NT-I7, a Long-Acting Interleukin-7 Molecule, Promotes T Cell Reconstitution Following Radiation Injury through Thymic-Dependent and Independent Pathways

Total body irradiation (TBI) causes suppression of hematopoiesis and T cell depletion. T cell lymphopenia following radiation exposure increases the risk of opportunistic infections. Currently, therapeutic options for promoting T cell recovery are not available. Interleukin-7 (IL-7) is a critical cytokine involved in the adaptive immune system. Previous murine and human studies have demonstrated the beneficial effects of exogenous IL-7 on T cell recovery and function. However, its clinical application has not been realized, at least partially due to its short half-life in circulation. In the current study, we examined the effect of NT-I7, a novel and long-acting human IL-7 molecule, in facilitating T cell reconstitution following TBI.

C57BL/6 mice were first exposed to 5 Gy of TBI. After irradiation, mice received a weekly dose of NT-I7 subcutaneously at a dose of 10 mg/kg until day +21 (24 hr, day +7, +14, +21). T cell recovery was monitored in peripheral blood by flow cytometry. TBI rapidly depleted both CD4+ and CD8+ T cells. In the vehicle control group, it took at least 8 weeks for the T cells to recover back to the baseline levels. In contrast, CD4+ and CD8+ T cell counts recovered back to normal in 2 and 3 weeks respectively in NT-I7 treated mice. NT-I7 increased all T cell subsets including naïve, central and effector memory T cells. NT-I7 efficacy was similar when the treatment was initiated at day +7, when both CD4+ and CD8+ T cell counts were at the nadir. In addition, similar results were observed in mice irradiated with 2 and 7 Gy and in older animals. Using a novel T cell-specific luciferase reporter mouse strain, we further demonstrated that NT-I7 significantly enhanced systemic T cell recovery after TBI. The positive effect of NT-I7 on systemic T cell reconstitution was further confirmed by measuring T cell recovery in spleen and bone marrow directly.

The positive effect of NT-I7 on T cell reconstitution post-TBI is a result of enhanced thymopoiesis because NT-I7 increased the number of both CD4+ and CD8+ recent thymic emigrants (Qa2+CD24+) in peripheral blood. More importantly, the numbers of total thymocytes and all thymocyte subsets including DN, DP, SP4, and SP8 were higher in the NT-I7 group compared with the vehicle control group when measured at day +21. Enhanced thymopoiesis is likely due to increased production of common lymphoid progenitors in the bone marrow since both the frequency and total number of common lymphoid progenitors were increased in the NT-I7 treated mice. In addition, NT-I7 further promoted T cell recovery through a thymic-independent pathway as we demonstrated in an adoptive transfer model that NT-I7 expanded irradiated T cells directly.

In vitro functional assays suggested that expression of granzyme B, IFNg, and TNFa, were preserved in the NT-I7 group, in addition to preservation of proliferation capacity in response to TCR stimuli. Given these promising in vitro data, we next used a skin transplantation model to test whether NT-I7-driven T cell recovery also restored functionality after irradiation in vivo. C57BL/6 mice were irradiated with 5 Gy of TBI and then treated with NT-I7. On day 7 after irradiation, skin grafts from MHC-mismatched BALB/c mice were transplanted into these mice. While 5 Gy of TBI significantly prolonged skin graft survival compared with the non-irradiated control, treatment with NT-I7 significantly shortened the time required for rejection of the skin graft compared with the vehicle control group, demonstrating that NT-I7 promotes functional primary T cell responses in vivo. To test whether NT-I7 can promote functional memory T cell recovery, we performed a 2^nd skin transplantation. Eight weeks after the 1^st skin transplantation, primed C57BL/6 mice underwent additional sub-lethal irradiation at 5 Gy, followed by NT-I7 injections. On day 7 (9 weeks after the 1^st skin transplantation), a 2^nd skin graft from BALB/c mice was transplanted. The result demonstrated that treatment with NT-I7 also significantly shortened the time required for rejection of the 2^nd skin graft compared with the vehicle control group, demonstrating that NT-I7 can also promote functional memory T cell responses in vivo. These results indicate that NT-I7 is a promising therapeutic for significantly accelerating recovery of functional T cells after radiation exposure.