Index of Bone Marrow Output and Imbalance of B-Lymphocyte Homeostasis before and after Transplantation Correlate Differently with Graft-Versus-Host Disease and Relapse

Introduction

The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT.

Methods

Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients.

Results

Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006).  Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (p<0,05):

-values of regulatory effector memory T-cells at +30, percentage of CD8+TEMRA cells at +90,   values of immature B cells and levels of KRECs at +180 (positive correlation)

-percentage of CD4+ central memory and CD8+ effector memory cells at +90 (negative correlation).

The relapse rate (27%; median time: 5,5 months, range 3-12) was used as clinical index of ineffective GVT. The following cluster of immunological parameters at day +90 correlated positively with relapse: CD8+ effector memory cells, immature B cells, naïve, switched memory B cells, memory double negative (IgD-CD27-) B cells (p=0,006).

Discussion

Different clusters of immunological parameters at different time points were evidenced as predictors of GVHD and ineffective GVT, allowing a clear-cut distinction between these immunological reactions. Changes in pre- and post-transplant B-lymphopoietic microenvironment and specific imbalances in the subset of B-cells may be involved in acute and chronic GVHD development. The atypical association of regulatory T-cells with GVHD may be explained by the relative efficiency of different subsets of regulatory T-cells (naïve>effector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward “senescent” memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies.