Comparison of reference values for immune recovery between event-free patients receiving haploidentical allografts and those receiving human leukocyte antigen-matched sibling donor allografts

Abstract

To establish optimal reference values for recovered immune cell subsets, we prospectively investigated post-transplant immune reconstitution (IR) in 144 patients who received allogeneic stem cell transplantation (allo-SCT) and without showing any of the following events: poor graft function, grades II‒IV acute graft-versus-host disease (GVHD), serious chronic GVHD, serious bacterial infection, invasive fungal infection, or relapse or death in the first year after transplantation. IR was rapid in monocytes, intermediate in lymphocytes, CD3⁺ T cells, CD8⁺ T cells, and CD19⁺ B cells, and very slow in CD4⁺ T cells in the entire patient cohort. Immune recovery was generally faster under HLA-matched sibling donor transplantation than under haploidentical transplantation. Results suggest that patients with an IR comparable to the reference values display superior survival, and the levels of recovery in immune cells need not reach those in healthy donor in the first year after transplantation. We suggest that data from this recipient cohort should be used as reference values for post-transplant immune cell counts in patients receiving HSCT.

Keywords

immune reconstitution / hematopoietic stem cell transplantation / event-free patients / reference range

Introduction

Allogeneic stem cell transplantation (allo-SCT) is an effective and sometimes the only curative therapy for patients with hematologic malignancies. Immune reconstitution (IR) is strongly associated with clinical outcomes after allo-SCT, and some approaches to improve post-transplant immune recovery are currently employed. However, no optimal reference values for recovered immune cell subsets after HSCT have been reported, and approaches to enhance post-transplant immune recovery based on data from health donors are limited. Thus, an important question has been raised: what kind of candidates are suitable for interventions or aggressive approaches to improve immune recovery?

Studies have described the kinetics of IR after transplantation, and findings indicate that different immune cells recover at different phases [¹–⁶]. However, most of these studies were retrospective, and nearly all of them have focused on patients with different pre-transplant, peri-transplant, and post-transplant characteristics. Given that graft failure, graft-versus-host disease (GVHD), and serious infections, as well as treatment of these events, exert profound effects on IR, data from these studies could not be used as reference values to evaluate IR.

In the present study, we prospectively investigated the kinetics of post-transplant IR in event-free patients; these patients received allo-SCT but did not experience poor graft function (PGF), II‒IV acute GVHD, serious chronic GVHD, serious bacterial infection, invasive fungal infection (IFI), relapse, or death 1 year after transplantation. Given that the patients included in this study displayed excellent survival after HSCT, the immune cell counts were acceptable and may be used as reference values to evaluate IR at sequential time points after transplantation.

Patients and methods

Patients

This study include patients with hematologic malignancies and who underwent HLA-identical sibling (MSDT) or haploidentical stem cell transplantation (haplo-SCT) between January 2011 and December 2013. A total of 706 consecutive patients participated in this IR study. Full engraftment in all of these patients were determined using chimerism assessment. To assess IR, we excluded patients who experienced transplant-related complications, such as PGF, II‒IV acute GVHD, serious chronic GVHD, serious bacterial infections (especially severe pneumonia), or IFI in the first year after transplantation. Patients who relapsed or died during the first year were also excluded from the analysis. In the end, 144 patients who did not experience any of the events described above were included for immune cell subset testing performed between days 30 and 365 in this study (Fig. 1). In addition, 41 healthy patients were included in an immune analysis performed in a single time point, and data were used as normal control. This study was approved by the Institutional Review Board of Peking University Institute of Hematology.

Transplant procedure

Transplantations were performed as described previously [⁷,⁸]. In brief, patients with HLA-identical sibling donors received myeloablative regimen (modified Bu/Cy regimen), including a combination of cytosine arabinoside (Ara-C; 4 g/(m²∙d), on days -10 and -9), busulfan (3.2 mg/(kg∙d), administered intravenously on days -8 to -6), cyclophosphamide (1.8 g/(m²∙d), on days -5 and -4), and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Me-CCNU, 250 mg/m², once on day -3). For patients with haploidentical donors, the conditioning therapy used was the Bu/Cy regimen and anti-human thymocyte immunoglobulin (ATG, Sang Stat, Lyon, France; 2.5 mg/(kg∙d), administered on days -5 to -2). All of the haploidentical and HLA-identical sibling transplant recipients received a mixture of allografts consisting of granulocyte colony-stimulating factor (G-CSF)-mobilized bone marrow and peripheral blood stem cell harvests. The patients in both transplantation cohorts received the same GVHD prophylaxis regimen consisting of cyclosporine A, mycophenolate mofetil, and short-term methotrexate [⁸].

After allo-HSCT, recombinant human G-CSF was subcutaneously administered in HLA-mismatched transplant recipients at 5 mg/(kg∙d) from day 6 until neutrophil count reached 0.5 × 10⁹/L for three consecutive days. G-CSF was not used after performing allo-HSCT with matched sibling transplants [⁹]. Cytomegalovirus and Epstein–Barr virus levels were monitored, and these infections were treated as described previously [⁹]. Moreover, immunosuppressive therapy was performed as described previously [⁸,⁹].

Immunophenotype analysis

The absolute lymphocyte and monocyte counts were determined by routine blood examination in the clinical hematology laboratory at each of the following time points: 30, 60, 90, 180, and 365 days after transplantation. Immune cell subsets were identified by flow cytometry as described previously [³].

Definitions and evaluation

In this study, an event-free condition is defined as the absence of primary or secondary PGF, II‒IV acute GVHD, serious chronic GVHD, serious infections, proven or probable IFI, and relapse or death in the first year after transplantation. Primary PGF is defined as slow or incomplete recovery of blood counts as indicated by an absolute neutrophil cell count of≤0.5 × 10⁹/L and platelet count of≤20 × 10⁹/L at least 28 days after allo-SCT. Secondary PGF is defined as recurrent pancytopenia at levels satisfying the diagnostic criteria for PGF after successful engraftment in the absence of severe GVHD or hematologic relapse [¹⁰]. Diagnosis and grading of acute and chronic GVHD was assigned by a transplant center according to standard criteria [¹¹,¹²].

Serious infections included bacterial infection of any organ site requiring intravenous therapy and/or hospitalization. Bacterial infections were either proven or presumed based on clinical manifestation, etiological evidence, and response to treatment with antibiotics [¹³–¹⁵]. IFI was defined as proven or probable according to the criteria used by the European Organization for Research and Treatment of Cancer/Mycoses Study Group [¹⁶].

Statistical analysis

The baseline characteristics of the patients were compared using the Mann–Whitney test. Immune cell counts and proportions were summarized as medians and 25th–75th percentiles. Differences in immune cell counts between groups at each time point were compared using the Mann–Whitney test or independent t-test. Univariate and multivariate analyses of pre-transplant factors and immune recovery were performed using the Prentice–Williams–Peterson conditional approach for ordered multiple events. All variables achieving a P-value of<0.1 in univariate analysis were considered for multivariate analysis. For most analyses, P-values of<0.05 (2-tailed) were considered significant. To minimize chances of spurious associations during analysis of factors associated with immune recovery, we considered P-values of<0.005 as significant unless the associations appeared significant for two adjacent time points (P<0.05 for both time points) [⁴]. All analyses were performed using SPSS 22.0 and GraphPad Prism 6.0 software packages.

Results

Patient characteristics and clinical outcomes

A total of 144 patients were evaluated in this study with a median follow-up of 1256 days (range: 392‒1813 days). The median age was 30 years (range: 3–59 years old). Underlying diseases included acute myeloid leukemia (AML, n = 72), acute lymphoblastic leukemia (ALL, n = 44), myelodysplastic syndrome (MDS, n = 12), and chronic myeloid leukemia (CML, n = 16). The MSDT and haplo-SCT modalities were used in 40.97% (59/144) and 59.03% (85/144) of the study population, respectively. Table 1 shows the characteristics of recipients and donors.

In this cohort, six patients relapsed 1 year after transplantation, and the median relapse time was 760 days (range: 420–1234 days). Three patients died, two of them died due to relapse and one due to serious infection. The 141 other patients survived until the end of the follow-up period. The 4-year probabilities of relapse, non-relapse mortality (NRM), leukemia-free survival (LFS), and overall survival (OS) were 5% (95% CI: 1%–9%), 1% (95% CI: 0%–2%), 95% (95% CI: 90%–99%), and 98% (95% CI: 96%–100%), respectively (Fig. 2). The clinical outcomes of late relapse and death were comparable between patients receiving MSDT and haplo-SCT.

Kinetics of IR in event-free patients after allogeneic HSCT

Fig.3 shows that monocytes recovered rapidly, and the recovery level remained higher than normal in the first year after transplantation. Compared with monocytes, total lymphocytes recovered slowly, and their recovery levels were lower than those of healthy donors until 180 days after transplantation. The recovery of CD19⁺ B cells was delayed, and they did not reach 100 cells/µL until 180 days after transplantation. The total CD3⁺ T cell counts were very low in the first 30 days, but they normalized by 90 days after transplantation. CD4⁺ helper T cells recovered very slowly and did not reach 400/µL 1 year after transplantation. CD8⁺ cytotoxic T cells recovered rapidly. The absolute numbers of CD8⁺ T cells were higher after 90 days after transplantation than those in healthy donors. Consequently, a significant inversion of the CD4:CD8 ratio was observed up to 1 year after transplantation. CD4⁻CD8⁻ T cells recovered slowly and did not reach the normal value until 180 days after transplantation. Regular T cells called CD4⁺CD25⁺ T cells recovered slowly and never reached the normal level 365 days after transplantation. Considering that CD28/B7 co-stimulation plays a significant role in T cell alloreactivity, we investigated the expression of CD28/B7 co-stimulatory molecules on recovered CD4⁺ and CD8⁺ T cells. As shown in Fig. 3, the number of CD4⁺CD28⁺ T was lower compared with that of healthy donors in the first year after transplantation. Moreover, CD28 expression was higher in CD8⁺ T cells than in healthy donors from day 60 to day 365 after transplantation.

Factors influencing IR

To provide reference values that can be used for all recipients, we investigated the effects of recipients’ age, gender, and underlying disease on IR in this cohort of event-free patients.

Monocytes

In univariate analysis, no association was found between age, gender, or underlying disease of the recipients and monocyte recovery, except that adult recipients showed faster monocyte recovery 30 days after transplantation. The reconstitution of monocytes was comparable between recipients after MSDT and haplo-SCT on days 30, 90, and 180 after transplantation. When considering multivariate analyses, recipient gender and haplo-SCT modality no longer exerted a definitive effect on monocyte recovery.

Total lymphocytes

Univariate analysis showed that the total lymphocyte counts were significantly higher in adult recipients than in pediatric cases 30, 60, and 180 days after transplantation. We found no effect of gender on IR for lymphocytes, except that female recipients showed higher lymphocyte counts on days 90 and 180 than the male recipients. Within the first 90 days after transplantation, lymphocytes recovered faster in patients after MSDT than those who received haplo-SCT. This trend continued until 1 year after transplantation, but the difference was no longer significant. Multivariate analysis demonstrated the effects of haplo-SCT on total lymphocyte recovery from days 30 to 90 (Table 2).

B lymphocytes (CD19⁺ B cells)

Univariate analysis showed that the absolute counts of CD19⁺ B cells were higher in adult recipients than in pediatric cases on days 30 and 90 after transplantation. Patients in the MSDT group experienced faster reconstitution of CD19⁺ B cells than those in the haplo-SCT group during the first year after transplantation. Multivariate analysis showed that the haplo-SCT modality was the only factor affecting CD19⁺ B cell recovery from days 30 to 180 (Table 2).

CD3⁺T cells

Compared with that in the adult cohort, CD3⁺ T cell recovery was delayed in the child cohort on days 30, 60, and 180 after transplantation. The recovery of CD3⁺ T cells from days 30 to 180 was affected by the recipients’ gender. In addition, reconstitution of CD3⁺ T cells was significantly delayed in haploidentical recipients compared with that in the HLA-identical recipients in the first 90 days after transplantation. Multivariate analysis showed that the haplo-SCT modality was the only factor that influenced CD3⁺ T cell recovery from days 30 to 90 (Table 2).

Helper T lymphocytes (CD3⁺CD4⁺ T cells)

In univariate analysis, CD4⁺ T cells recovered faster in adults than in children within the first 6 months after transplantation. Female recipients showed faster CD4⁺ T cell recovery from days 60 to 360. Patients in the MSDT group showed significantly faster CD4⁺ T cell recovery than those in the haplo-SCT group within the first year after transplantation. Multivariate analysis confirmed the effects of haplo-SCT modality and recipients’ gender on reconstitution of CD4⁺T cells (Table 2).

Cytotoxic T lymphocytes (CD3⁺CD8⁺ T cells)

Comparable CD8⁺ T cell counts between pediatric and adult patients were obtained, except that CD8⁺ T cell counts were higher in adult cases on day 60. The absolute counts of recovered CD8⁺ T cells were lower in haploidentical recipients than in HLA-identical sibling recipients within the first 60 days after transplantation. Multivariate analysis showed that the haplo-SCT modality was the only factor affecting CD8⁺ T cell recovery on days 30 and 60 after transplantation (Table 2).

CD4:CD8 ratio

The CD4:CD8 ratio was higher in adult recipients than in pediatric cases 90 days after transplantation. The univariate analysis revealed the effect of the underlying disease on CD4:CD8 ratio. Given the slow recovery of CD4⁺ T cells in the haploidentical cohort, the CD4:CD8 ratio was significantly lower in the haplo-SCT group than in the MSDT group 1 year after transplantation. Multivariate analysis showed that the haplo-SCT modality was the only factor affecting the CD4:CD8 ratio on day 30 and from days 90 to 365 following transplantation (Table 2).

Reference values for immune cell subset counts post-transplant

Based on the above-mentioned analysis, the haplo-SCT modality was confirmed to be associated with immune recovery. The reference values for the recovered immune cells and their subsets were calculated and listed in Table 3 and Fig. 4 according to modality.

Discussion

Compared with previous studies, the present study evaluated the kinetics of IR after allo-SCT in event-free patients who displayed excellent survival. The main findings of the current study are: (1) immune recovery is fast for monocytes, intermediate for lymphocytes, CD3⁺ T cells, CD8⁺ T cells, and CD19⁺ B cells, and very slow for CD4⁺ T cells in the entire patient cohort; (2) all immune cell subsets, except monocytes, recovered faster after MSDT than after haplo-SCT; and (3) reference values were obtained for recovered immune subsets in event-free patients following MSDT or haplo-SCT. Our study suggests that to achieve promising survival rates, the recovery of immune cells need not reach the recovery levels for healthy donor in the first year after transplantation. Furthermore, patients with IR comparable to the reference values displayed superior survival.

Studies have reported that early recovery of total monocytes and lymphocytes is associated with better outcomes [¹⁷–²⁴]. DeCook et al. [¹⁸] showed that absolute monocyte counts with a cutoff value of 300 cells/µL on day 100 were independently associated with survival (risk ratio: 0.22, 95% CI: 0.07–0.73, P = 0.01). In the present prospective study, we found that total monocyte counts were consistently higher than 400 cells/µL in the first year after transplantation, indicating that recovery of monocytes to the reference values resulted in superior survival. Powles et al. [²³] found that rapid lymphocyte recovery of≥0.2 × 10⁹/L on day 29 after MSDT appeared to be associated with a lower risk of relapse in AML patients. Savani et al. [²¹] analyzed 157 patients with hematologic malignancies and received MSDT, and they concluded that rapid lymphocyte recovery on day 30 (>450 cells/µL) is associated with improved survival (71%±5% versus 38%±6%, P<0.0001), reduced relapse (21%±5% versus 44%±7%, P<0.009), reduced NRM (9%±3% versus 36%±6%, P<0.0001), and reduced acute GVHD (34%±5% versus 51%±6%, P<0.025). Furthermore, Kim [²⁵] and colleagues conducted a retrospective study of 1109 adult patients who underwent allo-SCT (41% HLA-matched related recipients, 51% HLA-matched unrelated recipients, and 8.5% HLA-mismatched recipients). Regardless of time point (1, 2, or 3 months after HSCT), a high absolute lymphocyte count (>200 cells/µL) was associated with superior OS. Our previous data showed that patients with a higher absolute lymphocyte counts (≥300 cells/µL) on day 30 showed improved OS and LFS after haplo-SCT [²²]. In this study, event-free patients showed early recovery of lymphocytes (median total lymphocyte count on day 30: 557.60 cells/µL in MSDT and 289.20 cells/µL in haplo-SCT). Moreover, our data confirmed our previous observations indicating the importance of lymphocyte recovery as a prognostic factor in transplant outcome; more importantly, they confirm that the reference range for lymphocytes provided by our study was acceptable and reliable.

Studies suggest that rapid recovery of CD4⁺ T cells is strongly correlated with better outcome [²⁶–³⁰]. Berger et al. [²⁹] assessed the kinetics of lymphocyte subset recovery in 758 allo-SCT recipients (502 MSDT recipients and 256 family mismatched or unrelated recipients); they found that CD4⁺ T cell counts of>86 cells/µL on day 35 protected patients from transplant-related mortality. In our current study, the median CD4⁺ T cell count on day 30 was 72.53 cells/µL, slightly lower than that reported by Berger et al. [²⁹]. This difference may be explained by the following. First, more haploidentical recipients were included in our study. Second, the evaluation time was 5 days earlier in our study. Kim et al. [³⁰] reported that CD4⁺ T cell reconstitution at 3 months is strongly correlated with OS, NRM, and opportunistic infections at the cutoff value of 200 cells/µL after allo-SCT (66 HLA-matched SCT recipients and three partially mismatched SCT recipients). In our cohort, the median CD4⁺ T cell count in HLA-matched recipients was 281.68 cells/µL at 3 months. Our results suggest that patients with CD4⁺ T cell reconstitution comparable to the reference values provided in our current study will display superior transplant outcomes.

Rapid recovery of CD8⁺ T cells improves survival in different transplant settings [³¹]. As reported by Giannelli et al.[³²], significantly prolonged OS was observed in patients regaining the 10th and 50th percentile of age-adjusted, normal, absolute CD8⁺ T lymphocyte counts. Data from our center (in revision) showed that CD8⁺ T cell reconstitution 3 months after haploidentical transplantation is associated with infection rate and OS at a cutoff value of 375 cells/µL. In our current study, an extremely high CD8⁺ T cell count (684 cells/µl) was observed at 3 months in event-free patients, confirming that the reference range for CD8⁺ T cells provided in our study is acceptable and reliable.

Overall, the available data suggest that patients with IR comparable to the reference values provided in our current study will display superior survival. Therefore, identification of candidates suitable for interventions or aggressive approaches to improve immune recovery based on the reference values, instead of using data from healthy donors, may have practical significance given that the post-transplant status of immune system varies between patients and healthy donors.

This study has some limitations. First, we only analyzed the main immune cells in this study; other immune cells, such as natural killer cells, were not included in the analysis. Second, this is a single-center study and the sample size may not be sufficiently large to determine a reference range for all transplant platforms. However, we suggest that the ideal immune reconstitution for each transplant platform should be determined, and a multicenter study that includes a larger number of cases is warranted to establish a standard reference range for all patients receiving different modalities of transplantation. Third, an association may be found between recipient age, gender, or underlying diseases and IR when more cases are included, and we should provide reference values for IR based on multiple pre-transplant factors.

In summary, our study is the first to investigate the kinetics of IR in event-free patients. By analyzing factors influencing IR, we classified the patients into two groups and provide immune cell subset counts at sequential time points after transplantation based on haplo-SCT modality. Our results suggest that patients with IR comparable to the reference values display superior survival, and the immune cells may not have to recover to healthy donor levels in the first year after transplantation. Although this study is possibly insufficiently large to determine a reference range for all transplant platforms, we suggest that each transplant center should determine the ideal immune reconstitution, and a multicenter study that includes a larger number of cases is warranted to establish a standard reference range for all patients receiving different modalities of transplantation.

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