Date of Award
Spring 5-2024
Degree Name
Master of Science (MS)
Degree Type
Thesis
Abstract
HSV infection is broadly spread all over the world with some patients having severe and/or recurrent HSV infections. Our lab studies human Natural Killer (NK) cells, which are important in innate immune responses to viral infections and tumors. A publication in 2013 by Ornstein et al from our lab studied HSV+ pediatric patients and found some associations between severe infection and defects in NK cytolytic function. PLCG2 haploinsufficient variants found in 2 HSV patients causing PLCγ2 hypophosphorylation, and loss of cytolytic function in NK cells is a novel finding recently published by Alinger et al from our lab in 2023. Prior to that study, PLCG2 gain of function variants in B cells had been well studied but loss of function had not been reported in the context of human NK cells in patients with recurrent HSV infection. This project is a continuing study to determine whether PLCγ2 hypophosphorylation in NK cells is shared by many HSV+ patients. Our focus is to look for any differences in PBMC immune profiles and NK profiles in the patients and perform cytotoxic assay and PLCγ2 phosphorylation status of NK cells using flow cytometry.
This project required learning spectral flow and processing the results to analyze the high-dimensional data. We aimed to analyze an extensive immune profiling panel containing 19 colors for patients' PBMCs using spectral flow cytometry. The Cytek Aurora is a state-of-the-art cytometer which has 5 lasers and 64 channels. An additional NK panel will delineate the maturation and activation state of NK cells. Spectral flow cytometry is a powerful immunoprofiling tool detecting up to 40 markers on a single blood sample, which enables us to collect large datasets using a minimal number of cells.
All patient samples in this thesis are collected from children who have severe or unusually recurrent herpesvirus (HSV) infections. We analyzed 3 sets of patients each compared to an age- and sex- matched control. Unfortunately, in two of three sets, the age-matched controls had unusually low cytolytic activity, making it difficult to draw any conclusions about the two sets of corresponding patient profiles. In the other set where the control cells were normal range for cytolytic ability, we found one pediatric HSV patient with a significant defect in their ability to lyse K562 targets. This young female patient with the cytolytic defect surprisingly had PLCγ2 hyper-phosphorylation in NK cells instead of hypophosphorylation as expected.
She also has a normal immune cell profile, but a more terminally mature NK profile (CD57+) compared to the healthy control. This finding correlates to Alinger’s characterization of a female pediatric HSV+ patient with PLCγ2 defects in their work. In general, NK-cell immunodeficiency may be related to abnormal PLCγ2 functions and an increased number of CD57+ NK cells.
Language
English (en)
Chair
Jai Rudra, Biomedical Engineering Department
Committee Members
Michael Vahey, Katherine Schreiber