The Maternal Immune Response to in Utero Hematopoietic Stem Cell Transplantation.
Authors: MacKenzie Tippi , Jarvis Erin , Nijagal Amar , Le Tom , Wegorzewska Marta , Tang Qizhi 
Published: October 2019

Abstract
Abstract 64
In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising treatment strategy for many congenital hematopoietic disorders such as immunodeficiencies. However, clinical applications have been hampered by lack of engraftment, possibly secondary to a host immune response. This has been a conundrum in the field, since the fetus can also be tolerized to allogeneic cells in some circumstances. We hypothesized that it is the maternal immune response which limits engraftment of in utero transplanted cells.

Methods:
Fetal BALB/c mice at 14 days' gestation were transplanted with age-matched fetal liver (FL) cells (2.5 × 106 cells/fetus) from allogeneic C57B6 mice and levels of circulating donor cell chimerism were determined serially starting at 4 weeks after in utero transplantation. Rates of engraftment (number of chimeric pups/number of surviving pups) and levels of chimerism (donor CD45 cells/total CD45 cells) were compared to controls in which animals were transplanted with congenic cells (C57B6 (CD45.2) fetal hosts transplanted with C57B6 (CD45.1) FL). In order to determine the role of the maternal adaptive immune system, immunodeficient BALB/c.Rag−/− mothers (deficient in T and B cells) were bred to wild type BALB/c males, such that the fetuses (BALB/c.Rag+/−) would be immunocompetent. These fetuses were transplanted with C57B6 FL and rates of engraftment and levels of chimerism in these transplants were compared to those in wild type allogeneic transplants. In order to determine whether the maternal influence is caused by maternal lymphocytes trafficking into the fetus, C57B6 (CD45.2) females were bred to C57B6 (CD45.1) males, such that the fetal cells (CD45.1+/CD45.2+) could be distinguished from maternal cells (CD45.1−/CD45.2+). Fetal blood and tissues were examined for the presence of maternal cells by flow cytometry at various gestational ages.

Results:
The rate of engraftment after IUHSCTx in control animals transplanted with congenic cells was 14/16 (88%) and average levels of chimerism were 9.9±8.4%. In contrast, the rate of engraftment in wild-type BALB/c fetuses transplanted with allogeneic B6 cells was 11/25 (44%; p<0.05 compared to congenic), and levels of chimerism were 21±19 (p=NS), confirming there is an adaptive immune response to fetal stem cell transplantation. As expected, chimeric animals were tolerant to the donor strain by mixed lymphocyte reaction while injected, non-chimeric animals were sensitized. However, in the absence of a maternal adaptive immune system, rates of chimerism (in immunocompetent BALB/c.Rag+/− pups) increased to 100% (n=10, p<0.05 compared to wild type allogeneic) and levels of chimerism were significantly higher (44±18, p<0.05). Levels of chimerism in engrafted animals declined over time after allogeneic transplantation but not after congenic transplantation, indicating there is a second, late phase immune response to allogeneic cells. However, chimerism levels did not decline in the BALB/c.Rag+/− recipients, suggesting that the maternal immune system has long-lasting effects on the success of fetal transplantation, perhaps by priming the host immune system. In our analysis of maternal/fetal cellular trafficking, we detected maternal lymphocytes in the blood of midgestation fetuses (14±7% at E12.5–E14.5, n=9) which declined gradually and was undetectable after birth. Lineage analysis demonstrated that 45±15 % of maternal cells are Gr-1+ granulocytes and 21±15% are B cells. Trafficking of maternal cells into the fetus was increased following fetal manipulation (injection of PBS < injection of allogeneic HSC).

Conclusions:
There is an adaptive immune response which limits early engraftment after in utero transplantation of allogeneic cells and leads to a gradual decline in levels of chimerism in engrafted animals. However, in the selective absence of maternal T and B cells, all fetuses transplanted with allogeneic FL cells show long-term, multilineage engraftment and demonstrate donor-specific tolerance. These results indicate that the maternal immune system plays a significant role in the success of fetal HSC transplantation. Cellular trafficking between the mother and fetus may be a mechanism by which maternal lymphocytes encounter cells transplanted into the fetus. Our findings have clinical implications in that the success of IUHSCTx may be improved by harvesting cells from the mother or HLA-matching cells to the mother.

Disclosures:
No relevant conflicts of interest to declare.