Mtb-specific CD4 T cells in the lungs of PD-1 KO mice are less differentiated, more parenchymally localized and produce increased amounts of IFN-γ.

<p>(<b>A</b>) Survival of WT and PD-1 KO mice after Mtb infection. Data are representative of at least three independent experiments. (n = 5-6/experiment). (<b>B</b>) IFN-γ levels in the lung homogenates of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (<b>C</b>) PD-1 expression on the parenchymal and intravascular I-A^bESAT-6_4–17 or I-A^bEsxG_46–61–specific CD4 T cells in WT lung on day 30 p.i. (<b>D</b>) Iv-staining of I-A^bESAT-6_4–17–specific CD4 T cells in the lung of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (<b>E</b>) Phenotypic analysis of I-A^bESAT-6_4–17–specific CD4 T cells in WT and PD-1 KO lungs on day 30 p.i. (<b>F</b>) Intracellular IFN-γ staining of lung CD4 T cells in WT and PD-1 KO mice after in vitro stimulation with ESAT-6_1–20 peptide on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). (<b>G</b>) Direct ex vivo IFN-γ staining for I-A^bESAT-6_4–17–specific CD4 T cells in the lungs of WT and PD-1 KO mice on day 30 p.i. Data are pooled from three independent experiments (n = 3-4/experiment). Cells in (<b>A</b>) and (<b>E</b>) were pooled from n = 3/experiment for FACS analysis. ***, <i>P</i><0.0005; ****, <i>P</i><0.0001.</p

Increased IFN-γ–production by CD4 T cells exacerbates pulmonary Mtb infection and leads to the early host mortality, despite enhancing bacterial control in the spleen.

<p>RAG1 KO mice were infected with Mtb 7 days earlier and reconstituted with CD4 T cells from uninfected donors at increasing ratios of either WT or ARE-Del CD4 T cells mixed with IFN-γ KO CD4 T cells (<b>A</b>). All mice received the same total number of donor CD4 T cells, as only the fractions of IFN-γ–producing CD4 T cells varied (either WT or IFN-γ–overproducing). On day 42 IFN-γ concentrations in the lung homogenates (<b>B</b>) and bacterial load in the tissues were measured (<b>C</b>). Data are representative of two independent experiments (n = 5/group/experiment). (<b>D</b>) A mixture of WT and ARE-Del CD4 T cells (at 1:1 ratio) were co-transferred into day-7 infected TCRα KO mice, and on day 60 IFN-γ production by donor CD4 T cells was measured by DrxICS. Data are pooled from two independent experiments (n = 6/experiment) and each connecting line represents an individual animal. ****, <i>P</i><0.0001 (<b>E</b>) Correlation between IFN-γ levels and bacterial numbers in the lungs of RAG1 KO mice. Data shown are replotted from the values shown in (<b>B and C</b>) to illustrate the correlation. (<b>F</b>) TCRα KO mice were infected with Mtb 7 days before and received with WT, ARE-Del or a 1:1 mixture of WT and ARE-Del naïve CD4 T cells and mouse survival was monitored. Data are representative of three independent experiments. (n = 4-5/group/experiment). **, <i>P</i><0.002; compared to control group received WT CD4 T cells alone.</p

CD4 T cell-derived TNF has a minor contribution to inhibition of Mtb growth in both lung and spleen.

<p>CD4 T cells isolated from naïve WT or TNF KO mice were adoptively transferred into day-7 infected RAG1 KO recipients at increasing ratios of WT cells to TNF KO cells. All mice received the same total number of donor CD4 T cells, as only the fractions of TNF–producing CD4 T cells varied (<b>A</b>). TNF levels in the tissue homogenates (<b>B</b>) and bacterial load in the tissues (<b>C</b>) were measured on day 42 p.i. Data are representative of two independent experiments (n = 5/group/experiment).</p

PD-1 KO CD4 T cells require IFN-γ production to drive early mortality after Mtb infection.

<p>(<b>A and B</b>) Parenchymal iv^- and intravascular iv⁺ effector CD44^hi CD4 T cells were FACS purified from the lungs of day-30 infected WT mice and adoptively transferred into TCRα KO mice that had been infected with Mtb 7 days previously (<b>A</b>) and mouse survival was monitored (<b>B</b>). **, <i>P</i><0.001. (<b>C and D</b>) Parenchymal iv^- effector CD44^hi CD4 T cells were FACS purified from the lungs of WT and PD-1 KO mice on day 30 p.i., and adoptively transferred into day-7 infected TCRα KO mice (<b>C</b>) and mouse survival was monitored (<b>D</b>). Data are representative of two independent experiments (n = 5/group/experiment). **, <i>P</i><0.002; compared to mice received iv^- WT CD4 T cells alone. (<b>E and F</b>) TCRα KO mice infected with Mtb 7 days earlier were reconstituted with WT, PD-1 KO or a mixture of WT and PD-1 KO or PD-1/IFN-γ double KO CD4 T cells (<b>E</b>) and survival was monitored (<b>F</b>). **, <i>P</i><0.002 for WT + PD-1 KO (dotted red line) versus WT + PD-1/IFN-γ double KO (blue line). Data are representative of three independent experiments (n = 4-5/group/experiment).</p

PD-1 expression on CD4 T cells inhibits accumulation of and IFN-γ production by lung parenchymal CD4 T cells during Mtb infection.

<p>(<b>A</b>) Correlation between bacterial load and frequency of parenchymal CD4 T cells in the lungs of Mtb infected mice. Data are pooled from experiments performed at different times p.i. ranging from day 0 to day 180. (<b>B-F</b>) FACS purified naïve CD4 T cells from WT (CD45.1) and PD-1 KO (Thy1.1) mice were mixed at a 1:1 ratio and co-transferred into day-7 infected WT mice <b>(B)</b>. On day 28 an iv-stain was performed in the recipient mice and donor CD4 T cells were identified by their congenic markers (<b>C</b>). The frequency of donor total CD44^hi effector CD4 T cells in the recipient lungs (<b>D</b>) and in the lung parenchyma (<b>E</b>). (<b>F</b>) The frequency of KLRG1^- cells in the donor CD44^hi effector CD4 T cells. (<b>G</b>) The frequency of donor naïve CD4 T cells accumulating in the lung parenchyma. (<b>H</b>) IFN-γ production of donor effector CD4 T cells was determined by DrxICS. Data are representative of two independent experiments (n = 5/experiment) and each connecting line represents an individual mouse (n = 5/experiment). *, <i>P</i><0.02; **, <i>P</i><0.006.</p

CD4 T Cell-Derived IFN-γ Plays a Minimal Role in Control of Pulmonary <i>Mycobacterium tuberculosis</i> Infection and Must Be Actively Repressed by PD-1 to Prevent Lethal Disease

<div><p>IFN-γ–producing CD4 T cells are required for protection against <i>Mycobacterium tuberculosis</i> (Mtb) infection, but the extent to which IFN-γ contributes to overall CD4 T cell-mediated protection remains unclear. Furthermore, it is not known if increasing IFN-γ production by CD4 T cells is desirable in Mtb infection. Here we show that IFN-γ accounts for only ~30% of CD4 T cell-dependent cumulative bacterial control in the lungs over the first six weeks of infection, but >80% of control in the spleen. Moreover, increasing the IFN-γ–producing capacity of CD4 T cells by ~2 fold exacerbates lung infection and leads to the early death of the host, despite enhancing control in the spleen. In addition, we show that the inhibitory receptor PD-1 facilitates host resistance to Mtb by preventing the detrimental over-production of IFN-γ by CD4 T cells. Specifically, PD-1 suppressed the parenchymal accumulation of and pathogenic IFN-γ production by the CXCR3⁺KLRG1^-CX3CR1^- subset of lung-homing CD4 T cells that otherwise mediates control of Mtb infection. Therefore, the primary role for T cell-derived IFN-γ in Mtb infection is at extra-pulmonary sites, and the host-protective subset of CD4 T cells requires negative regulation of IFN-γ production by PD-1 to prevent lethal immune-mediated pathology.</p></div

Molecular Assembly of Zinc Chlorophyll Derivatives by Using Recombinant Light-Harvesting Polypeptides with His-tag and Immobilization on a Gold Electrode

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials