Modulating the PI3K Signalling Pathway in Activated PI3K Delta Syndrome: a Clinical Perspective.

Activated phosphoinositide-3-kinase (PI3K) δ syndrome (APDS) is an inborn error of immunity characterised by immune dysregulation. Since the discovery of genetic mutations resulting in PI3Kδ overactivation, treatment of APDS patients has begun to focus on modulation of the PI3K pathway in addition to supportive therapies. The mTOR inhibitor sirolimus has been used effectively for some clinical manifestations of this condition, however the arrival of specific PI3Kδ inhibitor leniolisib has shown promising early results and may provide a more targeted approach. This review summarizes key aspects of PI3K pathway biology and discusses potential options for nuanced modulation of the PI3K pathway in APDS from a clinical perspective, highlighting differences from PI3K inhibition in haematological malignancies.

Activating PIK3CD mutations impair human cytotoxic lymphocyte differentiation and function and EBV immunity.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Activating mutations in PIK3CD disrupt the differentiation and function of human and murine CD4+ T cells.

BACKGROUND: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8+ T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4+ T cells in disease pathogenesis. OBJECTIVE: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4+ T-cell function. METHODS: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4+ T cells and a novel murine disease model caused by overactive PI3K signaling. RESULTS: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (TFH) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human TFH cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a TFH cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4+ T cells also acquired an aberrant TFH phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and TFH cells was largely CD4+ T-cell extrinsic, whereas changes in cytokine production and TFH cell function were cell intrinsic. CONCLUSION: Our studies reveal that CD4+ T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations.

Gastric Cancer Screening in Common Variable Immunodeficiency.

Individuals with common variable immunodeficiency (CVID) have an increased risk of gastric cancer, and gastrointestinal lymphoma, yet screening for premalignant gastric lesions is rarely offered routinely to these patients. Proposed screening protocols are not widely accepted and are based on gastric cancer risk factors that are not applicable to all CVID patients. Fifty-two CVID patients were recruited for screening gastroscopy irrespective of symptoms or blood results and were compared to 40 controls presenting for gastroscopy for other clinical indications. Overall, 34% of CVID patients had intestinal metaplasia (IM), atrophic gastritis or moderate to severe non-atrophic gastritis, which can increase the risk of gastric cancer, compared to 7.5% of controls (p < 0.01). Focal nodular lymphoid hyperplasia, a precursor lesion for gastrointestinal lymphoma, was seen in eight CVID patients (16%), one of whom was diagnosed with gastrointestinal lymphoma on the same endoscopy. High-risk gastric pathology was associated with increased time since diagnosis of CVID, smoking, Helicobacter pylori, a low-serum pepsinogen I concentration, and diarrhea, but not pepsinogen I/II ratio, iron studies, vitamin B12 levels or upper gastrointestinal symptoms. There was a lower rate of detection of IM when fewer biopsies were taken, and IM and gastric atrophy were rarely predicted by the endoscopist macroscopically, highlighting the need for standardized biopsy protocols. The prevalence of premalignant gastric lesions in patients with CVID highlights the need for routine gastric screening. We propose a novel gastric screening protocol to detect early premalignant lesions and reduce the risk of gastric cancer and gastric lymphoma in these patients.

IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts.

B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.

IL-21 is the primary common γ chain-binding cytokine required for human B-cell differentiation in vivo.

SCID resulting from mutations in IL2RG or JAK3 is characterized by lack of T and natural killer cells; B cells are present in normal number, but antibody responses are defective. Hematopoietic cell transplantation (HCT) is curative for SCID. However, B-cell dysfunction persists in a substantial proportion of patients. We hypothesized that impaired B-cell responses after HCT in IL2RG/JAK3 deficiency results from poor donor B-cell engraftment and defective γc-dependent cytokine signaling in host B cells. To test this, and to identify which γc cytokine(s) is critical for humoral immunity, we studied 28 transplanted patients with IL2RG/JAK3 deficiency. Lack of donor B-cell engraftment associated with persistent humoral dysfunction and significantly reduced memory B cells. B-cell proliferation induced by CD40L alone or together with CpG, anti-Ig, IL-4, IL-10, or IL-13 was comparable in healthy controls and in post-HCT SCID patients, irrespective of their chimerism status. However, in vitro stimulation with CD40L/IL-21 induced B-cell proliferation, plasmablast differentiation, and antibody secretion in patients with donor B cells, but not in patients with autologous B cells. These data imply that IL-21-mediated signaling is critical for long-lived humoral immunity and to restore antibody responses in IL2RG/JAK3-deficient patients after HCT. Furthermore, in vitro stimulation with CD40L/IL-21 can predict in vivo B-cell immunity in IL2RG/JAK3 SCID after transplantation.

Duodenal plasma cells correspond to serum IgA in common variable immunodeficiency.

Common variable immunodeficiency (CVID) can be associated with a range of serum IgA concentrations, from absent, to variably reduced, and in some patients classified as 'possible CVID', even normal. The aim of this study was to assess the proportion of duodenal plasma cells in patients with CVID and determine whether there was an association with serum IgA concentration. Duodenal biopsies obtained at upper endoscopy from 35 patients with CVID were assessed for the presence of plasma cells and compared with serum IgA concentrations. A reduction or absence of duodenal plasma cells in 60% of patients with CVID and an association between the proportions of duodenal plasma cells and serum IgA concentrations was demonstrated. The presence of duodenal plasma cells associated with numbers of isotype switched memory B cells in the peripheral blood. A reduction in serum IgA over time was observed in 19% of CVID patients. The gastrointestinal tract provides a window into the immune system in CVID, and these results reinforce the association between gastrointestinal plasma cells and serum IgA concentrations. Preservation of gastrointestinal plasma cells and serum IgA in some patients with CVID, and the sequential decline of both in others, highlight the heterogeneity of this disorder.

Use of Infliximab to Treat Paradoxical Tuberculous Meningitis Reactions.

We documented dramatic responses to infliximab in 4 tuberculous meningitis cases with severe paradoxical reactions after effective antibacterial treatment, despite high-dose steroids. In every instance, infliximab was used as a last resort after all other options were exhausted, resulting in delayed initiation that may have adversely affected patient outcomes.

Activated PI3Kδ breaches multiple B cell tolerance checkpoints and causes autoantibody production.

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

B-cell maturation defects in common variable immunodeficiency and association with clinical features.

AIMS: Patients with common variable immunodeficiency (CVID) often have defects in post-antigenic B-cell differentiation with fewer memory B cells and impaired isotype switching. We aimed to classify CVID patients according to these defects and determine whether this predicted clinical manifestations. METHODS: We analysed the memory marker CD27, maturation marker CD21, and IgD on peripheral blood B cells from 31 CVID patients and 23 controls using a whole-blood lysis technique, allocated patients according to two classifications ('Freiburg' and 'Paris') and correlated results with clinical manifestations. RESULTS: CVID patients had fewer memory (CD27(+)) B cells and isotype-switched (IgD(-)) memory B cells in absolute number and proportion. Many CVID patients had increased immature (CD21(-)) B cells. Lymphoproliferation and autoimmune cytopenias were found almost exclusively in these patients, including Freiburg group Ia (decreased switched memory and increased immature B cells), but also those with normal switched memory and increased immature B cells. The Paris classification was less useful in predicting clinical manifestations. CONCLUSIONS: CVID is associated with defects in memory B-cell differentiation. Subclassification helps identify patients with clinical manifestations, particularly lymphoproliferation and autoimmune cytopenias in those with impaired B-cell maturation and isotype switching. Routine B-cell phenotyping may assist clinicians in predicting these clinical features.

Germline-activating mutations in PIK3CD compromise B cell development and function.

Gain-of-function (GOF) mutations in PIK3CD, 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 PIK3CD GOF mutations and established a novel mouse model using CRISPR/Cas9-mediated gene editing to introduce a common pathogenic mutation in Pik3cd 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.

Pregnancy in a patient with congenital analbuminaemia.

Congenital analbuminaemia is a rare autosomal recessive disorder that is characterised by a severe reduction or total absence of serum albumin. This condition has implications for therapeutics as a large proportion of commonly used drugs are plasma protein bound where albumin is the primary component of plasma protein. This is the first case report of pregnancy in a patient with congenital analbuminaemia in the medical literature. In the absence of drug dosage guidelines for patients with congenital analbuminaemia, a list of drugs which may be required for this patient during pregnancy, delivery and/or emergency situations were compiled by a multidisciplinary team. Our patient suffered from polyhydramnios during her pregnancy which was successfully managed with albumin transfusions and had a normal vaginal delivery with no complications in the intrapartum or postpartum period. The management and unique challenges of pregnancy in a patient with congenital analbuminaemia are discussed.

Migratory large vessel vasculitis preceding acute myeloid leukemia: a case report.

BACKGROUND: Large vessel vasculitis is a rare disorder usually occurring in the context of the autoimmune conditions of giant cell arteritis and Takayasu's arteritis. Case reports have described large vessel vasculitis occurring in individuals with myelodysplastic syndrome, preceding transformation to acute myeloid leukemia. CASE PRESENTATION: A 56-year-old Afghanistan-born woman presented with fever, a tender left carotid artery, and raised inflammatory markers. Computed tomography revealed thickening of the wall of her left carotid artery. Her symptoms resolved spontaneously; however, they recurred weeks later on the contralateral side, along with abdominal pain after eating. Further imaging with computed tomography and positron emission tomography demonstrated resolution of her left carotid artery abnormality, but new wall thickening and inflammation in her right carotid artery, abdominal aorta, and superior mesenteric artery. She was diagnosed as having large vessel vasculitis, which resolved with corticosteroids and methotrexate. Five months later, she developed acute myeloid leukemia. She had no known history of myelodysplastic syndrome at the time of diagnosis with vasculitis. CONCLUSIONS: Large vessel vasculitis in older individuals presenting with atypical clinical features, such as a migratory pattern of affected vessels, vessel wall tenderness, and marked systemic inflammation, should prompt a search for underlying myelodysplasia. Clinicians should be vigilant for progression to acute myeloid leukemia.

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells.

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell-dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10- and IL-21-mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21-induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.