Amino acid requirements of proliferating chicken lymphocytes

Upon activation, T and B lymphocytes undergo cell proliferation resulting in increased protein synthesis for which amino acids are required. T and B lymphocytes have different functions that result in the synthesis of different proteins and the need for different amino acids. The present work determined a method for the proliferation of separated chicken T and B lymphocytes, the effect of amino acid supplementation on proliferating T lymphocytes, and amino acid utilization by proliferating lymphocytes. Proliferation of T and B lymphocytes was optimal when 0.1% chicken serum was used as the serum source in the media. Phytohemagglutinin (PHA) was determined to be a T lymphocyte mitogen, lipopolysaccharide was determined to be a B lymphocyte mitogen, and pokeweed mitogen (PWM) was determined to be both a T and B lymphocyte mitogen. The relationship between PHA stimulated T lymphocyte proliferation and individual amino acid concentrations were determined. Asparagine was not required for T lymphocyte proliferation. Stimulation indexes (SI) were higher when arginine and tryptophan concentrations in the media were above what is normally found in RPMI. Linear relationships existed between SIs and threonine and methionine media concentrations. A custom media amino acid formulation did not produce statistically higher SIs than RPMI, but results suggest that RPMI amino acid concentrations are not optimal for T lymphocyte proliferation. The amino acid depletion in the media for PWM stimulated T and B lymphocytes was measured to determine if the amino acid requirements for proliferation were being met. PWM stimulation led to increased cell entry into the S phase of the cell cycle, increased cell death, and release of amino acids into the media. Media from non-stimulated lymphocytes had higher amino acid concentrations for the majority of amino acids than RPMI. Media from PWM stimulated lymphocytes had essential amino acid concentrations similar to those in RPMI. Glutamine and asparagine in media from PWM stimulated T lymphocytes and serine, glutamine, glycine, isoleucine, leucine, tyrosine, and tryptophan concentrations in media from PWM stimulated B lymphocytes were lower than in RPMI. This suggests that the majority of amino acids required for lymphocyte proliferation are supplied by cell death.