Associations of cortical SPP1 and ITGAX with cognition and common neuropathologies in older adults.

INTRODUCTION: The secreted phosphoprotein 1 (SPP1) gene expressed by CD11c+ cells is known to be associated with microglia activation and neuroinflammatory diseases. As most studies rely on mouse models, we investigated these genes and proteins in the cortical brain tissue of older adults and their role in Alzheimer's disease (AD) and related disorders. METHODS: We leveraged protein measurements, single-nuclei, and RNASeq data from the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) of over 1200 samples for association analysis. RESULTS: Expression of SPP1 and its encoded protein osteopontin were associated with faster cognitive decline and greater odds of common neuropathologies. At single-cell resolution,  integrin subunit alpha X (ITGAX) was highly expressed in microglia, where specific subpopulations were associated with AD and cerebral amyloid angiopathy. DISCUSSION: The study provides evidence of SPP1 and ITGAX association with cognitive decline and common neuropathologies identifying a microglial subset associated with disease.

A narrow T cell receptor repertoire instructs thymic differentiation of MHC class Ib-restricted CD8+ regulatory T cells.

Although most CD8+ T cells are equipped to kill infected or transformed cells, a subset may regulate immune responses and preserve self-tolerance. Here, we describe a CD8 lineage that is instructed to differentiate into CD8 T regulatory cells (Tregs) by a surprisingly restricted set of T cell receptors (TCRs) that recognize MHC-E (mouse Qa-1) and several dominant self-peptides. Recognition and elimination of pathogenic target cells that express these Qa-1-self-peptide complexes selectively inhibits pathogenic antibody responses without generalized immune suppression. Immunization with synthetic agonist peptides that mobilize CD8 Tregs in vivo efficiently inhibit antigraft antibody responses and markedly prolong heart and kidney organ graft survival. Definition of TCR-dependent differentiation and target recognition by this lineage of CD8 Tregs may open the way to new therapeutic approaches to inhibit pathogenic antibody responses.

Definition of the contribution of an Osteopontin-producing CD11c+ microglial subset to Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid ß (Aß) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aß. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.

Antibody-mediated blockade of the IL23 receptor destabilizes intratumoral regulatory T cells and enhances immunotherapy.

Regulatory T cells (Treg) can impede antitumor immunity and currently represent a major obstacle to effective cancer immunotherapy. Targeting tumor-infiltrating regulatory Treg while sparing systemic Treg represents an optimal approach to this problem. Here, we provide evidence that the interleukin 23 receptor (IL23R) expressed by tumor-infiltrating Treg promotes suppressive activity. Disruption of the IL23R results in increased responsiveness of destabilized Treg to the IL12 cytokine, the production of γ-interferon, and the recruitment of CD8 T cells that inhibit tumor growth. Since the Treg destabilization pathway that is initiated by IL23R blockade is distinct and independent from the destabilization pathway coupled to glucocorticoid-induced TNFR-related protein (GITR) activation, we examined the impact of the coordinate induction of the two destabilization pathways on antitumor immune responses. Combined GITR and IL23R antibody treatment of mice inoculated with MC38 tumors resulted in robust and synergistic antitumor responses. These findings indicate that the delineation of independent Treg destabilization pathways may allow improved approaches to the development of combination immunotherapy for cancers.

Definition of a mouse microglial subset that regulates neuronal development and proinflammatory responses in the brain.

Expression of Itgax (encoding the CD11c surface protein) and Spp1 (encoding osteopontin; OPN) has been associated with activated microglia that can develop in healthy brains and some neuroinflammatory disorders. However, whether CD11c and OPN expression is a consequence of microglial activation or represents a portion of the genetic program expressed by a stable microglial subset is unknown. Here, we show that OPN production in the brain is confined to a small CD11c+ microglial subset that differentiates from CD11c- precursors in perinatal life after uptake of apoptotic neurons. Our analysis suggests that coexpression of OPN and CD11c marks a microglial subset that is expressed at birth and persists into late adult life, independent of environmental activation stimuli. Analysis of the contribution of OPN to the intrinsic functions of this CD11c+ microglial subset indicates that OPN is required for subset stability and the execution of phagocytic and proinflammatory responses, in part through OPN-dependent engagement of the αVß3-integrin receptor. Definition of OPN-producing CD11c+ microglia as a functional microglial subset provides insight into microglial differentiation in health and disease.

From antibody specificity to T cell recognition.

Landsteiner's definition of human blood groups and the genetic rules that govern blood transfusion represents a milestone in human genetics and a historic event in public health. His research into the specificity of serological reactions, although less well known, has had a critical influence on the development of contemporary views on immune recognition, clonal selection, and immunological self-tolerance.

Mechanism of EBV inducing anti-tumour immunity and its therapeutic use.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein-Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein-Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers.

A new chapter in the CD8 T reg story.

CD8+ T reg cells play an important role in the maintenance of self-tolerance and can inhibit the development of autoimmune disease. In this issue of JEM, Mishra et al. (https://doi.org/10.1084/jem.20200030) reveal that TGF-ß signaling and an Eomes-dependent genetic program contribute to CD8 T reg cell differentiation and function.

Overcoming Immune Checkpoint Blockade Resistance via EZH2 Inhibition.

Recent progress in cancer immunotherapy highlights the power of the immune system to control tumors, although a small patient subset responds to current immunotherapies. Additional approaches to mobilize antitumor immunity are required to overcome primary and acquired resistance to immunotherapy such as immune checkpoint blockade (ICB). Emerging evidence shows that targeting epigenetic elements that promote tumor progression and inhibit immune cell activity can enhance antitumor immunity by reshaping the tumor microenvironment (TME). Here, we review the pleiotropic functions in tumor and immune cells of enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), with a focus on EZH2 inhibition as a potentially promising approach to enhance current immunotherapies and improve patient outcomes for certain cancers.

Regulatory CD8 T cells that recognize Qa-1 expressed by CD4 T-helper cells inhibit rejection of heart allografts.

Induction of longstanding immunologic tolerance is essential for survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.

NK Cells Regulate CD8+ T Cell Mediated Autoimmunity.

Elucidating key factors that regulate immune-mediated pathology in vivo is critical for developing improved strategies to treat autoimmune disease and cancer. NK cells can exhibit regulatory functions against CD8+ T cells following viral infection. Here we show that while low doses of lymphocytic choriomeningitis virus (LCMV-WE) can readily induce strong CD8+ T cell responses and diabetes in mice expressing the LCMV glycoprotein on ß-islet cells (RIP-GP mice), hyperglycemia does not occur after infection with higher doses of LCMV. High-dose LCMV infection induced an impaired CD8+ T cell response, which coincided with increased NK cell activity during early time points following infection. Notably, we observed increased NKp46 expression on NK cells during infection with higher doses, which resulted in an NK cell dependent suppression of T cells. Accordingly, depletion with antibodies specific for NK1.1 as well as NKp46 deficiency (Ncr1gfp/gfp mice) could restore CD8+ T cell immunity and permitted the induction of diabetes even following infection of RIP-GP mice with high-dose LCMV. Therefore, we identify conditions where innate lymphoid cells can play a regulatory role and interfere with CD8+ T cell mediated tissue specific pathology using an NKp46 dependent mechanism.

Control of Germinal Center Localization and Lineage Stability of Follicular Regulatory T Cells by the Blimp1 Transcription Factor.

Follicular regulatory T (TFR) cells are a specialized suppressive subset that controls the germinal center (GC) response and maintains humoral self-tolerance. The mechanisms that maintain TFR lineage identity and suppressive activity remain largely unknown. Here, we show that expression of Blimp1 by FoxP3+ TFR cells is essential for TFR lineage stability, entry into the GC, and expression of regulatory activity. Deletion of Blimp1 in TFR cells reduced FoxP3 and CTLA-4 expression and increased pro-inflammatory cytokines and spontaneous production of autoantibodies, including elevated IgE. Maintenance of TFR stability reflected Blimp1-dependent repression of the IL-23R-STAT3 axis and activation of the CD25-STAT5 pathway, while silenced IL-23R-STAT3 or increased STAT5 activation rescued the Blimp1-deficient TFR phenotype. Blimp1-dependent control of CXCR5/CCR7 expression also regulated TFR homing into the GC. These findings uncover a Blimp1-dependent TFR checkpoint that enforces suppressive activity and acts as a gatekeeper of GC entry.

New Insights Into the Biology of CD8 Regulatory T Cells.

Regulatory T cells are central mediators of immune regulation and play an essential role in the maintenance of immune homeostasis in the steady state and under pathophysiological conditions. Disruption of CD8 Treg-dependent recognition of Qa-1-restricted self-antigens can result in dysregulated immune responses, tissue damage, autoimmune disease and cancer. Recent progress in studies on regulatory T cells of the CD8 lineage has provided new biological insight into this specialized regulatory T cell subpopulation. Identification of the Helios transcription factor as an essential control element for the differentiation and function of CD8 regulatory T cells has led to a better understanding of the unique genetic program of these cells. Recent analyses of T-cell receptor usage and antigen recognition by Qa-1-restricted CD8 Treg have provided additional insight into the unusual biological function of this regulatory CD8 lineage. Here we summarize recent advances in our understanding of CD8 regulatory T cells with emphasis on lineage commitment, differentiation and stability.

Chromatin remodeling by the NuRD complex regulates development of follicular helper and regulatory T cells.

Lineage commitment and differentiation into CD4+ T cell subsets reflect an interplay between chromatin regulators and transcription factors (TF). Follicular T cell development is regulated by the Bcl6 TF, which helps determine the phenotype and follicular localization of both CD4+ follicular helper T cells (TFH) and follicular regulatory T cells (TFR). Here we show that Bcl6-dependent control of follicular T cells is mediated by a complex formed between Bcl6 and the Mi-2ß-nucleosome-remodeling deacetylase complex (Mi-2ß-NuRD). Formation of this complex reflects the contribution of the intracellular isoform of osteopontin (OPN-i), which acts as a scaffold to stabilize binding between Bcl6 and the NuRD complex that together regulate the genetic program of both TFH and TFR cells. Defective assembly of the Bcl6-NuRD complex distorts follicular T cell differentiation, resulting in impaired TFR development and skewing of the TFH lineage toward a TH1-like program that includes expression of Blimp1, Tbet, granzyme B, and IFNγ. These findings define a core Bcl6-directed transcriptional complex that enables CD4+ follicular T cells to regulate the germinal center response.

Comparative transcriptome analysis reveals distinct genetic modules associated with Helios expression in intratumoral regulatory T cells.

Regulatory T cells (Tregs) are key modulators of immune tolerance, capable of suppressing inflammatory immune responses and promoting nonlymphoid tissue homeostasis. Helios, a transcription factor (TF) that is selectively expressed by Tregs, has been shown to be essential for the maintenance of Treg lineage stability in the face of inflammatory conditions that include autoimmune disease and cancer. Helios-deficient Tregs within tumors acquire effector T cell function and contribute to immune responses against cancer. However, the underlying genetic basis of this Treg reprogramming is not well understood. Here, we report that Helios-deficient Tregs within the chronic inflammatory tumor microenvironment (TME) derepress genetic programs associated with T helper (Th) cell differentiation by up-regulating Th cell-associated TFs and effector cytokines. These genetic changes of Helios-deficient Tregs are most apparent in a Treg subpopulation with high affinity for self-antigens, as detected by both increased GITR/PD-1 expression and increased responsiveness to self-antigens. Their combined effects may promote a phenotype conversion of Tregs into effector T cells within the TME, where TCR engagement and costimulatory receptor expression by Tregs are increased. These data provide a genetic basis for the unstable phenotype of Helios-deficient Tregs within the inflammatory environment of tumors and suggest that immune milieu-dependent alterations in gene expression are a central feature of Treg conversion.

Signaling by the Epstein-Barr virus LMP1 protein induces potent cytotoxic CD4+ and CD8+ T cell responses.

The B-lymphotropic Epstein-Barr virus (EBV), pandemic in humans, is rapidly controlled on initial infection by T cell surveillance; thereafter, the virus establishes a lifelong latent infection in the host. If surveillance fails, fatal lymphoproliferation and lymphomagenesis ensue. The initial T cell response consists of predominantly CD8+ cytotoxic T cells and a smaller expansion of CD4+ cells. A major approach to treating EBV-associated lymphomas is adoptive transfer of autologous or allogeneic T cells that are stimulated/expanded on EBV-transformed B cells. Strikingly, the clinical response correlates with the frequency of CD4 cells in the infused T cells. Although in vitro studies suggested that EBV-specific CD4 cells develop cytotoxicity, they have not been comprehensively characterized and the molecular mechanism underlying their formation remains unknown. Our recent work, using a transgenic approach in mice, has revealed a central role for the EBV signaling molecule LMP1 in immune surveillance and transformation of EBV-infected B cells. The mouse model offers a unique tool for uncovering basic features of EBV immunity. Here, we show that LMP1 expression in B cells induces potent cytotoxic CD4 and CD8 T cell responses, by enhancing antigen presentation and costimulation by CD70, OX40 ligand, and 4-1BB ligand. Our data further suggest that cytotoxic CD4 cells hold superior therapeutic value for LMP1 (EBV)-driven lymphomas. These findings provide insights into EBV immunity, demonstrating that LMP1 signaling alone is sufficient to induce a prominent cytotoxic CD4 response, and suggest strategies for immunotherapy in EBV-related and other cancers.

γδ T Cells Contribute to Injury in the Developing Brain.

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αßT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.