Document

Report
Dale and Betty Bumpers
Vaccine Research Center
National Institute of Allergy and Infectious Diseases
National Institutes of Health
CD8 T cells in germinal centers are
functionally capable of mediating
bispecific antibody mediated killing
Richard A. Koup, MD
July 19, 2014
Bispecific Antibody Concept
HIV-expressing CD4 T cell
Bispecific antibody
VRC07
Fab (Gly Ser )
4
NN-
HIV Env
Amar Pegu
VL
1 3
anti-CD3
Linker scFv
VH
CL
CD8 T cell (not HIV-specific)
VL
-C
S
S
VH
CH1
-C
Redirected lysis
CD3
Highest copy
number of HIV DNA
HIV DNA copies/106 cells
Germinal Center TFH: Major Source of
Active and Inducible HIV Replication
PD-1
CXCR5
PD-1
Source of inducible
HIV replication
CXCR5
Perreau et al, J Exp Med, 2013
CD8 CTL are Rare in Germinal Centers
2007
Objectives
• Evaluate the distribution of CD8 T cells in T
and B cell zones of lymph nodes and tonsils
– Frequency
– Phenotype
– Changes with HIV infection?
• Determine ability of B cell zone CD8 T cells to
mediate bispecific antibody-directed killing of
HIV-infected CD4 T cells
– In comparison to CD8 T cells in other LN zones
Memory CD8 T cells accumulate in HIVinfected human LN
Aqualow
3
0
50K
0
4
104
10
3
0
0 10
50K 100K 150K 200K 250K
10
2
10
3
10
4
10
5
10
2
10
2
104
103
0
10
3
10
4
10
5
0
*
80
60
40
10
3
10
4
5
10
CD4
CD3
***
100
3
5
0
0
SSC
FSC-A
10
10
0
10
3
10
4
10
5
CD45RO
***
100
80
60
40
20
0
T
HIVLN
HIV+
LN
**
100
80
60
40
20
0
T
20
0
T
HIVLN
HIV+
LN
CD27loCD45ROhi
[% of CD8 T cells]
0
10
CD27hiCD45ROhi
[% of CD8 T cells]
10
5
CD27
100K
CD20
Aqua
104
150K
CD8 T cells
[% of CD3 T cells]
FSC-H
200K
5
10
* p < 0.05
** p < 0.001
CD45RO/CD27
CD4-CD8+
CD20-CD3+
5
CD8
10
CD27hiCD45ROlo
[% of CD8 T cells]
Singlets
250K
HIVLN
HIV+
LN
***
100
*
80
60
40
20
0
T
HIVLN
HIV+
LN
CCR7loCXCR5hi (follicular) CD8 T cells
accumulate in HIV+ LNs
10
3
0
10
3
10
4
10
10
5
105
10
4
10
4
104
103
103
0
0
0 102
5
103
104
105
CD45RO
10
3
0
0 102
103
104
105
0 10
2
10
3
10
4
CXCR5
% CCR7hiCXCR5lo
0
5
100
*
80
60
40
20
0
T
HIVLN
HIV+
LN
*
100
*
*
80
60
40
20
0
T
HIVLN
% CCR7hiCXCR5hi
4
CD27loCD45ROhi
10
% CCR7loCXCR5hi
10
CCR7
10
CD27hiCD45ROhi
CD27hiCD45ROlo
% CCR7loCXCR5lo
CD27
CD45RO/CD27
5
HIV+
LN
*
100
*
80
60
40
20
0
T
HIVLN
HIV+
LN
*
100
*
*
80
60
40
20
0
T
HIVLN
HIV+
LN
10
5
* p < 0.05
** p < 0.001
CD8 T cells in human LN
CD20
CD20
CD4
CD8
CD8
CD20 CD4 CD8
CD20 CD8
CXCR5
Tonsil
HIV- LN
HIV+ LN
Quantification of GC CD4 and CD8 T cells
+
HIV+HIV
LN
GC defined as
Ki67+CD20+
Position y
Position y
LN
HIVHIV
CD8
CD4
Ki67 + CD20
CD20
Position x
CD8
CD8
Position x
CD4
CD4
Michael Gerner
Follicular CD8 T cells express
cytolytic potential
150K
100K
10
4
CD8
Aqua
FSC-H
200K
103
50K
0
50K 100K 150K 200K 250K
FSC-A
5
10
104
10
3
10
2
0
10
2
10
3
10
4
10
5
104
10
3
0 10
2
10
3
10
4
10
0 10
5
2
10
3
10
4
10
5
CXCR5
CD4
CD3
CD3/CD28/CD2 Beads
5h stimulation
5
102
0
0
0
0
CCR7 vs CXCR5
CD4-CD8+
10
10
105
10
4
10
4
10
3
10
2
103
0
80
20
% CD107a+Perforin+
MFI Granzyme
loCXCR5hiB
(CCR7(ex
vivo) )
3
10
4
10
5
0 10
1510000
40
10
10
20
5
TT
HIVHIVLN
LN
HIV+
HIV+
LN
LN
5000
5
0
0
4
10
3
10
2
10
3
10
4
10
5
0 10
2
10
3
10
CCR7hi
CCR7lo
lo
TCXCR5
HIV- CXCR5
HIV+hi
LN
LN
2000
1500
1000
500
0
CCR7hi
CXCR5lo
4
Perforin
2500
15000
20
60
15
2
GrzB
MFI Perforin
(ex vivo)
% CD107a+Granzyme
% Granzyme B+ B+
loCXCR5hi)
(CCR7
Perforin+
(ex vivo)
10
10
0
GrzB
Newly
Ex vivoformed
0
0
105
0
0
Perf+107a+
GrzB+107a+
5
CD107a
Perforin
GrzB+Perf+
Ex vivo
CD107a
AqualoCD3+
105
CCR7
Singlets
250K
CCR7lo
CXCR5hi
10
5
Function
CD20
CD8
GrzB
CD8 GrzB
CD8 GrzB CD20
HIVLN
HIV+
LN
HIV+
LN
(GC)
% Lysi
Bispecific-mediated Killing (Specificity)
40
20
P1
0
P9
60
VRC07
control
aCD3/VRC07
aCD3/isotype
40
10:1 Effectors:Target
8 hours
Quantification ofVRC07
Aqua+AnexinV+ CEM
Isotype
control
20
CD45RO
CXCR5
P9
P8
CCR7lo lo
CD27hi hi
CCR7hi hi
CCR7hi
hi
CD27
CCR7
CCR7
CCR7
CXCR5hi
CD45ROlo lo CXCR5lo lo CXCR5hi
hi
hi
P7
0
CXCR5
P1
0
% Lysis CEM
% Aqua+AnnexinV+
P7
0
CXCR5
Bispecific-mediated Killing in HIV+ LNs
Memory markers
10
104
CD27hiCD45ROhi
5
10
103
CCR7
CCR7
104
103
0
0
103
0
104
CD45RO
50
105
5
CCR7hi
CXCR5lo
104
CCR7hi
CXCR5hi
103
CCR7lo
CXCR5hi
0
0
102
103
104
105
CXCR5
0
102
103
104
105
CXCR5
Tonsil
HIV+ LN
40
% Lysis
CD27
10
CD27hiCD45ROlo
5
30
20
10
0
CD27 hi
CD45ROlo
CCR7 hi
CXCR5lo
CCR7lo
CXCR5hi
Bispecific-mediated Killing (Mechanism)
Memory markers
10
104
CD27hiCD45ROhi
5
10
103
CCR7
104
CCR7
CD27
10
CD27hiCD45ROlo
5
103
0
0
0
103
104
105
CD45RO
5
CCR7lo
CXCR5hi
0
0
102
103
104
105
0
102
CXCR5
103
104
105
CXCR5
Supernatants
CCR7loCXCR5hi
Control
+ Z-VAD (50 uM)
0.25
80
8 hours
19 hours
0.20
60
pg/mL
% Lysis
CCR7hi
CXCR5hi
103
Caspase inhibitor
40
20
0
CCR7hi
CXCR5lo
104
0.15
0.10
0.05
CD27 hi
CD45ROlo
CCR7 hi
CXCR5lo
CCR7lo
CXCR5hi
0.00
sFasL
Granzyme B
Perforin
Conclusions
• Recruitment of CD8 T cells into the B cell follicles (germinal
centers) during HIV infection
– Defined by high CXCR5 and low CCR7 by flow cytometry
– Confirmed by confocal imaging
• Increased cytolytic potential of CD8 T cells in B cell follicles
compared to extrafollicular CD8 T cells, especially in HIVinfected LNs
– CD107a, granzyme, and perforin
– Co-localization of granzyme and CD8 T cells on confocal imaging
• CD8 T cells within the B cell follicle are capable of mediating
bispecific antibody-mediated killing of HIV-infected cells
– Caspase-dependent
– Associated with secretion of perforin and granzyme
Acknowledgments
Immunology Laboratory
Sara Ferrando-Martinez
Constantinos Petrovas
Kristin Boswell
Joseph Cassaza
Takuya Yamamoto
David Ambrozak
Irene Primmer
David Kotlyar
Virology Laboratory
Amar Pegu
Mangai Asokan
John Mascola
Laboratory of Systems Biology
NIAID
Michael Gerner
Ronald Germain
National Institute of Respiratory Diseases,
Mexico City
Gustavo Reyes-Teran
Perla del Rio
CIENI
Yuria Ablanedo Terrazas
Amaranta Rivero Arrieta
Hospital Civil de Guadalajara
Luz Alicia González
Jaime Andrade Villanueva
Laboratory of Immunovirology
Sevilla, Spain
Manuel Leal
Ezequiel Ruiz-Mateos
Children’s National Hospital, DC
Patients and donors

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