Ruxolitinib to Modulate CD4+ Memory T-cells in HIV+ Individuals Under Long-term Antitretroviral Therapy Open Access

Abstract

Despite the successful use of Antiretroviral Therapy (ART). physicians have still been unable to eradicate Human Immunodeficiency Virus (HIV) from the body of HIV+ individuals. ART is only able to target infected CD4+ T cells in which HIV is actively replicating, and not every cell infected with HIV will be actively producing more virus. These latently infected CD4+ T cells make up the HIV latent viral reservoir and prevent the ability to fully rid the body of an HIV infection. Central and stem cell memory (TCM and TSCM) CD4+ T cells, both of which are long-lived, younger T cell subtypes, make up a disproportionately large amount of this latent reservoir. This study analyzes the ability of ruxolitinib, a Jak-STAT inhibitor FDA approved for myelofibrosis and polycythemia vera, to cause the differentiation of these CD4+ T cells. This induced differentiation effectively pushes the latently infected cells to die. We analyzed blood samples from the A5336 AIDS Clinical Trials Group (ACTG) randomized controlled trial, in which participants received ruxolitinib for 5 weeks. Through flow cytometric analysis, we found conflicting results. We found that ruxolitinib treatment resulted in a reduction of the CD4+ TTM and TTE and CD8+ TCM, TTM, and TEM cell subsets. We also found significant correlations between the change in size of CD4+ TTM cell subset and the change in expression of Bcl-2 as well as the change in size of the CD8+ TCM cell subset and the change in expression of soluble CD14. Overall, our results do not provide an overarching conclusion of the ability of ruxolitinib to change the composition of the CD4+ T cell population in a way that would facilitate the eradication of the HIV latent viral reservoir. However, the correlation between the changes in Bcl-2 expression and relative CD4+ TTM cell population size could imply there are certain patients who can reduce the reservoir. This study provides the groundwork for future analyses on the effectiveness of the drug as a possible cure for HIV infections.

Table of Contents

Introduction……………………………………………………………………………….…1-9

History of Human Immunodeficiency Virus………..………………………………1-2

Transmission of HIV…………………………………………………………………..2

Structure of HIV…………………………………………………………….………2-3

Memory CD4+ T cells………………………………………………………………3-4

Memory CD8+ T cells………………………………………………………………4-5

The Latent Viral Reservoir………………………………………………….………5-6

Jak-STAT Pathway………………………………………………………………….6-7

Cellular and Soluble Markers of HIV Infection…………………………………….7-8

Ruxolitinib……………………………………………………………………..……8-9

Objectives and Hypotheses……………………………………………………………….10-11

Objectives……….……………………………………………………………………10

Hypotheses………………………………………………………………..………10-11

Methods………………………………………………………………………………..…12-15

Patient Selection…………………………………………………………………...…12

PBMC Selection………………………………………………………………….12-13

Flow Cytometric Analysis……………………………………………………………13

Cell Gating Strategies……………………………………………….……………13-14

Statistical Analyses…………………………………………………………….…14-15

Results…………………………………………………………………………………….16-20

Patient Sample Description………………………………………..…………………16

Overall trends in CD4+ and CD8+ T cell populations from baseline, 

week 5, and week 12……………………..…………………….…………...………..16

Overall trends in memory CD4+ T cell populations from baseline, 

week 5, and week 12…………………………………………………………...…16-17

Overall trends in memory CD8+ T cell populations from baseline, 

week 5, and week 12…………………………………………………………………17

Changes in CD4+ and CD8+ T cell populations from baseline to week 5……..……17

Changes in memory CD4+ T cell populations from baseline to week 5……..……...18

Changes in memory CD8+ T cell populations from baseline to week 5……..……...18

Correlations between the change in frequency of memory CD4+ and 

CD8+ T cell subsets and the change in levels of IL-6, CA-RNA, and 

totDNA from baseline to week 5……..…………..…………..…………………..18-19

Correlation between the change in frequency of memory CD4+ and 

CD8+ T cell subsets and the change in the percentage of cells expressing 

Bcl-2 from baseline to week five……..…………..…………..…………..………….19

Correlation between the change in frequency of memory CD4+ and CD8+ 

T cell subsets and the change in the expression of sCD14 from baseline to 

week five…………..…………………..…………………..…………………...…19-20

Discussion…………..…………………..…………………..…………………..…...……20-25

Future Directions…………..…………………..…………………..…………………..……..26

Figures………………………………………………………………………….…………27-40

Figure 1. Gating Strategies for memory CD4+ and CD8+ cell subsets……..……27-28  

Figure 2. Frequency Trends for CD4+ and CD8+ T Cells from Baseline to 

Week 12………………………………………………………………………………29

Figure 3. Frequency Trends for Memory CD4+ T Cell Subsets from Baseline 

to Week 12…………………………………………………………………...…...30-31

Figure 4. Frequency Trends for Memory CD8+ T Cell Subsets from Baseline

to Week 12……………………………………………………….………………32-33 

Figure 5. Change in CD4+ and CD8+ T Cell Percentages of the Total T Cell 

Population from Baseline to Week 5…………………………………………………34

Figure 6. Change in Memory CD4+ T Cell Subset Percentages from Baseline 

to Week 5…………………………………………………………………………35-36

Figure 7. Change in Memory CD8+ T Cell Subset Percentages from Baseline

to Week 5……………………………………………………………...…………37-38 

Figure 8. Correlation Between the Percentage Change in BCL2 Expression

 and CD4+ TTMCell Percentage from Baseline to Week 5…………………………..39

Figure 9. Correlation Between the Percentage Change in sCD14 Expression 

and CD8+ TCMCell Percentage from Baseline to Week 5…………………………..40 

References……………………………………………………..………………………….41-45

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Virology
Keyword	Jak-STAT Ruxolitinib Human Immunodeficiency Virus
Committee Chair / Thesis Advisor	Ann Chahroudi, Emory University
Committee Members	Megan Cole, Emory University Vincent Marconi, Emory University

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