We previously demonstrated that the C57Bl/6 mArt-/-
mouse model accurately reflects Artemis-deficient SCID in humans in that it is T and B cell deficient, non-leaky, has normal NK cell numbers and function, and is sensitive to ionizing radiation [17
]. It also compares to Artemis-deficient children in that the T cell but not the B cell defect can be corrected with a congenic HSCT, while allogeneic mismatched grafts (using BALB/c donors) are rejected when conditioning therapy is not used [17
In this study we found that when allogeneic mismatched HSC were co-injected with donor T cells that previously had been sensitized to recipient antigens and treated with psoralen and UVA light to prevent proliferation significant multilineage engraftment was achieved with full T and B cell reconstitution. Furthermore, in secondary transplants we showed that lethally irradiated wild type recipients could be reconstituted with marrow from primary engrafted mice, and we confirmed that multilineage engraftment was due to engraftment of donor hematopoietic stem cells.
We found that when PCT-4 STC or anti-NK1.1 mAb was employed independent of each other, there was limited engraftment of allogeneic mismatched HSC. The reason for the lack of efficacy of anti-NK1.1 mAb alone may be explained by the fact that there are sufficient niches for no more than a limited number of donor cells to engraft, even when NK-mediated graft resistance is reduced. It is also possible that because only about 60% of NK function was eliminated by anti-NK1.1 mAb treatment alone, graft resistance was not suppressed sufficiently. Interestingly, injecting higher doses of anti-NK1.1 mAb into control animals resulted in significant mortality but only modest additional reduction in cytotoxic function (data not shown). We speculate that PCT-4 STC has some anti-recipient NK cell activity, and when it is combined with anti-NK1.1 mAb treatment, there is sufficient suppression of graft resistance to allow robust engraftment.
Our in vitro
experiments showed that the PCT-4 sensitized BALB/c CD8a+
T cells directly target B6 HSC. However, when we tried CD8a+
PCT-4 STC in vivo
, there was no effect on engraftment (data not shown), comparable to what we previously saw in an in utero
transplant model using wild type mice [18
]. We also performed a competitive repopulation experiment to confirm that the co-injection of donor PCT-4 STC with HSC would target niches in the recipient marrow. Importantly, B6 mArt-/-
bone marrow that had been previously exposed in vivo
to PCT-4 STC 24 hours earlier and then injected into irradiated F1 recipients resulted in significantly reduced B6 HSC engraftment compared to B6 mArt-/-
bone marrow not exposed to PCT-4 STC. The difference suggested that PCT-4 STC can target host HSC and make niches in bone marrow.
When we evaluated varying doses of PCT-4 STC we found that the optimal dose for T, B and granulocyte engraftment was 4×105
cells. At the lowest dose (1×105
cells) of PCT-4 STC, we found minimal engraftment, possibly due to insufficient generation of niches in recipient bone marrow. Interestingly, at the highest dose (6×105
cells) there was significantly reduced T and B cell reconstitution in addition to the lowest survival. The reason for the reduced engraftment and survival at the highest dose used (6×105
cells) is due most likely to GvHD as indicated by elevated clinical GvHD scores and a greater extent of liver damage histologically. We believe that GvHD damaged the bone marrow niche early, including the stroma and osteoblasts, resulting in poor engraftment [27
While the dose of PCT-4 STC that resulted in the highest level of durable multilineage engraftment and T and B cell immune reconstitution was 4×105 cells, only 70% of these animals survived (>180 days) with 30% dying at 52.5±4.9 days. In contrast to what was seen in the 6×105 non-PCT control animals, we could find no consistent pathologic evidence for acute or chronic GvHD in these animals. In addition to inflammation and duct damage, cholestasis also is common in acute GvHD and was not seen in this treatment group (or any other). Further, the pathology results in the 4×105 PCT-4 STC experimental group indicated chronic inflammation in the liver, raising the possibility that chronic infection and/or hepatitis of unclear etiology might have accounted for the 30% mortality. However, we don't know whether Tregs were eliminated by PCT, which may lead to GvHD. It is also possible that the PCT-4 STC were not completely rendered incapable of causing GvHD. Photochemical treatment of donor STC resulted in a significant reduction in lymphocyte proliferation in vitro (while maintaining cytotoxic capability and expression of activation markers); however, there was a degree of residual proliferation, although no more than 4% of control. We were concerned that these remaining cells could have resulted in GvHD. However, injection of an equivalent number (2.5×104) of STC (without PCT-4) into anti-NK1.1 mAb pre-treated Artemis-deficient recipients resulted in no engraftment, immune reconstitution, or mortality. When we injected 6×105 CFSE-labeled PCT-4 STC into anti-NK1.1 mAb pre-treated animals, they were undetectable in the circulation or in various organs after 24 hours post transplant. In contrast, 6×105 STC alone (without PCT-4) increased in number over time following transplant and resulted in 100% mortality from GvHD.
Compared with other approaches [28
], our data demonstrate that major barriers to achieving efficient allogeneic donor engraftment in mArt-/-
mice include recipient NK cell mediated resistance and occupancy of host bone marrow niches. In this study we demonstrate that reduction of NK cell function combined with PCT-4 STC results in durable multilineage engraftment of MHC mismatched HSC and restoration of normal T and B cell immunity in Artemis-deficient mice. Not surprisingly, the dose of PCT-4 STC is critical. Too low a dose results in minimal engraftment while too high a dose results in GvHD and poor reconstitution. In this study the optimal dose in terms of producing significant chimerism and full immune reconstitution, was nonetheless associated with some mortality of unclear etiology. This study suggests that the challenge of this treatment strategy will be to find a balance between beneficial and harmful T cell effects.