DataSheet_1_IL-21 is required for the maintenance and pathogenesis of murine Vγ4+ IL-17-producing γδT cells.pdf

Murine IL-17-producing γδT (γδT17) cells are divided into two subsets: natural γδT17 (nγδT17) cells, whose development is restricted to the fetal thymus, and inducible γδT17 cells, which require antigen exposure for their IL-17 production and are presumed to develop from Rorc+Il17a-CCR9+ immature γδT17 cells in the adult thymus and whose T cell receptor (TCR) is biased toward Vγ4. Although IL-23 is known to be involved in developing γδT17 cells, the roles of other cytokines, such as IL-21, which is involved in developing Th17 cells like IL-23, in the development, maintenance, and pathophysiology of γδT17 cells remain unknown. Here, we show that IL-21 is dispensable for the fetal thymic development of nγδT17 cells but is required for the peripheral maintenance of Vγ4+nγδT17 cells. Upon stimulation with γδTCR, IL-1 plus IL-21 induces the proliferation of Vγ4+nγδT17 cells via STAT3 as effectively as IL-1 plus IL-23. Using bone marrow chimeric mice, we demonstrated that immature γδT17 cells are produced de novo in the adult mice from donor adult bone marrow cells and that IL-21 is dispensable for their development. Instead, IL-21 is required to expand newly induced Vγ4+γδT17 cells in the periphery upon immunization. Finally, using adoptive transfer experiments of γδT17 cells, we found that IL-21 receptors on γδT17 cells are involved in maintaining Vγ4+γδT17 cells, subsequent infiltration of Th17 cells into the spinal cord, and exacerbation of experimental autoimmune encephalomyelitis. Collectively, IL-21 plays a vital role in the maintenance and pathogenesis of Vγ4+γδT17 cells.</p

DataSheet_2_IL-21 is required for the maintenance and pathogenesis of murine Vγ4+ IL-17-producing γδT cells.doc

Murine IL-17-producing γδT (γδT17) cells are divided into two subsets: natural γδT17 (nγδT17) cells, whose development is restricted to the fetal thymus, and inducible γδT17 cells, which require antigen exposure for their IL-17 production and are presumed to develop from Rorc+Il17a-CCR9+ immature γδT17 cells in the adult thymus and whose T cell receptor (TCR) is biased toward Vγ4. Although IL-23 is known to be involved in developing γδT17 cells, the roles of other cytokines, such as IL-21, which is involved in developing Th17 cells like IL-23, in the development, maintenance, and pathophysiology of γδT17 cells remain unknown. Here, we show that IL-21 is dispensable for the fetal thymic development of nγδT17 cells but is required for the peripheral maintenance of Vγ4+nγδT17 cells. Upon stimulation with γδTCR, IL-1 plus IL-21 induces the proliferation of Vγ4+nγδT17 cells via STAT3 as effectively as IL-1 plus IL-23. Using bone marrow chimeric mice, we demonstrated that immature γδT17 cells are produced de novo in the adult mice from donor adult bone marrow cells and that IL-21 is dispensable for their development. Instead, IL-21 is required to expand newly induced Vγ4+γδT17 cells in the periphery upon immunization. Finally, using adoptive transfer experiments of γδT17 cells, we found that IL-21 receptors on γδT17 cells are involved in maintaining Vγ4+γδT17 cells, subsequent infiltration of Th17 cells into the spinal cord, and exacerbation of experimental autoimmune encephalomyelitis. Collectively, IL-21 plays a vital role in the maintenance and pathogenesis of Vγ4+γδT17 cells.</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

(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

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) 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

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

(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