B Lymphocytes Are Required during the Early Priming of CD4\u3csup\u3e+\u3c/sup\u3e T Cells for Clearance of \u3cem\u3ePneumocystis\u3c/em\u3e Infection in Mice

B cells play a critical role in the clearance of Pneumocystis. In addition to production of Pneumocystis-specific Abs, B cells are required during the priming phase for CD4+ T cells to expand normally and generate memory. Clearance of Pneumocystis was found to be dependent on Ag specific B cells and on the ability of B cells to secrete Pneumocystis-specific Ab, as mice with B cells defective in these functions or with a restricted BCR were unable to control Pneumocystis infection. Because Pneumocystis-specific antiserum was only able to partially protect B cell–deficient mice from infection, we hypothesized that optimal T cell priming requires fully functional B cells. Using adoptive transfer and B cell depletion strategies, we determined that optimal priming of CD4+ T cells requires B cells during the first 2–3 d of infection and that this was independent of the production of Ab. T cells that were removed from Pneumocystis-infected mice during the priming phase were fully functional and able to clear Pneumocystis infection upon adoptive transfer into Rag1−/− hosts, but this effect was ablated in mice that lacked fully functional B cells. Our results indicate that T cell priming requires a complete environment of Ag presentation and activation signals to become fully functional in this model of Pneumocystis infection

Aqueous Vernomia amygdalina Extracts Alter MCF-7 Cell Membrane Permeability and Efflux

Breast cancer is the second leading cause of cancer related deaths of women in the United States. Several treatment strategies have been developed over the past decade to reduce cancer morbidity and mortality rates. While mortality rates have declined in some ethnic populations, the overall cancer incidence continues to grow. Hence, chemotherapeutic agents are needed to improve cancer treatment outcome. Previous studies show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retard the proliferative activities of estrogen receptor positive (ER+) human breast cancerous cells (MCF-7) cells in vitro in a concentration-dependent fashion. The anti-proliferative activities of VA are extracellular signal-regulated kinases 1/2 (ERKs 1/2)-dependent. Cell culture and animal model studies, conducted by other investigators using other plant extracts, have also revealed that plant extract components called thionins may be responsible for their anticancer activities. These thionins are believed to interact with the cells in ways that compromise membrane potential/permeability resulting in the alteration of efflux, cytosolic activities, and subsequent cell death. Therefore, we hypothesized that VA exposure may compromise cell membrane as another mode of action to elicit its anticancer activities in MCF-7 cells. The exposure of cells to VA decreased [3H]thymidine uptake in a concentration-dependent (0, 30, and 100 μg/ml VA) manner (p < 0.05) but increased [3H]thymidine release, expressed as percent of [3H]thymidine incorporated, into the medium (p < 0.05). The amount of [3H]thymidine released into the medium was 1.7, 7.4, and 11.0 % for 0, 30, and 100 μg/ml VA respectively. Thus suggesting the membranes in VA-treated cells were compromised in a concentration-dependent fashion

Correction: IFN-γ and IL-21 Double Producing T Cells Are Bcl6-Independent and Survive into the Memory Phase in Plasmodium chabaudi Infection.

[This corrects the article DOI: 10.1371/journal.pone.0144654.]

IL-10 regulates generation of both IL-21⁺IFN-γ⁺ and IFN-γ⁺ GC Tfh cells.

<p>Wildtype (WT) and IL-10 deficient mice (IL-10 KO) were infected and 7 days later splenocytes were analyzed by intracellular staining. Contour plots and bar graphs from IL-10 KO and WT controls showing expression of (A) CXCR3 and T-bet, and (B) IL-12Rβ2 in the IFN-γ⁺ Teff (CD127^-) population; (C) IFN-γ and IL-21 in all Teff, and (D) PD-1 and CXCR5 in the IFN-γ⁺ Teff population. Data are representative of two independent experiments with 3–4 animals per group. Statistical significance from Students <i>t</i>-test. Error bars represent the SEM; ^★<i>p</i> < 0.05, ^★★<i>p</i> < 0.01, ^★★★<i>p</i> < 0.001), ns = not significant.</p

Expression of markers of Th1 differentiation is reduced at the peak of IFN-γ production.

<p><i>Ifng/Thy1</i>.<i>1</i> KI mice were infected with <i>P</i>. <i>chabaudi</i> iRBC, and splenocytes were analyzed. (A) Contour plots show expression of CXCR3, T-bet, and RUNX3 gated on CD4⁺<i>Ifng</i>/<i>Thy1</i>.<i>1</i>⁺ on days 5, 7, and 9 post-infection. The numbers depicted on the plots are average percentages. Gates were drawn using fluorescence minus one (FMO, CXCR3) or isotype (T-bet, Runx3) controls for each day, as shown to the left. (B) Bar graphs showing percentages of CXCR3, T-bet, and RUNX3 positive cells in Thy1.1⁺ Teff population on each day. Data is summarized in (C) pie charts of Boolean gating analysis of all possible combinations of CXCR3⁺, Runx3⁺, and T-bet⁺ within CD4⁺<i>Ifng</i>/<i>Thy1</i>.<i>1</i>⁺ effector T cells. <i>Ifng/Thy1</i>.<i>1</i>⁺ T cells expressing all three Th1 markers are shown in black. Two markers are shown in dark grey, and one marker is indicated by light grey. Data are representative of three independent experiments with three animals per timepoint. Statistical significance was obtained using Students <i>t</i> test. Error bar represents SEM; ^★<i>p</i> < 0.05, ^★★<i>p</i> < 0.01, <i>ns</i> = not significant.</p

Bcl6 controls generation of GC Tfh, but not cytokine profile, in responding effector cells.

<p>(A) Naïve (CD44^loCD25^-) CD4 T cells (2x10⁶) from either Bcl6^fl/fl CD4^Cre (Bcl6 cKO) or Bcl6^fl/fl (WT) were labeled with cell trace violet (CTV) and adoptively transferred into Ly5.1 (CD45.1) congenic mice, followed by <i>P</i>. <i>chabaudi</i> infection. On day 7 post-infection, splenocytes were harvested and stained with (B) CD4, CD45.2, CTV, (C, D) PD-1, CXCR5, (E) IFN-γ, IL-21, and (F) IFN-γ, IL-10. (B) Plots showing the gating on responding CD4⁺CD45.2⁺CTV^- T cells. (C) Graph shows percentages for individual recipients of effector T cell subsets. No CD45.2⁺ CXCR5^hiPD-1^hi GC Tfh cells were detected in any recipient of Bcl6 cKO T cells. (D) Bar graph shows CXCR5 MFI of CD4⁺CD45.2⁺CTV^- donor cells. (E) Plots and bar graph of average IFN-γ and IL-21 cytokine producers in the responding donor cells (CD45.2⁺CTV^-) in recipients of WT and Bcl6 cKO T cells. (F) Dot plot showing intracellular cytokine staining. Data are representative of three independent experiments with 4–5 animals per group. Numbers within plots represent mean percentages. Statistical significance was obtained using Students <i>t</i>-test. Error bars represent the SEM; ^★<i>p</i> < 0.05, ^★★★<i>p</i> < 0.001, ns = not significant.</p

IFN-γ⁺ CD4 memory T cells maintain CXCR5 expression, but little T-bet.

<p>BAC-In mice were infected and splenocytes were analyzed by flow cytometry on day 60 post-infection. (A) CD4⁺<i>Ifng</i>/<i>Thy1</i>.<i>1</i>⁺ memory (CD44^hiCD127⁺, green box, gate set on CD4⁺) T cells were gated and expression of CXCR3, T-bet, CXCR5, and Bcl6 was measured. Numbers represent mean percentages. (B, C) Surface staining of CD4⁺ T cells with CD4, CD44, CD127, and Thy1.1 (yellow) was followed by intracellular staining with Bcl-6 (green), T-bet (red), and nuclei with DAPI (purple). Cells were analyzed by imaging flow cytometry. (B) Histogram showing expression of <i>Ifng</i>/<i>Thy1</i>.<i>1</i> on single focused cells. Bar graphs show MFI of Bcl6 and T-bet within Thy1.1 gates (C) Representative images of individual <i>Ifng</i>/<i>Thy1</i>.<i>1</i> high and low cells showing Bcl6 and T-bet expression and localization in relation to the DAPI stained nucleus. Bright field (BF; left) and DAPI (nucleus)/Bcl6/T-bet merged images also shown (right). (D) Real time PCR analysis of <i>bcl6</i>, <i>tbx21</i>, <i>prdm1</i>, and <i>eomes</i> of CD4⁺Thy1.1⁺ sorted T cells. Results were normalized to control gene <i>rp18s</i>. RNA from FACS Sorted naïve (CD44^loCD25^-) cells from uninfected, aged-matched BAC-In mice was used as control. Data are representative of three (A) and one (B, C, D) independent experiments with 3–4 animals per group. Statistical significance shown using Students <i>t</i>-test. Error bars represent the SEM; ^★<i>p</i> < 0.05, ^★★<i>p</i> < 0.01, ns = not significant.</p