Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

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.

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.

The IL-15 receptor {alpha} chain cytoplasmic domain is critical for normal IL-15Ralpha function but is not required for trans-presentation.

IL-15 is critical for natural killer (NK)-cell development and function and for memory CD8(+) T-cell homeostasis. The IL-15 receptor consists of IL-15Ralpha, IL-2Rbeta, and the common cytokine receptor gamma chain (gamma(c)). IL-15Ralpha is known to "trans-present" IL-15 to an IL-2Rbeta/gamma(c) heterodimeric receptor on responding cells to initiate signaling. To investigate the importance of the IL-15Ralpha cytoplasmic domain, we generated a chimeric receptor consisting of the extracellular domain of IL-15Ralpha and intracellular domain of IL-2Ralpha (IL-15Ralpha(ext)/IL-2Ralpha(int)) and examined its function in 32D cells, in knock-in (KI) mice, and in adoptive-transfer experiments. The chimeric protein exhibited decreased cell-surface expression, and KI mice exhibited diminished NK, NKT, and CD8(+) T-cell development and defects in T-cell functional responses. However, 32D cells expressing the chimeric receptor had less IL-15-induced proliferation than wild-type (WT) transfectants with similar levels of IL-15Ralpha expression, indicating a signaling role for the IL-15Ralpha cytoplasmic domain beyond its effect on expression, and demonstrating that the IL-2Ralpha and IL-15Ralpha cytoplasmic domains are functionally distinct. Interestingly, adoptive-transfer experiments indicated that the chimeric IL-15Ralpha(ext)/IL-2Ralpha(int) receptor still supports trans-presentation. These experiments collectively indicate that IL-15Ralpha can act in cis in addition to acting in trans to present IL-15 to responding cells.

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.

Constitutive expression of IL-7 receptor alpha does not support increased expansion or prevent contraction of antigen-specific CD4 or CD8 T cells following Listeria monocytogenes infection.

Expression of IL-7Ralpha (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7Ralpha on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7Ralpha alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7Ralpha did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7Ralpha transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7Ralpha transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7Ralpha does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7Ralpha alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.

T-cell development and function are modulated by dual specificity phosphatase DUSP5.

Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates lymphocyte proliferation and peripheral tolerance. IL-2 activates mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and signal transducer and activator of transcription (STAT) pathways and modulates expression of target genes. Systematic analysis of IL-2 target genes has revealed regulation of potential feedback inhibitors of IL-2 signaling, including several suppressor of cytokine signaling (SOCS) family members as well as MAPK pathway-regulating dual specificity phosphatases (DUSPs). Here we have evaluated the in vivo actions of DUSP5, an extracellular signal-regulated kinase 1/2 (ERK1/2)-specific phosphatase, by generating transgenic mice overexpressing DUSP5 within the lymphoid compartment. We show that transgenic DUSP5 expression results in a block in thymocyte development at the double positive stage. We also demonstrate that DUSP5-expressing mature T cells exhibit decreased IL-2-dependent proliferation and defective IL-2-mediated induction of genes. Finally, DUSP5 transgenic mice develop autoimmune symptoms, suggesting a role for the MAPK pathway in the regulation of tolerance. Thus, proper regulation of DUSP5 activity is critical for normal immune system development, IL-2 actions, and tolerance.

Targeting the GA binding protein beta1L isoform does not perturb lymphocyte development and function.

GA binding protein (GABP) is a ubiquitously expressed Ets family transcription factor that consists of two subunits, GABPalpha and GABPbeta. GABPalpha binds to DNA, and GABPbeta heterodimerizes with GABPalpha and possesses the ability to transactivate target genes. Our previous studies using GABPalpha-deficient mice revealed that GABPalpha is required for the development of both T and B cells. Two splice variants of GABPbeta are generated from the Gabpb1 locus and differ in their carboxy-terminal lengths and sequences. The longer isoform (GABPbeta1L) can homodimerize and thus form alpha(2)beta(2) tetramers depending on the gene context, whereas the shorter isoform (GABPbeta1S) cannot. In this study, we generated mice that are deficient in GABPbeta1L but that retain the expression of GABPbeta1S. Surprisingly, GABPbeta1L-/- mice had normal T- and B-cell development, and mature T and B cells showed normal responses to various stimuli. In contrast, targeting both GABPbeta1L and GABPbeta1S resulted in early embryonic lethality. Because of its incapability of forming homodimers, GABPbeta1S has been suspected to have a dominant negative role in regulating GABP target genes. Our findings argue against such a possibility and rather suggest that GABPbeta1S has a critical role in maintaining the transcriptional activity of the GABPalpha/beta complex.

The transcription factor GABP is a critical regulator of B lymphocyte development.

GA binding protein (GABP) is a ubiquitously expressed Ets-family transcription factor that critically regulates the expression of the interleukin-7 receptor alpha chain (IL-7Ralpha) in T cells, whereas it is dispensable for IL-7Ralpha expression in fetal liver B cells. Here we showed that deficiency of GABPalpha, the DNA-binding subunit of GABP, resulted in profoundly defective B cell development and a compromised humoral immune response, in addition to thymic developmental defects. Furthermore, the expression of Pax5 and Pax5 target genes such as Cd79a was greatly diminished in GABPalpha-deficient B cell progenitors, pro-B, and mature B cells. GABP could bind to the regulatory regions of Pax5 and Cd79a in vivo. Thus, GABP is a key regulator of B cell development, maturation, and function.