Chlamydia is responsible for millions of new infections annually, and current efforts focus on understanding cellular immunity for targeted vaccine development. The Chlamydia-specific CD4 T cell response is characterized by the production of IFNγ, and polyfunctional Th1 responses are associated with enhanced protection. A major limitation in studying these responses is the paucity of tools available for detection, quantification, and characterization of polyfunctional, antigen-specific T cells. We addressed this problem by developing a TCR transgenic mouse with CD4 T cells that respond to a common antigen in Chlamydia muridarum and Chlamydia trachomatis. Using an adoptive transfer approach, we show that naïve transgenic CD4 T cells become activated, proliferate, migrate to the infected tissue, and acquire a polyfunctional Th1 phenotype in infected mice. Polyfunctional Tg Th1 effectors demonstrated enhanced IFNγ production compared to polyclonal cells, protected immune deficient mice against lethality, mediated bacterial clearance, and orchestrated an anamnestic response. Adoptive transfer of Chlamydia-specific CD4 TCR Tg T cells with polyfunctional capacity offers a powerful approach for analysis of protective effector and memory responses against chlamydial infection, and demonstrates that an effective monoclonal CD4 T cell response may successfully guide subunit vaccination strategies.
CD4 T cells and antibody are required for optimal acquired immunity to genital tract infection, and T cell-mediated gamma interferon (IFN-γ) production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice with CM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal for and mice, in which IFN-γ signaling was absent, and for mice, which lacked T and B cells and in which innate IFN-γ signaling was retained. In contrast, B cell-deficient muMT mice, which can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN-γ signaling survived. These data collectively indicate that IFN-γ prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell corequirement. Adoptive transfer of convalescent-phase immune serum but not naive IgM to mice infected with CM001 significantly increased the survival time, while transfer of naive B cells completely rescued mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T cell-independent B cell responses and innate IFN-γ in chlamydial host defense and suggest that interactions between T cell-independent antibody and IFN-γ are essential for limiting extragenital dissemination.
Pelvic inflammatory disease (PID) is a female upper genital tract inflammatory disorder that arises after sexually transmitted bacterial infections (STI). Factors modulating risk for reproductive sequelae include co-infection, microbiota, host genetics and physiology. In a pilot study of cervical samples obtained from women at high risk for STIs, we examined the potential for unbiased characterization of host, pathogen and microbiome interactions using whole transcriptome sequencing analysis of ribosomal RNA-depleted total RNAs (Total RNA-Seq). Only samples from women with STI infection contained pathogen-specific sequences (3 to 38% transcriptome coverage). Simultaneously, we identified and quantified their active microbial communities. After integration with host-derived reads from the same data, we detected clustering of host transcriptional profiles that reflected microbiome differences and STI infection. Together, our study suggests that total RNA profiling will advance understanding of the interplay of pathogen, host and microbiota during natural infection and may reveal novel, outcome-relevant biomarkers.
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.
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