Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells

Abstract

Th1 cells are critical for containment of Mycobacterium tuberculosis infection, but little else is known about the properties of protective CD4 T cell responses. In this study, we show that the pulmonary Th1 response against M. tuberculosis is composed of two populations that are either CXCR3(hi) and localize to lung parenchyma or are CX3CR1(hi)KLRG1(hi) and are retained within lung blood vasculature. M. tuberculosis-specific parenchymal CD4 T cells migrate rapidly back into the lung parenchyma upon adoptive transfer, whereas the intravascular effectors produce the highest levels of IFN-γ in vivo. Importantly, parenchymal T cells displayed greater control of infection compared with the intravascular counterparts upon transfer into susceptible T cell-deficient hosts. Thus, we identified a subset of naturally generated M. tuberculosis-specific CD4 T cells with enhanced protective capacity and showed that control of M. tuberculosis correlates with the ability of CD4 T cells to efficiently enter the lung parenchyma rather than produce high levels of IFN-γ.

FIGURE 1. Mtb-specific CD4 T cells are highly enriched in both the lung parenchyma and lung vasculature

(A) Iv staining of Mtb-infected mice. (B) Representative FACS plots of CD4 T cells on day 30 pi. Data are representative of at least three independent experiments. (C) Immunofluorescence microscopy of day 30 lung sections from anti-CD45 (red) injected mice. CD4 is green and CD31 is blue. Scale bar represents 100μm. (D) Frequency and total number of iv⁻ CD4 T cells after Mtb infection. Data are pooled from at least two independent experiments (n≥3 per experiment). (E) Quantification of I-A^bESAT-6_4–17 and I-A^bEsxG_46–61-specific cells by MHC class II staining on day 30 pi. Each connecting line shows an individual animal. Data are pooled from two independent experiments (n=5 per experiment).

FIGURE 2. Lung parenchymal and intravascular Mtb-specific CD4 T cells are functionally and phenotypically distinct subsets

(A) IFNγ staining after in vitro stimulation with ESAT-6_1–20 peptide on day 30 pi. Plots are gated on lung CD4 T cells, and data are pooled from three independent experiments (n=4 per experiment). (B) Direct ex vivo ICS for IFNγ–producing I-A^bESAT-6_4–17-specific CD4 T cells in the lung at 30 days pi. Each connecting line shows an individual animal (n=5). Data are representative of three independent experiments. (C) T-bet and (D) PD-1, CD69 and KLRG1 expression in total naïve (CD44^loFoxp3⁻) and iv⁺ or iv⁻ I-A^bESAT-6_4–17 tetramer⁺ lung CD4 T cells on day 30 pi. Numbers represent the MFI or percent⁺ for the iv⁻ (top) and iv⁺ (bottom). (E) CXCR3 or CX3CR1 staining gated on total lung CD4 T cells on day 30 pi (left panels), and expression of the chemokine receptors and KLRG1 gated on iv⁻ and iv⁺ I-A^bESAT-6_4–17 tetramer⁺ cells (middle and right panels). Cells in panels C–E were pooled from n≥4 mice per experiment for FACS analysis. Data are representative of three independent experiments.

FIGURE 3. KLRG1⁻ parenchymal effector CD4 T cells migrate most efficiently into the lung while intravascular KLRG1⁺ cells are the least efficient

FACS purified iv⁻ and iv⁺ CD4 T cells from lungs of day 30 infected mice (CD45.1) were transferred into infection-matched congenic recipient mice (CD45.2). After 1.5 days, migration of the donor cells into the lung tissue was monitored with a second iv stain in the recipient mice. (A) Migration of I-A^bESAT-6_4–17 tetramer⁺ donor CD4 T cells into the parenchyma. Plots are electronically concatenated from each group of mice (n=5). Data are representative of two independent experiments. (B) Gating strategy to identify naïve, KLRG1^hi and KLRG1^lo effector donor CD4 T cells. Plots are concatenated from each group of mice (n=5). (C) Summary of donor cells migrating into the lung parenchyma of recipients. Data are pooled from two independent experiments (n=5 per experiment).

FIGURE 4. Parenchyma homing CD4 T cells display greater control of Mtb infection compared to the intravascular cells

Iv⁻ and iv⁺ CD4 T cells were FACS purified from day 30 infected mice and adoptively transferred into TCRα^−/− mice that had been infected with Mtb 7 days before, and lungs were harvested on day 28 pi. (A) Absolute number of CD44^hiFoxp3⁻ effector and I-A^bESAT-6_4–17 tetramer⁺ donor CD4 T cells in the recipient lungs. (B) Direct ex vivo IFNγ staining of donor CD4 T cells. (C) Bacterial loads in the lungs of recipient mice. All data are representative of two independent experiments (n=5 per experiment).