Immunological State Of β-Thalassemia Major And Different Clinical Outcome And Immune Reconstitution Between CB+BM And PB+BM Cohort

BACKGROUND: β-thalassemia is one of the common nonmalignant blood disease,which was threatening the patients’ life and health.Allo-HSCT was the only way to cure this disease.Match sibling donor was the most common donor used in the past.The patient’s immune status directly affected the ways of the transplant,the pretreatment conditioning and transplantation outcome.Myeloablative conditioning can avoid GVHD effectively,but it would lead to a delay of immune recovery.Different immune reconstitution pattern were also observed in different recipient who received different graft.So it’s very important to look into the Immunological state of β-thalassemia major,the immune recovery of the β-thalassemia major patient who received myeloablative conditioning MSD-HSCT,and the different clinical outcome and immune reconstitution between CB+BM and PB+BM for β-thalassemia major.OBJECTIVES: 1.To describe the immunological state of β-thalassemia major;2.Prospective analysis of immune reconstitution of β-thalassemia major who received T cell depleted myeloablative MSD-HSCT;3.Compare clinical outcome and immune reconstitution of CB+BM with PB+BM in β-thalassemia major.METHODS: Between December 2015 and March 2018,47 match sibling SCT recipient(24 CB+BM cases,23 PB+BM cases)from Stem Cell Transplantation Center of our hospital were studied.All the patients accepted T cell depleted myeloablative conditioning with Bu+Cy+Flu+ATG before transplantation.Blood was drawn before starting conditioning and on 1m,3m,6m,12 m posttransplant,when clinical figures were also recorded.24 cases of healthy control were also enrolled.Techniques used included flow cytometry,real-time q-PCR,SYBR Green dye method realtime fluorescence quantitative PCR and ELISA.Testing indexes included:(1)T cell system: CD3+、CD4+ 、 CD8+T cell,CD4/CD8 ratio,Transcription factors T-bet and GATA3,TRECS;(2)B cell system: CD19+B cell,Breg cell,sj KRECS and cj KRECS,BAFF and BAFF/B ratio;(3)NK cell system: CD56+/CD16+ NK cell,CD56+ NK cell,CD56 dim cell 、CD56bright cell and CD56-CD16+ cell.We analyzed:(1)Used indexes mention above to describ the immunological state ofβ-thalassemia major;(2)Recorded all clinical figures including complications after HSCT,all measurement data at each timepoint will be compared with that before transplant;(3)Compared clinical outcome and immunological measurements in CB+BM with that in PB+BM before and after transplant.RESULT: 1.Immunological state of β-thalassemia major :T cell,B cell and NK cell subpopulation and their related indices mentioned above had no significant difference betweet the β-thalassemia major and the healthy control,except the CD56 bright was lower in β-thalassemia major.2.Clinical outcome of β-thalassemia major underwent T cell depleted myeloablative MSD-HSCT: Median follow-up was 19 months(1.8-31.9m),Conditioning regimen was myeloablative which included ATG in all patients.Median time to absolute neutrophil count >0.5× 109/L was 11(8-17)days,median time to PLT >20× 109/L was 16(10-49)days.Cumulative incidences of acute GVHD,chronic GVHD,HEMORRHAGIC CYSTISIS,VOD,bacterial /fungal infection,CMV reactivation,,EBV reactivation and PTLD were12.8%,14.9%,25.5%,21.3%,70.2%,36.2%,10.6%,4.2% respectively.Overall survival at 1 years was 93.6%,when thalassemia free survival was 93.6%3.The kinetics of immune recovery after T cell depleted myeloablative MSD-HSCT in β-thalassemia major:(1)T cell : The number of CD3+ T cells remained lower within 6m than that before transplantation,but become the same in 12 m.;CD4+ T cells remained lower throughout the year after transplantation;the number of CD8 + T cells showed a faster increase than CD4 after HSCT,they could reach the level before transplantation at 6m and 12 m.CD4/CD8 ratio was strongly suppressed after HSCT within 12m;T-bet was consistently increased since 1m,and became higher in 3m,6m,12 m than that before transplantation;GATA3 recovered within 1m,and become higher in 3m and 6m,than fall down to normal at 12 m.TRECS remained lower throughout the year after transplantation.(2)B cells: CD19+ B cells and Breg cells remained lower within 3m posttransplant,than recovered in 6m and 12m;sj KRECS remained lower within 1m posttransplant,and recovered in 3m and 6m,then increased higher in 12 m.;cj KRECS remained lower throughout the year posttransplant.BAFF recovered quickly and reached the peak in 1m,then decreased steadily tonormal level at 12 m.BAFF/B ratio reached the peak in 1m and returned to normal at 6m.(3)NK cell: Recovery of NK cell number was fast,NK cell recovered to normal within 1m,then reached a higher level in 12 m.In the subpopulations of NK cell,CD56 dim were less than that before transplantation in 1m and 12 m,when CD56 bright were higher in 3m,6m,12 m posttransplant,CD56-CD16+ cell were higher in 6m and and 12 m.(4)Correlation analysis:We found a better correlation for the CD3+ and CD4+ T cell subset with TREC level before and after transplantation;Breg cell as well as CD19+ B cells highly correlated with sj KREC levels,Breg cell but not CD19+ B correlated with cj KREC levels.4.Compared immune reconstitution of CB+BM with PB+BM cohorts:(1)T cell: Significantly lower levels of CD3+,CD4+T cells were observed in CB+BM recipients in 1m after transplantation,but CD4+T cell and CD4/CD8 ratio were higher in CB+BM at 6m.There were no significant different between CB+BM and PB+BM in CD8+ T cell,TRECS and T-bet throughout the year after transplantation.At 1m and 3m had a higher level of GATA3 in CB+BM recipient.(2)B cell: B cell count had no significant difference between CB+BM and PB+BM.at 1m,while it was high in CB+BM at 3m,6m and 12 m.Breg cell count had no significant difference between CB+BM and PB+BM.at 1m and3 m,while it was higher in CB+BM at 6m and 12 m.Sj KRECS had the same change and difference as the B cell.While the cj KRECS had the same change and difference as the Breg cells.BAFF reached the peak at 1m in both groups,and had no significant difference between these 2 group,but PB+BM recipient had higher BAFF and BAFF/B ratio at 3m,6m and 12 m.(3)NK cell:NK cell and its subpopulations had no significant difference between CB+BMand PB+BM.at 1m,3m and 12 m,except at 6m,NK cell and its CD56-CD16+subgroup were higher in CB+BM.CONCLUSION: 1.β-thalassemia major had almost normal immune state.2.T cell depleted myeloablative conditioning MDS-HSCT was a good method to cure β-thalassemia major,the most common complications of it were infection;3.The kinetics of immune recovery after T cell depleted myeloablative MSD-HSCT in β-thalassemia major:(1)T lymphocytes and their subpopulations showed delayed recovery after T cell depleted myeloablative conditioning MDS-HSCT.(2)Recovery of B cells was faster than T cell.Our results also suggest that possibly there was a mechanism of negative feedback regulation of B cell count on BAFF concentration.(3)An earliest NK cell recovery was observed,but the function was defective.4.Compared with PB+BM,CB+BM had a prolonged T lymphopenia and provided rapid B-cell recovery.But the expansion of B cell was relied mainly on Breg cell subset,KRECS showed the same difference as B cell and Breg cells.NK subpopulation differences in these two groups was mainly in non-function subset.