Th1 Differentiation Drives the Accumulation of Intravascular, Non-protective CD4 T Cells during Tuberculosis

Abstract

Recent data indicate that the differentiation state of Th1 cells determines their protective capacity against tuberculosis. Therefore, we examined the role of Th1-polarizing factors in the generation of protective and non-protective subsets of Mtb-specific Th1 cells. We find that IL-12/23p40 promotes Th1 cell expansion and maturation beyond the CD73⁺CXCR3⁺T-bet^dim stage, and T-bet prevents deviation of Th1 cells into Th17 cells. Nevertheless, IL- 12/23p40 and T-bet are also essential for the production of a prominent subset of intravascular CX3CR1⁺KLRG1⁺ Th1 cells that persists poorly and can neither migrate into the lung parenchyma nor control Mtb growth. Furthermore, T-bet suppresses development of CD69⁺CD103⁺ tissue resident phenotype effectors in lung. In contrast, Th1-cell-derived IFN-γ inhibits the accumulation of intravascular CX3CR1⁺KLRG1⁺ Th1 cells. Thus, although IL-12 and T-bet are essential host survival factors, they simultaneously oppose lung CD4 T cell responses at several levels, demonstrating the dual nature of Th1 polarization in tuberculosis.

Keywords: IL-12; T cell migration; T-bet; Th1 cells; terminal effector; tuberculosis.

Figure 1. Host protective Mtb-specific CD4 T cells differentiate into intravascular non-protective terminal effectors

(A and B) Kinetic analysis of the I-A^bESAT-6_4–17 tetramer specific response in the blood, MLN, iv⁻ and iv⁺ compartments of the lung using the MHCII tetramer pull-down assay. (A) Representative FACS plots shown are gated on I-A^bESAT-6_4–17⁺CD4⁺ cells. (B) Summary graphs of the I-A^bESAT-6_4–17⁺CD4⁺KLRG1⁺ cells in the blood, MLN, iv⁻ lung, and iv⁺ lung. The blood and lymph node are pooled from 3–4 mice at each time point. (C) The expression of indicated markers on the iv⁺ KLRG1⁻, iv⁻KLRG1⁻, iv⁻ KLRG1⁺, and iv⁺KLRG1⁺ lung cells. Gray histogram is the expression on naïve CD4 T cells. (D–G) CD44^hiFoxp3⁻CD4⁺ cells were sorted from D28PI Foxp3-GFP reporter mice into four populations: iv⁺KLRG1⁻, iv⁻KLRG1⁻, iv⁻KLRG1⁺, or iv⁺KLRG1⁺ cells. (D) Sorted cells transferred into congenic mice on D28PI (post infection) and lungs harvested on D42PI. The frequency of KLRG1⁺ donor cells recovered. Each point is an independent experiment with FACS plots concatenated from 2–5 mice. *p ≤ 0.05; **p ≤ 0.01;ns; paired t-test. (E) Sorted cells transferred into congenic mice on D28PI and lungs harvested on D30PI. The frequency of iv⁻ donor cells recovered. Data pooled from two experiments. *p ≤ 0.05; ****p ≤ 0.0001; one-way ANOVA, with Tukey’s multiple comparisons test (F and G) Sorted cells were transferred into TCRα^−/− mice on day 7PI, and harvested on D28PI. Log₁₀ transformed total donor cell numbers recovered (F) and log₁₀ transformed CFU counts (G) from the lung of TCRα^−/− recipient mice. The whiskers represent the min and max values and the lines represent the geometric mean. Data pooled from two experiments. *p ≤ 0.05; **p ≤ 0.01.; one-way ANOVA, with Tukey’s multiple comparisons test

Figure 2. Clonal competition for antigen inhibits the generation of terminally differentiated Th1 cells and facilitates development of Th17 cells during Mtb infection

(A) Schematic of experimental setup. The increasing concentrations of IL-12 and IL-2 used during the five day in vitro Th1 polarization are represented by triangles in B, C, D, and E. The recipient mice received 5×10⁴, 5×10⁵, or 5×10⁶ donor in vitro Th1 polarized C7 TCR-Tg CD4 T cells of each condition. Data is representative of two experiments, one performed on day 16 and the other on day 19 post-infection. (B) Frequency of donor C7 TCR-Tg CD4 T cells recovered from the recipient lung. (C) Representative FACS plots of T-bet and KLRG1 expression after recovery of C7 TCR Tg CD4 T cells polarized with IL-2/IL-12 at 30ng/ml in vitro. Each plot represents the number of cells administered to the recipient. Summary graphs of the frequency of donor KLRG1⁺T-bet^hi donor C7 TCR Tg CD4 T cells at each in vitro Th1 polarization condition and cell number administered. (D) Geometric MFI of T-bet in the KLRG1⁻ donor C7 TCR Tg CD4 T cells at increasing in vitro polarization conditions and cell number administered. (E) Representative FACS plots of expression of T-bet and either IFNγ or IL-17A of donor C7 TCR Tg CD4 T cells with IL-2/IL-12 at 30ng/ml in vitro. Each plot represents the number of cells administered to the recipient. Summary graphs of either IFNγ⁺ or IL-17A⁺ donor C7 TCR Tg CD4 T cells at each in vitro Th1 polarization condition and cell number administered. See also Figure S1.

Figure 3. IL-12/23p40 drives the generation of intravascular CD4⁺ T cells during Mtb infection

WT and p40^−/− mice infected with Mtb by aerosol infection. On day 28PI lungs and spleen were harvested. Log₁₀ transformed CFU counts of lung (A) and spleen (B) in WT and p40^−/− mice. The dotted line represents the limit of detection. Data representative of two experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (C) Representative FACS plots of lung I-A^bESAT-6_4–17⁺CD4⁺ T cells in WT and p40^−/− mice. Summary graph of the frequency of I-A^bESAT-6_4–17⁺CD4⁺ T cells in WT and p40^−/− mice. Data pooled from two experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (D) Summary graph of the frequency of iv⁺ I-A^bESAT-6_4–17⁺CD4⁺ T cells in WT and p40^−/− mice. Data pooled from two experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (E) Representative FACS plots of CD73, CD69, CXCR3, KLRG1, CX3CR1, and CD103 expression on iv⁻ and iv⁺ I-A^bESAT-6_4–17⁺CD4⁺ T cells in the lung. (F) SPICE analysis WT and p40^−/− I-A^bESAT-6_4–17⁺CD4⁺ T cells from the lung. Pie graphs depict the distribution of the different combinations of receptor expression. Arcs represent the expression pattern of receptors on intravascular (red) or CD73⁺ (green) cells. See also Figure S2A. (G) Summary graph of the frequency of lung iv⁺CX3CR1⁺I-A^bESAT-6_4–17⁺CD4⁺ T cells in WT and p40^−/− mice. Results pooled from two experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (H) t-SNE map of overlay of lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and p40^−/− mice. (I) t-SNE map of CD45iv, CX3CR1, KLRG1, CXCR3, CD69, CD73, TCF-1 and CD103 of the lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and p40^−/− mice. Circles are drawn to highlight cell populations discussed in the text. See also Figure S2B and S2C.

Figure 4. T-bet is essential for the differentiation of intravascular Th1 cells and inhibits the development of CD69⁺CD103⁺ parenchymal CD4 T cells

WT and T-bet^−/− mice infected with Mtb by aerosol infection. On day 28PI lungs and spleen were harvested. Log₁₀ transformed CFU counts of lung (A) and spleen (B) in WT and T-bet^−/− mice. The dotted line represents the limit of detection. Data pooled from two experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (C) Representative FACS plots of lung I-A^bESAT-6_4–17 versus CD45iv CD4 T cells from WT and T-bet^−/− mice. Summary graph of the frequency of I-A^bESAT-6_4–17⁺CD4⁺ T cells from WT and T-bet^−/− mice. Data pooled from three experiments. (D) Summary graphs of frequency of iv⁺I-A^bESAT-6_4–17⁺CD4⁺ T cells from WT and T-bet^−/− mice. Data pooled from three experiments. ****p ≤ 0.0001; unpaired two-tailed t test. (E) Representative FACS plots of CD69, CD103, KLRG1, and Ly6C versus CD45iv on I-A^bESAT-6_4–17⁺CD4⁺ T cells in the lung from WT and T-bet^−/− mice. (F) SPICE analysis of WT and T-bet^−/− I-A^bESAT-6_4–17⁺CD4⁺ T cells from D28PI lung. Pie graphs depict the distribution of the different combinations of receptor expression. See also Figure S3. (G) t-SNE map of overlay of lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and T-bet^−/− mice. (H) t-SNE map of CD45iv, CX3CR1, CXCR3, CD69, CD73, TCF-1 and CD103 of the lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and T-bet^−/− mice. Circles are drawn to highlight cell populations discussed in the text.

Figure 5. T cell intrinsic role for T-bet in host protection and CD4 T cell differentiation during Mtb infection

(A) A survival curve for WT, T-bet^−/− and T-bet^fl/fl CD4-Cre mice after aerosol Mtb infection. ****p ≤ 0.0001; Log-Rank Test (B) Experimental schematic of naïve adoptive transfer of sorted WT and T-bet^−/− cells into D9PI recipients and harvested on D28PI. Representative FACS plots of donor cell isolation from recipient lung on D28PI. (C) Representative concatenated FACS plots of KLRG1, CX3CR1, and CD69 versus CD45iv on WT and T-bet^−/− donor cells from three recipient mice. Data representative of two experiments.

Figure 6. T-bet inhibits RORγt expression and prevents deviation of Th1 cells into Th17 cells during Mtb infection

(A, B) Lung lymphocytes stimulated with ESAT-6_1–20 peptide and cytokine expression measured by intracellular cytokine staining. (A) Representative FACS plots of TNF, IFNγ, and IL-17A expression in iv⁻ CD4 T cells in WT and T-bet^−/− mice. (B) Summary graphs of frequency of IFNγ⁺ and IL-17A⁺ CD44^hiFoxp3⁻CD4⁺ T cells. **p ≤ 0.01, ****p ≤ 0.0001; unpaired two-tailed t test. Data representative of three experiments. (C) Representative FACS plots of direct ex vivo IFNγ, TNF, and IL-17A expression in iv⁻ lung effector CD4 T cells from T-bet ZsGreen reporter and T-bet^−/− T-bet ZsGreen reporter mice on D28PI. (D) Representative FACS plots of RORγt expression in iv⁻ lung effector CD4 T cells from T-bet ZsGreen reporter and T-bet^−/− T-bet ZsGreen reporter mice on D28PI. (E) t-SNE map of overlay of lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT, T-bet^−/−, and all RORγt⁺ cells. (F) Two independent survival curves of WT and T-bet^−/− mice treated with anti-IL-17A. Gray box indicates the time of treatment.

Figure 7. CD4 T cell derived IFNγ limits the accumulation of intravascular terminal effector Th1 cells during Mtb infection

WT and IFNγ^−/− mice infected with Mtb by aerosol infection. On day 28PI lungs and spleen were harvested. Data pooled from two experiments. Log₁₀ transformed CFU counts of lung (A) and spleen (B) in WT and IFNγ^−/− mice. The dotted line represents the limit of detection. ****p ≤ 0.0001; unpaired two-tailed t test. (C) Representative FACS plot and histograms of CD45iv staining of I-A^bESAT-6_4–17+CD4⁺ T cells from the lungs of WT and IFNγ^−/− mice. (D) Summary graph of two experiments of the frequency of I-A^bESAT-6_4–17⁺CD4⁺ T cells from the lungs of WT and IFNγ^−/− mice. (E) Summary graph of the frequency of iv⁺I-A^bESAT-6_4–17⁺CD4⁺ T cells from the lungs of WT and IFNγ^−/− mice. ***p ≤ 0.001; unpaired two-tailed t test. (F) Representative FACS plots of CX3CR1, KLRG1, CXCR3, CD69, CD103, CD73, and TCF-1 versus CD45iv on lung I-A^bESAT-6_4–17⁺CD4⁺ T cells from WT and IFNγ^−/− mice. (G) SPICE analysis of WT and IFNγ^−/− lung I-A^bESAT-6_4–17⁺CD4⁺ T cells. Pie graphs depict the distribution of the different combinations of receptor expression. See also Figure S4A. (H) t-SNE map of overlay of lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and IFNγ^−/− mice. (I) t-SNE map of CD45iv, CX3CR1, CXCR3, CD69, CD73, TCF-1 and CD103 of the lung I-A^bESAT-6_4–17⁺CD4⁺ T cells clusters in WT and IFNγ^−/− mice. (J) Summary graph of the frequency of donor WT or IFNγ^−/− KLRG1⁺iv⁺CD4 T cells with increasing numbers of T cells transferred. **p ≤ 0.01; unpaired two-tailed t test. (K) Representative FACS plot of donor cell recovery from recipient lung. Summary graph of the frequency of donor WT and ARE KLRG1⁺ intravascular CD4 T cells. **p ≤ 0.01; paired two-tailed t test. See also Figure S4B.