Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC, and metalloprotease-mediated cleavage.

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear how this downregulation is controlled.

Downregulation of PIK3IP1/TrIP on T cells is controlled by TCR signal strength, PKC and metalloprotease-mediated cleavage.

The protein known as PI3K-interacting protein (PIK3IP1), or transmembrane inhibitor of PI3K (TrIP), is highly expressed by T cells and can modulate PI3K activity in these cells. Several studies have also revealed that TrIP is rapidly downregulated following T cell activation. However, it is unclear as to how this downregulation is controlled.

Tim-3 Is Not Required for Establishment of CD8+ T Cell Memory to Lymphocytic Choriomeningitis Virus.

Tim-3 is a transmembrane protein that is best known for being highly expressed on terminally exhausted CD8+ T cells associated with chronic infection and tumors, although its expression is not limited to those settings. Tim-3 is also expressed by CD8+ T cells during acute infection and by multiple other immune cell types, including CD4+ Th1 and regulatory T cells, dendritic cells, and mast cells. In this study, we investigated the role of Tim-3 signaling on CD8+ T cell memory using a Tim-3 conditional knockout mouse model and mice lacking the signaling portion of the Tim-3 cytoplasmic domain.

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18).

Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment.

Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth.

The costimulatory activity of Tim-3 requires Akt and MAPK signaling and its recruitment to the immune synapse.

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein.

Resistance to PD1 blockade in the absence of metalloprotease-mediated LAG3 shedding.

Mechanisms of resistance to cancer immunotherapy remain poorly understood. Lymphocyte activation gene-3 (LAG3) signaling is regulated by a disintegrin and metalloprotease domain-containing protein-10 (ADAM10)- and ADAM17-mediated cell surface shedding. Here, we show that mice expressing a metalloprotease-resistant, noncleavable LAG3 mutant (LAG3) are resistant to PD1 blockade and fail to mount an effective antitumor immune response.

Neuropilin-1 is a T cell memory checkpoint limiting long-term antitumor immunity.

Robust CD8 T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8 T cell-restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth.

Treg-Cell-Derived IL-35-Coated Extracellular Vesicles Promote Infectious Tolerance.

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in Treg C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3 gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3 and Foxp3 (iTr35) T cells.

Tim-3 co-stimulation promotes short-lived effector T cells, restricts memory precursors, and is dispensable for T cell exhaustion.

Tim-3 is highly expressed on a subset of T cells during T cell exhaustion in settings of chronic viral infection and tumors. Using lymphocytic choriomeningitis virus (LCMV) Clone 13, a model for chronic infection, we found that Tim-3 was neither necessary nor sufficient for the development of T cell exhaustion. Nonetheless, expression of Tim-3 was sufficient to drive resistance to PD-L1 blockade therapy during chronic infection.

LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes.

Inhibitory receptors (IRs) are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (T) cell proliferation, viability, and function. However, the role of IRs on regulatory T cells (T) remains obscure because they could be required for suppressive activity and/or limit T function. We evaluated the role of lymphocyte activation gene 3 (LAG3; CD223) on T by generating mice in which LAG3 is absent on the cell surface of T in a murine model of type 1 diabetes.

Kinetics of Alloantigen-Specific Regulatory CD4 T Cell Development and Tissue Distribution After Donor-Specific Transfusion and Costimulatory Blockade.

Background: The influence of donor-side regulation toward recipient antigens on graft outcome is poorly understood.

Methods: Because this influence might be due in part to the accumulation of tissue-resident memory T cells in the donor organ, we used a standard murine tolerization model (donor-specific transfusion plus CD40L blockade) to determine the kinetics of development and peripheralization of allospecific regulatory T cell in lymphoid tissues and liver, a secondary lymphoid organ used in transplantation.

Results: We found that donor-specific transfusion and CD40L blockade leads to a progressive and sustained T regulatory allospecific response.

Interleukin-35 Limits Anti-Tumor Immunity.

Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function.

Verification of 2A peptide cleavage.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence.

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence.

Design and construction of 2A peptide-linked multicistronic vectors.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence.

The plasticity of regulatory T cell function.

Regulatory T cells (T(regs)) can suppress a wide variety of cell types, in diverse organ sites and inflammatory conditions. Whereas T(regs) possess multiple suppressive mechanisms, the number required for maximal function is unclear. Furthermore, whether any interrelationship or cross-regulatory mechanisms exist to orchestrate and control their utilization is unknown.

Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways.

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells.

Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3.

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells.

The development and function of regulatory T cells.

Regulatory T cells (Tregs) are a critical subset of T cells that mediate peripheral tolerance. There are two types of Tregs: natural Tregs, which develop in the thymus, and induced Tregs, which are derived from naive CD4(+) T cells in the periphery. Tregs utilize a variety of mechanisms to suppress the immune response.

Cutting edge: regulatory T cells do not require stimulation through their TCR to suppress.

The mechanism and stimulatory requirements of regulatory T cell (Treg)-mediated suppression are still unclear. To assess the requirement for Treg stimulation by cognate peptide:MHC, we used T cells from OTII and AND TCR transgenic mice that are specific for and restricted by distinct, noncrossreactive peptide:MHC combinations. This allowed us to independently activate Tregs and their conventional T cell (Tconv) targets.

The proline-rich sequence of CD3epsilon as an amplifier of low-avidity TCR signaling.

Engagement of peptide-MHC by the TCR induces a conformational change in CD3epsilon that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3epsilon ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3epsilon PRS independent.

Generation of T-cell receptor retrogenic mice.

T-cell receptor (TCR) transgenic (Tg) mice have revolutionized our understanding of many aspects of T-cell biology. Whereas they provide an almost unlimited source of T cells with a single specificity, breeding them onto different backgrounds and/or new knockout/knock-in mouse models is often time-consuming (6 months to several years), which can make the process costly and can significantly delay research. This protocol describes a new method for expressing defined TCR-alpha and TCR-beta proteins from a single 2A peptide-linked multicistronic retroviral vector in mice, using retrovirus-mediated stem cell gene transfer.