Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

The atypical two-subunit σ factor from Bacillus subtilis is regulated by an integral membrane protein and acid stress.

Extracytoplasmic function (ECF) σ factors constitute a major component of the physicochemical sensory apparatus in bacteria. Most ECF σ factors are co-expressed with a negative regulator called an anti-σ factor that binds to its cognate σ factor and sequesters it from productive association with core RNA polymerase (RNAP). Anti-σ factors constitute an important element of signal transduction pathways that mediate an appropriate transcriptional response to changing environmental conditions. The Bacillus subtilis genome encodes seven canonical ECF σ factors and six of these are co-expressed with experimentally verified anti-σ factors. B. subtilis also expresses an ECF-like atypical two-subunit σ factor composed of subunits SigO and RsoA that becomes active after exposure to certain cell-wall-acting antibiotics and to growth under acidic conditions. This work describes the identification and preliminary characterization of a protein (RsiO, formerly YvrL) that constitutes the anti-σ factor cognate to SigO-RsoA. Synthesis of RsiO represses SigO-RsoA-dependent transcription initiation by binding the N-terminus of SigO under neutral (pH 7) conditions. Reconstitution of the SigO-RsoA-RsiO regulatory system into a heterologous host reveals that the imposition of acid stress (pH 5.4) abolishes the ability of RsiO to repress SigO-RsoA-dependent transcription and this correlates with loss of RsiO binding affinity for SigO. A current model for RsiO function indicates that RsiO responds, either directly or indirectly, to increased extracytoplasmic hydrogen ion concentration and becomes inactivated. This results in the release of SigO into the cytoplasm, where it productively associates with RsoA and core RNAP to initiate transcription from target promoters in the cell.

Functional reconstitution of an unusual Firmicutes σ factor into a Gram-negative heterologous host.

Sigma (σ) factors are single-subunit proteins that reversibly bind RNA polymerase and play an important role in the transcription initiation process. An unusual 2-subunit σ factor, consisting of proteins SigO and RsoA, activates transcription from a group of related promoters in Bacillus subtilis. These 2 proteins specifically interact with each other and with RNA polymerase subunits. This system is widespread among species in several Bacillus-related genera, but otherwise appears restricted to the Firmicutes. Here, we reconstituted SigO-RsoA, and a cognate promoter, into the distantly related heterologous host Escherichia coli to examine whether this system can function in bacteria outside of the Firmicutes. We show that these proteins can productively associate with E. coli RNA polymerase and activate transcription, demonstrating that there are no structural barriers to function. In parallel, we tested a wide array of protein-protein interaction mutations and promoter mutations that impact SigO-RsoA function in both B. subtilis and E. coli and conclude that the SigO-RsoA system behaves, in most instances, similarly in both genetic backgrounds. These data raise the possibility of genetically isolating the system in this heterologous host and away from unknown B. subtilis factors that may also be playing a role in SigO-RsoA regulatory pathways, thus facilitating further study of the system. As a result of this work, we also provide a comprehensive mutational analysis of a SigO-RsoA promoter and report the preliminary identification of amino acids in SigO that play a role in mediating the SigO-RsoA protein-protein interaction.

Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy.

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8+ T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8+ T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8+ T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity.

IDH2 and TET2 mutations synergize to modulate T Follicular Helper cell functional interaction with the AITL microenvironment.

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.

Galectin-9 regulates the threshold of B cell activation and autoimmunity.

Despite the mechanisms of central and peripheral tolerance, the mature B cell compartment contains cells reactive for self-antigen. How these cells are poised not to respond and the mechanisms that restrain B cell responses to low-affinity endogenous antigens are not fully understood. Here, we demonstrate a critical role for the glycan-binding protein galectin-9 in setting the threshold of B cell activation and that loss of this regulatory network is sufficient to drive spontaneous autoimmunity. We further demonstrate a critical role for galectin-9 in restraining not only conventional B-2 B cells, but also innate-like B-1a cells. We show that galectin-9-deficient mice have an expanded population of B-1a cells and increased titers of B-1a-derived autoantibodies. Mechanistically, we demonstrate that galectin-9 regulates BCR and distinct TLR responses in B-1a cells, but not B-1b cells, by regulating the interaction between BCR and TLRs with the regulatory molecules CD5 and CD180, respectively. In the absence of galectin-9, B-1a cells are more readily activated and secrete increased titers of autoantibodies that facilitate autoantigen delivery to the spleen, driving autoimmune responses.

Galectin-Glycan Interactions as Regulators of B Cell Immunity.

Cell surface glycans and their glycan-binding partners (lectins) have generally been recognized as adhesive assemblies with neighbor cells or matrix scaffolds in organs and the blood stream. However, our understanding of the roles for glycan-lectin interactions in immunity has expanded substantially to include regulation of nearly every stage of an immune response, from pathogen sensing to immune contraction. In this Mini-Review, we discuss the role of the ß-galactoside-binding lectins known as galectins specifically in the regulation of B-lymphocyte (B cell) development, activation, and differentiation. In particular, we highlight several recent studies revealing new roles for galectin (Gal)-9 in the modulation of B cell receptor-mediated signaling and activation in mouse and man. The roles for cell surface glycosylation, especially I-branching of N-glycans synthesized by the glycosyltransferase GCNT2, in the regulation of Gal-9 binding activity are also detailed. Finally, we consider how dysregulation of these factors may contribute to aberrant immune activation and autoimmune disease.

Galectin-9 binds IgM-BCR to regulate B cell signaling.

The galectin family of secreted lectins have emerged as important regulators of immune cell function; however, their role in B-cell responses is poorly understood. Here we identify IgM-BCR as a ligand for galectin-9. Furthermore, we show enhanced BCR microcluster formation and signaling in galectin-9-deficient B cells. Notably, treatment with exogenous recombinant galectin-9 nearly completely abolishes BCR signaling. We investigated the molecular mechanism for galectin-9-mediated inhibition of BCR signaling using super-resolution imaging and single-particle tracking. We show that galectin-9 merges pre-existing nanoclusters of IgM-BCR, immobilizes IgM-BCR, and relocalizes IgM-BCR together with the inhibitory molecules CD45 and CD22. In resting naive cells, we use dual-color super-resolution imaging to demonstrate that galectin-9 mediates the close association of IgM and CD22, and propose that the loss of this association provides a mechanism for enhanced activation of galectin-9-deficient B cells.

Protein Tyrosine Phosphatase Inhibition Prevents Experimental Cerebral Malaria by Precluding CXCR3 Expression on T Cells.

Cerebral malaria induced by Plasmodium berghei ANKA infection is dependent on the sequestration of cytotoxic T cells within the brain and augmentation of the inflammatory response. Herein, we demonstrate that inhibition of protein tyrosine phosphatase (PTP) activity significantly attenuates T cell sequestration within the brain and prevents the development of neuropathology. Mechanistically, the initial upregulation of CXCR3 on splenic T cells upon T cell receptor stimulation was critically decreased through the reduction of T cell-intrinsic PTP activity. Furthermore, PTP inhibition markedly increased IL-10 production by splenic CD4+ T cells by enhancing the frequency of LAG3+CD49b+ type 1 regulatory cells. Overall, these findings demonstrate that modulation of PTP activity could possibly be utilized in the treatment of cerebral malaria and other CXCR3-mediated diseases.