Graft Versus Host Disease (GvHD) Is Attenuated By Administration of Pregnancy Specific Glycoproteins through Induction of Immune Tolerance

Hematopoietic stem cell transplantation is curative for many disorders; however, it can be associated with significant morbidity and mortality, often as a result of graft versus host disease (GvHD).  GvHD is an immune mediated reaction in which donor T-cells recognize the host antigens as foreign, causing donor T-cells to proliferate and attack host tissues.  Establishment of a tolerogenic immune environment while preserving immune response to infectious agents and malignancy is required for successful bone marrow transplantation and GvHD presents a significant obstacle to this.

Pregnancy specific glycoproteins (PSGs) are synthesized by the placenta at the onset of pregnancy and are believed to play a role in maintaining a tolerogenic immune environment to prevent rejection of the fetus by the maternal immune system.  Two specific PSG proteins, PSG1 and PSG9, are of particular note as they have both been shown to be involved in pathways devoted to induction of immune tolerance.  Both PSG1 and PSG9 are involved in activation of transforming growth factor-β (TGFβ), a cytokine essential to suppression of inflammatory T-cells and important for differentiation of tolerance inducing CD4⁺CD25⁺FoxP3⁺ regulatory T-cells (T_regs), a cell population shown to be important in the prevention of GvHD.  We thus hypothesized that PSG1 and PSG9 (PSG1/9) could be used to treat or prevent GvHD by inducing immune tolerance through TGFβ.  

Using surface plasmon resonance (SPR) analysis, we have shown that PSG1/9 bind directly to the latency associated peptide (LAP), a protein that confers latency to mature TGFβ, effectively blocking binding of TGFβ to its receptors.  Further, bioassays and ELISA data show an increase in the levels of bioactive TGFβ after treatment with PSG1/9, indicating that these proteins have a role in TGFβ activation through their interaction with LAP.  In vitro data using naïve mouse T-cells showed that upon treatment with PSG1, there was a significant increase of CD4⁺CD25⁺ cells expressing FoxP3 (18%±1.5; n=3) compared to only 2% in untreated controls (2%±0.4; n=6) (p<0.0001).  Similarly, PSG9 treatment also increased FoxP3 expression (11%±2.8; n=3) over controls (p<0.0001).  The increase in FoxP3 expression was also observed upon treatment of primary naïve human T-cells with both PSG1 and PSG9 (PSG1, p=0.0073; PSG9, p=0.0028).  When a TGFβ receptor inhibitor was added to cell culture, the increase in FoxP3 expression was effectively blocked, further supporting the hypothesis that PSG1/9 induce expression of FoxP3⁺ T_regs through regulation of TGFβ.  As IL-2 is important for the stability and differentiation of T_regs in vivo and its transcription is suppressed by TGFβ, we performed bioassays on CD4⁺ naïve T-cells with no added IL-2.  We observed that in the absence of added IL-2, treatment with PSG1/9 resulted in an inhibition of IL-2 secretion by activated CD4⁺ T-cells, and there was no increase in the number and percentage of FoxP3 expressing cells.

SBE-luc mice, which express luciferase in response to activation of the TGFβ-SMAD pathway, exhibited significantly increased luciferase expression in the abdomen when injected intraperitoneally with PSG1. This supports the idea that PSG1 induces expression of FoxP3 through the TGFβ receptor pathway in vivo. Finally, using a murine model of GvHD, we observed that mice receiving PSG1 had reduced numbers of infiltrating inflammatory CD3⁺ T-cells in the colon and showed a marked improvement physically and histologically over untreated controls.  In addition, PSG1 treated mice had significantly higher expression of FoxP3 in CD4⁺CD25⁺ splenic cells (30%±9.6; n=4) when compared to untreated GvHD controls (8.6%±3.2; n=3) (p=0.0287). 

Currently, we are studying the cytokine profiles of mice treated with PSG to determine the efficacy of PSG1/9 in reducing the release of pro-inflammatory cytokines during GvHD.  Overall, our data suggests that PSG1/9 induce immune tolerance in GvHD and may be a future treatment option for these patients.