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. Author manuscript; available in PMC: 2012 Jul 10.

Published in final edited form as: Immunity. 2008 Dec 8;29(6):876–887. doi: 10.1016/j.immuni.2008.09.019

(A). Bead-purified CD4⁺CD8⁻ cells (2 × 10⁶) from wild-type or Zbtb7b^t/t mice were adoptively transferred into Rag2^−/− recipients. Left: starting populations. Right: splenocytes from recipient mice were analyzed two weeks later by 4 color flow cytometry for expression of CD4, CD8α, TCRβ and CD44. Single parameter histograms identify TCR⁺ splenocytes, on which contour plots show expression of CD4 vs. CD8α and of CD4 vs. CD44. Data is representative of two separate experiments (the second one with β2m-deficient recipients), each including 5 recipients for each donor genotype.

(B). Expression of T-bet, Eomes, Perforin and GzmB mRNAs was assessed by qRT-PCR in sorted naïve (CD44^lo) CD4⁺CD8⁻ LN T cells from wild-type and Zbtb7b^t/t mice and in CD4⁻CD8⁺ LN cells from wild-type mice. Messenger RNA levels are normalized to β-actin mRNA and expressed as a ratio over those in wild-type CD8 cells (set to 1, not depicted). Error bars indicate experimental variation among triplicates within the experiment shown; data is representative of three separate such experiments.

(C). Expression of Runx3 mRNAs initiated at the distal (dis-Runx3) and proximal (prox-Runx3) promoters was analyzed by conventional RT-PCR in sorted naïve (CD44^lo) CD4⁺CD8⁻ LN T cells from wild-type and Zbtb7b^t/t mice and in CD4⁻CD8⁺ LN cells from wild-type mice. Wedges represent 3 fold dilutions (1.2; 0.4 and 0.12 × 10⁴ cells, respectively). β-actin mRNA expression was assessed in parallel as a control for mRNA preparation (bottom row). Data is representative of three separate experiments.

(D-F). Sorted CD44^lo CD4⁺CD8⁻ LN T cells from wild-type or Zbtb7b^t/t mice were activated by anti-CD3 and anti-CD28 on irradiated APCs in the presence of IL-2, IL-4 and antibodies against IL-12 and IFNγ (Th2 conditions) or IL-2 only (ThN conditions), and analyzed five days after stimulation. (D) Cells were stained for CD4, CD8 and intra-cellular GzmB; GzmB expression is shown on gated CD4⁺CD8⁻ Zbtb7b^t/t (plain line) or wild-type (gray filled histograms); the dashed lines show GzmB expression on wild-type CD8 cells stimulated in parallel in the same conditions. (E) IL-4 and IFNγ production was assessed by flow cytometry on cells restimulated with PMA and Ionomycin. Cells were stained for CD4, CD8 and intra-cellular IL-4 and IFNγ; contour plots are gated on CD4⁺CD8⁻ cells. Numbers indicate the percent of cells within quadrants. (F) Analyses of gene expression were conducted on Th2-differentiated effectors as in panel (B). Data is representative of three or more separate experiments for each panel.

(A). Bead-purified CD4⁺CD8⁻ cells (2 × 10⁶) from wild-type or Zbtb7b^t/t mice were adoptively transferred into Rag2^−/− recipients. Left: starting populations. Right: splenocytes from recipient mice were analyzed two weeks later by 4 color flow cytometry for expression of CD4, CD8α, TCRβ and CD44. Single parameter histograms identify TCR⁺ splenocytes, on which contour plots show expression of CD4 vs. CD8α and of CD4 vs. CD44. Data is representative of two separate experiments (the second one with β2m-deficient recipients), each including 5 recipients for each donor genotype.

(B). Expression of T-bet, Eomes, Perforin and GzmB mRNAs was assessed by qRT-PCR in sorted naïve (CD44^lo) CD4⁺CD8⁻ LN T cells from wild-type and Zbtb7b^t/t mice and in CD4⁻CD8⁺ LN cells from wild-type mice. Messenger RNA levels are normalized to β-actin mRNA and expressed as a ratio over those in wild-type CD8 cells (set to 1, not depicted). Error bars indicate experimental variation among triplicates within the experiment shown; data is representative of three separate such experiments.

(C). Expression of Runx3 mRNAs initiated at the distal (dis-Runx3) and proximal (prox-Runx3) promoters was analyzed by conventional RT-PCR in sorted naïve (CD44^lo) CD4⁺CD8⁻ LN T cells from wild-type and Zbtb7b^t/t mice and in CD4⁻CD8⁺ LN cells from wild-type mice. Wedges represent 3 fold dilutions (1.2; 0.4 and 0.12 × 10⁴ cells, respectively). β-actin mRNA expression was assessed in parallel as a control for mRNA preparation (bottom row). Data is representative of three separate experiments.

(D-F). Sorted CD44^lo CD4⁺CD8⁻ LN T cells from wild-type or Zbtb7b^t/t mice were activated by anti-CD3 and anti-CD28 on irradiated APCs in the presence of IL-2, IL-4 and antibodies against IL-12 and IFNγ (Th2 conditions) or IL-2 only (ThN conditions), and analyzed five days after stimulation. (D) Cells were stained for CD4, CD8 and intra-cellular GzmB; GzmB expression is shown on gated CD4⁺CD8⁻ Zbtb7b^t/t (plain line) or wild-type (gray filled histograms); the dashed lines show GzmB expression on wild-type CD8 cells stimulated in parallel in the same conditions. (E) IL-4 and IFNγ production was assessed by flow cytometry on cells restimulated with PMA and Ionomycin. Cells were stained for CD4, CD8 and intra-cellular IL-4 and IFNγ; contour plots are gated on CD4⁺CD8⁻ cells. Numbers indicate the percent of cells within quadrants. (F) Analyses of gene expression were conducted on Th2-differentiated effectors as in panel (B). Data is representative of three or more separate experiments for each panel.