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Early ART initiation in acute HIV infection generates long-lived memory HIV-specific CD8+ T cells endowed with efficient proliferative and cytolytic recall response
Hiroshi Takata
@TwitterHandle
Good afternoon. Thank for the introduction.
I also would like to thank the organizers for this opportunity to talk about our resent study.
Today, I would like to talk about “Memory HIV-specific CD8 T cells generated in acutely treated individuals”.
Share your thoughts on this presentation with #IAS2019
The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army, the Department of Defense, or HJF.

3. Disclosure The author has no conflict of interest to disclose.
I have no conflict of interest to disclose.
The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army, the Department of Defense, or HJF.

4. HIV-specific CD8+ T cells in acute and treated HIV infection
HIV-specific CD8+ T cells become dysfunctional after peak viremia in acute HIV infection (AHI)
Initiation of ART during AHI improves cytokine productivity and memory potential of HIV-specific CD8+ T cells after 1 month of treatment
Trautmann Blood 2012, Ndhlovu Immunity 2015, Takata Sci Transl Med 2017, Ndhlovu Sci Transl Med 2019
Treated from chronic HIV infection (CHI):
ART restores polyfunctionality, and partially normalizes activation, exhaustion markers, and IL-7R.
Rehr M, J Virol , Janbazian L, J Immunol , Vigano S, J Virol , Mahnke YD, Pathog Immun 2016.
Akondy R, et al. Nature 2017., Fuertes Marraco SA, et al. Sci Transl Med , Buggert M,et al. PLoS Pathog
Majority of HIV-specific CD8+ T cells are found in transitional memory subset in contrast to yellow fever vaccine induced memory CD8+ T cells which are found in long-lived memory subsets.
HIV-specific CD8 T cells play a critical role in controlling HIV viremia and could be important in reducing HIV-infected cells.
However, they become dysfunctional after peak viremia in acute HIV infection.
Initiation of ART during AHI improves cytokine productivity and memory potential of HIV-specific CD8+ T cells after short 1 month of treatment.
This suggests that early initiation of ART preserves functionality of HIV-specific CD8 T cells.
But, it is still not clear whether these preserved cells differentiate into functional memory CD8 T cells after long term ART.
In people treated from chronic infection, it has been reported that ”” of HIV-specific CD8 T cells.
But, they are still not as same as functional memory T cells which we can find after successful vaccination such as yellow fever vaccine.
Indeed, “”.
But, it is possible that early ART initiation in AHI overcomes this limitation by preserving immune memory.

5. Questions
Does early initiation of ART from different stages of AHI induce more functional memory CD8+ T cells compared to treating in chronic HIV infection?
Memory T cell differentiation status
Magnitude and quality of secondary response
All PBMC samples were obtained after more than 1 year of suppressive ART.
HLA-A*1101 restricted HIV-Nef/ Pol and EBV-specific CD8+ T cells were analyzed.
Study population
Fiebig stage ART initiated
Cohort
# of participants
Duration of ART (Wks)
Fiebig I-V
RV254 34
107 (48-192)
Chronic
SEARCH011 12
127 (48-196)
Since, little is known about memory HIV-specific CD8 T cells in people treated in acute HIV infection.
We asked “” “”.
Are there any difference in “”.
In this study, we analyzed PBMC samples from 34 participants treated from Fiebig I-V in Thai RV254 cohort and 12 participants treated from chronic infection in Thai Search011 cohort.
All PBMC samples were obtained after more than 1 year of suppressive ART.
Then, HLA-A*1101 restricted HIV-Nef/ Pol and EBV-specific CD8+ T cells were analyzed by peptide MHC-class I tetramer.

6. Secondary HIV-specific CD8 T cell response in vitro
CFSE
CD4 50 26 74
Specific lysis: 65%
Target only
With Effector
ET ratio: 2
Peptide+
Peptide-
Day 13
Expand with PHA+IL-2
ARVs
Isolate CD4+ T cells
Labeled with CFSE X
Coculture
Day 0
Day 6
Day 12
Label with
Cell Trace dye
+HIV epitope peptide,
Phenotyping
Phenotyping
PBMCs from
individuals on ART more than 1 year
HIV-specific CD8+ T cells
CD4+ T cells
38.2
Cell Trace Violet
HLA-A11 HIV Nef
0.4
CD8
2.4
To answer the questions, we performed assays to assess secondary HIV-specific CD8 T cell response in vitro.
First, PBMCs from individuals on ART more than 1 year were labeled with Cell Trace dye to see cell proliferation.
Then, they were stimulated with cognate HIV CD8 epitope peptides.
The cells were stained for peptide-MHC class I tetramer, T cell differentiation markers, and transcription factors on day 0 (ex vivo), day 6 and 12 days after the peptide stimulation.
As you can see below, HIV-specific CD8 T cells were detected as tetramer+ CellTrace dye low cells on day 6 and day 12.
We also performed CFSE based killing assay with day 13 peptide stimulated cells and PHA expanded autologous CD4 T cells as targets.
Cytolytic activity was evaluated by comparing ratio of epitope peptide pulsed CFSE low vs unpulsed CFSE high CD4 T cells in 4 hrs coculture with the CD8 T cells.
If there is a specific killing, CFSE low population decreases as in bottom right panel.

7. Participants treated from acute HIV infection have lower frequency and cell number of HIV-specific CD8+ T cells than treated chronic HIV infection
Percentage in total CD8+ T cells
Absolute cell number in peripheral blood
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
EBV-specific
First, we analyzed frequency of ex vivo tetramer+ HIV specific CD8 T cells in total CD8 on the left, and their absolute cell number on the right.
People treated from acute HIV infection Fiebig I and II-V had significantly lower frequency and cell number of HIV-specific CD8 T cells than treated from chronic HIV infection showing in black.
Especially, HIV-specific CD8 T cells from Fiebig I treated group in yellow were at the lowest, and even lower than EBV-specific CD8 T cells showing as open circles.

8. Initiation of ART in acute HIV infection promotes differentiation of memory CD8+ T cells with self-renewal and long-lived phenotype
Self-renewal
Long lived
Cell survival
IL-7 receptor
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
EBV-specific
Next, we looked into differentiation status of these ex vivo HIV-specific CD8 T cells.
As you can see, treated in acute Fiebig I-V group had significantly higher percentage of T cell subsets have potent self-renewal and long lived capacities such as stem cell memory and central memory cells compared to treated in chronic group.
In contrast, the cells from treated in chronic group were predominantly found in short lived T cell subset Transitional memory.
EBV-specific CD8 T cells, which persist life long, consisted of both long-lived central memory and transitional memory cells.
We also looked at a functional marker important for cell survival, IL-7 receptor CD127.
HIV-specific CD8 T cells in Treated from Fiebig I group showed the highest expression followed by treated from Fiebig II-V group.
These expression levels were significantly higher than that of treated from chronic group, but comparable to that of EBV-specific CD8 T cells.
These suggest that early initiation of ART in acute HIV infection promotes differentiation of memory cells with self-renewal and long lived phenotype.

9. HIV-specific CD8+ T cells from people treated from AHI expand better than those from people treated from chronic HIV infection
Day 6/ Day0
Day 12/ Day0
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
EBV-specific
Next, we analyzed magnitude of recalled HIV-specific CD8 T cell response after peptide stimulation.
Here, I am showing fold expansion of HIV-specific and EBV-specific CD8 T cells in cell number between day 0 and day 6 on the left, and day 0 and day 12 on the right.
Overall, treated from acute HIV infection group showed the highest expansion and it became significant until day 12 compared to treated from chronic group.
This suggests that “”.

10. Self-renewal and cell survival
Magnitude of the recall response correlates with characteristic of long-lived memory CD8+ T cells
Self-renewal and cell survival
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
We further looked for immune correlates in ex vivo memory HIV-specific CD8 T cells for the magnitude of recall response.
We found that the higher fold expansion of HIV-specific CD8 T cells corelated with higher expression of TCF1,which is a transcriptional factor important for self-renewal capacity in the left panel,
as well as percentage of long lived stem cell memory and central memory cells in the right panel.
These suggest that higher magnitude of the recall response correlates with characteristic of long-lived memory CD8 T cells generated in vivo.

11. Recalled HIV-specific CD8+ T cells from people treated from AHI have superior killing capacity than those from treated from CHI
CFSE killing assay with day 13 recalled A11 HIV Nef GK10-specific and EBV-specific CD8+ T cells
Recalled HIV-specific CD8+ T cells
Ex vivo HIV-specific CD8+ T cells
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
EBV-specific
Finally, we analyzed the most important effector function, cytolytic activity of the recalled HIV-specific CD8 T cells.
Before peptide stimulation,
ex vivo HIV-specific CD8 T cells from treated in chronic group had higher expression of cytolytic molecule Granzyme B than those in treated from acute and EBV-specific CD8 T cells.
However, according to CFSE killing assay, treated from acute group showed superior specific killing than those from treated from chronic group after recall.
This killing capacity of the acutely treated group was comparable to that of EBV-specific CD8 T cells.
For mechanism behind this difference,

12. High PD-1 expression status is imprinted in HIV-specific CD8+ T cells from treated from chronic infection even after secondary response
Fiebig I
Fiebig II
Fiebig III
Fiebig IV/V
Chronic
We looked at Check point protein PD-1 expression.
In both groups, PD-1 was upregulated on day 6 then downregulated on day 12 except for treated from chronic group.
The expression levels were always higher in treated from chronic group than those in treated from acute on day 0 ex vivo, day 6, and even day 12 of the culture.
This suggests that the high PD-1 expression status is imprinted even after secondary response and it might induce exhausted dysfunctional HIV-specific CD8 T cells.

13. Conclusion
Early ART in acute HIV infection promotes differentiation of long-lived memory CD8+ T cells that have higher expansion and cytotoxic capacity after recall than those in people treated from chronic infection
Even after suppressive ART, recalled HIV-specific CD8+ T cells from individuals treated from chronic infection are exhausted based on their upregulated PD-1 expression and dampened effector function
HIV remission strategies in people treated in chronic infection will likely require a combination of reversing T cell exhaustion and boosting the HIV-specific CD8+ T cell response
In conclusion, “”.

14. Acknowledgements RV254 and Search011 participants
MHRP Cellular
Immunology Section
Lydie Trautmann
Caroline Subra
Julie Mitchell
Noemia Lima
Faria Fatmi
Jueyon Kakazu
Shu-Wei Wu
Kelsi Jameson
Kenneth Dietze
Alexander Haregot
Nikiah Dawson
Industry
Thai Gov Pharm Organization
ViiV Healthcare
Gilead
Merck
Monogram
U.S. MHRP Nelson Michael Sandhya Vasan Merlin Robb Robert Gramzinski Jintanat Ananworanich Julie Ake Diane Bolton Morgane Rolland Sodsai Tovanabutra Rasmi Thomas Mark de Souza Linda Jagodzinski Shelly Krebs Trevor Crowell Suteera Pinyakorn Flow Core
AFRIMS
Robert O’ Connell
Alexandra Schuetz
Siriwat Akapirak
Denise Hsu
Rapee Trichavaroj
Bessara Nantapinit
Pornsuk Visudhiphan
COG
Thai Red Cross
Praphan Phanuphak
Nipat Teeratakulpisarn
Nittaya Phanuphak
Eugene Kroon
Donn Colby
Nitiya Chomchey
Carlo Sacdalan
James Fletcher
Pornpen Tantivitayakul
Phillip Chan
Jintana Intasan
Many more
Chulalongkorn U
Supranee Buranapraditkun
Sunee Sirivichayakul
Rungsun Rerknimitr
Sukalya Lerdlum
Phandee
Wattanaboon-
yongcharoen
Sopark Manasnayakorn
Montréal U
Nicolas Chomont
Louise Leyre
Remi Fromentin
Drexel U
Elias Haddad
Roshell Muir
UCSF
Victor Valcour
I would like to acknowledge all the people involved with this study, especially study participants from RV254 and Search011 and the funders.
Thank you very much for your attention.
Funding from U.S. MHRP Program, R01AI108433, R01MH106466