Immune Recovery

Immune reconstitution in patients with acquired severe aplastic anemia after haploidentical stem cell transplantation

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Abstract

Immune recovery (IR) after haploidentical stem cell transplantation (haplo-SCT) in severe aplastic anemia (SAA) patients remains relatively unknown. In this study, we examined immune cell subset counts and immunoglobulins in 81 SAA patients from day 30 to day 365 after haplo-SCT. Simultaneously, we determined which factors influence IR and analyzed the effects of immune cell subsets on transplant outcomes. We found that: (i) The reconstitution of different immune cell subsets occurred at different rates after haplo-SCT. Monocytes were the first to recover, followed by CD8+ T and CD19+ B cells, and finally CD4+ T cells. (ii) In the multivariate analysis, lower recipient age, female gender, high mononuclear cell counts in the graft and absence of CMV reactivation were associated with improved IR after transplant. (iii) A CD4/CD8 ratio less than 0.567 on day 30 post transplantation was associated with higher overall survival after haplo-SCT in SAA patients. In conclusion, SAA patients showed a faster recovery of monocytes and CD8+ T cells after haplo-SCT, whereas the recovery of the CD4+ T-cell subset was delayed. Our results may provide insight into methods for better predicting and modulating IR of SAA patients and subsequently improving outcomes after transplantation.

Introduction

Acquired severe aplastic anemia (SAA) is a rare disease that is characterized by bone marrow failure that results in pancytopenia and hypoplastic bone marrow.1, 2 Hematopoietic stem cell transplantation (SCT) is one of the main treatment strategies for SAA.3, 4, 5, 6 In recent years, for patients who require transplantation, but have no HLA-matched donors, haploidentical SCT (haplo-SCT) is an important alternative option.7, 8 Currently, researchers, including ourselves, have reported experiences of treating SAA with haplo-SCT. Long-term follow-up data showed that SAA patients who received haplo-SCT had promising clinical outcomes.8, 9, 10

Delayed immune reconstitution (IR) after haplo-SCT played a crucial role in infections and GVHD and was considered a barrier to the wider application of haplo-SCT in SAA.11, 12 The assessment of IR may provide tools to better predict and modulate adverse outcomes and subsequently improve survival after transplantation. Kinetics of IR in SAA patients after haplo-SCT had been previously evaluated by our group and others, however, mainly focused only on CD3+ T, CD4+ T and CD19+ B cells, and only limited data could be acquired.10, 13, 14 Moreover, factors that may affect IR, as well as the impact of IR on clinical outcomes have not been studied systematically in SAA patients.

In this study, we retrospectively analyzed IR in 81 SAA patients who received haplo-SCT. Simultaneously, we investigated the factors that may affect IR and assessed the impact of immune cell subset recovery on transplant outcomes.

Patients and methods

Patients and transplantation

Between January 2008 and December 2015, a total of 102 patients with acquired SAA who underwent haplo-SCT in our center were studied. The selection criteria were as follows: the patient was receiving their first allogeneic transplantation, and the patient’s data on immune cell subsets from at least one of the post-transplant time points were available. Patients who had no myeloid engraftment or had died by day 30 were excluded. Finally, 81 patients with acquired SAA were analyzed in this study. Forty-eight patients were reported in our pervious study; however, only IR kinetics of CD3+ T, CD4+ T and CD19+ B cells were analyzed. The number of patients analyzed at each time point varied. The latest evaluation of immune cells available at 30 days (n=66), 60 days (n=57), 90 days (n=63), 180 days (n=61), 270 days (n=41) and 365 days (n=44) was used for the analysis. Written informed consent was obtained from patients or their guardians and their donors. The study was approved by the Institutional Review Board of Peking University. As normal controls, 15 age-matched healthy donors were also analyzed in this study.

Transplantation procedures

The conditioning regimen, stem cell collection, GVHD prophylaxis and supportive care were described previously.9, 10 Briefly, the conditioning regimen was as follows: busulfan (0.8 mg/kg four times daily on days −7 and −6); cyclophosphamide (50 mg/kg once daily on days −5 to −2); rabbit antithymocyte globulin (ATG, Sangstat Lyon, France, 2.5 mg/kg once daily for days −5 to −2). All patients received G-CSF-primed bone marrow combined with G-CSF-primed peripheral blood. All patients received a GVHD prophylaxis regimen consisting of cyclosporine A, mycophenolate mofetil and short-term methotrexate (MTX). G-CSF (5 μg/kg/day) was administered subcutaneously from day +6 until myeloid engraftment. All patients were hospitalized in a laminar airflow room and given prophylactic antibiotics according to an established protocol.15

Multiparameter flow cytometric analysis of immune cell subsets

The absolute monocyte and lymphocyte counts were determined by a routine blood examination in the clinical hematology laboratory at each time point: 30, 60, 90, 180, 270 and 365 days after transplantation. Immune cell subsets were identified by multiparameter flow cytometry according to the method in previously published studies.16

Immunoglobulins

To evaluate total body plasma cell count and function, we measured IgA, IgM and IgG serum levels in a clinical laboratory on days 30, 60, 90, 180, 270 and 365 post-transplant. Notably, all of the 81 patients were supplemented by intravenous IgG at a dose of 0.4 g/kg on day 1, day 11 and day 21 post-transplant and 24 patients also received IgG after day 30 post-transplant.

Definitions and evaluation

A diagnosis of SAA, or very SAA, was made according to previously published studies.17, 18 Myeloid and platelet engraftment were defined according to previous reports.19 The diagnosis and grading of acute and chronic GVHD were assigned by the transplant center according to the standard criteria.20, 21

Statistical analysis

Immune cell counts and proportions were summarized as medians and 25th–75th percentiles. The Mann–Whitney rank sum test was used to compare subset counts between subject groups. Univariate and multivariate analyses of factors and IR were performed using logistic regression model.22, 23, 24 All variables achieving a P-value<0.1 in univariate analysis were considered for multivariate analysis. For most analyses, P-values<0.05 (two-tailed) were considered significant. To minimize chances of spurious associations when analyzing factors associated with IR, P-values<0.005 were considered significant unless the associations appeared significant for two adjacent time points (P<0.05 for both time-points).25 All analyses were performed using the SPSS 22.0 and GraphPad Prism 6.0 software packages.

Results

Patient characteristics and transplant outcomes

The patient and transplant characteristics are shown in Table 1. The patient’s median age was 14 years (range, 3–45). There were 46 children (<18 years old) and 35 adult patients included in this study. Among the 81 patients, 18 were diagnosed with very severe aplastic anemia. The median mononuclear cell counts in the grafts were 8.87 × 108/kg recipient body weight, and the median CD34+ cell counts were 2.76 × 106/kg recipient body weight.

Table 1 Patient and transplant characteristics

Transplant outcomes for the patients are shown in Table 2. All patients included in this study had a myeloid engraftment by day 30, and the median time for neutrophil engraftment was 12 (range, 10–22) days. Seventy-six patients had successful platelet engraftment, and the median time for platelet engraftment was 15 (range, 7–150) days. Of the 81 patients, the 100-day cumulative incidences of grades II–IV and grades III–IV aGVHD were 33.3±5.2% and 7.6±3.0%, respectively. Overall, 26 (35.62%) of the 73 evaluable patients developed cGVHD (21 limited and 5 extensive). A total of 10 patients died after transplantation, with a median time of 67 (range, 28–409) days. The estimated 3-year OS was 87.5±3.7%, with a median follow-up time of 835 (range, 267–1960) days.

Table 2 Transplant outcomes of patients

Immune recovery of lymphocytes and their subsets

The IR of lymphocytes and their subsets is shown in Figure 1. Monocytes recovered rapidly and persisted at higher levels than normal during the first year after transplantation. In contrast to monocytes, total lymphocytes recovered slowly, and the levels were lower compared to healthy donors until 180 days after transplantation. CD3+ T cell counts were very low in the first 30 days but normalized by 60 days post transplant. CD4+ helper T cells recovered slowly and did not reach a normal value until 1 year after transplantation. CD8+ cytotoxic T cells recovered much faster than CD4+ T cells. The median count of CD8+ T cells rose above the normal value after 60 days post transplant. A significant inversion of the CD4:CD8 ratio was observed up to 1 year after transplantation. CD4CD8 T cells reached up to normal value on 90 days after transplantation. CD19+ B lymphocytes almost disappeared within 90 days post transplant, slowly recovered from day 90 onward, and finally recovered to a normal value by day 270. For the CD4+ T cell subset, we found that CD4+ naive T cells did not start to recover until 180 day after transplantation, whereas CD4+ memory T cells recovered much faster and returned to normal values on day 180.

Figure 1
figure1

Median counts of immune cells and subsets in haploidentical transplant recipients with SAA on days 30, 60, 90, 180, 270 and 365 after transplantation. Error bars indicate the 25th to 75th percentiles. Normal values from healthy donors are shown as horizontal lines (broken line for the median and gray background for the 10th to 90th percentiles). *P-values <0.05, **P-values <0.005.

Co-stimulatory molecule expression in recovered T cells

As CD28/B7 co-stimulation played a significant role in T-cell alloreactivity. We also investigated the expression of CD28/B7 co-stimulatory molecules on recovered CD4+ and CD8+T cells. As shown in Figure 2, the expression of CD28 on CD4+ T cells and the CD4+CD28+ T cell counts were lower compared to those of healthy donors during the first year after transplantation. The expression of CD28 on CD8+ T cells was higher compared to healthy donors in the first 30 days and dropped to normal values from day 60 to day 365 (Figure 2a). Due to the quick recovery of CD8+ T cells, the total counts of CD8+CD28+ T cells were higher than the normal values from day 60 to day 365 after transplantation (Figure 2b).

Figure 2
figure2

Co-stimulatory molecule expression in recovered CD4+ and CD8+ T cells. (a) Percentage of CD28 expressed in CD4+ and CD8+ T cells. (b) Median counts of CD4+CD28+ T cells and CD8+CD28+T cells. Error bars indicate the 25th to 75th percentiles. Normal values from healthy donors are shown as horizontal lines (broken line for the median and gray background for the 10th to 90th percentiles). * P-values <0.05, **P-values <0.005.

Immune recovery of immunoglobulin

As shown in Figure 3, the IgA and IgM levels from patients were significantly lower compared to healthy controls and did not normalize by day 365. In contrast, the IgG levels were only mildly subnormal during the first 1 year after transplantation.

Figure 3
figure3

Recovery of immunoglobulin post-transplant. Error bars indicate the 25th to 75th percentiles. Normal values from healthy donors are shown as horizontal lines (broken line for the median and gray background for the 10th to 90th percentiles). All of the 81 patients were supplemented by intravenous IgG at a dose of 0.4 g/kg on day 1, day 11 and day 21 post-transplant and 24 patients also received IgG after day 30 post-transplant. *P-values <0.05, **P-values <0.005.

Factors influencing immune reconstitution

Factors that may have affected IR were evaluated. The results of multivariate analysis regarding the influence of patient-, disease-, graft-, and transplant-related variables (aGVHD, cGVHD, CMV and EBV) to IR are summarized in Table 3.

Table 3 Factors associated with immune recovery by multivariate analysis

Age of the recipient

Age was the main factor associated with IR. A lower recipient age resulted in increased counts of CD3+, CD4+, CD4+CD28+, CD4+ memory T cells and CD19+ B cells on day 30. Additionally, there was an inverse correlation between recipient age and CD4+, CD4+CD28+ and CD4+ memory T cells on day 60; CD19+ B cells on day 90; CD4+CD28+, CD4+ naive T cells and CD19+ B cells on day 180; CD4+, CD4+CD28+ and CD4+ naive T cells on day 270; CD4+, CD4+ memory and CD4+ naive T cells on day 365.

Gender of the recipient

In multivariate analysis, female recipients had a quicker reconstitution of CD4+ T cells as well as CD4+ memory T cells from day 60 to day 90.

Cell composition of the graft

An increased number of mononuclear cells collected in grafts correlated significantly with better recovery of CD3+ T cells from day 270 to day 360.

CMV reactivation

Patients with CMV reactivation had a slower recovery of CD4+ naive T cells from day 60 to day 90.

GVHD

There were also correlations between aGVHD, cGVHD and IR, however, these did not reach significant differences.

Impact of immune recovery on transplant outcomes in patients with SAA who received haplo-SCT

The cutoff value for every immune cell subset was calculated for transplant outcome prediction at different time points after SCT through a ROC curve. Importantly, we found that recipients with CD4/CD8<0.567 on day 30 had a higher overall survival and lower treatment-related mortality than did recipients with CD4/CD8>0.567 on day 30 (P=0.003 and P=0.003, respectively) (Figure 4). Except for the CD4/CD8 ratio on day 30, there was no significant difference in the other subsets.

Figure 4
figure4

Association of immune recovery and transplant outcomes. (a) Recipients with CD4/CD8<0.567 on day 30 had a survival advantage compared to recipients with CD4/CD8>0.567 (P=0.003). (b) Recipients with CD4/CD8<0.567 on day 30 had a lower treatment-related mortality compared to recipients with CD4/CD8>0.567 (P=0.015). A full color version of this figure is available at the Bone Marrow Transplantation journal online.

Discussion

To the best of our knowledge, this was the largest sample study to focus systematically on the IR in patients with SAA undergoing haplo-SCT. The most important findings of this study are as follows: (i) Monocytes were the first to recover, followed by CD8+ T cells and CD19+ B cells, and then finally CD4+ T cells. Early CD4+ T-cell recovery occurred at the expense of memory cells, whereas naïve CD4+ T cells rose only 6 months after SCT. (ii) In multivariate analysis, lower recipient age, female gender, high mononuclear cell counts in the graft and absence of CMV reactivation were associated with improved IR after transplant. (iii) A CD4/CD8 ratio less than 0.567 on day 30 post transplantation was associated with a higher overall survival after haplo-SCT in SAA patients.

The rapid establishment of the immune system is critical for reducing post-transplant morbidity and mortality. Currently, the kinetics of IR in patients with SAA after haplo-SCT have only been described for a relatively small number of cases.13, 14, 26, 27, 28 Koh et al.26 depicted CD3+, CD4+ and CD56+ cell reconstitution after haplo-SCT using CD3 or CD3/CD19 depletion conditioning in four SAA patients. Their data showed that three of the four patients achieved CD3+ cell counts of >400 cells/μL and CD4+ cell counts of >150 cells/μL on day 180. Similarly, Ho et al. evaluated CD3+ T, CD4+ T and CD19+ B-cell reconstitution after haplo-SCT by using CD3 depletion conditioning in 12 SAA patients and showed that the median CD3+, CD4+ and CD19+ cell counts at approximately 180 days were 519, 196 and 47 cells/μL, respectively.27 Compared to their data,26, 27 the patients in our study appeared to have more rapid IR in the first year post transplant (Figure 1), which may be the result of a difference in transplant mortality. It was reported that haplo-SCT with in vitro T-cell depletion was associated with delayed IR after transplantation.

In our previous study, we investigated IR in 50 patients who received haplo-SCT, including three patients diagnosed with SAA and 47 patients with malignant hematology diseases.16 However, we did not analyze IR in the three SAA patients alone in that research. Compared to that series of patients with mainly hematological malignancies,16 the reconstitution of T and B cells and subsets, except CD4+ naïve T cells, tended to be faster for the SAA patients in our current study. Another recent study from our group described CD3+, CD4+ and CD19+ cell recovery in SAA patients who received haplo-SCT.10 Compared with those data,10 IR occurred more rapidly in our current study. Several aspects may contribute to these promising results. First, more children were enrolled in the current study, and the median age of the patients was younger than that of the previous cohort. Second, more female patients were enrolled in the current study. As shown in our present study, lower recipient age and female gender were associated with improved IR after transplant (Table 3). Third, our previous study was a multicenter study; different therapeutic strategy may possibly influence IR.

Previous studies showed that the serum immunoglobulin levels usually decreased post transplant, followed by a gradual increase and normalization in a sequential pattern. As reported, initial recovery occurred in IgM levels (2–6 months), followed by IgG levels (3–8 months) and finally IgA levels (6–36 months).14 However, in our study, the IgG levels remained within a normal range during the first 1 month, followed by a gradual decrease, and returned to normal after 3 months. One of the possible reasons was that all of the patients in our study received intravenous IgG therapy for prevention or treatment with viral infection.

The CD4/CD8 ratio, as a routine measure of immune function and response, is genetically controlled in healthy people. The impact of the CD4/CD8 ratio on transplant outcomes has been reported by other studies.29, 30, 31 Data from our previous study showed that patients with a higher CD4/CD8 ratio in a G-CSF-primed bone marrow graft (1.16) had an increased risk of acute grades II–IV and a survival disadvantage.32 Similarly, Huttunen et al.30 reported that a higher early CD4/CD8 T-cell ratio was associated with the increased incidence of acute grades II–IV GVHD. In the current study, we found that a lower CD4/CD8 ratio on day 30 post transplant was associated with decreased treatment-related mortality and improved overall survival after haplo-SCT in SAA patients. However, we did not find any link between the CD4/CD8 ratio and aGVHD. The biological basis for this was not clear, and a further study may be warranted to clarify the involvement of the CD4/CD8 ratio in acute GVHD.

Our study had several limitations. First, our data were limited by single-center retrospective analyses, and IR data could not be acquired at each time point post transplant. Therefore, a prospective, multicenter study is needed to confirm the results observed in this study. Second, the nature killer cells and other subsets of T cells were not analyzed in this study.

In summary, we provided the kinetics for IR in SAA patients who received haplo-SCT. In general, our study demonstrated that the recovery of monocyte and CD8+ T cells was fast in SAA patients, whereas the recovery of the CD4+ T-cell subset was delayed. In addition, our data suggested that the CD4/CD8 ratio may be useful for predicting transplant outcomes in SAA patients after they complete haplo-SCT. Our results may be useful for making better predictions and modulating the IR of SAA patients, which would subsequently improve the outcomes after transplantation.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 81370666 and No. 81530046). The authors thank every faculty member of Peking University People’s Hospital, Institute of Hematology who has participated in this study.

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Correspondence to Y-J Chang or X-J Huang.

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