title: Cytokine-Independent Detection of Antigen-Specific Germinal Center T Follicular Helper Cells in Immunized Nonhuman Primates Using a Live Cell Activation-Induced Marker Technique.
abstract: A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein-immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function.
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037;
authors.forename: Colin
authors.initials: C
authors.lastname: Havenar-Daughton
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037;
authors.forename: Samantha M
authors.identifier: 0000-0001-5273-0898
authors.initials: SM
authors.lastname: Reiss
authors.affiliation: Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322;
authors.forename: Diane G
authors.initials: DG
authors.lastname: Carnathan
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037;
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Wu
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037;
authors.forename: Kayla
authors.identifier: 0000-0002-6215-9522
authors.initials: K
authors.lastname: Kendric
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;
authors.forename: Alba
authors.initials: A
authors.lastname: Torrents de la Peña
authors.affiliation: Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322;
authors.forename: Sudhir Pai
authors.identifier: 0000-0002-6421-2040
authors.initials: SP
authors.lastname: Kasturi
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Division of Infectious Diseases, University of California San Diego, La Jolla, CA 92093;
authors.forename: Jennifer M
authors.identifier: 0000-0002-1672-0657
authors.initials: JM
authors.lastname: Dan
authors.affiliation: Department of Surgery, University of California San Diego, San Diego, CA 92123; and Pediatric Otolaryngology, Rady Children's Hospital-San Diego, San Diego, CA 92123.
authors.forename: Marcella
authors.identifier: 0000-0001-9771-6221
authors.initials: M
authors.lastname: Bothwell
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;
authors.forename: Rogier W
authors.initials: RW
authors.lastname: Sanders
authors.affiliation: Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322;
authors.forename: Bali
authors.initials: B
authors.lastname: Pulendran
authors.affiliation: Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037; Yerkes National Primate Research Center, Emory University, Atlanta, GA 30322; Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322;
authors.forename: Guido
authors.initials: G
authors.lastname: Silvestri
authors.affiliation: Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037; shane@lji.org.
authors.forename: Shane
authors.identifier: 0000-0002-6484-6262
authors.initials: S
authors.lastname: Crotty
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis; D004195:Disease Models, Animal; D005434:Flow Cytometry; D018858:Germinal Center / Q000166:cytology / Q000276:immunology; D015658:HIV Infections / Q000276:immunology*; D006801:Humans; D007158:Immunologic Techniques / Q000379:methods*; D008253:Macaca mulatta; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*
chemical_list: D015415:Biomarkers; D016207:Cytokines