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

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.

Figure 1. Detection of LLOp:I-A^b-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-A^b+ 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-A^b+ cells. (C) CD4⁺ T cells from B6 mice at the indicated times after intravenous infection with Lm-2W bacteria with gates on LLOp:I-A^b+ 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-A^b+ 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. Identification of T_EM and T_CM cells

(A) Plots of CCR7 versus T-bet (left) or CXCR5 (right) on LLOp:I-A^b-specific CD44^low naïve cells from uninfected B6 mice (red dots) or LLOp:I-A^b-specific CD44^high 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, CXCR5^intermediate PD-1⁻ T_CM cells, and CXCR5^high PD-1⁺ T_FH 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), T_CM cells (gray line), T_FH cells (dashed line), or CD44^low 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-A^b-specific Th1 (filled circles), T_CM (open circles), or T_FH (gray circles) cells identified as shown in (B). (G) CCR7 and eYFP expression by 2W:I-A^b-specific CD4⁺ T cells in 2W:I-A^b 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-A^b-specific cells (H) or CCR7⁺ cells among eYFP⁺ 2W:I-A^b-specific cells (I) from individual mice.

Figure 3. T_CM 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, T_CM cells as CCR7⁺ cells, and T_FH cells as CCR7^low cells. (B) Post-sort analysis of the indicated populations. Four x 10⁶ Th1, 3 x 10⁶ T_CM, or 10⁶ T_FH 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 T_CM 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), T_CM precursor (91 cells), and T_FH (38 cells) located in the indicated areas one day after transfer.

Figure 4. Secondary responses by memory cells

(A) Contour plots showing T-bet and CXCR5 expression by LLOp:I-A^b-specific T cells (left panels), or intracellular IFN-γ and IL-2 staining (lower panels) in LLOp:I-A^b-specific T_EM (middle panels) or T_CM 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-A^b-specific T_CM or T_EM 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 CD44^high CD4⁺ CCR7⁺ CXCR5⁺ PD-1⁻ T or 4.3 x 10⁵ CD44^high CD4⁺ CCR7⁻ CXCR5⁻ CM PD-1⁻ T_EM 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-A^b-specific cells 6 days after challenge are shown, along with plots of PD-1 versus CXCR5 used to identify Th1 effector cells, T_CM precursors, and T_FH cells of donor (middle) or recipient (right) origin. (D) Percentage of Th1 effector, T_CM precursor, or T_FH cell progeny from donor T_EM, donor T_CM, or recipient naïve cells. **, p < 0.01.

Figure 5. Development of Th1 effector cells depends on CD25

(A) Dot plot of CD25 versus CXCR5 expression by LLOp:I-A^b-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-A^b-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-A^b-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-A^b-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-A^b 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-A^b-specific Th1, T_CM, and T_FH 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. Development of T_CM cells depends on Bcl6

(A) Plot of Bcl6 versus CXCR5 expression by wild-type (black dots) or Bcl6^−/− (red dots) LLOp:I-A^b-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-A^b-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-A^b-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-A^b-specific Th1 effector cells, T −/− CM precursor cells, and T_FH 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-A^b-specific cells in these chimeras is also shown. (D) Number of LLOp:I-A^b-specific Th1, T_CM, and T_FH 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. Development of T_CM cells depends on ICOS signals from B cells

(A-C) Number of LLOp:I-A^b-specific Th1, T_CM, and T_FH 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