e, even though their RBCs type D+, C+, and e+, respectively. To examine whether Rh alloimmunization was associated with inheritance of diverse RH alleles that encode altered Rh proteins, we characterized RHD and RHCE in 50 patients with SCD: 27 who were alloimmunized to at least one Rh antigen, and 23 patients who had received transfusions but were not Rh alloimmunized. RH testing consisted of PCR-RFLP, AS-PCR and/or amplification and sequencing of exons and Rh-cDNA. In the Rh alloimmunized group, the average number of unit exposures was 275, and the median was 189. Twelve patients had anti-e despite typing e+ serologically, and all 12 had genotypes associated with weak or altered e antigen suggesting the antibody formed in response to exposure to conventional e antigen. Three of these patients subsequently formed anti-E when placed on e-negative transfusion protocols. Of 12 patients with anti-C, 7 typed serologically as C+. Of these, 3 had a hybrid RHD-CE-D that expresses an altered C antigen. However, the remaining 5 had at least one RHCE encoding conventional C antigen. Interestingly, anti-D was reported in 14 patients who typed serologically as D+. Genotyping revealed weak and/or partial D variants in 3, but 11 patients had at least one conventional RHD. These Rh antibodies were almost all transient, particularly unusual for anti-D. In the non-Rh-alloimmunized group, the average number of unit exposures was 189, and the median was 120. RH genotyping of the non-alloimmunized group revealed significant genetic diversity as well, with most patients carrying altered RH alleles. Of 23 e+ patients, 15 had only variant RHce, while 8 had at least one conventional RHce (only 3 were homozygous for conventional RHce). All 4 C+ patients had conventional RHCe genotypes. All 23 non-Rh-alloimmunized patients typed serologically D+, of which 15 had at least one conventional RHD and 8 had altered RHD. In conclusion, we demonstrate that some but not all Rh alloimmunization in patients with SCD transfused with D, C, E, and K phenotype-matched RBCs from primarily African American donors is due to recipients with variant RH genes who are exposed to conventional Rh proteins. In this small study of 50 patients, all 12 allo anti-e occurred in patients with only variant RHce, likely due to mismatched e antigen. Three cases of anti-C in C+ patients and 3 anti-D in D+ patients were also explained by variant RH. However, we found a number of cases of anti-D and -C in patients who had at least one conventional allele and would not be expected to make alloantibodies. More studies are needed, but one possible explanation is that variations in Rhce or RhD proteins may alter the entire Rh antigenic complex in the RBC membrane, as most of these patients had changes in at least one RH allele. Alternatively, these transient Rh antibodies could be stimulated by exposure to units from African American donors expressing variant Rh antigens, and these antibodies could represent “mimicking” or “cross-reactive” specificities. Since the RH genotypes of most of the non-alloimmunized patients also demonstrated variant RH alleles, future studies should evaluate which Rh variants are immunogenic. These data suggest 1) RH genotyping is a valuable complement to serologic phenotyping, 2) it may become important to use molecularly characterized reagent cells for antibody identification, and 3) knowledge of donor genotypes will be important for understanding Rh alloimmunization and possibly for selecting blood for patients with SCD. Disclosures:Westhoff:Immucor scientific advisory board: Membership on an entity's Board of Directors or advisory committees.

The impact of anti-HLA antibodies on unrelated cord blood transplantations

AbstractThe majority of cord blood transplantations (CBTs) have human leukocyte antigen (HLA) disparities. We investigated the impact that patients' pretransplantation anti-HLA antibodies have on the outcome of CBTs. Testing for anti-HLA antibody and its specificity was performed retrospectively at the Japanese Red Cross Tokyo Blood Center with sensitive solid-phase antibody detection assays. Among 386 CBTs, which were first myeloablative stem cell transplantations for malignancies and used a single unit of cord blood, 89 tested positive. Among the antibody-positive group, the cord blood did not have the corresponding HLA type for the antibody in 69 cases (ab-positive), while 20 cases had specificity against the cord blood HLA (positive-vs-CB). Cumulative incidence of neutrophil recovery 60 days after transplantation was 83% (95% confidence interval [CI], 79%-87%) for the antibody-negative group (ab-negative), 73% (95% CI, 61%-82%) for ab-positive, but only 32% (95% CI, 13%-53%) for the positive-vs-CB (P < .0001, Gray test). With multivariate analysis, the ab-positive showed significantly lower neutrophil recovery than the ab-negative (relative risk [RR] = 0.69, 95% CI, 0.49-0.96, p = .027). The positive-vs-CB had significantly lower neutrophil recovery (RR = 0.23, 95% CI, 0.09-0.56, P = .001) and platelet recovery (RR = 0.31, 95% CI, 0.12-0.81, P = .017) than the ab-negative. Patients' pretransplantation anti-HLA antibodies should be tested and considered in the selection of cord blood.

Umbilical cord blood stem cells: what to expect

Umbilical cord blood (UCB) is a valuable alternative source of hematopoietic stem cells (HSCs). It has unique advantages of easy procurement, absence of risk to donors, low risk of transmitting infections, immediate availability, greater tolerance of human leukocyte antigen (HLA) disparity, and lower incidence of inducing severe graft-versus-host disease (GVHD). In the last several years, these features of UCB permit the field of UCB transplantation (UCBT) to move at a faster pace for both children and adults with malignancies and nonmalignancies. However, new strategies and novel developments are expected to improve engraftment and reconstitution, and to enable in utero transplantation for early therapy, as well as to allow the therapy for a wide spectrum of human diseases.

Crucial Role For Cross-Presentation In The Induction Of GVHD By T Cells Directed Against A Single Immunodominant Minor Histocompatibility Antigen Despite Lack Of Epitope Spreading

The current paradigm indicates that epitope speading is critical for GVHD after MHC matched minor antigen (miHA) mismatched BMT. Because clinical data suggest that sex mismatched antigens are relevant miHAs for GVH responses, we directly tested relevance of epitope spreading by inducing GVHD in the presence of only a single immuno-dominant Y antigen disparity. We utilized female (F)→ male(M) MHC matched BMT with anti-H-Y monospecific, H-2(b)-restricted T cell receptor transgenic (TCR Tg) MataHari CD8+T cells.

The Human “Treg MLR”: Immune Monitoring for FOXP3+ T Regulatory Cell Generation

Controversy exists about the conditions effecting the development of forkhead/winghead helix transcription factor P3 (FOXP3) expressing T cells and their relevance in transplant recipients. We generated carboxy-fluorescein diacetate succinimidyl ester-labeled CD4+CD25 high FOXP3+ cells in mixed lymphocyte reactions (MLRs) ("the Treg MLR"), with varying human leukocyte antigen (HLA) disparities and cell components. Five color flow cytometry and H-thymidine uptakes were the readouts. (1) Despite lower stimulation indices (SIs) than two DR-mismatched MLRs, 2 DR-matched MLRs generated more than twofold higher percentages when gating on proliferating CD4+CD25 high FOXP3+ cells; (2) Even with low numbers of proliferating cells, autologous and HLA identical MLRs generated the highest FOXP3+:FOXP3- cell ratios; (3) Elimination of either non-CD3+ responding cells (resulting in "direct presentation" only) or responding CD25+ (Treg generating) cells increased the SI but inhibited proliferating CD4+CD25 high FOXP3+ cell development; (4) MLR-generated CD4+CD25 high FOXP3+ cells added as third components specifically inhibited the same freshly set MLR SI and caused recruitment of new CD4+CD25 high FOXP3+ cells. As an example of the "Treg MLR" immune monitoring potential, addition of third component peripheral blood mononuclear cell containing high percentages of CD4+CD25 high FOXP3+ cells from an HLA identical kidney transplant recipient (in a tolerance protocol) caused donor-specific Treg MLR inhibition or recruitment. This was similar to the third component MLR Tregs generated entirely in vitro. In the Treg MLR, the generation of CD4+CD25 high FOXP3+ cells is more pronounced in the context of self-recognition (HLA matching, indirect presentation). These cells can be assayed for MLR inhibitory and Treg recruitment functions, so as to immunologically monitor the allospecific regulation after transplantation.

New perspectives on the biology of acute GVHD

The use of allogeneic hematopoietic cell transplantation (HCT) has increased as new techniques have been developed for transplantation in patients who previously would not have been considered HCT candidates. However, its efficacy continued to be limited by the development of frequent and severe acute GVHD. The complex and intricate pathophysiology of acute GVHD is a consequence of interactions between the donor and host innate and adaptive immune responses. Multiple inflammatory molecules and cell types are implicated in the development of GVHD that can be categorized as: (1) triggers that initiate GVHD by therapy-induced tissue damage and the antigen disparities between host and graft tissue; (2) sensors that detect the triggers, that is, process and present alloantigens; (3) mediators such as T-cell subsets (naive, memory, regulatory, Th17 and natural killer T cells) and (4) the effectors and amplifiers that cause damage of the target organs. These multiple inflammatory molecules and cell types that are implicated in the development of GVHD have been described with models that use stepwise cascades. Herein, we provide a novel perspective on the immunobiology of acute GVHD and briefly discuss some of the outstanding questions and limitations of the model systems.

Engraftment syndrome following allogeneic hematopoietic stem cell transplantation in children

ES is a complication that occurs immediately before or at the timing of neutrophil engraftment following autologous or allogeneic SCT. It is characterized by fever, skin rash, and non-cardiac pulmonary infiltrates. We evaluated the incidence, risk factors, and outcomes of ES following allogeneic SCT in children. Of 100 pediatric patients, 20 (20%) developed ES occurring at a median of 14 days (range 8-27 days) post-transplant. Patients presented with fever (100%), skin rash (100%), diffuse pulmonary infiltration (25%), and body weight gain (85%). On multivariate analysis, significant risk factors for ES included younger age (<8 yr old) and human leukocyte antigen disparity between donors and recipients. Univariate analysis showed that patients with ES had a higher incidence of developing chronic graft-versus-host disease and ES was not associated with other complications. Event-free survival did not significantly differ between patients with and without ES regardless of the presence of malignant or non-malignant diseases.

Improving cord blood transplantation in children

Umbilical cord blood transplantation (UCBT) is widely used to treat children affected by many disorders. In comparison to bone marrow transplantation, the advantages of UCBT are represented by lower incidence and severity of graft-versus-host disease, easier procurement and prompter availability of cord blood cells, and by the possibility of using donors showing human leucocyte antigen disparities with the recipient. Despite these advantages, the large experience gained over the last decade has clearly demonstrated that UCBT patients may be exposed to an increased risk of early fatal complications, due to the lower engraftment rate of donor haematopoiesis, delayed kinetics of neutrophil recovery and lack of adoptive transfer of pathogen-specific memory T-cells. An inverse correlation between the number of nucleated cord blood cells infused per kilogramme recipient body weight and the risk of dying from transplantation-related causes exists. Thus, it is not surprising that strategies aimed at increasing the number of cord blood progenitors, favouring stem cell homing, and transferring pathogen-specific lymphocytes, have been recently investigated. In particular, selection of the richest cord blood units, infusion of 2 units in the same recipient, intrabone injection of cord blood cells, and transplantation of ex-vivo expanded progenitors can contribute to improve the results of UCBT.

Disparate immune responses to Mi-HAg occur in vitro and in vivo (141.1)

Abstract Historically, insights into the mechanism of alloresponses to minor histocompatibility antigen (Mi-HAg) disparities have focused on T cell activity and used in vitro assays. It was believed that prior in vivo immunization was required for T cell responses to Mi-HAg to occur in vitro. Here, we investigated the generation of adaptive immune responses to Mi-HiAg using an in vivo model. We found that AKR (H-2k) mice rejected skin grafts from MHC-matched, Mi-HAg-disparate B10.BR (H-2k) mice and generated significantly high levels of anti-donor Ab, suggesting that skin graft rejection was associated with the initiation of strong adaptive humoral responses. As B cell activation requires signaling from activated T cells, in vitro and in vivo MLR assays were performed to examine the immune response to Mi-HAg. AKR cells exhibited no reactivity to Mi-HAg disparate stimulators in in vitro MLR assays. In contrast, T cells from AKR mice exhibited strong reactivity in in vivo MLR assays to Mi-HAg in B10.BR mice. The reason for this disparity in response to Mi-HAg in vitro vs. in vivo MLR is not clear. The in vitro culture environment may lack some factors that were present in vivo and that are critical for generation of T cell immune responses to Mi-HAg. T cells were indeed activated in response to Mi-HAg, as CD44highCD62L-/low effector/memory T cells were generated. 2:02 p.m.

Unrelated Transplants for High-risk Adult Acute Lymphoblastic Leukaemia

Standard chemotherapy has a poor outcome in adult patients with high-risk acute lymphoblastic leukaemia (ALL). Allogeneic haematopoietic stem cell transplant (alloSCT) is the treatment of choice, and SCT with alternative progenitors should be considered in the absence of a matched sibling donor. There is great heterogeneity in the data on unrelated SCT for adult patients with high-risk ALL. Many studies include both paediatric and adult patients, or provide combined data from ALL and acute myeloid leukaemia (AML) patients. Moreover, the follow-up of the reported series is highly variable, and the definition of high-risk criteria varies from one study to another. The reported disease-free survival (DFS) for adult ALL patients ranges from 30 to 70%, and leukaemia relapse (14–47%) is an important cause of treatment failure. Despite great improvements in recent years, transplant-related mortality (TRM) from unrelated SCT (USCT) remains unacceptably high (29–36%). The human leukocyte antigen (HLA) disparity in the unrelated donor setting with a high incidence of graft-versus-host disease (GvHD) and delayed immunological reconstitution with a high incidence of infectious events in the unrelated cord blood SCT are the most important causes of morbidity and mortality in unrelated transplants. Disease status at transplant and the presence of acute and/or chronic GvHD are the most important factors determining relapse in high-risk ALL adult patients undergoing USCT.

Selective Depletion of Alloreactive T Lymphocytes Using Patient-Derived Nonhematopoietic Stimulator Cells in Allograft Engineering

Selective depletion of alloreactive T cells in vitro results in efficient graft-versus-host disease prophylaxis in allogeneic hematopoietic stem-cell transplantation, but it is accompanied by increased recurrence of leukemia. To spare donor T-cell-mediated graft-versus-leukemia immunity against hematopoiesis-restricted minor histocompatibility (minor-H) antigens, we explored the use of patient-derived nonhematopoietic antigen-presenting cells (APC) as allogeneic stimulators for selective allodepletion in leukemia-reactive donor T-cell lines. Primary keratinocytes, dermal fibroblasts, and bone marrow fibroblasts were generated from skin biopsies and diagnostic bone marrow aspirates of acute myeloid leukemia patients in vitro. Cell cultures were analyzed for expansion, phenotype, and immunostimulatory capacity in comparison with CD40-activated B cells as professional APC. In addition, nonhematopoietic APCs were used for selective allodepletion in vitro. Patient-derived fibroblasts could be reliably expanded to large cell numbers, whereas keratinocytes had limited growth potential. Interferon-gamma-pretreated fibroblasts showed increased expression of human leukocyte antigen (HLA)-class I and II molecules, CD40, and CD54. Fibroblasts and CD40-activated B cells comparably stimulated HLA-A*0301-specific CD8 T cells after transient expression of HLA-A*0301 as a model alloantigen. Finally, fibroblasts could be effectively applied to selectively deplete alloreactivity within leukemia-reactive donor CD8 T-cell lines by targeting the activation-induced antigen CD137. Primary fibroblasts can be efficiently used as allogeneic nonhematopoietic APC for selective depletion of donor T cells reactive to HLA and ubiquitously expressed minor-H antigen disparities in leukemia-stimulated CD8 T-cell lines. Therefore, harnessing alloreactivity to hematopoietic minor-H antigens in addition to leukemia-associated antigens might increase graft-versus-leukemia immunity of donor lymphocyte grafts in allogeneic hematopoietic stem-cell transplantation.

Incidence, risks, and outcome of idiopathic pneumonia syndrome early after allogeneic hematopoietic stem cell transplantation

We retrospectively analyzed 23 cases with early-onset idiopathic pneumonia syndrome (IPS) of 192 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) from April 1997 to October 2007. Risk factors for IPS development were evaluated using Cox proportional hazards model, including age, gender, underlying disease, disease status at transplant, transplant type, conditioning regimens, donor type, acute graft-vs.-host disease (GVHD), severity of acute GVHD (aGVHD), human leukocyte antigen (HLA) disparity, and organ involvement of aGVHD. Factors that were significant at the 0.1 level on univariate analysis were evaluated by multivariate analysis. Twenty-three of 192 patients developed IPS (12.0%). Median time to IPS onset after allogeneic HSCT was 76 d (range 32-120 d); median time to death after the diagnosis of IPS was 9 d (range 3-92 d); 20 patients with IPS died because of the rapid progression of respiratory failure (87.0%). Nineteen patients with IPS developed aGVHD (82.6%), with grade III-IV aGVHD in 11 patients (47.8%) and aGVHD of gut in 16 patients (69.6%). The following six factors were associated with an increased risk of IPS by univariate analysis: not in remission, unrelated donor, HLA disparity, occurrence of aGVHD, grade III-IV aGVHD and aGVHD of gut. These risk factors were entered into a multivariate analysis model. Only unrelated donor, grade III-IV aGVHD and aGVHD of gut are identified as being significantly associated with the occurrence of IPS, and among them, aGVHD of gut was associated with the largest risk of IPS, suggesting that the lung may be a target organ of aGVHD.

Molecular Genetic Analysis of Interleukin-1 Promoter and Receptor Antagonist Tandem Repeat Polymorphisms Among HLA-Identical Renal Transplant Recipient and Donor Pairs

The cytokine gene polymorphisms as well as the minor histocompatibility antigen (mHAg) disparities have been thought to result in transplant graft rejection and graft-versus-host reaction between HLA-identical donor/recipient pairs. We detected interleukin (IL)-1 promoter and receptor antagonist tandem repeat polymorphisms among 21 HLA identical renal transplant recipient and donor pairs; of 21 pairs, 15 (71%) showed at least one locus difference between donor and recipient, and 73% of the pairs with the disparities at these loci showed either recipient to donor (R→D) or donor to recipient (D→R) positive CD4 + cell response measured by a modified Cylex assay developed in our laboratory. Allele 2 of IL-1 receptor antagonist genotype (IL-1ra VNTR), a gene associated with an increasing IL-1β production, was detected in the three recipients who developed rejections. One HLA-identical pair that had variations on all four loci of IL-1 gene cluster consistently showed positive CD4 + cell immune responses between the donor and the recipient. This study may provide information of the role of non-HLA genes participating in allograft rejection; this demonstrates that in addition to the disparities of the mHAgs, the non-HLA antigens have to be considered as risk factors in HLA-identical solid organ transplantation.