Batf3-Deficient Mice: Susceptibility to Toxoplasma gondii and Responses to IL-12 Treatment in vivo

CD8α+ dendritic cells are important in vivo for cross-presentation of antigens derived from intracellular pathogens and tumors. Additionally, stimulation of IL-12 production by CD8α+ DCs has suggested a role for these cells in response to Toxoplasma gondii antigens, although no experiments have yet shown an in vivo requirement for these cells against T. gondii infection. Towards this goal, we examined T. gondii infection of Batf3-/- mice, which selectively lack only lymphoid-resident CD8α+ DCs and related peripheral CD103+ DCs. Batf3-/- mice were extremely susceptible to T. gondii infection, with defective priming of CD8+ T cells, and decreased production of IL-12 and IFNγ. IL-12 administration restored resistance in Batf3-/- mice, and mice in which IL-12 production was ablated only from CD8α+ DCs failed to control infection. These results reveal that the function of CD8α+ DCs extends beyond a role in cross-presentation and includes a critical role for activation of innate immunity through IL-12 production during T. gondii infection. While investigating the immune responses of Batf3-/- mice to T. gondii infection, we made the surprising discovery that IL-12 treatment of infected Batf3-/- mice resulted in re-appearance of the CD8α+ DC population in the spleen. In addition, we show that IL-12-treatment alone in the absence of infection restored the CD8α+ DC population in Batf3-/- mice. Analysis of the restored cells by microarray revealed very few differences in gene expression between wild-type and IL-12-induced Batf3-/- CD8α+ DCs. Furthermore, IL-12 treatment of Batf3-/- mice restored their capacity for in vivo cross-presentation of necrotic cell-associated antigens. Finally, the restored CD8α+ DCs primed CD8+ T cells against T. gondii-derived antigen, and produced IL-12 in vivo in response to T. gondii infection. Thus, IL-12 can induce development of CD8α+ DCs through a Batf3-independent mechanism, and these cells can function to both prime T cells as well as produce IL-12 during infection in vivo

Targeting of B and T lymphocyte associated (BTLA) prevents graft-versus-host disease without global immunosuppression

One-time treatment with an antibody against BTLA provides long-term protection against graft-versus-host disease without affecting effector T cell responses to tumors or pathogens

Adipose Tissue in Persons With HIV Is Enriched for CD4+ T Effector Memory and T Effector Memory RA+ Cells, Which Show Higher CD69 Expression and CD57, CX3CR1, GPR56 Co-expression With Increasing Glucose Intolerance

Chronic T cell activation and accelerated immune senescence are hallmarks of HIV infection, which may contribute to the increased risk of cardiometabolic diseases in people living with HIV (PLWH). T lymphocytes play a central role in modulating adipose tissue inflammation and, by extension, adipocyte energy storage and release. Here, we assessed the CD4+ and CD8+ T cell profiles in the subcutaneous adipose tissue (SAT) and blood of non-diabetic (n = 9; fasting blood glucose [FBG] < 100 mg/dL), pre-diabetic (n = 8; FBG = 100–125 mg/dL) and diabetic (n = 9; FBG ≥ 126 mg/dL) PLWH, in addition to non- and pre-diabetic, HIV-negative controls (n = 8). SAT was collected by liposuction and T cells were extracted by collagenase digestion. The proportion of naïve (TNai) CD45RO−CCR7+, effector memory (TEM) CD45RO+CCR7−, central memory (TCM) CD45RO+CCR7+, and effector memory revertant RA+(TEMRA) CD45RO−CCR7− CD4+ and CD8+ T cells were measured by flow cytometry. CD4+ and CD8+ TEM and TEMRA were significantly enriched in SAT of PLWH compared to blood. The proportions of SAT CD4+ and CD8+ memory subsets were similar across metabolic status categories in the PLWH, but CD4+ T cell expression of the CD69 early-activation and tissue residence marker, particularly on TEM cells, increased with progressive glucose intolerance. Use of t-distributed Stochastic Neighbor Embedding (t-SNE) identified a separate group of predominantly CD69lo TEM and TEMRA cells co-expressing CD57, CX3CR1, and GPR56, which were significantly greater in diabetics compared to non-diabetics. Expression of the CX3CR1 and GPR56 markers indicate these TEM and TEMRA cells may have anti-viral specificity. Compared to HIV-negative controls, SAT from PLWH had an increased CD8:CD4 ratio, but the distribution of CD4+ and CD8+ memory subsets was similar irrespective of HIV status. Finally, whole adipose tissue from PLWH had significantly higher expression of TLR2, TLR8, and multiple chemokines potentially relevant to immune cell homing compared to HIV-negative controls with similar glucose tolerance

Livedoid Dermatitis Treated With Nifedipine

Intravenous injection of buprenorphine as a cause of livedoid dermatitis is a recently described phenomenon. This report reviews the brief literature of this finding, and presents a case of livedoid dermatitis of both heels following injection more than one day prior, and thesuccessful treatment with nifedipine monotherapy

The Polymorphic Pseudokinase ROP5 Controls Virulence in <em>Toxoplasma gondii</em> by Regulating the Active Kinase ROP18

<div><p>Secretory polymorphic serine/threonine kinases control pathogenesis of <em>Toxoplasma gondii</em> in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 <em>in vitro</em>, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δ<em>rop5</em> and Δ<em>rop18</em> mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3^−/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.</p> </div

ROP5 regulates the kinase activity of ROP18.

<p>(A) Expression of ROP18 and ROP5 detected by western blotting of parasite lysates with rabbit anti-ROP18 (Rb α-ROP18), rabbit anti-ROP5 (Rb αROP5), and rabbit anti-actin (Rb αACT1) as a loading control. Representative of 3 experiments with similar outcomes. (B) Quantification of ROP18 expression by phosphorimager analysis of western blots, normalized for loading by actin staining. Means ± S.E.M. n = 3 experiments. (C) Immunofluorescence localization of ROP18 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>ΔKu80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. ROP18 was localized based on the C-terminal Ty-1 epitope described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Taylor1" target="_blank">[7]</a> using mAb BB2 (directly conjugated to Alexa Fluor 488, green). The vacuole membrane was stained with polyclonal rabbit anti-GRA7, described previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002992#ppat.1002992-Dunn1" target="_blank">[64]</a>, (secondary: Alexa Fluor 594, red). Scale = 5 microns. (D) Immunofluorescence localization of ROP5 on the parasitophorous vacuole membrane in wild type (RH<i>Δku80</i>) and ROP5 deficient (RH<i>Δku80Δrop5</i>) parasites infecting HFF cells <i>in vitro</i>. The vacuole membrane was labeled with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). ROP5 was labeled with polyclonal Ab for ROP5 (secondary: Alexa Fluor 488, green). Scale = 5 microns. (E) <i>In vitro</i> kinase reaction using the kinase domain of ROP18 (ROP18-KD, 100 ng) and the heterologous substrate dMBP ± recombinant ROP5 (rROP5, 200 ng). (F) <i>In vitro</i> kinase reaction using full length ROP18 (ROP18-FL, 25 ng) and the natural substrate Irgb6 ± recombinant ROP5 (rROP5, 50 ng). Irgb6 was immunoprecipitated (∼10–20 ng/reaction) from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. (G) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with polyclonal rabbit anti-ROP18 (Rb anti-ROP18). Bound (denoted as B) and unbound (denoted as UB) samples were resolved by SDS-PAGE and blotted for ROP18 (Rb anti-ROP18 biotin). (H) <i>In vitro</i> kinase reaction of ROP18 using the heterologous substrate dMBP. ROP18 immunoprecipitations from (G) were incubated with or without substrate in the presence of ³²P ATP. Reactions were resolved by SDS-PAGE and subjected to phosphorimager analysis. (I) Immunoprecipitation of ROP18 (∼5 ng/reaction) from parasite lysates with rabbit anti-ROP18 as in (G). (J) <i>In vitro</i> kinase reaction of ROP18 and a natural substrate Irgb6. ROP18 immunoprecipitations from (I) were incubated with or without substrate in the presence of ³²P ATP. Irgb6 was immunoprecipitated from IFN-γ activated RAW cells with polyclonal rabbit anti-Irgb6. Reactions in E, F, H, and J were carried out in the presence of ³²P ATP, resolved by SDS-PAGE and subjected to phosphorimager analysis. E–J are representative of 3 or more experiments with similar outcomes.</p

ROP5 and ROP18 are required for avoidance of clearance and IRG recruitment.

<p><i>In vitro</i> clearance of parasites in naïve peritoneal macrophages (A) or Gr1⁺ inflammatory monocytes (B) was measured by immunofluorescence microscopy. Cells were stained at 0.5 and 20 h post infection for host cell surface markers (see methods) and the parasite surface marker SAG1. Infection rates at 20 h post infection were normalized to initial infection rates. Means ± S.E.M., n = 3 samples each, from 3 combined experiments. Student's <i>t</i> test, **<i>P</i><0.0005. (C) Immunofluorescence localization of Irgb6 on the parasitophorous vacuole membrane in Gr1⁺ inflammatory monocytes infected for 0.5 h <i>in vitro</i>. The vacuole membrane was visualized by staining with mAb Tg 17-113 for GRA5 (secondary: Alexa Fluor 594, red). Irgb6 was visualized with rabbit anti-Irgb6 (secondary: Alexa Fluor 488, green). Scale = 5 microns. (D) Quantification of Irgb6 localization to the vacuolar membrane in Gr1⁺ monocytes. Mean ± SEM, n = 3 samples each, from 3 combined experiments. Student's <i>t</i> test, *<i>P</i><0.005.</p