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Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function

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Science Immunology  11 Aug 2017:
Vol. 2, Issue 14, eaan1487
DOI: 10.1126/sciimmunol.aan1487
  • Fig. 1 Blood Tfr cells are indicators of ongoing humoral activity.

    (A) Frequency of total Treg cells, Tfr cells, and CXCR5 Treg cells in peripheral blood of SS patients and age-matched HDs (n = 25; unpaired Student’s t test with Welch’s correction for variance). Representative plots (left) and pooled data (right) are shown. (B) Blood Tfr/Tfh ratio in SS patients and HDs (n = 25; unpaired Student’s t test with Welch’s correction for variance). (C) Blood Tfr/Tfh ratio in SS patients with and without serum autoantibodies (AAb) (anti-SSA/Ro52, anti-SSB/Ro60, and anti-SSB/La) (n = 25; unpaired Student’s t test). (D) Variation of blood Tfr/Tfh ratio according to C-reactive protein (CRP). Analysis by linear regression was used. (E) Variation of blood Tfr/Tfh ratio according to disease severity score (ESSDAI). Analysis by linear regression was used. Bars represent SEM (A and C) or minimum and maximum values (in box and whisker graphs) (C).

  • Fig. 2 Blood Tfr cells show expression of follicular and regulatory markers.

    (A) CXCR5+ Tfr cells constitute 18.57 ± 6.55% of Treg cells (left) and 0.93 ± 0.56% of total CD4+ T cells (middle), representing 9985 ± 9043 cells per milliliter of blood (right) (n = 42; adult HDs). (B) Variation of blood Tfr cell frequency (left) and absolute number per milliliters of blood (right) according to age (age range, 22 to 92 years) (n = 42; linear regression). (C) Expression of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1, Bcl-6, and CD57 by Tfh cells (blue), CXCR5 Treg cells (black), and Tfr cells (red) in children blood (top) and tonsils (bottom). Naïve CD4+ T cells were used as control (gray). Representative plots from six healthy children are shown. CXCR5+ subsets in tonsils were defined as CXCR5+ICOS+ cells (fig. S2B). Tconv, conventional T cells. (D) Immunofluorescence microscopy of formalin-fixed, paraffin-embedded human tonsils stained for DAPI (blue), CXCR5 (yellow), CD4 (red), and Foxp3 (green). Top, middle, and bottom outlined areas indicate top, middle, and bottom enlarged areas on the right, respectively. Data are representative of tonsil sections from five healthy children. (E) Blood Tfr/Tfh ratio in adult blood, children blood (tonsil donors), and tissues (tonsils). Black and red dots represent blood and tonsil results, respectively (n = 42 for adults and n = 6 for children; Student’s t test). Error bars represent SEM.

  • Fig. 3 Blood Tfr cells are a distinct subset of suppressive Treg cells.

    (A) Schematic representation of in vitro suppression assay. FACS-sorted 25 × 103 CXCR5CD25CD127+CD4+ Tconv cells were cocultured with 25 × 103 CXCR5CD25+CD127CD4+ Treg cells or CXCR5+CD25+CD127CD4+ Tfr cells under stimulation by anti-CD3 (1 μg/ml) in the presence of 105 irradiated (25 Gy) allo-PBMCs. After 5 days, responder cells were analyzed for CTV dilution by flow cytometry. Sorting strategy is described in fig. S2 (A and B). (B) Proliferation of Tconv cells without Treg cells or in the presence of either CXCR5 Treg or Tfr cells. Representative plots (left) and pooled data (right) (n = 3, each with technical triplicates; one-way ANOVA with posttest Turkey’s multiple comparisons) are shown. ns, not significant. (C) Suppression curve of CXCR5 Treg and Tfr cells in different ratios using the same conditions described in (A) and (B) (n = 1, with technical triplicates; two-way ANOVA). (D) Stability of Foxp3 expression by sorted CXCR5 Treg cells and CXCR5+ Tfr cells after 5 days of in vitro culture under αCD3/αCD28 (1 μl per well) stimulation. Percentage (left) and cell number (right) (n = 5, each with technical triplicates; Student’s t test) are shown. (E) Relative expression of Foxp3 and CXCR5 by sorted Tconv cells, Tfh cells, CXCR5 Treg cells, and Tfr cells from blood by real-time reverse transcription PCR. Gene expression was normalized to housekeeping genes (B2M, G6PD, and ACTB) (n = 2, each with technical duplicates; Student’s t test). (F) Expression of Foxp3, CD25, CTLA-4, and CXCR5 by sorted CXCR5 Treg and Tfr cells at baseline [day 0 (d0)] and after 5 days of in vitro culture under αCD3/αCD28 (1 μl per well) stimulation. Data are representative histograms of three independent experiments, each one with technical triplicates. Error bars indicate SEM.

  • Fig. 4 Blood Tfr cells do not show specialized humoral regulatory capacity.

    (A) Proliferation of CXCR5+CD25CD127+CD4+ Tfh cells without Treg cells or in the presence of either CXCR5 Treg or Tfr cells after 5 days of in vitro culture as described in Fig. 3A. Representative plots (left) and pooled data (right) (n = 3, each with technical triplicates; one-way ANOVA with posttest Turkey’s multiple comparison) are shown. (B) Schematic representation of suppression coculture assay. FACS-sorted 25 × 103 CXCR5+CD25CD127+CD4+ Tfh cells (or CXCR5CD25CD127+CD4+ Tconv cells) were cocultured for 5 days with 25 × 103 CXCR5+CD25+CD127CD4+ Treg cells (or CXCR5CD25+CD127CD4+ Treg cells) under stimulation by SEB (1 μg/ml) and in the presence of 30 × 103 CD27IgD+CD19+ naïve B cells. (C) Up-regulation of CD38 and down-regulation of IgD by naïve B cells (top) and proliferation of Tfh cells by CTV dilution (bottom) without Treg cells or in the presence of either CXCR5 Treg or Tfr cells. Representative plots (left) and pooled data (right) (n = 5, each with technical triplicates; one-way ANOVA with posttest Turkey’s multiple comparisons) are shown. (D) Suppression curve of CXCR5 Treg and Tfr cells in different ratios using the same conditions described in (B) and (C) (n = 1, with technical triplicates; two-way ANOVA). (E) ELISA determination of IgA, IgM, and total IgG in supernatants after 10 days of in vitro coculture performed as described in (D) but using SEB (1 μg/ml) + SEA (10 g/ml) + SEE (10 ng/ml) + TSST-1 (10 ng/ml) as superantigen stimulation (n = 3, each with technical triplicates; one-way ANOVA with posttest Turkey’s multiple comparisons). (F) In vitro migration of 75 × 103 sorted Tconv cells, Tfh cells, CXCR5 Treg cells, and Tfr cells toward a CXCL13 gradient (2 μ/ml) expressed by chemotaxis index (n = 3, each with technical triplicates; one-way ANOVA with posttest Turkey’s multiple comparisons). Error bars indicate SEM. SA, superantigens.

  • Fig. 5 Blood Tfr cells are immature but are not committed in the thymus.

    (A) Backgate of CXCR5 and CXCR5+ Treg cells according to CD45RO and Foxp3 expression. (B) Expression of CD45RO, CD45RA, CCR7, CD62L, HLA-DR, and CD27 by Tfr cells (red) and CXCR5 Treg cells (black) in the blood. (C) Expression of Ki-67 by CXCR5+ Treg cells and CXCR5 Treg cells in the blood (n = 22; Student’s t test). (D) CD45RO+CCR7 effector memory, CD45RO+CCR7+ central memory, and CD45ROCCR7+ naïve subsets of Tfr cells and CXCR5 Treg cells in adult blood. Representative plots (left) and pooled data (right) (n = 22; Student’s t test). Tfh cells are represented in blue, CXCR5 Treg cells in black, and Tfr cells in red. (E) Variation of CD45ROCCR7+ naïve Tfr cell and CXCR5 Treg cell frequency in blood according to age (n = 22; linear regression). (F) Expression of CXCR5 by Foxp3+CD4+ thymocytes (n = 4). (G) Expression of CXCR5 by cord blood Foxp3+CD4+ T cells (n = 3). (H) Expression of CD45RO, CCR7, and ICOS by cord blood Treg cells (n = 3). Bars represent SEM.

  • Fig. 6 Blood Tfr cells are lymphoid tissue–derived Tfr precursors.

    (A) CD45RO+CCR7 effector memory, CD45RO+CCR7+ central memory, and CD45ROCCR7+ naïve subsets of Tfr cells and CXCR5 Treg cells in children blood (top) and tissues (bottom). Representative plots (left) and pooled data (right) (n = 6; Student’s t test) are shown. Tfh cells are represented in blue, CXCR5 Treg cells in black, and Tfr cells in red. CXCR5+ subsets in tonsils were defined as CXCR5+ICOS+ cells (fig. S2B). (B) Blood Tfh and Tfr cells from X-linked agammaglobulinemia (BTK-deficient) patients compared with sex- and age-matched HDs. Representative plots (left) and pooled data (right) (n = 5; Student’s t test) are shown. (C) Model of CXCR5+ Tfh and Treg cell generation and recirculation in humans upon antigen stimulation. Tfh cells are shown in red, and Tfr cells in blue. DC, dendritic cell. (D) Frequency of peripheral blood Tfr cells on the day of influenza vaccination (d0) and 7 days later in healthy volunteers. Schematic representation and representative plots (left) and pooled data (right) (n = 32; Student’s t test) are shown. Bars represent SEM.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/14/eaan1487/DC1

    Table S1. Clinical characteristics of 25 patients with primary (pSS) and secondary (sSS) Sjögren syndrome.

    Fig. S1. Gating strategy for Tfh cells, CXCR5 Treg cells, and Tfr cells in human blood.

    Fig. S2. Variation of blood Tfh cells and total Treg cells according to age.

    Fig. S3. Sorting strategy for human blood naïve B cells, Tfh cells, CXCR5 Treg cells, and Tfr cells.

    Fig. S4. Representative plots of CXCR5 Treg and Tfr cell suppression curves for coculture assay.

    Fig. S5. Expression of CD45RO, CD45RA, and CD31 by human blood Tfh and Tfr cells.

  • Supplementary Materials

    Supplementary Material for:

    Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function

    Valter R. Fonseca, Ana Agua-Doce, Ana Raquel Maceiras, Wim Pierson, Filipa Ribeiro, Vasco C. Romão, Ana Rita Pires, Susana Lopes da Silva, João Eurico Fonseca, Ana E. Sousa, Michelle A. Linterman, Luis Graca*

    *Corresponding author. Email: lgraca{at}medicina.ulisboa.pt

    Published 11 August 2017, Sci. Immunol. 2, eaan1487 (2017)
    DOI: 10.1126/sciimmunol.aan1487

    This PDF file includes:

    • Table S1. Clinical characteristics of 25 patients with primary (pSS) and secondary (sSS) Sjögren syndrome.
    • Fig. S1. Gating strategy for Tfh cells, CXCR5 Treg cells, and Tfr cells in human blood.
    • Fig. S2. Variation of blood Tfh cells and total Treg cells according to age.
    • Fig. S3. Sorting strategy for human blood naïve B cells, Tfh cells, CXCR5 Treg cells, and Tfr cells.
    • Fig. S4. Representative plots of CXCR5 Treg and Tfr cell suppression curves for coculture assay.
    • Fig. S5. Expression of CD45RO, CD45RA, and CD31 by human blood Tfh and Tfr cells.

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