Evaluation of a human in vitro skin test for predicting drug hypersensitivity reactions.

The occurrence of drug hypersensitivity reactions (DHRs) following administration of low molecular weight (LMW) drugs is an important health concern. However, in vivo animal models which could be used as tools for the prediction of DHRs are lacking. As a result, research has focused on development of in vitro tools for predicting DHRs. In this study a novel human in vitro pre-clinical skin explant test was used to predict T cell-mediated hypersensitivity responses induced by LMW drugs. Responses in the skin explant test for 12 LMW drugs associated with T cell-mediated hypersensitivity in the clinic (abacavir, amoxicillin, carbamazepine, diclofenac, lamotrigine, lapatinib, lumiracoxib, nevirapine, ofloxacin, phenytoin, propranolol, sulfamethoxazole) were compared with responses for 5 drugs with few/no reports of T cell-mediated hypersensitivity reactions (acetaminophen, cimetidine, flecainide, metformin, verapamil). Changes in skin histology following in vitro exposure to the drugs as well as T cell proliferation and interferon gamma (IFNγ) production were studied. The results of the skin explant assays showed a good positive correlation (r = 0.77, p < .001) between the test outcome (prediction of positive or negative) and the clinical classification of the tested drugs. The T cell proliferation assay showed a correlation of r = 0.60 (p < .01) and the IFNγ assay r = 0.51 (p < .04). The data suggest that the skin explant model could be a useful tool to predict the potential of LMW drugs to induce DHRs.

Autologous tolerogenic dendritic cells for rheumatoid and inflammatory arthritis.

OBJECTIVES: To assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities. METHODS: An unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood. RESULTS: There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high. CONCLUSION: IA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 tolDC but no systemic clinical or immunomodulatory effects were detectable. TRIAL REGISTRATION NUMBER: NCT01352858.

Impact of genomic risk factors on survival after haematopoietic stem cell transplantation for patients with acute leukaemia.

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.

An in vitro human skin test for assessing sensitization potential.

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process.

Non-HLA genomics: does it have a role in predicting haematopoietic stem cell transplantation outcome?

Haematopoietic stem cell transplantation (HSCT) remains the only cure for many haematological neoplasms; however, the mortality rate remains high, at around 30-80%. Complications after HSCT include relapse, graft-versus-host disease, graft rejection and infection. High-resolution HLA matching has improved survival in HSCT over recent years; however, GVHD still remains a serious complication. Single nucleotide polymorphisms (SNPS) within genes that are involved with an individual's capability to mount an immune response to infectious pathogens, residual leukaemia, alloantigens or genes involved in drug metabolism have been studied for their association with HSCT outcome. Indeed, over the last 15 years, several groups, including ourselves, have demonstrated that non-HLA gene polymorphisms can be predictive of HSCT outcome. Can genetic characteristics of the patient and donor be used in the future to tailor HSCT protocols and determine GVHD prophylaxis? This review summarizes some of the recent SNP association studies in HSCT and highlights some of the disparities therein, discussing the integral problems of performing genetic association studies on diseases with complex outcomes using heterogeneous cohorts. The review will comment on recent genomewide association studies (GWAS) and discuss their relevance in this field, and it will also comment on recent meta-analysis combining GWAS studies with other studies such as gene expression micro array data in the field of autoimmune disease and solid organ transplantation. It will mention possible novel candidate gene polymorphisms, for example SNPS in microRNAs. In addition, it will discuss some of the inherent problems associated with gene association studies including the GRIPs (genetic risk prediction studies) recommendations. In summary, this review will assess the usefulness of non-HLA genomic studies in HSCT with regard to predicting outcome and modifying therapy.

Mesenchymal stem cells from CD34(-) human umbilical cord blood.

Umbilical cord blood (UCB) is well known to be a rich source of stem cells especially for haematopoietic stem cells (HSCs). Recently, mesenchymal stem cells (MSCs) have also been shown to exist in cord blood. Although MSCs have been described by a subset of surface antigens after expansion, little is known about the cell surface phenotype of undifferentiated MSCs. The aim of this study therefore was to clarify whether undifferentiated MSCs are resident among CD34(-) UCB cells. CD34(+) cells were separated from UCB mononuclear cells (MNCs) by magnetic sorting and the CD34(-) cell fractions were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal calf serum (FCS) and basic-fibroblast growth factor. Isolated CD34(+) cells were also cultured in the same medium. Adherent fibroblast-like cells at passage 3-4 were analyzed by fluorescence-activated cell sorting (FACS) for MSC marker expression , and standard adipogenic, osteogenic and chondrogenic assays were used to investigate their differentiation potentials. After 4-5 weeks in culture, the cells from the CD34(-) fraction became confluent with flat and fibroblast-like morphology. These cells were positively stained for the mesenchymal cell markers CD29, CD73 and CD105. In adipogenic differentiation, the cells showed oil red O positive and expressed FABP4, adipsin and proliferation-activated receptor gamma-2 (PPARgamma2 genes) associated with adipogenesis. In osteogenic differentiation, calcium accumulation and osteocalcin were detected. The cells grown in chondrogenic conditions were positively stained for human aggrecan and expressed collagen type II and Sox-9 genes. In contrast, cells from the CD34(+) fraction failed to generate any cells with MSC morphology under the same culture conditions. Our results showed that UCB contained MSCs which are only resident in the CD34(-) fraction. The MSCs could be induced to differentiate into at least three lineage cell types, adipocytes, osteoblasts and chondrocytes.

Polymorphisms of cytokine and innate immunity genes 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.

Non-HLA genetics and predicting outcome in HSCT.

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.

The role of genetic variants of NOD2/CARD15, a receptor of the innate immune system, in GvHD and complications following related and unrelated donor haematopoietic stem cell transplantation.

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.

The proteome pattern cGvHD_MS14 allows early and accurate prediction of chronic GvHD after allogeneic stem cell transplantation.

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_MS14) established by capillary electrophoresis-mass spectrometry to predict onset and severity of cGvHD as an unbiased laboratory test. cGvHD_MS14 was evaluated on samples from 412 patients collected prospectively in four transplant centers. Sensitivity and specificity was 84 and 76% by cGvHD_MS14 classification. Sensitivity further increased to 93% by combination of cGvHD_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_MS14. cGvHD_MS14 consists of 14 differentially excreted peptides, six of those have been sequenced to date and are fragments from thymosin ß-4, eukaryotic translation initiation factor 4γ2, fibrinogen ß-chain or collagens. In conclusion, the cGvHD_MS14-pattern allows early, highly sensitive and specific prediction of cGvHD as an independent diagnostic criterion of clinical diagnosis potentially allowing early therapeutic intervention.

Interleukin-2 induction of lymphokine-activated killer activity in the peripheral blood of an acute lymphoblastic leukaemia patient--case study.

In this case study an acute lymphoblastic leukaemia (ALL) patient relapsing after autotransplant had remission reinduced with chemotherapy and consolidated after initial response by a course of therapy with recombinant interleukin-2 (rIL-2) given subcutaneously. Immunological parameters measured during therapy demonstrated an increase in the numbers of T cells and in lymphokine-activated killer (LAK) cell activity against autologous leukaemic blasts and LAK-sensitive cell lines. The therapy was well tolerated and administered on an out-patient basis. The patient has remained in haematological remission for over twelve months. Sustained remissions have not been observed previously in relapsed transplant patients using chemotherapy alone. The data suggests that rIL-2 deserves further evaluation in ALL patients who are immunologically intact with residual disease after primary or secondary chemotherapy.

Cytokine treatment of human bone marrow activates anti-leukaemia effector cells: monitoring of purging by polymerase chain reaction and DNA analysis.

The aims of this study were to investigate the role of cytokines (tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-2 (IL-2) in augmenting graft-versus-leukaemia (GVL). We have investigated the effector cells involved in GVL, by studying the role of these cells in purging of the cell line K562 in short-term bone marrow cultures. The effect of the addition in vitro of rGCSF was also studied. Monitoring of purging was achieved by cytotoxicity assays, DNA analysis and the use of the polymerase chain reaction for the detection of bcr/abl transcripts in the Philadelphia positive (Ph+) K562 cell line. Supernatants from IL-2-treated and non-treated bone marrow were tested for cytokine production (TNF alpha and IFN gamma). The results have shown that the main cytotoxic effector cells in the bone marrow generated by IL-2 have the CD56+ CD8+ phenotype. Overnight incubation of bone marrow was sufficient to generate cytotoxic cells as measured by Chromium51 (Cr51) release assays. Measurable levels of TNF alpha but not IFN gamma were also detected in supernatants. Addition of TNF alpha and IFN gamma to the IL-2 in the bone marrow cultures augmented the cytotoxicity but tended to inhibit progenitor cell growth as measured by granulocyte-macrophage colony-forming unit (GM-CFU) and erythroid blast-forming unit (BFU-e) assays. An estimate of the purging of the marrow could also be achieved by DNA analysis of K562 DNA in bone marrow. The bcr/abl transcript could still be detected by PCR analysis in marrow containing 1% K562 and treated with IL-2 for 24 h, but by 6 days of incubation the bcr/abl transcript was weak or undectable. The results suggest that although reduction in the proportion of leukaemia in contaminated marrow can be detected after incubation with IL-2 for 24 h, complete elimination of minimal residual disease requires longer incubation times.

Analysis of CD34 populations in mobilised peripheral blood stem cell harvests and in bone marrow by fluorescent in situ hybridisation for the bcr/abl gene fusion in patients with chronic granulocytic leukaemia.

For those patients ineligible for allogeneic bone marrow transplant and who are non-responsive to interferon, autotransplant with peripheral blood stem cells (PBSC) mobilised after intensive chemotherapy, may provide a novel approach to improve prognosis in patients with chronic granulocytic leukaemia. PBSC harvests are assessed for CD34-positive cell numbers, which serve as an indicator of engraftment potential, and are also analysed cytogenetically to ascertain tumour cell contamination. However, a more accurate assessment of PBSC harvest contamination requires investigation of the Philadelphia (Ph) status of the CD34pos population, in which the cells that provide long-term engraftment are contained. In this study, we have analysed these levels in mobilised PBSC and also in bone marrow (BM) harvests, taken several weeks prior to mobilising chemotherapy. Using fluorescent in situ hybridisation for the bcr/abl gene fusion, we have shown that the median number of Ph negative cells in CD34pos isolated populations was 14.95% in BM compared to 79.05% in PBSC harvests and that in all PBSC samples tested, Ph positivity in CD34pos populations was always detectable either by FISH or one round PCR methods. In paired assessments of both PBSC and BM harvests, higher levels of Ph negative CD34pos cells (> or = 14%) isolated from BM harvests, taken prior to intensive chemotherapy, correlated with higher levels of Ph negative CD34pos cells (> or = 78.5%) in PBSC harvests. These data may aid in the selection of patients for whom PBSC harvesting, after mobilisation, is more likely to achieve an autograft product containing predominantly Ph negative CD34pos cells and may exclude those patients for whom the risk, morbidity and expense of stem cell harvesting may have no apparent benefit over a chronic phase BM harvest.

Collection of Philadelphia-negative peripheral blood progenitor cells in unselected patients with chronic granulocytic leukaemia. Northern Regional Haematology Group.

Thirty unselected patients with chronic granulocytic leukaemia (CGL), age range 22-59 years, were treated with intensive chemotherapy and G-CSF to mobilize peripheral blood progenitor cells (PBPC). Chemotherapy was well tolerated and PBPC were collected by leukapheresis early during white cell recovery. PBPC collections considered adequate for engraftment were collected in 21 patients. Cytogenetic analysis of all collections in these patients showed >75% Ph negativity (range 79-100%) in 10. Successful collections, ie those >75% Ph negative and with total cell count of >1 x 10(6) CD34+ve cells/kg or >20 x 10(4) CFU-GM/kg were further analysed by Southern blot or RT-PCR. All samples were positive for the bcr/abl transcript. Patients with a low Sokal score (<0.8) were more likely to achieve a successful collection. In contrast, there was no association between transcript expression and likelihood of successful collection. We have confirmed that it is possible to mobilize and collect Ph-negative enriched PBPC in unselected patients with CGL. This procedure is more likely to be successful earlier rather than later in the course of the disease. Whether such collections will give an advantage over unmanipulated autologous bone marrow transplantation in CGL requires further study, but our experience suggests that suitable material for autologous rescue can be obtained from approximately one third of eligible, unselected young patients.

Dipyridamole potentiates antipurine antifolate activity in the presence of hypoxanthine in tumor cells but not in normal tissues in vitro.

The cytotoxicity of the antifolate inhibitors of de novo purine biosynthesis, lometrexol (LTX) and LY309887, can be abolished by hypoxanthine (HPX) salvage. The nucleoside transport inhibitor, dipyridamole (DP) can prevent HPX rescue from LTX growth inhibition in a cell line-specific manner. The studies described here have shown that, excluding colon and hematological malignancies, DP prevents HPX rescue from LTX growth inhibition in approximately one-third of cell lines with otherwise limited tissue specificity. The clinical dose-limiting toxicities of antipurine antifolates are to the bone marrow and gastrointestinal tract. In vitro models of these normal tissues were established, and the effect of DP on HPX rescue from LY309887 treatment was studied. Growth inhibition assays are not feasible in these primary cultures; therefore, an alternative assay, cellular ATP depletion, was validated in four tumor cell lines as a marker of de novo and salvage purine synthesis. In LY309887-treated cells, DP prevented HPX-mediated maintenance of ATP levels only in cell lines in which DP inhibited HPX rescue from antifolate cytotoxicity. Hence, ATP depletion is a reliable indicator of sensitivity of HPX transport to DP when direct cell growth measurement is impractical. In primary cultures of human hematopoetic progenitor cells and mouse small intestine, coincubation with HPX prevented LY309887-mediated ATP depletion, which was not blocked by DP. These data suggest that DP would not prevent HPX rescue from antipurine antifolate growth inhibition in sensitive normal tissues, whereas activity against certain solid human tumors would be maintained.

Identification and validation of biomarkers associated with acute and chronic graft versus host disease.

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.

Heat shock protein 70: correlation of expression with degree of graft-versus-host response and clinical graft-versus-host disease.

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.

T-cell frequency analysis does not predict the incidence of graft-versus-host disease in HLA-matched sibling bone marrow transplantation.

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.

Predicting graft-versus-host disease in HLA-identical bone marrow transplant: a comparison of T-cell frequency analysis and a human skin explant model.

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.

Cellular alloreactivity of human cord blood cells detected by T-cell frequency analysis and a human skin explant model.

BACKGROUND: Recent clinical data have demonstrated the success of allogeneic stem cell transplantation using HLA-mismatched unrelated human umbilical cord blood (CB). The incidence and severity of acute graft-versus-host disease (GVHD) in these mainly pediatric transplants is low. The immunological mechanisms by which CB transplants may result in reduced GVHD is not completely clear. In this study, the functional cellular alloreactivity of CB cells was investigated, by measuring the frequency of alloreactive helper and cytotoxic T lymphocyte precursors (HTLp and CTLp, respectively) in CB and detecting the ability of CB cells to induce graft-versus-host (GVH) type alloreactivity in vitro. METHODS: A human skin explant model was used to measure GVH type alloreactivity in vitro. A combined limiting dilution assay was carried out in parallel to determine alloreactive HTLp and CTLp frequencies. The cellular alloreactivity was compared between cord and HLA-haploidentical parental blood cells against the same HLA-mismatched unrelated stimulator. RESULTS: The results demonstrated that alloreactive CTLp frequency in CB mononuclear cells (CBMCs) was significantly lower (mean, 1:35,694, range, 1:1,667-<1:500,000) than that in adult peripheral blood mononuclear cells (PBMCs) (mean, 1:5,333, range, 1:544-1:47,619). Alloreactive HTLp frequencies, however, were comparable for CBMCs and PBMCs (mean, 1:7,586, range, 1:1,359-1:200,000; and mean, 1:5,976, range, 1:385-1:50,000, respectively). A significantly decreased ability to induce in vitro GVH type alloreactivity was observed for CBMCs and that was strongly associated with low alloreactive CTLp frequencies (P=0.001). CONCLUSIONS: The present study provides the first clear in vitro evidence to suggest that CBMCs are less able than PBMCs to induce skin GVH type alloreactivity in HLA-mismatched pairs. The severity of in vitro GVH type alloreactivity (graded as I-IV) was strongly associated with the levels of alloreactive CTLp frequencies. The low cellular alloreactivity of CBMCs detected in vitro suggests that in a proportion of cases HLA-mismatched unrelated CB may not give rise to severe GVHD in vivo after transplantation.