Single-cell sequencing resolves early changes in mice T cells after microtransplantation

Background Phenotypic and molecular characteristics of the immune cell population after microtransplantation (MST), heterogeneity within the population, and evolution of T cells are still unclear.

Objective To characterize the early changes of T cells in mice after MST and the corresponding mechanisms by applying single-cell sequencing.

Methods Female CB6F1 mice (H-2Kb/d) were used as recipients and male C57BL/6J mice (H-2Kb/b) were used as donors. The microtransplantation mouse model was established by infusing 6 × 10⁷ G-CSF-mobilized donor spleen cells (GDSC) into the recipients without any pretreatment and graft versus host disease (GVHD) prophylaxis. Single nucleated cells were collected from the peripheral blood of microtransplanted mice at 0 d, 7 d and 14 d (3 mice at 0 d, 6 mice at 7 d and 14 d). All data from each sample were combined after sorting CD3 ⁺ cells with a flow sorter to perform unsupervised clustering of the data based on highly variable genes, and cells were projected in two dimensions using uniform manifold approximation and projection (UMAP). The known sex genes Ddx3y, Eif2s3y, Xist and Tsix were used to differentiate donors and recipients. Validation was performed with single cell transcriptome sequencing and unbiased bioinformatics analysis.

Results Donor cells survived and proliferated in recipients as microchimeric chimeras after microtrans-plantation. Differential gene expression from scRNA-seq data classified T cells into six subpopulations, with CD4 ⁺ T cells comprising three subpopulations: CD4 ⁺ Naive, CD4 ⁺ Memory, CD4 ⁺ Treg, and CD8 ⁺ T cells also comprising three T cell subpopulations: CD8 ⁺ Naive, CD8 ⁺ Memory, CD8 ⁺ Effector. The expression of cytotoxic factors (NKG7, GZMA, CTSW, GZMB, GZMK, KLRC1) in the CD8 ⁺ Effector subpopulation was increased, and the difference in expression before and after microtransplantation was statistically significant (P＜0.05), which suggested that microtransplantation increased the killing and effector functions of the recipient mice, while the regulatory T cell subpopulation expressed a large number of immunosuppressive factors LGALS9, CD274, IL10, PDCD1LG2, CD48, IL2, CD200R1 and effector subpopulation receptors to negatively regulate cytotoxicity and eventually achieve immune stability.

Conclusion Under pretreatment-free conditions, infusion of donor GDSC alone can form donor microchimerism and successfully establish a mouse microtransplantation model under natural immune status. Further mechanistic studies showed that microtransplantation could trigger differential gene expression and signaling pathway enrichment of early T cells, especially CD8 ⁺ Effector population and CD4 ⁺ central Treg population in recipient mice, suggesting corresponding cytotoxic effects and immunomodulatory responses, which provides useful insights into the immunological and molecular mechanisms of microtransplantation and offers some new ideas for clinical applications of microtransplantation.