Normal Development and Function of T Cells in Proline Rich 7 (Prr7) Deficient Mice

Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling

Ly6G deficiency alters the dynamics of neutrophil recruitment and pathogen capture during Leishmania major skin infection.

Neutrophils represent one of the first immune cell types recruited to sites of infection, where they can control pathogens by phagocytosis and cytotoxic mechanisms. Intracellular pathogens such as Leishmania major can hijack neutrophils to establish an efficient infection. However the dynamic interactions of neutrophils with the pathogen and other cells at the site of the infection are incompletely understood. Here, we have investigated the role of Ly6G, a homolog of the human CD177 protein, which has been shown to interact with cell adhesion molecules, and serves as a bona fide marker for neutrophils in mice. We show that Ly6G deficiency decreases the initial infection rate of neutrophils recruited to the site of infection. Although the uptake of L. major by subsequently recruited monocytes was tightly linked with the concomitant uptake of neutrophil material, this process was not altered by Ly6G deficiency of the neutrophils. Instead, we observed by intravital 2-photon microscopy that Ly6G-deficient neutrophils entered the site of infection with delayed initial recruitment kinetics. Thus, we conclude that by promoting neutrophils' ability to efficiently enter the site of infection, Ly6G contributes to the early engagement of intracellular pathogens by the immune system

CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission.

The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection

Prr7 deficiency does not influence T cell response to <i>Listeria monocytogenes</i> infection.

<p>(A) <i>Prr7</i>^-/- mice and <i>Prr7</i>^+/+ control mice were i.v. infected with 1x10⁴ Lm ova. On day 9 post infection, colony forming units were determined in spleen and liver. (B) Representative dot plot of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (C) Frequency of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (D) Absolute number of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (E) Frequency of CD8⁺ naive (CD62L⁺CD44^-), activated (CD62L^-CD44⁺) and memory (CD62L⁺CD44⁺) T cells in the spleen of infected mice. (F) Absolute number of CD8⁺ naive, activated and memory T cells in the spleen of infected mice. (G-K) Splenocytes of infected mice were restimulated with Ova_257-264-peptide (SIINFEKL, 10⁻⁸ M) or left unstimulated for 12 h in the presence of Brefeldin A, to allow for the intracellular accumulation of cytokines. (G) Dot plot of IFN-γ and TNF-producing CD8⁺ T cells without restimulation. (H) Dot plot of TNF-producing CD8⁺ T cells after restimulation. (I) Dot plot of IFN-γ producing CD8⁺ T cells after restimulation. (J) Frequency of IFN-γ and TNF-producing CD8⁺ T cells in the spleen of infected mice. (K) Absolute number of IFN-γ and TNF-producing CD8⁺ T cells in the spleen of infected mice. Data are represented as mean ± SEM of 4–5 mice per group. n.s. not significant.</p

TCR response and AICD is unaffected in T cells from Prr7-deficient mice.

<p>(A) Flow cytometry analysis of the activation marker CD69 in Prr7-deficient CD4⁺ or CD8⁺ T cells stimulated with 1 μg of plate-bound anti-CD3 for 24 or 36 h. (B) Proliferation of Prr7^+/+ and Prr7^-/- splenocytes in response to TCR stimulation with plate-bound anti-CD3 measured as [³H]thymidine incorporation (DNA synthesis). cpm, counts per minute. (C) Schema of the <i>in vitro</i> AICD induction protocol. (D) Representative examples of flow cytometry analysis of AICD in T cells upon restimulation. Live = PI^-Annexin V^-, Apoptotic = PI^-Annexin V⁺, Dead = PI⁺Annexin V⁺ (E) Quantification and statistical analysis of AICD performed as shown in (D). (F) Immunoblotting of c-Jun total levels in restimulated T cells isolated from three different wild-type and knockout mice (#1, #2, #3). Tubulin served as a loading control. Data in (A, B, D) represent the mean + SEM of at least three animals per group. n.s., not significant.</p

T cell development is largely unaffected in Prr7-deficient mice.

<p>(A) Total numbers of nucleated cells in the thymus (left) and spleen (right) isolated from Prr7^+/+ and Prr7^-/- mice as counted using a haemocytometer. (B) Schematic representation of T cell developmental stages in the thymus. DN, double negative, DP, double positive, SP, single positive. Lower panels with dot plots are representative examples of flow cytometry analysis of thymocyte subpopulations. Percentages of DN subpopulations (C), DP subpopulations (D), and SP subpopulations (E) in thymi of Prr7^+/+ and Prr7^-/- mice as analysed by flow cytometry. (F) Flow cytometry analysis of CD4⁺ and CD8⁺ T cells subpopulations in the secondary lymphatic organs spleen and lymph nodes expressed as percentage of total. (G) Flow cytometry analysis of splenic CD3⁺ T cells expressing markers of naïve T cells (CD62⁺CD25^-), activated T cells (CD62L^-CD25⁺), or memory T cells (CD62L^-CD25⁺). Data in (A-G) represent the mean +SEM, n = 3–8. *p < 0.05, n.s., not significant.</p

Absence of Prr7 does not affect the susceptibility of mice to EAE.

<p>(A) Clinical EAE scores in 8–12 week old WT and Prr7 knockout female mice upon immunization with MOG peptide over time. (B) Cumulative EAE score from (A). (C) Maximum EAE scores from (A). Data are represented as mean +/- SEM of 13 (Prr7^-/-) and 14 (Prr7^+/+) mice per group from two independent experiments. n.s. not significant.</p

Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells

International audienceNitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells