Predictive Markers for Acute Graft-Versus-Host Disease in the Peripheral Blood - Surface Receptor Profile Defines Alloreactive T Cells In Vivo.

Acute graft-versus-host disease (aGVHD) still results in high morbidity and mortality in patients undergoing allogeneic hematopoietic cell transplantation (aHCT). Early diagnosis of aGVHD remains difficult and is made based on clinical symptoms and histological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestations. Therefore, it is important to develop predictive assays to identify patients at risk of developing aGVHD for improved disease prevention.

Using bioluminescence imaging (BLI) we have recently demonstrated that aGVHD pathogenesis is tightly spatially and temporally regulated in a murine model across major histocompatibility barriers. Therefore, we asked whether insights in the timing of aGVHD initiation and effector phase could allow for the development of a diagnostic test whereby specific cell surface profiles can predict the onset of aGVHD.

FVB/N (H-2^q, Thy1.1⁺, 4x10⁶) or C57Bl/6 (H-2^b, Thy1.1⁺, 4x10⁶) splenocytes plus bone marrow (BM) cells (5x10⁶) were transplanted into conditioned (2x400rad) allogeneic Balb/c (H-2^d, Thy1.2⁺) or syngeneic (2x450rad) C57Bl/6 (H-2^b, Thy1.2⁺) recipients. Allogeneic recipients developed the first clinical signs of aGVHD starting at day+6 which progressed rapidly to animal death typically by day+14. However, BLI revealed that aGVHD target organ infiltration had already occurred days earlier. On day+3 after aHCT, dividing (BrdU⁺) donor derived allogeneic T cells (Thy1.1⁺) were still confined to the T cell areas in secondary lymphoid organs. Between day+3 and day+4 T cells appeared in the red pulp of the spleen, indicating likely entry into the blood circulation. Thereafter, increasing aGVHD target organ infiltration became apparent. These findings prompted us to analyze peripheral blood (PB) samples from allogeneic vs. syngeneic recipients (day+1 to +6). Until day+3 after aHCT, no significant PB T cell numbers were detectable. However, by day+4 we found a dramatic increase of PB T cells (mostly CD4⁺, by day+5 mostly CD8⁺) that were CD44^hi and expressed the homing receptors α4β7 integrin, αEβ7 integrin, CCR9, E-selectin ligand, P-selectin ligand, CCR5, and CXCR3 in allogeneic recipients, but not in syngeneic or BM controls. These T cell populations could be verified as clonally expanded (CFSE^lo) donor-derived alloreactive T cell subsets (Thy1.1⁺).

In summary, alloreactive T cells could be identified by the timed up-regulation of a panel of distinct homing receptors. Furthermore, the transition between aGVHD initiation and effector phase emerged as an early diagnostic window for the detection of alloreactive T cells in the peripheral blood days before aGVHD onset. Taken together, this approach could predict aGVHD in order to tailor immunosuppressive therapy for individual patients.