BACKGROUND: Natural killer (NK) cells are traditionally considered part of the innate immune system but have recently been shown to possess adaptive qualities similar to T cells in response to an infection with a pathogen. In addition to possessing adaptive features, NK cells have also been found to reside in different organs such as the liver, spleen, and lymph nodes and differ based on phenotypic markers and their responses to different cytokines. Utilizing these findings, several groups have isolated and identified CD49a as a marker for tissue-resident NK cells. In the liver, CD49a has also been shown to be a positive indicator for NK cell memory-like responses in murine models. Building off work that demonstrated antigen-specific responses in rhesus macaques, this project focuses on characterizing the phenotypic markers and functional profile of CD49a+ NK cells in non-human primates. To better understand the role of CD49a in memory-like NK cells outside of the liver, this project utilized spleen samples from rhesus macaques infected with SIV/SHIV. This work aims to help us better understand the dysfunction NK cells experience as a result of HIV-1 infection in humans and also to demonstrate the changes NK cells experience as the disease progresses. A thorough understanding of the adaptive capabilities of NK cells can pave the way for targeted therapies to increase NK cell antiviral activity in HIV and other infections.
METHODS: To characterize the functional and phenotypic profiles of CD49a+ NK cells by multiparameter flow cytometry, thirteen samples of spleen from rhesus macaques were thawed and then stained with two different protocols. A phenotyping protocol involved staining with antibodies against surface markers as well as intracellular markers T-Bet and Eomes. For the functional characterization protocol, the same thirteen samples were stained intracellularly after being stimulated with a cocktail of PMA and ionomyocin. The antibodies used in this were for functional markers. Of the thirteen samples used, six were infected with SHIVSF162P3, two were infected with SIVmac239X, and the remaining five were uninfected.
After staining, these samples were analyzed on an BD LSRII from BD Biosciences. The data obtained were then analyzed using FlowJo software to study NK cells, which were characterized as CD45+CD14-CD20-CD3-CD159+.
RESULTS: The analysis compared NK cells with T cells, B cells, and NKB cells. Some increases were seen among CD49a+ NK cells in the frequency of CD336+ (NCR2/NKp44), CD337+ (NKp30), and CD366+ (Tim-3) after infection.
Although there were some mild increases in CD107a and TNF- in infected samples compared to uninfected, a significant increase was observed in the frequency of IFN-ɣ among infected CD49a+ NK cells compared to uninfected.
CONCLUSION: When comparing samples that were infected vs uninfected, it appears there were some mild decreases after infection in the ratio of NK cells to other lymphocytes. In addition, there did not appear to be a significant increase in the frequency of CD49a+ among these NK cells as a result of the infection. However, among the CD49a+ subpopulation, there were some observed non-significant decreases in CD56-CD16+ cells. Furthermore, there was found to be an almost significant increase in TNF- (p = 0.06) among CD49a+ cells after infection. These findings demonstrate an increase in cytotoxic activity in splenic NK cells associated with an adaptation to the virus. Although there does not appear to be significant changes in the ratio of NK cell populations in the spleen, the changes observed in phenotypic and functional markers associated with CD49a+ demonstrate an increase in the cytotoxic activity of NK cells as a result of infection with SIV/SHIV. However, it remains to be seen if CD49a is a direct indicator of this type of infection. Future work geared toward memory-like NK cells in non-human primate splenic tissue could look at the contrast in CD49a+ NK cells from different states of infection with HIV-1 and/or SIV (acute vs chronic) to better understand the integrin’s role in adaptation.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/38596 |
| Date | 09 October 2019 |
| Creators | Arias, Christian Fernando |
| Contributors | Reeves, Roger K., Taylor, Andrew W. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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