Figure 1.
TCR activation induced AR expression in human PBMC T cells.
(A) PBMC were treated as indicated and analyzed by ICS. The upper panels show the gating strategy to identify activated (CD69+) CD4 or CD8 T cells expressing AR. The lower panels show the induction of AR by different stimuli in CD4 or CD8 T cells. (B) AR and IL-2 mRNA were measured by RT-PCR in purified CD4 and CD8 T cells after activation by anti-CD3+anti-CD28 beads. Results in (A) and (B) are representative of at least three experiments.
Figure 2.
HB-EGF was also expressed by TCR activated human CD4 T cells.
(A) Purified CD4 T cells were incubated with medium alone or anti-CD3/CD28 beads. At 4 hours, the mRNA levels of EGF family members were measured by RT-PCR. * Indicates undetectable values. (B) The kinetics of HB-EGF mRNA expression were measured on purified CD4 T cells responding to anti-CD3/CD28 beads. All results are representative of three independent experiments.
Figure 3.
Release of AR from the T cell surface was blocked by the ADAM17 inhibitor TAPI-1.
(A) PBMC were stimulated with SEB in the presence or absence of TAPI-1 for variable times. After cell surface staining of AR, the percentage of CD69+AR+ cells within CD4 and CD8 T cells was analyzed. (B) Purified CD4 T cells were treated with medium alone or anti-CD3/CD28 beads with or without TAPI-1 for 24 hours. The concentration of AR in the supernatant was measured by ELISA. All results are representative of at least three experiments.
Figure 4.
Both naïve and memory human CD4 T cells expressed AR during TCR activation.
(A) PBMC were treated with medium alone or allogeneic EBV-transformed B cells for 10 hours and analyzed by ICS. The gating strategy to identify activated CD4+ and CD8+ T cells is shown. (B) AR, IL-2, IFNγ or IL-4 expression was measured in four subjects in CD45RA+ (open) and CD45RA- (solid) CD4+ and CD8+ T cells after allogeneic EBV-transformed B cell stimulation. Background values have been subtracted. (C) PBMC were treated with medium alone or SEB in the presence of TAPI-1 for 8 hours. Then six populations were sorted based on surface AR, CD69 and CD45RA expression (left). AR mRNA in each population was measured by RT-PCR (right). Results in (A) and (B) represent at least three experiments, (C) represents two experiments.
Figure 5.
Several human CD4 T cell subsets can produce AR.
(A) Allogeneic Th1 and Th2 cell lines from three subjects were stimulated with PMA + ionomycin for 6 hours. The percentage of cells expressing IFNγ, IL-4, and AR was analyzed by ICS. (B) The expression of AR and other cytokines was measured in SEB-stimulated PBMC from four subjects by ICS, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the CD154+ CD4+ T cells. The figure shows the ratio between the observed frequencies of double-producing T cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). Values represent the ratios for the double-producer combination defined by the row and column labels. Ratios above or below 1 are indicated by solid or open symbols, respectively. (C) IL-4, IFNγ and IL-2 mRNA levels were measured by RT-PCR in the sorted populations described in Figure 4C. (D) PBMC were treated with influenza H1N1 peptides or tetanus (five subjects each), or the allergens Fel d1 (solid symbols) or Der p1 (open symbols)(three subjects each). The numbers of memory CD4 T cells expressing AR and other cytokines were measured by ICS. The backgrounds (no antigen) have been subtracted. Each symbol represents one individual and the filled bar is the mean of all tested subjects. (E) CD69+ CD4+ T cells (Control_CD69+) were sorted from PBMC incubated in medium alone. CD69+IFNγ+ and CD69+IFNγ- CD4 T cells were sorted from influenza peptide-treated PBMC using the cytokine secretion assay. The mRNA levels of IFNγ and AR were measured by RT-PCR. Results in (A-C) are representative of at least three experiments, (D) represents two experiments using a total of 5 independent subjects, and (E) represents two experiments.
Figure 6.
AR is produced by T cell subsets expressing different chemokine receptors and surface markers.
PBMC were treated with medium alone, anti-CD3+ anti-CD28 antibodies, or SEB in the presence of TAPI-1 for 8 hours. Cells were stained for AR and cell-surface markers and analyzed by flow cytometry. Representative of two experiments.
Figure 7.
TCR and cAMP synergize to induce AR production in human CD4 T cells.
Purified CD4 T cells were incubated with or without TCR stimulation (anti-CD3/CD28 beads) and the cAMP agonist. (A) AR and HB-EGF mRNA expression was measured by RT-PCR. (B) The concentrations of AR in the supernatant and cell lysates were measured by ELISA. (C) Enriched CD45RA+CD45RO- (naïve) and CD45RA-CD45RO+ (memory) CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of cAMP agonist (1 ∼ 1000 µM). The concentration of AR in the supernatant at 24 hours was measured by ELISA. (D) Purified CD4 T cells were treated with medium alone, or anti-CD3/CD28 beads in the presence or absence of the cAMP-modifying agents shown. RNA was extracted at 4 hours, and AR mRNA was measured by RT-PCR. The concentration of AR in the 24-hour supernatant was measured by ELISA. (E) PBMC were treated with anti-CD3+ anti-CD28 antibodies in the presence or absence of cAMP agonist or antagonist for 8 hours. CD4 T cells were purified by cell sorting and RNA was extracted. The mRNA levels of AR and other cytokines were measured by RT-PCR. All results are representative of at least three experiments.