CD30 co-stimulation drives differentiation of protective T cells during Mycobacterium tuberculosis infection.

Control of Mycobacterium tuberculosis (Mtb) infection requires generation of T cells that migrate to granulomas, complex immune structures surrounding sites of bacterial replication. Here we compared the gene expression profiles of T cells in pulmonary granulomas, bronchoalveolar lavage, and blood of Mtb-infected rhesus macaques to identify granuloma-enriched T cell genes. TNFRSF8/CD30 was among the top genes upregulated in both CD4 and CD8 T cells from granulomas. In mice, CD30 expression on CD4 T cells is required for survival of Mtb infection, and there is no major role for CD30 in protection by other cell types. Transcriptomic comparison of WT and CD30-/- CD4 T cells from the lungs of Mtb-infected mixed bone marrow chimeric mice showed that CD30 directly promotes CD4 T cell differentiation and the expression of multiple effector molecules. These results demonstrate that the CD30 co-stimulatory axis is highly upregulated on granuloma T cells and is critical for protective T cell responses against Mtb infection.

Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD.

BACKGROUND: Although ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear. METHODS: We explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff). RESULTS: We found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators. CONCLUSIONS: We propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.

Neuroinflammation is associated with infiltration of T cells in Lewy body disease and α-synuclein transgenic models.

BACKGROUND: α-Synuclein (α-syn) is a pre-synaptic protein which progressively accumulates in neuronal and non-neuronal cells in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. Recent evidence suggests that aberrant immune activation may be involved in neurodegeneration in PD/DLB. While previous studies have often focused on the microglial responses, less is known about the role of the peripheral immune system in these disorders. METHODS: To understand the involvement of the peripheral immune system in PD/DLB, we evaluated T cell populations in the brains of α-syn transgenic (tg) mice (e.g., Thy1 promoter line 61) and DLB patients. RESULTS: Immunohistochemical analysis showed perivascular and parenchymal infiltration by CD3+/CD4+ helper T cells, but not cytotoxic T cells (CD3+/CD8+) or B cells (CD20+), in the neocortex, hippocampus, and striatum of α-syn tg mice. CD3+ cells were found in close proximity to the processes of activated astroglia, particularly in areas of the brain with significant astrogliosis, microgliosis, and expression of pro-inflammatory cytokines. In addition, a subset of CD3+ cells co-expressed interferon γ. Flow cytometric analysis of immune cells in the brains of α-syn tg mice revealed that CD1d-tet+ T cells were also increased in the brains of α-syn tg mice suggestive of natural killer T cells. In post-mortem DLB brains, we similarly detected increased numbers of infiltrating CD3+/CD4+ T cells in close proximity with blood vessels. CONCLUSION: These results suggest that infiltrating adaptive immune cells play an important role in neuroinflammation and neurodegeneration in synucleinopathies and that modulating peripheral T cells may be a viable therapeutic strategy for PD/DLB.

Immune system development and age-dependent maintenance in Klotho-hypomorphic mice.

Circulating Klotho peptide hormone has anti-aging activity and affects tissue maintenance. Hypomorphic mutant Klotho [kl/kl] mice on C57BL/6xC3H, BALB/c and 129 genetic backgrounds, show decreased Klotho expression that correlate with accelerated aging including pre-mature death due to abnormally high levels of serum vitamin D. These mice also show multiple impairments in the immune system. However, it remains unresolved if the defects in the immune system stem from decreased Klotho expression or high vitamin D levels in the serum. Transfer of the kl/kl allele to pure C57BL/6 genetic background [B6-kl/kl] significantly reduced expression of Klotho at all ages. Surprisingly, B6-kl/kl mice showed normalized serum vitamin D levels, amelioration of severe aging-related phenotypes and normal lifespan. This paper reports a detailed analysis of the immune system in B6-kl/kl mice in the absence of detrimental levels of serum vitamin D. Remarkably, the data reveal that in the absence of overt systemic stress, such as abnormally high vitamin D levels, reduced expression of Klotho does not have a major effect on the generation and maintenance of the immune system.

The Rate of CD4 T Cell Entry into the Lungs during Mycobacterium tuberculosis Infection Is Determined by Partial and Opposing Effects of Multiple Chemokine Receptors.

The specific chemokine receptors utilized by Th1 cells to migrate into the lung during Mycobacterium tuberculosis infection are unknown. We previously showed in mice that CXCR3+ Th1 cells enter the lung parenchyma and suppress M. tuberculosis growth, while CX3CR1+ KLRG1+ Th1 cells accumulate in the lung vasculature and are nonprotective. Here we quantify the contributions of these chemokine receptors to the migration and entry rate of Th1 cells into M. tuberculosis-infected lungs using competitive adoptive transfer migration assays and mathematical modeling. We found that in 8.6 h half of M. tuberculosis-specific CD4 T cells migrate from the blood to the lung parenchyma. CXCR3 deficiency decreases the average rate of Th1 cell entry into the lung parenchyma by half, while CX3CR1 deficiency doubles it. KLRG1 blockade has no effect on Th1 cell lung migration. CCR2, CXCR5, and, to a lesser degree, CCR5 and CXCR6 also promote the entry of Th1 cells into the lungs of infected mice. Moreover, blockade of G-protein-coupled receptors with pertussis toxin treatment prior to transfer only partially inhibits T cell migration into the lungs. Thus, the fraction of Th1 cell input into the lungs during M. tuberculosis infection that is regulated by chemokine receptors likely reflects the cumulative effects of multiple chemokine receptors that mostly promote but that can also inhibit entry into the parenchyma.

Noncoding variations in Cyp24a1 gene are associated with Klotho-mediated aging phenotypes in different strains of mice.

In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging-related phenotypes caused by Klotho down-regulation. The hypomorphic Klotho [kl/kl] allele that decreases Klotho expression in C3H, BALB/c, 129, and C57BL/6 genetic backgrounds substantially increases 1,25(OH)2D3 levels in the sera of susceptible C3H, BALB/c, and 129, but not C57BL/6 mice. This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super-enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6-kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.

Limited Pulmonary Mucosal-Associated Invariant T Cell Accumulation and Activation during Mycobacterium tuberculosis Infection in Rhesus Macaques.

Mucosal-associated invariant T cells (MAITs) are positioned in airways and may be important in the pulmonary cellular immune response against Mycobacterium tuberculosis infection, particularly prior to priming of peptide-specific T cells. Accordingly, there is interest in the possibility that boosting MAITs through tuberculosis (TB) vaccination may enhance protection, but MAIT responses in the lungs during tuberculosis are poorly understood. In this study, we compared pulmonary MAIT and peptide-specific CD4 T cell responses in M. tuberculosis-infected rhesus macaques using 5-OP-RU-loaded MR-1 tetramers and intracellular cytokine staining of CD4 T cells following restimulation with an M. tuberculosis-derived epitope megapool (MTB300), respectively. Two of four animals showed a detectable increase in the number of MAIT cells in airways at later time points following infection, but by ∼3 weeks postexposure, MTB300-specific CD4 T cells arrived in the airways and greatly outnumbered MAITs thereafter. In granulomas, MTB300-specific CD4 T cells were ∼20-fold more abundant than MAITs. CD69 expression on MAITs correlated with tissue residency rather than bacterial loads, and the few MAITs found in granulomas poorly expressed granzyme B and Ki67. Thus, MAIT accumulation in the airways is variable and late, and MAITs display little evidence of activation in granulomas during tuberculosis in rhesus macaques.

Host resistance to pulmonary Mycobacterium tuberculosis infection requires CD153 expression.

Mycobacterium tuberculosis infection (Mtb) is the leading cause of death due to a single infectious agent and is among the top ten causes of all human deaths worldwide1. CD4 T cells are essential for resistance to Mtb infection, and for decades it has been thought that IFNγ production is the primary mechanism of CD4 T-cell-mediated protection2,3. However, IFNγ responses do not correlate with host protection, and several reports demonstrate that additional anti-tuberculosis CD4 T-cell effector functions remain unaccounted for4-8. Here we show that the tumour-necrosis factor (TNF) superfamily molecule CD153 (encoded by the gene Tnfsf8) is required for control of pulmonary Mtb infection by CD4 T cells. In Mtb-infected mice, CD153 expression is highest on Mtb-specific T helper 1 (TH1) cells in the lung tissue parenchyma, but its induction does not require TH1 cell polarization. CD153-deficient mice develop high pulmonary bacterial loads and succumb early to Mtb infection. Reconstitution of T-cell-deficient hosts with either Tnfsf8-/- or Ifng-/- CD4 T cells alone fails to rescue mice from early mortality, but reconstitution with a mixture of Tnfsf8-/- and Ifng-/- CD4 T cells provides similar protection as wild-type T cells. In Mtb-infected non-human primates, CD153 expression is much higher on Ag-specific CD4 T cells in the airways compared to blood, and the frequency of Mtb-specific CD153-expressing CD4 T cells inversely correlates with bacterial loads in granulomas. In Mtb-infected humans, CD153 defines a subset of highly polyfunctional Mtb-specific CD4 T cells that are much more abundant in individuals with controlled latent Mtb infection compared to those with active tuberculosis. In all three species, Mtb-specific CD8 T cells did not upregulate CD153 following peptide stimulation. Thus, CD153 is a major immune mediator of host protection against pulmonary Mtb infection and CD4 T cells are one important source of this molecule.

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.

The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII(-/-) mice.

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc-FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR(-/-) strains. 2A-treated FcRγ(-/-) mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII(-/-) animals. These improved anti-tumor responses were associated with increased splenic CD8ß T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.

A high throughput method for enrichment of natural killer cells and lymphocytes and assessment of in vitro cytotoxicity.

In vitro assessment of lymphocyte and natural killer (NK) cell cytotoxicity typically employs density gradient centrifugation and magnetic cell separation to isolate effector cells, and chromium release to assess cytotoxicity. In order to improve the rapidity and scalability of in vitro cytotoxicity assessment, we evaluated the efficacy of a protocol utilizing tetrameric antibody complexes and SepMate™ isolation tubes to negatively select NK cells (TACs/Sep), and calcein-AM release to measure cytotoxicity. We compared the efficiency and accuracy of this protocol to a conventional approach employing density gradient centrifugation and magnetically labeled antibodies (DG/MACS) to isolate NK cells and chromium release to measure cytotoxicity. The TACs/Sep method significantly decreased the time required for NK cell isolation (1h vs. 4h), but resulted in higher red blood cell contamination. NK cell activation marker expression (including CD94, NKG2D, NKp30, NKp46, DNAM-1, 2B4, KIR2DL1/S1, KIR2DL2/L3, intracellular granzyme B, and perforin) was similar when comparing NK cells isolated by the TACs/Sep or DG/MACS methods, but the TACs/Sep method induced higher expression of CD16. In vitro cytotoxicity against HT29 colon cancer and K562 leukemia cells was not affected by the isolation method. Lastly, by combining the TACs/Sep NK cell isolation method with calcein-acetoxymethyl diacetylester (calcein-AM) release, the time required to assess in vitro cytotoxicity was reduced by 33% (4h) compared to protocols employing DG/MACS and chromium release. Altogether, these results provide the foundation for the development of a rapid, high throughput functional assay, and make it practical for the multiplexing of downstream applications, such as flow cytometric analysis and enzyme-linked immunosorbent assays (ELISAs).

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.

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-gamma 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-alpha and -beta. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.