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Results: 4

1.
Fig. 3

Fig. 3. From: Late Interleukin-6 escalates T follicular helper cell responses and controls a chronic viral infection.

Late blockade of IL-6 or IL-6R reduces T follicular helper responses, B cell responses and delays viral clearance. (A–E) WT mice were infected with LCMV Cl 13. Mice received either 150μg of IL6R mAb i.p. every 5 days between days 20 and 45 p.i. or an initial dose of 0.5 mg followed by 0.25 mg i.p. every 2 days of IL6 mAb between days 20 and 35 p.i. Control groups were given the equivalent dose, isotype, and regime of Ab treatment. (A–C) At day 30 p.i. splenocytes were analyzed for Tfh virus specific CD4+ T cells by flow cytometry (A), Bcl6 expression in sorted I-Ab GP67–77 tetramer+ CD4+ T cells by qPCR (B), and germinal center B cell (CD19+GL7+CD38) formation by flow cytometry (C). (D&E) Viremia was determined in mAb treated mice at indicated timepoints p.i. by plaque assay. Representative of 2 experiments with n = 5 mice per group each. * P<0.05, ** P<0.01 and ***P<0.001.

James A. Harker, et al. Science. ;334(6057):825-829.
2.
Fig 1

Fig 1. From: Late Interleukin-6 escalates T follicular helper cell responses and controls a chronic viral infection.

Biphasic IL-6 is produced by radiation resistant cells and is essential for virus control during chronic LCMV infection. (A) C57BL/6 WT mice were infected with LCMV Cl 13 and serum IL-6 concentrations were determined by enzyme-linked immunosorbant assay (ELISA) throughout infection. (B) WT or IL-6 ko mice were infected with LCMV Cl 13 and virermia determined by plaque assay. (C&D) WT or IL-6 ko mice were lethality irradiated and reconstituted with BM from either WT or IL-6 ko mice. 8 weeks later mice were infected with LCMV Cl 13. Serum IL-6 levels (C) and viremia (D) were determined. (E) Il6 expression relative to gapdh was determined in FACS isolated PICD45, PICD45FDCM1CD21/35 and PICD45FDCM1+CD21/35+ splenocytes from either WT mice either naïve, at day 1 or day 25 post Cl 13 infection. (A–D) are the mean ± SEM of ≥4 mice per group representative of >2 independent experiments and statistical comparison were performed by two-way ANNOVA. *WT>WT versus WT>IL- 6ko or #WT>WT versus IL-6ko>IL-6ko. (E) Data is pool of >2 mice per group and representative of 3 independent experiments. * P<0.05, ** P<0.01 and ***P<0.001.

James A. Harker, et al. Science. ;334(6057):825-829.
3.
Fig. 2

Fig. 2. From: Late Interleukin-6 escalates T follicular helper cell responses and controls a chronic viral infection.

T follicular helper cell and germinal center responses are increased in an IL-6 dependent fashion at late stages of chronic LCMV infection. WT or IL-6 ko mice were infected with LCMV Cl 13 and splenocytes analyzed at day 30 p.i. (A) The number and percentages of CD4+ I-Ab GP66–77 tetramer+ CXCR5+ ICOS+ SLAM CD200+ PD1 T follicular helper cells (Tfh) was determined by flow cytometry. (B) Sorted WT and IL-6ko I-Ab GP67–77 tetramer+ and total CD4+ T cells were isolated and bcl6 expression, relative to gapdh, determined by qPCR. (C&D) BCL6 protein levels within WT and IL-6ko I-Ab GP67–77 tetramer+ CD4+ T cells (C) and germinal center B cell (CD19+GL7+CD38) formation (D) were determined by flow cytometry. (E&F) LCMV specific IgG1 and IgG2a (E) and Ig avidity (F) were determined by ELISA in serum from WT and IL-6ko mice at day 30 p.i.. Data are presented as individual mice or as mean ± SEM of ≥ 4 mice/group and representative of ≥ 2 experiments, with indicative FACS plots and % gated population shown where necessary. * P<0.05, ** P<0.01 and ***P<0.001.

James A. Harker, et al. Science. ;334(6057):825-829.
4.
Fig. 4

Fig. 4. From: Late Interleukin-6 escalates T follicular helper cell responses and controls a chronic viral infection.

Cell-intrinsic IL-6 signaling on virus-specific CD4 T cells upregulates BCL-6 and Tfh responses during chronic LCMV infection. (A&B) Adoptively transferred CD45.1+ SMARTA CD4 T cells (A) or LCMV specific I-Ab GP67–77 tetramer+ CD4+ (B) were FACS isolated from WT Cl 13 infected mice at day 8 and 18 p.i. and stimulated with rmIL-6 ex-vivo. Phosphorylation of STAT3 was determined by flow cytometry 1h post-stimulation (A). Levels of socs3, bcl6 (12 hrs post stimulation) and Il21 (6 hrs post stimulation) were determined by qPCR and data shown as fold increase over unstimulated (B). (C–E) CD45.1 WT mice were lethality irradiated and reconstituted with matched BM from CD45.1 and IL6-Rko. 8 weeks later mixed chimeras were infected with LCMV Cl13 and analyzed at day 30 p.i. The proportion of splenic CD45.1 and CD45.2 IAb GP67–77 tetramer+ CD4 Tfh cells was determined by flow cytometry (C). IAb GP67–77 tetramer+ CD4 T cells were FACS isolated and bcl6 expression, relative to gapdh, determined by qPCR (D). Bcl6 protein levels were determined in IAb GP67–77 tetramer+ by flow cytometry (E). (F) 2 × 106 CD4+SLAMCD62L (Tfh enriched) or CD4+SLAM+ (non-Tfh) cells from day 30 post LCMV Cl13 infected WT mice were transferred i.v. into infection matched IL6ko mice (day 30 p.i.), control IL6ko mice received PBS. Serum viremia was determined by plaque assay and significance determined by 2-way ANNOVA. Changes in the CD4 T cell compartment were assessed by paired t tests between the two populations in individual mice. (A&B) are representative of 2 experiments with ≥4 mice/group. (C–E) are representative of 2 experiments of ≥7 mice/group each. (F) represents 1 experiment with 5 mice per group. * P<0.05, ** P<0.01 and *** P<0.001.

James A. Harker, et al. Science. ;334(6057):825-829.

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