Post on 23-Feb-2016
description
Dale and Betty Bumpers
Vaccine Research CenterNational Institute of Allergy and Infectious DiseasesNational Institutes of Health
Richard A. Koup, MDJuly 19, 2014
CD8 T cells in germinal centers are functionally capable of mediating
bispecific antibody mediated killing
Bispecific Antibody ConceptHIV-expressing CD4 T cell CD8 T cell (not HIV-specific)Bispecific antibody
HIV Env CD3Redirected lysis
VRC07Fab
anti-CD3scFv
N-
N-
-C
-C
(Gly4Ser1)3 Linker
VL CL
VH CH1
VH VLSS
Amar Pegu
Germinal Center TFH: Major Source of Active and Inducible HIV Replication
Perreau et al, J Exp Med, 2013
Highest copy number of HIV DNA
PD-1
CXCR5HI
V DN
A co
pies
/106 c
ells
PD-1CXCR5
Source of inducible HIV replication
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 HIV-infected human LN
* p < 0.05** p < 0.001
* p < 0.05** p < 0.001
CCR7loCXCR5hi (follicular) CD8 T cells accumulate in HIV+ LNs
CD8 T cells in human LNCD20 CD4 CD8 CD20 CD4 CD8
Tonsil
HIV- LN
HIV+ LN
CD20 CD8 CD20 CD8CXCR5
Quantification of GC CD4 and CD8 T cellsHIV-
CD4
CD8
GC defined as Ki67+CD20+
CD8CD4Ki67 + CD20CD20
CD4
CD8
HIV+
Michael Gerner
Follicular CD8 T cells express cytolytic potential
CD3/CD28/CD2 Beads5h stimulation
Ex vivoNewly formed
FunctionCD20 GrzBCD8 CD8 GrzB
HIV- LN
HIV+ LN
CD8 GrzB CD20
HIV+ LN(GC)
Bispecific-mediated Killing (Specificity)
10:1 Effectors:Target8 hours
Quantification of Aqua+AnexinV+ CEM
CD27hi
CD45ROloCCR7hi
CXCR5loCCR7hi
CXCR5hi
CCR7lo
CXCR5hi
aCD3/VRC07aCD3/isotype
Bispecific-mediated Killing in HIV+ LNs
Caspase inhibitor Supernatants
Bispecific-mediated Killing (Mechanism)
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 HIV-infected 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
AcknowledgmentsImmunology LaboratorySara Ferrando-MartinezConstantinos Petrovas
Kristin BoswellJoseph Cassaza
Takuya YamamotoDavid AmbrozakIrene PrimmerDavid Kotlyar
Virology LaboratoryAmar Pegu
Mangai AsokanJohn Mascola
Laboratory of Systems Biology NIAID
Michael GernerRonald Germain
Children’s National Hospital, DCPatients and donors
Laboratory of ImmunovirologySevilla, Spain
Manuel LealEzequiel Ruiz-Mateos
National Institute of Respiratory Diseases, Mexico City
Gustavo Reyes-TeranPerla del Rio
CIENIYuria Ablanedo TerrazasAmaranta Rivero Arrieta
Hospital Civil de Guadalajara Luz Alicia González
Jaime Andrade Villanueva