Doctor of Philosophy, Stanford University, IMMUN-PHD (2022)
Bachelor of Science, The University of Chicago, Biological Sciences (2014)
Markus Covert, Postdoctoral Faculty Sponsor
Mass Cytometry Phenotyping of Human Granulocytes Reveals Novel Basophil Functional Heterogeneity.
2020; 23 (11): 101724
Basophils, the rarest granulocyte, play critical roles in parasite- and allergen-induced inflammation. We applied mass cytometry (CyTOF) to simultaneously asses 44 proteins to phenotype and functionally characterize neutrophils, eosinophils, and basophils from 19 healthy donors. There was minimal heterogeneity seen in eosinophils and neutrophils, but data-driven analyses revealed four unique subpopulations within phenotypically basophilic granulocytes (PBG; CD45+HLA-DR-CD123+). Through CyTOF and fluorescence-activated cell sorting (FACS), we classified these four PBG subpopulations as (I) CD16lowFcepsilonRIhighCD244high (88.5± 1.2%), (II) CD16highFcepsilonRIhighCD244high (9.1± 0.4%), (III) CD16lowFcepsilonRIlowCD244low (2.3± 1.3), and (IV) CD16highFcepsilonRIlowCD244low (0.4± 0.1%). Prospective isolation confirmed basophilic-morphology of PBG I-III, but neutrophilic-morphology of PBG IV. Functional interrogation via IgE-crosslinking or IL-3 stimulation demonstrated that PBG I-II had significant increases in CD203c expression, whereas PBG III-IV remained unchanged compared with media-alone conditions. Thus, PBG III-IV could serve roles in non-IgE-mediated immunity. Our findings offer new perspectives in human basophil heterogeneity and the varying functional potential of these new subsets in health and disease.
View details for DOI 10.1016/j.isci.2020.101724
View details for PubMedID 33205028
IN-DEPTH CHARACTERIZATION OF GESTATIONAL IMMUNE DYNAMICS USING MASS CYTOMETRY
W B SAUNDERS CO LTD. 2019: E87–E88
View details for Web of Science ID 000483998500276
An Immune Atlas of Mid to Late Mouse Gestation.
SAGE PUBLICATIONS INC. 2019: 345A–346A
View details for Web of Science ID 000459610400837
- Proliferation tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells NATURE BIOTECHNOLOGY 2019; 37 (3): 259-+
Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis.
Methods in molecular biology (Clifton, N.J.)
2019; 1989: 55–81
The advent of mass cytometry (CyTOF®) has permitted simultaneous detection of more than 40 antibody parameters at the single-cell level, although a limited number of metal-labeled antibodies are commercially available. Here we present optimized and scalable protocols for conjugation of lanthanide as well as bismuth ions to immunoglobulin (Ig) using a maleimide-functionalized chelating polymer and for characterization of the conjugate. The maleimide functional group is reactive with cysteine sulfhydryl groups generated through partial reduction of the Ig Fc region. Incubation of Ig with polymer pre-loaded with lanthanide ions produces metal-labeled Ig without disrupting antigen specificity. Antibody recovery rates can be determined by UV spectrophotometry and frequently exceeds 60%. Each custom-conjugated antibody is validated using positive and negative cellular control populations and is titrated for optimal staining at concentrations ranging from 0.1 to 10 μg/mL. The preparation of metal-labeled antibodies can be completed in 4.5 h, and titration requires an additional 3-5 h.
View details for PubMedID 31077099
Chemical inhibitors of Candida albicans hyphal morphogenesis target endocytosis.
2017; 7 (1): 5692
Candida albicans is an opportunistic pathogen, typically found as a benign commensal yeast living on skin and mucosa, but poised to invade injured tissue to cause local infections. In debilitated and immunocompromised individuals, C. albicans may spread to cause life-threatening systemic infections. Upon contact with serum and at body temperature, C. albicans performs a regulated switch to filamentous morphology, characterized by emergence of a germ tube from the yeast cell followed by mold-like growth of branching hyphae. The ability to switch between growth morphologies is an important virulence factor of C. albicans. To identify compounds able to inhibit hyphal morphogenesis, we screened libraries of existing drugs for inhibition of the hyphal switch under stringent conditions. Several compounds that specifically inhibited hyphal morphogenesis were identified. Chemogenomic analysis suggested an interaction with the endocytic pathway, which was confirmed by direct measurement of fluid-phase endocytosis in the presence of these compounds. These results suggest that the activity of the endocytic pathway, which is known to be particularly important for hyphal growth, represents an effective target for hyphae-inhibiting drugs.
View details for DOI 10.1038/s41598-017-05741-y
View details for PubMedID 28720834
View details for PubMedCentralID PMC5515890
Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis.
journal of allergy and clinical immunology
Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63(hi) basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.BATs to measure upregulation of basophil CD203c and induction of a CD63(hi) basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.
View details for DOI 10.1016/j.jaci.2016.04.060
View details for PubMedID 27527263
View details for PubMedCentralID PMC5237629