Immune Profile of Sickle Cell Anemia Patients Treated with Allogeneic Stem Cell Transplantation

Abstract

Sickle cell disease (SCD) is one the most common recessive monogenic disease in the world. In the last years, SCD patients have been successfully treated with allogeneic hematopoietic stem cell transplantation (HSCT), the only curative option to date. However, the immune reconstitution mechanisms involved in therapeutic response of HSCT in SCA patients are not elucidated yet. For that, blood samples from SCD patients (N=24) were collected before (pre-tx) and until 1 year after transplantation (1 month, 3, 6 and 12 months) and immune cell subpopulations were analyzed by flow cytometry. In addition, peripheral blood mononuclear cells from 4 SCD patients and 4 healthy individuals were isolated, RNA was isolated and cDNA synthetized, and expression of genes associated with inflammatory response was evaluated by PCR array. Serum cytokines/chemokine levels were also measured in SCD patients by luminex assays. We observed decreased number of total lymphocytes until 3 months after HSCT (mean±SD: 1496±1012 cells/uL) compared to pre-tx (2671±924.8; p<0.05), as well as B lymphocytes (pre-tx: 305.7±239; 3 months: 132.2±193.7; p<0.05). The T lymphocytes counts decreased until 1 month after transplantation (pre-tx: 788.3±537.3; 1 month: 327.9±461; p<0.05) and CD4 T cells numbers reduced until 6 months (pre-tx: 487±337.3; 6 months: 281.3±222.7; p<0.05), while CD8 T cells counts did not change, resulting in significant inversion of CD4/CD8 ratio until 1 year after transplantation (1.11±0.58) compared to Pre-tx (2.05±0.77; p<0.05). Specifically, naïve CD4+ T cell number decreased until 6 months after transplantation (pre-tx: 182.6±146.9; 6 months: 91.33±90,42; p<0.05) and central memory CD4+ T cell until 3 months (pre-tx: 224.7±160; 3 months: 77.42±75.74; p<0.05). Interestingly, the numbers of RTE was lower until 3 months after HSCT (55.43±59.88) than pre-tx (136±108.7; p<0.05), but baseline value was achieved after this period, suggesting the generation of new naïve T cells by thymus after transplantation. Effector CD4+ T cell increased 6 months after HSCT (pre-tx: 20.03±18.67; 6 months: 39.89±31.24; p<0.05) and effector CD8+ T cell 12 months (pre-tx: 64.83±64.97; 12 months: 174.7±193.2; p<0.05). In addition, the reconstitution of regulatory T cell subsets was also studied. The absolute number of CD4+CD25+CTLA-4+ T cell (pre-tx: 118.5±95.16; 6 months: 54.37±42.25) and CD4+CD25+Foxp3+ T cell (pre-tx: 112.4±78.96; 6 months: 56.55±46.84) decreased until 6 months after transplantation compared to pre-tx (p<0.05), but reached baseline counts 1 year after HSTC. The number of CD4+CD25+GITR+ T cell and CD8+CD28-Foxp3+ T cell did not change after transplant. However, increased number of CD8+CD28- T cells was observed 6 (307.6±382.6) and 12 months (373.3±357.5) after transplantation compared to pre-tx (120.8±133.8) and CD8+CD28-CD57+ T cells raised 12 months (pre-tx: 52.69±92.86; 12 months: 160.5±175.9; p<0.05). Clustering analysis demonstrated that gene expression profiling of SCD patients at 1 year after transplantation was similar to healthy individuals. Expressions of chemokine genes (such as CCL2, CCL7, CXCL1, CXCL2, CXCL3, CXCL8) and/or chemokine receptors (MIP-1alpha, MCP-3, CCL5, IL-8) important for leukocytes recruitment were increased in SCD patients before transplant compared to healthy subjects. Indeed, the serum concentration of MIP-1α (control: 3.64±1.07pg/mL; pre-tx: 6.4±2.54) and IL-8 (control: 0±0; pre-tx: 7.4±8.69) was also higher in patients before transplantation compared to controls (p<0.05). However, one month after transplantation the expression of chemokine genes CXCL1, CXCL2, CXCL3, CXCL8, CCL2 and CCL7 was decreased when compared to pre-transplantation levels. One year after transplantation, the expression of chemokine genes (CCL4, CCL5 and CCL16) and proinflammatory cytokines (IL-6, TNF and IFN-γ) increased compared to pre-tx, however the gene expression of chemokine CCL7, CXCL1, CXCL2, CXCL3, CXCL8 remained decreased. In addition, serum levels of MIP-1α decreased until 12 months after transplantation (3.82±3.34) compared to pre-transplant (6.4±2.54). Thus, our data suggest that HSCT induces alterations in immune and inflammatory profile of SCD patients after HSCT, such as downregulation of the chemokine genes expression and increase of regulatory T cell subsets, besides the correction of the genetic disease. DisclosuresHermine:AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Hybrigenics: Research Funding; Novartis: Research Funding; INatherys: Equity Ownership, Research Funding; Celgene: Research Funding.