Immunologic and Genetic Contributors to CD46-Dependent Immune Dysregulation.

Mutations in CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly-but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.

Real-Time Volatile Metabolomics Analysis of Dendritic Cells.

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by end-point measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature and activated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation. Distinct metabolic signatures of DCs treated with different supernatants (SNs) of bacterial cultures were detected during real-time analyses over 6 h compared to their respective controls (SN only). Furthermore, the technique allowed for the detection of 13C-incorporation into volatile metabolites, opening the possibility for real-time tracing of metabolic pathways in DCs. Moreover, differences in the metabolic profile of naïve and activated DCs were discovered, and pathway-enrichment analysis revealed three significantly altered pathways, including the TCA cycle, α-linolenic acid metabolism, and valine, leucine, and isoleucine degradation.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

Memory CD8+ T Cells Balance Pro- and Anti-inflammatory Activity by Reprogramming Cellular Acetate Handling at Sites of Infection.

Serum acetate increases upon systemic infection. Acutely, assimilation of acetate expands the capacity of memory CD8+ T cells to produce IFN-γ. Whether acetate modulates memory CD8+ T cell metabolism and function during pathogen re-encounter remains unexplored. Here we show that at sites of infection, high acetate concentrations are being reached, yet memory CD8+ T cells shut down the acetate assimilating enzymes ACSS1 and ACSS2. Acetate, being thus largely excluded from incorporation into cellular metabolic pathways, now had different effects, namely (1) directly activating glutaminase, thereby augmenting glutaminolysis, cellular respiration, and survival, and (2) suppressing TCR-triggered calcium flux, and consequently cell activation and effector cell function. In vivo, high acetate abundance at sites of infection improved pathogen clearance while reducing immunopathology. This indicates that, during different stages of the immune response, the same metabolite-acetate-induces distinct immunometabolic programs within the same cell type.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Long-lived T follicular helper cells retain plasticity and help sustain humoral immunity.

CD4+ memory T cells play an important role in protective immunity and are a key target in vaccine development. Many studies have focused on T central memory (Tcm) cells, whereas the existence and functional significance of long-lived T follicular helper (Tfh) cells are controversial. Here, we show that Tfh cells are highly susceptible to NAD-induced cell death (NICD) during isolation from tissues, leading to their underrepresentation in prior studies. NICD blockade reveals the persistence of abundant Tfh cells with high expression of hallmark Tfh markers to at least 400 days after infection, by which time Tcm cells are no longer found. Using single-cell RNA-seq, we demonstrate that long-lived Tfh cells are transcriptionally distinct from Tcm cells, maintain stemness and self-renewal gene expression, and, in contrast to Tcm cells, are multipotent after recall. At the protein level, we show that folate receptor 4 (FR4) robustly discriminates long-lived Tfh cells from Tcm cells. Unexpectedly, long-lived Tfh cells concurrently express a distinct glycolytic signature similar to trained immune cells, including elevated expression of mTOR-, HIF-1-, and cAMP-regulated genes. Late disruption of glycolysis/ICOS signaling leads to Tfh cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of Tfh and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct long-lived T cell subsets and establish Tfh cells as an attractive target for the induction of durable adaptive immunity.

Co-Occurrence of ANCA-Associated Vasculitis and Sjögren's Syndrome in a Patient With Acromegaly: A Case Report and Retrospective Single-Center Review of Acromegaly Patients.

Background: ANCA-associated vasculitis (AAV) and Sjögren's syndrome (SS) are uncommon autoimmune diseases. The co-occurrence in the same patient has been rarely described. Acromegaly has been associated with autoimmune thyroiditis, but the prevalence of other autoimmune disorders such as AAV and SS has not been evaluated in acromegaly. Methods: Characterization of a patient with acromegaly and two rare autoimmune diseases-SS and AAV (microscopic polyangiitis (MPA))-by autoantibody-array and whole exome sequencing (WES). Single-center retrospective review of medical records of acromegaly patients to explore the prevalence of diagnosed autoimmune diseases. Results: We report a Caucasian woman in her 50's with a serologically (anti-SSA/Ro, anti-MPO-ANCA antibodies) and histologically confirmed diagnosis of symptomatic SS and MPA. SS with MPO-ANCA positivity preceded MPA. An exploratory autoantigen array detected a broad spectrum of autoantibodies. WES revealed heterozygous carrier status of the PTPN22 mutation R620W, which is associated with an increased risk for autoimmunity. A similar combination of positive anti-SSA/Ro autoantibodies and ANCA was only present in 5/1184 (0.42%) other patients tested for both antibodies in our clinic over six years. Amongst 85 acromegaly patients seen at our clinic in a 20-year period, 12% had a clinically relevant associated immunological disease. Conclusion: We present a rare case of SS and AAV in a patient with acromegaly and multiple autoantibody specificities. Patients with SS and ANCA should be closely monitored for the development of (subclinical) AAV. Whether acromegaly represents a risk for autoimmunity should be further investigated in prospective acromegaly cohorts.

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Prophylactic and Therapeutic Effects of Interleukin-2 (IL-2)/Anti-IL-2 Complexes in Systemic Lupus Erythematosus-Like Chronic Graft-Versus-Host Disease.

Murine chronic graft-versus-host-disease (cGvHD) induced by injection of parental lymphocytes into F1 hybrids results in a disease similar to systemic lupus erythematosus. Here, we have used DBA/2 T cell injection into (C57BL/6 × DBA/2)F1 (BDF1) mice as a model system to test the prophylactic and therapeutic effects of interleukin-2 (IL-2)/anti-IL-2 immune complexes on the course of cGvHD. Our findings demonstrate that pretreatment with Treg inducing JES6/IL-2 complexes render BDF1 mice largely resistant to induction of cGvHD, whereas pretreatment with CD8+ T cell/NK cell inducing S4B6/IL-2 complexes results in a more severe cGvHD. In contrast, treatment with JES6/IL-2 complexes 4 weeks after induction had no beneficial effect on disease symptoms. However, similar treatment with S4B6/IL-2 complexes led to a significant amelioration of the disease. This therapeutic effect seems to be mediated by donor CD8+ T cells. The fact that a much stronger cGvHD is induced in BDF1 mice depleted of donor CD8+ T cells strongly supports this conclusion. The contrasting effects of the two different IL-2 complexes are likely due to different mechanisms.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.