A. E. Lee1, A. M. Alhajjat2, B. S. Strong1, L. E. Turner1, R. K. Wadhwani1, T. Newkold1, A. F. Shaaban1 1Cincinnati Children’s Hospital Medical Center,Center For Fetal Cellular And Molecular Therapy,Cincinnati, OH, USA 2University Of Iowa,Department Of Surgery,Iowa City, IA, USA
Introduction:
In utero hematopoietic cellular transplantation (IUHCT) has the potential to treat congenital benign cellular disease without the need for chemotherapy or radiation. Prenatal tolerance is essential in this regard. Previous studies have shown that allospecific prenatal natural killer (NK) tolerance requires a threshold level of circulating chimerism (> 1.8%) during the critical phases of NK cell education resulting in the elimination of alloreactive NK cell phenotypes. However, it remains unclear if sub-threshold levels of chimerism have any impact on the education of developing NK cells. Given the lack of sustained tolerance, we hypothesized that exposure to sub-threshold levels of hematopoietic chimerism does not lead to durable changes in NK cell selection.
Methods:
To challenge this hypothesis, we compared NK cell education in stable murine prenatal chimeras (engrafters) to littermates that rejected their grafts (rejecters) in a Balb/c → B6 murine model of IUHCT. Following the prenatal transplantation of allogeneic fetal liver hematopoietic cells at E14, the level of chimerism and the frequency of alloreactive NK cell phenotypes were measured in the peripheral blood of either group and compared to naïve age-matched controls at serial time points during the critical phases of NK cell education. In particular, we measured frequency of naturally occurring donor-reactive NK cells in wild-type B6 mice that express the donor-reactive Ly49D activating receptor without co-expression of donor-specific Ly49A, L49F or Ly49G inhibitory receptors (hostile NK cells).
Results:
As expected, all of the engrafter mice exhibited stable long-term engraftment while the rejecter mice universally rejected their grafts several weeks after birth. This pattern was unaffected by fostering of the recipients by naive pregnant dams suggesting no contribution of the maternal humoral immune response to these outcomes. At weeks 3 and 4 after birth, both engrafter and rejecter mice exhibited low frequencies of phenotypically hostile NK cells (Ly49D+AFG-) when compared to age-matched controls (4.4 and 5.2% vs. 19.6%). Both engrafter and control mice maintained a stable level of hostile NK cells through the 14 week time point (4.7% and 22.3% respectively) while rejecter mice displayed a steady rise in the frequency of hostile NK cells throughout the study period (6 weeks: 12.5%, 10 weeks: 12.9%, 14 weeks: 18%) eventually reaching levels similar to control mice.
Conclusion:
From these findings, we conclude that: 1) durable prenatal NK cell selection requires the stable persistence of a threshold level of circulating tolerogen; 2) transient reductions in the frequency of hostile NK cells are inadequate to maintain long-term engraftment; and 3) the emergence of either tolerance or rejection is intrinsic to the education of developing lymphocytes in prenatal chimeras and is independent of the maternal humoral immune response. Further study is needed to examine functional response of hostile NK cells in rejecter mice and the potential for donor-specific memory to develop after rejection.