Anti-HLA Antibodies In Allogeneic Hematopoietic Stem Cell Transplantation

PART Ⅰ Impact of anti-HLA antibodies detected dynamicly and HLA-DP loci mismatches on outcomes of HLA-12/12 matched unrelated-donor of allo-HSCTObjectives: Investigate the impact of anti-HLA antibodies and HLA-DP loci mismatches on the outcomes of HLA-A, B, C, DRB1, DQB1, DQA1(12/12) matched unrelated-donor allogeneic hematopoietic stem cell transplantation(MUD-HSCT) for hematologic malignancies patients, in order to promote the establishment of anti-HLA antibodies clinical system containing pre-transplantation antibodies screening, donor selection, prognosis evaluation, intervention treatment and follow-up inspection, to improve the outcomes of HSCT for hematologic malignancies.Methods: The 123 patients with hematologic malignancies were received HLA-12/12 MUD-HSCT between October 2011 and March 2014. Recipients and donors pairs were performed HLA-A, B, C, DRB1, DQA1, DQB1, DPA1, DPB1 loci high-resoluction typing using sequence-based testing(SBT), sequence specific oligonucleotide probes(SSOP) and sequence specific primers(SSP) methods. Anti-HLA antibodies screening using the Luminex method was applied to 123, 117 and 106 serum samples collected before and 1 month and 3 months after transplantation. The cut-off value of mean fluorescence intensity(MFI) for the mixed antigen bead assay is 500. MFI < 500 was considered negative, 500-1000 weakly positive, 1000-2000 positive, 2000-5000 intermediately positive, and > 5000 strongly positive. Statiscial analysis was performed to evaluate the impact of anti-HLA antibodies and HLA-DP loci mismatches on HSCT outcomes.Results: At the last follow-up, the percentage of OS and DFS in 2 years was 73.2% and 69.1%, respectively. The 2-year probability of OS for AML/MDS, ALL and CML patients were 56.4%, 65.7%, and 88.9%, respectively(P=0.278). The 2-year probability of DFS for AML/MDS, ALL, and CML patients were 53.1%, 58.2%, and 88.9%, respectively(P=0.217).The presences of anti-HLA antibodies at before and 1 month and 3 months after transplantation were 37.4%(46/123), 40.2%(47/117), and 22.6%(24/106). And we found that 6(4.9%) patients had DSA(anti-HLA-DPB1 antibodies) before transplantation. The recipients who at 1 and 3 month post-transplantation had anti-HLA antibodies were 30.7% and 15.1%(P=0.030), respectively, in the anti-HLA Abs-negative group before transplantation and were 57.1% and 35.0%(P = 0.044), respectively, in the anti-HLA Abs-positive group before transplantation. Patients who had anti-HLA antibodies before transplantation were more likely to have persistent antibodies at 1 month(P = 0.005) and 3 months(P = 0.018) after transplantation.There were significances in platelets recovery(14d vs. 13 d, P=0.033), grade II-IV a GVHD(43.3%vs.20.4%, P=0.006) and OS(50.2% vs. 65.7%, P=0.029) between anti-HLA antibodies positive and negative groups before transplantation. No significant differences in relapse(29.8% vs. 17.5%, P=0.125) and TRM(33.1% vs.15.8%, P=0.224) were observed between the two groups. Among acute myeloid leukemia and myelodysplastic syndrome patients, the presence of anti-HLA antibodies before transplantation were associated with lower OS(35.3% vs. 74.3%, P=0.006) and DFS(36.3% vs. 68.3%, P=0.025) compared with without antibodies.At 1 month after transplantation, the incidences of grade II-IV a GVHD were 21.2%, 17.4%, 33.3% and 55.6%(P=0.02), respectively, in continuously negative antibodies group, antibodies from negative turned to positive group, continuously positive group and antibodies from positive turned to negative group. OS were 84.6%, 73.9%, 62.5% and 61.1%(P=0.082),DFS were 78.8%, 69.6%, 58.3% and 61.1%(P=0.24), respectively, in the four groups. At 3 month after transplantation, the incidences of grade II-IV a GVHD were 23.4%, 11.1%, 35.7% and 55.6%(P=0.05), OS were 83.0%, 88.9%, 64.3% and 61.1%(P=0.15),DFS were 78.8%, 7.8%, 57.1% and 61.1%(P=0.29), respectively, in continuously negative antibodies group, antibodies from negative turned to positive group, continuously positive group and antibodies from positive turned to negative group.A lower rate of c GVHD was observed in pre-existing anti-HLA Abs-negative group(32.4%), whereas Abs-positive group had a higher rate(52.4%, P=0.036). The incidence of extensive c GVHD was also significantly different between the two groups(9.9% vs. 23.8%, P=0.045). During follow-up, patients with continuously positive antibodies had a trend of higher c GVHD compared to patients with continuously negative antibodies(61.5% vs. 32.6%, P=0.061).92 recipients and donors pairs of 110 were mismatched in HLA-DPA1 and/or HLA-DPB1 locus. Among them 69 pairs were mismatched at both DPB1 and DPA1 loci, 18 pairs were mismatched at the HLA-DPB1 loci, and 5 pairs were mismatched at the HLA-DPA1 loci. No significant differences in II-IV a GVHD(24.6% vs. 32.3%, P=0.610) and OS(43.2% VS. 34.2%, P=0.778) were observed in patients receiving grafts from either HLA-DP loci matched donors or HLA-DP loci mismatched donors. In the anti-HLA Abs-negative group, patients with HLA-DP loci mismatches had a trend of lower in OS(59.5% vs. 80.8%, P=0.575) and relapse(12.8% VS. 18.2%, P=0.639) compared to patients matched at HLA-DP.Multivariate analysis showed that pre-existing anti-HLA antibodies was a risk factor for a GVHD(P=0.03, HR=2.876, 95% CI 1.106-7.475) and c GVHD(P=0.004, HR=4.062, 95% CI 1.580-10.440) and OS(P=0.04, HR=2.199, 95% CI 1.037-4.661). In addition, advanced stage of the disease at the time of transplantation was a risk factor for OS(P=0.026, HR=2.391, 95% CI 1.112-5.139) and DFS(P=0.017, HR=2.397, 95% CI 1.172-4.903). And CD34+ cell number more than 4*106/kg(P=0.003, HR=4.908, 95% CI 1.743-13.824) and ABO blood type(HR=3.120, 95% CI 1.153-8.444, P=0.026) were risk factors for a GVHD.Conclusions: Our results suggest that dynamic changes of anti-HLA antibodies before and after transplantation are an important index for outcomes of HLA-12/12 MUD-HSCT. And anti-HLA antibodies independently predicted for negative outcome of HSCT independent of HLA-DP loci mismatches. We want to emphasize that patients whose anti-HLA antibodies was positive should accept more intensive therapeutic strategies with dynamic monitoring of anti-HLA antibodies, in order to improve the condition of patients. PART Ⅱ In vitro study of mechanism of endothelial cell injury caused by the donor-specific anti-HLA antibodiesObjectives: To establish the model of donor-specific anti-HLA antibodies(DSA) in vitro, to discuss whether DSA effect the proliferation and apoptosis of PI3K/AKT signaling pathway, to clarify that the cell proliferation is an important factor of causing impaired function of endothelial cells(EC), and further to provide the evidence of DSA affect prognosis of HSCT.Methods: There are human umbilical vein endothelial cells I(HUVEC-I) whose HLA-I typing is A* 02:01, 03:01 B* 15:15, 35:03 C* 01:02, 04:01 and HUVEC-II whose HLA-I typing is A* 24:02, 68:03 B* 51:01, 51:01 C* 14:02, 15:021. The corresponding antibodies were anti-HLA-A2、B35、Cw1、A24、B51 antibodies. The stock solution of DSA was serial diluted and the value of MFI of DSA was tested using Luminex. HUVEC treated with mouse isotype Ig G was used as a control. Positive, intermediately positive, strongly positive and super-strongly positive were in the treatment group. The cells were trained with DSA of different intensity levels for 15 minutes after hungered 6 hours, and were lysed in buffer for 30 min on ice to collect protein. The total protein concentration was measured using microplate spectrophotometer method using BCA as standard. The lysates prepared from HUVEC were subjected to Western Blotting to measure the phosphorylation of FAK-Tyr576/577、PDK1-Ser241、AKT-Thr308、AKT-Ser473、p70S6K-Thr421/Ser424、S6RP-Ser235/236, and the expression of Bcl-2 and beta-actin in PI3K/AKT signal pathway. Meanwhile, HUVEC and DSA whose final concentration were 0.01μg/ml, 0.1μg/ml, 1.0μg/ml and 10.0μg/ml were co-cultured 15 minutes, then the procedures were same as above mentioned.Results:1. The association between MFI and concentration of DSAWhen the concentration of anti HLA-A2, A24, B51 antibodies were 0.01μg/ml, 0.011μg/ml and 0.094 μg/ml, respectively, MFI value of their were 2000 and positive level; and when concentration were 0.031μg/ml, 0.023μg/ml and 0.25μg/ml, respectively, MFI value were 7000 and intermediately positive level; and when concentration were 0.077μg/ml, 0.05μg/ml and 0.75μg/ml, respectively, MFI value were 10000 and strongly positive level; and when concentration were 0.153μg/ml, 0.5μg/ml and 1.5μg/ml, respectively, MFI value were 14000-20000 and super-strongly positive level. In a certain concentration range, with the increase of antibody concentration, higher MFI value; More than a certain concentration, MFI value no longer continued to rise and showed a trend of saturation. So the curve of anti HLA antibodies combined with HLA antigen is rising gradually.2. DSA-mediated endothelial cells proliferation and survival via the PI3K/Akt pathway2.1 Antibody ligation of HLA-A molecule active EC proliferation and inhibit apoptosisTreatment of HUVEC-1 with various MFI of anti-class A2 antibody for 15 min stimulated phosphorylation of PDK1 at Ser241. Then, the percentage of phosphorylation of AKT Thr308, p70S6 K Thr421/Ser424 and S6 RP Ser235/236 of proliferation pathways in the control, positive, intermediately positive and super-strongly positive group were 54%, 73%, 100%, 62%, 37% and 55%, 72%, 94%, 100%, 50% and 51%, 65%, 68%, 100%, 82%(P﹤0.001). Meanwhile, the percentage of phosphorylation of Akt Ser473 and expression of Bcl-2 in anti-apoptosis pathways were 80%, 31%, 32%, 100%, 56% and 39%, 58%, 100%, 97%, 60%(P﹤0.001) in the five groups. There were similar effects on the highest expression levels of proliferation and anti-apoptosisExposured of HUVEC-1 to various concentrations of anti-class A2 antibody for 15 min, phosphorylation of FAK Tyr576/577 and PDK1 Ser241 were early actived by ligation of antigen and antibody. Consequently, a dose dependent fashion in phosphorylation of AKT Thr308 and p70S6 K Thr421/Ser424 and Akt Ser473 were observed at concentrations ranging from 0.01 to 10μg/ml. Importantly, the percentage of pathway terminal proteins of phosphorylation of S6 RP Ser235/236 and expression of Bcl-2 were 34%, 42%, 60%, 64%, 100%(P ﹤ 0.001) and 80%, 79%, 84%, 100%, 91%(P ﹤ 0.001). Those results demonstrated that anti-HLA A2 antibody may induce HUVEC proliferation and anti-apoptosis by binging directly to HLA A2 molecules and stimulating PI3K/AKT signal pathway.HUVEC-2 were treated with different concentration of anti-HLA A24 antibody for 15 min. The percentage of phosphorylation of S6 RP Ser235/236 were 76%, 95%, 100%, 90%, 93%, 88%, 90%, 94% and 93% in the control, 0.005μg/ml, 0.01μg/ml, 0.05μg/ml, 0.1μg/ml, 0.5μg/ml, 1μg/ml, 5μg/ml and 10μg/ml groups. There was no obvious increase of S6 RP Ser235/236 when the concentration of antibody was higher.2.2 Anti-HLA-B antibodieies stimulate phosphorylation of S6 RP at sites Ser235/236HUVEC-2 and anti-HLA B35 antibody were cocultured at 15 min. The expression of S6 RP Ser235/236 was 70% in the control group. In contrast, there were 36%, 100%, 85% and 96% in 0.01μg/ml, 0.1μg/ml, 1.0μg/ml, 10μg/ml groups. When the concentration was more than 0.1μg/ml, the expression of S6 RP Ser235/236 was increased. But the ascending trend was not obvious.The percentage of phosphorylation of S6 RP Ser235/236, which was stimulated by ligation of anti-HLA B51 antibody and molecule, were 91%, 98%, 100%, 90% and 99% in the control, 0.01μg/ml, 0.1μg/ml, 1.0μg/ml, 10μg/ml groups. Raised the concentration of antibody, the expression of S6 RP Ser235/236 were 89%, 88%, 98%, 92%, 96%, 100% and 97% in the control, 1μg/ml, 5μg/ml, 10μg/ml, 15μg/ml, 30μg/ml, 45μg/ml groups. The datas showed that the ascending trend of phosphorylation of S6 RP Ser235/236 were not obvious when EC were treated with anti-HLA B antibody. We thought preliminary that the antigenicity may be different in HLA-I molecures. The antigenicity of B loci is inferior to A and C loci.2.3 The effect of anti-HLA-C antibody on S6 RP Ser235/236 in ECExposure of HUVEC-2 to varying concentrations of anti-HLA Cw1 antibody resulted in elevated S6 RP Ser235/236 protein levels with a dose dependent increased. The percentages of phosphorylation of S6 RP Ser235/236 were 37%, 44%, 88%, 97% and 100%(P﹤0.001) in the control, 0.01μg/ml, 0.1μg/ml, 1.0μg/ml, 10μg/ml groups. These results are consistent with the effect of anti-HLA A2 antibody on phosphorylation of S6 RP Ser235/236 and indicate that different dose anti-HLA Cw1 antibody induce proliferation in EC.Conclusions: This study established the model of donor-specific anti-HLA antibodies(DSA) in vitro and confirmed that DSA could activate PI3K/AKT signal pathway of endothelial cells, and induce cells proliferation and apoptosis. In addition, our results found preliminary that there may exist different antigenicity in HLA-I. A and C loci have a stronger antigenicity compared with B loci. Thus, our studies provide the theoretical basis for future clinical research, and will lay the foundation of EC functional experiment.