Multilineage Engraftment with Minimal Graft-Versus-Host Disease Following In Utero Transplantation of S-59 Psoralen/Ultraviolet A Light-Treated, Sensitized T Cells and Adult T Cell-Depleted Bone Marrow in Fetal Mice

Abstract

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 × 105 C57BL/6 (B6) naive CD3+ cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 × 105 CD4+ or CD8+ cells were used, low levels of engraftment were seen vs recipients of 1.5 × 105 CD3+ cells (2.4 ± 1.1 and 6.6 ± 3.9 vs 20.4 ± 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60–0%), but was also associated with a significant reduction in engrafted donor cells (53.4 ± 4.2 to 1.7 ± 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 ± 2.4% H2Kb+ cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.

Targeted Disruption of the Artemis Murine Counterpart Results in SCID and Defective V(D)J Recombination That Is Partially Corrected with Bone Marrow Transplantation

“…Single cell suspensions were prepared from the thymus, spleen, and bone marrow (BM), and cells were stained with mAbs for flow cytometric analysis on a FACScan (BD Biosciences), as described (17). The conjugated mAbs (BD Pharmingen) were used in combination, as indicated in the figure legends.…”

“…The conjugated mAbs (BD Pharmingen) were used in combination, as indicated in the figure legends. In addition, we evaluated the lymphocyte proliferative response to Con A (5 g/ml), solid-phase anti-CD3 (2 g/ml), and LPS (5 g/ml), as well as NK cytotoxicity, as described (17).…”

“…BM cells were freshly harvested, and several donor cell preparations were used, including whole BM (WBM), T cell-depleted BM (TCDBM), lineage-depleted (lin Ϫ ) BM, and BM that was lin Ϫ and positively selected for Sca-1 (17). TCDBM was prepared using an anti-CD3 mAb and immunomagnetic beads, as previously described (17).…”

Allogeneic T Cells Treated with Amotosalen Prevent Lethal Cytomegalovirus Disease without Producing Graft-versus-Host Disease Following Bone Marrow Transplantation

“…First, amotosalen appeared to stabilize the infused T cell repertoire by preventing clonal expansion without inhibiting T cell function. In the presence of UVA light, 1 nM amotosalen covalently cross-links dsDNA every 10 5 -10 6 bp or 3,000 -30,000 times per cell (26), effectively blocking T cell proliferation (19,27). In the present study, concentrations of 2-20 nM likewise prevented proliferation, but did not inhibit production of the antiviral cytokine IFN-␥ by CD8 ϩ T cells (Fig.…”

“…Consistent with this mechanism, splenocytes from MCMV-immune mice, which have an expanded antiviral compartment including memory CTLs (28), were more effective at preventing viral lethality than lymphocytes from naive mice (data not shown). Other investigators have also noted that amotosalen-treated T cells from immune donors are more effective than cells from naive donors, and that effector CTLs retain potent cytolytic activity following amotosalen treatment (19,27).…”