Acute Gvhd Induced by In Utero Hematopoietic Cell Transplantation Is Reversed by a Regulatory Response Resulting in a Potent Graft Vs. Hematopoietic Effect without Clinical Gvhd

In Utero Hematopoietic Cell Transplantation (IUHCT) is a potential treatment for congenital hematologic disorders. The rationale is to utilize normal events during hematopoietic and immunologic development to facilitate hematopoietic engraftment. While the risk of graft versus host disease (GVHD) after IUHCT would be anticipated to be high, based on Medawar's tenets, surprisingly little GVHD has been observed after experimental IUHCT and IUHCT induced GVHD has not been previously characterized. To better understand the potential risk of IUHCT induced GVHD prior to clinical application, we attempted to intentionally induce GVHD pathophysiology in the allogeneic murine model of IUHCT.

Bone marrow (BM) cells (10×10⁶) from C57/BL6 (B6, H2k^b, GFP⁻) mice were co-injected with increasing doses of splenocytes from B6-GFP (H2k^b, GFP⁺) mice intravenously into Balb/c (H2k^d) fetuses at embryonic day 14 (E14). Control groups included: 1) 10×10⁶ B6 BM cells alone; 2) same doses of B6-GFP splenocytes alone; and 3) IUHCT of congenic donor cell populations into B6 recipients. Surviving pups were assessed using a clinical GVHD scale and separate cohorts were harvested at pre- and post-natal time-points for analysis by fluorescence stereoscopic microscopy (FSM), flow cytometry for assessment of donor and host cell populations in hematopoietic tissues, and histologic assessment of target tissues for GVHD.

Prenatal Analysis - In the allogenic groups that received 10×10⁶ BM cells plus increasing doses of splenocytes (0.1×10⁶ to 10×10⁶ splenocytes/fetus) a consistent distribution of GFP positive cells could be seen by whole body FSM in lymphohematopoietic sites (spleen, lymph nodes, thymus and fetal liver) at E16 that did not differ significantly from congenic controls. By E19 – E20 however, marked proliferation of the cells occurred with clear infiltration of GVHD target organs (skin, lungs, liver, intestine). This expansion was proportionate to the dose of splenocytes and was not seen in congenic or BM only controls. Increased mortality was not observed until doses greater than 5 × 10⁶ splenocytes were given (approx. 5 × 10¹¹ splenocytes/kg est. fetal wt.) with perinatal death likely attributable to respiratory insufficiency.

Postnatal Analysis - In the allogeneic groups there was minimal clinical evidence of GVHD up to and inclusive of the 5×10⁶ splenocyte dose. A few pups showed poor growth relative to the normal growth curve and mild fur ruffling, but these resolved by P15, and there was no further clinical evidence of GVHD and no postnatal deaths up to P60. In harvested tissues many GFP⁺ cells persisted in GVHD target organs, however, no GVHD histopathology was observed at any time. Flow cytometric analysis confirmed a proliferation of donor splenocytes that peaked around P5-P10. Lymphocyte subset analysis revealed an expansion of predominantly CD8⁺ T-cells. Alkaline phosphatase in the host liver, as a marker of GVHD was increased during the same time course and returned to normal by P15. An increase of host and donor BM derived CD4⁺ cells was also observed that peaked around P15-17. Further analysis confirmed an increase in both T-regulatory cells and activated CD4⁺ T-cells compared to normal or BM only controls. The most remarkable observation was a dramatic graft versus hematopoietic (GVH) effect with an increase in donor BM derived chimerism at P60 to > 80% with no clinical GVHD. These findings were seen only in the allogenic group that received both splenocytes and bone marrow cells, not in the other groups. In the group that received only bone marrow cells, the chimerism remained at low levels (Figure 1).

Our data demonstrates that the fetus is in fact resistant to GVHD relative to myeloablated postnatal BMT recipients. While there is an aggressive alloimmune activation and expansion of donor T-cells, clinical manifestations are mild and are reversed by what appears to be a T-regulatory cell expansion. In our model, the alloimmune activation phase is accompanied by a potent GVH response providing a competitive advantage for donor cells and resulting in marked enhancement of donor cell engraftment without clinical evidence of GVHD. Further work in this model is directed toward defining regulatory and effector mechanisms involved in controlling GVHD and the GVH effect.

No relevant conflicts of interest to declare.