Evolving Approach to Clinical Cytometry for Immunodeficiencies and Other Immune Disorders.

Primary immunodeficiencies were initially identified on the basis of recurrent, severe or unusual infections. Subsequently, it was noted that these diseases can also manifest with autoimmunity, autoinflammation, allergy, lymphoproliferation and malignancy, hence a conceptual change and their renaming as inborn errors of immunity. Ongoing advances in flow cytometry provide the opportunity to expand or modify the utility and scope of existing laboratory tests in this field to mirror this conceptual change. Here we have used the B cell subset, variably known as CD21low B cells, age-associated B cells and T-bet+ B cells, as an example to demonstrate this possibility.

Flow cytometry for B-cell subset analysis in immunodeficiencies.

B cells are one of the fundamental components of the adaptive immune system and are best known for their ability to produce antibodies. Among the various types of inborn errors of immunity, antibody deficiencies represent the largest group in terms of the number of affected individuals. Not all antibody deficiencies are due to B cell intrinsic defects but investigating B cell number and function are a critical part of the diagnostic process. B cells studies in clinical practice almost always rely on flow cytometry as the main tool of investigation. The advantage of flow cytometry is that it allows absolute and relative counting of B cells, and their phenotypic and functional evaluation at a single-cell level, while allowing the analysis of a large number of cells. Although versatile and broad in its utility, clinical flow cytometry has both theoretical and practical limitations. These include lack of consensus about definitions and classifications, and the use of non-standardized methods. Patients in all age groups, from newborns to the elderly, may require testing, yet B cells show significant changes in both numbers and subset distribution over the lifespan, requiring distinct reference ranges for narrowly defined age brackets for accurate interpretation. Sampling for testing is usually restricted to peripheral blood samples, and the number of markers routinely used are limited. This paper will provide a brief overview of flow cytometry and B cell biology, describe the human peripheral B cell subsets most commonly identified in clinical flow cytometry and discuss their clinical relevance in different settings.

Development of a multiomics model for identification of predictive biomarkers for COVID-19 severity: a retrospective cohort study.

BACKGROUND: COVID-19 is a multi-system disorder with high variability in clinical outcomes among patients who are admitted to hospital. Although some cytokines such as interleukin (IL)-6 are believed to be associated with severity, there are no early biomarkers that can reliably predict patients who are more likely to have adverse outcomes. Thus, it is crucial to discover predictive markers of serious complications. METHODS: In this retrospective cohort study, we analysed samples from 455 participants with COVID-19 who had had a positive SARS-CoV-2 RT-PCR result between April 14, 2020, and Dec 1, 2020 and who had visited one of three Mayo Clinic sites in the USA (Minnesota, Arizona, or Florida) in the same period. These participants were assigned to three subgroups depending on disease severity as defined by the WHO ordinal scale of clinical improvement (outpatient, severe, or critical). Our control cohort comprised of 182 anonymised age-matched and sex-matched plasma samples that were available from the Mayo Clinic Biorepository and banked before the COVID-19 pandemic. We did a deep profiling of circulatory cytokines and other proteins, lipids, and metabolites from both cohorts. Most patient samples were collected before, or around the time of, hospital admission, representing ideal samples for predictive biomarker discovery. We used proximity extension assays to quantify cytokines and circulatory proteins and tandem mass spectrometry to measure lipids and metabolites. Biomarker discovery was done by applying an AutoGluon-tabular classifier to a multiomics dataset, producing a stacked ensemble of cutting-edge machine learning algorithms. Global proteomics and glycoproteomics on a subset of patient samples with matched pre-COVID-19 plasma samples was also done. FINDINGS: We quantified 1463 cytokines and circulatory proteins, along with 902 lipids and 1018 metabolites. By developing a machine-learning-based prediction model, a set of 102 biomarkers, which predicted severe and clinical COVID-19 outcomes better than the traditional set of cytokines, were discovered. These predictive biomarkers included several novel cytokines and other proteins, lipids, and metabolites. For example, altered amounts of C-type lectin domain family 6 member A (CLEC6A), ether phosphatidylethanolamine (P-18:1/18:1), and 2-hydroxydecanoate, as reported here, have not previously been associated with severity in COVID-19. Patient samples with matched pre-COVID-19 plasma samples showed similar trends in muti-omics signatures along with differences in glycoproteomics profile. INTERPRETATION: A multiomic molecular signature in the plasma of patients with COVID-19 before being admitted to hospital can be exploited to predict a more severe course of disease. Machine learning approaches can be applied to highly complex and multidimensional profiling data to reveal novel signatures of clinical use. The absence of validation in an independent cohort remains a major limitation of the study. FUNDING: Eric and Wendy Schmidt.

Anti-cytokine autoantibodies and inborn errors of immunity.

The past quarter of a century has witnessed an inordinate increase in our understanding of primary immunodeficiencies / inborn errors of immunity. These include a significant increase in the number of identified conditions, broadening the phenotypes of existing entities, delineation of classical inborn errors of immunity from those with a narrow phenotype, and a gradual shift from supportive to definitive care in patients afflicted with these diseases. It has also seen the discovery of conditions broadly defined as phenocopies of primary immunodeficiencies, where somatic mutations or autoantibodies mimic a recognised primary immunodeficiency's presentation in the absence of the underlying genetic basis for that disease. This article will provide a review of the anti-cytokine autoantibody-mediated phenocopies of inborn errors of immunity and discuss the therapeutic and laboratory aspects of this group of diseases.

A Toolkit and Framework for Optimal Laboratory Evaluation of Individuals with Suspected Primary Immunodeficiency.

Knowledge related to the biology of inborn errors of immunity and associated laboratory testing methods continues to expand at a tremendous rate. Despite this, many patients with inborn errors of immunity suffer for prolonged periods of time before identification of their underlying condition, thereby delaying appropriate care. Understanding that test selection and optimal evaluation for patients with recurrent infections or unusual patterns of inflammation can be unclear, we present a document that distills relevant clinical features of immunologic disease due to inborn errors of immunity and related appropriate and available test options. This document is intended to serve the practicing clinical immunologist and, in turn, patients by describing best available test options for initial and expanded immunologic evaluations across the disease spectrum. Our goal is to demystify the process of evaluating patients with suspected immune dysfunction and to enable more rapid and accurate diagnosis of such individuals.

Highlights of the 33rd annual scientific meeting of the Association of Medical Laboratory Immunologists (AMLI).

The annual meeting of the Association of Medical Laboratory Immunologists (AMLI) was convened virtually over the month of August. Prior to the emergence of the COVID-19 pandemic, AMLI's scientific committee had chosen the following topics as the focus of its 2020 meeting: Histocompatibility Testing and Transplant Immunology; Secondary Immunodeficiency and Immunotherapy Monitoring; ANA Update; and Emerging Infectious Diseases and New Algorithms for Testing. Given the central role of the discipline in the evaluation of the host response to infection, it was apt to add a separate session on antibody testing for SARS-CoV-2 infections to the original program. The current report provides an overview of the subjects discussed in the course of this meeting.

Asymptomatic Infant With Atypical SCID and Novel Hypomorphic RAG Variant Identified by Newborn Screening: A Diagnostic and Treatment Dilemma.

The T-cell receptor excision circle (TREC) assay detects T-cell lymphopenia (TCL) in newborns and is especially important to identify severe combined immunodeficiency (SCID). A spectrum of SCID variants and non-SCID conditions that present with TCL are being discovered with increasing frequency by newborn screening (NBS). Recombination-activating gene (RAG) deficiency is one the most common causes of classical and atypical SCID and other conditions with immune dysregulation. We present the case of an asymptomatic male with undetectable TRECs on NBS at 1 week of age. The asymptomatic newborn was found to have severe TCL, but normal B cell quantities and lymphocyte proliferation upon mitogen stimulation. Next generation sequencing revealed compound heterozygous hypomorphic RAG variants, one of which was novel. The moderately decreased recombinase activity of the RAG variants (16 and 40%) resulted in abnormal T and B-cell receptor repertoires, decreased fraction of CD3+ TCRVα7.2+ T cells and an immune phenotype consistent with the RAG hypomorphic variants. The patient underwent successful treatment with hematopoietic stem cell transplantation (HSCT) at 5 months of age. This case illustrates how after identification of a novel RAG variant, in vitro studies are important to confirm the pathogenicity of the variant. This confirmation allows the clinician to expedite definitive treatment with HSCT in an asymptomatic phase, mitigating the risk of serious infectious and non-infectious complications.

Aging and the immune system: An overview.

The world is witnessing a rapid demographic shift towards an older population, a trend with major medical, social, economic and political implications. Aging is a multifaceted process, involving numerous molecular and cellular mechanisms in the context of different organ systems. A crucial component of aging is a set of functional and structural alterations in the immune system that can manifest as a decreased ability to fight infection, diminished response to vaccination, increased incidence of cancer, higher prevalence of autoimmunity and constitutive low-grade inflammation, among others. In addition to cell-intrinsic changes in both innate and adaptive immune cells, alterations in the stromal microenvironment in primary and secondary lymphoid organs play an important role in age-associated immune dysfunction. This article will provide a broad overview of these phenomena and point out some of their clinical and therapeutic implications.

Interleukin-22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice.

Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.

Acute infection of mice with Clostridium difficile leads to eIF2α phosphorylation and pro-survival signalling as part of the mucosal inflammatory response.

The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with Clostridium difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and signal transducer and activator of transcription 3 (STAT3), an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the interleukin-22-pSTAT3-RegIIIγ axis as two of the pathways that could be used to contain and counteract the damage inflicted on the intestinal epithelium.

Interleukin-17 drives pulmonary eosinophilia following repeated exposure to Aspergillus fumigatus conidia.

Previous research in our laboratory has demonstrated that repeated intranasal exposure to Aspergillus fumigatus conidia in C57BL/6 mice results in a chronic pulmonary inflammatory response that reaches its maximal level after four challenges. The inflammatory response is characterized by eosinophilia, goblet cell metaplasia, and T helper T(H)2 cytokine production, which is accompanied by sustained interleukin-17 (IL-17) expression that persists even after the T(H)2 response has begun to resolve. T(H)17 cells could develop in mice deficient in gamma interferon (IFN-γ), IL-4, or IL-10. In the lungs of IL-17 knockout mice repeatedly challenged with A. fumigatus conidia, inflammation was attenuated (with the most significant decrease occurring in eosinophils), conidial clearance was enhanced, and the early transient peak of CD4(+) CD25(+) FoxP3(+) cells blunted. IL-17 appeared to play only a minor role in eosinophil differentiation in the bone marrow but a central role in eosinophil extravasation from the blood into the lungs. These observations point to an expanded role for IL-17 in driving T(H)2-type inflammation to repeated inhalation of fungal conidia.

Use of direct gradient analysis to uncover biological hypotheses in 16s survey data and beyond.

This study investigated the use of direct gradient analysis of bacterial 16S pyrosequencing surveys to identify relevant bacterial community signals in the midst of a "noisy" background, and to facilitate hypothesis-testing both within and beyond the realm of ecological surveys. The results, utilizing 3 different real world data sets, demonstrate the utility of adding direct gradient analysis to any analysis that draws conclusions from indirect methods such as Principal Component Analysis (PCA) and Principal Coordinates Analysis (PCoA). Direct gradient analysis produces testable models, and can identify significant patterns in the midst of noisy data. Additionally, we demonstrate that direct gradient analysis can be used with other kinds of multivariate data sets, such as flow cytometric data, to identify differentially expressed populations. The results of this study demonstrate the utility of direct gradient analysis in microbial ecology and in other areas of research where large multivariate data sets are involved.

Repeated exposure to Aspergillus fumigatus conidia results in CD4+ T cell-dependent and -independent pulmonary arterial remodeling in a mixed Th1/Th2/Th17 microenvironment that requires interleukin-4 (IL-4) and IL-10.

Pulmonary arterial remodeling is a pathological process seen in a number of clinical disease states, driven by inflammatory cells and mediators in the remodeled artery microenvironment. In murine models, Th2 cell-mediated immune responses to inhaled antigens, such as purified Aspergillus allergen, have been reported to induce remodeling of pulmonary arteries. We have previously shown that repeated intranasal exposure of healthy C57BL/6 mice to viable, resting Aspergillus fumigatus conidia leads to the development of chronic pulmonary inflammation and the coevolution of Th1, Th2, and Th17 responses in the lungs. Our objective was to determine whether repeated intranasal exposure to Aspergillus conidia would induce pulmonary arterial remodeling in this mixed Th inflammatory microenvironment. Using weekly intranasal conidial challenges, mice developed robust pulmonary arterial remodeling after eight exposures (but not after two or four). The process was partially mediated by CD4+ T cells and by interleukin-4 (IL-4) production, did not require eosinophils, and was independent of gamma interferon (IFN-γ) and IL-17. Furthermore, remodeling could occur even in the presence of strong Th1 and Th17 responses. Rather than serving an anti-inflammatory function, IL-10 was required for the development of the Th2 response to A. fumigatus conidia. However, in contrast to previous studies of pulmonary arterial remodeling driven by the A. fumigatus allergen, viable conidia also stimulated pulmonary arterial remodeling in the absence of CD4+ T cells. Remodeling was completely abrogated in IL-10-/- mice, suggesting that a second, CD4+ T cell-independent, IL-10-dependent pathway was also driving pulmonary arterial remodeling in response to repeated conidial exposure.

Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity.

Previous work from our laboratory has shown that primary fibroblasts from long-lived Snell dwarf mice display a higher sensitivity to the lethal effects of endoplasmic reticulum (ER) stressors, such as thapsigargin, than cells from normal mice. Here we show that thapsigargin induces higher expression of CHOP, enhanced cleavage of caspase-12, higher caspase-3 activity, and increased phosphorylation of c-JUN, all indicators of enhanced apoptosis, in dwarf fibroblasts. Dwarf and normal fibroblasts show no genotypic difference in up-regulating BiP, GRP94, and ERp72 proteins after exposure to thapsigargin. However, dwarf fibroblasts express lower basal levels of a number of putative XBP1 target genes including Armet, Edem1, Erdj3, p58(IPK) and Sec61a1, as well as Ire1α itself. Furthermore, when exposed to thapsigargin, dwarf fibroblasts display attenuated splicing of Xbp1, but similar phosphorylation of eIF2α, in comparison to normal fibroblasts. These data support the notion that IRE1/XBP1 signaling is set at a lower level in dwarf fibroblasts. Diminished Xbp1 splicing in dwarf-derived fibroblasts may tilt the balance between prosurvival and proapoptotic signals in favor of apoptosis, thereby leading to higher induction of proapoptotic signals in these cells and ultimately their increased sensitivity to ER stressors. These results, together with recent findings in Caenorhabditis elegans daf-2 mutants, point to a potential interplay between insulin/IGF-1 signals and unfolded protein response signaling.

Life-span extension in mice by preweaning food restriction and by methionine restriction in middle age.

Life span can be extended in rodents by restricting food availability (caloric restriction [CR]) or by providing food low in methionine (Meth-R). Here, we show that a period of food restriction limited to the first 20 days of life, via a 50% enlargement of litter size, shows extended median and maximal life span relative to mice from normal sized litters and that a Meth-R diet initiated at 12 months of age also significantly increases longevity. Furthermore, mice exposed to a CR diet show changes in liver messenger RNA patterns, in phosphorylation of Erk, Jnk2, and p38 kinases, and in phosphorylation of mammalian target of rapamycin and its substrate 4EBP1, HE-binding protein 1 that are not observed in liver from age-matched Meth-R mice. These results introduce new protocols that can increase maximal life span and suggest that the spectrum of metabolic changes induced by low-calorie and low-methionine diets may differ in instructive ways.

Fibroblasts from naked mole-rats are resistant to multiple forms of cell injury, but sensitive to peroxide, ultraviolet light, and endoplasmic reticulum stress.

Fibroblasts from long-lived mutant mice are resistant to many forms of lethal injury as well as to the metabolic effects of rotenone and low-glucose medium. Here we evaluated fibroblasts from young adult naked mole-rats (NMR; Heterocephalus glaber), a rodent species in which maximal longevity exceeds 28 years. Compared to mouse cells, NMR cells were resistant to cadmium, methyl methanesulfonate, paraquat, heat, and low-glucose medium, consistent with the idea that cellular resistance to stress may contribute to disease resistance and longevity. Surprisingly, NMR cells were more sensitive than mouse cells to H(2)O(2), ultraviolet (UV) light, and rotenone. NMR cells, like cells from Snell dwarf mice, were more sensitive to tunicamycin and thapsigargin, which interfere with the function of the endoplasmic reticulum (ER stress). The sensitivity of both Snell dwarf and NMR cells to ER stress suggests that alterations in the unfolded protein response might modulate cell survival and aging rate.

Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells.

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.

Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.