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Background: Chronic graft-versus-host disease (cGvHD) is a complex disorder that typically manifests after allogeneic hematopoietic stem cell transplantation (HSCT). It is a major cause of non-relapse mortality, which makes finding biomarkers associated with its occurrence a priority. Recent studies increasingly indicate that microRNAs (miRNAs, short regulatory RNA molecules) can be used as biomarkers of various disorders. They can circulate in patients' bodies encapsulated within extracellular vesicles (EVs)., Objectives: To identify miRNAs associated with the occurrence of cGvHD in EVs isolated from the plasma of patients after allogeneic HSCT., Material and Methods: We performed global miRNA expression profiling in a pilot cohort of 3 cGvHD cases and 4 non-cGvHD patients without disease symptoms 90 days after the transplantation (control group)., Results: The 2 groups were naturally clustered according to their miRNA profiles using unsupervised hierarchical clustering analysis. We identified 3 miRNAs that were differentially expressed in the cGvHD patients compared to the non-cGvHD patients. The levels of hsa-miR-630 and hsa-miR-374b-5p were lower in the cGvHD patients: 4.1-fold (p = 0.002) and 2.7-fold (p = 0.044), respectively. In contrast, the levels of hsa-miR-29c-3p were 5.8-fold higher (p = 0.004)., Conclusions: Our results suggest that miRNA profiles from plasma EVs may act as markers of cGvHD onset.

Hematopoietic allogeneic stem cell transplantation (allo-SCT) is a curative option for patients with hematological malignancies. However, due to disparities in major and minor histocompatibility antigens between donor and recipient, severe inflammatory complications can occur, among which chronic graft-versus-host disease (cGVHD) can be life-threatening. A classical therapeutic approach to the prevention and treatment of cGVHD has been broad immunosuppression, but more recently adjuvant immunotherapies have been tested. This review summarizes and discusses immunomodulatory approaches with T cells, including chimeric antigen receptor (CAR) and regulatory T cells, with natural killer (NK) cells and innate lymphoid cells (ILCs), and finally with mesenchymal stromal cells (MSC) and extracellular vesicles thereof. Clinical studies and pre-clinical research results are presented likewise., Competing Interests: AD was employed by Alcyomics Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Doglio, Crossland, Alho, Penack, Dickinson, Stary, Lacerda, Eissner and Inngjerdingen.)

Introduction: Acute graft vs. host disease (aGvHD) is a frequent complication following allogeneic haematopoeitic transplantation (HSCT). Despite recent advances, there are no universally accepted biomarkers to determine development of aGvHD. MicroRNAs miR-146a and miR-155 have been previously associated with aGvHD and show promise as clinically translatable biomarkers. In this study, we performed comprehensive expression profiling of miR-146a, miR-155, and miR-155 * expression in aGvHD target tissue and biofluids and relate expression to post-HSCT outcomes. Materials and Methods: MicroRNA expression was assessed by qRT-PCR in gastrointestinal ( n = 31) and skin ( n = 31) biopsies as well as serum (exploratory cohort n = 34, verification cohort n = 81, diagnostic cohort n = 65) and urine (exploratory cohort n = 30, verification cohort n = 56, diagnostic cohort n = 20) biofluids, including extracellular vesicle (EV) cohorts (serum EV n = 15, urine EV n = 30). Expression was related to aGvHD incidence, severity and overall survival. Results: In GI samples, expression of miR-155 ( p = 0.03) and miR-146a ( p = 0.03) was higher at aGvHD onset compared to patients with no GvHD. In skin biopsies, expression of miR-155 ( p = 0.004) was upregulated in aGvHD patients compared to normal control skin. Expression of miR-146a was higher in aGvHD compared to no aGvHD biopsies ( p = 0.002). In serum, miR-155 ( p = 0.03) and miR-146a ( p = 0.02) expression was higher at day 14 (D14), while in urine expression was elevated at D7 post-HSCT in patients who developed aGvHD compared to those disease-free. This was verified in an independent serum (miR-155 p = 0.005, miR-146a p = 0.003) and urine (miR-155 p = 0.02, miR-146a p = 0.04) cohort, where both microRNAs were also associated with aGvHD by ROC analysis. In serum and urine samples taken at the time of aGvHD symptoms, expression of miR-155 and miR-146a was also elevated (serum miR-155 p = 0.03, miR-146a p < 0.001; urine miR-155 p = 0.02, miR-146a p = 0.02). In contrast, miR-146a and miR-155 were downregulated at D14 in serum EVs and at D7 in urine EVs in patients who developed aGvHD compared to those that remained disease-free, in both an exploratory (serum miR-155 p = 0.02, miR-146a p = 0.06; urine miR-155 p = 0.02, miR-146a p = 0.07) and an independent cohort (serum miR-155 p = 0.01, miR-146a p = 0.02). Conclusions: These results further support a role for miR-155 and miR-146a as non-invasive, clinically relevant biomarkers for aGvHD. However, the link between their involvement in generalized inflammation and in specific pathophysiology requires further investigation at a systemic level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Crossland, Norden, Ghimire, Juric, Pearce, Lendrem, Collin, Mischak-Weissinger, Holler, Greinix and Dickinson.)

Prognostic, diagnostic or predictive biomarkers are urgently needed for assessment of chronic graft-versus-host disease (cGvHD), a major risk for patients undergoing allogeneic hematopoietic stem cell transplantation. The main goal of this review generated within the COST Action EUROGRAFT "Integrated European Network on Chronic Graft Versus Host Disease" was to identify potential novel biomarkers for cGvHD besides the widely accepted molecular and cellular biomarkers. Thus, the focus was on cellular biomarkers, alloantibodies, glycomics, endothelial derived particles, extracellular vesicles, microbiome, epigenetic and neurologic changes in cGvHD patients. Both host-reactive antibodies in general, and particularly alloantibodies have been associated with cGvHD and require further consideration. Glycans attached to IgG modulate its activity and represent a promising predictive and/or stratification biomarker for cGVHD. Furthermore, epigenetic changes such as microRNAs and DNA methylation represent potential biomarkers for monitoring cGvHD patients and novel targets for developing new treatment approaches. Finally, the microbiome likely affects the pathophysiology of cGvHD; bacterial strains as well as microbial metabolites could display potential biomarkers for dysbiosis and risk for the development of cGvHD. In summary, although there are no validated biomarkers currently available for clinical use to better inform on the diagnosis, prognosis or prediction of outcome for cGvHD, many novel sources of potential markers have shown promise and warrant further investigation using well characterized, multi-center patient cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BB declared a shared affiliation, with no collaboration, with one of the authors, FP, to the handling editor at the time of review., (Copyright © 2020 Crossland, Perutelli, Bogunia-Kubik, Mooney, Milutin Gašperov, Pučić-Baković, Greinix, Weber, Holler, Pulanić, Wolff, Dickinson, Inngjerdingen and Grce.)

Alemtuzumab conditioning is highly effective at reducing the incidence of acute and chronic graft-versus-host disease (GVHD) in reduced-intensity fludarabine and melphalan transplantation with cyclosporine monotherapy. Less frequent and lower dose scheduling may be used with sibling donors, but an optimal regimen for matched unrelated donors has not been defined. In this retrospective observational study of 313 patients, the incidence and severity of GVHD was compared in patients receiving 3 different dose schedules: the standard 100-mg regimen (20 mg on days -7 to -3), 60 mg (30 mg on days -4 and -2), or 50 mg (10 mg on days -7 to -3). Patients treated with 100 mg, 60 mg, or 50 mg developed acute GVHD grades I to IV with an incidence of 74%, 65%, and 64%, respectively, whereas 36%, 32%, and 41% developed chronic GHVD. An excess of severe acute grades III/IV GVHD was observed in the 50-mg cohort (15% versus 2% to 6%; P = .016). The relative risk of severe acute grade GVHD remained more than 3-fold higher in the 50-mg cohort compared with the 100-mg cohort after adjustment for differences in HLA match, age, gender mismatch, cytomegalovirus risk, and diagnosis (P = .030). The findings indicate that the 60-mg alemtuzumab schedule was comparable with the 100-mg schedule, but more attenuated schedules may increase the risk of severe grade GVHD., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) may be curative, but is associated with significant morbidity and mortality. Chronic graft-versus-host disease (cGvHD), characterized by inflammation and fibrosis of multiple target organs, considerably contributes to the morbidity and mortality even years after allo-HSCT. Diagnosis of cGvHD is based on clinical features and histology of biopsies. Here, we report the generation of a urinary cGvHD-specific proteome-pattern (cGvHD&#95;MS14) established by capillary electrophoresis-mass spectrometry to predict onset and severity of cGvHD as an unbiased laboratory test. cGvHD&#95;MS14 was evaluated on samples from 412 patients collected prospectively in four transplant centers. Sensitivity and specificity was 84 and 76% by cGvHD&#95;MS14 classification. Sensitivity further increased to 93% by combination of cGvHD&#95;MS14 with relevant clinical variables to a logistic regression model. cGvHD was predicted up to 55 days prior to clinical diagnosis. Acute GvHD is not recognized by cGvHD&#95;MS14. cGvHD&#95;MS14 consists of 14 differentially excreted peptides, six of those have been sequenced to date and are fragments from thymosin β-4, eukaryotic translation initiation factor 4γ2, fibrinogen β-chain or collagens. In conclusion, the cGvHD&#95;MS14-pattern allows early, highly sensitive and specific prediction of cGvHD as an independent diagnostic criterion of clinical diagnosis potentially allowing early therapeutic intervention.

The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival., (© 2016 John Wiley & Sons Ltd.)

Graft versus host disease (GVHD) is a major complication of haematopoietic SCT (HSCT). A number of inflammatory cytokines/chemokines are implicated in GVHD and have been identified in numerous single centre studies as potential biomarkers for acute and/or chronic GVHD. In this study, we analysed candidate inflammatory biomarkers (B-cell activating factor (BAFF), interleukin 33 (IL-33), CXCL10 and CXCL11) in a two-centre study. Biomarkers were evaluated pre-transplant and at serial time points post transplant in acute and chronic GVHD patient sera with time-matched control samples from patients without GVHD. Further validation was performed using the human skin explant assay, clinical GVHD biopsies and mRNA expression analysis. BAFF was significantly increased pre-transplant. BAFF, IL-33, CXCL10 and CXCL11 showed increased levels in acute GVHD patient sera and high protein expression in grades II-III of the in vitro skin explant graft versus host reaction (GVHR) group. BAFF, CXCL10 and CXCL11 also showed increased mRNA expression levels in clinical biopsies compared with the no/low-grade GVHD group. BAFF, CXCL10 and CXCL11 levels were increased in chronic GVHD patient sera. The results identify BAFF and CXCL10 as predictive biomarkers for acute GVHD and BAFF, CXCL10 and CXCL11 as useful diagnostic biomarkers for acute GVHD and chronic GVHD.

Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100 days) or chronic (cGVHD: after 100 days) based on the time-point of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p = 0.008) up-regulated at 28 days post-transplant in cGVHD patients with a significant area under the curve (AUC = 0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3 months post-transplant in both the aGVHD grade II-III (p = 0.012) and cGVHD (p = 0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Atg16L1 mediates the cellular degradative process of autophagy and is considered a critical regulator of inflammation based on its genetic association with inflammatory bowel disease. Here we find that Atg16L1 deficiency leads to an exacerbated graft-versus-host disease (GVHD) in a mouse model of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Atg16L1-deficient allo-HSCT recipients with GVHD displayed increased T cell proliferation due to increased dendritic cell (DC) numbers and costimulatory molecule expression. Reduced autophagy within DCs was associated with lysosomal abnormalities and decreased amounts of A20, a negative regulator of DC activation. These results broaden the function of Atg16L1 and the autophagy pathway to include a role in limiting a DC-mediated response during inflammatory disease, such as GVHD.

Allogeneic hematopoietic stem cell transplantation is one curative treatment for hematological malignancies, but is compromised by life-threatening complications, such as severe acute graft-versus-host disease (aGvHD). Prediction of severe aGvHD as early as possible is crucial to allow timely initiation of treatment. Here we report on a multicentre validation of an aGvHD-specific urinary proteomic classifier (aGvHD&#95;MS17) in 423 patients. Samples (n=1106) were collected prospectively between day +7 and day +130 and analyzed using capillary electrophoresis coupled on-line to mass spectrometry. Integration of aGvHD&#95;MS17 analysis with demographic and clinical variables using a logistic regression model led to correct classification of patients developing severe aGvHD 14 days before any clinical signs with 82.4% sensitivity and 77.3% specificity. Multivariate regression analysis showed that aGvHD&#95;MS17 positivity was the only strong predictor for aGvHD grade III or IV (P<0.0001). The classifier consists of 17 peptides derived from albumin, β2-microglobulin, CD99, fibronectin and various collagen α-chains, indicating inflammation, activation of T cells and changes in the extracellular matrix as early signs of GvHD-induced organ damage. This study is currently the largest demonstration of accurate and investigator-independent prediction of patients at risk for severe aGvHD, thus allowing preemptive therapy based on proteomic profiling.

Graft-versus-host disease (GvHD) remains the major barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) is a potent immunomodulatory treatment option for GvHD. In contrast to conventional immunosuppressants, ECP is considered not to increase relapse and infection rates resulting from generalised immunosuppression. ECP involves the mechanical separation of 5-10% of patient peripheral blood mononuclear cells, which are then exposed to psoralen and UVA light (PUVA) before they are returned to the patient. ECP has been shown to induce apoptosis in various cell types, in particular lymphocytes. Several studies describe downregulation of pro-inflammatory cytokines as well as promotion of peripheral tolerance through enhanced production of T regulatory cells in the course of ECP-treatment. Modulation of antigen-presenting cells such as dendritic cells (DC) by PUVA-treated lymphocytes might be implicated in these regulatory processes. We evaluated the impact of PUVA-treated lymphocytes on immature DC and further demonstrated the functional capacity of such modified DC to modulate GVH reactions using a well-established human skin-explant model of GvHD. Addition of immature DC isolated after co-culture with PUVA-treated but not untreated MLR cells significantly downregulated skin-GvH reactions (p=0.023, Mann-Whitney-Test). IFN-gamma levels were non-significantly decreased in MLR and skin supernatants. We observed a non-significant increase in PD-L1 expression in iDC after co-culture with PUVA-treated MLR cells whereas expression levels of IDO and ILT-3 were not affected. We conclude that iDC modulated by PUVA-induced apoptotic cells potently downregulate allogeneic immune responses possibly through PD-L1- dependent signaling.

Background: Graft-versus-host disease (GVHD) is an important complication occurring after hematopoietic stem-cell transplantation (HSCT). Animal model studies have shown the involvement of the Fas (APO-1/CD95)/Fas-Ligand pathway in GVHD pathogenesis, but its association with cutaneous GVHD in human remains to be established., Methods: In the present study, Fas involvement in skin damage was assessed using a human skin explant model of GVHD. Fas and FasL expression were measured by immunohistochemistry and blockade of Fas pathway was investigated using an antagonistic anti-human Fas monoclonal antibody. In addition, levels of soluble Fas (sFas) were determined in the serum of patients receiving allogeneic HSCT with and without GVHD., Results: The results showed that Fas up-regulation in the epithelium of human skin explants correlated with graft-versus-host reaction (GVHR) in the skin explant model (P<0.001). Decreased GVHR grades were observed by using a Fas blocking monoclonal antibody. Levels of sFas were increased post-HSCT (P<0.001) but rather than being associated with the severity of GVHD, sFas levels differed with the conditioning treatments the patients received before the HSCT., Conclusions: Higher GVHR grades were associated with increased Fas expression in the epithelium of the skin explants. In addition, by blocking Fas-mediated apoptosis, the GVHR grades were decreased. Our study thus shows the involvement of Fas in cutaneous GVHD damage, and supports the potential use of Fas as a therapeutic target.

Background: The major histocompatibility complex (MHC) is the most important genomic region that contributes to the risk of graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Matching of MHC class I and II genes is essential for the success of transplantation. However, the MHC contains additional genes that also contribute to the risk of developing acute GVHD. It is difficult to identify these genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes and other genes involved in the pathophysiology of GVHD by mRNA expression profiling., Methodology/principal Findings: To reduce the complexity of the task, we used genetically well-defined rat inbred strains and a rat skin explant assay, an in-vitro-model of the graft versus host reaction (GVHR), to analyze the expression of MHC, natural killer complex (NKC), and other genes in cutaneous GVHR. We observed a statistically significant and strong up or down regulation of 11 MHC, 6 NKC, and 168 genes encoded in other genomic regions, i.e. 4.9%, 14.0%, and 2.6% of the tested genes respectively. The regulation of 7 selected MHC and 3 NKC genes was confirmed by quantitative real-time PCR and in independent skin explant assays. In addition, similar regulations of most of the selected genes were observed in GVHD-affected skin lesions of transplanted rats and in human skin explant assays., Conclusions/significance: We identified rat and human MHC and NKC genes that are regulated during GVHR in skin explant assays and could therefore serve as biomarkers for GVHD. Several of the respective human genes, including HLA-DMB, C2, AIF1, SPR1, UBD, and OLR1, are polymorphic. These candidates may therefore contribute to the genetic risk of GVHD in patients.

Background: Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically., Methods: To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, "donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase), then primed "donor" CD8 T cells were co-cultured with "recipient" skin to induce GVH tissue damage (effector phase). "Donor"-derived Treg were added at the priming or effector phase of the GVH response. Histopathologic changes in the skin were evaluated using a clinically validated GVHD scoring system., Results: "Donor"-derived Treg significantly reduced the severity of GVH histopathologic damage when present during T-cell priming. In contrast, Treg failed to prevent GVH tissue damage when added to the skin co-culture (effector phase), concurrently with primed T cells. Importantly, "donor" Treg alone did not induce GVH tissue damage. Delayed Treg addition led to reduced and impaired Treg suppression of CD8 T-cell activation and their cytolytic function., Conclusion: "Donor"-derived Treg effectively suppress CD8 T-cell-mediated GVH tissue damage but are critically required during priming of effector T cells. "Donor"-derived Treg seem to be safe and do not induce GVH histopathologic damage.

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell-dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed (51)Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Previous studies from our group indicated a role of SNPs within the innate immunity receptor NOD2/CARD15 as a risk factor for GvHD and treatment-related mortality allogeneic stem cell transplantation from HLA-identical siblings. We now extended these studies to assess the role of NOD2/CARD15 SNPs in 342 unrelated donor transplants. Overall, presence of any SNPs in patients or donor resulted in an increased risk of severe GvHD (25% in wildtype versus 38% in recipients and donors with variants, P= 0.01), which did not translate in increased mortality. When the analysis was broken down to individual SNPs, the presence of a SNP13 in the donor turned out to be the only highly significant risk factor (GvHD III/IV 22% wt, 42% SNP13 donor, P < 0.004; TRM 33% wt versus 59% SNP13 donor, P= 0.01; overall survival 49% wt versus 26% SNP13 donor, P= 0.007). This association was confirmed in multivariate analysis. Analysis of clinical risk factors suggested that this effect was most prominent in patients receiving any form of T cell depletion. Thus our observation indicates that the presence of a defect in innate immunity signalling in donor monocytes and possibly antigen presenting cells is most prominent in patients having additional T cell deficiency.

Over the last 10 years, an increasing number of studies have demonstrated the role of non-human leukocyte antigen (HLA) genes in predicting outcome in haematopoietic stem cell transplantation (HSCT). These studies have included investigations into 'single nucleotide polymorphisms (SNPs)' or microsatellites of cytokines, cytokine receptor genes, or genes associating with innate immunity. These polymorphisms give rise to functional differences in the production of e.g. cytokines, or altered function of genes which are reflected in potential up- or down- regulating of the 'cytokine storm' of GvHD. This review summarises some of the studies and relates the results to their potential for improving HSCT outcome by predicting transplant-related mortality and GvHD.

In the last 10 years, non-HLA genotypes have been investigated for their potential roles in the occurrence and severity of graft-versus-host disease (GVHD) as well as for their contribution to overall transplant-related mortality, infectious episodes, and overall survival. This chapter will review the latest results of cytokine gene polymorphisms between patient and donor which may cause the production of high or low levels of cytokines during the three-stage process of the GVHD 'cytokine storm'. More recent investigations into innate immunity and the interaction with subsequent downstream cytokine production and ultimate tissue damage are discussed. The potential of these non-HLA genetics to aid in predicting GVHD and post-transplant survival and the relevance of this information to the clinic are reviewed.

Haematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for many patients with malignant and non-malignant haematological diseases. The success of HSCT is greatly reduced by the development of complications, which include graft-versus-host disease (GVHD), relapse and infection. Human leukocyte antigen (HLA) matching of patients and donors is essential, but does not completely prevent these complications; non-HLA genes may also have an impact upon transplant outcome. Polymorphisms within genes that are associated with an individual's capability to mount an immune response to alloantigen and infectious pathogens and/or response to drugs (pharmacogenomics) are all currently being studied for their association with HSCT outcome. This review summarises the potential role of non-HLA polymorphisms in predicting HSCT outcome, from studies on retrospective transplant cohorts of HLA-identical siblings and matched unrelated donors. The clinical relevance and interpretation of non-HLA genetics, and how these could be used alongside clinical risk factors in HSCT, are also discussed.

CD1a, an antigen-presenting molecule related to major histocompatibility complex (MHC) class I, is frequently described as nonpolymorphic. In humans it is dimorphic, due to two linked amino acid substitutions in the alpha1 domain (Ile13Thr and Trp51Cys). The CD1a gene on chromosome 1 is not linked to MHC and may be mismatched between human leukocyte antigen-identical siblings. We analyzed 155 donor-recipient pairs of the Eurobank cohort, 141 matched for CD1a and 14 unmatched in the graft-versus-host disease (GVHD) direction. The burden of GVHD was not increased by CD1a mismatching. The incidence of GVHD in matched and unmatched groups was respectively: grade I-IV: 81% and 86% (P = 0.492); II-IV 61% and 57% (P = 0.495); III-IV 23% and 21% (P = 0.608). Adjusting for age, sex mismatch, GVHD prophylaxis, and conditioning did not reveal any significant difference. This suggests that, unlike conventional class I molecules, CD1a does not function as a transplantation antigen and does not require matching in hematopoietic stem cell transplantation.

A human skin explant model has been used to predict the clinical outcome and to study the immunopathology of human graft-versus-host disease (GVHD). Whether the model gives the same predictive effect for GVHD in different hematopoietic stem cell transplantation (HSCT) settings has not been assessed. It is also unknown whether the skin explant result reflects the known biological risk factors for clinical GVHD. In this study, the skin explant model was used to detect graft-versus-host reactions (GVHR) in vitro for 225 eligible patient/donor pairs. The predicted skin GVHR grade was correlated with the outcome of clinical GVHD, as well as HLA matching status, sex mismatches, and patient age. In sibling HSCT under either myeloablative or reduced-intensity conditioning, a significant correlation was observed between the predicted skin GVHR and clinical GVHD (P < .001 and P = .033, respectively). In HSCT using unrelated donors, the involvement of T-cell depletion led to a sharp increase in false-positive GVHR results, and no correlation was observed between the predicted skin GVHR and clinical GVHD. The skin GVHR grade correlated significantly with the HLA matching status (HLA-matched sibling pairs, HLA-matched unrelated pairs, and HLA-unmatched unrelated pairs). Furthermore, HLA-matched sibling pairs with a female-to-male sex mismatch had a significantly higher overall skin GVHR grade and a higher ratio of high- versus low-grade skin GVHR than the sibling pairs with all other sex combinations. Patient age was not reflected in the skin explant result. In conclusion, the predictive value of the skin explant model for aGVHD varies depending on the clinical transplant protocols, such as the type of GVHD prophylaxis used. Nevertheless, the skin explant model remains a unique in vitro system that provides an in situ histopathologic readout for studying alloreactivity and human GVHD. The model has also the potential to aid the development of novel prophylaxis and treatment for GVHD.

Graft-versus-host disease (GvHD), a life-threatening complication of bone marrow transplantation, is initiated by donor T cells reacting to recipient dendritic cells (DC). GvHD can be controlled by attenuating donor T cells, but few strategies exist to target DC, particularly resident tissue DC, despite recent evidence of their importance. In this report, CMRF-44, a mouse monoclonal IgM reactive to human DC, is tested against human Langerhans cells (LC) in vitro. CMRF-44 antigen is expressed at low level on fresh LC but is up-regulated 40-60-fold during migration. CMRF-44 and complement kill more than 97% of migratory LC in vitro and inhibit allostimulation by LC up to 95%. In comparison, alemtuzumab, which binds CD52, reacts weakly with primary LC and fails to induce significant lysis with complement (less than 5%). These results highlight the potential of new therapeutic antibodies active against tissue DC to control graft-versus-host reactions.

Graft-versus-host disease (GvHD) can be a major complication after allogeneic stem cell transplantation (SCT) especially when donor and recipient are unrelated. The latter serious complication, together with the growing number of available unrelated stem cell donors, demand a simple in vitro assay for functional stem cell donor selection. Activated donor cytotoxic T lymphocytes (CTLs) and natural killer cells produce granzymes (Gr) that are involved in the pathogenesis of GvHD. We measured granzymes A and B (GrA and GrB) production levels in the supernatants of 96 h pretransplant mixed lymphocyte cultures (MLC) of 26 sibling and 31 unrelated patient/donor pairs by enzyme-linked immunosorbent assay (ELISA). In detail, the GrA and GrB production levels from a selected cohort of 37 potential patient/donor pairs were correlated with relative responses (RR) of MLC and with human leukocyte antigen (HLA) class II mismatches and with the development of acute GvHD in a second, consecutive cohort of 20 sibling SCT recipients. In vitro measurement of GrA and GrB production levels significantly correlated with the RR of pretransplant MLC (r=0.492, p< or =0.01 and r=0.853, p< or =0.01, respectively) and increased with the number of HLA class II mismatches between patient and donor. Pretransplant GrA production levels were significantly associated with the in vivo development of acute GvHD grades II-IV in patients transplanted with an HLA-identical sibling donor (p< or =0.001). In conclusion, in vitro GrA and GrB production levels can be measured by a quantitative and sensitive ELISA. This novel and simple method may be used for functional selection of unrelated stem cell donors and for the identification of patients who are at risk for acute GvHD grades II-IV.

Background: A single nucleotide polymorphism in the tumor necrosis factor type II receptor (TNFRII) gene, codon 196, results in the substitution of arginine (R allele) for methionine (M allele). The 196R allele is reportedly associated with an increased susceptibility to autoimmune disease, and donor 196R allele carriage correlates with increased severity of acute graft-versus-host disease (GVHD) after matched unrelated bone marrow transplantation (BMT)., Methods: We investigated the impact of donor and recipient TNFRII genotype on GVHD incidence and severity among 104 adult recipients of myeloablative sibling BMTs., Results: 196R allele frequency was 0.28 among recipients, donors, and controls. There was an increased incidence of acute GVHD among 196R-positive recipients (odds ratio [OR] 3.6, P=0.05). This association was confirmed in multivariate analysis (relative risk 4, P=0.04), correcting for previously established clinical and genetic risk factors. Donor 196R homozygosity was associated with an increased incidence of extensive chronic GVHD (OR 18.5, P=0.02). This association was also confirmed in multivariate analysis (OR 11, P=0.02). To investigate the functional impact of the TNFRII 196 M/R polymorphism, 79 volunteer blood donors were genotyped at this locus, by polymerase chain reaction and single-strand conformational polymorphism analysis, and plasma soluble TNFRII (sTNFRII) levels were measured by ELISA. Mean plasma sTNFRII levels (pg/mL: +/-SEM) were 1224 (+/-26) and 1063 (+/-65) for 196M-postive (196 M homozygous or heterozygous) individuals and 196R homozygotes, respectively (P=0.02)., Conclusions: Because sTNFRIIs can act as TNF antagonists, the association between recipient and donor TNFRII 196R allele status and acute or extensive chronic GVHD incidence, respectively, may reflect reduced circulating sTNFRII.

Background: The heat shock proteins are increasingly becoming associated with immunopathologic phenomena, being induced in response to inflammation. They are highly immunogenic and are postulated as playing a role in both innate and adaptive immunity. Their proposed role in peptide binding and antigen presentation could suggest a potential role in the alloreactive process that leads to graft-versus-host disease (GVHD) after bone marrow transplantation., Methods: In this study we examined the expression of the widely studied heat shock protein 70 (hsp70) in an in vitro-generated graft-versus-host reaction in human skin, using streptavidin biotin immunohistochemistry and laser scanning confocal microscopy., Results: Hsp70 expression was correlated with high graft-versus-host responses (P<0.001) and was confirmed using laser scanning confocal microscopy. Increased expression of hsp70 was further defined due to increases in the inducible form of hsp70. Expression of inducible hsp70 was predictive of both clinical acute GVHD (P=0.001) and incidence of chronic GVHD (P<0.001)., Conclusions: This investigation has demonstrated for the first time the expression of hsp70 in a human model of GVHD, suggesting involvement in the pathogenesis of the disease and providing the basis for further investigation. Increased expression of inducible hsp70 in the model could provide a biologic marker for the prediction of clinical acute and chronic GVHD.

The interleukin-1 (IL-1) family of cytokines is widely involved in inflammatory processes and diseases with an inflammatory component. Polymorphisms of the IL-1alpha, IL-1beta and IL-1Ra genes have been implicated in a number of autoimmune or inflammatory conditions, with polymorphism of the IL-1Ra gene showing association with severity of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). We compared the clinical outcomes (GVHD and survival) of 115 patients after human leucocyte antigen (HLA)-identical sibling allogeneic BMT with their genotype for two polymorphisms present in the IL-1alpha gene, which have been implicated in immune-related pathology. Possession of allele 2 of the IL-1alpha-889 polymorphism and allele 2 of the IL-1alpha variable number tandem repeat (VNTR) polymorphism in the donor genotype was associated with the occurrence of chronic, but not acute GVHD. A local normal population was also genotyped for these polymorphisms, and subsequent analysis identified conserved haplotypes in this gene region. Haplotypes containing allele 2 at both IL-1alpha-889 and IL-1alpha VNTR loci were extremely uncommon, suggesting that both risk alleles would be inherited independently. Both loci could therefore function as independent disease association markers. The polymorphisms of the IL-1alpha gene could be used to predict chronic GVHD in HLA-matched sibling transplants alongside clinical risk factors.

Oestrogen receptors mediate the cellular response to oestrogens and related compounds and promote a wide range of effects on haemopoiesis. Polymorphisms of the oestrogen receptor genes have previously been associated with variation in bone mineral density, likelihood of fractures, risk of developing Alzheimer's disease, endometrial cancer and response to hormone replacement therapy. We examined the polymorphisms in both ERalpha and ERbeta genes in 108 patients receiving a bone marrow transplant from an HLA-matched sibling donor, and compared ER genotype with outcomes of occurrence of graft-versus-host disease (GVHD) and survival using logistic regression analysis. Polymorphism of ERalpha (presence of the PX haplotype (PvuII-XbaI RFLP) of intron 1), but not ERbeta, in the patient genotype associates with occurrence of acute GVHD and with lower overall survival, following correction for known clinical and genotypic risk features. Analysis of ER genotype prior to transplant might therefore inform on a patient's likelihood of developing post-transplant complications. Variation in transplant performance because of ER genotype suggests an underlying role for oestrogens in the pathophysiology of transplant-related complications, and suggests that oestrogen-related therapy may offer a new modality of post-transplant support.

Background: In HLA-identical sibling bone marrow transplantation, certain recipient cytokine gene polymorphism genotypes and minor histocompatibility differences influence the occurrence and severity of acute graft-versus-host disease (aGvHD). The present study investigated the role of cytokine tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 gene polymorphisms HY, HA-1, and CD31 minor histocompatibility antigen (mHag) mismatch in the development of aGvHD after unrelated cord blood (CB) transplant (CBT)., Methods: DNA samples of 115 CB recipients and their unrelated CB grafts were analyzed for genotype associated with TNF-alpha (TNFd3/d3) and IL-10 (IL-10(-1064), 11-16) and for disparities in major and three minor histocompatibility antigens, HY, HA-1, and CD31 codon 125. Results were correlated with the incidence of aGvHD grades II to IV., Results: Neither the donor nor the recipient GvHD risk alleles TNFd3/d3 and IL-10(-1064) (11-16) were associated with the development of aGvHD grades II to IV and I to IV. Because of the heterogeneity of CBTs, the data were reanalyzed separately for patients with malignancies (n=83) or with inborn errors (n=24). No significant association was observed between the severity of aGvHD and the possession of either TNFd3/d3 or IL-10 (11-16) genotypes. Mismatches for the mHags HY, HA-1, and CD31 exon 125 between donor and recipient did not associate with aGvHD grades II to IV., Conclusions: In contrast to HLA-identical sibling bone marrow transplantation, in mismatched unrelated CBT, neither the cytokine genotypes TNFd3/d3 alone or in combination with IL-10(-1064) alleles nor the minor histocompatibility antigens HY, HA-1, and CD31 exon 125 were associated with aGvHD grades II to IV. Further determination of the cytokine gene polymorphism genotypes in CBTs compared with bone marrow transplants may identify those polymorphisms that could be potential predictive markers for the occurrence of aGvHD.

We investigated the role of polymorphism of the vitamin D receptor (VDR) gene in HLA-matched sibling BMT for polymorphisms previously associated with human disease pathology. In intron 8 of the VDR gene, the B and A alleles of the BsmI and ApaI RFLPs were found to associate with reduced aGVHD when present in the patient's genotype. Logistic regression analysis demonstrated that patient VDR genotype, along with previously identified IL-10(-1064) and IFN-gamma genotype to be risk factors for severe acute GVHD. The A allele also associates with increased likelihood of death when present in the donor genotype (AA vs Aa or aa, hazard ratio 2.03, P = 0.0232). In patients who received increased prophylaxis with multi-agent therapy, patients whose graft was from a donor with an AA genotype had a substantially worse survival than patients whose graft was from a donor with a non-AA genotype (hazard ratio 12.93, P < 0.0001). Analysis of VDR genotype in prospective BMT recipients could indicate patients at risk of severe aGVHD. Analysis of VDR genotype in prospective BMT donors may identify individuals who have greater transplant-related mortality, and also allow appropriately restricted use of increased immunosuppressive prophylaxis.

Aims: Keratinocyte apoptosis is a major pathogenic mechanism in dermal complications, such as graft versus host disease (GVHD), after allogeneic bone marrow transplantation. However, the mechanisms by which recipient target cells undergo apoptosis in GVHD are still unclear, but may result from DNA damage caused by chemotherapeutic agents and/or by direct cytokine action. The basis of this investigation was to correlate keratinocyte apoptosis with (1) the severity of graft versus host reactions (GVHR) in vitro and (2) the clinical grade (0--III) of GVHD., Methods: Skin sections generated from an in vitro skin explant model for detecting experimental or clinically relevant GVHR were investigated for the detection of apoptotic nuclei using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique. This investigation also aimed to establish whether the TUNEL assay could be used as an additional, predictive method for the severity of GVHD before transplantation in potential patient/donor pairs given standard GVHD prophylaxis (cyclosporin A and methotrexate)., Results: By comparing mean values of apoptosis for each GVHR grade in a cohort of 83 retrospective skin sections it was shown that as the severity of GVHR increased there was a parallel increase in the percentage of apoptotic cells (p < 0.0001). However, the correlation between clinical GVHD grade II--III and overall keratinocyte apoptosis (> 2.6%) did not reach this degree of significance (chi(2): 4.2; degrees of freedom, 1; p = 0.04; Fisher's exact test: p = 0.06)., Conclusions: The detection of apoptosis correlated with degree of GVHR using an in vitro assay and a higher degree of apoptosis tended to correlate with more severe GVHD. Further studies in a larger cohort of patients, using other methods to detect apoptosis in conjunction with the TUNEL assay, may give additional insight into the complex immunopathophysiology of GVHD.

This present review concentrates on the recent results investigating the role of certain cytokine gene polymorphisms, including tumor necrosis factor alpha, interferon gamma, interleukin-6 (IL-6), IL-10, and IL-1 receptor antagonist, in allogeneic stem cell transplantation. The review discusses their potential role in predicting outcome and the development of a genetic risk index for graft-versus-host disease in human leukocyte antigen matched sibling transplants. By the comparative use of an in vitro human skin explant model, initial results suggest that certain polymorphisms may be associated with more severe disease.

Acute graft-versus-host disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT) with an incidence of 40-60% and a mortality of up to 50%. Several assays have been developed to try to predict the development of GVHD including the mixed lymphocyte culture reaction, cytotoxic and helper T lymphocyte precursor frequency assays. In the Northern region of England we have used an in vitro skin explant model for predicting GVHD in MHC compatible bone marrow transplant recipients since 1988. The aims of the present study was to test the reproducibility of the model in two other bone marrow transplant centers in Europe. The assay consists of incubating patient skin explants with effector cells from mixed donor versus recipient lymphocyte cultures and the subsequent detection of graft-versus-host reactions by histopathological grading (0-IV) of the skin explants. 503 slides from 134 patients were evaluated. All were graded for negative GVHR grade 0-I or positive grade II-IV. Results from control and test slides significantly correlated between centers to the p value of 0.0001 by Fisher's exact probability test. These results show that the skin explant assay is reproducible between centers and supports the continued use of the assay to predict GVHD in allogeneic stem cell transplant recipients.

Proinflammatory cytokines including interferon-gamma (IFNgamma), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha) are implicated in the pathogenesis of acute graft-versus-host disease (aGVHD). Cytokine gene polymorphism is associated with functional differences in cytokine regulation and altered clinical performance in a variety of diseases. Polymorphism in the IFNgammaIntron1 microsatellite (CA)n repeat has been linked with in vitro IFNgamma production and renal transplant rejection. The IL-6(-174)(G/C) single nucleotide polymorphism has been linked to in vitro and in vivo IL-6 production, juvenile chronic arthritis, and renal transplant rejection. This study examined the potential association of GVHD with IFNgamma and IL-6 polymorphisms in 80 sibling bone marrow transplant (BMT) donor/recipient pairs. Patients homozygous for the IFNgammaIntron1 allele 3 had more severe (grade III-IV) aGVHD. Patients possessing the IL-6(-174)G allele had a trend toward higher grades of aGVHD, and those homozygous for the IL-6(-174)G allele were more likely to develop chronic GVHD (cGVHD). The associations of previously identified aGVHD severity-associated cytokine gene polymorphisms (TNFd and IL-10(-1064)) with severe aGVHD were reconfirmed. Logistic regression analysis confirmed the association of severe aGVHD with recipient genotype at IFNgammaIntron1 (odds ratio [OR] 3.92; P =.02), IL-10(-1064) (OR 4.61; P =.026) and TNFd (OR 3.29; P =.039), and that of cGVHD with recipient IL-6(-174) genotype (OR 4.25; P =.007), in addition to age, gender mismatch, and underlying disease. Assessment of cytokine genotype may potentially allow more accurate prediction of GVHD and appropriate adjustment of GVHD prophylaxis, as well as indicating novel areas for future studies of GVHD pathogenesis.

Severe acute graft-versus-host disease (GvHD) remains a serious complication of allogeneic stem cell transplantation. An in vitro skin explant assay was used to predict the occurrence and severity of acute GvHD in a cohort of 30 pediatric patients undergoing human leucocyte antigen (HLA)-matched sibling transplants (20 patients) and matched or one antigen mismatched unrelated donor transplants (10 patients). In the cohort of HLA-matched sibling transplants, the result appeared to reflect the degree of GvHD prophylaxis. The skin explant assay correlated with GvHD outcome in 12 of 20 children, but this did not reach statistical significance (chi-square 0.95, d.f.=1, p=0.32). These results support previous observations. In this present cohort, patients were treated either with cyclosporin A (CsA) monotherapy (n=7) or with CsA plus additional methotrexate (MTX) (n=13). We have previously demonstrated that the skin explant assay was not as predictive in patients receiving CsA plus additional MTX compared to cohorts treated with CsA alone. In the group of patients treated with CsA alone, four of five patients (80%) predicted to develop GvHD developed acute GvHD of grade II or above; of two patients predicted to develop only grade 0-I GvHD, one patient developed no GvHD and the other grade II GvHD. In the CsA plus MTX group, nine patients were predicted to develop GvHD. Five of nine (55%) developed acute GvHD of grade II or above, while three of four with grade 0 or I skin explant assay results developed only grade 0-I GvHD. In a cohort of 10 patients who received unrelated donor transplants, the skin explant assay correlated with GvHD outcome in all 10 patients (Fisher's exact test p=0.008). Hence, the skin explant assay is a pretransplant in vitro GvHD predictive test that predicts the occurrence and severity of acute GvHD in pediatric unrelated donor transplants and to varying degrees, depending on the GvHD prophylaxis protocols, in HLA-matched sibling cohorts.

Interleukin 1 (IL-1) is involved in various autoimmune and inflammatory diseases. IL-1 receptor antagonist (IL-1Ra) is the naturally occurring antagonist to IL-1alpha and -1beta. Polymorphisms of IL-1beta have been associated with variations in IL-1beta production (nucleotides +3953 and -511). A variable number tandem repeat (VNTR) polymorphism in the IL-1Ra gene has been associated (allele 2) with increased IL-1Ra production. We examined these polymorphisms in human leucocyte antigen (HLA)-matched allogeneic bone marrow transplant patients and donors. IL-1Ra VNTR (allele 2) in the donor genotype was more frequent with milder acute graft-versus-host disease (aGvHD) grades 0-II (29 out of 59 transplants) than severe GvHD grades III-IV (2 out of 18 transplants) (P = 0.0032). This association was confirmed in a subgroup with cyclosporine monotherapy prophylaxis: donor possession of allele 2 was again associated with milder aGvHD, grades 0-II (19 out of 38 transplants), than grades III-IV (1 out of 14) (P = 0.0042) transplants. No association was found between the IL-1beta -511 or IL-1beta +3953 polymorphism and severity of GvHD. Recipient IL-1Ra VNTR genotype (allele 2) showed a strong trend towards association with aGvHD severity (P = 0.0697). Thus, the donor genotype for the IL-1Ra polymorphism has an apparent protective role against acute GvHD following transplantation and may be an additional factor for individual risk assessment for complications, including GvHD, post transplant.

Background: Acute graft versus host disease (GvHD) remains a severe complication of allogeneic haematopoietic stem cell transplantation (HSCT). Our study summaries results of skin explant assay (SEA) as a pretransplant GvHD predictive test in a cohort of paediatric (n = 33) and adult (a = 8) patients receiving grafts from their HLA identical siblings (n = 28), haploidentical relatives (n = 3) and unrelated donors (n = 10). Results GvHD prediction are correlated with the occurrence and severity of acute GvHD posttransplant and effect of GvHD prophylaxis on GvHD clinical outcome is evaluated., Methods and Results: SEA utilises responding lymphocytes of the donor, which are sensitized firstly in vitro by mononuclears cells of patient in allogeneic mixed lymphocyte culture (MLC) and subsequently co-cultured with recipient's skin. Histopathological changes found in patients' skin explants are evaluated according to standard Lerner classification for acute GvHD. In general, GvHD predictive results in SEA correlated with GvHd clinical outcome in 28 out of 41 tested patients (68%, p = 0.015). In a cohort of HLA identical sibling transplants GvHD predictive results correlated with clinical manifestation of acute GvHD only in 15 out of 28 patients on individual GvHD prophylaxis. GvHD prophylaxis in the form of cyclosporine A (CsA) combined with short-term methotrexate (MTX) reduced the risk of acute GvHD in 10 out of 14 transplanted patients (71%) meanwhile CsA alone prophylaxis only in 1 out of 5 patients (20%). In a cohort of unrelated pairs on CsA/MTX prophylaxis combined with horse anti-lymphocyte globuline (ALG) correlated the GvHD prediction with GvHD clinical outcome (100%, p = 0.003). In all patients transplanted with the grafts from their haploidentical relatives the occurrence of severe GvHD was predicted., Conclusion: Skin explant assay helps identify pretransplant patients at higher risk of severe acute GvHD. GvHD predictive results enable the transplantation team to individualise GvHD prophylaxis and to optimise selection of the donor.

Background: Graft-versus-host disease (GVHD) is a major and sometimes fatal complication of allogeneic bone marrow transplantation (BMT). The prediction of GVHD remains an important issue in preventing morbidity and mortality after allogeneic BMT. In the past 10 years, there has been great interest in using the frequency analysis of alloreactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp) to detect recipient-specific alloreactivity and thus predict GVHD in HLA-matched related and unrelated BMT. However, the results remain controversial. The intention of the present study was to investigate whether alloreactive HTLp and CTLp frequencies measured in donor peripheral blood before BMT would be a useful predictor for the occurrence of acute GVHD after HLA-matched sibling BMT., Method: A combined limiting dilution assay was used to determine alloreactive HTLp and CTLp frequencies for 42 HLA-matched sibling patient/donor pairs. The pretransplantation host-reactive HTLp and CTLp frequencies were then correlated with post-transplantation clinical outcomes of acute GVHD. The association between HTLp/CTLp frequencies and the incidence of acute GVHD was determined using the Fisher's exact test., Results: The mean values of HTLp and CTLp frequencies for this cohort of HLA-matched sibling patient/donor pairs were 1:321,322 (range 1:71,000 to 1:1000,000) and 1:195,260 (range 1:3,717 to 1:1000,000), respectively. Acute GVHD (> or =II) was observed in one of four patients with high (>1:100,000) HTLp frequencies and 20 of 36 patients with low (<1:100,000) HTLp frequencies. Similarly, 6 of 10 patients with high (>1: 100,000) CTLp frequencies and 14 of 29 patients with low (<1:100,000) CTLp frequencies developed acute GVHD (> or =II). The overall correlation between hostreactive HTLp/CTLp frequencies and the incidence of acute GVHD in this cohort of patients was 42.5% and 53.9%, respectively. There was no significant difference in the incidence of acute GVHD between the patients with either high or low host-reactive HTLp/ CTLp frequencies (P=0.331 and 0.716, respectively). The data were also analyzed separately for the adult patient group based on GVHD prophylaxis with either cyclosporine alone or the combination of cyclosporine and methotrexate. Within these two prophylaxis groups, neither HTLp nor CTLp frequencies correlated with acute GVHD., Conclusion: Host-reactive HTLp and CTLp frequency analysis did not provide informative prediction for the occurrence of acute GVHD after HLA-matched sibling BMT.

Graft-versus-host disease (GVHD) is the most serious complication following bone marrow transplantation, with an incidence of 40-60%. The disease can be fatal in 50% of cases, even in patients receiving marrow from an HLA identical sibling. Several assays have been developed to try to predict the development of GVHD, including mixed lymphocyte culture reaction, cytotoxic T lymphocyte precursor, and helper T lymphocyte precursor frequency assays. This review describes an in vitro skin explant model which has been used since 1988 for both predicting acute GVHD in HLA identical sibling bone marrow transplantation and studying the pathophysiology of the disease. The model involves sensitising donor lymphocytes in vitro in a primary mixed lymphocyte reaction and then evaluating the secondary response on patient skin biopsies by grading the graft-versus-host reactivity (grades I-IV) histopathologically. From analysis of collective data the model is a clear predictor of GVHD and superior to the other assays widely used, with a correlation of 82% with clinical outcome. The skin explant model allows the investigator to study the pathogenesis of GVHD. The cytokines TNF alpha and IFN gamma are shown to be important mediators of cellular damage in graft-versus-host reactions. Recent work has also involved using the model to study the alloreactivity of cord blood. The model is currently being assessed in several bone marrow transplantation centres in Europe on different patient groups including those who receive marrow from haploidentical and matched unrelated donors.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is strongly implicated in graft-versus-host disease (GVHD) and other acute bone marrow transplant (BMT) complications. The antiinflammatory interleukin-10 (IL-10) antagonizes TNF-alpha and reduces GVHD. We previously showed association of recipient TNF (TNFd) and IL-10 (IL-10(-1064)) gene polymorphisms with acute GVHD severity in matched sibling BMT using only cyclosporin A monotherapy. The current study tested association of GVHD with TNFd and IL-10(-1064/-1082) polymorphisms in a large cohort (144 matched sibling donor/recipient pairs) given both cyclosporine A (CyA) and methotrexate (MTX) prophylaxis. Genotype results were correlated with acute and chronic GVHD and mortality. Patients homozygous for the TNFd microsatellite allele 3 had higher early mortality: 23.7% of TNFd3/d3 homozygotes died before day 30, compared with 6.80% of non-d3/d3 recipients (P =.013). Recipients possessing longer IL-10(-1064) microsatellite alleles developed more severe acute GVHD: 22.3% of recipients possessing alleles 12 to 15 developed grade III to IV GVHD, versus 3.92% of those with smaller alleles (P <.01). Other recipient or donor genotypes tested did not significantly affect GVHD or mortality. We conclude that recipient TNFd and IL-10(-1064) polymorphisms associate with early mortality and severe acute GVHD in matched sibling BMT with dual prophylaxis. This supports the hypothesis of genetic predisposition towards GVHD and other BMT complications other than histocompatibility antigen disparity.

It is now well known that the initial phase of graft-versus-host disease (GVHD) involves cytokine release during preconditioning of the recipient of an allogeneic bone marrow transplant (BMT). Tumor necrosis factor (TNF), in particular, has been implicated in pathological damage and is released pretransplant due to irradiation and cytotoxic preconditioning regimens. Interleukin-10 (IL-10), a natural immunosuppressant of TNF, may be involved in downregulation of these responses, which may be an individual patient-specific effect. In this study, we determined the genotype for polymorphisms associated with TNF and IL-10 in 80 potential allo-BMT recipients and correlated the genotype with the severity of GVHD in 49 patients for whom clinical data relating to GVHD was available. The widely studied TNF -308 polymorphism does not show any significant associations, but the d3 homozygous allele of the TNFd microsatellite is preferentially associated with grade III/IV GVHD (7 of 11 patients) compared with its occurrence in 8 of 38 patients with grade 0/II GVHD (P =.006). Alleles of the IL-10 (-)1064 promoter region microsatellite polymorphism that possess greater numbers of dinucleotide (CA) repeats also significantly associate with more severe GVHD. This region has been demonstrated to be important in the regulation of the IL-10 promoter. Eighteen of 38 patients with grade 0-II GVHD possessed alleles with greater numbers (12 or more) of dinucleotide repeats, compared with 9 of 11 cases with grade III-IV GVHD (P <.02). Of the 38 patients with grade 0-II GVHD, 3 of 38 had a both TNFd3/d3 and IL-10 (12-15) genotype, compared with 6 of 11 patients with grade III-IV GVHD (P <.001). There was no association of either the TNFd or IL-10 microsatellite polymorphisms with mortality (P =.43 and.51, respectively). Our results suggest that patient cytokine gene polymorphism genotypes may influence GVHD outcome by affecting cytokine activation during the pretransplant conditioning regimens, and these results are the first to suggest a genetic predisposition to this important transplant-related complication.

Background: Graft-versus-host disease (GVHD) occurring after HLA-identical sibling bone marrow transplantation (BMT) is considered to be mainly caused by minor histocompatibility antigen (mHag) disparities between the recipient and donor. In our laboratory, a human skin explant model has been successfully used to predict acute GVHD in HLA-identical sibling BMT. More recently, the frequency analysis of host-reactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp, respectively) has been shown to have potential application for predicting GVHD. In the present study, HTLp and CTLp frequency analysis and the skin explant model were directly compared for their ability to predict acute GVHD in HLA-identical sibling BMT., Methods: Host-reactive HTLp and CTLp frequencies were determined using a combined limiting dilution assay. A human skin explant model was used to detect graft-versus-host reactions in vitro. The results from the skin explant model (graft-versus-host reaction grades I-IV) and T cell frequency analysis (>/< 1:100,000) were correlated with posttransplant GVHD outcome, respectively., Results: The skin explant model correctly predicted GVHD outcome in 77% of cases (P=0.03). HTLp frequencies were very low in all patient/donor pairs tested. None of them exceeded 1:100,000, although 9/18 recipients developed GVHD (> or =clinical grade II) after transplant. In all patients tested, the relationship between either high (>1:100,000) or low (<1:100,000) CTLp frequency and occurrence of GVHD appeared to be random (P=1.0)., Conclusions: HTLp and CTLp frequency analysis did not predict the occurrence of acute GVHD after HLA-identical sibling BMT. The human skin explant model, however, remained an accurate indicator of acute GVHD and probably detects mHag disparities.

Graft vs host disease (GVHD) following allogeneic marrow transplant in humans is known to be mediated in part by cytokines. Tumour necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are considered to be critical in the mechanism. A commercially produced antibody to TNF-alpha (CB0006) was tested in vitro to assess its ability to suppress mixed lymphocyte reactions (MLR). Peripheral blood mononuclear cells treated and untreated with CB0006 were also tested for their ability to elicit a graft vs host reaction (GVHR) in vitro in a human skin-explant model for GVHD. The results showed that CB0006 could inhibit mixed lymphocyte culture (MLC) proliferative responses and that in vitro antibody treated responder cells were less effective in causing GVHR in vitro skin-explant assays. CB0006 was also shown to inhibit MLC supernatant induced GVHR.

An in vitro skin explant model has been used to predict the severity of acute graft-versus-host disease (GVHD) in 34 HLA-identical bone marrow transplant recipients (correlation coefficient 0.6 p < 0.001). Supernatants from HLA-matched patient/donor mixed lymphocyte cultures (MLCs) were analysed for levels of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). High levels of both cytokines correlated with the development of GVHD grades II or above (p < 0.05). The supernatants were also tested for induction of class II MHC antigen expression on third party skin and results correlated with clinical outcome in 17 of 22 cases (77%) (correlation coefficient 0.65, p < 0.001). The results suggest that measurement of both TNF alpha and IFN gamma in HLA-matched MLC supernatants is of predictive value and that the skin explant model is a useful model for studying the aetiology of GVHD in humans.

An in vitro skin explant model has been used in an attempt to predict the severity of graft-versus-host disease (GVHD) in HLA-identical donor-recipient pairs. The skin explant model involves the use of donor lymphocytes which have been sensitized against recipient lymphocytes in vitro and then co-cultured with the recipient's skin. Thirteen patients were studied in a prospective manner and results from the skin explant model compared with the clinical status of the patient post-transplant showed good correlation (p less than 0.001). Results from T cell-depletion studies indicated a role for both CD4 and CD8 positive cells in GVHD. In conclusion the results confirmed that the skin explant assay is a useful and predictive test of GVHD in humans.

An in vitro skin explant model for graft-versus-host disease (GVHD) in humans has been used to study the role of effector T cells in the histological pathogenesis of GVHD. In 11 of 12 experiments clear GVHD changes of grades II-IV were induced in HLA-mismatched skin explants cultured with allogeneic T cells sensitized by in vitro mixed lymphocyte culture. The role of effector T cells was investigated by comparing results before and after removal of CD3 positive cells, and CD4 positive and CD8 positive T cell-subsets by antibody and complement cytolysis from responder populations. Only total removal of CD3 positive T cells prevented histopathological lesions of GVHD in the skin biopsy specimens. The results also demonstrated that the CD4 positive population caused the greatest degree of GVHD in vitro in skin biopsy specimens and direct infiltration into skin by cells is not required for changes to become evident. These results confirm the early results on animal models and demonstrate the use of the skin explant model as a tool for studying the biology of GVHD in humans.