Functional assays for the diagnosis of primary defects in lymphocyte cytotoxicity

Cytotoxic lymphocytes encompass natural killer (NK) cells and cytotoxic T lymphocytes (CTL). These cells detect and kill virus-infected as well as malignant cells. Primary defects in lymphocyte cytotoxicity are associated with development of a hyperinflammatory syndrome termed hemophagocytic lymphohistiocytosis (HLH). This thesis investigates cytotoxic lymphocyte function in HLH cases as well as the creation of new assays to improve diagnostics. An NK cell degranulation assay has been developed to quantify NK cell responses as an alternative to the radioactive chromium release assay. CD107a is a transmembrane protein that in unstimulated cells is contained within the inner membrane of perforin-containing cytotoxic granules but is exposed on the cell surface upon cytotoxic granule exocytosis. In a pan-European effort, we established and validated a consensus protocol for the diagnosis of primary HLH patients with defective degranulation (Paper II). NK cell degranulation below 5% predicted a primary defect in exocytosis leading to defective lymphocyte cytotoxicity with 96% sensitivity and 88% specificity. We also provided further optimized protocols for NK cell phenotyping and degranulation (Paper I). Highlighting the importance of reliable functional assays in primary immunodeficiency discovery, we described novel non-coding aberrations in UNC13D as a cause of HLH and defective degranulation (Paper III, IV). Point mutations were found in a highly conserved intronic region, while a 253kb inversion was identified as the most frequent cause of HLH in Swedish infants. ORAI1 and STIM1 mediate store-operated Ca2+ entry in T cells, which is required for induction of cytokine expression. However, the role of store-operated Ca2+ entry was not clear. In Paper V, we studied NK cell cytotoxicity using ORAI1 and STIM1-deficient patients, confirming abrogated Ca2+ entry upon target cell stimulation. Importantly, ORAI1 and STIM1-deficient NK cells did not degranulate nor produced proinflammatory cytokines, demonstrating that storeoperated Ca2+ entry is required for overall cytotoxic lymphocyte effector functions. Primary HLH diagnostics have relied on NK cell functional assays but little had been done to evaluate T cell responses in these patients. In Paper VI, we described how the surface marker CD57 can be used to identify bone fide cytotoxic T lymphocytes, readily allowing identification of a T cell subset that expresses intracellular perforin and can efficiently kill target cells upon TCR engagement. The CD8+CD57+ T cell subset, similar to CD56dim NK cells, was found to display defective degranulation in FHL types 3-5, suggesting CTL and NK cell exocytosis has similar molecular requirements. In paper VII, we compared the established K562 cell-induced NK cell degranulation assay (Paper II) against the newly developed T cell degranulation assay (Paper VI) prospectively evaluating all primary immunodeficiency patients sent to our laboratory during a 3 year period. The T cell assay excelled with respect to sensitivity and specificity (97% and 95%, respectively) for predicting a primary defect in degranulation. Combining NK cell and T cell assays further increased assay specificity. This thesis demonstrates the power of functional assays in evaluating cytotoxic lymphocyte activity and how this expands our understanding of their biological role and the primary HLH syndrome. These simple, rapid and accurate assays give us the power to quickly diagnose a lifethreatening disease and initiate treatment, thus saving lives

Probing the Local Velocity Distribution of WIMP Dark Matter with Directional Detectors

We explore the ability of directional nuclear-recoil detectors to constrain the local velocity distribution of weakly interacting massive particle (WIMP) dark matter by performing Bayesian parameter estimation on simulated recoil-event data sets. We discuss in detail how directional information, when combined with measurements of the recoil-energy spectrum, helps break degeneracies in the velocity-distribution parameters. We also consider the possibility that velocity structures such as cold tidal streams or a dark disk may also be present in addition to the Galactic halo. Assuming a carbon-tetrafluoride detector with a 30-kg-yr exposure, a 50-GeV WIMP mass, and a WIMP-nucleon spin-dependent cross-section of 0.001 pb, we show that the properties of a cold tidal stream may be well constrained. However, measurement of the parameters of a dark-disk component with a low lag speed of ~50 km/s may be challenging unless energy thresholds are improved.Comment: 38 pages, 15 figure

Homologous recombination suppresses transgenerational DNA end resection and chromosomal instability in fission yeast

Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8(+) Trm cells on a compartmental and functional basis. In human skin epithelia, CD8(+)CD49a(+) Trm cells produced interferon-γ, whereas CD8(+)CD49a(−) Trm cells produced interleukin-17 (IL-17). In addition, CD8(+)CD49a(+) Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8(+)CD49a(+) Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8(+)CD49a(–) Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8(+) Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases

Antibiotic mediated synthesis of gold nanoparticles with potent antimicrobial activity and their application in antimicrobial coatings

We report a one-pot synthesis of spherical gold nanoparticles (52-22 nm) and their capping with cefaclor, a second-generation antibiotic, without use of other chemicals. The differently sized gold nanoparticles were fabricated by controlling the rate of reduction of gold ions in aqueous solution by varying the reaction temperature (20-70 C). The primary amine group of cefaclor acted as both the reducing and capping agent for the synthesis of gold nanoparticles leaving the b-lactam ring of cefaclor available for activity against microbes. Antimicrobial testing showed that cefaclor reduced gold nanoparticles have potent antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as compared to cefaclor or gold nanoparticles alone. The minimum inhibition concentrations (MICs) of cefaclor reduced gold nanoparticles were 10m gmL1 and 100m gmL1 for S. aureus and E. coli respectively. The cefaclor reduced gold nanoparticles were further coated onto poly(ethyleneimine) (PEI) modified glass surfaces to obtain antimicrobial coatings suitable for biomedical applications and were tested against E. coli as an exemplar of activity. The antimicrobial coatings were very robust under adverse conditions (pH 3 and 10), inhibited the growth of E. coli on their surfaces, and could be used many times with retained activity. Results from a combined spectroscopic (FTIR) and microscopic study (AFM) suggest that the action of these novel particles is through the combined action of cefaclor inhibiting the synthesis of the peptidoglycan layer and gold nanoparticles generating "holes" in bacterial cell walls thereby increasing the permeability of the cell wall, resulting in the leakage of cell contents and eventually cell death

Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rapid-onset, potentially fatal hyperinflammatory syndrome. A prompt molecular diagnosis is crucial for appropriate clinical management. Here, we validated and prospectively evaluated a targeted high-throughput sequencing approach for HLH diagnostics. Methods: A high-throughput sequencing strategy of 12 genes linked to HLH was validated in 13 patients with previously identified HLH-associated mutations and prospectively evaluated in 58 HLH patients. Moreover, 2504 healthy individuals from the 1000 Genomes project were analyzed in silico for variants in the same genes. Results: Analyses revealed a mutation detection sensitivity of 97.3 %, an average coverage per gene of 98.0 %, and adequate coverage over 98.6 % of sites previously reported as mutated in these genes. In the prospective cohort, we achieved a diagnosis in 22 out of 58 patients (38 %). Genetically undiagnosed HLH patients had a later age at onset and manifested higher frequencies of known secondary HLH triggers. Rare, putatively pathogenic monoallelic variants were identified in nine patients. However, such monoallelic variants were not enriched compared with healthy individuals. Conclusions: We have established a comprehensive high-throughput platform for genetic screening of patients with HLH. Almost all cases with reduced natural killer cell function received a diagnosis, but the majority of the prospective cases remain genetically unexplained, highlighting genetic heterogeneity and environmental impact within HLH. Moreover, in silico analyses of the genetic variation affecting HLH-related genes in the general population suggest caution with respect to interpreting causality between monoallelic mutations and HLH. A complete understanding of the genetic susceptibility to HLH thus requires further in-depth investigations, including genome sequencing and detailed immunological characterization.Peer reviewe

A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge

The rock-hosted, oceanic crustal aquifer is one of the largest ecosystems on Earth, yet little is known about its indigenous microorganisms. Here we provide the first phylogenetic and functional description of an active microbial community residing in the cold oxic crustal aquifer. Using subseafloor observatories, we recovered crustal fluids and found that the geochemical composition is similar to bottom seawater, as are cell abundances. However, based on relative abundances and functional potential of key bacterial groups, the crustal fluid microbial community is heterogeneous and markedly distinct from seawater. Potential rates of autotrophy and heterotrophy in the crust exceeded those of seawater, especially at elevated temperatures (25 °C) and deeper in the crust. Together, these results reveal an active, distinct, and diverse bacterial community engaged in both heterotrophy and autotrophy in the oxygenated crustal aquifer, providing key insight into the role of microbial communities in the ubiquitous cold dark subseafloor biosphere

Dominant TOM1 mutation associated with combined immunodeficiency and autoimmune disease

Mutations in several proteins functioning as endolysosomal components cause monogenic autoimmune diseases, of which pathogenesis is linked to increased endoplasmic reticulum stress, inefficient autophagy, and defective recycling of immune receptors. We report here a heterozygous TOM1 p.G307D missense mutation, detected by whole-exome sequencing, in two related patients presenting with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. The index patient suffered from recurrent respiratory tract infections and oligoarthritis since early teens, and later developed persistent lowcopy EBV-viremia, as well as an antibody deficiency. Her infant son developed hypogammaglobulinemia, autoimmune enteropathy, interstitial lung disease, profound growth failure, and treatment-resistant psoriasis vulgaris. Consistent with previous knowledge on TOM1 protein function, we detected impaired autophagy and enhanced susceptibility to apoptosis in patient-derived cells. In addition, we noted diminished STAT and ERK1/2 signaling in patient fibroblasts, as well as poor IFN-gamma and IL-17 secretion in T cells. The mutant TOM1 failed to interact with TOLLIP, a protein required for IL-1 recycling, PAMP signaling and autophagosome maturation, further strengthening the link between the candidate mutation and patient pathophysiology. In sum, we report here an identification of a novel gene, TOM1, associating with early-onset autoimmunity, antibody deficiency, and features of combined immunodeficiency. Other patient cases from unrelated families are needed to firmly establish a causal relationship between the genotype and the phenotype.Peer reviewe

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15 -/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies