Diverse Roles For T-Bet In The Immune Response To Toxoplasma Gondii

The T-box transcription factor T-bet is most prominently known for its role in production of the cytokine interferon-γ (IFNγ) by effector T cells after infection with Th1-inducing pathogens. Here, we demonstrate additional roles for T-bet during effector T cell responses including an essential function in T cell trafficking to secondary sites of infection during toxoplasmosis. Mice that are deficient in T-bet are unable to survive infection with the intracellular parasite Toxoplasma gondii, and this mortality is caused by uncontrolled parasite replication at secondary sites of infection and is associated with a paucity of T cells at these sites. Additionally, we provide evidence that T-bet is also involved in early events of T cell priming and expansion. Together, the data presented in this thesis provide a better understanding of the diverse roles that T-bet plays in the generation and function of T cell responses during the immune response to T. gondii

Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses

Increased T cell responses to <i>Toxoplasma</i> antigen and crude commensal antigen in <i>Ahr</i>^<i>-/-</i> mice following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with 100 <i>T</i>. <i>gondii</i> cysts for 7 days. <b>(A, B)</b> Splenocytes were stimulated with the indicated antigen preparations for 5 hours and then incubated overnight with brefeldin A. The cells were stained to assay IFN-γ expression by CD4⁺ T cells. The graph shows pooled data from 3 independent experiments. <b>(C)</b> Parasite burdens in the terminal ileum were assayed by RT-PCR. Results are pooled from 2 separate experiments.</p

<i>Ahr</i>^<i>-/-</i> mice exhibit increased T cell activation following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with <i>T</i>. <i>gondii</i> for 9 days. <b>(A)</b> Group 3 ILC frequency in the lamina propria of wild type or <i>Ahr</i>^<i>-/-</i> mice following infection. The plots on the left are gated on live CD90.2⁺CD11c^-B220^- cells. (<b>B)</b> Weight loss was monitored at various days post-infection. Data are pooled from 2 experiments with 6–8 mice per group. <b>(C)</b> H&E staining of small intestinal tissue sections. <b>(D)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse. The Peyer’s patch exhibits severe lymphocytolysis (*) and the lamina propria of adjacent villi is expanded by primarily lymphocytes and plasma cells (➜). <b>(E)</b> A higher magnification image of the section in Fig 2C shows that the Peyer’s patch exhibits severe lymphocytolysis characterized by pyknosis and other cellular debris. <b>(F)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse shows crypt loss (*) and multifocal necrotic enterocytes (➜). The lamina propria of the villi is expanded by lymphocytes and plasma cells (❋). <b>(G)</b> Expression of T-bet and Ki67 by FoxP3^- CD4⁺ T cells in the mesenteric lymph nodes of infected mice. Results are pooled from 3 separate experiments. <b>(H)</b> Cytokine production by CD4⁺ T cells following stimulation with PMA/ionomycin. Data are pooled from 2 experiments.</p

Treg phenotype and IL-10 production in orally infected <i>Ahr</i>^<i>-/-</i> mice.

<p><i>Ahr</i>^<i>-/-</i> mice or wild type controls were infected orally with 20 Me49 cysts for 9 days. Results are pooled from 2–3 separate experiments. <b>(A)</b> Frequency of Tregs in the indicated tissues of wild type or <i>Ahr</i>^<i>-/-</i> mice. In the lamina propria, the plots shown are gated on live CD45⁺CD3⁺CD4⁺ cells, and Tregs were gated as live CD45⁺CD3⁺CD4⁺FoxP3⁺ cells. For the mesenteric lymph node and spleen, the plots shown are gated on CD3⁺CD4⁺ cells, and Tregs were gated as CD3⁺CD4⁺FoxP3⁺ cells. <b>(B)</b> Expression of T-bet, CXCR3, and Ki67 on Tregs in the spleens of wild type or <i>Ahr</i>^<i>-/-</i> mice. The plots are gated on CD3⁺CD4⁺FoxP3⁺ cells. <b>(C)</b> IL-10 secretion by cells isolated from the lamina propria or the spleen following restimulation with soluble <i>Toxoplasma</i> antigen. Results are pooled from 2 separate experiments with a total of 5–7 mice per group.</p

Spontaneous partial loss of the OT-I transgene.

Nat Immunol 2016 May; 17(5):471

IL-27 Limits Type 2 Immunopathology Following Parainfluenza Virus Infection

<div><p>Respiratory paramyxoviruses are important causes of morbidity and mortality, particularly of infants and the elderly. In humans, a T helper (Th)2-biased immune response to these infections is associated with increased disease severity; however, little is known about the endogenous regulators of these responses that may be manipulated to ameliorate pathology. IL-27, a cytokine that regulates Th2 responses, is produced in the lungs during parainfluenza infection, but its role in disease pathogenesis is unknown. To determine whether IL-27 limits the development of pathogenic Th2 responses during paramyxovirus infection, IL-27-deficient or control mice were infected with the murine parainfluenza virus Sendai virus (SeV). Infected IL-27-deficient mice experienced increased weight loss, more severe lung lesions, and decreased survival compared to controls. IL-27 deficiency led to increased pulmonary eosinophils, alternatively activated macrophages (AAMs), and the emergence of Th2 responses. In control mice, IL-27 induced a population of IFN-γ⁺/IL-10⁺ CD4⁺ T cells that was replaced by IFN-γ⁺/IL-17⁺ and IFN-γ⁺/IL-13⁺ CD4⁺ T cells in IL-27-deficient mice. CD4⁺ T cell depletion in IL-27-deficient mice attenuated weight loss and decreased AAMs. Elimination of STAT6 signaling in IL-27-deficient mice reduced Th2 responses and decreased disease severity. These data indicate that endogenous IL-27 limits pathology during parainfluenza virus infection by regulating the quality of CD4⁺ T cell responses and therefore may have therapeutic potential in paramyxovirus infections.</p></div

Composition of total cell populations, mCherry^+veViolet^−ve cell populations, and mCherry^+veViolet^+ve populations from the PECS of naïve and vaccinated mice.

<p>Mice were vaccinated with 10⁶ Violet-labeled, mCherry-expressing <i>cpsII</i> parasites intraperitoneally and sacrificed 18 hours post-vaccination. Cell type composition of total peritoneal cell populations in naïve and vaccinated mice, and the cell type composition of mCherry^+veViolet^−ve cells and mCherry^+veViolet^+ve cells in vaccinated mice were examined. Representative flow plots demonstrating infected cells and cells that have phagocytosed <i>T. gondii</i> for each major cell type present in the PECS are shown (a). The composition of the PECS in naïve mice and vaccinated mice, and the composition of infected cells (mCherry^+veViolet^+ve) and cells that have phagocytosed <i>T. gondii</i> (mCherry^+veViolet^−ve) are depicted (b). Percentages of macrophages and dendritic cells in the total peritoneal cell population in vaccinated mice are compared to the percentages of infected cells that are macrophages and dendritic cells (c). T/B/NK cells are identified by expression of CD3, CD19, or NK1.1. Dendritic cells were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^HI,MHCII^HI. Monocytes and neutrophils were defined as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^+ve. Macrophages were identified as CD3^−ve,CD19^−ve,NK1.1^−ve,CD11c^LOW-INT,Gr-1^−ve,CD11b^INTorHI. *p<0.05; ***p<0.0005. AVG±STDEV. A paired, two-tailed student's t test was used to analyze the data in (c). Results shown are from one representative experiment. Similar results were obtained over the course of seven separate experiments.</p