Signal Transducing Adaptor Protein (STAP) Family Accelerates Gut and Thymic Graft-Versus-Host-Disease in Murine Model

Graft-versus-host-disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most frequent complication and one of the major causes of non-relapse mortality. However, its pathogenesis has not yet been fully understood. We cloned signal-transducing adaptor protein (STAP)-2 as a c-fms binding protein from a fetal liver library in 2003. The family that contains STAP-1 and STAP-2 has a pleckstrin homology (PH) and Src-homology 2 (SH2)-like domains, suggesting that this adapter protein functions as an immune and inflammatory regulator. Indeed, STAP-2 regulates adhesion and chemotaxis in T cells (Sekine et al., J Immunol. 2009). In this study, we aimed to elucidate the roles of STAP family in GVHD.

First, we examined the expression of STAP-1 and STAP-2 mRNA in various human hematopoietic subsets, including CD34⁺ CD38^- hematopoietic stem cells (HSCs), CD34⁺ CD38⁺ hematopoietic progenitor cells (HPCs), CD19⁺ CD27^- naïve B cells, CD19⁺ CD27⁺ memory B cells, CD3⁺ CD4⁺ helper T-cells, and CD3⁺ CD8⁺ cytotoxic T lymphocytes, using real-time PCR. As a result, STAP-1 and STAP-2 were expressed in lymphoid cells, as well as HSCs and HPCs. STAP-2 mRNA was highly expressed in T cells. Next, to investigate the role of STAPs in GVHD, we made an experimental murine model. To study the pathogenesis of immune reconstitution and tolerance after allo-HSCT, lethally irradiated BALB/c mice were injected with T and B cell-depleted bone marrow cells (5×10⁶ cells) derived from syngeneic BALB/c or allogeneic C57BL/6 mice on day 0. Co-transplantation of splenocytes was not adapted in this model. Survival and clinical degree of GVHD were assessed by a scoring system that sums changes in 5 clinical parameters: body weight (BW) loss, posture, activity, fur texture, and skin integrity. Recipients transplanted from allogenic wild type (WT) C57BL/6 donor survived without suffering from severe GVHD symptoms, owing to development of immune tolerance against allogeneic antigens. However, compared to syngeneic transplanted mice, these recipients started to show gradual BW loss and GVHD score was increased approximately 28 days after allo-HSCT, indicating the existence of an allogeneic immune reaction. To evaluate the role of STAPs in GVHD, we generated transgenic mice (Tg) that overexpress STAP under the control of Em enhancer and Lck proximal promoter. The promoter could drive expression of the inserted cDNA in lymphoid lineage cells from the common lymphoid progenitor (CLP) stage. When STAP-2 Tg marrow was used as a donor source, we found that the overall survival of STAP-2 Tg recipients was significantly lower than that of WT recipients (22.2% and 91.7%, respectively; p<0.001) on day 60. STAP-2 Tg recipients showed decreased BW and had a higher clinical GVHD score with statistical significance compared to control WT recipients. The overexpression of STAP-1 also exacerbated the severity of GVHD. At day 42, BW was decreased by 16.3% in WT recipients. In contrast, recipients of STAP-1 Tg and the STAP-2 Tg donor showed more severe BW loss along with diarrhea (23.3% and 29.8%, respectively). STAP-1 as well as STAP-2 Tg recipients showed significantly worsened GVHD scores, and this lasted until day 90 at the end of follow-up. In histologic examination of both STAP Tg recipients, inflammatory damages with lymphocyte infiltration were most notably observed in the colon. Interestingly, we found that thymus was atrophic or indistinguishable and the cortico-medullary junction disappeared. Moreover, compared to control WT recipients, the number of CD4⁺ CD25⁺ regulatory T (Treg) cells in the peripheral blood was significantly low in STAP-2 Tg recipients on day 60 (WT vs STAP-2 Tg; 44.0 /μL vs 18.2 /μL; p<0.05).

In this study, we show that STAPs in reconstituted lymphocytes after allo-HSCT regulate the pathogenesis of GVHD. Our results suggest that STAP activation in lymphocytes during immune reconstitution accelerates gut and thymic GVHD. Severe thymic damage induced by STAP overexpression might contribute to impairment of immune tolerance such as a decreased number of Treg cells as well as dysfunction of thymic negative selection of host-reactive T cells after allo-HSCT, which is involved in persistence of GVHD. Future study should further elucidate the detailed molecular mechanisms involved.