TCR SEQUENCING REVEALS CLONAL EXPANSIONS OF INDUCIBLE RESERVOIRS IN SPECIFIC SUBSETS
Pierre GANTNER1, Amélie PAGLIUZZA2, Marion PARDONS1, Moti RAMGOPAL3, Jean-Pierre ROUTY4, Rémi FROMENTIN2, Nicolas CHOMONT1,2.1Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montreal, Quebec, Canada 2Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada3Midway Immunology & Research Center, Fort Pierce, Florida, USA 4Division of Hematology & Chronic Viral Illness Service, McGill University Heath Centre, Montreal, Quebec, Canada
Correspondance to Dr Nicolas Chomont:Centre de recherche du CHUM, 900, rue St-Denis, Tour Viger,
Montréal, QC, H2X 0A9 - CanadaTel:(+1) 514-890-8000 #[email protected]
Blood samples from 8 individuals on suppressive ART for at least 2 years were collected longitudinally.Clonotype characterization of HIV-infected cells was determined by combining index single-cell sortingof HIV-infected cells by HIV-Flow and TCR sequencing (Figure 1A). TCR clonotypes were retrieved fromTCR sequences using the IMGT database (example for participant #1, 1st sample in Figure 1B) and theirmemory phenotype was assessed post hoc (Figure 1C).A subset of p24- cells was analyzed to determine TCR diversity in the CD4+ T-cell compartment.
Clonal expansions occur in the persistent HIV reservoir as demonstrated by the duplication of HIV genesand/or integration sites reported in several studies. However, these approaches do not permit tophenotypically analyze these expanded clones of infected cells nor the inducibility of the proviruses.We took advantage of the uniqueness of the T-cell receptor (TCR) expressed by a given T-cell clone totrack HIV-infected cells.
Introduction
Methods
Results
TCM50,9
TEM10,9
TTM19,4
Y18,8
0-10 3 10 3 10 4 10 5
Comp-BV421-A
0
-10 3
10 3
10 4
10 5
Com
p-Pe
rCP-
Cy5
-5-A
Sample Name Subset Name Count TUBE NAME
ST105 Clone 5_1.fcs Ungated 1,00 5
ST105 Clone 4_1.fcs Ungated 1,00 4
ST105 Clone 3_1.fcs Ungated 2,00 3
ST105 Clone 2_1.fcs Ungated 1,00 2
ST105 Clone 1_1.fcs Ungated 8,00 1
Specimen_001_ST105_Concat_1.fcs CD45RA- 1,39E6 ST105
0-10 3 10 3 10 4 10 5
Comp-PE-A
0
-10 3
10 3
10 4
10 5
Com
p-AP
C-A
Sample Name Subset Name Count TUBE NAME
ST105 Clone 5_1.fcs Ungated 1,00 5
ST105 Clone 4_1.fcs Ungated 1,00 4
ST105 Clone 3_1.fcs Ungated 2,00 3
ST105 Clone 2_1.fcs Ungated 1,00 2
ST105 Clone 1_1.fcs Ungated 8,00 1
Specimen_001_ST105_Concat_1.fcs p24- 2,46E6 ST105
p24
28B7
p24 expressionCD8-CD45RA-
p24 KC57
CC
R7
CD27
Well ID# V segment CDR3 sequence (amino-acids) J segment
A1 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A2 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A3 V4-1*01 CASSLTEAYGYTF J1-2*01A4 V20-1*01 CSAKDRVIETQFF J2-5*01A5 V29-1*01 CSVKDSYNEQFF J2-1*01A6 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A7 V10-3*01 CGVRDPFYPPHF J1-5*01A8 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A9 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A10 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A11 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01A12 V4-1*01 CASSLTEAYGYTF J1-2*01B1 V6-5*01 CASRRAWRGALSNSPLHF J1-6*01
TCRβ sequencing and clonotype identification
ST105 ST105B ST105C0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 11Clone 10Clone 9Clone 8Clone 7Clone 6Clone 5Clone 4Clone 3Clone 2Clone 1
p24- cells
Memory phenotype
We obtained the TCRβ sequences from 636 p24+ and 357 p24- single-sorted cells, among those, weidentified 98 and 353 different TCR clonotypes, respectively. Clonal expansions were detected in the poolof p24+ cells from all participants (Figure 2) and accounted for the majority of reservoir cells (mean,74%). We observed a median of 2 independent clonal expansions per sample (range, 1-5 expandedclonotype per sample), each expansion being detected in a median of 5 p24+ cells (range, 2-187).
Over time on ART, clonally-expanded p24+ cells could whether be further detected, indicating persistentclonal expansion, or not, indicating transient clonal expansion.Expanded infected clonotypes persisted on ART in 7/8 individuals (except participant #5).However, the dynamic of the HIV reservoir on ART greatly varied between individuals, with someparticipants showing a stable repertoire, whereas others displayed emergence of new clonotypes overtime (Figure 3).
B C Post hoc analysis of the memory phenotype of TCR clonotypes
Figure 1. Strategy for analyzing the TCR repertoire of single-sorted HIV-infected cells.A: Overview of methods; B: Example of TCR clonotypes identification for participant #1 (1st sample) after single-cellsorting of p24+ cells; C: Dot plots of the memory phenotypes of the TCR clonotypes.
A
Figure 4. V and J segment association in the pool of p24+ and p24- cells.Association of V and J segment (links) for both p24+ (A) and p24- cells (B), each color represent a participant.
BA
The majority of the translation-competent reservoir is clonally expanded
Expanded clonotypes in p24+ cells often persist on ART Clonally-expanded p24+ cells display multiple memory phenotypes
Expanded p24+ clonotypes systematically displayed at least two different memory phenotypes (Figure 5A).Expanded infected clones were overrepresented in the most differentiated cells (i.e. transitional, TTM; andeffector memory, TEM). Nonetheless, these expanded clones were also identified within the central memory(TCM) compartment from the majority of the participants, albeit at lower frequencies (Figure 5B).Importantly, the memory phenotype of these expanded reservoir cells was maintained over time on ART
The TCR repertoire of p24+ cells is biased when compared to p24- cells
There was a bias in the selection of V and J segments in p24+ cells (Figure 4A) when compared to p24-cells (Figure 4B). This bias was mainly introduced by clonal expansions in the HIV reservoir.
Figure 2. Clonal expansion proportion among p24+ cells.A: Proportion of clonally expanded cells in the pool ofp24+ and p24- cells from for each participant.B: Proportion of clonal expansion in the pool of p24+ cellsfor each participant according to the study visit.
BA
Figure 3. Clonal expansion proportion among p24+ cells.Proportion of TCRβ clones among the pool of p24+ cells among the different studyparticipants (ranked #1 to #8) and according to the study.
Conclusions
Acknowledgements
We developed a method to obtain TCR sequencesfrom single HIV-infected cells in individuals on ART.Using this approach, we demonstrate that:(1) clonal expansions highly contribute to thepersistence of the translation-competent HIVreservoir, (2) clonally-expanded cells displaymultiple memory phenotypes, and (3) the dynamicof the repertoire of the HIV reservoir variesbetween individuals.Our results suggest that:à Antigen stimulation is a major driver of the HIV
reservoir dynamics during ART.à Infected T cell clonotypes displaying a
differentiated phenotype are the progeny ofinfected central memory cells undergoing clonalexpansion during ART (Figure 6).
The study team is grateful to the individuals who volunteered to participate in this study. The authors thank JoséeGirouard and Brenda Jacobs for recruitment and clinical assistance with study participants. We thank the flow cytometrycore at the CRCHUM, managed by Dominique Gauchat and Philippe St-Onge.
HIV-Flow Single-cellsorting
PMA/iono
CD4+ T-cell TCRs p24 protein HIV genome Anti-p24 antibody
CASRRAWRGALSNSPLHF
CASSLTEAYGYTF
TCRβ sequencing
Unique clones
Transient clonal expansion
Persistent clonal expansion
CHO24 CHO24B0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 1
Clone 3Clone 4
Clone 7Clone 8Clone 9Clone 10Clone 11
Clone 5Clone 6
#6 Clone 2
Clone 12Clone 13Clone 14Clone 15Clone 16Clone 17
-6 -4 -2 0 2
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 ) H
IV-RN
A (log10 copies / m
L)
DRV/r+ETR+RAL+ABC/3TC DRV/r
#6
n=19 n=30
ST125 ST125B0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 1Clone 2Clone 3Clone 4
Clone 7Clone 8Clone 9
Clone 5Clone 6
#8
Clone 10Clone 11Clone 12Clone 13Clone 14Clone 15Clone 16
-2 0 2
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 )
FPV/r+RAL+TDF/FTC DTG+TDF/FTC
HIV-R
NA (log
10 copies / mL)
#8
n=10 n=10
CHO34 CHO34B CHO34C0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 2Clone 3
Clone 5
Clone 1
#7Clone 6Clone 7Clone 8Clone 9Clone 10Clone 11Clone 12Clone 13Clone 14Clone 15Clone 16Clone 17Clone 18Clone 19Clone 20Clone 21
Clone 4
-4 -2 0 2 4
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 ) H
IV-RN
A (log10 copies / m
L)
RPV/TDF/FTC
#7
n=22 n=15 n=36
ST146 ST146B0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 1Clone 2Clone 3Clone 4
Clone 8Clone 9
Clone 6Clone 7
#5
Clone 5
-2 0 2 4 6 8
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 )
EFV/TDF/FTC EVG/c/TDF/FTC EVG/c/TDF/FTC
HIV-R
NA (log
10 copies / mL)
#5
n=16 n=10
ST137 ST137B0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 4Clone 3Clone 2Clone 1
#2
-2 0 2
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 )
EFV/TDF/FTC
HIV-R
NA (log
10 copies / mL)
#2
n=141 n=194
ST121 ST121B0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 2Clone 3Clone 4
Clone 7Clone 8
Clone 5Clone 6
#4
Clone 1
Clone 9Clone 10
-4 -2 0 2 4 6 8
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 )
MVC+ETR+RAL
HIV-R
NA (log
10 copies / mL)
#4
n=14 n=9
ST109 ST109B ST109C0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 10Clone 9Clone 8Clone 7Clone 6Clone 5Clone 4
Clone 2Clone 1
#3
Clone 3
-8 -6 -4 -2 0 2 4 60
1000
2000
2500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 ) H
IV-RN
A (log10 copies / m
L)
EFV+TDF+FTC
#3
n=29n=16
ST105 ST105B ST105C0
25%
50%
75%
100%
% o
f TC
R c
lone
s on
p24
+ ce
lls
Clone 8Clone 7Clone 6Clone 5Clone 4Clone 3Clone 2Clone 1
Clone 9Clone 10
#1
Clone 11Clone 12Clone 13
-4 -2 0 2 4
0
500
1000
1500
0
2
4
6
Time (years)
Abs
olut
e C
D4
coun
t (ce
lls/m
m3 ) H
IV-RN
A (log10 copies / m
L)
FPV/r+ABC/3TC DRV/r+ABC/FTC
#1
n=13 n=13 n=39
#1V1 #1V2 #1V3
#3V1 #3V2
#4V1 #4V2
1
2
MET
1020
30
4050
60 70 80
1020
3040
50
1020
30
40
50607080
90
100
110
120
130140
1
1
2
MET
1020
30
4050
60 70 80
1020
3040
50
1020
30
40
50607080
90
100
110
120
130140
1
1
2
MET
1020
30
4050
60 70 80
1020
3040
50
1020
30
40
50607080
90
100
110
120
130140
1
1
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MET
1020
30
4050
60 70 80
1020
3040
50
1020
30
40
50607080
90
100
110
120
130140
1
#2V1 #2V2
1
12
3
45
6 7 8
3 1
21
24
15
1
MET
12
3
45
67
MTT
12
3
MCT
12
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612
3
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19
1
MET
12
3
45
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MTT
12
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2
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MTT
12
5
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1520
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12
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MET
MTT
1 212
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MET
MTT
12
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MTT
1
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MET
MTT
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MTT
1
12
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MET
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12
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1520
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12
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MET
MTT
12
5
312
3
21
2 11
2 3 4 56
7
89
4 12
.htO
12
3
MET 12MTT
12345
6
7
89
MCT1
2
2
12
34
56
1
12
34
5 6 7 8 9 10 11 12 13 1415 16
6 17 1
5 12
8 1
9 110 1
.htO
12
3
MET
12
3
45
MTT 123456789
1011
1213
14
MCT
12
34
56
7
1
2
34
MET
1020
3040
50
1020 30 40 50 60
7080
90
100110
120130
1020
30
40
50
6070
8090100110120
130
140
150
160
170180
190
1
2
34
MET
1020
3040
50
1020 30 40 50 60
7080
90
100110
120130
1020
30
40
50
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8090100110120
130
140
150
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170180
190
1
2
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MET
1020
3040
50
1020 30 40 50 60
7080
90
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120130
1020
30
40
50
6070
8090100110120
130
140
150
160
170180
190
1
2
34
MET
1020
3040
50
1020 30 40 50 60
7080
90
100110
120130
1020
30
40
50
6070
8090100110120
130
140
150
160
170180
190
1
2
34
MET
1020
3040
50
1020 30 40 50 60
7080
90
100110
120130
1020
30
40
50
6070
8090100110120
130
140
150
160
170180
190
51
11
23
61
7 1
8 19
110
1
.htO
1
2
3
4
MET
12
3
4M
CT1
1
2
3
4
METMTT
MCT
12
3
45
61
2
3
4
METMTT
MCT
1 23
41
2
3
4
METMTT
MCT
1
1
2
3
4
METMTT
MCT
12
3
1
2
3
4
METMTT
MCT
12
3
4
567
1
2
3
4
METMTT
MCT
12
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1
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METMTT
MCT
1
#7V1 #7V2
#6V1 #6V2
#5V1 #5V2
#8V1 #8V2
TCM
TTM
TEM
Others
Unique clones
Transient clonal expansion
Persistent clonal expansion
1
2
34
56
MET
MTT
MCT
.htO
12
34
56
78 9 10 11 12 13
1
2
34
56
MET
MTT
MCT
.htO
11
2
34
56
MET
MTT
MCT
.htO
11
2
34
56
MET
MTT
MCT
.htO
12
1
2
34
56
MET
MTT
MCT
.htO
1
1
2
34
56
MET
MTT
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.htO
1
1
2
34
56
MET
MTT
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1
1
2
34
56
MET
MTT
MCT
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12
3
4
1
2
34
56
MET
MTT
MCT
.htO
123456
78
9
10
1
2
34
56
MET
MTT
MCT
.htO
12
34
1
12
34
56
7
89
10
1
11
12
1 2 3
7
1 2 3 4 5
8
12
3
91
212
113 1
14 1
15 1
16 1
17 1
MET
12
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7
8
MTT 12
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1213
1415
1617
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MCT
12
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1
12
34
5
67
89 10 11 12 13 14 15
16
1718
1920
212
1
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12
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5
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MTT
12
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78
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#7V3
3
12
3
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41
61
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8 1
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1
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12
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MTT
1
2
34
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3
12
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1
13
12
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45
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89 10 11 12 13 14 15 16 17 18 19
2021
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1
16 1
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20 1
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MET
12
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1112
1314
1516171819202122
2324
2526
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MTT
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7
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2 2
1 31
41
51
6 1
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1
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1
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3MTT
123
MCT
1
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11 10
1 111 12
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1
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2
14 115 1
161
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1
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MTT 123
MCT
1
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1
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METMTT
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12
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1
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METMTT
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METMTT
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1
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456
7
8MT
T1
2
20%
80%
p24+n=65
100%
p24-n=59
23%
77%
p24+n=26
96%
p24-n=51
4%
1%
99%
p24+n=335
100%
p24-n=35
24%
76%
p24+n=49
86%
p24-n=35
14%
11%
89%
p24+n=45
100%
p24-n=25
22%
78%
p24+n=73
100%
p24-n=71
30%
70%
p24+n=23
100%
p24-n=39
30%
70%
p24+n=20
100%
p24-n=42
16%
74%
p24+
100%
p24-
5 6
Unique clonotypes
Expandedclonotypes
7 8 Mean
1 2 3 4
Participant #ID:
1 2 3 4 5 6 7 80
20%
40%
60%
80%
100%
Participants ID#Pro
porti
on o
f clo
nal e
xpan
sion
s in
the
pool
of p
24+
cells
(%)
Visit 1, n=8
Visit 2, n=8
Visit 3, n=2
1 2 3 1 2 1 2 1 2 1 2 1 2 1 2 3 1 20
20%
40%
60%
80%
100%
Pro
porti
on o
f the
diff
eren
t mem
ory
subs
ets
in c
ells
th
at w
ere
clon
ally
-exp
ande
d in
the
pool
of p
24+
cells
(%)
TCM
TTM
TEM
Others
Visit:
Participant #ID: 1 2 3 4 5 6 7 8
Participant #ID:
1
2
3
4
5
6
7
8
TRBV10−3TRBV11−2TRBV11−3
TRBV12−3
TRBV13TRBV14TRBV15TRBV18TRBV19
TRBV2
TRBV20−1
TRBV23−1TRBV24−1
TRBV25−1
TRBV27
TRBV29−1TRBV3−1TRBV30
TRBV4−1
TRBV4−2
TRBV5−4TRBV6−1TRBV6−2
TRBV6−5
TRBV6−6TRBV7−2TRBV7−3TRBV7−9
TRBV9
TRBJ1−1
TRBJ1−2
TRBJ1−3
TRBJ1−4
TRBJ1−5
TRBJ1−6
TRBJ2−1
TRBJ2−2
TRBJ2−3
TRBJ2−4
TRBJ2−5
TRBJ2−6
TRBJ2−7
0
1
2
3
4
5
6
7
8
13
21
22
23
25
45
147
187
X.ID TRBV TRBJ FreqParticipant #ID TRBV TRBJ
1
2
3
4
5
6
7
8
TRBV10−3
TRBV11−2TRBV11−3
TRBV12−3
TRBV12−5TRBV13TRBV14TRBV15
TRBV18
TRBV19
TRBV2
TRBV20−1
TRBV22TRBV24−1TRBV25−1
TRBV27TRBV28TRBV29
TRBV29−1
TRBV3−1
TRBV30
TRBV4−1
TRBV4−2TRBV5−4TRBV5−5
TRBV5−6
TRBV5−7
TRBV6−1
TRBV6−2
TRBV6−5
TRBV6−6
TRBV7−2
TRBV7−3TRBV7−4TRBV7−9
TRBJ1−1
TRBJ1−2
TRBJ1−3
TRBJ1−4
TRBJ1−5
TRBJ1−6
TRBJ2−1
TRBJ2−2
TRBJ2−3
TRBJ2−4
TRBJ2−5
TRBJ2−6
TRBJ2−7
0
1
2
3
4
6
X.ID TRBV TRBJ Freq
p24+ cells (n=636) p24- cells (n=357)
Participant #ID TRBV TRBJ
TCM
HIV genome
TCR
TTM
TEM
HIV particle
Antigen
Legend:
Figure 6. TCM cells represent a long-lived source of reservoircellsTTM and TEM cells represent the main contributors to the poolof infected cells, but are the progeny of infected TCM cells thathave long survival and self-renewal capacities, and canexpand upon antigen stimulation and replenish the pool ofinfected cells.
A
BFigure 5. Expanded p24+ clonotypes display multiple memoryphenotypes.A: Distribution of TCRβ clones from p24+ among memory subsets eachstudy participant (ranked #1 to #8) and according to the study visit (V1to V2 or V3). This circular representation shows the link between aspecific clone (top half of the circle) and its memory subset(s) (bottomhalf of the circle). The circular axis represents the number of isolatedand sequenced p24+ cells in each clone/subset.The clone color code is defined as follows: expanded clones persistingover time in green; expanded clones detected at a single visit in blue;and unique clonotypes in grey. The memory subset color code is definedas follows: TCM in blue; TTM in orange; TEM in red; and others in grey.B: Proportion of the different memory phenotypes among cells thatwere clonally-expanded cells in the pool of p24+ cells for all participantsand all study visits.
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