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![Page 1: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/1.jpg)
Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes
Michael ImbeaultLaboratory of Dr. Michel J. TremblayUniversité Laval, Québec CanadaAIDS 2010 Vienna
![Page 2: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/2.jpg)
Goal
•Describe the transcriptomic profile of primary CD4+ T cells exposed to HIV-1 in vitro
•Compare infected cells and bystander cells
![Page 3: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/3.jpg)
Infection of primary lymphocytes in vitro
0 1 2 3 40123456789
% of infected cells
Time (days)
% i
nfe
cte
d c
ell
s (G
FP
)
![Page 4: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/4.jpg)
Problem
Mock Control Infected (10%)
Quantification of RNA - 10 copies vs 12 copies = 1.2 foldBut in infected cells, its a 3 fold induction
![Page 5: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/5.jpg)
NL4-3-IRES-HSA
• Express all viral genes
• Allow for separation of infected cells using magnetic beads
• More sensitive than the parental GFP virus (Levy & al)
• Details published in Virology. 2009 Oct 10;393(1):160-7
![Page 6: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/6.jpg)
NL4-3-IRES-HSA
![Page 7: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/7.jpg)
NL4-3-IRES-HSA
![Page 8: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/8.jpg)
Human Exon 1.0 ST array• Latest offering from
Affymetrix
• 1.4 millions probesets
• 1 million exons
• Covers▫ All known human genes▫ in silico predicted genes▫ ESTs
• Allow for quantification of expression and determination of alternative splicing events
![Page 9: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/9.jpg)
Human Exon 1.0 ST array
![Page 10: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/10.jpg)
Protocol
![Page 11: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/11.jpg)
Separation of infected cells
•Isolate a mean of 500 000 infected cells starting from▫50 million cells at day 1▫25 million cells at day 2 and 3
•Extraction of RNA using a dual Trizol – Qiagen custom protocol
•Quantification of purity by Taqman qRT-PCR against Tat-spliced
![Page 12: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/12.jpg)
Tat-spliced PCR
Mock 24h
HIV Neg 24h
HIV Pos 24h
Mock 48h
HIV Neg 48h
HIV Pos 48h
Mock 72h
HIV Neg 72h
HIV Pos 72h
0
0.5
1
1.5
2
2.5
3
ABC
Rela
tive
Tat s
plic
ed m
RNA
leve
ls
norm
aliz
ed o
n 18
S
![Page 13: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/13.jpg)
Analysis
•Strict statististical analysis using Bioconductor’s LIMMA
•FDR 1%
•1.7 fold minimum
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Results
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Results
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Automated litterature analysis
•Automated analysis of literature using Genomatix Bibliosphere
•Citation of 2 genes in the same sentence in at least 3 different abstracts
•Exported to graph management software Gephi▫Gephi.org
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Automated analysis of literature
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Main features
•AP-1 (FOS and JUN, some other related genes)
•A group of genes related to activated / effector T cells
▫Many cytokines associated to Th1, Th2, Th17
▫Th17 related genes have higher surexpression values, perhaps indicating a higher susceptibility
•p53 related genes
![Page 19: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/19.jpg)
AP-1
FOS JUN
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Promoter analysis
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Cytokine related
Th1 (IFNG, TNF-a, TNF-b, IL1A, IL3)
Th2 (IL4, IL5, IL-10, IL13)
Th17 (IL17A, IL17F, IL21, IL22, IL23R, IRF4)
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Th17 related genes
IL17A IL17F
![Page 23: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/23.jpg)
Th17 related genes
IL22 IL23R
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p53 related genes
![Page 25: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/25.jpg)
Automated analysis of literature
![Page 26: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/26.jpg)
Alternative splicing analysis• Used the combination of two of the best
algorithms▫MADS and PECA-SI
• P < 0.01
• Splicing index of at least 0.6
• Exon can be detected in at least 1/3 of arrays
• Filter out exons not currently associated with genes
![Page 27: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/27.jpg)
Alternative splicing results
•547 probes in 372 transcripts
•52% of these are in non-coding UTRs
•48% in coding exons
![Page 28: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/28.jpg)
PTPRC
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Alternative splicing - PTPRC
Mock A
Mock B
Mock C
Bystander A
Bystander B
Bystander C
Infected A
Infected B
Infected C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
24h48h72h
Rel
ativ
e n
orm
aliz
ed le
vels
of
exon
4 o
f C
D45
![Page 30: Exon-level transcriptome analysis of HIV-1 infected and bystander primary CD4+ T lymphocytes Michael Imbeault Laboratory of Dr. Michel J. Tremblay Université.](https://reader034.fdocuments.in/reader034/viewer/2022051517/5697bfc61a28abf838ca6f6c/html5/thumbnails/30.jpg)
Conclusion
• Infected cells have a transcriptomic profile of highly activated / effector / memory T cells
•No effect at all in bystander cells
•Confirmed a lot of things▫Role of p53 in HIV-1 pathogenenis
•Many interesting candidates▫Potential susceptibility and restriction factors
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Acknowledgements
• Michel J. Tremblay
• Project Leaders• Corinne Barat, Ph.D.• Réjean Cantin, Ph.D.• Robert Lodge, Ph.D.• Michel Ouellet, Ph.D.
• Postdoctoral Fellows• Ravendra Garg, Ph.D.• Pranav Kumar, Ph.D.• Guadalupe Andreani,
Ph.D.• Masayuki Fujino, Ph.D.• Sandra Côté, Ph.D.
• Ph.D. Students• Rémi Fromentin, M.Sc.• Alexandra Lambert, M.Sc.• Lise-Andrée Gobeil, M.Sc.• Jonathan Bertin, M.Sc.• Pascal Jalaguier, M.Sc.• Anissa Cheikh, M.Sc.• Alejandro Martin Gomez
Lic.
• M.Sc. Students• Alexis Danylo, B.Sc.• Katia Giguère, B.Sc.• Audrey Plante, B.Sc.• Jean-François Bolduc, B.Sc.• Véronique Veillette, B.Sc.
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Questions