Cutting edge: A double-mutant knockin of the CD28 YMNM and PYAP motifs reveals a critical role for the YMNM motif in regulation of T cell proliferation and Bcl-xL expression

Abstract

CD28 is a critical regulator of T cell function, augmenting proliferation, cytokine secretion, and cell survival. Our previous work using knockin mice expressing point mutations in CD28 demonstrated that the distal proline motif was primarily responsible for much of CD28 function, whereas in marked contrast to prior studies, mutation of the PI3K-binding motif had little discernible effect. In this study, we examined the phenotype of mice in which both motifs are simultaneously mutated. We found that mutation of the PYAP motif unmasks a critical role for the proximal tyrosine motif in regulating T cell proliferation and expression of Bcl-xL but not cytokine secretion. In addition, we demonstrated that, although function is more severely impaired in the double mutant than in either single mutant, there remained residual CD28-dependent responses, definitively establishing that additional motifs can partially mediate CD28 function.

Figure 1

Synergistic impairment of proliferation in CD28-Y170F/AYAA double mutant mice. A) Splenocytes were isolated from mice of each genotype and stimulated with graded doses of α-CD3. B) Splenocytes were stimulated as in panel B but with the inclusion of α-CD28 (1 μg/ml). C) Splenocytes were stimulated with α-CD3 (1 μg/ml) and α-CD28 (1 μg/ml) for 48 hours. D) Splenocytes were stimulates with either PMA (5 ng/ml) alone, in combination with ionomycin (0.4 μg/ml) or α-CD28 (1 μg/ml) for 48 hours. E) Splenocytes were stimulated with either α-CD3 (1 μg/ml) alone, or in the presence of murine CTLA4Ig (10 μg/ml) with graded doses of α-CD28 (0, 0.01, 0.1 or 1.0 μg/ml) for 48 hours. F) Naïve CD4+ T cells from the D011.10 transgenic on the Balb/c background were isolated by MACS separation (final purity >95% for each strain (data not shown)) and stimulated with graded doses of OVA_323-339 peptide for 72 hours in the presence of irradiated T-depleted splenocytes as a source of APC. For all assays shown, proliferation was measured by tritiated thymidine incorporation. The mean ± s.d. of quadruplicate wells is shown. Representative data from 3 independent experiments is presented. *= p< 0.05, ** = p < 0.005, ***= p<0.0001. For panel A, the statistical comparisons shown are for 1 and 10 μg/ml α-CD3 comparing the Y170F/AYAA vs AYAA and the AYAA vs wild type. For panel B the statistical comparisons shown are for 1 and 10 μg/ml α-CD3 comparing between CD28 −/− vs Y170F/AYAA and CD28-Y170F/AYAA vs CD28-AYAA. For panel F, the statistical comparisons are as for panel B but at 0.3 and 3.0 μg/ml OVA peptide.

Figure 2

Cytokine secretion is regulated predominantly by the PYAP motif. Splenocytes were isolated from mice of each genotype and stimulated with α-CD3 (0.25 μg/ml) and α-CD28 (1.0 μg/ml). Culture supernatant was collected at 48 following stimulation and assayed for IL-2 and IFN-γ. Presented is the combined means ± standard error of 3 independent experiments, where each experiment was performed in triplicate. *= p< 0.05, ** = p < 0.01. *** p < 0.005.

Figure 3

Synergistic regulation of Bcl-X_l expression by the Y170 and PYAP motifs of CD28. Splenocytes were isolated from mice of each genotype then stimulated with α-CD3 (0.25 μg/ml) and α-CD28 (1 μg/ml) for 48 hours. Viability was measured by 7-AAD staining. Expression of Bcl-X_l was determined by intracellular staining. All samples were stained with α-CD4 results of the CD4+ cells are shown. The combined mean ± standard error of 3 independent experiments is shown. *= p< 0.05 ** = p < 0.005.

Figure 4

Allergic airway inflammation is regulated by alternative motifs whereas germinal center formation depends primarily on the PYAP motif. Mice of each genotype were immunized and challenged with OVA as described in the Methods. A) Total cell counts and B) cell differentials obtained from bronchoalveolar lavages. C) Representative 10 × fields of tissue sections stained with H & E. D) Sections of spleen were stained with PNA and α-IgD and evaluated for germinal centers. The # of germinal centers per 10× field is presented. Representative data from 3 independent experiments are shown. * = p< 0.05 as compared to wild type.