Regulated costimulation in the thymus is critical for T cell development: dysregulated CD28 costimulation can bypass the pre-TCR checkpoint

Abstract

Expression of CD28 is highly regulated during thymic development, with CD28 levels extremely low on immature thymocytes but increasing dramatically as CD4- CD8- cells initiate expression of TCRbeta. B7-1 and B7-2, the ligands for CD28, have a restricted distribution in the thymic cortex where immature thymocytes reside and are more highly expressed in the medulla where the most mature thymocytes are located. To determine the importance of this regulated CD28/B7 expression for T cell development, we examined the effect of induced CD28 signaling of immature thymocytes in CD28/B7-2 double-transgenic mice. Strikingly, we found that differentiation to the CD4+ CD8+ stage in CD28/B7-2 transgenics proceeds independent of the requirement for TCRbeta expression manifest in wild-type thymocytes, occurring even in Rag- or CD3epsilon- knockouts. These findings indicate that signaling of immature thymocytes through CD28 in the absence of TCR- or pre-TCR-derived signals can promote an aberrant pathway of T cell differentiation and highlight the importance of finely regulated physiologic expression of CD28 and B7 in maintaining integrity of the "beta" checkpoint for pre-TCR/TCR-dependent thymic differentiation.

Figure 1

CD28 expression during thymocyte development A. Thymocytes from B6 mice were stained with biotin-conjugated anti-CD4, FITC anti-CD8 and PE anti-CD28. Histograms show CD28 staining on CD4⁻CD8⁻ DN, CD4⁺CD8⁺ DP, CD4⁺SP and CD8⁺SP thymocytes (black). Staining with an isotype-matched control antibody is also shown (gray). For the CD28 staining, the mean fluorescence intensity (MFI) for bracketed regions is indicated to the right of each figure. B. Thymocytes were labeled with anti-CD3, -TCRβ, -CD4, -CD8, -B220, -TCRγ, -MAC-1, -NK1.1, -GR.1, TER-119 and –CD11c (all biotinylated and detected with strepavidin-PerCP), FITC anti-CD25, PE-anti-CD28 and APC anti-CD44. PerCP-negative cells were analyzed for expression of CD25 versus CD44. Histograms show CD28 staining on CD44⁺CD25⁻ (DN1), CD44⁺CD25⁺ (DN2), CD44⁻CD25⁺ (DN3) and CD44⁻CD25⁻ (DN4) thymocytes. Staining with an isotype-matched control antibody is also shown (gray). For the CD28 staining, the percent population (when other than 100%) and MFI for bracketed regions are indicated to the right of each figure.

Figure 2

Coordinate expression of TCRβ and CD28 in DN thymocytes. A. Thymocytes were labeled with anti-CD3, -TCRβ, -CD4, -CD8, -B220, -TCRγ, -MAC-1, -NK1.1, -GR.1, TER-119 and –CD11c (all biotinylated and detected with strepavidin-PerCP), PE-anti-CD28 and APC-anti-CD25 followed by intracellular staining with FITC anti-TCRβ. PerCP-negative, CD25⁺ cells were analyzed for expression of CD28 and intracellular TCRβ. Histograms show CD28 and intracellular TCRβ staining on TCRβ^neg(gray) and TCRβ^hi (black) cells. B. Increased expression of CD28 induced on Rag-2 KO thymocytes by in vivo anti-CD3 treatment. 12-week old Rag-2 KO mice were injected with 300 μg of anti-CD3 (2C11) i.p. Thymocytes were labeled with FITC anti-CD8, APC anti-CD4 and PE anti-CD28. Histograms in the left panel show CD28 staining on CD4⁻CD8⁻ DN thymocytes from a Rag-2 KO 8 days after injection of anti-CD3 (solid black line), a control Rag-2 KO (dotted black line) and a wildtype B6 (gray filled). Staining with an isotype-matched PE control antibody is shown for each mouse (dashed, stippled and solid gray lines). Histograms in the right panel show CD28 staining on CD4⁺CD8⁺ DP thymocytes from a Rag-2 KO 8 days after injection of anti-CD3 (solid black line) and a control B6 (solid gray line). Staining with an isotype-matched PE control antibody is shown for each mouse (dashed and solid gray lines).

Figure 3

Thymic B7-2 expression patterns in wildtype, B7-1/B7-2 knockout, irradiated B7-2 tg and B7-2 tg mice. Sections of frozen thymus tissue from 7–14 week old mice or from an irradiated B7-2 tg mouse 7–14 days after reconstitution with CD28.FL tg bone marrow were stained with anti-B7-2. Medullary regions of the thymus are identified by staining with anti-keratin 14 and are identified in the figure with an “M”. B7-2 reactivity was detected with Texas Red conjugated anti-Ig and keratin 14 reactivity was detected with FITC conjugated anti Ig as described in Materials and Methods

Figure 4

CD28 expression on thymocytes from CD28.FL transgenic mice. A. Thymocytes were labeled as described in Fig. 1B. Histograms show CD28 staining on CD44⁺CD25⁻ (DN1), CD44⁺CD25⁺ (DN2), CD44⁻CD25⁺ (DN3) and CD44⁻CD25⁻ (DN4) thymocytes from wildtype (solid gray line) and CD28.FL (solid black line) mice. Staining with an isotype-matched PE control antibody is shown for the CD28.FL thymocytes (dashed gray lines). For CD28 staining, the mean fluorescent intensity (MFI) of wildtype and CD28.FL transgenic thymocytes is indicated to the right of each figure. B. Thymocytes from wildtype and CD28.FL transgenic mice were stained with biotin-conjugated anti-CD4, FITC anti-CD8 and PE anti-CD28. Histograms show CD28 staining on CD4⁺CD8⁺ DP, CD4⁺SP and CD8⁺SP thymocytes from wildtype (solid gray line) and CD28.FL (solid black line) mice. The MFI for CD28-stained cells are indicated to the right of each figure. Staining with an isotype-matched PE control antibody is shown for the CD28.FL thymocytes (dashed gray lines).

Figure 5

Altered thymocyte profile in CD28.FL/B7-2 double transgenic mice. Thymocytes from wildtype, CD28.FL, B7-2 Line 7, and CD28.FL/B7-2, double transgenic mice were stained with PE anti-CD4 and FITC anti-CD8 mAb. Results are representative of four independent experiments.

Figure 6

Altered frequency of thymocyte subpopulations in CD28.FL/B7-2 double transgenic mice. A. Analysis of thymus cellularity and frequency of DN, DP, CD4 SP, and CD8 SP thymocytes in wildtype (striped), B7.2 tg (white), CD28.WT tg (gray) and CD28.FL/B7.2 double transgenic mice (black). Data shown are derived from analysis of 3–5 mice per group and represent the mean ± s.e.m. B. Increased proportion of DN 4 thymocytes in CD28.FL/B7.2 transgenic mice. Thymocytes from wildtype, B7 tg, CD28.FL tg, and CD28.FL/B7.2 double transgenic mice were labeled with anti-CD3, -TCRβ, -CD4, -CD8, -B220, -TCRγ, -MAC-1, -NK1.1, -GR.1, TER-119 and –CD11c (all biotinylated and detected with strepavidin-PerCP), PE anti-CD25, and APC anti-CD44. PerCP-negative cells were analyzed for expression of CD25 versus CD44.

Figure 7

The majority of CD28.FL/B7-2 DP thymocytes do not express TCRβ. A. Thymocytes from wildtype, CD28.FL/B7-2, CD28.TM/B7-2, and CD28.Y198F/B7.2 double transgenic mice were surface stained with antibodies specific for CD4, CD8 and TCRβ. TCRβ expression on CD4⁺CD8⁺ gated cells is shown. The gray histogram shows staining with an isotype-matched biotinylated control antibody. Results are representative of four independent experiments. B. Thymocytes from wild type, TCRβ KO, and CD28.FL/B7-2 double transgenic mice were stained with antibodies specific for CD4 and CD8 followed by intracellular staining for TCRβ. Total TCRβ expression in CD4⁺CD8⁺ gated cells is shown. Results are representative of three independent experiments.

Figure 8

CD28.FL/B7-2 transgenes rescue development to the DP stage in Rag-2 and CD3ɛ KO mice. A. CD8 versus CD4 thymic profiles from Rag-2 KO and CD28.FL/B7-2 double transgenic mice on a Rag-2 KO background. B. CD8 versus CD4 thymic profiles from CD3ɛ KO and CD28.FL/B7-2 double transgenic mice on a CD3ɛ KO background. Total thymocyte numbers are indicated for each mouse. Results are representative of three independent experiments for each KO background.

Figure 9

A. Development to the DP stage is rescued in CD28.FL/CD3ɛ KO→B7-2/CD3ɛ KO chimeras. Chimeras were generated by reconstituting B7-2/CD3e KO mice with CD28.FL/CD3ɛ KO bone marrow. Controls chimeras were generated by reconstituting CD3ɛ KO mice with CD3ɛ KO bone marrow. Results are representative of two independent experiments. B. Development to the DP stage in CD28.FL/CD3ɛ KO mice. Results are representative of 4 independent experiments.