Multipotent adult progenitor cells can suppress graft-versus-host disease via prostaglandin E2 synthesis and only if localized to sites of allopriming

Abstract

Multipotent adult progenitor cells (MAPCs) are nonhematopoietic stem cells capable of giving rise to a broad range of tissue cells. As such, MAPCs hold promise for tissue injury repair after transplant. In vitro, MAPCs potently suppressed allogeneic T-cell activation and proliferation in a dose-dependent, cell contact-independent, and T-regulatory cell-independent manner. Suppression occurred primarily through prostaglandin E(2) synthesis in MAPCs, which resulted in decreased proinflammatory cytokine production. When given systemically, MAPCs did not home to sites of allopriming and did not suppress graft-versus-host disease (GVHD). To ensure that MAPCs would colocalize with donor T cells, MAPCs were injected directly into the spleen at bone marrow transplantation. MAPCs limited donor T-cell proliferation and GVHD-induced injury via prostaglandin E(2) synthesis in vivo. Moreover, MAPCs altered the balance away from positive and toward inhibitory costimulatory pathway expression in splenic T cells and antigen-presenting cells. These findings are the first to describe the immunosuppressive capacity and mechanism of MAPC-induced suppression of T-cell alloresponses and illustrate the requirement for MAPC colocalization to sites of initial donor T-cell activation for GVHD inhibition. Such data have implications for the use of allogeneic MAPCs and possibly other immunomodulatory nonhematopoietic stem cells for preventing GVHD in the clinic.

Figure 1

MAPCs potently inhibit allogeneic T-cell proliferation and activation. (A) MLR was performed by mixing B6-purified T cells with irradiated BALB/c stimulators (1:1) and B6 MAPCs (1:10, 1:100). These cultures were pulsed with ³H-thymidine on the indicated days and harvested 16 hours later. Proliferation was determined as a measure of radioactive uptake. MLR reaction was performed as above using BALB/c T cells plus B6 stimulators and BALB/c MAPCs (B), or BALB/c T cells plus B10.Br stimulators and B6 MAPCs (C). FACS analysis of B6 > BALB/c MLR plus B6 MAPCs was performed on the indicated days and gated on CD4⁺ T cells (D) or CD8⁺ T cells (E) in conjunction with activation markers.

Figure 2

MAPC-mediated suppression in vitro is independent of Tregs. B6 > BALB/c MLR culture was performed using purified T cells or T cells that were CD25-depleted and MAPCs at 1:10 ratios. ³H-thymidine was added on the indicated days, and proliferation was measured (A). FACS analysis was performed on day 5 on the non-CD25–depleted (B) and the CD25-depleted (C) MLR cocultures to determine the percentage of CD4⁺FoxP3⁺ T cells.

Figure 3

MAPCs mediate suppression via a soluble factor. B6 > BALB/c MLR plus MAPCs at 1:10 ratio was arranged by placing T cells and stimulators in the lower well of a Transwell insert and MAPCs in the upper chamber, or by placing MAPCs in direct contact with stimulators and responders (A). (B) Supernatant taken from MAPC or control cocultures on day 3 was added in 1:1 ratio with fresh media to a B6 > BALB/c MLR. Results of MAPCs at 1:10 and 1:100 ratios in direct contact with responding T cells are shown for comparison. Proliferation was assessed using ³H-thymidine uptake. Enzyme-linked immunosorbent assay was performed on MLR supernatant harvested on the indicated day to determine the amount of proinflammatory (C) and anti-inflammatory (D) cytokines in culture with MAPCs at 1:10 and 1:100 ratios.

Figure 4

MAPCs inhibit T-cell alloresponses through the secretion of PGE₂. B6 > BALB/c MLR cultures were arranged as before. MAPCs, either untreated, treated overnight with 5 μM indomethacin to inhibit production of PGE₂, or treated with vehicle, were titrated in at 1:10 ratios. Proliferation was assessed as above (panel A).

Figure 5

The capacity of MAPCs to delay GVHD mortality and limit target tissue destruction is dependent on anatomic location of the cells and their production of PGE₂. BALB/c mice were lethally irradiated and then given 10⁶ BM cells from B6 mice on day 0, followed by 2 × 10⁶ purified CD25-depleted whole T cells on day 2. On day 1, mice were given 5 × 10⁵ untreated B6 MAPCs or PBS delivered via IC injections. Kaplan-Meier survival curve is representative of 1 experiment in which BM only and BM plus T group had n = 6, and MAPC group had n = 8 (A). (B) BMT was performed as in panel A, except mice were given PBS or 5 × 10⁵ MAPC IS on day 1. The survival curve is representative of 3 pooled experiments (BM only, n = 18; BM + T, n = 20; MAPCs, n = 26; MAPCs vs BM + T, P < .001). (C) Survival curve representative of 1 experiment in which mice received BMT plus untreated MAPCs or MAPCs pretreated overnight with indomethacin before IS injection (BM only, n = 5; BM + T, n = 5; MAPCs, n = 10; MAPC indo, n = 10; MAPCs vs MAPC indo, P = .002). Tissue taken from cohorts of mice from panel B was harvested on day 21 and embedded in OCT, followed by freezing in liquid nitrogen. Sections (6 μM) were stained with hematoxylin and eosin and analyzed for histopathologic evidence of GVHD. Representative images are shown. (D) Magnification × 200. (E) The average GVHD score for BM only, BM plus T, and BM plus T plus MAPC (IS) cohorts is shown. (F) Spleens were harvested from BMT plus MAPC IS transplanted mice on day 21 and snap frozen in OCT compound. Tissue sections were cut and stained using anti-luciferase and anti-PGE synthase antibodies. Confocal analysis reveals that MAPCs are found in the spleen at this time point and retain their ability to produce PGE₂. (F) Top shows luciferase alone, and bottom shows colocalization of PGE synthase with luciferase.

Figure 6

MAPCs dampen T-cell proliferation and activation within the local environment. In vivo MLR was performed by administering lethally irradiated BALB/c mice with 5 × 10⁵ B6 MAPCs IS (day 0), followed by 15 × 10⁶ B6 CFSE-labeled CD25-depleted T cells (intravenously; day 1). Control mice were given labeled T cells alone plus sham surgeries. Spleens and LNs were harvested on day 4 and analyzed via FACS for CD4 and CD8 expression and percentage of CFSE dilution (A). The proliferative capacity for CD4⁺ and CD8⁺ T cells in the spleen (B) and LNs (C) of transplanted mice was calculated, as previously published. (D-E) Activation markers for CD4⁺ and CD8⁺ T cells in the spleen and LNs were analyzed using FACS and graphed.

Figure 7

MAPCs affect costimulatory molecule expression on T cells and DCs in the spleen. FACS analysis of spleen cells harvested from transplanted mice on day 4 was performed to determine the percentage of CD4⁺ (A), CD8⁺ (B), and CD11c⁺ (C) cells that expressed the indicated costimulatory molecules. In this transplant, MAPCs were untreated or pretreated with indomethacin, as described, before their application.