Effect of reducing Lck or Fyn levels on polyclonal γδ T cell development.

<p>A. Demonstration of the reduction of Lck or Fyn levels in CD4⁺ thymocytes and LN cells from Lck^+/− and Fyn^+/− mice. The MFI of the i.c. levels of Lck and Fyn in CD4⁺ lineage cells from heterozygous mice are expressed as a percentage of the MFI of the i.c. levels of Lck and Fyn in CD4⁺ lineage cells from WT mice. A dashed line marks the expected 50% reduction in WT Lck and Fyn levels. B. Number of DN γδ thymocytes and LN γδ T cells in WT, Lck^+/−, and Fyn^+/− mice. Data represent at least 6 mice per genotype. C. Quantifying the reduction of Lck and Fyn expression levels in DN γδTCR⁺ thymocytes and LN cells from Lck^+/− and Fyn^+/− mice. The MFI of the i.c. levels of Lck and Fyn in γδ lineage cells from heterozygous mice are expressed as a percentage of the MFI of the i.c. levels of Lck and Fyn in γδ lineage cells from WT mice. A dashed line marks the expected 50% reduction in WT Lck and Fyn levels. D. Relative expression levels of Lck and Fyn in WT DN2 (lin⁻ CD25⁺ CD44⁺) thymocytes, where lin⁻ is defined as CD4⁻ CD8⁻ CD11b⁻ TCRβ⁻ TCRγδ⁻ CD19⁻ NK1.1⁻ IA^b− Ly6-G/Ly6-C⁻. E. Quantifying the reduction of Lck and Fyn expression levels in DN2 thymocytes from Lck^+/− and Fyn^+/− mice. The MFI of the i.c. levels of Lck and Fyn in DN2 thymocytes from heterozygous mice are expressed as a percentage of the MFI of the i.c. levels of Lck and Fyn in DN2 thymocytes from WT mice. A dashed line marks the expected 50% reduction in WT Lck and Fyn levels. In A, B, C, and E, the bars represent mean ± SEM. *<i>p≤0.05</i>, **<i>p≤0.01</i>, #<i>p≤0.001</i>.</p

Effect of reducing Lck or Fyn levels on αβ/γδ lineage commitment and γδ T cell development.

<p>A. Dot plots show representative CD4 versus CD8 staining profiles for WT γδTCR Tg, Lck^+/− γδTCR Tg, and Fyn^+/− γδTCR Tg thymocytes. Numbers in the quadrants represent percentage of cells in each quadrant. The mean thymus cell number ± SEM for each genotype are displayed above the respective two-color plot. B. Mean number of DN (DN γδTCR⁺; γδ lineage) and DP (αβ lineage) thymocytes in WT γδTCR Tg, Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice. Data represent at least 6 mice per genotype. C. Mean number of DN γδ T cells in the LNs of WT γδTCR Tg, Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice. Data represent at least 5 mice per genotype. In B and C, the bars represent mean ± SEM. *<i>p≤0.05</i>, #<i>p≤0.001</i>.</p

Flow cytometric analysis of the intracellular levels of Lck and Fyn in γδ lineage cells.

<p>A. Histograms show representative staining of the i.c. levels of Lck and Fyn in gated populations of DP thymocytes and of thymic and LN CD4⁺ CD3⁺ and DN γδTCR⁺ cells from WT (B6) mice. Staining of cells from Lck^−/− and Fyn^−/− mice are shown as negative controls for i.c. staining of Lck and Fyn, respectively. B. Comparison of the relative expression levels of Lck and Fyn in gated DN γδTCR⁺ thymocytes and LN cells. C. Quantifying the change in the relative expression levels of Lck and Fyn in DN γδTCR⁺ thymocytes and LN cells and, for comparison, CD4⁺ CD3⁺ thymocytes and LN cells. Lck and Fyn expression levels in immature and mature subsets were normalized to those of DP thymocytes, as this population had, in every experiment, consistently lower levels of Lck and Fyn than any other thymocyte or T cell subset (see A). Data are presented as fold change relative to DP thymocytes (set to 1). Data are representative of at least 6 independent experiments. Bars represent mean ± SEM. *<i>p≤0.05</i>, **<i>p≤0.01</i>, #<i>p≤0.001</i>.</p

Percentage of Ki-67⁺ DN γδTCR⁺ thymocytes^a.

<p><i>^a</i>Ki-67 expression marks cells in late G₁ phase through mitosis and is used as marker of active cell cycling.</p><p>**<i>p≤0.01</i>.</p

Phenotypic analysis of γδ lineage cells from Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice.

<p>A. Comparison of CD5 and TCRγδ surface levels on DN γδ thymocytes from WT γδTCR Tg, Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice. MFIs of CD5 and TCRγδ surface levels on DN γδ thymocytes from heterozygous mice are presented as a percentage of the MFIs of CD5 and TCRγδ surface levels on DN γδ thymocytes from WT γδTCR Tg mice. Data represent at least 6 mice per genotype. B. Percentage of CD24⁺ and CD44⁺ γδ T cells in WT γδTCR Tg, Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice. Data represent at least 3 mice per genotype. C. Comparison of CD5 and TCRγδ surface levels on DN γδ T cells from the LNs of WT γδTCR Tg, Lck^+/− γδTCR Tg and Fyn^+/− γδTCR Tg mice. MFIs of CD5 and TCRγδ surface levels on peripheral DN γδ T cells from heterozygous mice are presented as a percentage of the MFIs of CD5 and TCRγδ surface levels on peripheral DN γδ T cells from WT γδTCR Tg mice. Data represent at least 5 mice per genotype. In A, B, and C, the bars represent mean ± SEM. *<i>p≤0.05</i>, **<i>p≤0.01</i>, #<i>p≤0.001</i>.</p

Effects of CD28 deficiency on γδ thymocyte proliferation, cell death and lineage commitment.

<p>(<b>A</b>) Bars represent the mean ± SEM of the percentage of Ki-67⁺ DN TCRγδ⁺ thymocytes from CD28^+/+ (n = 8) and CD28^−/− (n = 8) mice. (<b>B</b>) Bars represent the mean percent ± SEM of Annexin V⁺ DN TCRγδ⁺ thymocytes from CD28^+/+ (n = 4) and CD28^−/− (n = 4) mice. (<b>C</b>) Phenotypic analysis of CD28^+/+ γδTCR Tg and CD28^−/− γδTCR Tg mice. Dot plots show representative CD4 versus CD8 staining profiles on total thymocytes. Numbers in quadrants of the two-color plots represent the percentage of cells in each quadrant. Adjacent dot plots show representative TCRγδ versus CD3 staining on gated DN thymocytes. Numbers represent percentage of cells in each gate. Data are representative of three independent experiments. (<b>D</b>) Mean number ± SEM of DN (DN TCRγδ⁺; γδ lineage) and DP (αβ lineage) thymocytes in CD28^+/+ γδTCR Tg and CD28^−/− γδTCR Tg mice. Data represent seven mice per genotype.</p

γδ T Cells Acquire Effector Fates in the Thymus and Differentiate into Cytokine-Producing Effectors in a Listeria Model of Infection Independently of CD28 Costimulation

<div><p>Both antigen recognition and CD28 costimulation are required for the activation of naïve αβ T cells and their subsequent differentiation into cytokine-producing or cytotoxic effectors. Notably, this two-signal paradigm holds true for all αβ T cell subsets, regardless of whether they acquire their effector function in the periphery or the thymus. Because of contradictory results, however, it remains unresolved as to whether CD28 costimulation is necessary for γδ T cell activation and differentiation. Given that γδ T cells have been recently shown to acquire their effector fates in the thymus, it is conceivable that the contradictory results may be explained, in part, by a differential requirement for CD28 costimulation in the development or differentiation of each γδ T cell effector subset. To test this, we examined the role of CD28 in γδ T cell effector fate determination and function. We report that, although IFNγ-producing γδ T (γδ-IFNγ) cells express higher levels of CD28 than IL-17-producing γδ T (γδ-17) cells, CD28-deficiency had no effect on the thymic development of either subset. Also, following Listeria infection, we found that the expansion and differentiation of γδ-17 and γδ-IFNγ effectors were comparable between CD28^+/+ and CD28^−/− mice. To understand why CD28 costimulation is dispensable for γδ T cell activation and differentiation, we assessed glucose uptake and utilization by γδ T cells, as CD28 costimulation is known to promote glycolysis in αβ T cells. Importantly, we found that γδ T cells express higher surface levels of glucose transporters than αβ T cells and, when activated, exhibit effector functions over a broader range of glucose concentrations than activated αβ T cells. Together, these data not only demonstrate an enhanced glucose metabolism in γδ T cells but also provide an explanation for why γδ T cells are less dependent on CD28 costimulation than αβ T cells.</p></div

Glucose uptake by, and expression of GLUT isoforms on, γδ T cells.

<p>(<b>A</b>) Purified γδ T cells from either CD28^+/+ γδTCR Tg or CD28^−/− γδTCR Tg mice were cultured in glucose-free medium supplemented with 30 µM 2-NBDG in the presence or absence of 1 µg/mL plate-bound anti-CD3 mAb. γδ T cells were harvested from the plate at various time points and 2-NBDG uptake was measured by flow cytometric analysis. Graph shows MFI of 2-NBDG as a function of time for unstimulated (black line) and stimulated (dashed line) γδ T cells. Data are representative of three independent experiments; the data from the CD28^−/− γδ T cells are shown. (<b>B</b>) Top panel: GLUT1 and GLUT3 expression levels on neutrophils (CD11b⁺ Ly-6G⁺; blue histograms) and DP thymocytes (shaded histograms) from CD28^+/+ mice. Bottom panel: GLUT1 and GLUT3 expression levels on CD4⁺ CD44^– αβ T cells, CD8⁺ CD44^– αβ T cells, and DN γδ T cells. Staining on DP thymocytes (shaded histogram) is also shown as a negative control. Data are representative of three independent experiments, using at least 9 CD28^+/+ mice. (<b>C</b>) Distribution of γδ-17 (CCR6⁺ CD27^–) and γδ-IFNγ (CCR6^– CD27⁺) within all γδ T cells and within the GLUT1^hi and GLUT3^hi γδ T cell subsets. (<b>D</b>) Comparison of glucose uptake between unstimulated αβ and γδ T cells. Purified γδ T cells from CD28^+/+ γδTCR Tg mice and purified αβ T cells from CD28^+/+ mice were cultured in glucose-free medium supplemented with 30 µM 2-NBDG for various periods of time. 2-NBDG uptake as well as CD4 and CD8 expression was measured by flow cytometric analysis. Graph shows MFI of 2-NBDG as a function of time for γδ T cells (black line), CD4⁺ αβ T cells (dashed line) and CD8⁺ αβ T cells (dotted line). Data are representative of three independent experiments.</p

Effect of glucose concentration on γδ T cell proliferation and cytokine production.

<p>Purified γδ T cells from CD28^+/+ γδTCR Tg and CD28^−/− γδTCR Tg mice were labeled with CFSE and then cultured in glucose-free medium or glucose-free medium supplemented with increasing concentrations of glucose in the presence of 1 µg/mL (<b>A</b>) or 0.2 µg/ml (<b>B</b>) of plate-bound anti-CD3 mAb. 48 h later, cells were harvested and their proliferative response was measured by flow cytometric analysis. Supernatants were also collected and cytokine production was measured by ELISA. Effect of glucose concentration on cellular proliferation, IL-2 production, IL-17A production, and IFNγ production. Data are representative of at least three mice per genotype. *<i>p</i> ≤ 0.05, **<i>p</i> ≤ 0.01, #<i>p</i> ≤ 0.001.</p

Comparison of CD28 expression levels on γδ T cell subsets in the thymus and periphery.

<p>Analysis of CD28 expression on various gated subsets in the thymus (<b>A</b>) and pLNs (<b>B</b>) of CD28^+/+ (i.e., IL-23R^gfp/+) mice. Black histograms show representative staining of CD28 on total, IL-23R⁺ (GFP⁺) and CD27⁺ DN TCRγδ⁺ subsets as well as on CD4⁺ TCRαβ⁺ subsets. Staining of thymocytes (<b>A</b>) and pLN cells (<b>B</b>) from CD28^−/− mice are shown as negative controls (shaded histograms). Numbers in the plots represent the mean fluorescent intensity (MFI) of CD28 expression. Data are representative of six mice in three independent experiments.</p