Chlamydia trachomatis infection of the human Fallopian tubes can lead to damaging inflammation and scarring, ultimately resulting in infertility. To study the human cellular responses to chlamydial infection, researchers have frequently used transformed cell lines that can have limited translational relevance. We developed a primary human Fallopian tube epithelial cell model based on a method previously established for culture of primary human bronchial epithelial cells. After protease digestion and physical dissociation of excised Fallopian tubes, epithelial cell precursors were expanded in growth factor-containing medium. Expanded cells were cryopreserved to generate a biobank of cells from multiple donors and cultured at an air-liquid interface. Culture conditions stimulated cellular differentiation into polarized mucin-secreting and multi-ciliated cells, recapitulating the architecture of human Fallopian tube epithelium. The polarized and differentiated cells were infected with a clinical isolate of C. trachomatis, and inclusions containing chlamydial developmental forms were visualized by fluorescence and electron microscopy. Apical secretions from infected cells contained increased amounts of proteins associated with chlamydial growth and replication, including transferrin receptor protein 1, the amino acid transporters SLC3A2 and SLC1A5, and the T cell chemoattractants CXCL10, CXCL11, and RANTES. Flow cytometry revealed that chlamydial infection induced cell surface expression of T cell homing and activation proteins including ICAM-1, VCAM-1, HLA class I and II, and IFNγR. This human Fallopian tube epithelial cell culture model is an important tool with translational potential for studying cellular responses to Chlamydia and other sexually-transmitted pathogens.

Summary Memory CD8+ T cells are an essential component of anti-tumour and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumours and infections are not well known. Activation of co-stimulatory receptors, including CD28 and natural killer group 2D (NKG2D), activate phosphatidylinositol-3 kinase and subsequently may activate the mTOR pathway in CD8 + T cells. This study compared the activation of the mTOR signalling pathway after co-stimulation through CD28 or NKG2D receptors in murine effector CD8 + T cells. Compared with CD28 co-stimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1, as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6 and eukaryotic initiation factor 4E binding protein 1. NKG2D co-stimulation also showed increased gene expression of tuberous sclerosis protein 2, a negative regulator of mTORc1, whereas CD28 co-stimulation increased gene expression of Ras homologue enriched in brain, an activator of mTORc1, and hypoxia-inducible factor-1a and vascular endothelial growth factor-a, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-co-stimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, B lymphocyte-induced maturation protein (BLIMP-1), interferon regulatory factor 4, and inhibitor of DNA binding 2, whereas low levels of mTORc1 activation allowed for the expression of Eomes, B-cell lymphoma 6 (BCL6), and inhibitor of DNA binding 3 during NKG2D stimulation, and increased expression of memory markers CD62 ligand and CD127. These data show that compared with CD28, co-stimulation through the NKG2D receptor leads to the differential activation of the mTOR signalling pathway and potentially supports memory CD8 + T-cell differentiation.

Memory CD8+ T cells are an essential component of anti-tumor and anti-viral immunity. Activation of the mammalian/mechanistic target of rapamycin (mTOR) pathway has been implicated in regulating the differentiation of effector and memory T cells. However, the mechanisms that control mTOR activity during immunity to tumors and infections are not well known. Activation of costimulatory receptors, including CD28 and NKG2D, activate PI3K and subsequently may activate the mTOR pathway in CD8+ T cells. Therefore this study compared the activation of the mTOR signaling pathway after costimulation through CD28 or NKG2D receptors in murine effector CD8+ T cells. Compared to CD28 costimulation, activation through CD3 and NKG2D receptors had weaker activation of mTORc1 as shown by decreased phosphorylation of mTORc1 targets S6K1, ribosomal protein S6, and 4EBP-1. NKG2D costimulation also showed increased gene expression of TSC2, a negative regulator of mTORc1, whereas CD28 costimulation increased gene expression of Rheb, an activator of mTORc1, and HIF-1α and VEGFα, pro-angiogenic factors downstream of mTORc1. Strong mTORc1 activation in CD28-costimulated cells also increased expression of transcription factors that support effector cell differentiation, namely T-bet, and BLIMP1, whereas low levels of mTORc1 activation allowed for the expression of Eomes and BCL6 during NKG2D stimulation, and increased expression of central memory markers CD62L and CD127. These data show that compared to CD28, costimulation through the NKG2D receptor leads to the differential activation of the mTOR signaling pathway and potentially supports memory CD8+ T cell differentiation. Citation Format: Bryan McQueen, Kelsey Trace, Emily Whitman, Amorette Barber. NKG2D and CD28 receptors differentially activate mTOR to alter murine effector CD8+ T cell differentiation. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A05.

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with TLR-signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 in immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3 -/- mice had no difference in chlamydial burden or duration of lower genital tract infection. We also observed a similar incidence of oviduct hydrosalpinx 45 days post-infection in trem1,3 -/- compared to WT mice. However, compared to WT, trem1,3 -/- mice developed significantly fewer uterine horn hydrometra. Early in infection, trem1,3 -/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased G-CSF. Trem1,3 -/- mice also had reduced erosion of the luminal epithelium. These data indicate TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the development of uterine hydrometra in infected mice.

Chlamydia trachomatis (Ct) causes the most prevalent bacterial sexually transmitted disease leading to ectopic pregnancy and infertility. Swine not only have many similarities to humans, but they are also susceptible to Ct. Despite these benefits and the ease of access to primary tissue from this food animal, in vitro research in swine has been underutilized. This study will provide basic understanding of the Ct host–pathogen interactions in porcine oviduct epithelial cells (pOECs)—the counterparts of human Fallopian tube epithelial cells. Using NanoString technology, flow cytometry, and confocal and transmission-electron microscopy, we studied the Ct developmental cycle in pOECs, the cellular immune response, and the expression and location of the tight junction protein claudin-4. We show that Ct productively completes its developmental cycle in pOECs and induces an immune response to Ct similar to human cells: Ct mainly induced the upregulation of interferon regulated genes and T-cell attracting chemokines. Furthermore, Ct infection induced an accumulation of claudin-4 in the Ct inclusion with a coinciding reduction of membrane-bound claudin-4. Downstream effects of the reduced membrane-bound claudin-4 expression could potentially include a reduction in tight-junction expression, impaired epithelial barrier function as well as increased susceptibility to co-infections. Thereby, this study justifies the investigation of the effect of Ct on tight junctions and the mucosal epithelial barrier function. Taken together, this study demonstrates that primary pOECs represent an excellent in vitro model for research into Ct pathogenesis, cell biology and immunity.