Intrathymic δ Selection Events in γδ Cell Development Lorena Passoni, Eric S Hoffman, Sylvia Kim, Tessa Crompton, William Pao, Meng-Qiu Dong, Michael J Owen, Adrian C Hayday  Immunity  Volume 7, Issue 1, Pages 83-95 (July 1997) DOI: 10.1016/S1074-7613(00)80512-9

Figure 1 A Scheme for Intrathymic αβ T Cell Development in Adult Mice The scheme denotes cell surface markers commonly used to define αβ T cell progenitors and stages at which known mechanisms of developmental regulation occur, namely β selection and αβ T cell positive/negative selection. Numbers indicate the approximate representation of the respective subsets as a percentage of total cells in the thymus. Note that in total, TCRγδ cells (not depicted here) usually represent <2% of total thymocytes. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 2 Flow Cytometric Analysis of Thymocyte Subsets (A) Adult C57.BL/6, TCRβ −/−, and TCR(β×δ)−/− thymocytes were four- color–stained with antibodies specific for CD4, CD8, CD25 and CD44. CD4 versus CD8 expression (first row), CD25 versus CD44 expression on DN thymocytes (second row), and cell size from the DN CD44loCD25+ subset to show E and L cells (third row) are plotted. Quadrant percentages are indicated in rows 1 and 2, and the percentage of L cells in the DN CD44loCD25+ subset is indicated in the third row. (B) Adult TCRβ−/− littermate thymocytes from eight littermates were stained with antibodies specific for CD4 and CD8. CD4 versus CD8 expression is plotted, and DP percentages are indicated. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 3 PCR-RFLP Analysis of Vδ4-Jδ1, Vδ5-Jδ1, and Vδ6-Jδd1 Joins in TCRβ−/− Thymocyte Subsets (A) Sorted DN CD25− thymocytes were analyzed for TCRδ join status. Lane 1, DN TCRδ+HSAhi thymocytes; lane 2, DN TCRδ+ HSAlo thymocytes; lane 3, DN HSAhiCD44lo CD25− thymocytes. (B) Sorted DP thymocytes were analyzed for TCRδ join status. (C) Sorted E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes were analyzed for TCRδ join status. In each case, in-frame join lengths are indicated, as determined by comparison to the sequence ladder (ACTG); ∼ indicates a nonpolymorphic boundary fragment that is generated by the restriction digestion strategy and that migrates in the same region of the gel. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 3 PCR-RFLP Analysis of Vδ4-Jδ1, Vδ5-Jδ1, and Vδ6-Jδd1 Joins in TCRβ−/− Thymocyte Subsets (A) Sorted DN CD25− thymocytes were analyzed for TCRδ join status. Lane 1, DN TCRδ+HSAhi thymocytes; lane 2, DN TCRδ+ HSAlo thymocytes; lane 3, DN HSAhiCD44lo CD25− thymocytes. (B) Sorted DP thymocytes were analyzed for TCRδ join status. (C) Sorted E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes were analyzed for TCRδ join status. In each case, in-frame join lengths are indicated, as determined by comparison to the sequence ladder (ACTG); ∼ indicates a nonpolymorphic boundary fragment that is generated by the restriction digestion strategy and that migrates in the same region of the gel. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 4 DNA Content and Flow Cytometric Analysis of Thymocytes Subsets from C57.BL/6 and TCRβ−/− Mice Total and TCRδ+ DP thymocytes from (A) C57.BL/6 and (B) TCRβ−/− mice; (C) DN HSAhiCD44loCD25− thymocytes from C57.BL/6 and TCRβ−/− mice; E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes from (D) C57.BL/6 and (E) TCRβ−/− mice. Each subset was sorted, stained with propidium iodide, and analyzed by FACS to determine DNA content. Percentages of cells from each population with greater than 2N DNA content (and therefore in S/G2/M) are indicated. The values in parentheses in the C57.BL/6 panel of (C) indicate the range of values found from multiple experiments. (F) Thymocytes from TCRβ−/− mice were stained with antibodies specific for CD4 and CD8 and sorted for DN cells, which were then four-color–stained with antibodies specific for CD25, CD44, HSA, and TCRδ. CD25 versus CD44 expression (top) is shown and was used to establish gates. HSA vs. TCRδ expression on gated CD44−CD25+ (bottom left) and CD44−CD25− (bottom right) thymocytes is shown. Quardant percentages are indicated. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 4 DNA Content and Flow Cytometric Analysis of Thymocytes Subsets from C57.BL/6 and TCRβ−/− Mice Total and TCRδ+ DP thymocytes from (A) C57.BL/6 and (B) TCRβ−/− mice; (C) DN HSAhiCD44loCD25− thymocytes from C57.BL/6 and TCRβ−/− mice; E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes from (D) C57.BL/6 and (E) TCRβ−/− mice. Each subset was sorted, stained with propidium iodide, and analyzed by FACS to determine DNA content. Percentages of cells from each population with greater than 2N DNA content (and therefore in S/G2/M) are indicated. The values in parentheses in the C57.BL/6 panel of (C) indicate the range of values found from multiple experiments. (F) Thymocytes from TCRβ−/− mice were stained with antibodies specific for CD4 and CD8 and sorted for DN cells, which were then four-color–stained with antibodies specific for CD25, CD44, HSA, and TCRδ. CD25 versus CD44 expression (top) is shown and was used to establish gates. HSA vs. TCRδ expression on gated CD44−CD25+ (bottom left) and CD44−CD25− (bottom right) thymocytes is shown. Quardant percentages are indicated. Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 5 PCR-RFLP Analysis of Vγ1-Jγ4, Vγ4-Jγ1, and Vγ7-Jγ1 Joins in TCRβ−/− Thymocyte Subsets (A) Sorted E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes were analyzed for TCRγ join status. (B) Sorted DN CD25− thymocytes were analyzed for TCRγ join status. Lane 1, DN TCRδ+HSAhi thymocytes; lane 2, DN TCRδ+ HSAlo thymocytes; lane 3, DN HSAhiCD44lo CD25− thymocytes. (C) Sorted DP thymocytes were analyzed for TCRγ join status. The in-frame join lengths are indicated in each case, as determined by comparison to the sequence ladder (ACTG). Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 6 Temporal PCR-RFLP Analysis of Vγ5-Jγ1 Joins in Thymocytes from TCRβ−/− Mice (A) Sorted thymocytes were analyzed for TCR Vγ5-Jγ1 join status at 1 day, 1 week, or 2 weeks of age. Lanes 1, sorted E (small DN HSAhiCD44loCD25+) thymocytes; lanes 2, HSAhiCD44loCD25− DN thymocytes; lanes D, Dendritic epidermal T cells. (B) (Left) Sorted E (small DN HSAhiCD44loCD25+) and L (larger DN HSAhiCD44loCD25+) thymocytes analyzed for TCR Vγ5-Jγ1 join status at 4–6 weeks of age. (Right) Sorted DN CD25− thymocytes analyzed for TCR Vγ5-Jγ1 join status. Lane 1, DN TCRδ+HSAhi thymocytes; lane 2, DN TCRδ+HSAlo thymocytes; lane 3, DN HSAhiCD44loCD25− thymocytes. The canonical in-frame join length of 52 bp was determined from the sequence ladder (ACTG) and the migration of the DEC band is indicated in each case (D). Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)

Figure 7 The Developmental Pathways of Thymocytes Mediated by δ Selection Immunity 1997 7, 83-95DOI: (10.1016/S1074-7613(00)80512-9)