Background Follicular T helper (Tfh) cells play a critical role in follicular B cell activation and maturation during antibody responses to allograft. Here we report on an adoptive transfer model in our previously established model of allosensitization to better characterize the mechanisms by which Tfh cells impact humoral immunity during HLA-sensitization.

Methods Alloantigen-primed T cells from a C57BL/6 recipient of HLA.A2+ skin allograft were isolated via FACS sorting. Resulting T cells were adoptively transferred through tail vein injection into a nude mouse (B6.Cg-Foxn1nu), which was also sensitized with deactivated HLA.A2+ splenocytes. Constitution of transferred T-cells in the nude spleens was studied using FACS analysis. Antibody responses were monitored by measuring donor-specific antibodies (DSA).

Results Cell sorting via FACS using a negative selection strategy to exclude B (B220+), monocyte/macrophages (CD11b+) and NK (NK1.1+) cells from total splenocytes resulted in highly enriched CD3+ cell populations (90% purity and 85% livability). The splenocytes of the nude mice at designated days post T-cell transfer were analyzed in FACS. Results showed that the nude mouse spleen had a small population of CD4+ cells (2.78%, slightly leaky). No change observed in CD4+ population in the HLA.A2-sensitized nude spleens (2.84% at Day 21), indicating that allosensitization has no impact on native CD4+ cells. The nude mice receiving T-cell transfer exhibited increases in the CD4+ cell populations, accounting for 6.49% at Day 4, 3.5% at Day 14 and 5.54% at Day 21(p=0.05 vs. Control). Serologic studies found that naïve and HLA.A2-primed nude mice without T-cell transfer did not develop anti-HLA.A2 donor-specific antibodies (DSA). The nude mice with T-cell adaptive transfer, however, exhibited significantly increased DSA IgG at Day 14 (33.8+-1.22 MFI vs. naïve 24.45+-0.46, p=0.0009; and vs. HLA-primed 24.4+-0.82, p=0.0008), and Day 21 (40.96+-1.32 vs. naïve 24.45+-0.46, p=0.0017; and vs. HLA-primed, 26.98+-0.36, p=0.0031).

Conclusion Our preliminary data demonstrate that allo-immunities of T-deficient nude mice can be rescued by adoptive transfer of alloantigen-primed T-cells. The adoptively transferred T-cells can act as Tfh to promote DSA responses by B cells. Thus, this model can be used for investigating Tfh and B cell interactions and pharmaceutical interventions.

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