Precursor T cells were generated in a feeder-free culture system
Hematopoietic progenitor cells from the BM of male C57BL/6-tomato mice were stimulated with DL4-Fc that was used to precoat the cell culture plate and cultured for about ten days. Large numbers of precursor T cells, including double negative (DN) 2 stage cells (CD4-CD8-CD44 + CD25+) and DN3 stage cells (CD4-CD8-CD44-CD25+) were harvested (Fig. 1a-b). And the highly expression of CCR9 and RANKL was found in precursor T cells (Fig. 1c).
Stable donor chimerism was achieved in recipient mice after cell infusion
Complete donor chimerism in recipient mice peripheral blood, bone marrow and thymus was achieved within 4 weeks after transplantation (Fig. 1d-f). To eight weeks after transplantation, different degrees of GVHD symptoms including wrinkled hair, rash and weight loss were observed in MNC group and MNC + preT group.
Tracking precursor T cells migration in recipient mouse thymus
The highly expressed CCR9 and RANKL in precursor T cells contributed to thymus homing and crosslinking with RANK, which was expressed in thymic epithelial cells (TEC). The thymocytes originating from infused precursor T cells could be detected from 3 days to 8 weeks in the recipient mice thymus. Three days after cell infusion, precursor T cell chimerism was still low (3.1 ± 2.4%) (Fig. 1g). Then, the chimerism increased to more than 60% just two weeks later and slowly decreased to less than 1% at + 8 week. Thus, a temporary wave of T cell development was observed (10)。
Then the migration of precursor T cells in recipient mice thymus were tracked. A few precursor T cells (purple) entered the thymus cortex from 3 days to 1 week after infusion, while they were still absent in the thymus medulla at this time (Fig. 1h). After 2 weeks, more precursor T cells were found in the thymus cortex and a certain amount of precursor T cells appeared on the thymus medulla. Therefore, allogeneic precursor T cells could migrate from the cortex to the medulla inside the recipient thymus, just the same as normal lympho-progenitor thymic migration and T-cell differentiation.
The phenotype of precursor T cells was then measured (Fig. 1i). They were still at the DN stage 3 days after infusion (93.8 ± 2.9%). After one week, less than 10% of preT cells had differentiated into DP thymocytes and two weeks later, a rather high percentage of preT cells had differentiated into the DP stage (70.6 ± 10.6%). The single positive (SP) thymocytes, including CD4 + and CD8 + cells, were less than 20% at this time. From week 4 to 8, the percentage of CD4 + and CD8 + SP cells were gradually increased to 36.4 ± 10.6% and 7.9 ± 4.4%. While the percentage of DP cells were decreased to 52.2 ± 14.4%. Combining phenotype change and migration pathway, we presumed that allogeneic precursor T cells could enter irradiated recipient mice thymus, start T-cell generation from the cortex to the medulla, and finally exit the thymus after differentiation to naive T cells.
Impaired thymus structure was restored after preT cell infusion
Through HE pathological observation, the irradiated thymus of mice in the control group was recovered 8 weeks after transplantation, with an intact basic structure, and a normal number of lymphocytes and thymus corpuscles (Fig. 2a).
Unsurprisingly, the thymus of mice in the MNC group were severely impaired and disorganized. The number of lymphocytes and thymus corpuscles were largely decreased. Instead, more blood vessels and fibrous tissue emerged. After precursor T cell infusion, although the thymus structure was not as good as the control group, it was much better organized than the MNC group. Immunofluorescence pathology is a valuable method for studying the areas of the thymus cortex and medulla. The thymus of the control and MNC + preT groups had a cortex, medulla, and a defined corticomedullary boundary, while the thymus of the MNC group exhibited a rather poor boundary between the cortex and medulla and the medulla was even absent in some samples (Fig. 2b).
Eight weeks after precursor T cell infusion, higher RANK expression of thymic epithelial cells (TEC) was found in MNC + preT group, comparing to that in MNC group. No significantly difference could be found between MNC + preT group and control group. (Fig. 2c)
The KGF level in the MNC + preT group was significantly higher than that in the MNC group (14.1 ± 2.8 vs 8.4 ± 5.1, P = 0.0243) (Fig. 2d). It proved the thymus of mice in the MNC + preT group actively initiated restoration after precursor T cell infusion.
Immune reconstitution was accelerated upon precursor T cell infusion
The total number of thymocytes and the T-cell development level represented by the proportion of cells at DP stage are vital indexes of central immune reconstruction. One week after transplantation, the number of thymocytes were significantly decreased in both the MNC + preT and MNC groups compared to the control group (1.8 ± 1.2×106 and 2.7 ± 1.9×106 vs 47.1 ± 1.9×106, P༜0.01). And the percentage of DP cells in MNC + preT group and MNC group were 14.1 ± 1.7% and 15.5 ± 4.5%, respectively, which were significantly lower than 71.5 ± 0.3% in Control group (P༜0.01). Proving that the thymus was severely impaired and T-cell regeneration was severely delayed after donor splenic cell infusion (Fig. 3a-b). At 3 weeks after transplantation, the number of thymocytes and percentage of DP cells in MNC + preT group were higher than that in MNC group (11.5 ± 6.9×106 vs 2.3 ± 0.8×106, P = 0.08;75.5 ± 0.6% vs 60.4 ± 4.9%, P༜0.01). To 8 weeks after cell infusion, the indices of the MNC + preT group were significantly higher than those of the MNC group (37.9 ± 24.0×106 vs 12.3 ± 15.8×106, P = 0.008; 56.2 ± 10.4% vs 28.4 ± 24.6%, P = 0.005). This indicates that central immune reconstruction was significantly accelerated with precursor T cells’ help at the cellular level.
IL-7 is a key cytokine in the development of T-cell generation in the thymus, and its secretion level is closely related to the maturation process of T cells. Serum IL-7 levels in the MNC + preT and control groups were significantly higher than those in the MNC group (55.1 ± 31.6 vs 14.5 ± 14.3, P = 0.0167), suggesting that infusion of preT cells promoted T-cell regeneration in the thymus (Fig. 3c).
TCR Vβ expressions of several DN and DP thymocytes in the MNC + preT group were higher than those in the MNC group, and the expression of Vβ9 was significantly different among them (0.93 ± 0.05 vs 0.37 ± 0.30, P = 0.015) (Fig. 3d). This suggested that precursor T cells enriched the diversity of the TCR repertoire. However, there was no significant difference in TCR expression between the two groups for SP CD4 + and SP CD8 + T cells. Then, these two transplant groups were compared with the control group. TCR expression in the former two groups was higher than those of Vβ9 and Vβ12. No significant differences were found in other loci.
The status of peripheral immune reconstitution was closely related to central immune reconstitution due to the continued export of naive T cells from the thymus. The peripheral blood CD4:CD8 ratio, which reflects immune homeostasis, was normal in the control group, and no inverted lymphocyte subsets were found (Fig. 3e). At 4 weeks and 8 weeks after transplantation, the CD4:CD8 ratio of mice in the MNC + preT group was significantly higher than that in the MNC group (1.2 ± 07 vs 0.6 ± 0.5, P = 0.018; 2.3 ± 1.2 vs 1.6 ± 0.6, P = 0.013). The percentage of peripheral blood CD3 cells 8 weeks after was also significantly higher in the former group (15.1 ± 6.7 vs 7.0 ± 5.1, P = 0.04) (Fig. 3f). It demonstrated that the precursor T cells accelerated T-cell regeneration of peripheral blood and reconstitution of peripheral immune cells.
Immune tolerance induced by infusing preT cells attenuated donor GVHD or GVH effects in thymus
We assumed that a more severe immune response occurred in this group. As shown in Fig. 3e, the peripheral blood CD4:CD8 ratio was lower in the MNC group compared with MNC + preT group. As decreased CD4:CD8 ratio is closely related to GVHD, and the GVH effect mediated by CD8 + T cells is the main reason for recipient tissue damage (11), we measured the FasL and IFN-γ expression in thymocytes, which was positively correlated with the GVH effect (12). Percentages of FasL-positive CD4 + and CD8 + cells in the MNC + preT group were significantly lower than those in the MNC groups (0.36 ± 0.19 vs 1.84 ± 1.45, P = 0.03; 0.58 ± 0.17 vs 4.65 ± 2.46, P = 0.003) (Fig. 4a-b). The percentage of IFN-γ-positive CD8 + cells was also lower than that in the MNC group (0.92 ± 0.66 vs 2.42 ± 1.38, P = 0.02) (Fig. 4b). As a result, GVH effects within the recipient thymus were attenuated after precursor T cell infusion. Owing to this, the thymus structure and function soon recovered to normal immune status.