(A) Single pTα CD4CD8CD25 TCRγδ cells were sorted onto OP9-DL1 monolayers

On day 7, the contents of each well were divided into 3 parts. One part was analyzed by FACS for surface marker expression (CD45, CD4, CD8, TCRγδ, and TCRβ), the other two parts were transferred to wells with fresh OP9-DL1 monolayers coated with TCRγδ antibodies or uncoated. On day 14 cells were again analyzed for surface marker expression, and cells from anti-TCRγδ–coated wells were further transferred to uncoated wells with fresh OP9DL1 monolayers and analyzed for marker expression on day 19. (B) The proportion of wells containing DN or both DN and DP cells is shown. Numbers represent percentages of particular wells among the total number of wells that contained CD45 cells at any time point. (C) A representative well containing γδ lineage cells only is shown. CD4CD8TCRγδ cells are detected at least at one time point, and CD4CD8 cells are absent at all time points. (D) A representative well containing αβ and γδ lineage cells only is shown. CD4CD8 cells are present before anti-TCRγδ stimulation. Only CD4CD8TCRγδ cells are detected in the presence of anti-TCRγδ antibody and after its removal. Histogram below shows comparison of surface expression of TCRγδ on DN and DP cells in the same well on day 14 of culture without antibody. Numbers in quadrants indicate the percentage of cells. Red numbers above plots refer to absolute cell number.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p

(A) TCRγδ cells derived from spleen (left) or thymus (right) of WT mice were co-cultured on OP9-DL1 monolayers

On day 7 of co-culture CD4 and CD8 expression was analyzed by flow cytometry. (B) Thymocytes from pTα (middle and right) or TCRδ (left) mice were stained with CD4, CD8, TCRγδ, CD25, and CD24 antibodies. TCRγδCD25, TCRγδCD25CD24, and TCRγδCD25CD24 cells were sorted from the thymi of pTα mice. Post-sort reanalysis of TCRγδCD25 cells is shown on the right. The applied gates are shown above each plot. Unstained thymocytes were used as negative control for CD24 staining (shaded). (C) Sorted subsets (as described in B) were co-cultured with OP9-DL1 in wells coated with anti-TCRγδ antibodies (C, bottom row) or in uncoated wells (C, top row). On day 5, cells were transferred to a fresh OP9-DL1 monolayer in uncoated wells. On day 8, cells were harvested and analyzed as in A. (D) 5,000 of the TCRγδCD25 thymocytes were cultured on OP9-DL1 (left) or OP9-GFP (right) monolayers with (bottom) or without (top) anti-TCRγδ coating. On day 4, cells were transferred to a fresh feeder of the same type in uncoated wells. On day 8, cells were harvested and analyzed as in A. Absolute cell numbers are shown in red on the top of each plot. (E) TCRγδCD25 thymocytes were cultured on an OP9-DL1 monolayer, as in D. CD24 expression on TCRγδ DN cells was analyzed on day 8 of culture. Unstained thymocytes were used as negative control for CD24 staining (shaded). Numbers in each gate indicate the percentage of cells.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p

TNPs have robust T lineage potential.

<p>BM-derived MPPs, CLPs, TNPs and thymic ETPs were sorted from C57BL/6 mice and 500 cells of each population were cultured on OP9-DL1 cells in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. A) Cells were gated as CD4⁻CD8⁻ (DNs) and analyzed for expression of CD44 and CD25 at the indicated time points. One representative experiment out of 2 independent experiments is shown. B) Cells were analyzed for expression of CD4 and CD8 at the indicated time point. C, D) Quantification of data obtained from panel B. E) Relative expansion of cultures based on an input cell number of 500. Combined data of two independent experiments with 6 wells per experiment. Error bars indicate SEM.</p

(A) Surface expression of CD4 and CD8α by ex vivo pTα thymocytes (left) or by cells derived from TCRγδCD25 pTα thymocytes cultured for 8 d on OP9-DL1 monolayer (right)

(B) CFSE dilution profiles of TCRγδCD25 pTα thymocytes cultured on an OP9-DL1 monolayer for 4 d with (red) or without (blue and green) TCRγδ antibody. Gated on all live cells (blue and red) or on CD4CD8 cells only (green). (C) Surface expression of TCRβ (left), CD8β (middle), and TCRγδ (right) on ex vivo (top) or generated in cell culture (as in A; bottom) pTα DP cells (red). Unstained (for TCRβ and CD8β) thymocytes or thymocytes from Rag2 TCRβ transgenic mice (for TCR γδ) were used as negative control (shaded). TCRγδ expression on DN pTα thymocytes is shown as positive control (dotted). (D, left) Kinetics of expansion and differentiation of TCRγδCD25 pTa in OP9-DL1 co-cultures. (D, right) CD4CD8 cells were sorted from OP9-DL1 co-culture (no antibody) on day 14 and cultured for an additional 7 d. CD4/CD8 expression was analyzed.<p><b>Copyright information:</b></p><p>Taken from "T cell receptor–instructed αβ versus γδ lineage commitment revealed by single-cell analysis"</p><p></p><p>The Journal of Experimental Medicine 2008;205(5):1173-1186.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2373848.</p><p></p

TNPs display limited non-T lineage potential.

<p>BM derived MPPs, CLPs, TNPs and thymic ETPs were sorted from C57BL/6 mice and 500 cells of each population were cultured on OP9 cells in the presence of 5 ng/ml IL-7 and 5 ng/ml Flt3L. A) Cells were analyzed for the expression of CD11c and CD11b at day 7 of culture. B) Cells were analyzed for the expression of CD19 and B220 at day 11 of culture. C) Relative expansion of myeloid cells after 7 days of culture (from 500 input cells). D) Relative expansion of B cells after 11 days of culture (from 500 input cells). C, D) Numbers above columns indicate fold expansion. E) TNP-derived cultures. Analysis was performed as in panel A) after 17 and 24 days. F) TNP-derived cultures. Analysis was performed as in panel B) after 17 and 24 days. A, B, E, F) One representative experiment out of 2 independent experiments is shown. C, D) Combined data of two independent experiments with 6 wells per experiment. Error bars indicate SEM.</p

TNPs do not efficiently differentiate upon intrathymic transfer.

<p>BM derived MPPs, CLPs and TNPs from C57BL/6 mice were sorted and 20,000 cells were intrathymically injected into non-irradiated CD45.1 mice or CD45.1/2 mice. A) Donor derived cells were analyzed in thymus by FACS for the expression of CD45.1 and CD45.2 at 3 weeks after transfer. B) Donor derived cells from MPPs, CLPs and TNPs were also analyzed for CD4 and CD8 surface expression for identification of DN (CD4⁻CD8⁻), DP (CD4⁺CD8⁺) and SP (CD4⁺ or CD8⁺) cells. C) Donor derived electronically gated DN cells from MPPs, CLPs and TNPs were analyzed for CD117 and CD25 surface expression for identification of DN subsets (DN1: CD117⁺CD25⁻; DN2: CD117⁺CD25⁺; DN3: CD117⁻CD25⁺). One representative experiment out of three independent experiments with 3 mice each is shown. D) Combined analysis of 3 experiments with each 3 mice per group. Error bars indicate SEM.</p

Characterization of TNPs from BM and blood.

<p>Lineage depleted BM and blood cells from C57BL/6 mice were stained for lineage markers, CD27, CD135, CD117, CD127 and CD90. A) Gating strategy for the identification of TNPs in BM and blood. Representative data from 1 out of 4 mice (BM) and pooled cells from 10 mice (blood). B) Relative quantification of MPPs (lin⁻CD27⁺CD135⁺CD127⁻CD117^hi), CLPs (lin⁻CD27⁺CD135⁺CD127⁺CD117^+/low), TNPs (lin⁻CD27⁺CD135⁺CD127⁻CD117^−/lowCD90⁻) and CD90⁺ (lin⁻CD27⁺CD135⁺CD127⁻CD117^−/lowCD90⁺) candidate T cell precursors within lin⁻CD27⁺CD135⁺ cells. Combined data of 4 mice and 20 mice for BM and blood, respectively, from 2 independent experiments. Error bars indicate SEM.</p

TNPs do not efficiently differentiate upon intravenous transfer.

<p>BM derived MPPs, CLPs and TNPs from C57BL/6 mice were sorted and 20,000 cells were intravenously injected into non-irradiated <i>Il7ra</i>-deficient mice expressing CD45.1 or CD45.1 and CD45.2. Donor derived cells were analyzed in thymus and BM by FACS for the expression of CD45.1 and CD45.2 at A) 2 weeks and B) 4 weeks after transfer. One representative experiment out of four and three (2 weeks and 4 weeks, respectively) independent experiments with 2 mice each is shown. C) Combined analysis of frequencies of donor-derived thymocytes of 4 and 3 individual experiments 2 and 4 weeks after transfer, respectively. Error bars indicate SEM. D) CD4 vs. CD8 surface expression of donor-derived thymocytes 2 and 4 weeks after transfer. One representative experiment out of four and three (2 weeks and 4 weeks, respectively) independent experiments with 2 mice each is shown.</p

Thymic γδ T cells in the absence of miR-181a/b-1.

<p>(A) Expression analysis of miR-181a in FACS-sorted thymocytes pooled from 5 adult or 8 neonatal TcrdH2BeGFP mice. Expression levels of the indicated cell populations were analyzed by quantitative RT-PCR and normalized to snoRNA 412. Error bars show range of relative expression levels from triplicates. (B) Bar graph shows absolute γδ T cell numbers in miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to TcrdH2BeGFP and miR-181a/b-1^+/–x TcrdH2BeGFP controls (ctrl.), pooled data from five independent experiments with each 2–5 mice per group, mean + SD. (C) Expression analysis of miR-181d in FACS-sorted thymocytes pooled from 5 miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) and TcrdH2BeGFP controls (ctrl.). One representative experiment of two independent experiments that gave similar results. Expression levels of the indicated cell populations were analyzed by quantitative RT-PCR and normalized to snoRNA 412. Error bars show range of relative expression levels from triplicates. (D–I) FACS analysis of thymic γδ T cells in–/–mice compared to ctrl mice (D, F-I) and mixed bone marrow chimeras (E). (D) Vγ usage of thymic γδ T cells (gated on Tcrβ^–GFP^hi cells). Scatter plot shows pooled data from five experiments with 3–6 mice per group, one dot represents one mouse, mean. (E) Flow cytometric analysis of 1:1 mixed bone marrow chimeras. Scatter plot shows ratios of miR-181a/b-1^–/–(KO) and miR-181a/b-1 sufficient wild type (WT) donor Vγ1⁺ and Vγ4⁺ cells among all lymphocytes, respectively. Data are pooled from two independent experiments with each 3 mice per group, harmonic mean. (F) Scatter plot shows absolute numbers of NK1.1⁺ cells, pooled data from five independent experiments with each 2–5 mice per group. (G) Scatter plot shows absolute numbers of NK1.1⁺ γδ T cells, pooled data from five independent experiments with each 2–5 mice per group. (H) Representative contour plots of cluster B (CD44^hiCD24^–) and cluster A (CD44^–/loCD24⁺) γδ thymocytes (gated on Tcrβ^–GFP^hi cells), numbers indicate mean +/–SD of pooled data from four independent experiments with each 2–5 mice per group. (I) Representative contour plots of CCR6⁺ and NK1.1⁺ cluster B cells, numbers indicate mean +/–SD of pooled data from four independent experiments with each 2–6 mice per group. Statistical analyses were performed using the Mann-Whitney test.</p

Unchanged peripheral lymph node γδ T cell compartment in the absence of miR-181a/b-1.

<p>FACS analysis of γδ T cells in pLN of miR-181a/b-1^–/–x TcrdH2BeGFP mice (–/–) compared to miR-181a/b-1 sufficient controls, TcrdH2BeGFP and miR-181a/b-1^+/–x TcrdH2BeGFP mice (here referred to as ctrl.). (A) Total γδ T cells numbers in pLN of the indicated phenotypes. Scatter plot shows pooled data from five independent experiment with n = 2–5 mice per group, mean. (B) Scatter plot shows absolute numbers of NK1.1⁺ γδ T cells, pooled data from five independent experiments with each 2–5 mice per group, mean. (C) Vγ usage of γδ T cells (gated on Tcrβ^–GFP^hi cells). Bar graph shows pooled data from 5 experiments with 3–6 mice per group, mean + SD. (D + E) Intracellular cytokine staining for IFN-γ and IL-17A gated on γδ T cells. (D) Representative contour plots of two independent experiments with similar outcome, with each n = 2–5 mice per group. Numbers indicate mean +/–SD from pooled data. (E) Bar gaph shows pooled data from the two independent experiments, mean + SD. Statistical analyses were performed using the Mann-Whitney test.</p