Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.

Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings

Exacerbated susceptibility to infection-stimulated immunopathology in CD1d-deficient mice.

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms

Cognate interaction with iNKT cells expands IL-10-producing B regulatory cells

Successful induction of B-cell activation and memory depends on help from CD4(+) T cells. Invariant natural killer T (iNKT) cells (glycolipid-specific, CD1d-restricted innate lymphocytes) provide both cognate (direct) and noncognate (indirect) helper signals to enhance B-cell responses. Both forms of iNKT-cell help induce primary humoral immune responses, but only noncognate iNKT-cell help drives humoral memory and plasma cells. Here, we show that iNKT cognate help for B cells is fundamentally different from the help provided by conventional CD4(+) T cells. Cognate iNKT-cell help drives an early, unsustained germinal center B-cell expansion, less reduction of T follicular regulatory cells, an expansion of marginal zone B cells, and early increases in regulatory IL-10–producing B-cell numbers compared with noncognate activation. These results are consistent with a mechanism whereby iNKT cells preferentially provide an innate form of help that does not generate humoral memory and has important implications for the application of glycolipid molecules as vaccine adjuvants

Zika virus infection in immunocompetent pregnant mice causes fetal damage and placental pathology in the absence of fetal infection

<div><p>Zika virus (ZIKV) infection during human pregnancy may cause diverse and serious congenital defects in the developing fetus. Previous efforts to generate animal models of human ZIKV infection and clinical symptoms often involved manipulating mice to impair their Type I interferon (IFN) signaling, thereby allowing enhanced infection and vertical transmission of virus to the embryo. Here, we show that even pregnant mice competent to generate Type I IFN responses that can limit ZIKV infection nonetheless develop profound placental pathology and high frequency of fetal demise. We consistently found that maternal ZIKV exposure led to placental pathology and that ZIKV RNA levels measured in maternal, placental or embryonic tissues were not predictive of the pathological effects seen in the embryos. Placental pathology included trophoblast hyperplasia in the labyrinth, trophoblast giant cell necrosis in the junctional zone, and loss of embryonic vessels. Our findings suggest that, in this context of limited infection, placental pathology rather than embryonic/fetal viral infection may be a stronger contributor to adverse pregnancy outcomes in mice. Our finding demonstrates that in immunocompetent mice, direct viral infection of the embryo is not essential for fetal demise. Our immunologically unmanipulated pregnancy mouse model provides a consistent and easily measurable congenital abnormality readout to assess fetal outcome, and may serve as an additional model to test prophylactic and therapeutic interventions to protect the fetus during pregnancy, and for studying the mechanisms of ZIKV congenital immunopathogenesis.</p></div

Maternal ZIKV infection induces placental damage.

<p>Pregnant dams at E9.5 were infected with 3.4 × 10⁵ PFU of ZIKV and sacrificed at 3 dpi. Paraffin-embedded placental tissues were stained with H&E. Representative images are shown. (<b>A</b>) Labyrinth trophoblast hyperplasia in the placentas of infected dams. Arrows indicate mitotic labyrinth trophoblasts. Scale bar, 50 μm or 10 μm. (<b>B</b>) Trophoblast giant cell necrosis in the junctional zone. Arrows indicate normal tissue (left) or necrotic cell debris (right). D, decidua; JZ, junctional zone. Scale bar, 25 μm. (<b>C</b>) Thrombi in maternal or embryonic blood vessels in the labyrinth. Arrows indicate thrombi. Scale bar, 50 μm. (<b>D</b>) Loss of embryonic blood vessels, characterized by the absence of nucleated embryonic red blood cells, in the labyrinth of infected dams. Arrows indicate nucleated embryonic red blood cells. Scale bar, 50 μm.</p

ZIKV infection of pregnant C57BL/6J mice results in fetal demise caused by live infectious virus.

<p>(<b>A</b>) Scheme of infection and the follow up analyses. For <b>B</b> and <b>C</b>, pregnant dams at embryonic day 9.5 (E9.5) were infected with 3.4 × 10⁵ PFU of ZIKV by intravenous route. Control mice were left untreated (uninfected), or inoculated with Vero cell culture supernatant (Mock) or 3.4 × 10⁵ PFU-equivalent of heat-inactivated ZIKV (Inact. ZIKV). For <b>D</b> and <b>E</b>, ZIKV was left untreated (ZIKV) or pretreated with mouse IgG2a isotype (Isotype), the neutralizing anti-ZIKV mAb (mAb ZK-67), or the immune serum harvested from ZIKV-infected adult mice (ZIKV immune serum). Pregnant dams at E9.5 were then infected with 3.4 × 10⁵ PFU-equivalent of treated ZIKV by intravenous route. Control mice were inoculated with Vero cell culture supernatant (Mock). Mice were sacrificed 8 days post infection (dpi) at E17.5, and maternal tissues, placentas and embryos were harvested and examined. (<b>B</b>) Representative images of E17.5 uteri. The uteri of ZIKV-infected mice showed complete or partial fetal demise. Arrows indicate the placental residues of resorbed embryos. Arrowheads indicate the remaining intact embryos. (<b>C</b>) Impact of ZIKV infection on dams and embryos at E17.5. The percentage of dams showing abnormal pregnancy (i.e. at least one embryo in the litter showed morphological abnormality or suffered demise/resorption) is shown in the left panel. The percentage of embryos that were affected (i.e. had undergone demise/resorption, or exhibited any sign of growth restriction, malformation, or anatomical developmental defect by gross morphological examination) is shown in the right panel. Numbers on bars indicate normal pregnancy/embryo (top) or abnormal pregnancy/affected embryo (bottom). (ns, not significant; **** p<0.0001 compared with numbers in uninfected group by Fisher’s exact test). (<b>D</b>) Representative images of uteri and embryos or placental residues carried by dams infected with pretreated ZIKV. The uteri and embryos of dams infected with mAb- or immune serum-treated ZIKV showed no abnormality compared with those of mock infection. (<b>E</b>) Impact of infection with pretreated ZIKV on dams and embryos at E17.5, as described in (C). (ns, not significant; ** p<0.01; **** p<0.0001 compared with numbers in Mock group by Fisher’s exact test). Data for all panels are pooled from 5 (B and C) or 2 (D and E) independent experiments.</p

ZIKV antigen co-localizes with embryonic endothelial cells in the placental labyrinth and associates with damage.

<p>Pregnant dams at E9.5 were infected with 3.4 × 10⁵ PFU of ZIKV and sacrificed at 3 and 4 dpi. Paraffin-embedded placental tissues were stained with H&E (<b>A</b> and <b>C</b>) or ZIKV E protein (red) with hematoxylin counterstain (blue) (<b>B</b> and <b>C</b>). Representative images of the embryonic blood vessels in the placental labyrinth are shown. (<b>A</b>) Necrosis within the embryonic blood vessels. (<b>B</b>) ZIKV antigen was detected within the embryonic endothelial cells in embryonic blood vessels of the placental labyrinth. Arrows indicate nucleated embryonic red blood cells. Arrowheads indicate the staining of ZIKV antigen and highlight morphology of infected cells and antigen staining pattern. Asterisks indicate areas of necrotic cell debris. (<b>C</b>) Tissues from uninfected dams at the same gestational age were used as negative controls. MS, maternal sinus; EV, embryonic blood vessel. Scale bar, 50 μm or 25 μm.</p

Newborns of dams infected at E12.5 exhibit a significant rate of early postnatal death.

<p>Pregnant dams were infected with 3.4 × 10⁵ PFU at E12.5, and sacrificed 5 dpi at E17.5 or allowed to carry to term. Both dams and newborns were examined within 24 h after giving birth. (<b>A</b>) Scheme of infection. (<b>B</b>) E17.5 embryo and newborn weight. Data shown are mean with SD. (ns, not significant; **** p<0.0001 compared with the uninfected group by Student <i>t</i> test) (<b>C</b>) Newborn viability. Numbers on bars indicate newborns that were alive (top) or born alive but dead within 24 h after birth (bottom). (ns, not significant; ** p<0.01 compared with numbers in uninfected group by Fisher’s exact test). Data for all panels are pooled from 2–3 independent experiments. N/A, not applicable.</p