Fig 1.
IFN-γ-producing effector cells expand and contract with kinetics similar to parasitemia.
Ifng/Thy1.1 Knock-In (KI) mice were infected with P. chabaudi infected RBC (iRBC). Splenocytes were harvested and analyzed by flow cytometry on various days post-infection (p.i.) and (A) were gated on CD4+ Ifng/Thy1.1+, shown at day 0 and 9 p.i. Average percentages from three animals are shown on plots. (B) CD4+ Ifng/Thy1.1+ (as gated in A) showing CD4+CD127- effector gate (red box) and Thy1.1+CD127- effector gate (blue box) at day 9 p.i. (C) Total numbers of CD4+ effector T cells and CD4+Ifng/Thy1.1+ T cells (as gated in B) per spleen overlaid with parasitemia curve (%infected RBCs/total RBCs). Data are representative of eight independent experiments with three animals per timepoint. Error bars represent SEM.
Fig 2.
Expression of markers of Th1 differentiation is reduced at the peak of IFN-γ production.
Ifng/Thy1.1 KI mice were infected with P. chabaudi iRBC, and splenocytes were analyzed. (A) Contour plots show expression of CXCR3, T-bet, and RUNX3 gated on CD4+Ifng/Thy1.1+ 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+Ifng/Thy1.1+ effector T cells. Ifng/Thy1.1+ 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 t test. Error bar represents SEM; ★p < 0.05, ★★p < 0.01, ns = not significant.
Fig 3.
The Ifng/Thy1.1+ T cell population expresses Tfh markers.
Splenocytes from day 7 p.i (A) C57Bl/6J mice were analyzed for expression of CXCR5, Blimp-1, ICOS, SLAM, and BTLA in naïve (CD44loCD127+, grey), IFN-γ+ (black line), and IFN-γ- (dotted line) effector T cells (CD127-) as determined by intracellular cytokine staining. Bar graphs show mean fluorescence intensity (MFI). (B) Contour plots of PD-1 and CXCR5 from Ifng/Thy1.1 knock-in (KI) mice effector T cells (CD4+CD127-, as shown in Fig 2) are gated on naïve T cells from uninfected, Ifng/Thy1.1+, and Ifng/Thy1.1- mice. Bar graph shows percentages of Ifng/Thy1.1+/—subsets out of total effector T cells. (C) Contour plot of IL-21 and CXCR5 expression. (D) Percentages of IL-21 within effector subsets. (E) Histogram showing Bcl6 expression in CXCR5-IL-21+ (C, red box), CXCR5+IL-21+ (C, blue box), and CXCR5+IL-21- (C, green box) subsets relative to naïve and Ifng/Thy1.1- GC Tfh T cells. Bar graph shows average MFI of Bcl6 staining shown for each subset. (F) Contour plots for IFN-γ, IL-21 and IL-10 by intracellular cytokine staining in C57Bl/6J splenocytes day 7 p.i. Data are representative of four independent experiments with 3 mice per group. Statistical significance was obtained using Students t test. Error bar represents SEM; ★p < 0.05, ★★p < 0.01, ★★★p < 0.001, ns = not significant.
Fig 4.
Level of T-bet in the nucleus correlates with Ifng/Thy1.1 expression.
Ifng/Thy1.1 BAC-In reporter mice were infected with P. chabaudi and splenocytes were analyzed on day 7 post-infection. After positive selection of Thy1.1 cells, cells were stained for surface CD4, CD127, and Thy1.1 (yellow), followed by intracellular staining for Bcl6 (green) and T-bet (red). Just before analysis, nuclei are stained with DAPI (purple), and analyzed by imaging flow cytometry. (A) Histogram showing Ifng/Thy1.1 expression is gated on single focused cells. Cells were gated on CD4+CD127- Teff and (B) expression of Bcl6 and T-bet was measured within Ifng/Thy1.1 high and low gates as shown in (A). (C) Representative images of individual cells from imaging flow cytometry. Bright Field (BF) and DAPI/Bcl-6/T-bet merged images are shown. Statistical significance was obtained using Students t test. Error bar represents SEM; ★p < 0.05, ★★p < 0.01.
Fig 5.
Bcl6 controls generation of GC Tfh, but not cytokine profile, in responding effector cells.
(A) Naïve (CD44loCD25-) CD4 T cells (2x106) from either Bcl6fl/fl CD4Cre (Bcl6 cKO) or Bcl6fl/fl (WT) were labeled with cell trace violet (CTV) and adoptively transferred into Ly5.1 (CD45.1) congenic mice, followed by P. chabaudi 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+ CXCR5hiPD-1hi 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 t-test. Error bars represent the SEM; ★p < 0.05, ★★★p < 0.001, ns = not significant.
Fig 6.
IL-10 regulates generation of both IL-21+IFN-γ+ and IFN-γ+ GC Tfh cells.
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 t-test. Error bars represent the SEM; ★p < 0.05, ★★p < 0.01, ★★★p < 0.001), ns = not significant.
Fig 7.
IFN-γ+ CD4 memory T cells maintain CXCR5 expression, but little T-bet.
BAC-In mice were infected and splenocytes were analyzed by flow cytometry on day 60 post-infection. (A) CD4+Ifng/Thy1.1+ memory (CD44hiCD127+, 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 Ifng/Thy1.1 on single focused cells. Bar graphs show MFI of Bcl6 and T-bet within Thy1.1 gates (C) Representative images of individual Ifng/Thy1.1 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 bcl6, tbx21, prdm1, and eomes of CD4+Thy1.1+ sorted T cells. Results were normalized to control gene rp18s. RNA from FACS Sorted naïve (CD44loCD25-) 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 t-test. Error bars represent the SEM; ★p < 0.05, ★★p < 0.01, ns = not significant.
Fig 8.
IFN-γ+ memory cells co-produce IL-21, and Ifng-accessibility and T-bet are maintained by division during chronic malaria infection.
(A) BAC-In mice (CD45.2) were infected and at day 7 post-infection, CD4+ Ifng/Thy1.1+ T cells were purified, CTV labeled, and adoptively transferred into infection-matched CD45.1 recipients. At day 60 post-infection, splenocytes were analyzed by flow cytometry. (B) Dot plot and bar graph shows expression of Ifng/Thy1.1 in recovered donor cells. (C) Contour plots gated on CD4+CD45.2+ show expression of CXCR3 and T-bet in recovered Ifng/Thy1.1+ and Ifng/Thy1.1- populations. Bar graph shows T-bet MFI. (D) Ifng/Thy1.1 and T-bet within the CTV- maximally divided cells. Bar graph shows T-bet MFI in each division (E) IFN-γ and IL-21 expression ex vivo in CD4+CD45.2+ donor cells. Data are representative of two independent experiments with 3–4 mice per experiment. Statistical significance shown using Students t-test. Error bars represent SEM; ★p < 0.05, ★★★p < 0.001.