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Monocyte subsets in bone marrow grafts may contribute to a low incidence of acute graft‐vs‐host disease for young donors

Abstract Young donors are associated with a lower cumulative incidence of acute graft‐vs‐host disease (aGVHD) after allogenic haematopoietic stem cell transplantation (allo‐HSCT) than old donors. Although grafts are harvested from healthy donors, it is unclear whether donor age is associated with aGVHD occurrence owing to its effect on cell compositions in grafts. Moreover, the differences in monocyte subsets in grafts between young and old donors and the association between monocyte subsets in bone marrow (BM) grafts and aGVHD remain to be elucidated. In the current study, non‐classical monocytes and the CD4+/CD8+ T cell ratio were remarkably decreased in BM grafts in donors <30 years old. Multivariate analysis further revealed that the level of non‐classical monocytes in BM grafts (≥0.31 × 106/kg) was an independent risk factor for the occurrence of II‐IV aGVHD. In summary, our data indicate that non‐classical monocytes in BM grafts may help identify patients at high risk for aGVHD after allo‐HSCT. Although further validation is required, our results suggest that the low level of non‐classical monocytes and a low ratio of CD4+/CD8+ T cell in BM grafts may be correlated with the lower incidence of aGVHD in young donors.


| INTRODUC TI ON
Allogeneic haematopoietic stem cell transplantation (allo-HSCT) provides a potential curative therapy for patients with haematological diseases. However, acute graft-vs-host disease (aGVHD) remains a major complication after allo-HSCT. [1][2][3] The consensus for donor selection suggests that young donors are a better choice for patients, as they are associated with a lower incidence of aGVHD after allo-HSCT than old donors. [4][5][6] Several studies in HLA-matched transplants have shown a lower incidence of aGVHD using grafts from young donors. 7,8 The impact of donor age has been confirmed in the setting of haploidentical stem cell transplantation (haplo-SCT). 6 Wang et al reported a lower incidence of aGVHD associated with young donors (<30 years old) in haplo-SCT based on immune tolerance induced by granulocyte colony-stimulating factor (G-CSF) and antithymocyte globulin (ATG). 9 González-Vicent et al demonstrated a lower incidence of aGVHD after T cell-depleted haplo-SCT when using grafts from younger donors (<40 years old). 10 Nevertheless, the underlying reason why young donors are associated with a lower incidence of aGVHD is still unknown.
The pathogenesis of aGVHD is commonly believed to be caused by exaggerated and undesirable immune responses in which there is a complex interplay between the donor cells and recipient cells. It has been reported that the different cell compositions in donor grafts are involved in the pathogenesis of aGVHD. [11][12][13][14] The increased ratio of CD4 + /CD8 + T cells in donor bone marrow (BM) grafts is often utilized as a biomarker for a high incidence of aGVHD. Moreover, our recent study reported that an imbalance in macrophage polarization in donor BM grafts, characterized by a high M1/M2 macrophage ratio, exhibited a high incidence of aGVHD. 15 These studies suggest that the cell compositions in donor grafts may help to identify patients who are at high risk for aGVHD.
Given that grafts are harvested from healthy donors, donor age has been reported to be associated with the cell compositions in donor grafts. Yakoub-Agha et al reported that CD8 + -naïve T cells in grafts are negatively associated with donor age, whereas the ratio of CD4 + /CD8 + T cells and CD8 + effector memory T cells in grafts are positively associated with donor age. 16 Furthermore, a high percentage of CD14 + monocytes was reported in grafts of young donors. 17 In humans, circulating monocytes are classified into three subsets: classical, intermediate and non-classical monocytes. 18,19 Classical monocytes are highly phagocytic and are important scavenger cells. Intermediate monocytes have antigen presentation and angiogenesis functions. Non-classical monocytes demonstrate proinflammatory behaviour and secrete inflammatory cytokines in response to infection. In this regard, the imbalance in monocyte subsets has been reported to play a critical role in the occurrence and development of many inflammatory disorders. These findings suggest that the imbalance in monocyte subsets is a promising predictor for risk stratification in inflammatory diseases. [20][21][22][23][24][25][26] However, the differences in monocyte subsets between young and old donors and the association between monocyte subsets in BM grafts and aGVHD remain to be elucidated. Therefore, the current study was performed to determine whether donor age is associated with aGVHD occurrence owing to its effect on cell compositions in BM grafts. Our aim was to provide new insights into why young donors are a better choice for patients undergoing allo-HSCT than old donors.

| Patients and their healthy donors
A total of 83 patients who underwent allo-HSCT and their own healthy donors were enrolled at Peking University People's Hospital. The donor cohort comprised 59 males and 24 females, aged 16-63 years old (median, 39 years old). As shown in Table 1, the enrolled donors were designated into young (age < 30 years), middleaged (30 years ≤ age≤45 years) and old (age > 45 years) donor groups.
Blood cell counts including white blood cell(WBC), neutrophils, lymphocytes and monocytes in peripheral blood (PB) of healthy donors are analysed at three-time points: before G-CSF mobilization, before G-CSF-mobilized BM (G-BM, on the fourth day after G-CSF mobilization) harvesting and before G-CSF-mobilized peripheral blood (G-PB, on the fifth day after G-CSF mobilization) apheresis. Most of the characteristics including the underlying diseases of their related patients showed no significant differences among the three donor age groups, whereas the lymphocyte counts were significantly lower in middle-aged donor group (Table 1). Subsequently, the effect of the monocyte subsets in BM grafts on the occurrence of aGVHD was evaluated.
The current study was approved by the Ethics Committee of Peking University People's Hospital, and written informed consent was obtained from all patients and donors in compliance with the Declaration of Helsinki.

| Transplantation protocols
Donor selection, conditioning therapy, graft harvesting and the prevention of GVHD have been described previously. [27][28][29] Donors were ranked based on the best HLA match, age (younger preferred) and donor-recipient sex (same preferred). Donors were injected subcutaneously with G-CSF at 5 μg/kg daily for five consecutive days. For haplo-SCT, recipients were treated with a modified busulfan/cyclophosphamide plus ATG regimen before the infusion of unmanipulated G-BM and G-PB. GVHD prophylaxis was performed with cyclosporine, mycophenolate mofetil and short-course methotrexate. All transplantation recipients received cyclosporine A (CsA), mycophenolate mofetil (MMF) and short-term methotrexate (MTX) as GVHD prophylaxis. The dosage of CsA was 2.5 mg/kg/d IV from day 9 until bowel function returned to normal, at which point, patients were switched to oral CsA. Every 12 hours, 0.5 g of MMF was administered orally from day 9 and was discontinued after engraftment. MTX was administered intravenously at 15 mg/m 2 on day 1 and then at 10 mg/m 2 on days 3, 5 and 11 in haplo-SCT.

| Clinical definitions and assessments
aGVHD was diagnosed and graded based on clinical symptoms and/ or skin, oral mucosa, liver or gut biopsy, and disease severity was scored using published consensus criteria. [30][31][32] Relapse was defined by morphologic evidence of disease in PB, BM, or extramedullary sites or by the recurrence and sustained presence of pre-transplantation chromosomal abnormalities. Disease-free survival (DFS) was defined as the probability of being alive and free of disease at any point in time, with death or disease relapse considered events.
Overall survival (OS) was defined as the time from transplantation to death from any cause.

| Identification and analysis of cell compositions in donor grafts
Samples from G-BM grafts were labelled with the following mono-
*P-value between young and middle-aged donors.
**P-value between young and old donors.
***P-value between middle-aged and old donors.

| Characterization of monocyte subsets
As previously described, 19,33,34 classical monocytes, intermediate monocytes and non-classical monocytes were identified as CD14 high CD16 − , CD14 + CD16 + and CD14 + CD16 high , respectively. The relative frequencies of these monocyte subsets are expressed as a fraction of the CD14 + monocyte subset. Samples from G-BM grafts were labelled with CD14 and CD16 for monocyte subset analyses.
Multiparameter flow cytometric analyses were performed using a BD LSRFortessa cell analyser (BD Biosciences). The data were analysed using BD LSRFortessa software (BD Biosciences). The absolute numbers of monocyte subsets in BM grafts were calculated as the percentages of these cells multiplied by the percentages of total CD14 + cells multiplied by the total nucleated cell and divided by the actual patient weight to calculate the numbers of cells per kilogram. P-values < .05 were considered statistically significant.

| The percentages and numbers of classical and non-classical monocytes in BM grafts were different among young, middle-aged and old donors
The representative gating strategy for classical, intermediate and nonclassical monocytes in BM grafts is shown in Figure 1A. The enrolled donors were designated into young (age < 30 years), middle-aged were significantly higher in old group.

| Different immune cell subsets in BM grafts among donors of different ages
The number of lymphocytes ( Figure 2B than in young group. Therefore, the ratio of CD4 + /CD8 + T cells in BM grafts was highest in old donor group among the three age groups.

| WBC counts before G-CSF mobilization predicted the percentages of classical and nonclassical monocytes in BM grafts
Positive correlations were demonstrated between WBC counts before G-CSF mobilization and the percentage of classical monocytes ( Figure 2G; r = .23 (95% confidence interval (CI), 0.02, 0.43); P = .03).
However, inverse correlations were found between WBC counts be-

| Donor age was independently correlated with monocyte subsets and CD4 + /CD8 + T cells in BM grafts
To clarify the relationship between donor characteristics and monocyte subsets, the ratio of CD4 + /CD8 + T cells in BM grafts, donor age, sex, weight, WBC counts, neutrophils, lymphocytes and monocytes was analysed with univariate and multivariate analyses.
As shown in

| Percentages and numbers of classical, intermediate and non-classical monocytes in BM grafts of grade II-IV aGVHD patients
As shown in Table 3, most of the demographic and clinical characteristics showed no significant differences between patients with grade 0-I aGVHD and those with grade II-IV aGVHD.

| The monocyte subsets in BM grafts were associated with the incidence of grade II-IV aGVHD but did not have a significant influence on relapse or survival
The showed no significant differences between the different monocyte subsets groups.

| Non-classical monocytes in BM grafts were an independent risk factor for the occurrence of grade II-IV aGVHD
As shown in Table 4, the association between donor characteristics and the occurrence of grade II-IV aGVHD was analysed with a univariate analysis. The percentage of classical monocytes in BM grafts was negatively correlated with the incidence of grade II-IV aGVHD.
However, the percentage of non-classical monocytes in BM grafts was positively correlated with the incidence of grade II-IV aGVHD.
a To avoid potential confounding factors, logistic regression was assessed for interaction terms with covariates. The variables included in the logistic regression analyses exhibited P < .10 after univariate analyses. The final multivariate models were constructed using a forward stepwise selection approach.
*The continuous variables were compared using the Mann-Whitney U test, and the differences between the two groups were compared using the chi-square test. The criterion for statistical significance was P < .05. In summary, the current study shows that donor age is positively correlated with the percentage of non-classical monocytes and the ratio of CD4 + /CD8 + T cells, whereas negatively correlated with the percentage of classical monocytes in BM grafts. Moreover, our data indicate that non-classical monocytes in BM grafts may help to identify patients who are at high risk for aGVHD after allo-HSCT.

TA B L E 3
Although further validation is required, our results suggest that the low level of non-classical monocytes and a low ratio of CD4 + /CD8 + T cell in BM grafts may be correlated with the lower incidence of aGVHD in young donors.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.  .04

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Abbreviations: aGVHD, acute graft-vs-host disease; allo-HSCT, allogeneic haematopoietic stem cell transplantation;CI, confidence interval; HR, hazard ratio. a To avoid potential confounding factors, multivariate Cox proportional hazard models were assessed for interaction terms with covariates. The variables included in the Cox models exhibited P < .10 after univariate analyses. The final multivariate models were constructed using a forward stepwise selection approach.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.