Immunological Profile of Patients after HSCT Using the FCC Conditioning Regimen for Treatment of Severe Aplastic Anemia Shows Sustained Mixed T-Cell Chimerism Is Due to Persistence of Recipient CD8 T Cells and Indicates Potential Basis for Tolerance and Extremely Low Incidence of Graft Versus Host Disease

Mixed T-cell chimerism is frequent and persists despite discontinuation of immunosuppression after hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) using the fludarabine, cyclophosphamide and alemtuzumab/campath (FCC) conditioning regimen. To investigate the basis for long-term co-existence of donor and patient T cells and remarkably low rates of graft versus host disease (GVHD), the lymphocyte composition and lymphocyte subset chimerism of adult patients transplanted for SAA using FCC was assessed.

Patients

We have transplanted 42 patients (pts) for acquired idiopathic SAA using FCC conditioning from 2007- March 2015 at King's College Hospital. Median age was 33 years (range 15-63). BM was stem cell source in 7(17%) pts and PBSC in 30 (83%). Median cell dose was 6.85 (1.9-12.4) CD34+ cells x10⁶/Kg. Donor was matched sibling in 11(26%) and unrelated in 31 (74%) pts, 6 of whom also received 2 Gy TBI for 9/10 match. Post graft immunosuppression comprised ciclosporin (CSA) alone. Primary graft failure occurred in 1 pt (2%). Acute GVHD was seen in 6 (14%) pts (all grade I-II), and chronic GVHD in 5 (12%) patients (all skin, only 1 severe in a 9/10 match patient). Factors associated with cGVHD were previous aGVHD (p=0.035) and CD3+ chimerism at day+100 ³ 90% (p=0.006). Median follow-up of survivors was 41 months (4.3-96), with 93% overall survival and 90% event-free survival. At 1, 2 and 3 years post HSCT, 4/30, 20/23 and 16/16 patients, respectively, had discontinued CSA.

Results

Comparison to 11 healthy age-matched individuals showed prolonged lymphopenia and abnormal proportions of T, B and NK cells. T cells were profoundly deficient comprising only 1.76% of lymphocytes at day 30, rising to 43.8% by day 360 but still significantly below normal (66%, p=0.018). CD8 T cells recovered faster than CD4 T cells producing inversion of the normal CD4 to CD8 T cell ratio. Composition of na•ve, memory and effector subsets within the CD4 T cell population was near normal by day 360 and na•ve CD4 T cells expressed CD31 indicative of renewed thymopoiesis. In contrast, CD8 T-cell subset composition remained abnormal at day 360 due to a high proportion of effector T cells (57.2% of CD8 T cells compared to 9.3% for healthy individuals, p=<0.005). T-cell subsets from five patients were isolated by fluorescence-activated cell sorting, and chimerism determined by analysis of informative alleles from polymorphic short tandem repeat loci. Results shown in the figure below revealed that mixed T-cell chimerism at day 360 was principally due to persistence of patient CD8 T cells, with notable contribution of the effector subset.  Follow-up analysis of three patients at >2 years after HSCT showed the mixed chimerism profile within T-cell subsets was stable. A significant correlation was observed between lower donor T-cell chimerism at day 360 and CMV reactivation or EBV viremia early after HSCT (p=0.036), suggesting that antigen-driven expansion of virus-specific patient CD8 effector T cells may substantially contribute to persistent mixed T-cell chimerism. 

Sustained co-existence of donor and patient T cells and low rates of GVHD indicates conditions favor mutual tolerance. Findings suggested a potential role for cells with immunomodulatory properties. B cell recovery was robust, with normal numbers by day 100. Notably, the B cell population present early after HSCT contained a significantly increased proportion of cells with an immature transitional phenotype (CD24⁺ CD38^+, CD27^- IgM^high IgD^high 17.1% compared to 2.7% for healthy individuals, p=<0.005) and significantly more B cells produced IL-10 after in vitro stimulation with CD40L (14.6% compared to 6.3% for healthy individuals, p=0.013), consistent with the known immunosuppressive activity of immature B cells. Furthermore, proportions of regulatory T cells (CD4+ CD25^high CD27⁺ FoxP3⁺) within the CD4 T-cell population were normal (range 4.2-7.4% compared to 5.3% in healthy controls).

Conclusions

Patient-derived effector CD8 T cells shape mixed T-cell chimerism after FCC conditioning and presence of regulatory B and T cells may contribute to long-term tolerance of mixed T-cell chimerism with associated low incidence of GVHD in the absence of post graft immunosuppression.

Note: Marsh JCW and Barber LD joint last authors