Towards an Understanding of Protective Cellular Immunity - a TCR Transgenic Mouse Reactive with Chlamydia muridarum

Abstract

Abstract A major limitation in understanding the development of protective T cell memory against Chlamydia is the difficulty in characterizing low frequencies of antigen-specific T cells. To better understand the kinetics and phenotype of specific CD4 T cells, we developed a C. muridarum-specific T-Cell Receptor (TCR) transgenic (Tg) mouse. Primed T cells from C57BL/6 mice immune to C. muridarum were stimulated with EB/RB for 5 days and fused with BW5147 cells. Specific clones producing IL-2 and IFN-γ were harvested and screened for TCR Vα and Vβ by qPCR. Cloned transgenes were extracted, purified, and co-microinjected into fertilized oocytes. PCR and FACS confirmed integration and expression. Tg T cells were analyzed ex vivo for activation markers and cytokine production after EB/RB stimulation. 90% of peripheral CD4+ T cells express TCR transgenes and exhibited enhanced proliferation and increased expression of IFN-γ, IL-2, and CD69, compared to controls. Naive T cells were adoptively transferred from CD45.2 Tg mice into CD45.1 mice one day before intravaginal inoculation with C. muridarum. Tg cells showed enhanced proliferation and TNF-α, IL-2, and IFN-γ production by day 5 compared to controls. Tg T cells collected from SLOs and oviducts on days 5, 8, 12, 22, 44, and 65 post-infection exhibited enhanced expression of memory and activation markers. Adoptive transfer of Tg or WT CD4 T cells to Rag1−/− mice rescued an otherwise lethal C. muridarum genital infection. WT and Tg CD4 T cells exhibited comparable chlamydial clearance from the genital tract. Adoptive transfer of Chlamydia-specific CD4+ T cells offers a powerful approach to characterize cellular activation, differentiation, and memory development.