Dale and Betty Bumpers Vaccine Research Center National

Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health CD 8 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 CD 4 T cell Bispecific antibody CD 8 T cell (not HIV-specific) VRC 07 anti-CD 3 Fab (Gly Ser ) Linker sc. Fv 4 NN- HIV Env Amar Pegu VL VH CL CH 1 S S 1 3 VH VL -C -C Redirected lysis CD 3

Highest copy number of HIV DNA copies/106 cells Germinal Center TFH: Major Source of Active and Inducible HIV Replication PD-1 CXCR 5 PD-1 Source of inducible HIV replication CXCR 5 Perreau et al, J Exp Med, 2013

CD 8 CTL are Rare in Germinal Centers 2007

Objectives • Evaluate the distribution of CD 8 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 CD 8 T cells to mediate bispecific antibody-directed killing of HIV-infected CD 4 T cells – In comparison to CD 8 T cells in other LN zones

Memory CD 8 T cells accumulate in HIVinfected human LN * p < 0. 05 ** p < 0. 001

CCR 7 lo. CXCR 5 hi (follicular) CD 8 T cells accumulate in HIV+ LNs * p < 0. 05 ** p < 0. 001

CD 8 T cells in human LN CD 20 CD 4 CD 8 CD 20 CD 8 CXCR 5 Tonsil HIV- LN HIV+ LN

Quantification of GC CD 4 and CD 8 T cells HIV+ HIV- GC defined as Ki 67+CD 20+ CD 8 CD 4 Ki 67 + CD 20 CD 4 Michael Gerner

Follicular CD 8 T cells express cytolytic potential CD 3/CD 28/CD 2 Beads 5 h stimulation Newly Ex vivoformed

Function CD 20 CD 8 Grz. B CD 20 HIVLN HIV+ LN (GC)

Bispecific-mediated Killing (Specificity) a. CD 3/VRC 07 a. CD 3/isotype 10: 1 Effectors: Target 8 hours CD 27 hi CD 45 ROlo Quantification of Aqua+Anexin. V+ CEM CCR 7 hi CXCR 5 lo CCR 7 hi CXCR 5 hi CCR 7 lo CXCR 5 hi

Bispecific-mediated Killing in HIV+ LNs

Bispecific-mediated Killing (Mechanism) Caspase inhibitor Supernatants

Conclusions • Recruitment of CD 8 T cells into the B cell follicles (germinal centers) during HIV infection – Defined by high CXCR 5 and low CCR 7 by flow cytometry – Confirmed by confocal imaging • Increased cytolytic potential of CD 8 T cells in B cell follicles compared to extrafollicular CD 8 T cells, especially in HIVinfected LNs – CD 107 a, granzyme, and perforin – Co-localization of granzyme and CD 8 T cells on confocal imaging • CD 8 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