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Dale and Betty Bumpers Vaccine Research Center

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.

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Dale and Betty Bumpers Vaccine Research Center

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  1. 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

  2. Bispecific Antibody Concept HIV-expressing CD4 T cell Bispecific antibody CD8 T cell (not HIV-specific) VRC07 Fab anti-CD3 scFv Redirected lysis HIV Env CD3 (Gly4Ser1)3 Linker -C N- VH VL VL CL S S VH CH1 N- -C Amar Pegu

  3. Germinal Center TFH: Major Source of Active and Inducible HIV Replication HIV DNA copies/106 cells Highest copy number of HIV DNA PD-1 CXCR5 PD-1 CXCR5 Source of inducible HIV replication Perreau et al, J Exp Med, 2013

  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. CCR7loCXCR5hi (follicular) CD8 T cells accumulate in HIV+ LNs *p < 0.05 **p < 0.001

  8. CD8 T cells in human LN CD20 CD4 CD8 CD20CD4CD8 CD20 CD8 CD20 CD8 CXCR5 Tonsil HIV- LN HIV+ LN

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

  10. Follicular CD8 T cells express cytolyticpotential CD3/CD28/CD2 Beads 5h stimulation Newly formed Ex vivo

  11. Function CD20 CD8 GrzB CD8GrzB CD8GrzBCD20 HIV- LN HIV+ LN HIV+ LN (GC)

  12. a C D 3 / VR C 0 7 aCD3/ i s o ty p e Bispecific-mediated Killing (Specificity) 10:1 Effectors:Target 8 hours Quantification of Aqua+AnexinV+ CEM CCR7lo CXCR5hi CD27hi CD45ROlo CCR7hi CXCR5lo CCR7hi CXCR5hi

  13. Bispecific-mediated Killing in HIV+ LNs

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

  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 ConstantinosPetrovas Kristin Boswell Joseph Cassaza Takuya Yamamoto David Ambrozak Irene Primmer David Kotlyar National Institute of Respiratory Diseases, Mexico City Gustavo Reyes-Teran Perla del Rio CIENI YuriaAblanedoTerrazas AmarantaRiveroArrieta Hospital Civil de Guadalajara  Luz Alicia González Jaime Andrade Villanueva Virology Laboratory Amar Pegu MangaiAsokan John Mascola Laboratory of Immunovirology Sevilla, Spain Manuel Leal Ezequiel Ruiz-Mateos Laboratory of Systems Biology NIAID Michael Gerner Ronald Germain Children’s National Hospital, DC Patients and donors

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