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

IFN-γ–producing CD4 T cells are required for protection against Mycobacterium tuberculosis (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 \u3e80% 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

CD137 promotes proliferation and survival of human B cells

CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig–stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-g and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-a and -b. Our study suggests that CD137 costimulation may play a role in defining the fate of Agstimulated human B cells.Fil: Zhang, Xiaoyu. University of Maryland; Estados UnidosFil: Voskens, Caroline J.. University of Maryland; Estados UnidosFil: Sallin, Michelle. University of Maryland; Estados UnidosFil: Maniar, Amudhan. University of Maryland; Estados UnidosFil: Montes, Carolina Lucia. University of Maryland; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Zhang, Yue. University of Maryland; Estados UnidosFil: Lin, Wei. University of Maryland; Estados UnidosFil: Li, Guoyan. University of Maryland; Estados UnidosFil: Burch, Erin. University of Maryland; Estados UnidosFil: Tan, Ming. University of Maryland; Estados UnidosFil: Hertzano, Ronna. University of Maryland; Estados UnidosFil: Chapoval, Andrei I.. University of Maryland; Estados UnidosFil: Tamada, Koji. University of Maryland; Estados UnidosFil: Gastman, Brian R.. University of Maryland; Estados UnidosFil: Schulze, Dan H.. University of Maryland; Estados UnidosFil: Strome, Scott E.. University of Maryland; Estados Unido

Cell Type–Specific Transcriptome Analysis Reveals a Major Role for Zeb1 and miR-200b in Mouse Inner Ear Morphogenesis

Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type–specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants

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

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-betdim 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

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

IFN-γ–producing CD4 T cells are required for protection against Mycobacterium tuberculosis (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

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

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

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