Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia

Abstract

Background: Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood.

Design and methods: The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor.

Results: Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro.

Conclusions: Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.

Figure 1.

IL-10 production by T cells of transplanted thalassemic patients with persistent mixed chimerism (PMC). (A) Cytokine production by PBMC of patients with PMC (n=7), patients with complete chimerism (n=5), and normal donors (n=8), was determined by intra-cytoplasmic staining after polyclonal activation with phorbol myristate acetate/ionomycin. The percentages of IL-10-producing T cells within the gated CD4⁺ T cells for each patient and normal donors (●), and the corresponding averages (–) are indicated in the graph. (B) IL-10-producing cells of a patient with PMC and of a normal donor were counted by ELISPOT. The IL-10-positive spots were measured in the unstimulated cultures and after anti-CD3/CD28 stimulation.

Figure 2.

Kinetics of engraftment of a patient with PMC 8 years after HSCT. (A) Long-term stable mixed chimerism was evaluated by short tandem repeat analysis in the peripheral blood and in the bone marrow of the patient. High levels of the adult donor β-globin were found at all time points, as determined by high performance liquid chromatography. (B) Mixed chimerism was evaluated in PBMC and in different lymphoid sub-populations (CD4⁺, CD4⁻, CD3⁺, CD19⁺, CD56⁺) isolated from the peripheral blood of the PMC patient, at three different time points: 76 months, 91 months and 101 months after HSCT. Chimerism in the erythroid compartment was determined 91 and 101 months following the transplant. (C) Average values of IL-4, IL-10 and IFN-γ produced by Tr1 cell clones of the PMC patient (light gray columns for the first T-cell cloning [n=20], dark gray columns for the second [n=11]) and of the normal donor (black columns [n=7]). (D) Intracellular cytokine production by Tr1 cell clones of the patient with PMC, following TCR-mediated polyclonal activation. Two representative Tr1 cell clones from the second T-cell cloning of the patient are shown. (E) Intracellular expression of granzyme-A (black line) and granzyme-B (gray line) in the patient’s T-cell clones. Filled histograms represent the isotype control. The mean fluorescence intensity (MFI) for granzyme-A and granzyme-B is shown. Three representative Tr1 cell clones (top panel) and three Th cell clones (bottom panel) are shown.

Figure 3.

Characterization of T-cell clones obtained in a patient with PMC. T-cell clones were established from PBMC of the PMC patient (pt PMC) at two different time points (76 and 91 months after HSCT), of a tha-lassemic patient prior to transplantation (Pt PRE), and of a normal donor (ND). Based on their cytokine production upon TCR-mediated polyclonal stimulation, T-cell clones were distinguished into Th0, Th1, Th2, and Tr1. (A) The percentages of Th and Tr1 clones are shown for the PMC patient (light gray columns for the first T-cell cloning, dark gray columns for the second), for the normal donor (black columns), and for the thalassemic patient before HSCT (white columns). (B) Cytokine production by T-cell clones obtained from the first (top panel) and the second (middle panel) T-cell cloning of the PMC patient and from the normal donor (bottom panel). The absolute IL-10 production is reported on the x-axis, whereas the y-axis shows the ratio between IL-10 and IL-4 production (left panel), and between IL-10 and IFN-γ production (right panel). White circles represent Tr1 cell clones; black circles indicate all the other subsets of Th cell clones.

Figure 4.

Allo-antigen specificity of the PMC patient’s T-cell clones. Cytokine production profile of the patient’s host/donor-derived T-cell clones in response to allogeneic donor/host or third party mature dendritic cells. (A) Host-derived T-cell clones were stimulated with donor-derived mature dendritic cells (top panel) and with third party mature dendritic cells (bottom panel). (B) Donor-derived T-cell clones were stimulated with host-derived mature dendritic cells (top panel) and with third party mature dendritic cells (bottom panel).

Figure 5.

Suppressive activity of host and donor Tr1 cell clones. Responder cells were activated with anti-CD3 and anti-CD28 monoclonal antibodies in the presence or absence of Tr1 cell clones, and the supernatant was collected after 5 days of stimulation. Different responder cells were tested: recipient PBMC and donor PBMC (A), a host-derived Th cell clone and a donor-derived Th cell clone (B). The ability to suppress IFN-γ production by responder cells of host and donor-derived Tr1 cell clones was evaluated. The percentage of inhibition of IFN-γ production was calculated as follows: [(amount of cytokine by activated responder cells-amount of cytokine by activated responder cells in the presence of T-cell clones)/amount of cytokine by activated responder cells]*100”. (C) Patient’s PBMC labeled with CFSE were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies in the absence or presence of Tr1 cell clones at a 1:1 ratio. After 5 days of cell culture, the proliferation of responder cells was determined by flow cytometry analysis. (D) PBMC of the PMC patient and of a normal donor (ND) were stimulated with host, donor and third party mature dendritic cells (mDC) in the presence or absence of anti-IL10R monoclonal antibody. Proliferative responses were evaluated after 4 days of culture by adding ³H-thymidine for an additional 16 h. The increased proliferation in the presence of anti-IL10R monoclonal antibody is indicated above each histogram. The results from one representative normal donor out of two tested are shown.