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. 2016 Aug 1;197(3):983-93.
doi: 10.4049/jimmunol.1600318. Epub 2016 Jun 24.

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

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Free PMC article

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

Jennifer M Dan et al. J Immunol. .
Free PMC article

Abstract

Detection of Ag-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 Ag-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 from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining 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 Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus-specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4(+) T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine-specific CD4(+) T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers.

Figures

FIGURE 1
FIGURE 1
Limited cytokine production by GC Tfh cells after antigen stimulation. (A) Representative flow cytometry plot of GC Tfh (CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh (CXCR5CD45RACD4+) cell gating with representative flow cytometry plots of IL-21 production by live GC Tfh cells from 3 different tonsils. (B) Median intracellular cytokine production of TNF+IL-21+, TNF+IFNγ+, and TNF+CD40L+ by Strep-specific GC Tfh, mTfh, and non-Tfh cells. Representative FACS plots following 18-hour culture of tonsil cells with 10ug/mL Strep or PMA/Ionomycin, as a positive control. Limit of detection denoted by the grey dotted line. Data are from 9 donors. The response by antigen-specific cells was background subtracted for each donor. (C) Median intracellular cytokine production of IL-4+IL-13+, IL-10+, and IL-17+ by Strep-specific GC Tfh, mTfh, and non-Tfh cells. Limit of detection denoted by the grey dotted line. Data are from 19 donors.
FIGURE 2
FIGURE 2
Proliferation of GC Tfh cells upon restimulation. (A) Representative histograms of proliferating GC Tfh, mTfh, and non-Tfh cells from 2 different tonsils following stimulation of whole tonsil cells with 100ng/mL Staphylococcal enterotoxin B (SEB). (B) Representative flow cytometry plots of proliferating GC Tfh, mTfh, and non-Tfh cells from a donor following stimulation of whole tonsil cells with 100ng/mL SEB. Cells were gated on CTV+CD25+ or CTV+OX40+. Data are representative of a total of 11 samples from 2 independent experiments. (C) Representative flow cytometry plots of sorted GC Tfh (live CXCR5hiPD-1hiCD45RACD4+), mTfh (CXCR5+PD-1+CD45RACD4+), and non-Tfh cells (CXCR5CD45RACD4+) from 3 different tonsils. Cells were co-cultured with autologous irradiated EBV-transformed lymphoblastoid cell lines for 96 hours, stimulated with 100ng/mL SEB, and analyzed for CTV+CD25+ or CTV+OX40+ expression. FACS plots show only 20% of collected events, for easier visualization. Data are representative of a total of 21 samples from 6 independent experiments.
FIGURE 3
FIGURE 3
Activation Induced Marker induction by GC Tfh cells. (A) Experimental design for human tonsillar AIM assay (B) Representative flow cytometry plots of CD25+OX40+ upregulation by live GC Tfh cells (CXCR5hiPD-1hiCD45RACD4+) from 3 different tonsils following 18 hours stimulation with 1μg/mL Staphylococcal enterotoxin B (SEB). (C) Median CD25+OX40+ expression by live GC Tfh cells following stimulation with SEB. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (D) Comparison of Strep-specific GC Tfh by ICS (TNF+CD40L+) and AIM (CD25+OX40+). The % Strep-specific GC Tfh responses were background subtracted. Data are from 9 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (E) CD25+OX40+ GC Tfh cells are also PD-L1+. Representative FACS plots of CD25+PD-L1+ co-expression following stimulation with either Strep or SEB. Strep-specific PD-L1+CD4+ GC Tfh cells (black contour plot) were overlaid onto Strep-specific CD25+OX40+ GC Tfh cells (grey dots). SEB responsive PD-L1+CD4+ GC Tfh cells (black contour plot) were overlaid onto SEB responsive CD25+OX40+ GC Tfh cells (grey dots). Data are from 14 samples from 2 independent experiments. (F). Representative flow cytometry overlay plots demonstrating CD25+OX40+ cells (black dots) within each sorted population (grey dots) for each condition (Strep-stimulated, and SEB-stimulated). This is representative of 3 independent experiments consisting of 7 donors.
FIGURE 4
FIGURE 4
Activation Induced Marker induction by mTfh and non-Tfh cells. (A) Representative flow cytometry plots of CD25+OX40+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) following stimulation with Strep or 1μg/mL SEB. Median CD25+OX40+ expression by live mTfh following stimulation with Strep. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (B) Representative flow cytometry plots of and CD25+PD-L1+ upregulation by live mTfh (CXCR5+PD-1+CD45RACD4+) following stimulation with Strep or 1μg/mL SEB. Median CD25+PD-L1+ expression by live mTfh following stimulation with Strep. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (C) Representative flow cytometry plots of CD25+OX40+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+OX40+ expression by live non-Tfh following stimulation with Strep. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor. (D). Representative flow cytometry plots of CD25+PDL1+ upregulation by non-Tfh cells (CXCR5CD45RACD4+). Median CD25+PD-L1+ expression by live non-Tfh following stimulation with Strep. Responses were background subtracted. Data are from 14 samples from 2 independent experiments. The response by antigen-specific cells was background subtracted for each donor.
FIGURE 5
FIGURE 5
Detection of EBV/CMV-specific CD4+ T cells in peripheral blood. (A) Three individuals were tested by AIM for response to a Class II EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RO+CD4+), naïve CD4+ T cells (CD45ROCD4+), memory CD8+ T cells (CD45RO+CD8+), and naïve CD8+ T cells (CD45ROCD8+) were quantified. (**, p=0.0079). (B) An LTBI DRB5*01:01 donor with defined DRB5*01:01 Mtb epitope was stimulated with Mtb and EBV/CMV peptide pool. AIM+ (CD25+OX40+) memory CD4+ T cells were quantified. (C) Representative FACS plot demonstrates that EBV/CMV-specific CD4+ T cells (CD25+OX40+) were not positive for Mtb-tetramer (black dots).
FIGURE 6
FIGURE 6
Detection of Mtb-specific CD4+ T cells in peripheral blood. Eight QuantiFERON-TB Gold Positive patients with latent tuberculosis (LTBI) and eight QuantiFERON-TB Gold negative healthy controls (HC) were tested for response to a pool of ESAT-6 or CFP10 epitopes. (A) IFNγ ELISPOT of PBMCs from LTBI patients and healthy controls (*, p=0.0035 for ESAT-6, *, p=0.013 for CFP10). Limit of detection denoted by the grey dotted line. (B) AIM assay (CD25+OX40+) of PBMCs from LTBI patients and healthy controls similarly had higher CD25+OX40+ expression by AIM assay (*, p=0.027 for ESAT-6, ****, p<0.0001 for CFP10). Limit of detection denoted by the grey dotted line. Representative flow cytometry plots of CD25+OX40+ by an LTBI patient and a HC patient.
FIGURE 7
FIGURE 7
Detection of dengue-specific CD4+ T cells in peripheral blood. Representative FACS plots of CD4+ and CD8+ T cells following stimulation with dengue peptides or PHA, as a positive control. Cumulative data from five dengue seropositive patients (DENV+) and five dengue seronegative patients (DENV) with known expression of the HLA DRB1*0401 molecule. PBMCs were stimulated with dengue DRB1*0401 restricted peptide for 24 hours. (*, p=0.016). (**, p=0.0079).
FIGURE 8
FIGURE 8
Detection of pertussis-specific CD4+ T cells in peripheral blood using a pertussis peptide megapool. (A). AIM+ (CD25+OX40+) memory CD4+ T cells (CD45RACCR7+CD4+) naïve CD4+ T cells (CD45RA+CCR7+CD4+), memory CD8+ T cells (CD45RACCR7+CD8+), and naïve CD8+ T cells (CD45RA+CCR7+CD8+) were quantified. (****, p<0.0001). (B). CD25+PD-L1+ memory CD4+ T cells were quantified (****, p<0.0001) in ten individuals. (C). Quantification of AIM+ (CD25+OX40+) pertussis-specific within CD4+ T cell subsets. Pertussis-specific cells were predominantly in the CD45RACCR7 (**, p=0.0079). (D). Comparison of AIM+ (CD25+OX40+) pertussis-specific and ELISPOT pertussis-specific responses in individuals pre and post acellular pertussis booster vaccination.

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