tcr sequencing reveals clonal expansions of ......5 comp-bv421-a 0-10 3 10 3 10 4 10 5...

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





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






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 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


#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 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


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


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


-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

2

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

3

612

3

112

3 4 56

7

7 18

19

1

MET

12

3

45

6

MTT

12

3

4

5M

CT1

2

12

1011

1213

MET

MTT

12

5

10

1520

25

12

1011

1213

MET

MTT

1 212

1011

1213

MET

MTT

12

5

12

1011

1213

MET

MTT

1

12

1011

1213

MET

MTT

1

12

1011

1213

MET

MTT

1

12

1011

1213

MET

MTT

12

5

10

1520

25

30

12

1011

1213

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

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

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

3

4

56

1

2

3

4

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

MCT

.htO

1

1

2

34

56

MET

MTT

MCT

.htO

1

1

2

34

56

MET

MTT

MCT

.htO

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

34

56

7

8

MTT 12

34567891011

1213

1415

1617

1819

MCT

12

3

1

12

34

5

67

89 10 11 12 13 14 15

16

1718

1920

212

1

MET

12

34

5

67

89101112

MTT

12

34

56

78

910

#7V3

3

12

3

45

12 3

41

61

71

8 1

9 110 1

11 112

1

MET

12

3

45

6789

MTT

1

2

34

56

3

12

34 4

1

13

12

3

45

67

89 10 11 12 13 14 15 16 17 18 19

2021

22 23

14

115

1

16 1

17 1

18 1

19 1

20 1

21 1

MET

12

34

56

78

910

1112

1314

1516171819202122

2324

2526

27

28

29

MTT

12

34

56

7

11

2 2

1 31

41

51

6 1

7 18

19

1

MET

1

2

3MTT

123

MCT

1

2

34

11 10

1 111 12

113

1

21

2

14 115 1

161

.htO

1

MET1

MTT 123

MCT

1

2

3

45

1

23

4

METMTT

MCT

12

3

45

67 8 9 10

1112

13

1

23

4

METMTT

MCT

1

1

23

4

METMTT

MCT

1

1

23

4

METMTT

MCT

1

1

23

4

METMTT

MCT

12

3

4

56

789

1

23

4

METMTT

MCT

12

3

45

6

1

23

4

METMTT

MCT

1

61

2 7

1 51 2 3

4

58

19

1

MET

1

2

3

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


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


TRBV5−6

TRBV5−7

TRBV6−1

TRBV6−2

TRBV6−5

TRBV6−6

TRBV7−2


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.

CASRRAWRGALSNSPLHF

tcr sequencing reveals clonal expansions of ......5 comp-bv421-a 0-10 3 10 3 10 4 10 5...

Documents