Better treatment of autoimmune diseases requires an improved understanding of the cellular and molecular mechanisms that lead to the breakdown of immune tolerance. The discovery of individuals with ...germline mutations in PIK3CD (which encodes the p110δ catalytic subunit of PI3K) has revealed the importance of regulated PI3Kδ activity to maintain tolerance. These patients display a range of symptoms including both immunodeficiency and autoimmunity. Here, we discuss recent advances in our understanding of how dysregulated PI3Kδ signaling affects the activation and differentiation of multiple cell types leading to the production of autoantibodies in these patients. This has lessons, not only for the treatment of these patients, but also for the potential role of dysregulated PI3Kδ in other patients with autoimmune conditions.
Germline gain-of function (GOF) mutations in PIK3CD, encoding the catalytic p110δ subunit of phosphoinositide 3-kinase (PI3K), result in hyperactivation of the PI3K–AKT–mechanistic target of ...rapamycin pathway and underlie a novel inborn error of immunity. Affected subjects exhibit perturbed humoral and cellular immunity, manifesting as recurrent infections, autoimmunity, hepatosplenomegaly, uncontrolled EBV and/or cytomegalovirus infection, and increased incidence of B-cell lymphoproliferation, lymphoma, or both. Mechanisms underlying disease pathogenesis remain unknown.
Understanding the cellular and molecular mechanisms underpinning inefficient surveillance of EBV-infected B cells is required to understand disease in patients with PIK3CD GOF mutations, identify key molecules required for cell-mediated immunity against EBV, and develop immunotherapeutic interventions for the treatment of this and other EBV-opathies.
We studied the consequences of PIK3CD GOF mutations on the generation, differentiation, and function of CD8+ T cells and natural killer (NK) cells, which are implicated in host defense against infection with herpesviruses, including EBV.
PIK3CD GOF total and EBV-specific CD8+ T cells were skewed toward an effector phenotype, with exaggerated expression of markers associated with premature immunosenescence/exhaustion and increased susceptibility to reactivation-induced cell death. These findings were recapitulated in a novel mouse model of PI3K GOF mutations. NK cells in patients with PIK3CD GOF mutations also exhibited perturbed expression of differentiation-associated molecules. Both CD8+ T and NK cells had reduced capacity to kill EBV-infected B cells. PIK3CD GOF B cells had increased expression of CD48, programmed death ligand 1/2, and CD70.
PIK3CD GOF mutations aberrantly induce exhaustion, senescence, or both and impair cytotoxicity of CD8+ T and NK cells. These defects might contribute to clinical features of affected subjects, such as impaired immunity to herpesviruses and tumor surveillance.
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Gain-of-function (GOF) mutations in
, encoding the p110δ subunit of phosphatidylinositide 3-kinase (PI3K), cause a primary immunodeficiency. Affected individuals display impaired humoral immune ...responses following infection or immunization. To establish mechanisms underlying these immune defects, we studied a large cohort of patients with
GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in
In both species, hyperactive PI3K severely affected B cell development and differentiation in the bone marrow and the periphery. Furthermore, PI3K GOF B cells exhibited intrinsic defects in class-switch recombination (CSR) due to impaired induction of activation-induced cytidine deaminase (AID) and failure to acquire a plasmablast gene signature and phenotype. Importantly, defects in CSR, AID expression, and Ig secretion were restored by leniolisib, a specific p110δ inhibitor. Our findings reveal key roles for balanced PI3K signaling in B cell development and long-lived humoral immunity and memory and establish the validity of treating affected individuals with p110δ inhibitors.
Introduction
Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell ...lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood.
Methods
Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11
M365K
mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL.
Results and discussion
Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11
M365K
mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11
M365K
mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (T
REG
), T follicular (T
FH
) and T follicular regulatory (T
FR
) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of T
FH
, T
REG
and T
FR
cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.
Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been ...shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease.
Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-γ. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-γ) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher.
Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dendritic cells (DCs), the most important professional antigen-presenting cells (APC), play crucial role in both immunity and tolerance. It is well known that DCs are able to mount immune responses ...against foreign antigens and simultaneously tolerate self-antigens. Since DCs can be modulated depending on the surrounding microenvironment, they can act as a bridge between innate and adaptive immunity. However, the mechanisms that support this dual role are not entirely clear. Recent studies have shown that DCs can be manipulated ex vivo in order to trigger their tolerogenic profile, what can be a tool to be used in clinical trials aiming the treatment of various diseases and the prevention of transplant rejection. In this sense, the blockage of costimulatory molecules on DC, in the attempt of inhibiting the second signal in the immunological synapse, can be considered as one of the main strategies under development. This review brings an update on current therapies using tolerogenic dendritic cells modulated with costimulatory blockers with the aim of reducing transplant rejection. However, although there are current clinical trials using tolerogenic DC to treat allograft rejection, the actual challenge is to modulate these cells in order to maintain a permanent tolerogenic profile.
Abstract
Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of ...phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many surface receptors expressed by T cells and has been implicated in the control of CD4+ T cell helper differentiation, activation, and proliferation. APDS patients have increased PI3K activity leading to several immune manifestations with the some of the most common features being lymphoproliferation, respiratory tract infections, Th2-related pathologies, and impaired antibody responses.
We previously analysed a CRISPR/Cas9 mouse model (Pik3cdE1020K). This showed intrinsic changes impacting on T cell function (increased Th2 cytokines and poor Tfh function) but also extrinsic factors driving a decrease in frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation. Here we investigated these extrinsic components further. We found that PI3K B cells are driving Tfh differentiation. We also explored the role of PI3K in maintaining Treg function and identity and determined if Treg dysregulation was driving T cell hyperactivation.
These studies reveal an important role of PI3K in modulating T cell differentiation, activation, and function through both T cell intrinsic and extrinsic mechanisms. An improved understanding of T cell hyperactivation can provide insight into the lymphoproliferation that affects most APDS patients. Overall, these results reveal the potential to dissect the mechanisms underlying disease, providing grounds to define new drug targets and ultimately improve the clinical management of these patients.
Abstract
APDS is a primary immunodeficiency condition caused by heterozygous gain-of-function mutations in PIK3CD (APDS1), which encodes the leukocyte-restricted p110-δ catalytic subunit of ...phosphoinositide 3-kinase (PI3K) or heterozygous loss-of-function mutations in PIK3R1 (APDS2), which encodes the regulatory subunit of PI3K. PI3K is activated downstream of many receptors expressed by lymphocytes and plays important roles in their activation and differentiation. The increased PI3K activity in cells from APDS patients leads to multiple immune manifestations including lymphoproliferation, respiratory tract infections, Th2-related pathologies, impaired Ab responses and autoimmunity.
We previously dissected the cellular defects that lead to disease in APDS1 by studying patient cells and our novel mouse model. This revealed changes in lymphocytes including increased memory T cells, defective Tfh function, decreased isotype switching and a break in B cell tolerance and production of autoantibodies. We have now generated a mouse model of APDS2 (Pik3r1E11SpD) and use this together with studies of patients’ cells to determine whether increased PI3K signalling due to loss of regulatory function causes the same cellular dysregulation. While we observed many of the same changes previously observed in APDS1, including altered cytokine production and decreased isotype switching, we also identified multiple differences in the cellular phenotype in APDS2 compared to APDS1.
Together, these studies reveal that although APDS1 and 2 both cause increased PI3K signalling and result in similar clinical phenotypes, there are distinctions between how these two types of mutations affect cellular function.
Supported by grants from the NHMRC and an AAI Careers in Immunology Fellowship
Abstract
Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of ...phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many T cell receptors and has been implicated in the control of CD4+ T cell helper differentiation, activation and proliferation. APDS patients have increased PI3K activity that leads to several immune manifestations including lymphoproliferation, Th2-related pathologies and impaired Ab responses.
Analysis of a CRISPR/Cas9 mouse model (Pik3cdE1020K) showed a decreased frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation, as well as changes in cytokine expression and T cell help. Our studies investigated CD4+ T cell intrinsic and extrinsic alterations contributing to the dysregulation of the immune response.
We identified cell intrinsic features resulting in increased IL-4 and IL-5, explaining the Th2 mediated disease in these patients. Additionally, cell intrinsic changes in Tfh generation and function were shown in the in vivo mouse model, contributing to an impaired ability to sustain GCs and alter isotype switching. Furthermore, additional studies have shown that the overactive T cells result from both intrinsic and extrinsic factors, with contributions from both dysregulated T cells and B cells exerting effects on T cell activation.
Together, these studies reveals an important role of PI3K in modulating T cell differentiation and function through both T cell-intrinsic and extrinsic mechanisms. This provides new insights into the APDS pathophysiology with potential to improve the clinical management of these patients.
Primary immune deficiency is caused by genetic mutations that result in immune dysfunction and subsequent susceptibility to infection. Over the last decade there has been a dramatic increase in the ...number of genetically defined causes of immune deficiency including those which affect B‐cell function. This has not only identified critical nonredundant pathways that control the generation of protective antibody responses but also revealed that immunodeficiency and autoimmunity are often closely linked. Here we explore the molecular and cellular mechanisms of these rare monogenic conditions that disrupt antibody production, which also have implications for understanding the causes of more common polygenic immune dysfunction.
Many single‐gene defects have been described that lead to defective B‐cell responses and infectious susceptibility. Here we review these conditions and discuss the insights these provide into the key signaling pathways critical for effective antibody responses.
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