T-bet is required for optimal proinflammatory CD4+ T-cell trafficking

Abstract

Inflammatory responses are controlled by T helper 1 (Th1) lymphocytes. An important function of this polarity is the ability of T cells to traffick appropriately in vivo. This differential trafficking is dependent upon the binding of P-selectin glycoprotein ligand-1 to P- and E-selectin on inflamed endothelium as well as the expression of specific chemokine receptors. Here we show that in the absence of T-box expressed in T cells (T-bet), selective migration of T cells in vivo is completely abrogated and that T-bet regulates the binding of CD4(+) T cells to P-selectin. T-bet is also required for the expression of the chemokine receptor CXCR3. Thus, T-bet controls Th1-cell migration to inflammatory sites, which has fundamental consequences for the control of immunologic disease.

Figure 1.

In vivo trafficking of adoptively transferred WT and T-bet^–/– antigen-specific T cells activated under Th1-polarizing conditions. (A-B) Flow cytometric analysis of WT (DO11.10) and T-bet^–/– (DO11.10 × T-bet^–/–) CD4⁺ T cells. Percentages of cells positive for CD4 and the clonotypic antibody KJ1-26 are indicated in various secondary lymphoid organs and in inflamed peritoneum (ILN indicates inguinal lymph nodes; MLN, mesenteric lymph nodes; and PL, peritoneal lavage). (C) Cell counts of secondary lymphoid organs and peritoneal lavage from BALB/c mice adoptively transferred with DO11.10 (WT) and DO11.10 × T-bet^–/– (T-bet^–/–) CD4⁺ T cells (mean ± SEM, *P < .001) activated with OVA peptide and mitomycin C–treated syngeneic splenocytes. □ indicates WT; ▪, T-bet^–/–.

Figure 2.

Mechanistic analysis of the selectin binding properties of WT, T-bet^–/–, DO11.10, and DO11.10 × T-bet^–/– primary CD4⁺ T cells under conditions of shear flow. (A-B) Interaction of primary CD4⁺ T cells of different genotypes with immobilized P-selectin (left columns) and E-selectin (right columns) under conditions of laminar shear stress as indicated. (A) Wild-type (WT, □) and T-bet^–/– (▪) CD4⁺ Th1 cells activated by polyclonal stimulation with CD3 and CD28 antibodies. (B) CD4⁺ T cells generated from DO11.10 and DO11.10 × T-bet^–/– TCR-Tg animals and activated in an antigen-specific manner. (C-D) Interaction of WT (□) and T-bet^–/– (▪) Th1 and Th2 cells, activated with plate-bound CD3 and CD28 antibodies, with (C) immobilized P-selectin and (D) E-selectin under conditions of laminar shear stress as indicated. All data are expressed as mean ± SEM.

Figure 3.

Characterization of a transgenic mouse expressing T-bet under the human CD2 promoter. (A) Western blot of T-bet expression by CD4⁺ T cells from either WT BALB/c (WT) or T-bet CD2-transgenic (TG) mice (also on the BALB/c background) activated with anti-CD3 and anti-CD28 antibodies under different polarizing conditions. (B-D) Cytokine profiles measured by enzyme-linked immunosorbent assay (ELISA) secreted by CD4⁺ T cells activated under different polarizing conditions. (B) IFN-γ production during primary stimulation from WT (□) and TG (▪) T cells. (C) IFN-γ production during secondary stimulation from WT and TG T cells. (D) IL-4 production during secondary stimulation from WT and TG T cells. (E-F) Interaction of WT and TG CD4⁺ T cells with immobilized P-selectin (left columns) and E-selectin (right columns) under conditions of laminar shear stress as indicated. Cells were by stimulated with plate-bound CD3 and CD28 antibodies in the presence of appropriate skewing cytokines. (E) CD4⁺ Th1-cell interactions. (F) Th2-cell interactions. All data are expressed as mean ± SEM.

Figure 4.

Posttranslational modification of selectin ligands. (A) Flow cytometric surface staining of CD43a (top), CD43c (middle), and PSGL-1 (bottom) on WT and T-bet^–/– T cells activated under Th1- or Th2-polarizing conditions (gated on live CD4⁺ cells; black = isotype, red = WT, and green = T-bet^–/–). (B) Real-time PCR analysis of mRNA levels of FucTVII expressed in CD4⁺ T cells under Th1- and Th2-polarizing conditions (normalized to β-actin). (C) Real-time PCR analysis of mRNA levels of TPST-1 and TPST-2 expressed in CD4⁺ T cells under Th1-polarizing conditions (normalized to β-actin). □ indicates WT; ▪, T-bet^–/–. (D) Autoradiograph of ³⁵S incorporation into PSGL-1, with or without removal of O- and N-linked glycans. (E) Western blot for PSGL-1 in WT and T-bet^–/– (knock out [KO]) CD4⁺ T cells activated under Th1-polarizing conditions (top panel = PSGL-1 dimer; bottom panel = PSGL-1 monomer). (F) Quantification of protein expression of PSGL-1 either with or without removal of O- and N-linked glycans. Results are expressed as mean ± SEM. WB indicates Western blot.

Figure 5.

Analysis of chemokine receptor expression in the context of altered T-bet levels in primary CD4⁺ T cells. Real-time PCR analysis (A,C) and flow cytometric (B,D) surface staining of mRNA levels of CXCR3 in T cells activated under different polarizing conditions (mean ± SEM). (A-B) WT and T-bet^–/– CD4⁺ T cells. (C-D) WT and T-bet CD2-Tg CD4⁺ T cells. (E) Real-time PCR analysis of CXCR3 expression in primary T-bet^–/– and T-bet^–/– × IFN-γ^–/– CD4⁺ T cells retrovirally transduced with empty retrovirus (RV) or T-bet RV (mean ± SEM). (F) Flow cytometric surface staining of CXCR3 in retrovirally transduced T cells. (G) mRNA levels of CCR5 in WT and T-bet^–/– T cells. All real-time PCR results are normalized to β-actin and are expressed as mean ± SEM (*P < .01). Shaded areas represent isotype staining.

Figure 6.

Responses of primary CD4⁺ T cells to chemokine ligands in the context of altered T-bet levels. (A-C) Transmigration of CD4⁺ T cells in response to recombinant chemokine in the lower chamber of a transwell. (A-B) Chemotaxis of WT, T-bet^–/–, and T-bet^–/– × IFN-γ^–/– T cells to recombinant chemokines. (A) Chemotactic response to CXCL11 (I-TAC, 100 nM). □ indicates WT; ▪, T-bet ^–/–; , T-bet^–/–/IFN-γ^–/–.(B) Chemotactic response to CCL4 (MIP-1β, 10 nM). □ indicates WT; ▪, T-bet^–/–; , T-bet^–/–/IFN-γ^–/–. (C) Chemotaxis of retrovirally transduced T-bet (or empty vector control) into T-bet^–/– and T-bet^–/– × IFN-γ^–/– T cells to CXCR3 ligands, CXCL11 (100 nM), and CXCL10 (IP-10, 100 nM). □ indicates empty virus; ▪, T-bet^–/–; , T-bet^–/–/IFN-γ^–/–. (D) Attachment of WT and T-bet^–/– T cells to unstimulated endothelial cells under shear flow in the absence or presence of CXCL10 (40 ng/mL) (mean ± SEM). □ indicates control; ▪, CXCL10. **P < .05.