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. Author manuscript; available in PMC: 2020 Aug 20.
Published in final edited form as: Br J Haematol. 2017 Feb 7;176(6):950–960. doi: 10.1111/bjh.14448

Allogeneic transplantation using CD34+ selected peripheral blood progenitor cells combined with non-mobilized donor T cells for refractory severe aplastic anaemia

Enkhtsetseg Purev 1, Xin Tian 2, Georg Aue 1, Jeremy Pantin 3, Phuong Vo 1, Reem Shalabi 4, Robert N Reger 1, Lisa Cook 1, Catalina Ramos 1, Elena Cho 1, Tat’yana Worthy 1, Hanh Khuu 5, David Stroncek 5, Neal S Young 1, Richard W Childs 1
PMCID: PMC7440634  NIHMSID: NIHMS826195  PMID: 28169418

Summary

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia (SAA) unresponsive to immunosuppressive therapy. To reduce chronic graft-versus-host disease (GVHD), which occurs more frequently after peripheral blood stem cell (PBSC) transplantation compared to bone-marrow transplantation (BMT), and to prevent graft rejection, we developed a novel partial T-cell depleted transplant that infuses high numbers of granulocyte colony-stimulating factor-mobilized CD34+ selected PBSCs combined with a BMT-equivalent dose of non-mobilized donor T-cells. Fifteen patients with refractory SAA received cyclophosphamide, anti-thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8×106 CD34+ cells/kg and 2×107 non-mobilized CD3+ T-cells/kg from human leucocyte antigen-matched sibling donors. All achieved sustained engraftment with only two developing acute and two developing chronic GVHD. With a 3.5-year median follow-up, 86% of patients survived and were transfusion-independent. When compared to a retrospective cohort of 56 bone-marrow failure patients that received the identical transplant preparative regimen and GVHD prophylaxis with the exception that the allograft contained unmanipulated PBSCs, partial T-cell depleted transplant recipients had delayed donor T-cell chimerism and relative reduction of 75% in the incidence of acute grade II–IV GVHD (13% vs. 52%; P = 0.010) and of 82% in chronic GVHD (13% vs. 72%; P=0.0004). In multivariate analysis, partial T-cell depleted transplants remained significantly associated with a reduced risk of GVHD. In conclusion, for patients with refractory SAA, this novel transplant strategy achieves excellent engraftment and survival when compared to unmanipulated PBSC transplants and dramatically reduces the incidence of both acute and chronic GVHD.

Keywords: severe aplastic anaemia, allogeneic stem cell transplantation, bone marrow transplantation, CD34+ selected PBSC, partial T-cell depleted transplant

Introduction

Severe aplastic anaemia (SAA) is a life-threatening disease characterized by pancytopenia and hypocellular bone marrow (BM) occurring most commonly as a consequence of immune-mediated destruction of BM stem and progenitor cells (Scheinberg and Young 2012). Allogeneic haematopoietic stem cell transplantation (HCT) is curative for SAA and other BM failure syndromes (BMFS). Based on historical data, allogeneic HCT is recommended as upfront therapy for younger patients with SAA who have a human leucocyte antigen (HLA) identical relative (Bacigalupo, et al 2000, Bacigalupo 2014).

Immunosuppressive therapy (IST) with equine anti-thymocyte globulin (ATG) and ciclosporin (CSA) is typically used for older patients with SAA or for younger patients who lack an HLA identical BM donor. Furthermore, recent data suggest adding the thrombopoietin mimetic, eltrombopag, to conventional ATG/CSA improves response rates significantly compared to ATG/CSA alone (Townsley, et al 2015). As a consequence, an increasing number of patients may opt for upfront treatment with IST, given lower treatment-related mortality rates compared to allogeneic HCT, which historically is associated with a 15–25% risk of mortality following treatment (Locasciulli, et al 2007, Chu, et al 2011, Bacigalupo, et al 2012, Bacigalupo, et al 2015). Furthermore, many patients with SAA come from regions of the world where access to centres performing allogeneic transplantation is limited, and/or where financial constraints make the procedure prohibitively expensive. These patients often receive upfront-treatment with IST, proceeding to HCT only in the context of treatment failure.

Recent studies of BM transplantation (BMT) for SAA have showed superior outcome compared to peripheral blood stem cell (PBSC) transplants, including a lower incidence of chronic graft-versus-host disease (GVHD) and improved survival (Schrezenmeier, et al 2007, Chu, et al 2011, Bacigalupo, et al 2012, Bacigalupo, et al 2015). However, BM allografts contain lower numbers of CD34+ progenitor cells and T-cells compared to PBSC allografts, which are factors that historically may have increased the risk of graft rejection, particularly in heavily transfused and HLA allo-immunized SAA patients (Storb, et al 1983, Champlin, et al 1989, Margolis and Cammita 1998, Horowitz 2000, Urbano-Ispizua, et al 2001). In fact, patients who do not proceed to transplant as a frontline treatment and who have failed prior IST, are at increased risk of graft failure, with a recent trial describing early or late rejection in 17% of patients (Kobayashi, et al 2006). Therefore, for patients who do not receive HCT upfront and who have failed one or more IST trials, innovative new transplant strategies are needed to reduce the risk of graft failure in this high-risk cohort.

We, and others, have shown that fludarabine-based PBSC transplantation overcomes the risk of graft rejection in heavily transfused and allo-immunized SAA patients refractory to IST (Gomez-Almaguer, et al 2006, Srinivasan, et al 2006, Maury, et al 2009, Novitzky, et al 2009, Maury and Aljurf 2013, Pantin, et al 2013, Pantin, et al 2014). However, improvement in engraftment with PBSC transplantation was offset by a higher incidence of acute and chronic GVHD compared with BMT. The increase in GVHD observed with PBSC transplants is probably due to these grafts containing an average 20-fold higher dose of T-cells that have undergone granulocyte colony-stimulating factor (G-CSF)-induced T-helper cell type 2 (Th2) cytokine polarization (Ferrara and Krenger 1998, Sloand, et al 2000, Skert, et al 2009). Furthermore, with PBSC transplantation for SAA, rapid donor T-cell engraftment frequently occurs, which is significantly associated with an increased risk of chronic GVHD (Pantin, et al 2013).

Despite the higher risk of chronic GVHD, the majority of allogeneic transplants performed world-wide continue to utilize PBSCs as opposed to BM because PBSC allografts contain higher CD34+ cell numbers, are easier to collect from donors without the need for BM harvesting, and have faster engraftment that is more likely to be sustained (Anasetti, et al 2012, Pasquini and Zhu 2015). To harness the advantage of larger CD34+ progenitor cell numbers in PBSC allografts while overcoming the limitation related to their increased incidence of chronic GVHD, we developed a transplant approach that infuses high numbers of G-CSF-mobilized T-cell depleted CD34+ cells co-infused with a BMT equivalent dose of non-mobilized donor T-cells. These T-cells were collected prior to G-CSF mobilization, and thus had not undergone Th2 cytokine polarization that contributes to the increased incidence of chronic GVHD associated with unmanipulated PBSC allografts. Here we report the results of 15 heavily-transfused patients with BMFS, most with ATG-refractory SAA at high risk for graft rejection, who underwent this new transplant approach. We compare their outcomes with a cohort of 56 patients with SAA and other BMFS who received an unmanipulated PBSC transplant using the identical conditioning regimen and GVHD prophylaxis (Pantin, et al 2013).

Methods

Patients and study design

Patients with SAA, refractory anaemia myelodysplastic syndrome (RA-MDS), or other BMFS aged between 4 and 80 years who had a ≥9/10 HLA-matched (HLA-A/B/C/DR/DQ) related-donor were eligible. Fanconi anaemia was excluded in all SAA patients by the diepoxybutane or mitomycin-C chromosomal breakage test. The study protocol was approved by the National Heart, Lung and Blood Institutional Review Board and all patients gave informed consent in accordance with the Declaration of Helsinki. This trial was registered at www.clinicaltrials.gov, NCT#01174108.

The conditioning regimen consisted of intravenous cyclophosphamide 60 mg/kg/day (days −7 and −6) followed by intravenous fludarabine 25 mg/m2/day (days −5 to −1) and equine ATG 40 mg/kg/d (days −5 to −2). GVHD prophylaxis consisted of CSA beginning on day −4, titrated to achieve a target trough concentration between 200 and 400 ng/ml, and intravenous methotrexate (MTX; 5 mg/m2/day on days +1, +3 and +6). On Day 0, a previously collected and cryopreserved G-CSF-mobilized PBSC allograft containing CD34+ selected cells (Miltenyi-CliniMACS system; target CD34+ cell dose 8×106 cells/kg) was thawed and infused in combination with 2 × 107 CD3+ T-cells/kg (recipient body weight) that also were previously collected by apheresis and cryopreserved from the same donor prior to G-CSF mobilization.

End point definitions

Neutrophil recovery was defined as the first of three consecutive days with an absolute neutrophil count (ANC) of ≥0.5 × 109/l. Platelet recovery was defined as the first of 3 consecutive days with a platelet count of ≥20 × 109/l and no platelet transfusions for 7 days immediately preceding this date. Donor T-cell and myeloid chimerism were determined on post-transplant blood samples by polymerase chain reaction analysis of short tandem repeats as previously described on days 15, 30, 45, 60 and 100, and continued monthly until full donor chimerism was achieved in both lineages (Carvallo, et al 2004, Griffith, et al 2005). Full donor chimerism was defined as ≥95% donor derived cells in peripheral blood in a specific lineage. Acute GVHD was graded using established criteria (Przepiorka, et al 1995). Chronic GVHD was evaluated prospectively based on National Institutes of Health consensus criteria (Filipovich, et al 2005) and was retrospectively reclassified as limited or extensive for comparison between patients in this study and a cohort of previously reported BMFS patients who received an unmanipulated PBSC transplant using the identical conditioning regimen and GVHD prophylaxis (Pantin, et al 2013). Overall survival was defined as the time from transplantation to death from any cause. Data cut-off for study analysis was 1 March 2016.

Statistical analysis

Data were presented as median (range) or frequency (percentage). Overall survival was calculated by the Kaplan-Meier method, censoring at the last follow-up visit (Kaplan and Meier 1958). Cumulative incidences of engraftment, donor chimerism, acute and chronic GVHD were estimated by considering deaths without the specified event as competing risk events (Gooley, et al 1999). Comparisons were carried out between these 15 patients receiving this CD34+ selected PBSCs combined with non-mobilized T-cells (partial T-cell depleted PBSCT cohort) and our historical cohort of 56 BMFS patients who received an unmanipulated PBSCT following the identical conditioning regimen. For pre-transplant characteristics, Fisher’s exact test and Wilcoxon rank-sum test were used to compare categorical variables and continuous variables between the two cohorts of patients, respectively. For transplant outcomes, the log-rank test and the Gray test were used to compare survival probabilities and cumulative incidences of events between the two cohorts, respectively (Klein and Moeschberger 2003). Multivariate analysis based on the Cox proportional hazards regression was used to compare the cause-specific hazards for acute and chronic GVHD between the two transplant cohorts, controlling for pre-transplant risk factors (age, female donor/male patient, serum ferritin level, HLA-allo-immunization and CD34+ allograft cell dose) and early occurrence of full-donor T-cell chimerism (Cox 1972). All tests were two-sided and P values < 0.05 were considered statistically significant. Analysis was performed using the R statistical software, version 3.2.3 (R Foundation for Statistical Computing, Vienna, Austria).

Results

Patient and graft characteristics

Between February 2011 and April 2015, 15 patients with BMFS underwent conditioning and received a CD34+ selected PBSC transplant combined with a BMT-equivalent dose of non-mobilized donor T-cells. Fourteen SAA patients had failed prior IST and one had evolved to MDS prior to transplantation. Patient characteristics are summarized in Table I. The median age was 22 years (range 12–67). Patients were heavily transfused, had elevated ferritin levels, and were highly HLA allo-immunized. The pre-transplant serum ferritin level was markedly elevated at a median of 2267 µg/l (range 161–13928) and 10 (67%) patients were HLA allo-immunized with 53% having HLA class-I antibodies (median panel reactive antibody [PRA] 21%) and 67% having HLA class-II antibodies (median PRA 49%). Patients received a G-CSF-mobilized CD34+ selected PBSC allograft from a 10/10 (n=14) or 9/10 (n=1, due to crossover of a single HLA A locus mismatch) HLA-matched sibling donor containing a median 8×106 CD34+ cells/kg (range 5.3–12) that was co-infused with a median 1.8×107 non-mobilized CD3 cells/kg (range 1.4–2.1).

Table I.

Patient characteristics

Partial T-cell depleted
PBSC cohort
(N=15)		 Historical unmanipulated
PBSC cohort
(N= 56)		 P
Age, median (range) years 22 (12–67) 28.5 (9–66) 0.72
Gender, n (%) 0.56
  Male 8 (53) 35 (62)
  Female 7 (47) 21 (38)
Female donor/male patient, n (%) 2 (13) 17 (30) 0.32
Diagnosis, n (%) 0.13
  SAA 13 (87) 30 (54)
  SAA/PNH 1 (7) 17 (30)
  RA-MDS 1 (7)   6 (11)
  Other BMFS 0 (0)   3 (5)
Previous IST, n (%)* 14 (93) 56 (100) 0.21
HLA match, n (%) >0.99
  6/6 14 (93) 52 (93)
  5/6   1 (7)   4 (7)
HLA alloimmunization, n (%) 10 (67) 23 (43) 0.15
Ferritin (µg/l), median (range) 2267 (161–13928) 2159 (18–11267) 0.59
Ferritin>1000 µg/l, n (%) 12 (80) 43(78) >0.99
CMV recipient at risk, n (%) 10(67) 50 (89) 0.047
Transplanted cell dose, median (range)
  CD34+ cells ×106/kg 8 (5.3–12) 6.6 (1.7–21.1) 0.005
  CD3+ T-cells ×107/kg 1.8 (1.4–2.1) 26 (5–69) <0.0001
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BMFS, bone marrow failure syndrome; CMV, cytomegalovirus; ATG, atithymocyte globulin; CSA, ciclosporin; HLA, human leucocyte antigen; IST, immunosuppressive therapy; PBSC, peripheral blood stem cells; PNH, paroxysmal nocturnal haemoglobinuria; RA-MDS, refractory anaemia myelodysplastic syndrome; SAA, severe aplastic anaemia;

*

For the current partial T-cell depleted PBSC cohort: prior treatment with ATG + CSA (n=12) or cyclophosphamide + CSA (n=2); for historical unmanipulated PBSC cohort: prior treatment with ATG + CSA (n=39), alemtuzumab (n=2) and other immunosuppressive agents (n=15).

Engraftment and chimerism

All 15 (100%) patients receiving the above partial T-cell depleted transplant achieved sustained engraftment with neutrophil recovery occurring at a median time of 14 days (range 10–23). Fourteen (93%) patients achieved platelet recovery at a median time of 18 days (range 9–321). Despite a high proportion of patients being highly HLA allo-immunized and heavily transfused, none experienced graft rejection (Table II and Fig 1A–B).

Table II.

Transplant Outcomes

Partial T-cell depleted
PBSC cohort
(N=15)		 Historical unmanipulated
PBSC cohort
(N= 56)		 P
Neutrophil recovery, n (%) 15 (100) 55 (98)
  median (range), days 14 (10–23) 15 (6–24) 0.46
Platelet recovery, n (%) 14 (93) 50 (93)
  median (range), days 18 (9–321) 12 (5–168) 0.17
Full-donor T-cell chimerism, n (%) 15 (100) 55 (98)
  Time to achieve ≤ 30 days, n (%) 8 (53) 45 (82) 0.039
    ≥ 45 days, n (%) 7 (46) 10 (18)
  median (range), days 30 (15–730) 30 (15–168) 0.014
Full-donor myeloid chimerism, n (%) 15 (100) 54 (96)
  median (range), days 15 (15–30) 30 (15–100) 0.13
 CMV reactivation among at-risk patients, n (%) 9 (90) 31 (62)
  Day of onset, median (range) 33(3–117) 49 (12–62) 0.16
Acute GVHD: Grade II–IV, n (CI%) 2 (13) 29 (52) 0.0099
    Grade III–IV, n (CI%) 2 (13) 17 (30) 0.16
All chronic GVHD, n (CI %) 2 (13) 40 (72) 0.0004
Extensive chronic GVHD, n (CI %) 0 (0) 27 (49) 0.002
Overall survival at last follow-up, n (%) * 13 (86) 49 (87) 0.86
  Overall survival at 200 days, n (%) 14 (93) 52 (93) 0.96
  Follow-up, median (range), months 42 (11–61) 54 (21–132)
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CI, cumulative incidence; CMV, cytomegalovirus; GVHD, graft-versus-host disease; PBSC, peripheral blood stem cells.

*

Overall survival was estimated by the Kaplan-Meier method.

Figure 1.

Figure 1

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Cumulative incidence of neutrophil recovery (A) and platelet recovery (B). The percentage of donor T-cell chimerism (C) and donor myeloid chimerism (D).

All patients achieved full-donor (≥95%) T-cell and myeloid chimerism at a median time of 30 days (range 15–730) and 15 days (range 15–30) post-transplant, respectively (Table II and Fig 1C–D).

Viral reactivation, GVHD and survival

Cytomegalovirus (CMV) reactivation occurred in 9/15 (60%) patients including 9/10 (90%) patients at risk for reactivation at a median of 33 days (range 3–117) (Table II): all 9 responded to antiviral therapy with none developing CMV disease. Epstein–Barr virus (EBV) reactivation associated with the development of post-transplant lymphoproliferative disease (PTLD) occurred in one patient and resolved after rituximab therapy.

Two patients each developed acute grade II–IV and chronic GVHD. The cumulative incidence of acute grade II–IV GVHD at day 100 and chronic GVHD was 13% (95% confidence interval [CI]: 2–35) and 13% (95% CI: 2–35), respectively (Table II and Fig 2A–B). Both acute and chronic GVHD resolved completely with treatment.

Figure 2.

Figure 2

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Cumulative incidence of acute (A) and chronic (B) graft-versus-host disease (GVHD). Kaplan-Meier estimate of overall survival (C).

At day 200, all but one of the 15 patients (93%) survived. With a median follow-up of 42 months (range 11–61), 13/15 patients survived for an estimated probability of overall survival of 86% (95% CI: 71–100) (Table II and Fig 2C). Two patients died during the study: one patient died on day 46 from Klebsiella pneumoniae carbapenemase (KPC) bacteraemia (this infection predated the transplant), and another patient with chronic obstructive pulmonary disease (COPD) (a 67-year-old woman with a heavy smoking history who had this diagnosis pre-transplant) died 18 months post-transplant from complications of a bacterial pneumonia.

Comparisons with historical unmanipulated PBSCT

The characteristics and transplant outcomes of these 15 patients receiving this partial T-cell depleted transplant were compared to our historical cohort of 56 BMFS patients who received an unmanipulated PBSC transplant following the identical conditioning regimen (Tables I, II; and Fig 3). Compared to the unmanipulated PBSC cohort, the characteristics of patients in the two cohorts were similar except that partial T-cell depleted transplant recipients received a higher number of CD34+ cells (median 8 vs. 6.6 × 106 cells/kg; P=0.005) and a lower number of CD3+ T-cells (1.8 vs. 26 × 107 cells/kg; P<0.0001) (Figure S1). Both cohorts had rapid and sustained donor engraftment with similar patterns in the recovery of neutrophils and platelets and donor myeloid engraftment. However, partial T-cell depleted transplant recipients had a significant delay in the time to achieve full-donor T-cell chimerism (53% vs. 82% by day 30; P=0.014), which was associated with dramatic relative reductions of 75% in the incidence of grade II–IV acute GVHD (13% vs. 52%; P=0.010) and of 82% in the incidence of chronic GVHD (13% vs. 72%; P = 0.0004). The overall survival of partial T-cell depleted transplant recipients was comparable to survival in the historical unmanipulated PBSC transplant cohort (86% vs. 87%, P=0.86).

Figure 3.

Figure 3

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Outcomes in severe aplastic anaemia patients following transplantation of CD34+ Selected peripheral blood stem cells combined with non-mobilized donor T-cells (partial T-cell depleted) compared to a historical unmanipulated peripheral blood stem cell transplant (PBSCT) cohort. Cumulative incidence of neutrophil recovery (A), full donor T-cell chimerism (B) and full donor myeloid chimerism (C), acute GVHD (D) and chronic GVHD (E). Overall survival (F). GVHD, graft-versus-host disease.

Finally, in the multivariate analysis adjusted for multiple pre-transplant factors (Table III), partial T-cell depleted transplantation remained significantly associated with a reduced risk of acute GVHD (adjusted hazard ratio [aHR] = 0.22, P=0.043) and chronic GVHD (aHR = 0.13, P=0.007) compared to unmanipulated PBSCT. When early full-donor T-cell chimerism by day 30 was added to the multivariate model (Table III), partial T-cell depleted transplantation remained a significant factor for chronic GVHD (aHR = 0.16, P=0.014), while early full-donor T-cell chimerism showed a trend towards an increased risk of chronic GVHD (aHR = 2.56, P=0.055).

Table III.

Multivariate analysis of risk factors for acute and chronic GVHD.

Model 1
acute GVHD* 		Model 2
chronic GVHD		 Model 3
chronic GVHD
Factors Adjusted HR
(95% CI)		 P Adjusted HR
(95% CI)		 P Adjusted HR
(95% CI)		 P
Partial T-cell depleted PBSCT vs.
historical unmanipulated PBSCT		 0.22 (0.05–0.95) 0.043 0.13 (0.03–0.57) 0.007 0.16 (0.04–0.69) 0.014
Patient age at transplant,
 per 10 years		 1.05 (0.77–1.42) 0.76 1.05 (0.80–1.37) 0.72 1.01(0.77–1.31) 0.96
Female donor/male patient 1.55 (0.72– 3.38) 0.27 1.20 (0.60–2.42) 0.60 1.23 (0.60–2.51) 0.57
Ferritin, log-transformed 1.01 (0.77–1.33) 0.93 1.16 (0.89–1.51) 0.28 1.17 (0.87–1.58) 0.29
HLA-alloimmunization 0.86 (0.62–1.20) 0.38 1.08 (0.93–1.27) 0.31 1.07 (0.92–1.25) 0.36
CD34+ allograft cell dose, log-
transformed		 0.76(0.31–1.87) 0.55 0.56 (0.24–1.30) 0.18 0.60 (0.27–1.38) 0.23
Full-donor T-cell chimerism
≤day 30 vs. ≥day 45		 – – – – 2.56 (0.98–6.69) 0.055
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95% CI, 95% confidence interval; GVHD, graft-versus-host disease; HLA, human leucocyte antigen; HR, hazard ratio; PBSCT, peripheral blood stem cell transplant.

*

Because 13 acute GVHD events occurred before day 30, the post-transplant variable (full-donor T-cell chimerism ≤ day 30 vs ≥ day 45) was not included in the regression model for acute GVHD.

Discussion

Allogeneic HCT is considered the optimal upfront therapy for younger patients with SAA who have an HLA-identical relative to serve as a stem cell donor (Bacigalupo 2014). However, because IST therapy for SAA is associated with responses in a majority of patients with a low-risk of mortality (Scheinberg, et al 2011, Townsley, et al 2015), some patients will opt to receive IST first, reserving allogeneic transplantation as a salvage treatment should IST fail or in the event of disease relapse. Furthermore, many patients with SAA come from geographic areas where access to allogeneic HCT does not exist. Although allogeneic HCT can cure patients with SAA who fail to respond to IST, these patients tend to be heavily transfused, are often HLA allo-immunized, and have an increased risk of graft rejection. While HLA allo-antibodies in the setting of an HLA-identical sibling transplant does not directly lead to rejection, HLA antibodies are a surrogate for patients that have received multiple transfusions, which is clearly associated with an increased risk of worse transplant outcomes, including graft rejection, GVHD and reduced survival (Storb, et al 1983, Pullarkat, et al 2008, Deeg, et al 2009).

Allogeneic HCT can be performed utilizing stem cells collected from either the BM or the peripheral blood following G-CSF mobilization. Currently, BMT is considered by most to represent the optimal transplant approach for SAA as it is associated with a lower incidence of chronic GVHD and a similar rate of graft rejection compared to PBSC transplantation (Schrezenmeier, et al 2007, Chu, et al 2011, Bacigalupo, et al 2012, Bacigalupo, et al 2015). However, historically, the risk of graft rejection is increased with BMT compared to PBSC transplant, an often fatal complication which occurs more frequently in SAA patients who have failed prior IST (Storb, et al 1983, Champlin, et al 1989, Margolis and Cammita 1998, Horowitz 2000, Urbano-Ispizua, et al 2001, Kobayashi, et al 2006). In an effort to ensure sustained engraftment and to minimize the incidence of chronic GVHD, we developed a novel transplant regimen that uses an allograft containing high numbers of CD34+ selected PBSCs combined with a BMT-equivalent dose of non-mobilized T-cells. The first 15 patients treated with this regimen were at high-risk for graft rejection, with all but one having SAA that was refractory to IST, and the majority being heavily transfused and HLA allo-immunized. Remarkably, all had sustained donor engraftment with neutrophil and platelet recovery occurring at a median 14 and 18 days, respectively (Fig 1). Survival of recipients undergoing partial T-cell depleted transplant was excellent, with 93% of patients surviving at day 200 and, at a median follow-up of 3.5 years, 86% of patients survived, all transfusion-free and without chronic GVHD. Although two died from infections, both had serious predisposing underlying illnesses (KPC bacteremia and COPD) that predated the transplant, making it unlikely that partial T-cell depletion contributed to their infection- related deaths.

While this partial T-cell depleted transplant approach involves removing mobilized T-cells from the allograft, it differs from other strategies that have also been successful in preventing GVHD, such as through a) the use of post-transplant alemtuzumab to induce in vivo T-cell depletion (Marsh, et al 2011), b) the transplantation of allografts combining G-CSF-mobilized BM stem cells and PBSC in haploidentical donors combined with ATG as a lympho-depleting agent (Wang, et al 2013), c) the use of post-transplant cyclophosphamide administration to kill alloreactive T-cells (Luznik, et al 2008), or d) by removing GVHD-causing cells through ex vivo depletion of select T-cell subsets, such as CD8+ T-cells, alpha/beta T-cells or naïve T-cells (Bertaina, et al 2014). In contrast to these strategies, the transplant approach we utilized was designed to prevent GVHD by infusing T-cells that had not undergone Th2 polarization, as Th2 polarized T-cells are typically contained in large numbers in G-CSF mobilized allografts. Studies by Sloand et al (2000) and others have demonstrated that CD4+ T-cells collected from donors mobilized with G-CSF have decreased γ-interferon (IFNγ) and increased interleukin 4 (IL4) expression, highlighting the effect of this cytokine on altering the Th1/Th2 balance. Further, studies evaluating patients who developed chronic GVHD after PBSC transplantation have revealed their T-cells have a significant decrease in IFNγ and increased IL10 levels, strongly indicating a role of transplanted Th2 polarized T-cells in the development of chronic GVHD (Skert et al 2009).

Further, it is also important to consider that the increase in chronic GVHD observed with PBSC transplants compared to BMT probably occurs in part as a consequence of these grafts containing an average 20-fold higher dose of T cells. Higher T-cell doses may accelerate donor T-cell engraftment, which has been shown to increase the risk of GVHD following HCT (Santos, et al 1985, Childs, et al 1999, Marsh, et al 2011). Previously, we observed in a multivariate analysis that rapid donor T-cell engraftment was an independent risk factor for chronic GVHD in SAA patients receiving PBSC allografts (Pantin, et al 2013). To overcome this hurdle, the transplant approach described herein was developed to reduce the number of transplanted T cells to what would typically be transplanted with a BMT, in the hope this manoeuvre would slow the speed of donor T-cell engraftment. Remarkably, this partial T-cell depleted transplant achieved this goal; when compared to our historical cohort of SAA patients who received an unmanipulated PBSC transplant containing substantially higher numbers of G-CSF-mobilized T-cells (median 2.6×108 CD3+ T-cells/kg), partial T-cell depleted transplant recipients receiving a median of 2 × 107 CD3 + T-cells/kg had a significant delay in the time to achieve full-donor T-cell chimerism (53% vs. 82% by day 30; P=0.014). This delay in donor T-cell engraftment was associated with a significant reduction in the risk of acute and chronic GVHD by 75% and 82%, respectively, compared to unmanipulated PBSC transplant recipients. In multivariate analysis adjusted for pre-transplant risk factors, partial T-cell depleted transplants remained significantly associated with a reduced risk of both acute and chronic GVHD. These differences in outcomes are clinically meaningful as prior studies have demonstrated that quality of life is superior and long-term risk of transplant-related medical morbidities are lower in patients who do not develop chronic GVHD after allogeneic transplantation (Pidala, et al 2011, Atsuta, et al 2014). When examining the correlation between donor T-cell chimerism and the transplanted CD34+ and CD3+ cell doses in the graft for the patients from both cohorts, we found that the CD34+ cell dose did not correlate with T-cell chimerism (P=0.72), although we did observe that a lower T-cell dose in the graft significantly prolonged the time until full donor T cell chimerism was achieved (P=0.012). However, this difference was not observed when this analysis was restricted to recipients of partial T-cell depleted transplants, where T-cell variability, as per protocol, was minimal. Finally, partial T-cell depletion did not come at the expense of compromising myeloid engraftment nor did it lead to graft rejection: neutrophil and platelet recovery as well as donor myeloid chimerism were similar between SAA patients receiving a partial T-cell depleted transplant and recipients of an unmanipulated PBSC transplant (Fig 1). It is important to point out that engraftment kinetics following HCT for SAA are probably affected by multiple other donor/host variables, including the status of the recipient’s BM stroma and microenvironment (Chen, et al 2005, Takaku, et al 2010).

As would be expected with any T-cell reducing transplant manoeuvre, reactivation of CMV appeared higher among patients at risk for this complication in the partial T-cell depleted cohort (9/10; 90%) compared to our historical unmanipulated PBSC cohort (31/50; 62%, P=0.16) (Couriel, et al 1996). Importantly, despite a higher CMV reactivation rate, none of the patients developed CMV disease and all responded promptly to pre-emptive antiviral treatment.

Of note, besides evaluating individual outcomes post-transplantation, recent studies have suggested a composite endpoint of GVHD-free survival, with events including grade 3–4 acute GVHD, chronic GVHD or death, as being useful for comparing HCT approaches (Holtan, et al 2015). Using this definition, recipients of the partial T-cell depleted transplant had an estimated GVHD-free survival of 65%, which was far superior to that of our historical unmanipulated transplant cohort, who had a 21% GVHD-free survival (P=0.003, Figure S2).

Our study is not without limitations: although infusing high numbers of G-CSF-mobilized T-cell depleted CD34+ cells co-infused with non-mobilized T-cells appears to facilitate engraftment while simultaneously reducing GVHD, the transplant requires graft manipulation and two separate apheresis procedures. Therefore, this strategy would be limited to transplant centres that have cell-processing capabilities that include ex vivo CD34 selection. Further, our historical cohort, which received unmanipulated PBSCs, had a higher incidence of acute and chronic GVHD than has been observed in other large recent studies of PBSC transplantation for SAA (Schrezenmeier, et al 2007, Chu, et al 2011, Bacigalupo, et al 2012). Aside from differences in the patients, grafts and transplantation centres, our historical higher GVHD rate may be partly related to adding fludarabine to the conditioning regimen, which accelerates donor T-cell engraftment. Although the patient and pre-transplant characteristics are comparable between our current and historical cohorts that used the identical transplant preparative regimen and GVHD prophylaxis, the findings from comparisons with historical data are hypothesis generating and will require validation in a larger cohort.

In conclusion, for IST-refractory SAA patients, transplantation of a PBSC allograft containing high numbers of CD34+ selected cells co-infused with a BMT-equivalent dose of non-mobilized T-cells achieves excellent engraftment and survival. Compared to SAA patients receiving unmanipulated PBSCs, this novel transplant strategy results in delayed donor T-cell engraftment, and a reduction in both acute and chronic GVHD without increasing the risk of graft rejection. Although survival rates were similar between these two different transplant approaches, the substantial reduction in chronic GVHD in partial T-cell depleted allograft recipients would be expected to result in a substantial improvement in quality of life and a reduction in the long-term risk of medical morbidities associated with allogeneic transplantation. The results of our novel PBSC-based transplant approach are provocative as they describe outstanding clinical outcomes in a high-risk cohort of ATG-refractory SAA patients, providing a promising alternative to BMT, which currently is the recommended approach for SAA patients undergoing transplantation. We believe the encouraging results observed in this high-risk cohort, who were heavily transfused, HLA allo-immunized and had disease that was refractory to IST, warrant studies exploring the upfront use of this regimen in lower risk SAA patients with treatment-naïve disease.

Supplementary Material

Supp Fig S1-S2

Acknowledgments

The authors would like to thank our patients for participating in this study. This research was supported by the Intramural Research Program of the National Heart, Lung and Blood Institute and the Clinical Center, National Institutes of Health.

Footnotes

Author Contributions

J.P., R.W.C. and X.T. designed and wrote the protocol. E.P., J.P., R.W.C., G.A., L.C., C.R., E.C., T.W., P.V., R.S. and N.S.Y. participated in subject screening, enrolment and patient care. H.K., D.S. and R.N.R. processed the samples. E.P., J.P., H.K., R.W.C. and X.T. analysed the data. E.P., J.P., X.T. and R.W.C. wrote the paper. All authors reviewed and approved the final manuscript.

Conflict of interest:

The authors declare no conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1. Allograft cell doses in SAA patients following transplantation of CD34+ Selected PBSCs combined with non-mobilized donor T-cells (partial T-cell depleted PBSC) compared to a historical unmanipulated PBSCT cohort.(A) CD34+ allograft dose; (B) CD3+ allograft dose.

Figure S2. GVHD-free survival. The events included grade 3–4 acute GVHD, chronic GVHD, or death.

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