Post-Transplant G-CSF Restores T Cell Homeostasis By Inhibiting Alloreactive T Cells and Promoting Thymic Regeneration

T cell homeostasis restoration in recipients following allogeneic hematopoietic stem cell transplantation (HSCT) not only indicates that T cells reconstitute to defend against foreign pathogens and exert graft-versus-leukemia (GVL) effects, but also recognize the recipient as ‘self-antigen’, establishing self-tolerance. During the early stages after transplantation, the proliferation of donor mature T cells through the peripheral pathway results in a restricted T cell pool and alloreactive response, leading to graft-versus-host disease (GVHD). This, together with conditioning regimens, impairs recipient thymic function and delays complete T cell reconstitution through the central pathway, which is essential for restoring T cell homeostasis following HSCT. Therefore, it is imperative to explore therapies that boost thymic regeneration and restore T cell homeostasis following HSCT. Post-transplant granulocyte colony-stimulating factor (G-CSF) administration is widely used to accelerate neutrophil engraftment. In our study, we demonstrated that post-transplant G-CSF inhibits peripheral reconstituted precursor exhausted T cells, facilitates thymic regeneration, and promotes the restoration of T cell homeostasis. By employing R26-CAG-EGFP (C57BL/6 background) transgenic mice to establish MHC-matched (CD45.2 to CD45.1) and MHC-haplomatched (C57BL/6 to CB6F1) transplantation models, we delineated the dynamics of T cell reconstitution profiles from the peripheral pathway (GFP+) and the central pathway (GFP-) using spectrum flow cytometry and single cell RNA-seq (scRNA-seq). Compared to the MHC-matched group, peripheral reconstituted T cells in MHC-haplomatched mice were mainly composed of effector memory T cells and exhibited high activation and cytotoxicity, leading to thymic impairment, restrained T cell reconstitution from the central pathway, and lethal GVHD damage. Interestingly, we identified a cluster of precursor exhausted T cells (Tpex) with high expression levels of Tcf7 but lower expression level of Tox, Pdcd1 and Tim3 compared to terminally exhausted T cells, which were highly expanded in MHC-haplomatched mice. To explore whether G-CSF could restore T cells homeostasis, we administrated G-CSF to MHC-haplomatched mice daily from day +10 to +16 post-transplantation. The results showed that MHC-haplomatched mice treated with G-CSF experienced delayed GVHD progression and prolonged survival. In the G-CSF treated group, both CD4 and CD8 effector/effector memory T cells reconstituted from the peripheral pathway showed a decreased percentage, inhibited activation (CD69), and reduced cytokine production (CD107a and Granzyme B) compared to PBS group. Meanwhile, we found that TCF1+PD-1+ Tpex cells exhibited higher proliferative and cytotoxic capacities compared to TCF1-PD-1+ cells, consistent with our scRNA-seq findings. G-CSF administration mainly inhibited TCF1+PD-1+ Tpex cells while having minimal impact on TCF1-PD-1+ cells, indicating that G-CSF can inhibit T cell activation without promoting terminal exhausted T cell differentiation. Importantly, G-CSF administration boosted thymus regeneration, as evidenced by improved thymic cellularity, and facilitated T cell reconstitution from the central pathway, as shown by the increased number of naïve T cells in the G-CSF treated group compared to PBS group. To further elucidate the role of G-CSF in thymus regeneration, we employed a haploidentical transplantation model that received only T-cell depleted bone marrow cells (TCD-BM), thereby excluding the interference of peripheral alloreactive T cells. The results showed that G-CSF administration improved thymic cellularity and promoted T cell development in the thymus. This effect was mainly achieved by enhancing positive and negative selection processes in double-positive thymocytes and promoting the maturation of CD4 single-positive thymocytes. Additionally, we also found that G-CSF administration promoted the expansion of Aire+ medulla TEC (mTEC) and increased the infiltration of plasmacytoid DC (pDCs) in the thymus, both of which are crucial for negative selection and T cell maturation. In conclusion, our results reveal a novel function of post-transplant G-CSF administration in restoring T cell homeostasis through its dual role of inhibiting peripheral T cell alloreactivity and promoting thymic regeneration.