1. TCR Signal Strength Influences αβ/γδ Lineage Fate
Sandra M. Hayes, LiQi Li, Paul E. Love 
Immunity 
Volume 22, Issue 5, Pages (May 2005)
DOI: /j.immuni
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Generation of αβ and γδ Lineage Cells in γδTCR Tg Mice
(A) Phenotypic analysis of γδTCR Tg thymocytes. Two-color plot shows expression of CD4 versus CD8 and adjacent histograms show TCRγδ or TCRβ surface expression on total thymocytes. Numbers in quadrants of the two-color plots represent percentage of cells in each quadrant.
(B) γδTCR surface expression on and intracellular (ic) TCRβ expression in immature DN thymocytes from γδTCR Tg, TCRβ−/− and B6 or TCRδ−/− mice. DN thymocytes are defined as thymocytes lacking expression of CD4 and CD8 surface antigens. CD25 expression was used to stage maturation of DN thymocytes, with CD25hi cells being the most immature. A representative gating of CD25hi and CD25lo DN thymocytes from a B6 mouse is shown.
(C) Semiquantitative RT-PCR for CD25, Cα and Vγ6/Jγ1/Cγ1 transcripts in purified DN and DP thymocytes from γδTCR Tg and DP thymocytes from B6 mice. β−actin RT-PCR was also performed to demonstrate that equivalent amounts of RNA were analyzed.
(D) Phenotypic analysis of DP thymocytes from γδTCR Tg, TCRβ−/−, and TCRδ−/− mice. Histograms show TCRγδ, TCRβ (surface and intracellular), and CD25 expression on gated DP thymocytes. In panels (B), (C), and (D), γδTCR Tg thymocytes are represented as a blue histogram, TCRβ−/− thymocytes as a magenta histogram, B6 thymocytes as a dark gray histogram, and TCRδ−/− thymocytes as a shaded histogram.
(E) Phenotypic analysis of thymocytes from mixed bone marrow chimeras. Two-color plots show expression of CD4 versus CD8 on gated Ly5.2+ thymocytes and expression of Ly5.1 versus Ly5.2 on gated DP thymocytes. Numbers in quadrants of the two-color plots represent percentage of cells in each quadrant. The degree of chimerism in the bone marrow was 50% (Ly 5.2+) while that in the thymus was <4% (Ly 5.2+).
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 γδTCR Surface Levels Affect αβ/γδ Lineage Fate
(A) Comparison of TCR surface levels on CD8 immature single positive (ISP) and DP thymocytes from B6 and γδTCR Tg mice. CD8 ISP thymocytes are defined as CD5lo/− thymocytes whereas CD8 mature single positive (MSP) thymocytes are defined as CD5hi thymocytes. Staining with a hamster isotype control is also shown (shaded histogram).
(B) TCRγδ expression on gated CD25+ DN thymocytes and TCRγδ and TCRβ expression on gated DP thymocytes from γδTCR Tg TCRζ+/+ (blue histogram), γδTCR Tg TCRζ+/− (magenta histogram), and γδTCR Tg TCRζ Tg TCRζ+/− (purple histogram) mice. Staining with a hamster isotype control is also shown (shaded histogram). Mean fluorescence intensities (MFIs), representing γδTCR expression levels, are 487 for γδTCR Tg TCRζ+/−, 1461 for γδTCR Tg TCRζ+/+, and 3240 for γδTCR Tg TCRζ Tg TCRζ+/−.
(C) Top panel represents CD4 versus CD8 staining profiles for γδTCR Tg TCRζ+/+, γδTCR Tg TCRζ+/−, and γδTCR Tg TCRζ Tg TCRζ+/− thymocytes. Bottom panel represents CD4 versus CD8 staining profiles on Ly5.2+ thymocytes generated by γδTCR Tg TCRζ+/+, γδTCR Tg TCRζ+/−, or γδTCR Tg TCRζ Tg TCRζ+/− bone marrow cells in a mixed bone marrow chimera. Numbers in quadrants of the two-color plots represent percentage of cells in each quadrant. The mean percentage (± standard error of the mean) of Ly5.2+ DP thymocytes was 21.6 ± 1.7 from γδTCR Tg TCRζ+/+ progenitors, 12.8 ± 2.8 from γδTCR Tg TCRζ Tg TCRζ+/− progenitors, and 43.4 ± 8.6 from γδTCR Tg TCRζ+/− progenitors. The mean percentage (± standard error of the mean) of Ly5.2+ γδTCR+ DN thymocytes was 55.5 ± 1.1 from γδTCR Tg TCRζ+/+ progenitors, 69.8 ± 2.8 from γδTCR Tg TCRζ Tg TCRζ+/− progenitors, and 36.5 ± 6.4 from γδTCR Tg TCRζ+/− progenitors.
(D) Mean number of γδTCR+ DN thymocytes and DP thymocytes in γδTCR Tg TCRζ+/+ (n = 13), γδTCR Tg TCRζ+/− (n = 5), and γδTCR Tg TCRζ Tg TCRζ+/− (n = 5) mice. Bars represent standard errors of the mean; *p ≤ 0.05 and **p ≤ 0.01.
(E) TCRγδ expression on CD25+ DN thymocytes from γδTCR Tg LAT+/+ (magenta histogram) and γδTCR Tg LAT4YF (blue histogram) mice. In (B) and (E), CD25+ DN thymocytes are defined as CD25hi/lo CD4− CD8− thymocytes.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3 Effect of Weakening the γδTCR Signal on αβ/γδ Lineage Fate
(A) CD5 expression on CD25+ γδTCR+ DN thymocytes from γδTCR Tg LAT−/− mice (shaded histogram) and from γδTCR Tg TCRζ−/− mice reconstituted with a TCRζ full-length (ζ FL) (dark gray histogram) or tailless (ζ TL) (black histogram) transgene.
(B) Phenotypic analysis of thymocytes from γδTCR Tg TCRζ−/− mice reconstituted with a ζ FL or ζ TL transgene or reconstituted with a FcϵR1γ (FcRγ) transgene. Two-color plots show CD4 versus CD8 staining profiles on total thymocytes, and numbers in the quadrants represent percentage of cells in each quadrant. Adjacent histograms show TCRγδ expression on gated CD25+ DN thymocytes or DP thymocytes. DN thymocytes are defined as CD4−, CD8−, TCRβ−, CD19−, and NK1.1− cells, while CD25+ cells are defined as CD25hi/lo. Staining with a hamster isotype control is also shown (shaded histogram). γδTCR expression levels are represented by MFIs.
(C) Mean number of γδTCR+ DN thymocytes and DP thymocytes in γδTCR Tg TCRζ−/− mice reconstituted with the ζ FL (n = 5), ζ TL (n = 5), or FcRγ (n = 4) transgene compared to γδTCR Tg TCRζ+/+ (n = 13) mice. Bars represent standard errors of the mean; **p ≤ 0.01.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4 Effect of Loss of CD5 on αβ/γδ Lineage Commitment
(A) Phenotypic analysis of thymocytes and lymph node cells from CD5+/+, CD5+/−, and CD5−/− γδTCR Tg mice. Two-color plots show CD4 versus CD8 staining profiles on total thymocytes or lymph node cells, and numbers in the quadrants represent percentage of cells in each quadrant. Adjacent histograms show expression levels of TCRγδ on gated DN cells (defined as CD4− CD8−). γδTCR expression levels are represented by MFIs.
(B) Mean number of γδTCR+ DN thymocytes and DP thymocytes in CD5+/+ (n = 13), CD5+/− (n = 4), and CD5−/− (n = 4) γδTCR Tg mice. Bars represent standard errors of the mean; **p ≤ 0.01.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
Comparison of Intracellular pERK and pZAP-70 Levels in and CD5 Surface Expression on Ex Vivo αβ and γδ Lineage Cells
TCRα−/− thymocytes were immediately fixed after harvesting, stained with antibodies against surface molecules (CD4, CD8, CD25, CD5), permeabilized, and then stained with the IgG isotype control, anti-pERK or anti-pZAP-70. To detect γδ lineage cells, TCRα−/− thymocytes were stained for surface expression of the γδTCR (sTCRγδ+). To detect αβ lineage cells, TCRα−/− thymocytes were stained for intracellular expression of TCRβ (icTCRβ+). >1 x 106 cells were acquired on a Becton Dickinson LSR II.
(A) Sample histograms for intracellular pERK (top) and pZAP-70 (bottom) levels in CD25+ icTCRβ+ DN thymocytes (violet histogram) and CD25lo sTCRγδ+ DN thymocytes (blue histogram). Staining with a mouse IgG isotype is also shown (shaded histogram).
(B) MFIs are shown as a measurement of the levels of each signaling molecule as well as the isotype control in several thymocyte subsets. Data shown are representative of four mice.
(C) CD5 expression on CD25+ icTCRβ+ DN thymocytes (violet histogram), CD25lo sTCRγδ+ DN thymocytes (blue histogram), and CD25+ icTCRβ− DN thymocytes (shaded histogram) (representing putative pre-committed cells). MFIs for CD5 expression are 144 for CD25+ icTCRβ− DN thymocytes, 395 for CD25+ icTCRβ+ DN thymocytes, and 1646 for CD25lo sTCRγδ+ DN thymocytes.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Schematic Representation of the Effects of Altering γδTCR Signal Strength on αβ/γδ Lineage Fate in γδTCR Tg mice
The upper panel depicts γδTCR surface expression on pre-committed DN thymocytes. The solid vertical lines depict relative thresholds for αβ and γδ lineage fate. Cells that are γδTCRlo, and therefore receive a weak signal, develop into αβ lineage cells. Cells that are γδTCRhi, and therefore receive a strong signal, develop into γδ lineage cells. The genetic alterations experimentally imposed in this study and their effects on TCR surface expression or TCR signal strength are shown in the lower four panels. Hypothetical effects of changes in signal strength on the cohort of γδTCR+ cells that develop into αβ and γδ lineages are depicted by the solid (new thresholds) and dotted (old thresholds) lines. Expression of a TCRζ transgene on the γδTCR Tg TCRζ+/− background increases γδTCR surface expression on pre-committed DN thymocytes and results in the preferential development of γδ lineage cells. The TCRζ+/− mutation reduces γδTCR surface expression on pre-committed DN thymocytes, favoring development to the αβ lineage. Reducing the γδTCR signaling potential (ζ TL Tg or FcRγ Tg) results in commitment of cells with higher γδTCR surface levels to the αβ lineage. The ultimate effect of this alteration is that more cells adopt the αβ lineage fate and fewer cells adopt the γδ lineage fate. Removal of the negative regulator CD5 indirectly increases the γδTCR signaling potential, causing cells with lower γδTCR surface levels to commit to the γδ lineage. The ultimate effect of this alteration is that there are more cells in the γδ lineage and fewer cells in the αβ lineage.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 Signal Strength Model for αβ/γδ Lineage Choice
Immature DN thymocytes have the potential to become either an αβ or a γδ lineage cell, depending on the strength of the TCR signal. The surface level of the TCR (pre- or γδTCR) on immature DN thymocytes determines its signal strength. Typically, the pre-TCR is expressed at low levels on immature DN thymocytes, whereas surface expression of the γδTCR is relatively high. Therefore, if a cell expresses the pre-TCR, then it will receive a “weak” signal, choose the αβ lineage fate, undergo a strong proliferative burst and transition to the DP stage. However, if a cell expresses high levels of the γδTCR, then it will receive a “strong” signal, choose the γδ lineage fate and remain DN. Conversely, if it expresses low levels of the γδTCR, then it will receive a “weak” signal and choose the αβ lineage fate. Because the choice to become an αβ lineage cell by a γδTCR+ DN thymocyte occurs infrequently in wild-type mice, it is denoted by a dotted line.
Immunity  , DOI: ( /j.immuni )
Copyright © 2005 Elsevier Inc. Terms and Conditions