We also have shown that there is an important gene dosage effect in the mutant phenotype as a twofold change in the expression level of the mutant protein was sufficient to essentially normalize cell proliferation, but not IL-2 production

We also have shown that there is an important gene dosage effect in the mutant phenotype as a twofold change in the expression level of the mutant protein was sufficient to essentially normalize cell proliferation, but not IL-2 production. concomitant with T cell receptor engagement. CD28 is perhaps the most important and best-studied costimulatory receptor and is unique in its potency to augment IL-2 production. Ligation of CD28 increases cell proliferation and enhances cell survival as well as regulating production of multiple T cellCderived cytokines (1). Consistent with this, T cells from mice deficient in CD28 have impaired proliferative responses, make little IL-2, and have increased susceptibility to apoptosis (2C5). Although the importance of CD28 in T cell function is well established, many questions remain as to the mechanism by which CD28 mediates its biological effects. CD28 is most critical in the activation of resting, naive T cells. Furthermore, the primary outcomes of CD28 signaling are changes in cell proliferation and survival. Previous work examining the structural motifs responsible for CD28 function have relied on either transformed cell lines, retroviral transduction of primary T cells, or reconstitution of CD28-deficient mice with transgenic SR1078 constructs under the control of heterologous promoters. Although these have led to important insights, the results have often been conflicting and each has experimental limitations that make extrapolation to primary, naive T cells problematic. CD28 possesses no intrinsic enzymatic activity; however, discrete regions within the cytoplasmic tail interact with intracellular adaptor proteins and enzymes to initiate signaling. Mutagenesis studies have identified two regions of the tail as being of particular importance. A tyrosine-based motif in the membrane proximal region of the cytoplasmic domain binds and activates PI-3 kinase, as well as interacting with the adaptor proteins Grb-2 and GADS (6C11). SR1078 Less well characterized is a proline-based motif in the distal LEPR portion of the cytoplasmic domain. This region can interact with SH3 domain containing proteins including the Src family kinases Lck and Fyn to initiate signaling (7, 12). Recent studies have been conflicting as to the importance of this motif in CD28-dependent responses, with some suggesting that it is critical for enhancing proliferation and IL-2 secretion, whereas others indicate that it is dispensable for these functions but required for IL-4 production (13C16). Moreover, the requirement for this motif in the generation of a complex, in vivo immune response is unknown. Given the importance of CD28 in the activation of naive T cells and that CD28 expression is itself regulated by cell activation, we generated a targeted knockin mouse expressing a mutation of the distal proline motif to determine its function. The CD28 P187,190A knockin (CD28-AYAA KI) mice thus express only the mutant form of CD28. In contrast with other experimental approaches, the genetic elements that regulate transcription are preserved and thus the mutant protein is expressed and regulated in a manner identical to the endogenous gene product. This strategy permits both in vitro and in vivo analysis of the functional consequences of the mutation with less concern that the results are the result of aberrant patterns of expression. In these studies, we find that the proline motif is critical for normal CD28-dependent regulation of IL-2 production and is absolutely required for antibody production and germinal center formation in vivo. Furthermore, we demonstrate that gene dosage is important in determining the phenotype of this mutation. RESULTS Generation of CD28 P187,190A knockin mice To generate mice expressing mutant CD28 under endogenous regulatory control, we SR1078 constructed a gene targeting vector containing exon IV of CD28 and flanking intronic sequences (Fig. 1 A). Specific mutations were introduced to substitute alanine for proline at residues 187 and 190, thus disrupting the motif.