Autoimmune diseases affect roughly 5-10% of the total population, with women affected more than men. The standard treatment for autoimmune or autoinflammatory diseases had long been immunosuppressive ...agents until the advent of immunomodulatory biologic drugs, which aimed at blocking inflammatory mediators, including proinflammatory cytokines. At the frontier of these biologic drugs are TNF-α blockers. These therapies inhibit the proinflammatory action of TNF-α in common autoimmune diseases such as rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn's disease. TNF-α blockade quickly became the "standard of care" for these autoimmune diseases due to their effectiveness in controlling disease and decreasing patient's adverse risk profiles compared to broad-spectrum immunosuppressive agents. However, anti-TNF-α therapies have limitations, including known adverse safety risk, loss of therapeutic efficacy due to drug resistance, and lack of efficacy in numerous autoimmune diseases, including multiple sclerosis. The next wave of truly transformative therapeutics should aspire to provide a cure by selectively suppressing pathogenic autoantigen-specific immune responses while leaving the rest of the immune system intact to control infectious diseases and malignancies. In this review, we will focus on three main areas of active research in immune tolerance. First, tolerogenic vaccines aiming at robust, lasting autoantigen-specific immune tolerance. Second, T cell therapies using Tregs (either polyclonal, antigen-specific, or genetically engineered to express chimeric antigen receptors) to establish active dominant immune tolerance or T cells (engineered to express chimeric antigen receptors) to delete pathogenic immune cells. Third, IL-2 therapies aiming at expanding immunosuppressive regulatory T cells
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Conventional type 1 dendritic cells (DC1) contribute to the development of pathogenic T helper type 1 (Th1) cells in part
the production of the proinflammatory cytokine interleukin-12. Thus, ...depletion of DC1 has the potential to dampen autoimmune responses. Here, we developed X-C motif chemokine receptor 1 (XCR1)-specific chimeric antigen receptor (CAR)-T cells and CAR-Tregs that specifically targeted DC1. XCR1 CAR-T cells were successfully generated as CD4
and CD8
T cells, expressed XCR1 CAR efficiently, and induced XCR1-dependent activation, cytokine production and proliferation. XCR1 CAR-T cells selectively depleted DC1 when transferred into RAG2
mice with a compensatory increase in conventional type 2 DC (DC2) and plasmacytoid DC (pDC). XCR1 CAR-T cell-mediated depletion of DC1 modestly suppressed the onset of Th1-driven experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Diphtheria toxin-mediated DC1 depletion in XCR1-diphtheria toxin receptor mice also suppressed EAE, suggesting that DC1 depletion was responsible for EAE suppression. XCR1 CAR-Tregs were successfully generated and suppressed effector T cells in the presence of XCR1
cells. Therapeutic treatment with XCR1 CAR-Tregs suppressed Th1-driven EAE. Therefore, we conclude that depletion of DC1 with XCR1 CAR-T cells or immune suppression with XCR1 CAR-Tregs can modestly suppress Th1-driven EAE.
The molecular mechanisms that govern thymocyte development and maturation are incompletely understood. The P21-activated kinase 2 (Pak2) is an effector for the Rho family GTPases Rac and Cdc42 that ...regulate actin cytoskeletal remodeling, but its role in the immune system remains poorly understood. In this study, we show that T-cell specific deletion of Pak2 gene in mice resulted in severe T cell lymphopenia accompanied by marked defects in development, maturation, and egress of thymocytes. Pak2 was required for pre-TCR β-selection and positive selection. Surprisingly, Pak2 deficiency in CD4 single positive thymocytes prevented functional maturation and reduced expression of S1P1 and KLF2. Mechanistically, Pak2 is required for actin cytoskeletal remodeling triggered by TCR. Failure to induce proper actin cytoskeletal remodeling impaired PLCγ1 and Erk1/2 signaling in the absence of Pak2, uncovering the critical function of Pak2 as an essential regulator that governs the actin cytoskeleton-dependent signaling to ensure normal thymocyte development and maturation.DOI: http://dx.doi.org/10.7554/eLife.02270.001.
Conventional type 1 dendritic cells (cDC1s) are superior in antigen cross-presentation and priming CD8 + T cell anti-tumor immunity and thus, are a target of high interest for cancer immunotherapy. ...Type I interferon (IFN) is a potent inducer of antigen cross-presentation, but, unfortunately, shows only modest results in the clinic given the short half-life and high toxicity of current type I IFN therapies, which limit IFN exposure in the tumor. CD8 + T cell immunity is dependent on IFN signaling in cDC1s and preclinical studies suggest targeting IFN directly to cDC1s may be sufficient to drive anti-tumor immunity. Here, we engineered an anti-XCR1 antibody (Ab) and IFN mutein (IFN mut ) fusion protein (XCR1Ab-IFN mut ) to determine whether systemic delivery could drive selective and sustained type I IFN signaling in cDC1s leading to anti-tumor activity and, in parallel, reduced systemic toxicity. We found that the XCR1Ab-IFN mut fusion specifically enhanced cDC1 activation in the tumor and spleen compared to an untargeted control IFN. However, multiple treatments with the XCR1Ab-IFN mut fusion resulted in robust anti-drug antibodies (ADA) and loss of drug exposure. Using other cDC1-targeting Ab-IFN mut fusions, we found that localizing IFN directly to cDC1s activates their ability to promote ADA responses, regardless of the cDC1 targeting antigen. The development of ADA remains a major hurdle in immunotherapy drug development and the cellular and molecular mechanisms governing the development of ADA responses in humans is not well understood. Our results reveal a role of cDC1s in ADA generation and highlight the potential ADA challenges with targeting immunostimulatory agents to this cellular compartment.
Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit ...predominantly CD4
T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8
and CD4
T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP-Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4
T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP-Lpx stimulated both CD4
and CD8
T cells and suppressed tumor growth in a CD8
T cell-dependent manner. Combination of the SLP-Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4
and CD8
T cell responses that inhibit tumor growth.
Dendritic cells (DCs) are critical for immune homeostasis. To target DCs, we generated a mouse line with Flip deficiency in cells that express cre under the CD11c promoter (CD11c-Flip-KO). ...CD11c-Flip-KO mice spontaneously develop erosive, inflammatory arthritis, resembling rheumatoid arthritis, which is dramatically reduced when these mice are crossed with Rag(-/-) mice. The CD8α(+) DC subset is significantly reduced, along with alterations in NK cells and macrophages. Autoreactive CD4(+) T cells and autoantibodies specific for joint tissue are present, and arthritis severity correlates with the number of autoreactive CD4(+) T cells and plasmablasts in the joint-draining lymph nodes. Reduced T regulatory cells (Tregs) inversely correlate with arthritis severity, and the transfer of Tregs ameliorates arthritis. This KO line identifies a model that will permit in depth interrogation of the pathogenesis of rheumatoid arthritis, including the role of CD8α(+) DCs and other cells of the immune system.
BackgroundTumor necrosis factor receptor (TNFR) superfamily member CD40 plays key roles in mediating innate and adaptive immune responses against tumors and requires trimerization to induce agonistic ...signaling. Anti-CD40 therapeutic IgG antibodies have demonstrated limited anti-tumor activity in the clinic, perhaps due to inefficient multimerization through FcγR engagement in the tumor microenvironment. We generated and evaluated the functional properties of proof-of-concept agonistic anti-CD40 IgM antibodies and have compared their activities to corresponding IgGs.MethodsAnti-CD40 IgM and IgG antibodies were generated by inserting the same variable domains into an IgM or IgG heavy chain framework and co-expressing with light chain as well as J chain for IgM. Antibody binding and subsequent activation was evaluated by flow cytometry using human dendritic cells (DCs) and B cells. Mixed lymphocyte reaction (MLR) assay was used to evaluate antibody induced proliferation and cytokine release by human T cells.ResultsAgonistic anti-CD40 IgM antibodies demonstrated superior binding to primary human cells (DCs and B cells), as well as cell lines (overexpressing human and cyno CD40), as compared to IgG antibodies. Improved binding was due to the enhanced avidity of the multivalent IgMs compared to bivalent IgGs. In NF-κB-luciferase reporter assays, a significant increase in potency was observed with the IgMs in both EC50 and max activity compared to IgG in the presence or absence of cross-linking. In primary human DC and B cell activation assays, IgMs significantly enhanced the activation of DCs and B cells, as compared to IgGs. In an MLR assay, co-culture of agonistic anti-CD40 activated DCs with T cells, IgM antibodies showed significant T cell activation, as measured by inflammatory cytokine production, whereas IgGs had little to no effect.ConclusionsWe have discovered that IgM antibodies bind to and signal efficiently through CD40 as compared to the corresponding IgGs, even when IgGs were cross-linked. Efficient multimerization and agonism of CD40 with IgM antibodies may therefore enhance the anti-tumor responses by providing immunostimulatory signals to DCs, thereby promoting anti-tumor responses of T cells.
T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell ...adhesion but the contribution of LFA-1—induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1—dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.
Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the ...molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein-coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2(-/-) double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2(-/-) thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.
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