T Cell Repertoire Diversity of Sickle Cell Anemia Patients Treated with Allogeneic Hematopoietic Stem Cell Transplantation and Conventional Treatments
Authors: Jarduli Luciana Ribeiro , Marques Adriana Aparecida , de Azevedo Julia Teixeira Cottas , de Lima Keli Cristina , Júnior Wilson Araújo da Silva , Covas Dimas Tadeu , Simoes Belinda Pinto , Pinto Ana Cristina Silva , de Farias Kelen Cristina Ribeiro Malmegrim 
Published: October 2019

Abstract
Introduction:Sickle cell anemia (SCA) is a hereditary disorder caused by a mutation in the hemoglobin gene that produces an abnormal hemoglobin S (HbS). The polymerization of the HbS in the red cells is a key of SCA pathophysiology, leading to vaso-occlusive crisis, chronic inflammation, infections, organ ischemia and deficiencies (Hebbel, 2011). SCA is a disease with a highly variable phenotype. Clinical manifestations of the each patient and predictors of disease progression might guide therapeutic decisions. Currently, the available treatments for SCA are hydroxyurea (HU), chronic transfusions of red blood cells (RBC) and the allogeneic hematopoietic stem cell transplantation (allo-HSCT). The occurrence of thymic tissue micro-infarctions and its consequence to patient immunity has never been evaluated. A functional thymus ensures the generation of naive T cells and a consequent diverse T-cell repertoire. The generation and maintenance of a diverse T-cell repertoire is a critical element of immune competence. We hypothesized that SCA patients may have thymic dysfunction caused by micro-infarctions of the thymic tissue, leading to defective output of new naïve T cells, decreased diversity of the peripheral T cell repertoire, and altered adaptive immunity. The objective of this study was to evaluate the peripheral T cell repertoire diversity of SCA patients without treatment and under treatment with hydroxyurea, or chronic transfusions, or allogeneic hematopoietic stem transplantation.
Methods and Patients: Peripheral blood mononuclear cells were isolated from patients patients with SCA patients without treatment (n= 9) and under treatment with hydroxyurea (n=10), or chronic transfusions (n = 9), or allogeneic hematopoietic stem transplantation (n = 20), and in a group of heath afro-descendants individuals (n = 9). The diversity of the T cell repertoire was evaluated by TCRBV CDR3 Length Spectratyping method, which is based on the amplification of CDR3 region of TCR by two polymerase chain reactions, followed by capillary electrophoresis of CDR3 segments on automated DNA sequencer (ABI 3500xL Genetic Analyzer, Applied Biosystems, Foster City, CA, USA) and analyzed by Gene Mapper™ software (Applied Biosystems, Foster City, CA, USA) (Pannetier et al, 1993). The results were analyzed by the GraphPad Prism 5 software (La Jolla, CA, USA). Values were expressed as mean ± standard error of the mean. The differences were evaluated using analysis of variance (ANOVA) with the Bonferroni posttest orMann-Whitney nonparametric test as appropriate. The results were considered statistically significant when p < 0.05.
Results:Our findings demonstrated that patients with SCA have altered T cell repertories. Patients treated with hydroxyurea showed improved diversity of the T-cell repertoire compared to patients without any treatment. In addition, SCA patients treated with allo-HSCT showed a restricted T-cell repertoire diversity before transplantation, which even decreased at three months after transplantation, compared to pre-transplantation score. SCA patients showed an increase in the percentage of T cells expressing Vβ22 at one year and at ≥ 2 years after transplantation. Patients without any treatment showed a more diverse T cell repertoire diversity than patients who underwent allo-HSCT. Therefore, the TCR diversity was lower in SCA patients with more severe clinical manifestations and refractory to other treatments, which were selected to undergo allo-HSCT.
Conclusions:Our initial data suggest that patients with SCA might have thymic dysfunction, probably due to micro-infarctions of the thymic tissue by sickled cells. Analyses of the pattern of CDR3 peak distribution for each TCRVβ provided information about the T-cell generation via thymic-dependent pathway in patients with SCA under different kind of treatments. In conclusion, we demonstrated that SCA patients have decreased diversity of their peripheral T cell repertoire, which might affect their adaptive immunity. Further studies are necessary to confirm the peripheral T cell repertoire alterations in SCA patients and to investigate if current treatments are effective to improve thymic function and T cell repertoire diversity in SCA patients.

Disclosures
No relevant conflicts of interest to declare.