(A) Purified CD4 T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with IL-6 for 5 d

Cells were restimulated for another 5 d under the same condition (top) or Th1-, Th2-, or Th17-polarizing conditions (bottom). On day 10, cells were stimulated with anti-CD3 mAb and intracellular staining for indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments. (B) As positive controls, CD4 T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, or Th17-polarizing conditions for 5 d. Cells were restimulated under the same conditions for another 5 d. On day 10, cells were stimulated with anti-CD3 mAb and intracellular staining for indicated cytokines was performed.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

(A) IL-21 and -17A are produced by activated CD4 T cells under Th17-polarizing conditions

Purified CD4 T cells from lymph nodes of C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, and Th17-polarizing conditions. On day 5, 10 cells were restimulated with anti-CD3 mAb/anti-CD28 mAb for 24 h. The levels of cytokines in the culture supernatants were measured by ELISA. Data are the means ± the SD from four independent experiments. *, P < 0.01. (B) Establishment of a single-cell analysis of IL-21–producing cells. Ba/F3 cells were infected with pMX-IL-21-IRES-GFP retrovirus or control retrovirus (pMX-IRES-GFP). Nine clones of pMX-IL-21-IRES-GFP retrovirus-infected Ba/F3 cells (Ba/F3-IL-21-GFP cells) and one clone of control retrovirus-infected Ba/F3 cells (Ba/F3-GFP cells) were selected by limiting dilution. IL-21 in the culture supernatants was measured by ELISA (left). Ba/F3-GFP cells and Ba/F3-IL-21-GFP clone #6 cells were fixed, permeabilized, and incubated with IL-21R-Fc or PBS. After washing, cells were visualized with anti-Fc PE (right).<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

Purified CD4 T cells from Smad3 mice or littermate WT mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb, anti–IFN-γ mAb, and anti–IL-2 mAb with 100 ng/ml IL-6 or IL-6 plus 1 or 5 ng/ml TGF-β for 3 d

Cells were then stimulated with PMA/ionomycin and intracellular staining for IL-21 versus IL-17A was performed. Shown are representative FACS profiles from three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb (neutral condition) with 100 ng/ml IL-6 or IL-6 plus 1 ng/ml TGF-β

At indicated times after stimulation, cells were stimulated with PMA/ionomycin, and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

(A and B) Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of 10 μg/ml anti–IL-4 mAb and 10 μg/ml anti–IFN-γ mAb (neutral condition) with or without 100 ng/ml IL-6

Where indicated, 0.2–2 ng/ml TGF-β was added. (A) 4 d later, cells were stimulated with PMA/ionomycin, and intracellular staining for the indicated cytokines was performed. Shown are representative FACS profiles from three independent experiments. (B) 4 d later, cells were washed and stimulated with PMA/ionomycin for 8 h at 2 × 10 cells/ml. The levels of IL-21 in the culture supernatants were measured by ELISA. Data are the mean ± the SD ( = 3). *, P < 0.05; **, P < 0.01.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

Naive CD4 T cells from lymph nodes of C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-, Th2-, and Th17-polarizing conditions for 5 d

Cells were evaluated for the expression of the indicated cytokines by intracellular cytokine staining as described in the Materials and methods. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3 mAb/anti-CD28 mAb in the presence of anti–IL-4 mAb and anti–IFN-γ mAb with IL-6, IL-6 plus TGF-β, or TGF-β alone for 60 h

As controls, naive CD4 T cells were stimulated with anti-CD3 mAb/anti-CD28 mAb under Th1-polarizing conditions or Th2-polarizing conditions for 60 h. The expression of RORγt, Foxp3, T-bet, and GATA3 was assessed by real-time PCR. Data are representative of three independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Development and characterization of IL-21–producing CD4 T cells"</p><p></p><p>The Journal of Experimental Medicine 2008;205(6):1369-1379.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2413034.</p><p></p

Distinct Roles for CXCR6⁺ and CXCR6⁻ CD4⁺ T Cells in the Pathogenesis of Chronic Colitis

<div><p>CD4⁺ T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4⁺ T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4⁺ T cells expressed CXCR6 in the CD45RB^high T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn’s disease. Although surface marker analysis demonstrated that both CXCR6⁺ and CXCR6⁻ CD4⁺ T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6⁺ subset produced IFN-γ and TNF-α compared to CXCR6⁻ subset, and only the CXCR6⁺ subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6⁺ T cells into <i>Rag1</i>^−/− recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6⁻ cells evoked colitis similar to that observed in CD4⁺CD45RB^high T cell-transferred mice, and resulted in their conversion into CXCR6⁺ cells. Collectively, these observations suggest that the CXCR6⁺CD4⁺ T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6⁻CD4⁺ T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6⁺CD4⁺ T cells.</p></div

Expression of CXCL16 increased in the inflamed mucosa of CD45RB^high transfer colitis.

<p>(<b>A</b>) <i>Cxcl16</i> mRNA levels in colonic epithelium (CEC) and distal colon tissues were analyzed by Q-PCR 8 weeks after transfer. The expression of <i>Cxcl16</i> mRNA increased in both epithelium and colon tissues of the transfer model compared with healthy <i>Rag1^−/−</i> mice. Data were normalized to expression of <i>Gapdh</i> mRNA. (<i>n = </i>5; mean and s.d.). **, <i>P</i> < 0.01. (<b>B</b>) CXCL16 immunostaining of the distal colon in colitic and healthy <i>Rag1^−/−</i> mice. Scale bars, 100 µm. Data are representative of two independent experiments. (<b>C</b>) CXCR6 expression on CD4⁺ T cells was analyzed by flow cytometry using a mouse CXCL16-human IgG-Fc fusion protein or control human IgG-Fcγ at 8-week post transfer. CXCR6 was expressed at high levels by the majority of colonic LP CD4⁺ T cells in the colitic mice, by about half of the SP and MLN CD4⁺ T cells, and by ∼20% of BM cells. (<b>D</b>) Absolute numbers of CXCR6⁺ and CXCR6⁻ CD4⁺ T cells in each of tissues were calculated based on the flow cytometric analysis described in (<b>C</b>). Data are representative of three independent experiments (mean and s.d.). **, <i>P</i> < 0.01.</p

CXCR6 expression is not required for development of transfer colitis.

<p>(<b>A</b>) Body weight of <i>Rag1^−/−</i> recipients of i.v. injected purified CD45RB^highCD4⁺ T cells form <i>Cxcr6</i>^+/Egfp or <i>Cxcr6</i>^Egfp/Egfp (CXCR6-deficient) mice on day 0, presented as percent of original weight. (<b>B</b>) Colon weight of the mice in (<b>A</b>) on week 7. Data are representative of two independent experiments (mean and s.d.). (<b>C</b>) Histology of colon tissues from the mice in <b>B</b>. (<b>D</b>) CXCR6 expression by LP CD4⁺ T cells was analyzed by flow cytometry using CXCL16-hFc at 7-week post transfer. (<b>E</b>) Expression levels of indicated cytokines in distal colon were analyzed by Q-PCR at 7 weeks after the transfer. Data were normalized to expression of <i>Gapdh</i>. (<i>n = </i>4 or 5; mean and s.d.).</p