B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4 - PubMed (original) (raw)

B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4

D A Mandelbrot et al. J Clin Invest. 2001 Apr.

Abstract

To examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4(-/-) mice resemble CD28(-/-) mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4(-/-) mice. The proliferation of CD28/CTLA4(-/-) T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4(-/-) T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4(-/-) cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

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Figures

Figure 1

Figure 1

Generation of mice lacking both CD28 and CTLA4. (a) Structure of the CD28 targeting construct. The hygromycin-resistance gene is flanked by small parts of exon 2 and 3. The thymidine kinase gene (TK) lies external to the 5′ genomic fragment. The intron-exon organization of the genomic clone is shown. Exons coding for the extracellular, transmembrane, and intracellular domain of CD28 are depicted as filled rectangles. The 0.9-kb cDNA probe used for Southern blot analysis is indicated as a thin bar and is located in exon 4, external to the genomic DNA fragment used in the targeting construct. Gene targeting in ES cells by homologous recombination results in the replacement of part of exon 2, the intron between exon 2 and 3, and part of exon 3 by the hygromycin gene. Southern blot analysis uses the acquisition of an additional _Eco_RV site in the hygromycin gene (shown in bold) to distinguish genomic and targeted DNA. (b) Southern blot analysis of the CD28 locus. Tail DNA from wild-type (+/+), heterozygotes (+/–), and CD28/CTLA4 double-knockout (–/–) littermates were digested with _Eco_RV and hybridized with the probe indicated in Figure 1a, demonstrating the two bands indicative of the endogenous (12 kb) or targeted (5 kb) configuration. (c) Analysis of CD28 and CTLA4 expression. Cell-surface staining of CD28 is assayed on naive CD4 T cells; CTLA4 is stained intracellularly in activated CD4 cells. Thick lines depict staining with anti-CD28 or anti–CTLA4; thin lines depict staining with the isotype-matched hamster IgG control Ab.

Figure 2

Figure 2

T-cell activation in naive mice. Freshly isolated lymph node cells from the indicated strains were stained for CD4 and CD69 and analyzed by flow cytometry. The percentages to the right of the dot plots are the proportion of CD4+ cells that are CD69+. This experiment is representative of five mice of each strain.

Figure 3

Figure 3

Proliferation of CD4+ T cells from wild-type (WT), CD28–/–, and CD28/CTLA4–/– mice in the presence of wild-type APCs. (a) Primary stimulation of CD4+ T cells with the indicated dilutions of anti-CD3. Proliferation on day 3 is shown. Data are representative of five experiments. (b) Secondary stimulation of CD4+ T cells with the indicated dilutions of anti-CD3. Proliferation on day 2 is shown. Data are representative of three experiments.

Figure 4

Figure 4

Cytokine production on day 2 after secondary stimulation of wild-type and CD28/CTLA4–/– CD4+ T cells with the indicated dilutions of anti-CD3. Data are representative of four experiments.

Figure 5

Figure 5

Proliferation of CD4+ T cells from wild-type, CD28–/–, and CD28/CTLA4–/– mice in the presence of syngeneic APCs. T cells from the indicated strains were stimulated in the presence of wild-type APCs with or without CTLA4-Ig or in the presence of B7-deficient APCs. Proliferation on day 3 is shown. Data are representative of five experiments.

Figure 6

Figure 6

Proliferation of CD4+ T cells from wild-type, CD28–/–, and CD28/CTLA4–/– mice in the presence of the indicated concentration of anti-CD3 and CHO cells transfected with I-Ad or I-Ad plus the indicated B7 molecules. Proliferation on day 3 is shown. Data are representative of three experiments.

Figure 7

Figure 7

Cardiac transplantation studies. (a) BALB/c (H-2d) hearts were transplanted to B6 (H-2b) wild-type or CD28/CTLA4–/– recipients with or without CTLA4-Ig treatment. Graft survival was slightly prolonged in CD28/CTLA4–/– recipients (MST 15.5 days, n = 10), but there was no statistical significance compared with wild-type recipients (MST 9.4 days, n = 5; P = NS). CTLA4-Ig treatment in wild-type recipients prolonged graft survival significantly (MST > 90 days, n = 3; P < 0.02) compared with nontreated wild-type recipients. CTLA4-Ig treatment also prolonged graft survival in CD28/CTLA4–/– recipients significantly (MST 31.5 days, n = 11; P < 0.05) compared with nontreated CD28/CTLA4–/– recipients; however, that prolongation was much less than in wild-type recipients (P < 0.005). (b) Vascularized B6 (H-2b) hearts were transplanted to BALB/c (H-2d) wild-type, CD28–/–, or CD28/CTLA4–/– recipients. The graft survival in CD28–/– recipients was prolonged (MST 22.6 days, n = 5) compared with both wild-type (MST 9.0 days, n = 7; P < 0.001) and CD28/CTLA4–/– recipients (MST 10.3 days, n = 6; P < 0.002). CD28/CTLA4–/– recipients rejected B6 heart grafts at the same tempo as wild-type recipients.

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