Mixed T Cell Chimerism After Allogeneic Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia Using an Alemtuzumab-Containing Regimen Is Shaped by Persistence of Recipient CD8 T Cells

Highlights • Clinical outcomes are excellent using an alemtuzumab-containing hematopoietic stem cell transplantation regimen for aplastic anemia.• Outcomes are excellent despite prolonged abnormality of the T cell profile.• Recipient-derived CD8 T cells shape persistent mixed chimerism.

established, but incidence of viral disease was low suggesting that anti-viral immunity is maintained. The study showed that despite abnormality of the T-cell profile after allogeneic HSCT for SAA using the FCC regimen, it is conducive for excellent clinical outcome.

Introduction
In contrast to hematologic malignancies where GVHD has an important graft versus leukemic effect, there is no advantage for any GVHD in allogeneic HSCT for nonmalignant diseases. In vivo T-cell depletion (TCD) is used to reduce GVHD. We pioneered the use of alemtuzumab instead of antithymocyte globulin (ATG) for allogeneic HSCT with TCD to treat SAA. Using the 'FCC' conditioning regimen of fludarabine, low dose cyclophosphamide with alemtuzumab ('Campath-1H'), we previously reported in a retrospective multi-center study an OS of 85-90% and a remarkably low incidence of chronic GVHD 1 2 . Subsequent studies have confirmed the benefit of alemtuzumab over ATG in reducing both acute and chronic GVHD in SAA HSCT 3 4 5 . A recent large multicenter study examining cyclophosphamide dose de-escalation in unrelated donor HSCT for SAA using ATG, confirmed excellent OS but the 1-year incidence of chronic GVHD was 22.5-31.7%, depending on cyclophosphamide dose used 6 . Allogeneic HSCT with alemtuzumab for TCD has also been applied to the treatment of sickle cell anemia and excellent clinical outcomes reported 7 .
Previous studies of chimerism post HSCT for SAA examining unfractionated blood mononuclear cells have demonstrated a significant incidence of mixed chimerism.

Chimerism
Chimerism was assessed in unfractionated peripheral blood, CD3+ T cells and CD15+ granulocytes. Full donor chimerism was defined as > 95% donor hematopoietic cells.
Genomic DNA was extracted using a QIAamp DNA Micro kit (Qiagen). Percentage donor chimerism was determined using the PowerPlex 16 HS (Promega) multiplex short tandem repeat system for DNA typing. Genetic analysis was performed using an Applied Biosystems 3130xl and results interpreted using ChimerMarker (Version 3.0.9).

Statistical analysis
Univariate comparisons and multivariate analysis was performed using the Cox proportional hazards regression model with Statistical Package for the Social Science (SPPS) software. Comparisons of lymphocyte subset composition in patients and healthy volunteers were made with the Mann-Whitney U test using GraphPad Inc.
software, Prism version 6.0. All tests were 2-tailed, with P-values <0.05 considered to be statistically significant. All data were censored as of 30 December 2015.

Clinical outcomes after FCC HSCT
In this single center study, we have confirmed the excellent clinical outcomes as previously reported following HSCT using FCC conditioning for SAA 1 ( Table 2).
The median patient follow-up after HSCT was 31.4 months (range 3-93) with excellent OS and event-free survival (EFS) at five years of 93 and 91% respectively.
Importantly there was no significant difference in outcomes for MSD compared to UD transplants or for patients aged > 50 years (n=14), compared to younger patients.
Three patients died, resulting in a TRM rate of 7% at one year. The rate of GVHD was very low; the majority of which was mild. Reliable and sustained engraftment was observed with only one graft failure noted in a patient who received a suboptimal bone marrow infusion cell dose. Sequential chimerism data were available for 42 (93%) patients that confirmed the persistent mixed T-cell chimerism previously reported with the FCC conditioning regimen 1 ( Figure 1A). thrombocytopenia and 1 who had thyroiditis) and 1 with probable immune-mediated neutropenia that responded to granulocyte colony stimulating factor (supplementary Table 1. All 4 cases of pathology classified as warm type autoimmune hemolytic anemia (AIHA) responded to prednisolone but 3 relapsed, 2 of 2 responded to rituximab. The other patient, who received a 9/10 HLA-match HSCT, had refractory and fulminant hemolysis in association with severe GVHD and multiple thromboses.
Of the 4 cases of pure red cell aplasia (PRCA), 3 received a major ABO mis-matched transplant that is the likely cause of their PRCA. Three of four patients recovered attaining transfusion independence while in one patient the PRCA was refractory to steroids, intravenous immunoglobulin, rituximab, and donor lymphocyte infusion.
This patient has received a second HSCT from an alternative ABO matched MUD.

Lymphocyte reconstitution after FCC HSCT
Serial analysis of peripheral blood lymphocytes of 29 patients after FCC HSCT showed the expected lymphopenia associated with use of alemtuzumab, and total number of lymphocytes remained significantly below normal (P<0.0001) at one year and beyond ( Figure 1B). NK cells were the predominant lymphocyte population present early post HSCT comprising 49.3% of lymphocytes at day 30 ( Figure 1C). B cells were not detected at day 30, but rapidly recovered comprising 31.7 of lymphocytes at day 60 ( Figure 1C) and frequency remained significantly above normal at day 360 (23.7% compared to 10.5% for healthy volunteers, P=0.002%).

T-cell deficiency is prolonged and the CD8 T-cell population is skewed to an effector phenotype
T cells were profoundly deficient, comprising only 11.3% of lymphocytes at day 30, increasing to 43.8% at one year, but still significantly below normal (67.2%, P=0.018) ( Figure 1C). Rapid recovery of B cells with prolonged T-cell deficiency produced abnormal dominance of B cells over T cells, maintained long term.
Frequencies of CD4 T cells within the T-cell population were lower than normal producing inversion of the usual CD4 to CD8 T-cell ratio ( Figure 1D). Memory and effector T cells predominated early post HSCT ( Figure 1E). Naïve T cells began to recover from 6 months ( Figure 1E)

Discussion
Using the FCC conditioning regimen for allogeneic HSCT to treat SAA at a single center and with long-term follow up, we confirm the excellent clinical outcomes previously reported in our multi-center study 5 . Incidence of chronic GVHD was remarkably low, particularly given the high proportion of MUD in this cohort. The comparable outcome of older (>50 years of age) and younger patients is also encouraging, given that increasing age is typically associated with worse outcome 13 .        Median time to platelets > 20 x 10 9 /l 12 days (Range 9 -61) Primary graft failure 1 (2.2%) Acute GVHD:  Grade I/II  Grade III/IV 6 (13.3%)  6 of 6 (100%)  0 of 6 Chronic GVHD:  Mild  Moderate  Severe 6 (13.3%)  4 of 6 (66%)  1 of 6 (17%)  1 of 6 (17%) 1-year TRM 3 (6.6%)