King’s College Hospital FCC Conditioning for Severe Aplastic Anemia Induces Tolerance with Mixed T-Cell Chimerism and Extremely Low Incidence of Gvhd

We have previously shown that HSCT conditioning regimen for severe aplastic anemia (SAA) using fludarabine, cyclophosphamide and alemtuzumab (FCC), is associated with very low rate of GvHD (especially chronic, cGVHD), and excellent overall survival (OS) of 83% and 95% for matched unrelated (MUD) and matched sibling donor (MSD) HSCT, respectively. Sustained full donor myeloid chimerism occurred most frequently with mixed T-cell chimerism, and this persisted despite discontinuation of post graft immunosuppression with ciclosporin (CSA). We proposed that prolonged mixed T-cell chimerism with alemtuzumab conditioning may facilitate tolerance and hence a low risk of GVHD.

We have investigated the basis for the mixed T-cell chimerism in a cohort of 35 patients, median age 32y (range 15-63), transplanted for acquired idiopathic SAA from 2007 - 2014 at King’s College Hospital. FCC conditioning comprised fludarabine 30 mg/m² iv (day -7 to -4), cyclophosphamide 300 mg/m² iv (day -7 to -4) and alemtuzumab (median dose 70mg, range 45-100). CSA was used as post graft immune suppression for 9 months, followed by dose tapering to zero over the next 3 months depending on PB chimerism. Stem cell source was BM in 7(20%) patients and 28(80%) PBSC, with a median cell dose of 6.8 (1.9-12.4) CD34+ cells x 10⁶/Kg. Donor type was MSD in 9(26%) pts and UD in 26(74%), of whom 20 were matched (MUD) and 6 were 9/10 match ( received FCC with 2 Gy TBI). Median time to neutrophils >0.5x10⁹/l was 12(10-20) days and to platelets >50x10⁹/l was 10(9-61) days. Primary graft failure occurred in only one patient (2%). Acute GvHD was seen in 6(17%) pts (all grade I-II), and chronic GvHD in 5(14%) pts (3 mild, 1 moderate, 1 severe, all with only skin involvement). On univariate analysis with Cox regression model, factors significantly predicting cGVHD were previously diagnosed acute GVHD (p=0.035) and CD3+ chimerism at day+100 ≥90% (p=0.006). Four patients developed autoimmune anemia post HSCT (2 hemolytic anemia, 2 PRCA). Median follow-up was 30 months (4.3-86.8), OS 94% and event free survival 90%. One year TRM was 6%.

Of 32(91%) patients evaluable for chimerism, 8 (23%) were fully donor chimeric for unfractionated BM, PB CD15+ and PB CD3+, while 24 (77%) patients had persistent mixed CD3+ chimerism. Median CD3+ chimerism values were 77% at day+30, 60% at day+60, 41% at day +100,57% at +day180, 70,5% at 1 year, 77% at 2 years and 80% at 3 years. The proportion of evaluable patients no longer taking CSA was 0/23 at 1yr, 9/19 at 1.5yr, 12/16 at 2yr, 12/12 at 2.5yr and 4/4 at 3yr. PB immunophenotyping was performed in 19 patients. During the first year post HSCT, there was profound and prolonged lymphopenia, NK cells recovered first but numbers remained below normal. T cells comprised only 2.6% of lymphocytes at day 30, rising slowly to 41% at day 360 but still significantly below normal (66% in 11 aged-matched healthy individuals), with deficiency greatest for CD4+ T cells. Despite slow recovery of CD4+ T cells, percentage of T-regulatory cells (CD4+ CD25^high CD27⁺ FoxP3⁺) within this population was normal (range 4.2-7.4% for all time points compared to 5.3% for healthy individuals). Patients aged <50y showed recovery of naïve T cells that expressed markers of recent thymic emigrants (CD31⁺ CD62L⁺ CD45RA⁺), indicating renewed thympoiesis and implementation of central tolerance B cell recovery was notably robust, with numbers comparable to healthy individuals for most patients by day 100. Subset analysis showed absence of memory B-cells but significantly increased proportion of immature transitional (T1 and T2) B cells (CD24⁺ CD38^+, CD27^-, IgM ^high IgD ^high) for 6 months after FCC HSCT. Cells within this subset are known to possess immune suppressive activity.

The mechanism for prolonged mixed T-cell chimerism and associated low incidence of chronic GVHD following FCC conditioning for SAA HSCT may be multifactorial, but we here report novel findings: apart from a markedly low proportion of T cells, we show for the first time in SAA post HSCT the presence of T and B cell subsets with potential regulatory properties and establishment of central tolerance of naïve T-cells in younger patients. Our results also confirm the excellent outcomes of UD HSCT using alemtuzumab with reduced intensity conditioning in SAA, and we propose it is now time to re-think the up-front MUD transplant at all ages in eligible patients.