Identification and Characterization of CD4 T cells actively transcribing HIV RNA in Peripheral Blood
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1 Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Identification and Characterization of CD4 T cells actively transcribing HIV RNA in Peripheral Blood Richard A. Koup, MD June 29, 2013
2 Quantifying Intracellular HIV RNA Quantification of cell-associated HIV DNA is often used as a measure of past infection. Often used as a measure of the latent reservoir However, a large proportion of that DNA may be replication incompetent, and certainly transcriptionally inactive. Quantifying cell-associated HIV RNA could tell us: Which cells are transcriptionally active with respect to HIV Which cells are contributing to the plasma virus pool What is the viral burst size of HIV from a peripheral CD4 T cell How HIV antigens are expressed on the CD4 T cell surface in vivo What cellular processes are affected by active HIV transcription We therefore sought to quantify active HIV transcription in PBMC directly ex vivo
3 Which HIV RNA Should Be Measured?
4 9 Kb (genomic/gag) RNA without 2 Kb (Tat/Rev) RNA expression 9 Kb (genomic/gag) RNA and 2 Kb (Tat/Rev) RNA expression Viral protein expression and budding should be associated with CD4 down regulation
5 CD4 (+) Bright, Dim and Null Gating Strategy
6 Determining Active Expression of HIV RNA in CD4 T Cell Populations Sort CD4 Bright, Dim, and Null cells into multiple wells at varying numbers of cells per well Perform quantitative PCR for HIV Gag, Rev, and Tat RNA on each well Determine precursor frequency of cells actively expressing HIV Gag, Rev, and Tat RNA Use wells that are statistically likely to contain only a single HIV RNA expressing cell to determine the copy number of HIV RNA per cell in CD4 Bright, Dim, and Null cells + RNA PCR 9 Kb 2 Kb Gag Rev Tat
7 Determining Active Expression of HIV RNA CD4 Bright 3000 cells/well in CD4 T Cell Populations 1000 cells/well 300 cells/well Gag RNA 1:850 cells Rev RNA 1:3936 cells Tat RNA 1:4449 cells + RNA PCR 9 Kb 2 Kb Gag Rev Tat
8 Determining Active Expression of HIV RNA in CD4 T Cell Populations CD4 Bright CD4 Dim 3000 cells/well 30 cells/well 1000 cells/well 10 cells/well 300 cells/well Gag RNA 1:850 cells Rev RNA 1:3936 cells Tat RNA 1:4449 cells Gag RNA 1:64 cells Rev RNA 1:66 cells Tat RNA 1:80 cells + RNA PCR 9 Kb 2 Kb Gag Rev Tat
9 Determining Active Expression of HIV RNA in CD4 T Cell Populations CD4 Bright CD4 Dim 3000 cells/well 30 cells/well CD4 Null 500 cells/well 1000 cells/well 300 cells/well 10 cells/well Gag RNA 1:4746 cells Rev RNA 1:4746 cells Tat RNA 1:14749 cells Gag RNA 1:850 cells Rev RNA 1:3936 cells Tat RNA 1:4449 cells Gag RNA 1:64 cells Rev RNA 1:66 cells Tat RNA 1:80 cells + RNA PCR 9 Kb 2 Kb Gag Rev Tat
10 HIV Gag RNA copy number increases with decreasing CD4 surface expression Proportion of CD4 T cells Frequency of cells expressing 2kb RNA Copy No/2kB RNA + Cell 87% 1: % 1:58 11% 1:4746 VL = 47,259 CD4 = 244 Composite from 9 subjects
11 Activation Markers Further Identify Cells Expressing Spliced HIV RNA CD4 ICOS Rev RNA + = 1:34 CD8 CD38 Active HIV transcription is greatest in activated CD4 T cells
12 Question: Can we detect HIV-producing T cells using broadly neutralizing antibodies? Kwong, P.D, Mascola, J.R Immunity 37(3):
13 Broadly Neutralizing mab Staining of In Vitro BAL Infected CD4 T cells Mab concentration 3-5µg/ml
14 Can Broadly Neutralizing mabs Be Used to Sort In Vitro HIV-infected CD4 T Cells?
15 HIV RNA Copy Number Is Associated with CD4 Down-Regulation
16 Ex Vivo Index Sorting for HIV RNA PCR Index sorted 168 T cells into individual PCR wells (VL: >500,000, CD4: 24) 99 CD4 dim, 69 null CD56 - TCRγδ - Also stained with VRC07, PG9, CD27, CD45RO, ICOS and CD38 Fluorescence profile of each cell was stored Single-cell Sorting (e.g., CD4 dim vs null) CD4 CD27 PG9 CD45RO
17 Detecting HIV Transcription Ex Vivo Four wells were positive for HIV Tat, Rev, and Gag RNA One was positive for only Gag RNA All were CD4 dim (not null) HIV RNA + cells were PG9 dim and CD27 - Cells were variably positive for CD38 and ICOS
18 Conclusions Cell-associated viral replication in vivo can be quantified using cell sorting and Q-PCR for spliced and unspliced forms of HIV RNA Full HIV RNA transcription is associated with CD4 down-regulation in vivo The number of HIV RNA copies per cell increases with CD4 down-regulation HIV transcription is most frequent in T cells that express known activation markers The level of HIV env expression during active HIV replication in vivo is far less than it is on in vitro activated and infected T cells, making their detection by broadly neutralizing monoclonal antibodies difficult
19 Acknowledgements Immunology Laboratory Joseph Casazza David Ambrozak Alex Vostal Human Immunology Section Daniel Douek Brenna Hill Eli Boritz Special thanks to: Frank Maldarelli John Mellors John Coffin
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