The Construction and Characterization of a CD28 Mutant

By Grace Messah Mentor: Dr. Jonathan Green Pulmonary Division, Washington University School of Medicine, St. Louis, MO

T lymphocytes play a vital role in the body's ability to detect and respond to the invasion of foreign and potentially harmful particles or organisms. As a foreign substance, or antigen, enters the body, it is engulfed by an antigen presenting cell (APC), which has class II major histocompatability complexes (MHCs) on its surface. The APC breaks down the antigen, combines parts of it with the MHCs, and transports the MHC/antigen complexes to the cell surface where they can be recognized by T cell receptors (TCRs). However, in order for the T lymphocyte to be optimally activated and perform its effecter functions, another surface co-stimulatory receptor, CD28, must be simultaneously engaged. This model, called the 2-signal hypothesis, states that a signal must be transduced through both the TCR and CD28 for the T cell to be activated. If both signals are not transmitted, the T lymphocyte will go into a state where it will not be completely activated and will become unresponsive to a similar stimulus.

The means by which CD28 performs its co-stimulatory and signaling functions is one of the topics currently under investigation in Dr. Green's Lab. Experiments performed previously identified a region in the cytoplasmic tail of CD28 that contains two proline amino acids which, when mutated to alanines, resulted in loss of CD28 function. However, between the two prolines is a tyrosine, an amino acid that may be involved in signaling pathways because of its ability to be phosphorylated. The question thus arises as to whether the loss of function should be attributed to the mutation of the prolines, or whether it may have resulted from a possible disruption of the tyrosine.

The research that I performed this summer was designed to answer this question through the creation of a mutant construct of CD28 in which only the tyrosine was mutated. This was accomplished through PCR based site directed mutagenesis. By changing a single base pair in the sequence of CD28, the tyrosine was converted into a phenylalanine, an amino acid that shares the same structure as tyrosine except for the hydroxyl group that allows the tyrosine to be phosphorylated. This mutant construct was then cloned and sequenced to confirm the mutation. Once successful mutagenesis was confirmed, the construct was then cloned on a large scale, and the plasmid containing the mutant CD28 was purified before being transfected into phoenix cells. The phoenix cells are designed to produce retroviruses containing the mutant construct, which are then used to transfect T lymphocytes lacking CD28. These cells will then be analyzed using a proliferation assay to determine whether they behave as normal T cells or whether the mutation in the tyrosine results in a loss of CD28's co-stimulatory function.