The impact of CD4+Foxp3+ Treg on immunity to murine cytomegalovirus after bone marrow transplantation depends on the peripheral or thymic source of T cell regeneration
Introduction
Graft-versus-host disease (GVHD) and infectious complications are major factors contributing to morbidity and mortality following allogeneic hematopoietic stem cell transplantation [1], [2]. Since GVHD is mainly caused by donor-derived T cells recognizing genetically disparate host cells, an effective method to prevent GVHD is T cell depletion (TCD) of the graft. However, TCD may also ameliorate the beneficial graft-versus-leukemia (GVL) effect and results in a prolonged period of T cell lymphopenia [3], which is an important risk factor for viral reactivations, disease and treatment-related mortality [4], [5]. The recovery of virus-specific T cells has been shown to be pivotal for the control of cytomegalovirus (CMV)- and Epstein–Barr virus infection and disease [6], [7], [8], [9].
One of the more promising pre-clinical treatment modalities for GVHD is the adoptive transfer of regulatory T cells (Treg) [10], [11]. Treg are essential for the induction and maintenance of peripheral immunological tolerance and are characterized as CD4+CD25+Foxp3+ T cells. In vitro, Treg suppress the activation, proliferation and cytokine secretion of conventional T cells [12]. Accumulating data in several experimental bone marrow transplantation (BMT) models demonstrate the critical role of Treg in dampening GVHD and facilitating engraftment. Depletion of Treg from the graft or in vivo depletion of CD25+ T cells of the recipient before BMT augments GVHD [13], [14], [15]. In contrast, adoptive transfer of Treg at the time of BMT ameliorates GVHD [13], [14], [15], [16] and prevents bone marrow graft rejection [16]. As the suppressive effect of Treg is antigen non-specific [17], concerns have been raised about their possible bystander suppression on GVL and anti-viral immunity after SCT. Murine studies show that adoptive transfer of Treg in host mice with leukemia can ameliorate the development of GVHD while concurrently preserving GVL effects [18], [19], [20]. However, little is known about the impact of adoptively transferred Treg on anti-viral immunity after SCT. Opportunistic human CMV infection remains an important infectious complication following SCT [21], [22] and the murine cytomegalovirus (mCMV) model has served as an excellent model for T-cell mediated immunity [23], [24]. As the anti-viral immunity critically depends on the kinetics of T-cell reconstitution, we assessed the effects of Treg on both sources of T cell regeneration after BMT, i.e. lymphopenia-induced proliferation (LIP) of mature T cells and thymopoiesis. In addition we assessed the effects of Treg on T cell-mediated immunity against murine cytomegalovirus (mCMV). We show that adoptive transfer of CD4+CD25+Foxp3+ Treg suppresses LIP of T cells, while de novo generation of T cells in the thymus after BMT appears unaffected. Consequently, T cell-mediated immunity against mCMV is not altered by Treg when thymopoiesis provides the main source of T cell reconstitution.
Section snippets
Mice
Balb/c mice were purchased from Charles River Laboratory. Rag-2−/− common cytokine gamma chain (γc)−/− mice on a mixed background (provided by Dr. H Spits, Netherlands Cancer Institute, Amsterdam) were inbred on a Balb/c background. Wild-type Balb/c-Thy1.1 mice were provided by Dr. Busch (Munich, Germany). Mice were bred under specific pathogen-free conditions at the Erasmus MC Experimental Animal Centre. Mice were used at 10–12 weeks of age. Food and water were available ad libitum. Housing,
CD4+CD25+Foxp3+ Treg remain suppressive following in vitro expansion and inhibit LIP in a dose-dependent manner
As it was previously shown that CD4+CD25+ Treg, in contrast to CD4+CD25- non-Treg, can inhibit LIP of T cells [27], [28], [29], [30], [31], we first wanted to determine the effect of Treg on LIP of T cells in our model in the absence of mCMV. To generate sufficient number of Treg for adoptive transfer, CD4+CD25high Treg were purified and subsequently expanded using αCD3/αCD28 beads and high concentrations of exogenous IL-2 [32]. After enrichment and expansion, 90% of the cells co-expressed CD4
Discussion
Regulatory T cells are considered candidates for immunotherapy after BMT as they may reduce GVHD while maintaining GVL effects [18], [19], [20]. However, concerns have been raised about their possible bystander effect on infectious immunity after BMT. Infectious immunity correlates with T cell recovery after transplantation [5], [6], [7], [8], which may occur via two independent production routes, i.e. de novo generation of T cells in the thymus and LIP of mature T-cells. To distinguish the
Authorship and disclosures
MB and EJW designed the study, performed the laboratory work, performed the statistical analysis and wrote the paper. BL wrote the paper. JJC designed the study and wrote the paper. EB was the principal investigator and takes primary responsibility for the paper. All authors read and approved the final manuscript. The authors reported no potential conflicts of interest.
Role of the funding source
This work was financially supported by the Landsteiner Foundation for Blood Transfusion Research (grant no. 0221) and by the Dutch Cancer Society “Koningin Wilhelmina Fonds” (grant 2002-2694). The sponsors had no role in the study design, collection/analysis and interpretation of data, in writing the report and the decision to submit the paper for publication.
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MB and EJW contributed equally to the study.