A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood

Abstract

Detection of antigen-specific CD4+ T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of antigen-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be ‘stingy’ cytokine producers, fundamentally different than Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of antigen-specific cells by intracellular cytokine staining (ICS) relies on the ability of the CD4+ T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation induced marker (AIM) methodology to identify antigen-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus (Strep)-specific GC Tfh cells produced minimal detectable cytokines by ICS, the AIM method identified 85-fold more antigen-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 (PD-L1) upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare antigen-specific CD4+ T cells in human peripheral blood. Dengue-, tuberculosis-, and pertussis-vaccine-specific CD4+ T cells were readily detectable by AIM. In sum, cytokine assays missed 98% of antigen-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by co-expression of TCR-dependent activation markers.

Waning Immunity and Microbial Vaccines—Workshop of the National Institute of Allergy and Infectious Diseases

“…Alessandro Sette (La Jolla, CA, USA) explored the mechanism of waning immunity by comparing human T cell responses to ACV versus WCV at least 17 years after the original priming and following repeated ACV boosts (21)(22)(23). By studying ex vivo responses and a "pertussis megapool" of defined human T cell epitopes, 601 epitopes were defined.…”

Comment on “A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood”

“…1 T cell responses to Mycobacterium tuberculosis Ags (2). Similarly, we also note that in a parallel publication, the same group has used the OX40 assay to detect Ag-specific CD4…”

“…Levings, unpublished data) providing a simple and robust method to obtain viable Ag-specific cell populations for functional analysis even when present at low levels (2,7,8). Furthermore, the frequency and phenotype of these responses have been associated with the effectiveness of immune responses and response to therapy in several diseases (7, 10, 11).…”

“…Furthermore, the frequency and phenotype of these responses have been associated with the effectiveness of immune responses and response to therapy in several diseases (7, 10, 11). A technique to efficiently identify Ag-specific GC Tfh cells had not been available, due to intrinsically stingy cytokine production by human and non-human primate GC T follicular helper (Tfh) cells, as described in our two papers (1,2). Since this has been a major impediment for the field, we performed an RNA-seq experiment to identify non-cytokine proteins that are upregulated by GC Tfh cells in response to Ag.…”

Strategies to Study T Cells and T Cell Targets in Allergic Disease

“…As an alternative to CD154, other activation markers, such as Ox40 and CD25 have also been used to detect and isolate antigen-specific cells after short-term antigen stimulation [46]. The main advantage of this approach is that both Ox40 and CD25 are stably expressed on the cell surface and therefore cells can be detected and isolated in viable form without the need of fixation or permeabilization.…”