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. 2011 Oct 28;35(4):583-95.
doi: 10.1016/j.immuni.2011.09.009. Epub 2011 Oct 20.

Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells

Marion Pepper  1 , Antonio J Pagán, Botond Z Igyártó, Justin J Taylor, Marc K Jenkins
Affiliations

Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells

Marion Pepper et al. Immunity. .

Abstract

Listeria monocytogenes infection generates T helper 1 (Th1) effector memory cells and CC chemokine receptor 7 (CCR7)(+) cells resembling central memory cells. We tracked endogenous L. monocytogenes-specific CD4(+) T cells to determine how these memory cells are formed. Two effector cell populations were already present several days after infection. One highly expressed the T-bet transcription factor and produced Th1 memory cells in an interleukin-2 (IL-2) receptor-dependent fashion. The other resided in the T cell areas, expressed CCR7 and CXC chemokine receptor 5 (CXCR5), and like follicular helper cells depended on the Bcl6 transcription factor and inducible costimulator ligand on B cells. The CCR7(+)CXCR5(+) effector cells produced similar memory cells that generated diverse effector cell populations in a secondary response. Thus, Th1 effector memory and follicular helper-like central memory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells.

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Figures

Figure 1
Figure 1. Detection of LLOp:I-Ab-specific CD4+ T cells
(A) Gate used to identify CD3+ non-T cell lineage− T cells (left) and CD4+ and CD8+ T cells within that population (right) from the bound fraction after enrichment with LLOp:I-Ab+ tetramer. (B) CD4+ T cells (left) or CD8+ T cells (right) identified as in (A) from an uninfected B6 mouse with gates on LLOp:I-Ab+ cells. (C) CD4+ T cells from B6 mice at the indicated times after intravenous infection with Lm-2W bacteria with gates on LLOp:I-Ab+ cells. The percentages of cells in the indicated gates are shown. (D) Mean number (± SEM, n ≥ 3 for each data point) of CD4+ LLO:I-Ab+ T cells in the spleen and lymph nodes over the first 8 (left) or 440 days (right) after intravenous infection with Lm-2W bacteria.
Figure 2
Figure 2. Identification of TEM and TCM cells
(A) Plots of CCR7 versus T-bet (left) or CXCR5 (right) on LLOp:I-Ab-specific CD44low naïve cells from uninfected B6 mice (red dots) or LLOp:I-Ab-specific CD44high memory cells from mice infected with Lm-2W bacteria 60 days before analysis (black contours). (B) Plots of CXCR5 versus PD-1 used to identify CXCR5− PD-1− Th1 cells, CXCR5intermediate PD-1− TCM cells, and CXCR5high PD-1+ TFH cells on days 8 or 60 after intravenous infection with Lm-2W bacteria, with histograms of T-bet, CCR7, and Bcl6 on Th1 cells (black line), TCM cells (gray line), TFH cells (dashed line), or CD44low naïve cells (shaded histogram). (C-E) Mean fluorescence intensities (MFI) of T-bet (C), CCR7 (D), and Bcl6 (E) on the indicated cell types from 3 individual mice on days 8 (filled circles) or 60 (open circles) after intravenous infection with Lm-2W bacteria. (F) Mean number (± SEM, n ≥ 3) of LLO:I-Ab-specific Th1 (filled circles), TCM (open circles), or TFH (gray circles) cells identified as shown in (B). (G) CCR7 and eYFP expression by 2W:I-Ab-specific CD4+ T cells in 2W:I-Ab tetramer-enriched samples from r7UP mice infected with Lm-2W bacteria, treated with tamoxifen on days 4-8 post-infection, and analyzed on days 8 or 254 post-infection. (H, I) Percentage of eYFP+ cells among 2W:I-Ab-specific cells (H) or CCR7+ cells among eYFP+ 2W:I-Ab-specific cells (I) from individual mice.
Figure 3
Figure 3. TCM precursor cells are located in the T cell areas
(A) Gates used to sort purify the indicated subsets from the spleen and lymph nodes of B6 mice 10 days after Lm-2W infection. CCR7 staining was also used as a sorting parameter (not shown) with Th1 cells sorted as CCR7− cells, TCM cells as CCR7+ cells, and TFH cells as CCR7low cells. (B) Post-sort analysis of the indicated populations. Four x 106 Th1, 3 x 106 TCM, or 106 TFH cells were transferred into CD45.1+ recipients. (C) CD4 (green), IgD (purple), and CD45.2 (red) expression in a spleen section from a naïve B6 mouse that received TCM cells one day before analysis. CD45.2+ cells that were also CD4+ appeared orange. A T cell area (T), follicle (F), and red pulp area (RP) are indicated. Three CD4+ CD45.2+ transferred T cells in the T cell area and one in the follicle are labeled with asterisks. (D) Percentage of Th1 (151 cells), TCM precursor (91 cells), and TFH (38 cells) located in the indicated areas one day after transfer.
Figure 4
Figure 4. Secondary responses by memory cells
(A) Contour plots showing T-bet and CXCR5 expression by LLOp:I-Ab-specific T cells (left panels), or intracellular IFN-γ and IL-2 staining (lower panels) in LLOp:I-Ab-specific TEM (middle panels) or TCM cells (right panels) from day 92 Lm-2W-infected mice before (upper panels) or 2 hours after intravenous injection of LLOp (lower panels). (B) Percentage of LLOp:I-Ab-specific TCM or TEM cells producing the indicated lymphokines before (filled circles) or 2 hours after intravenous injection of LLOp (open circles). **, p < 0.01. (C) 4.8 x 105 CD44high CD4+ CCR7+ CXCR5+ PD-1− T or 4.3 x 105 CD44high CD4+ CCR7− CXCR5− CM PD-1− TEM cells were sorted from CD45.2+ B6 mice infected 30 days earlier with Lm-2W bacteria, and transferred into CD45.1+ mice, which were then challenged with Lm-2W bacteria. Representative plots used to identify donor (upper gates, left panels) or recipient (lower gates, left panels) 2W:I-Ab-specific cells 6 days after challenge are shown, along with plots of PD-1 versus CXCR5 used to identify Th1 effector cells, TCM precursors, and TFH cells of donor (middle) or recipient (right) origin. (D) Percentage of Th1 effector, TCM precursor, or TFH cell progeny from donor TEM, donor TCM, or recipient naïve cells. **, p < 0.01.
Figure 5
Figure 5. Development of Th1 effector cells depends on CD25
(A) Dot plot of CD25 versus CXCR5 expression by LLOp:I-Ab-specific cells from naïve mice (red dots) or from mice 3 days after intravenous infection with Lm-2W bacteria (black dots). The values on the plots represent the percentage of cells in the indicated quadrants from naïve (red) or Lm-2W-infected (black) mice. The scatter plot shows the percentage of CD25+ total naïve CD4+ T cells from uninfected B6 mice (diamonds) or CXCR5− (filled circles) or CXCR5+ (open circles) LLOp:I-Ab-specific cells from mice 3 days after intravenous infection with Lm-2W bacteria. ***, p < 0.001. (B) T-bet expression in total naïve CD4+ T cells from uninfected B6 mice (shaded), or CXCR5− (black) or CXCR5+ (gray) LLOp:I-Ab-specific cells from mice 4 days after intravenous infection with Lm-2W bacteria, with a scatter plot showing the fold increase of T-bet mean fluorescence intensity (MFI) of the indicated LLOp:I-Ab-specific populations over the T-bet MFI of naïve CD4+ T cells. ***, p < 0.001. (C) Plots of CD45.1 versus CD45.2 expression and CD44 expression versus LLOp:I-Ab tetramer staining of CD4+ T cells in a tetramer-enriched sample from a radiation chimera produced with CD45.2+ wild-type and CD45.1+ CD45.2+ Il2ra−/− bone marrow, 5 days after intravenous infection with Lm-2W bacteria. (D) Number of LLOp:I-Ab-specific Th1, TCM, and TFH cells identified as in Figure 2B of wild-type (filled circles) or Il2ra−/− (open circles) origin from individual mice, 5 days after intravenous infection with Lm-2W bacteria. **, p < 0.01.
Figure 6
Figure 6. Development of TCM cells depends on Bcl6
(A) Plot of Bcl6 versus CXCR5 expression by wild-type (black dots) or Bcl6−/− (red dots) LLOp:I-Ab-specific cells from a wild-type plus Bcl6−/− mixed radiation bone marrow chimera 3 days after intravenous infection with Lm-2W bacteria with a scatter plot showing the percentage of Bcl6+ naïve CD4+ T cells from uninfected B6 mice (diamonds) or CXCR5− (filled circles) or CXCR5+ (open circles) LLOp:I-Ab-specific cells from wild-type mice 3 days after infection. (B) Number of wild-type (filled circles) or Bcl6−/− (open circles) CXCR5− or CXCR5+ LLOp:I-Ab-specific cells from 3 wild-type plus Bcl6−/− mixed radiation bone marrow chimeras, 3 days after intravenous infection with Lm-2W bacteria. *, p < 0.05. (C) Plots of CXCR5 versus PD-1 staining used to identify wild-type or Bcl6−/− LLOp:I-Ab-specific Th1 effector cells, T −/− CM precursor cells, and TFH cells from wild-type plus Bcl6 mixed radiation bone marrow chimeras 7 days after Lm-2W infection. T-bet expression by wild-type or Bcl6−/− LLOp:I-Ab-specific cells in these chimeras is also shown. (D) Number of LLOp:I-Ab-specific Th1, TCM, and TFH cells identified as in (C) from individual wild-type plus Bcl6−/− (left) or wild-type plus Bcl6+/- (right) mixed radiation bone marrow chimeras, 7 days after intravenous infection with Lm-2W bacteria. *, p < 0.05, ***, p < 0.001.
Figure 7
Figure 7. Development of TCM cells depends on ICOS signals from B cells
(A-C) Number of LLOp:I-Ab-specific Th1, TCM, and TFH cells identified as in Figure 2B from individual (A) wild-type or B cell-deficient μMT mice, (B) wild-type plus Icos−/− mixed radiation bone marrow chimeras, or (C) wild-type plus μMT or Icosl−/− plus μMT mixed radiation bone marrow chimeras, at the indicated times after intravenous infection with Lm-2W bacteria. *, p < 0.05, **, p < 0.01, ***, p < 0.001
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