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In utero hematopoietic cellular transplantation (IUHCT) has the potential to treat congenital benign cellular disease without chemotherapy or radiation. Prenatal tolerance is essential in this regard. Previous studies involving prenatally created chimeras have shown that immunologically tolerant NK cells (friendly, fNK) from engrafter mice suppress allo Tc1 responses, while intolerant NK cells (hostile, hNK) from rejecter mice enhance allo Tc1 responses. Given that Th17 cells are also involved in allograft rejection, we sought to determine if rejecter hNK cells induce/expand allo-specific Th17/Tc17 cells. We examined the helper potential of prenatally educated hNK cells in non-tolerant prenatal Balb/c --- > B6.Thy1.2 rejecter chimeras. Naïve Thy1.1+ responder T cells and allogenic target cells were adoptively transferred into rejecter or naïve hosts that had been depleted of endogenous T cells and fNK cells. We found increased Th17/Tc17 alloimmunity in the responder Thy1.1+T cell population in rejecters when compared to naïve hosts. Additionally, through ELISPOT, we confirmed increased IL-17A production by responder T cells when co-cultured with allo-specific hNK cells from rejecter mice. Hostile NK cells from rejecter mice produce higher levels of Th17 driving cytokines, TGF-beta 1, IL-6 and GM-CSF when compared to naïve hNK. From these results, we conclude that prenatal rejecter hNK cells have an enhanced helper effect on Th17/Tc17 alloimmunity in vivo and in vitro. These findings support an evolving paradigm that prenatally educated NK cell can regulate the balance between Th1/Th17 and Treg functions. Supported by NIH R01HL103745 Lurie Children’s Hospital Research Foundation (to A.F.S.)

Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a friendly phenotype that is not cytotoxic to allogeneic donor cells. We wondered if prenatally educated friendly NK cells influenced allospecific T cell responses as an additional mechanism supporting prenatal tolerance. Therefore, we hypothesized that prenatally educated friendly NK cells exert suppressive effects on the induction of allospecific T cell responses. To challenge this hypothesis, we examined the suppression potential of prenatally educated friendly NK cells through gain and loss of function experiments in stable prenatal Balb/c into B6.Thy1.2 chimeras (engrafter mice). We first depleted allospecific NK cells in host engrafter mice and then adoptively transferred Thy1.1+ naïve responder T cells along with CFSE-labeled allogenic or syngeneic target cells into the engrafter hosts. We found that the depletion of allospecific Ly49D+ NK cells in engrafter mice increased Thy1.1+ T cell alloimmunity in vivo. Conversely, adoptive transfer of friendly NK cells suppressed the induction of allospecific T cell responses in host mice. Furthermore, cultured friendly NK cells from engrafter mice showed stronger suppression on the induction of T cell alloimmunity in vitro than that of naïve mice. Lasltly, NK cell-mediated suppression of allospecific T cells required cell contact. From these results, we conclude that prenatally educated friendly NK cell exert enhanced suppressive effects on T cell alloimmunity in vivo and in vitro. Depending on the context of their prenatal education, NK cells can differentially influence T cell responses in prenatal chimeras.

Allospecific NK cell hyporesponsiveness develops after in utero hematopoietic cellular transplantation (IUHCT) in mice who exhibit stable long-term engraftment. Conversely, allospecific NK cell responses are normal in mice that reject their graft. We hypothesized that these alternate NK cell responses also differentially influence T cell responses after IUHCT. To challenge this hypothesis, we compared the helper or suppression potential of prenatally educated NK cells between prenatal recipients with stable engraftment (engrafter mice) and littermates that rejected their graft (rejecter mice) in a Balb/c --- > a B6 model of IUHCT. Allospecific NK cells from engrafter, rejecter, or naïve mice were co-cultured with naïve responder T cells (B6) and irradiated allogeneic target cells (Balb/c) in an in vitro IFN-g assay. Intracellular production of IFN-g was measured by flow cytometry of the naïve responder T cells after 66 hrs of co-culture. We found that responder T cells displayed increased IFN-gamma production when co-cultured with allospecific NK cells from rejecter mice compared to co-culture with allospecific NK cells from naive control mice. Conversely responder T cells displayed decreased IFN-g production when co-cultured with allospecific NK cells from engrafter mice compared to co-culture with NK cells from naïve control mice. From these results, we conclude that allospecific NK cells can influence the allospecific response of T cells in vitro depending on the context of their prenatal education. Further studies are needed to determine if similar mechanisms support engraftment or rejection in vivo.