Student Work

Determining how infected macrophages evade natural killer cell ADCC elimination

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While HIV mainly infects CD4+ T cells, macrophages are also infected and have been seen to harbor virus during antiretroviral therapy (ART) posing a challenge for HIV clearance. Previous work from Clayton et al., 2021 found that HIV-infected macrophages were eliminated less efficiently compared to HIV-infected CD4+ T cells by natural killer (NK) cell antibody dependent cellular cytotoxicity (ADCC). ADCC is a mechanism used by NK cells where they bind to the Fc portion of an antibody bound to the antigen on the target cell which results in lysis of the target cell. My project is investigating the mechanism by which macrophages evade NK cell ADCC elimination. Previous work by Prevost et al., 2023 shows that HIV accessory protein, Vpu, downregulates expression of CD4 preventing engagement of the HIV env so that it remains in the closed conformation. This suggests that when CD4 is expressed, it binds the env in cis. Additionally, Gravina et al., 2022 found that over expression of CD64 helps cancer cells escape ADCC. CD64 binds the Fc portion of IgG while the Fab portion occupies the target antigen in cis, preventing ADCC in trans. Based off these published works, I hypothesize that Fc receptors in cis on infected macrophages inhibit ADCC in trans by binding to the Fc portion of IgG, whose Fab portion is bound to the HIV env. To test this, I created lentiviral constructs that express Fc receptors (CD16a, CD32a and CD64) and viral glycoproteins (HIV env, Ebola GP and Flu HA). Since there is variability in promoter-driven GFP expression in different cell types, I screened 11 different promoters to determine which promoter drives optimal transgene expression in Jurkats, CD4+ T cells, macrophages and CD8+ T cells. I determined that MSCVU3 was the optimal promoter in the Jurkats, CD4+ T cells and CD8+ T cells while CAGJ is the promoter that best drives GFP expression in macrophages. Since Jurkats do not endogenously express Fc receptors, they were used in transductions to express the viral glycoproteins and Fc receptors. Antibodies against the Ebola GP and Flu HA detected the expressed glycoproteins, I found that the human EBOV GP 15878 antibody responded strongly to Ebola GP and the F10 antibody responded well to the Flu HA. The Fc receptor lentiviral constructs transduced Jurkats with high Fc receptor expression which was confirmed by Fc antibody staining run with flow cytometry. As a positive control for ADCC assays, I tested the recognition of the PGT121 chimeric antigen receptor (CAR) T cell. CAR T cells express receptor proteins that have been engineered to target a specific antigen. The PGT121 CAR specifically recognized the HIV env, validating its specificity. The next steps for this project include improving the HIV env construct and creating functional CAR T cells specific for Ebola GP and Flu HA to be used in NK ADCC assays. With these constructs, performing NK ADCC assays with co-transduced Jurkats expressing the viral glycoproteins and Fc receptors will either support or refute my hypothesis.

  • This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.
Creator
Publisher
Identifier
  • E-project-042623-162433
  • 105496
Keyword
Advisor
Year
  • 2023
Sponsor
Date created
  • 2023-04-26
Resource type
Major
Source
  • E-project-042623-162433
Rights statement
Last modified
  • 2023-06-21

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