TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

The Road Less Taken: Less Appreciated Pathways for Manipulating CD8+ T Cell Exhaustion.

Exhausted CD8+ T (Tex) cells are a distinct cell population that arise during persistent antigen exposure in the context of chronic infections and cancers. Although characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression and distinct transcriptional and epigenetic programs, Tex cells are heterogeneous. Among these, a self-renewing TCF-1+ Tex population, having unique characteristics and the ability to respond to immune-checkpoint blockade, gives rise to TCF-1- terminally Tex cells. These TCF-1+ cells have stem cell-like properties similar to memory T cell populations, but the signals that regulate the developmental pathways and relationships among exhausted cell populations are still unclear. Here, we review our current understanding of Tex cell biology, and discuss some less appreciated molecules and pathways affecting T cell exhaustion. We highlight two co-stimulatory receptors, CD226 and CD137, and their role in inducing or restraining T cell exhaustion, as well as signaling pathways that may be amenable to pharmacological inhibition with a focus on Phosphoinositide-3 Kinase and IL-2 partial agonists. Finally, we discuss novel methods that may increase TCF-1+ populations and therefore improve immunotherapy responsiveness. Understanding features of and pathways to exhaustion has important implications for the success of immunotherapy, including checkpoint blockade and adoptive T-cell transfer therapies.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells.

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

PI3Kδ coordinates transcriptional, chromatin, and metabolic changes to promote effector CD8+ T cells at the expense of central memory.

Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS.

T and B-cell signaling in activated PI3K delta syndrome: From immunodeficiency to autoimmunity.

Phosphatidylinositol 3 kinases (PI3K) are a family of lipid kinases that are activated by a variety of cell-surface receptors, and regulate a wide range of downstream readouts affecting cellular metabolism, growth, survival, differentiation, adhesion, and migration. The importance of these lipid kinases in lymphocyte signaling has recently been highlighted by genetic analyses, including the recognition that both activating and inactivating mutations of the catalytic subunit of PI3Kδ, p110δ, lead to human primary immunodeficiencies. In this article, we discuss how studies on the human genetic disorder "Activated PI3K-delta syndrome" and mouse models of this disease (Pik3cdE1020K/+ mice) have provided fundamental insight into pathways regulated by PI3Kδ in T and B cells and their contribution to lymphocyte function and disease, including responses to commensal bacteria and the development of autoimmunity and tumors. We highlight critical roles of PI3Kδ in T follicular helper cells and the orchestration of the germinal center reaction, as well as in CD8+ T-cell function. We further  present data demonstrating the ability of the AKT-resistant FOXO1AAA mutant to rescue IgG1 class switching defects in Pik3cdE1020K/+ B cells, as well as data supporting a role for PI3Kδ in promoting multiple T-helper effector cell lineages.

Mg2+ regulation of kinase signaling and immune function.

Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2-inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity.

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Genetic Defects in Phosphoinositide 3-Kinase δ Influence CD8+ T Cell Survival, Differentiation, and Function.

Activated phosphoinositide 3-kinase delta syndrome (APDS), also known as p110 delta-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI), is an autosomal dominant primary human immunodeficiency (PID) caused by heterozygous gain-of-function mutations in PIK3CD, which encodes the p110δ catalytic subunit of PI3K. This recently described PID is characterized by diverse and heterogeneous clinical manifestations that include recurrent respiratory infections, lymphoproliferation, progressive lymphopenia, and defective antibody responses. A major clinical manifestation observed in the NIH cohort of patients with PIK3CD mutations is chronic Epstein-Barr virus (EBV) and/or cytomegalovirus viremia. Despite uncontrolled EBV infection, many APDS/PASLI patients had normal or higher frequencies of EBV-specific CD8+ T cells. In this review, we discuss data pertaining to CD8+ T cell function in APDS/PASLI, including increased cell death, expression of exhaustion markers, and altered killing of autologous EBV-infected B cells, and how these and other data on PI3K provide insight into potential cellular defects that prevent clearance of chronic infections.

X-Linked Lymphoproliferative Disease Type 1: A Clinical and Molecular Perspective.

X-linked lymphoproliferative disease (XLP) was first described in the 1970s as a fatal lymphoproliferative syndrome associated with infection with Epstein-Barr virus (EBV). Features include hemophagocytic lymphohistiocytosis (HLH), lymphomas, and dysgammaglobulinemias. Molecular cloning of the causative gene, SH2D1A, has provided insight into the nature of disease, as well as helped characterize multiple features of normal immune cell function. Although XLP type 1 (XLP1) provides an example of a primary immunodeficiency in which patients have problems clearing primarily one infectious agent, it is clear that XLP1 is also a disease of severe immune dysregulation, even independent of EBV infection. Here, we describe clinical features of XLP1, how molecular and biological studies of the gene product, SAP, and the associated signaling lymphocyte activation molecule family receptors have provided insight into disease pathogenesis including specific immune cell defects, and current therapeutic approaches including the potential use of gene therapy. Together, these studies have helped change the outcome of this once almost uniformly fatal disease.

PI3K Orchestrates T Follicular Helper Cell Differentiation in a Context Dependent Manner: Implications for Autoimmunity.

T follicular helper (Tfh) cells are a specialized population of CD4+ T cells that provide help to B cells for the formation and maintenance germinal centers, and the production of high affinity class-switched antibodies, long-lived plasma cells, and memory B cells. As such, Tfh cells are essential for the generation of successful long-term humoral immunity and memory responses to vaccination and infection. Conversely, overproduction of Tfh cells has been associated with the generation of autoantibodies and autoimmunity. Data from gene-targeted mice, pharmacological inhibitors, as well as studies of human and mice expressing activating mutants have revealed that PI3Kδ is a key regulator of Tfh cell differentiation, acting downstream of ICOS to facilitate inactivation of FOXO1, repression of Klf2 and induction of Bcl6. Nonetheless, here we show that after acute LCMV infection, WT and activated-PI3Kδ mice (Pik3cdE1020K/+) show comparable ratios of Tfh:Th1 viral specific CD4+ T cells, despite higher polyclonal Tfh cells in Pik3cdE1020K/+ mice. Thus, the idea that PI3K activity primarily drives Tfh cell differentiation may be an oversimplification and PI3K-mediated pathways are likely to integrate multiple signals to promote distinct effector T cell lineages. The consequences of dysregulated Tfh cell generation will be discussed in the context of the human primary immunodeficiency "Activated PI3K-delta Syndrome" (APDS), also known as "p110 delta-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency" (PASLI). Overall, these data underscore a major role for PI3K signaling in the orchestration of T lymphocyte responses.

Type I IFN induces binding of STAT1 to Bcl6: divergent roles of STAT family transcription factors in the T follicular helper cell genetic program.

CD4(+) T follicular helper cells (TFH) are critical for the formation and function of B cell responses to infection or immunization, but also play an important role in autoimmunity. The factors that contribute to the differentiation of this helper cell subset are incompletely understood, although several cytokines including IL-6, IL-21, and IL-12 can promote TFH cell formation. Yet, none of these factors, nor their downstream cognate STATs, have emerged as nonredundant, essential drivers of TFH cells. This suggests a model in which multiple factors can contribute to the phenotypic characteristics of TFH cells. Because type I IFNs are often generated in immune responses, we set out to investigate whether these factors are relevant to TFH cell differentiation. Type I IFNs promote Th1 responses, thus one possibility was these factors antagonized TFH-expressed genes. However, we show that type I IFNs (IFN-α/ß) induced B cell lymphoma 6 (Bcl6) expression, the master regulator transcription factor for TFH cells, and CXCR5 and programmed cell death-1 (encoded by Pdcd1), key surface molecules expressed by TFH cells. In contrast, type I IFNs failed to induce IL-21, the signature cytokine for TFH cells. The induction of Bcl6 was regulated directly by STAT1, which bound to the Bcl6, Cxcr5, and Pdcd1 loci. These data suggest that type I IFNs (IFN-α/ß) and STAT1 can contribute to some features of TFH cells but are inadequate in inducing complete programming of this subset.

Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency.

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

Cellular and molecular requirements for the selection of in vitro-generated CD8 T cells reveal a role for Notch.

Differentiation of CD8 single-positive (SP) T cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In the thymus and the OP9-DL1 system for T cell development, Notch signals are required for progenitors to commit to the T cell lineage and necessary for their progression to the CD4(+)CD8(+) double-positive (DP) stage of T cell development. However, it remains unclear whether Notch is a prerequisite for the differentiation of DP cells to the CD8 SP stage of development. In this study, we demonstrate that Notch receptor-ligand interactions allow for efficient differentiation and selection of conventional CD8 T cells from bone marrow-derived hematopoietic stem cells. However, bone marrow-derived hematopoietic stem cells isolated from Itk(-/-)Rlk(-/-) mice gave rise to T cells with decreased IFN-γ production, but gained the ability to produce IL-17. We further reveal that positive and negative selection in vitro are constrained by peptide-MHC class I expressed on OP9 cells. Finally, using an MHC class I-restricted TCR-transgenic model, we show that the commitment of DP precursors to the CD8 T cell lineage is dependent on Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout T cell differentiation, including the final step of CD8 SP selection.

T follicular helper cell diversity and plasticity.

CD4(+) T helper (Th) cells play an instrumental role in orchestrating adaptive immune responses to invading pathogens through their ability to differentiate into specialized effector subsets. Part of this customized response requires the development of T follicular helper (Tfh) cells, which provide help to B cells for the generation of germinal centers (GCs) and long-term protective humoral responses. Although initially viewed as terminally differentiated, we now recognize that Th cell subsets, including Tfh cells, display substantial flexibility and overlap in their characteristics. In this review, we highlight advances in our understanding of Tfh cell development, cytokine production, and the potential plasticity that allows Tfh cells to possess characteristics of other effector Th cell populations.

A role for Ly108 in the induction of promyelocytic zinc finger transcription factor in developing thymocytes.

The promyelocytic zinc finger transcription factor (PLZF) is required for the development of activated phenotypes in NKT and other innate T lymphocytes. Although strong TCR stimulation has been implicated in the induction of PLZF, the factors regulating PLZF expression are incompletely understood. We show in this study that costimulation of preselection double-positive thymocytes through the signaling lymphocyte activation molecule family receptor Ly108 markedly enhanced PLZF expression compared with that induced by TCR stimulation alone. Costimulation with Ly108 increased expression of early growth response protein (Egr)-2 and binding of Egr-2 to the promoter of Zbtb16, which encodes PLZF, and resulted in PLZF levels similar to those seen in NKT cells. In contrast, costimulation with anti-CD28 failed to enhance Egr-2 binding and Zbtb16 expression. Moreover, mice lacking Ly108 showed decreased numbers of PLZF-expressing CD4(+) T cells. Together, these results support a potential role for Ly108 in the induction of PLZF.

Positive and negative signaling through SLAM receptors regulate synapse organization and thresholds of cytolysis.

X-linked lymphoproliferative syndrome, characterized by fatal responses to Epstein-Barr virus infection, is caused by mutations affecting the adaptor SAP, which links SLAM family receptors to downstream signaling. Although cytotoxic defects in SAP-deficient T cells are documented, the mechanism remains unclear. We show that SAP-deficient murine CD8(+) T cells exhibited normal cytotoxicity against fibrosarcoma targets, yet had impaired adhesion to and killing of B cell and low-avidity T cell targets. SAP-deficient cytotoxic lymphocytes showed specific defects in immunological synapse organization with these targets, resulting in inefficient actin clearance. In the absence of SAP, signaling through the SLAM family members Ly108 and 2B4 resulted in increased recruitment of the SHP-1 phosphatase, associated with altered SHP-1 localization and decreased activation of Src kinases at the synapse. Hence, SAP and SLAM receptors regulate positive and negative signals required for organizing the T cell:B cell synapse and setting thresholds for cytotoxicity against distinct cellular targets.

The receptor Ly108 functions as a SAP adaptor-dependent on-off switch for T cell help to B cells and NKT cell development.

Humans and mice deficient in the adaptor protein SAP (Sh2d1a) have a major defect in humoral immunity, resulting from a lack of T cell help for B cells. The role of SAP in this process is incompletely understood. We found that deletion of receptor Ly108 (Slamf6) in CD4(+) T cells reversed the Sh2d1a(-/-) phenotype, eliminating the SAP requirement for germinal centers. This potent negative signaling by Ly108 required immunotyrosine switch motifs (ITSMs) and SHP-1 recruitment, resulting in high amounts of SHP-1 at the T cell:B cell synapse, limiting T cell:B cell adhesion. Ly108-negative signaling was important not only in CD4(+) T cells; we found that NKT cell differentiation was substantially restored in Slamf6(-/-)Sh2d1a(-/-) mice. The ability of SAP to regulate both positive and negative signals in T cells can explain the severity of SAP deficiency and highlights the importance of SAP and SHP-1 competition for Ly108 ITSM binding as a rheostat for the magnitude of T cell help to B cells.