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