There are many strategies for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC). T cells that have recently been activated through their T cell receptor (TCR) will express a series of activation markers at different time points following activation. Activation markers include receptors such as chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins. Some of these molecules have established functions in T cell biology, while the relevance or function of others remains elusive. Flow cytometry is the method of choice for evaluating various types of surface or intracellular markers that indicate the activation status of T cells. However, what are these markers, what is their function in T cell biology, what T cell populations will express them, and when can they be assessed are key questions to address when deciding which markers are best for a given assay and question of interest.
In this article, the first of a short series, I will discuss two of the most commonly used immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.
Immediate Early Activation Markers:
CD69 (AIM, Leu23, MLR3) is a signaling membrane glycoprotein involved in inducing T cell proliferation. CD69 is expressed at very low levels on resting CD4+ or CD8+ T cells in PBMC (<5-10%), and is one of the earliest assessable activation markers, being rapidly upregulated on CD4+ or CD8+ T cells within 1 hour of TCR stimulation or other T cell activators such as phorbol esters via a protein kinase C (PKC) dependant pathway. Expression of CD69 peaks by 16-24 hours and then declines, being barely detectable 72 hours after the stimulus has been withdrawn.
The inability to upregulate CD69 following TCR activation may be associated with T cell dysfunction. For instance, Critchley-Thorne et. al, showed that PBMC T cells from metastatic melanoma patients with lower responsiveness to interferons had reduced CD69 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients. Thus CD69 expression may be a measure of T cell dysfunction in human disease.
CD40L (CD154) is a member of the TNF-receptor superfamily that functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs). The CD40L-CD40 ligation results in the activation of multiple downstream pathways including the MAPK (JNK, p38, ERK1/2), NF-ĸB, and STAT3 transcription factors. CD40L expression is quickly upregulated within 1-2 hours after TCR stimulation via the transcription factors NFAT and AP-1. CD40L expression peaks near 6 hours after stimulation, and declines by 16-24hrs. CD40L expression however is biphasic, and the addition of anti-CD28 or IL-2 along with TCR stimulation leads to sustained expression for several days (Snyder et. al., 2007).
Expression of CD40L on resting PBMC CD4+ or CD8+ T cells from healthy donors is very low (<1%). However this percentage has been shown to be significantly increased on up to 17% of CD4+ T cells and 21% of CD8+ T cells in patients with active SLE, and these differences between healthy and SLE patients were also seen following anti-CD3 stimulation of PBMCs (Desai-Mehta, et. al, 1996). The review below by Daoussis et. al, discusses the role of CD40L expression in several other human diseases.
In summary, CD69 and CD40L are both rapidly induced following T cell activation and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in various human diseases. Understanding the biology of T cell activation markers will allow for the best application of these markers to specific experimental questions and assay types.
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