aGVHD In Patients Research Articles

Implication of Early Lymphocyte Recovery after Allogeneic Hematopoietic Stem Cell Transplantation in Children with Leukemia

PurposeThe repopulating lymphocytes after allogeneic hematopoietic stem cell transplantation have an important role not only on the prevention of serious infections in the early transplantation period, but also on the killing of residual leukemic cells by graft-versus-leukemia effect. The aim of this study was to analyze the impact of lymphocyte recovery after allogeneic stem cell transplantation in children with hematologic malignancies.Materials and MethodsWe evaluated 69 children transplanted for acute lymphoblastic leukemia (ALL) (n=34), acute myeloid leukemia (AML) (n=26), chronic leukemia (n=7) and juvenile myelomonocytic leukemia (n=2) between 1996 and 2008 at the Chonnam National University Hospital, Korea. The patients were grouped based on absolute lymphocyte counts (ALC) <500/µL or ≥500/µL at D+21 and D+30 after transplant.ResultsPatients with a High ALC at D+21 and D+30 had a faster neutrophil and platelet engraftment. The High at D+30 group had a better 5 year overall survival (71% vs. 53%, p=0.043) and event-free survival (72% vs. 53%, p=0.065) than the Low at D+30 group. The incidence of grade II-IV acute and chronic graft-versus-host disease (GVHD), and relapse rate did not differ by the ALC counts. However, the Low at D+30 group had a significantly increased risk for transplant-related mortality (p=0.019). The univariate analysis showed that the factors associated with decreased survival were a Low ALC at D+30, patients with high risk ALL, and grade II-IV aGVHD in patients with ALL and AML.ConclusionEarly posttransplant serial lymphocyte measurement would be a simple but useful method for predicting transplant outcomes.

Frequency of CD4+FOXP3+ regulatory T-cells at early stages after HLA-mismatched allogeneic hematopoietic SCT predicts the incidence of acute GVHD

Acute GVHD (aGVHD) is a major obstacle to allogeneic hematopoietic SCT (alloHSCT). Although it is thought that aGVHD is initiated in secondary lymphoid organs at a very early stage of alloHSCT, whether CD4(+)FOXP3(+) regulatory T-cells (Tregs) have an impact on aGVHD development during this period remains unclear. Here, we measured Tregs in peripheral blood as early as possible after HLA-mismatched alloHSCT, and assessed the incidence of aGVHD. Flow cytometric analyses revealed that at the second week after HSCT, patients with aGVHD had significantly (P=0.018) lower Treg:CD4(+)T-cell ratios than those without aGVHD. As these differences were seen before the development of aGVHD, these ratios can predict the incidence of aGVHD. The cumulative incidence of aGVHD in patients with ratios of <9% was significantly higher than that in patients with ratios of 9% (P=0.0082, log-rank test). Additionally, the specific ratio of Tregs:CD4(+)T-cells was the most significant value among all other possible lymphocyte-associated ratios and absolute cell counts. These findings suggest that the ratio of Tregs:CD4(+)T-cells at the second week post HLA-mismatched alloHSCT might be a potent predictor of aGVHD in these patients. The practical efficacy of this finding should be verified in further interventional studies.

Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

AbstractInvariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4− iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4− iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4− iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4− iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4− iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.

Graft-versus-Tumor Effect According to Type of Graft-versus-Host Disease Defined by National Institutes of Health Consensus Criteria and Associated Outcomes

The impact of National Institutes of Health consensus criteria (NCC) graft-versus-host disease (GVHD) on survival has rarely been investigated in a large cohort of patients with GVHD presenting before and after day 100 posttransplantation. We retrospectively investigated 775 patients who underwent allogeneic stem cell transplantation and assessed the GVHD effects on survival by the time-dependent covariates in Cox proportional hazards regression models. Using the NCC, the patients were classified into 4 groups: (1) no GVHD (n= 251); (2) acute GVHD (aGVHD) only (n = 199), including 26 patients with late aGVHD; (3) classic chronic GVHD (cGVHD; n = 232); and (4) overlap syndrome (OS; n = 93). Multivariate analyses showed that classic cGVHD (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.27-0.77) and OS (HR, 0.52; 95% CI, 0.28-0.96) were associated with significantly decreased risk of relapse, whereas aGVHD only was not associated with relapse rate (HR, 1.11; 95% CI, 0.76-1.63). All aGVHD events, including the period of aGVHD in patients who developed cGVHD after aGVHD, also did not affect the risk of relapse (HR, 0.74; 95% CI, 0.49-1.12). All types of GVHD were significantly associated with higher nonrelapse mortality in common. Finally, patients with aGVHD only had significantly lower overall survival and disease-free survival compared with those without GVHD, in contrast to favorable survival outcomes in patients with cGVHD without previous aGVHD. This study demonstrates that NCC GVHD type is associated with different graft-versus-tumor effects. Further studies are needed to investigate risk factors, pathogenesis, and biomarkers for each type of NCC GVHD.

Genome-Wide Association Study for Determinants of Acute Graft Vs Host Disease (aGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation: Development of 7 SNP Model Predicting aGVHD

Abstract 1010 Background:Numerous studies have been reported non-HLA genetic associations with acute graft vs host disease (aGVHD) developing after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, however, relatively few genes have been examined in each cohort. Meanwhile, aGVHD represents one of the most attractive targets to study the effect of normally silent genetic polymorphisms. We intended to elucidate genetic determinants predicting aGVHD in patients undergoing allo-HSCT by genome-wide screening of genetic polymorphisms, and develop a SNP model to predict development of aGVHD. Methods:Genetic polymorphisms predicting development of aGVHD were determined through successive 5 stages in a total of 429 patients who underwent allo-HSCT. Firstly, we screened genomic determinants predicting aGVHD using 109K SNP chip (Sentrix Human-1 GT Bead Chip, Illumina) in 15 patients (7 patients with aGVHD and 8 without aGVHD). At stage 2 and 3, low-density Immumina chips comprising 768 candidate SNPs and 96 SNPs, respectively, were manufactured and tested in independent cohorts of 91 patients (34 patients with aGVHD and 57 without aGVHD) and 84 patients (35 patients with aGVHD and 49 without aGVHD), respectively. At stage 4, 76 SNP chips comprising 33 SNPs discovered from the previous studies and also 43 SNPs reported to be associated with aGVHD in the literature were manufactured and evaluated in 36 patients (13 patients with aGVHD and 23 without aGVHD) undergoing allo-HSCT. Finally, 7 SNPs were chosen and validated in an independent cohort of 203 patients (50 patients with aGVHD and 153 without aGVHD) using TaqMan assay. Results:At stage 1, 955 SNPs were identified to be significantly associated with development of aGVHD by whole genome assay. At stage 2, candidate genetic determinants were narrowed down to 74 SNPs. Through stage 3 and 4, 7 SNPs including the two selected from the literature review (transforming growth factor beta receptor II (TGFBR2) and interleukin-18) were finally determined as predictive markers for aGVHD. The 7 SNP model was found to be effective in predicting the development of aGVHD after allo-HSCT. When decision tree model was applied, the sensitivity, specificity, accuracy, and area under ROC (AUR) of the 7 SNP model to predict aGVHD were 80.5%, 80.4%, 80.4% and 91.6%, respectively. Conclusion:A genome-wide association study approach was successful in elucidation of genetic determinants associated with development of aGVHD in patients undergoing allo-HSCT. The 7 SNP model was effective in predicting the development of aGVHD after allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Association Between MDSCs and Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation,

Abstract 4069Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cells, derived from bone marrow progenitor cells and immature myeloid cells. In tumor development and progression, MDSCs play a role of immunosuppressive effect. Recently, researchers found that granulocyte colony-stimulating factor (G-CSF) could mobilize MDSCs from bone marrow (BM) to peripheral blood (PB), but there is unknown about the effect of G-CSF to MDSCs in the human body and the relationship between MDSCs and graft-verse-host disease (GVHD). So we investigated the association between MDSCs and acute graft-verse-host disease (aGVHD).The expression of MDSCs and adhesion molecules in the BM, PB and peripheral blood stem cell grafts in 30 related donors, were detected by flow cytometry before and after recombined human G-CSF (rhG-CSF) mobilization. The expression of arginase (ARG) and inducible nitric oxide synthase (iNOS) were detected by enzyme-linked immunosorbent assay. According to the median number of MDSCs in peripheral blood stem cell harvests (9.60×106/kg), 30 leukemia patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were divided into two groups, high MDSCs group and low MDSCs group. The hematopoietic/immune reconstitution, incidence of aGVHD, relapse, and survival rate were compared between two groups.MDSCs could be detected in the blood and BM in healthy donors, the proportion in nuclear cells in the blood and BM was 0.30% ± 0.09% and 0.53% ± 0.16%, respectively. MDSCs in BM was higher than that in PB (P <0.001). After 5 days of rhG-CSF mobilization, the proportion in the blood and BM was 0.66% ± 0.28% and 0.72% ± 0.13%, respectively. The difference between proportion in the blood before and after mobilization was significant (P =0.017). Adhesion molecules on MDSCs surface after rhG-CSF mobilization were significant lower than that before rhG-CSF mobilization, both in blood and BM (P <0.001). The expression of ARG and iNOS after mobilization were significantly higher than that before mobilization (P<0.001), but not in BM (P =0.695, 0.073). There was a significant negative correlation between the number of transplanted MDSCs cells and the grading of aGVHD (correlation coefficient rs =0.445, P =0.014). The difference between the incidence of aGVHD in high MDSCs group and low MDSCs group (20.00% 315 vs 66.67% 1015, P =0.011), there was no significant difference between the hematopoietic/immune reconstitution, relapse rate (26.67% 415 vs 20.00% 315, P =0.671) and disease-free survival rate (46.7% ±14.2% vs 38.9%±15.8%, P =0.833) in two groups.In conclusion, in the human body, rhG-CSF could transfer more MDSCs from BM into the blood. RhG-CSF mobilization could increase the number of MDSCs in graft, which may be related to low incidence of aGVHD after allo-HSCT. The mechanism of rhG-CSF mobilized MDSCs to blood may be related to adhesion factor on MDSCs surface. The incidence of aGVHD in patients who accepted high MDSCs cells was lower; there was a significantly negative correlation between MDSCs transplant value and the grading of aGVHD. MDSCs cell infusion did not affect the function of immune reconstitution and the effect of graft versus leukemia. Disclosures:Liu:National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.

Association of HLA-G Low Expressor Genotype with Severe Acute Graft-Versus-Host Disease after Sibling Bone Marrow Transplantation

Background: Human leukocyte antigen-G (HLA-G) molecules play a prominent role in immune tolerance. Structurally similar to their classical HLA homologs, they are distinct by having high rate of polymorphism in the non-coding regions including a functionally relevant 14-base pair (bp) insertion/deletion (Ins/Del) allele in the 3′ untranslated region (3′UTR), rarely examined in a hematopoietic stem cell transplantation (HSCT) setting. Here, we analyzed the potential impact of HLA-G Ins/Del dimorphism on the incidence of acute graft-versus-host disease (aGvHD), transplant-related mortality (TRM), overall survival (OS), and incidence of relapse after HSCT using bone marrow (BM) as stem cell source from HLA-matched donors. Methods: One hundred fifty-seven sibling pairs, who had undergone HSCT, were studied for the distribution of the HLA-G 14 bp Ins/Del polymorphism using a polymerase chain reaction (PCR)-based technique. Potential genetic association with the incidence of aGvHD, TRM, and OS was analyzed by monovariate and multivariate analyses. Results: Monovariate analysis showed that the homozygous state for the 14-bp Ins allele is a risk factor for severe aGvHD (grade III and IV; P = 0.008), confirmed subsequently by multivariate analysis [hazard ratio (HR) = 3.5; 95% confidence interval (95%CI) = 1.3–9.5; P = 0.012]. We did not find any association between HLA-G polymorphism and the other studied complications. Conclusion: Our data suggest that the HLA-G low expressor 14 bp Ins allele constitutes a risk factor for the incidence of severe aGvHD in patients who received BM as stem cell source.

Diarrhea during the Conditioning Regimen Is Correlated with the Occurrence of Severe Acute Graft-versus-Host Disease through Systemic Release of Inflammatory Cytokines

Graft-versus-host disease (GVHD) is a major obstacle to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although many indicators have been used to predict GVHD, the prediction of GVHD remains very difficult. Determining how to predict the occurrence of acute GVHD (aGVHD) as exactly as possible has been a huge challenge so far. Systemic release of inflammatory cytokines during the conditioning regimen and early-phase post-HSCT plays a crucial role in the generation of aGVHD; additionally, the conditioning regimen causes the damage to the intestinal tract. Diarrhea is a common symptom of intestinal tract damage during the conditioning regimen. We therefore performed a prospective study to investigate the relationship between diarrhea related to the conditioning regimen, systemic release of inflammatory cytokines during the conditioning regimen and the early-phase post-HSCT, and the development of aGVHD. This study demonstrated that duration of diarrhea was >5.5 days, with a maximal volume >8.72 mL/kg and a mean volume of diarrhea for days -3 to 0 >7.94 mL/kg were risk factors of grade II to IV aGVHD. Diarrhea with any 1 risk factor was defined as severe diarrhea. Furthermore, this study first confirmed that the correlation between diarrhea and aGVHD was related to the serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 during the conditioning regimen and during the early phase after transplantation. Our study demonstrated that diarrhea related to the conditioning regimen could be used as a marker for the prediction of aGVHD and further explain the possible mechanism underlying the linkage described here. Therefore, for the patients with severe diarrhea related to the conditioning regimen, low-dose glucocorticoid may be used to reduce the levels of inflammatory cytokine release by a damaged intestinal tract, and possibly further reduce the occurrence of aGVHD. For these patients, prophylaxis of aGVHD may be need to be adjusted individually.

Genetic Variations of Interleukin-23R (1143A>G) and BPI (A645G), but Not of NOD2, Are Associated with Acute Graft-versus-Host Disease after Allogeneic Transplantation

Single nucleotide polymorphisms (SNPs) in genes of the immune system predict for aGVHD and mortality after allo-SCT. We investigated the effect of SNPs in the NOD2, BPI, and IL-23R genes on posttransplantation outcome in a cohort of 304 patients. NOD2 patient and donor genotype and BPI recipient genotype were not associated with the occurrence of aGVHD. However, IL-23R-SNP in the donor was correlated with less aGVHD. This association could be confirmed in multivariate analysis (odds ratio [OR], 0.39; P = .039), which identified in vivo T cell depletion (OR, 0.32; P < .001) and multiagent GVHD prophylaxis (OR, 0.51; P = .031) as other independent factors predicting for less-severe aGVHD. This multivariate model also revealed a trend toward less aGVHD in patients receiving a BPI G allele transplant (OR, 0.60; P = .067) and in those receiving a transplant from an HLA-matched donor (OR, 0.57; P = .058). Incontrast, relapse was more frequent in patients with NOD2-SNPs (46.2% for SNP vs 33.2% for wild-type; P= .020). This association was found to be of borderline significance in multivariate analysis. Neither BPI nor IL-23R genotype predicted for relapse, and none of the investigated SNPs was correlated with 5-year overall survival. In our analysis, NOD2 SNPs did not predict aGVHD, but IL-23R(1142A>G) and BPI(A645G) SNPs appeared to be promising markers in this regard. The importance of these markers in prediction models for GVHD and relapse remain to be defined in large prospective clinical trials.

Comparison between an artificial neural network and logistic regression in predicting acute graft-vs-host disease after unrelated donor hematopoietic stem cell transplantation in thalassemia patients

There is growing interest in the development of prognostic models for predicting the occurrence of acute graft-vs-host disease (aGVHD) after unrelated donor hematopoietic stem cell transplantation. A high number of variables have been shown to play a role in aGVHD, but the search for a predictive algorithm is still ongoing. Artificial neural networks (ANNs) represent an attractive alternative to multivariate analysis for clinical prognosis. So far, no reports have investigated the ability of ANNs in predicting HSCT outcome. We compared the prognostic performance of ANNs with that of logistic regression (LR) in 78 beta-thalassemia major patients given unrelated donor hematopoietic stem cell transplantation. Twenty-four independent variables were analyzed for their potential impact on outcomes. Twenty-six patients (33.3%) developed grade II to IV aGVHD. In multivariate analysis, homozygosity for donor KIR haplotype A (p = 0.03), donor age (p = 0.05), and donor homozygosity for the deletion of the human leukocyte antigen-G 14-bp polymorphism (p = 0.05) were independently significantly correlated to aGVHD. The mean sensitivity of LR and ANNs (capability of predicting aGVHD in patients who developed aGVHD) in test datasets was 21.7% and 83.3%, respectively (p < 0.001); the mean specificity (capability of predicting absence of aGVHD in patients who did not develop aGVHD) was 80.5% and 90.1%, respectively (p = NS). Although ANNs are unable to calculate the weight of single variables on outcomes, they were found to have a better performance than LR. A combination of these two methods could be more efficient in predicting outcomes and help tailor GVHD prophylaxis regimens according to the predicted risk of each patient. Whether ANN technology will provide better predictive performance when applied to other datasets remains to be confirmed.

Early onset of acute GVHD indicates worse outcome in terms of severity of chronic GVHD compared with late onset

Acute GVHD (aGVHD) is an important risk factor for predicting the incidence or severity of chronic GVHD (cGVHD). Transplant outcome can be influenced by the onset time of aGVHD in patients who have received allogeneic PBSC transplants (PBSCTs). The medical records of 134 patients who survived more than 3 months after myeloablative allogeneic PBSCT were retrospectively reviewed. In all, 38 patients (28.4%) developed grade II-IV aGVHD before day +28 (early aGVHD) and 25 patients (18.7%) after day +28 (late aGVHD). The 5-year cumulative incidence of cGVHD was 78.9% in the early-aGVHD group and 56.6% in the late-aGVHD group (P=0.034). The 5-year OS was 51.0% for the early-aGVHD and 80.8% for the late-aGVHD group (P=0.406). Infection was the primary cause of death for the early-aGVHD group (51.4 vs 16.7%, P=0.017), whereas relapse of the primary disease was higher among the patients with late aGVHD, although this was statistically insignificant (58.3 vs 25.7%, P=0.309). In a multivariate analysis, early aGVHD was identified as a risk factor for developing cGVHD (hazard ratio (HR) 2.278, P=0.004). The development of aGVHD early after allogeneic PBSCT increased the risk of cGVHD and infection-related death rate when compared with the late onset of aGVHD.

Early (Day −7) versus Conventional (Day −1) Inception of Cyclosporine-A for Graft-versus-Host Disease Prophylaxis after Unrelated Donor Hematopoietic Stem Cell Transplantation in Children. Long-Term Results of an AIEOP Prospective, Randomized Study

We carried out a randomized, multicenter study comparing the inception of cyclosporine- A (CsA) on day -7 to conventional CsA (on day -1) to evaluate the influence of this modification on graft-versus-host disease (GVHD), treatment-related mortality (TRM), relapse rate (RR), and event-free survival (EFS) in children with hematologic malignancies given unrelated donor (UD) hematopoietic stem cell transplantation (HSCT). Between 1997 and 2002, 152 children transplanted for acute leukemia (102), myelodysplastic syndromes (23), chronic myelogenous leukemia (20), and non-Hodgkin lymphoma (7) were enrolled in the study and randomized to receive either early CsA (group 1, N = 72) or conventional CsA (group 2, N = 80), after stratification according to HLA compatibility and disease phase. The cumulative incidence of both grade II-IV and grade II-IV acute GVHD (aGVHD), as well as of chronic GVHD (cGVHD), did not differ between the 2 groups. No significant differences were observed also with regard to TRM and RR. The 8-year Kaplan-Meier estimates of EFS were 56% in group 1, and 46% in group 2 (P = NS). In the Cox model, advanced disease phase, male recipient, older donor, and occurrence of grade III-IV aGVHD predicted inferior overall EFS. These data indicate that early inception of CsA does not improve posttransplantation outcome of children with hematologic malignancies given UD-HSCT.

Red Blood Cell Transfusion Dependence and Outcome after Allogeneic Peripheral Blood Stem Cell Transplantation in Patients with de Novo Myelodysplastic Syndrome (MDS)

The prognosis of patients with de novo myelodysplastic syndrome (MDS) who are red blood cell transfusion-dependent (TD) and receive supportive care is inferior to that of patients who do not require transfusions. Whether TD also affects outcome after allogeneic transplantation is unknown. Consequently, in 172 de novo MDS patients (median age, 51 years), we analyzed the impact of TD on outcome after high-dose conditioning and allogeneic peripheral blood stem cell transplantation (PBSCT). With a median follow-up of 37 months, the probability of 3-year overall survival (OS) did not differ significantly between patients who were TD and those who were not TD before PBSCT (P=.1); however, transfusion burden, as reflected by ferritin levels, was correlated with a greater probability of severe acute graft-versus-host disease (aGVHD; P=.03) and a higher comorbidity index (P=.01), and OS was inferior in those patients with a ferritin level>1000 microg/L before PBSCT (P=.03). In multivariate analysis, only marrow myeloblast count (P=.01) and comorbidity index (P=.001) had a significant impact on OS. Our data do not identify TD as an independent negative prognostic factor for outcome after allogeneic PBSCT' however, iron overload (presumably transfusion-related) may contribute to poor transplantation success by adding to the overall comorbidities. Whether clinical intervention in the form of iron chelation can improve the outcome of allogeneic PBSCT in TD patients with MDS remains to be determined.

Patients suffering from acute graft-versus-host disease after bone-marrow transplantation have functional CD4 +CD25 hiFoxp3 + regulatory T cells

Acute Graft-Versus-Host Disease (aGVHD), mediated by CD4 + and CD8 + effector T cells, is a life-threatening complication in hematopoietic stem cell (HSC) transplantation. Naturally-occurring CD4 +CD25 hi(Foxp3 +) regulatory T cells (T reg) have been shown to modulate tolerance to aGVHD in murine graft models. In this report, we investigated their role in the prevention of aGVHD in patients transplanted with bone-marrow-derived HSC. When CD4 +CD25 hiFoxp3 + T cells were isolated from bone-marrow grafts, they showed no suppressive activity. The analysis of their function in patients suffering from aGVHD after transplantation revealed a gain of suppressive activity indicating their inability to control the aGVHD induction. Thus, our findings clearly demonstrate that CD4 +CD25 + and CD4 +CD25 hiFoxp3 + T cells, when administered in steady-state physiological conditions, do not influence the outcome of aGVHD after bone-marrow transplantation.

Regulatory CD4 +CD25 hi T cells conserve their function and phenotype after granulocyte colony-stimulating factor treatment in human hematopoietic stem cell transplantation

Acute graft-versus-host disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell transplantation. CD4(+)CD25(hi) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine models. Based on these observations, we examined their role in the prevention of aGVHD in patients who underwent transplantation with peripheral blood-mobilized hematopoietic stem cells after administration of granulocyte colony-stimulating factor. The effects of the G-CSF on the phenotype, frequency, and function of CD4(+)CD25(hi) T cells were analyzed in grafts and after transplantation to determine whether these cells were regulatory T cells. CD4(+)CD25(hi) T cells could be detected at the same frequency before and after granulocyte colony-stimulating factor administration in the donors' peripheral blood. The isolation of these cells from the grafts or from the recipients' peripheral blood after transplantation revealed that they were suppressive to the same extent as T(reg) isolated from healthy volunteers. Their number and frequency were estimated in the grafts and the results indicated that protection against aGVHD was not dependent on the T(reg) amount transferred to the recipients. Similarly there was no correlation between the number of circulating CD4(+)CD25(hi) T cells in the recipients' peripheral blood during the early period after transplantation and the outcome of aGVHD.

The diversity of KIR gene in Chinese Northern Han population and the impact of donor KIR and patient HLA genotypes on outcome following HLA-identical sibling allogeneic hematopoietic stem cell transplantation for hematological malignancy in Chinese people

Killer cell immunoglobulin-like receptors (KIRs) are members of a group of molecules that specifically recognize HLA class I ligands and are found on subsets of human lymphopoetic cells. The number of KIR loci can vary between individuals, resulting in a heterogeneous array of possible KIR genes. The range of observed profiles has been explained by the occurrence of two haplotype families termed A and B, which can be distinguished on the basis of certain KIR sequences. Immunogenetic analysis of different ethnic populations shows significant differences in terms of the distribution for group A and group B haplotypes. Recently, attention has been focused on the role of killer cell immunoglobulin-like receptor (KIR)-ligand incompatibility in the graft-versus-host direction between donor and recipient in allogeneic hematopoietic stem-cell transplantation (ASCT). The goal of this study was to study the frequency of specific KIR genes in Chinese Northern Han population and evaluate the role of KIR-ligand mismatch in Chinese HLA-identical sibling hematopoietic stem cell transplantation patients with hematological malignancy. Here genomic DNA from 150 Northern Chinese Han individuals was typed for the presence or absence of KIR genes. Seventy-four allogeneic stem cell transplantation donor/recipient pairs were typed for HLA-A, B, C and KIR. Sixteen KIR genes were observed in the population, and framework genes 3DL3, 3DP1, 2DL4, and 3DL2 were present in all individuals. Twenty-two different genotypes were found. Group A haplotypes outnumbered group B haplotypes in frequency by approximately 3:1, with individuals having two group A haplotypes accounting for 51.9% (78/150). We observed that 57 out of 74 (77.3%) donor-recipient pairs could be characterized by lack of recipient HLA ligand for donor KIR. We observed that 36 out of 45 (80%) donor-recipient HLA-identical sibling transplant pairs could be characterized by lack of recipient HLA ligand for donor KIR. Cumulative incidence analysis of aGVHD in patients undergoing HLA-identical sibling hematopoietic stem cell transplantation in this study demonstrated a decreased incidence of severe aGVHD in patients lacking HLA ligand for donor-inhibitory KIR2DL1 (31.4 vs. 70%, P = 0.029). And also in AML (acute myeloid leukemia) patients lacking HLA ligand for donor-inhibitory KIR and KIR2DL1 (17.6 vs. 75%, P = 0.03). Our data demonstrated that the Chinese Han population is distinct in KIR gene frequencies and putative KIR haplotypes in comparison to some other populations. Almost all allogeneic donors could be characterized as having an inhibitory KIR for each of the three known class I ligands. KIR and KIR2DL1 mismatch is associated with lower aGVHD in Chinese after HLA-identical sibling hematopoietic stem cell transplantation.

Elevated Serum Interleukin-7 Levels Precede the Development of Acute Graft-Versus-Host Disease.

BACKGROUND: Despite advances in transplantation immunology, there is no clinically practical tool to predict acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Murine models indicate that aGVHD is promoted by interleukin-7 (IL-7), a homeostatic cytokine for naïve CD4+ and CD8+ T-cells. We hypothesized that serum IL-7 levels might be associated with the development of aGVHD in patients (pts) undergoing allogeneic HSCT, and evaluated this using serum samples obtained for this purpose in a prospective clinical trial.METHODS: Thirty-one pts underwent allogeneic HSCT from HLA-identical siblings. Pts received 1 to 3 pre-transplant chemotherapy cycles to induce profound, uniform host lymphopenia (CD4<100), followed by fludarabine-based reduced-intensity conditioning. GVHD prophylaxis was cyclosporine & methotrexate. Serum IL-7 was measured by ELISA at baseline and multiple time points from the day of transplant through 1 year after allogeneic HSCT. IL-7 levels were evaluated for associations with blood lymphocyte counts, aGVHD, and survival. Other variables examined for association with aGVHD were pt and donor age; CD3+ and CD34+ cell doses; donor gender; donor-recipient gender mismatch; donor or recipient CMV status, prior rituximab therapy; donor lymphoid or myeloid chimerism >95% at day +14; serum IL-15 levels; and levels of soluble tumor necrosis factor-α receptors 1 and 2 (sTNFR1 and sTNFR2).RESULTS: Grades I, II, and III aGVHD occurred in 3%, 23%, and 19%, respectively; none had grade IV. Median (range) IL-7 levels rose from baseline 12.1 (0–46.9) pg/ml to 37 (13.3–79.2) pg/ml on day 0 before allografting, then fell to 12.0 (1.3–74.7) pg/ml by day +14; these changes were inversely correlated with absolute lymphocyte counts, CD3, CD4, and CD8 counts at baseline and day +7. The development of aGVHD was associated with IL-7 levels at day +7 (p=0.01) and day +14 (p=0.000033) post-transplant (Figure), along with the allograft CD34+ cell dose (p=0.012). Higher IL-7 levels at day +14 corresponded to more severe grades of aGVHD (p<0.0001). [Display omitted] In logistic regression models, these factors jointly classified pts according to development or avoidance of aGVHD with a maximum sensitivity of 86% and a specificity of 100%. IL-7 levels were more strongly associated with aGVHD than were sTNFR levels or other parameters.CONCLUSIONS: Determination of serum IL-7 levels in the early post-transplant period accurately predicted the risk of developing aGVHD after allogeneic HSCT and holds promise as a simple, reproducible test to select pts for pre-emptive therapy. These data support preclinical observations that demonstrate a critical role for IL-7 in inducing aGVHD and provide a rational basis for novel approaches to GVHD prophylaxis through modulation of the IL-7 homeostatic pathway.

Deleterious Roles of the KIR Ligand Mismatch on Acute Graft Versus Host Disease in Unmanipulated HLA-Mismatched/Haploidentical Blood and Marrow Transplantation.

The beneficial effect of KIR ligand mismatch with a very low incidence of rejection, acute graft versus host disease (aGVHD), and leukemia relapse had been demonstrated by Perugia's group in the Haploidentical mismatched Hematopoitic cell transplantation (HCT) with extensive T cells depletion in vitro. However, many analytical results of the haploidentical and unrelated mismatched transplantation appeared to be inconsistent with Perugia's work. The disputes in the inconsistent roles of KIR ligand mismatch seem to be caused by the different transplant protocols with different extent of T cells depletion in vitro or in vivo. In recent years, we successfully established a novel protocol—conditioning including antithymocyte globulin followed by un-manipulated HLA-mismatched/haploidentical blood and marrow transplantation, which can achieve comparable outcomes to HLA-identical sibling transplantation. Following the contradictory results about the KIR ligand mismatch in the haploidentical related and mismatched unrelated HCT, we have evaluated the roles of the KIR ligand mismatch in 94 leukemia patients undergoing unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation. Multivariate analysis showed that both KIR ligand mismatch (HR 2.833, CI, 1.286–6.241, p=0.01) and doses of T cells (HR 3.059, CI, 1.292–7.246, p=0.011) were independent risk factors causing the acute graft versus host disease (aGVHD). In addition, compared to patients without KIR ligand mismatch, KIR ligand mismatch worsened the adverse effect of ‘high' dose T cells (>1.48×108/kg) on aGVHD (100% vs 63.3%, p=0.036), and increased the incidence of aGVHD in patients with HLA-C mismatch (80% vs 57.4, p=0.056). Since multivariate analysis demonstrated that high risk leukemia is the only predictor for TRM, relapse and OS, we further analyzed the effect of KIR ligand mismatch on prognosis in standard and high risk patients. The differences in TRM (50% vs 7.6%, p=0.005) and OS (50% vs 88.4%, p=0.014) between patients with and without KIR ligand mismatch were most striking for standard risk. Therefore we conclude that due to the presence of large dose T cells in the allograft, the alloreactivity of NK cells had been inhibited and KIR ligand mismatch directed alloreactive T cells played crucial roles in our model.

Fast Appearance of Donor Dendritic Cells in Human Skin: Dynamics of Skin and Blood Dendritic Cells after Allogeneic Hematopoietic Cell Transplantation

Both number and origin (donor vs. host) of dendritic cells (DC) are associated with acute graft-versus-host disease (aGvHD), relapse and graft failure after human allogeneic hematopoietic cell transplantation (aHCT). We prospectively and simultaneously investigated skin and blood DC subtypes, their donor/recipient origin, and the correlation of DC reconstitution kinetics with treatment, clinical outcome, and incidence of aGvHD in patients undergoing aHCT. A significant reduction of skin and a marked decrease of blood DC were observed in patients compared to healthy volunteers. A dominant donor chimerism of migratory Langerhans cells (LC) and dermal-dendritic-cells (DDC) was detected even early after transplantation, and developed independently from chemotherapy regimen, graft manipulation or time point after transplantation. Before start of the therapy patients showed significantly decreased numbers of peripheral blood CD123+ preDC2, whereas CD11c+ preDC1 numbers appeared to be diminished, but were statistically indistinguishable from controls. Host derived pB preDC were virtually absent following aHCT. After a further reduction in cell number around day 56 both preDC subtypes reconstituted and stabilized to pretransplant numbers by day 112. Occurrence of aGvHD and its treatment diminished numbers of both preDC subtypes. Furthermore conditioning therapy with Alemtuzumab apparently affected reconstitution of both preDC subsets negatively. Given that induction of GvHD in humans is as host DC dependent as in mouse models, investigation of DC chimerism and number at different sites and especially in GvHD target organs might provide important insights into the pathogenesis of the main obstacle of aHCT.

Clinical relevance of NK, NKT, and dendritic cell dose in patients receiving G-CSF-mobilized peripheral blood allogeneic stem cell transplantation

To analyze the relationship between the cellular composition of peripheral blood allografts and clinical outcome, we performed a prospective study in 45 adult patients who underwent allogeneic peripheral blood hematopoietic stem cell transplantation (HSCT) from a histocompatibility leukocyte antigen identical sibling donor for different hematological malignancies. The dose of CD34+, CD3+, CD4+, CD8+, and CD19+ lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, type 1 and type 2 dendritic cells (DC1 and DC2), as well as regulatory T (Treg) lymphocytes was analyzed. All patients were conditioned with busulphan and cyclophosphamide (BuCy2) +/- VP-16 and received a short course of methotrexate and cyclosporin-A as graft-versus-host disease (GVHD) prophylaxis. Acute GVHD (aGVHD) was present in 9 of 43 (21%) patients, and chronic GVHD (cGVHD) developed in 18 of 39 (46%) patients. There was a significantly higher incidence of aGVHD in patients receiving more than 6x10(6)/kg CD34+ cells. In univariate analysis, variables associated with better survival were as follows: a dose of less than 1.5x10(7)/kg NKT cells and less than 1.7x10(6)/kg DC2 for disease-free survival (DFS), and a dose of less than 3x10(7)/kg NK cells, less than 1.5x10(7)/kg NKT cells, less than 3x10(6)/kg DC1, and less than 1.7x10(6)/kg DC2 for overall survival (OS). In the Cox regression analysis, the dose of NKT cells was the only variable associated with better DFS, while the doses of NK, NKT, and CD34+ cells (less than 8x10(6)/kg) were associated with better OS. In conclusion, different circulating cell populations, other than CD34+ cells, are also of relevance in predicting the clinical outcome after allogeneic peripheral blood HSCT.