1. Dale and Betty Bumpers Vaccine Research Center National Institute of Allergy and Infectious Diseases National Institutes of Health Richard A. Koup, MD July 19, 2014 CD8 T cells in germinal centers are functionally capable of mediating bispecific antibody mediated killing

2. Bispecific Antibody Concept HIV-expressing CD4 T cellCD8 T cell (not HIV-specific)Bispecific antibody HIV EnvCD3 Redirected lysis VRC07 Fab anti-CD3 scFv N- -C (Gly 4 Ser 1 ) 3 Linker VLCL VHCH1 VHVL S S Amar Pegu

3. Germinal Center T FH : Major Source of Active and Inducible HIV Replication Perreau et al, J Exp Med, 2013 Highest copy number of HIV DNA PD-1CXCR5 HIV DNA copies/10 6 cells PD-1 CXCR5 Source of inducible HIV replication

4. CD8 CTL are Rare in Germinal Centers 2007

5. 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

6. Memory CD8 T cells accumulate in HIV- infected human LN * p < 0.05 ** p < 0.001

7. * p < 0.05 ** p < 0.001 CCR7 lo CXCR5 hi (follicular) CD8 T cells accumulate in HIV + LNs

8. CD8 T cells in human LN CD20CD4CD8CD20 CD4 CD8 Tonsil HIV- LN HIV+ LN CD20CD8CD20 CD8CXCR5

9. Quantification of GC CD4 and CD8 T cells HIV - CD4 CD8 GC defined as Ki67+CD20+ CD8 CD4 Ki67 + CD20 CD20 CD4 CD8 HIV + Michael Gerner

10. Follicular CD8 T cells express cytolytic potential CD3/CD28/CD2 Beads 5h stimulation Ex vivoNewly formed

11. Function CD20GrzBCD8CD8 GrzB HIV - LN HIV + LN CD8 GrzB CD20 HIV + LN (GC)

12. Bispecific-mediated Killing (Specificity) 10:1 Effectors:Target 8 hours Quantification of Aqua+AnexinV+ CEM CD27 hi CD45RO lo CCR7 hi CXCR5 lo CCR7 hi CXCR5 hi CCR7 lo CXCR5 hi aCD3/VRC07 aCD3/isotype

13. Bispecific-mediated Killing in HIV + LNs

14. Caspase inhibitor Supernatants Bispecific-mediated Killing (Mechanism)

15. 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

16. 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 Children’s National Hospital, DC Patients and donors Laboratory of Immunovirology Sevilla, Spain Manuel Leal Ezequiel Ruiz-Mateos 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