In Vitro Study Of Activation Mechanisms Of Human Skin αβT Cells And γδT Cells In The Process Of Immune Activation Associated With Wound Healing Startup Phase

Objectives The activation of skin T cells play important role in the initiatingstage of wound healing, and the activation failure of human skin T cells may be amajor cause of chronic wounds’ formation. It was reported that JunctionalAdhesion Molecule-Like protein (JAML) which expressed on the murine skin Tcells was a crucial costimulatory molecule for the activation of skin T cells in theinitiating stage of wound healing. JAML also can be found in human body, andthe role of JAML in human body is not verified. Hence, we isolated human skinαβT cells and γδT cells，interfere them with activators or retardants associatedwith JAML, then tested their reactions, observed whether JAML can be a crucialcostimulatory molecule for the activation of human skin T cells in the initiatingstage of wound healing. All those works are supposed to acquire more knowledgeabout human skin T cells activation in wound and insight into immunologicalmechanism in wound healing.Methods1.Isolating human skin T cells from tissue, observing cell viability,checking the quantity and phenotype of human skin T cells.2. Measuring IL-2and IGF-1production levels in peripheral T cells and human skin T cells after fullstimulation with100ng/ml PMA and1000ng/ml ionomycin.3. Amplifyingperipheral T cells and human skin T cells and then measuring their expansionpotential in vitro.4. Stimulating human skin T cells with different dose ofanti-CD3mAb（0.1μg/ml、0.5μg/ml、1μg/ml、2μg/ml、5μg/ml、10μg/ml）,measuring their expressions of the activation markers such as CD69, CD25andtheir secretion levels of IL-2and IGF-1.5. Stimulating human skin T cells in thepresence of0.5μg/ml anti-CD3mAb and4μg/ml recombinant human coxsackie and adenovirus receptor(CAR), or in the presence of4μg/ml CAR alone, andexpressions of costimulatory molecules including CD40, measuring theirexpressions of the activation markers such as CD69, CD25and their secretionlevels of IL-2and IGF-1.6. Isolating and purifying human skin αβT cells andhuman skin γδT cells, measuring their expressions of JAML and CD28.7.Purifying human skin γδT cells, stimulating them with0.5μg/ml anti-CD3mAband4μg/ml CAR, or with4μg/ml CAR alone, or with0.5μg/ml anti-CD3mAbalone, and then measuring their expressions of the activation markers andcostimulatory molecule such as CD69, CD25,CD80,CD86,JAML and theirsecretion levels of IL-2and IGF-1.8. Purifying human skin αβT cells, stimulatingthem with0.5μg/ml anti-CD3mAb and4μg/ml CAR, and then measuring theirexpressions of the activation markers and costimulatory molecule such as CD69,CD25,CD80,CD86,JAML and their secretion levels of IL-2and IGF-1. Finally,stimulating human skin αβT cells with4μg/ml CAR alone, or0.5μg/mlanti-CD3mAb alone, measuring their expressions of the costimulatory moleculeCD28.9. Stimulating human skin αβT cells with activational human skin γδT cellsand0.5μg/ml anti-CD3mAb, measuring their expressions of the activationmarkers such as CD69, CD25.Results1. The cell viability is good after human skin T cells were isolated fromtissue. Averagely, we can get （2.63±1.72）×107T cells from per gram epidermisand （1.95±1.31）×107T cells from per gram dermis. In epidermis, αβT cells: γδTcells was7.3±1.08, and in dermis, αβT cells: γδT cells was6.7±1.31. There wereno statistically significant differences between epidermis and dermis for their Tcells’ quantity and ratio. Moreover, all the human skin γδT cells were exclusiveVδ1+cells, no Vδ2+cells were found.2. After full stimulation with PMA andionomycin, the production levels of IL-2and IGF-1in peripheral T cells andhuman skin T cells rise significantly, but there were no statistically significantdifferences between them.3. Peripheral αβT cells, human skin αβT cells andperipheral γδT cells were amplified successfully, while human skin γδT cells could not be amplified. The proliferation multiple of Peripheral αβT cells andhuman skin αβT cells are more than that of peripheral γδT cells significantly.4.Human skin T cells were activated by the anti-CD3mAb when their dose wasmore than2μg/ml, the CD69, CD25expressions on the cells and the IL-2, IGF-1secretion levels from the cells rose significantly.5. Human skin T cells wereactivated by0.5μg/ml anti-CD3mAb and4μg/ml CAR, the CD69, CD25expressions on the cells and the IL-2, IGF-1secretion levels from the cells rosesignificantly. While4μg/ml CAR alone could not activate human skin T cells.6.Human skin αβT cells only expressed CD28, while did not express JAML. Theexpression percentage of CD28on human skin αβT cells was45.59±5.73%.Human skin γδT cells only expressed JAML, while did not express CD28. Theexpression percentage of JAML on human skin γδT cells was8.56±2.19%.7.0.5μg/ml anti-CD3mAb and4μg/ml CAR could activate human skin γδT cells, theexpressions of CD25、CD69、CD80、CD86、JAML on these cells and the IL-2,IGF-1secretion levels from these cells rose significantly.0.5μg/ml anti-CD3mAbalone or4μg/ml CAR alone could not activate human skin γδT cells, but0.5μg/mlanti-CD3mAb alone could induce JAML expression on the cells increase.8.0.5μg/ml anti-CD3mAb and4μg/ml CAR could not activate human skin αβT cells,but could induce CD28expression on the cells increase.0.5μg/ml anti-CD3mAbalone could induce CD28expression on the cells increase also, and4μg/ml CARalone had no effect on CD28expression on the cells.9. The activational humanskin γδT cells and0.5μg/ml anti-CD3mAb could activate human skin αβT cells,the expressions of CD25、CD69on human skin αβT cells rose significantly.Conclusions Human skin T cells are composed of αβT cells and γδT cells, JAMLis an important costimulatory molecule in the activation of human skin T cells, itcan transmit costimulatory signals to human skin γδT cells and promote them tobe activated, and the activational human skin γδT cells can transmit costimulatorysignals to human skin αβT cells and promote them to be activated. All the dataimply that the activation of human skin T cells in the initiating stage of wound healing may perform as such process, at first human skin γδT cells are activatedby the help of JAML, and then human skin αβT cells are activated by the help ofthe activational human skin γδT cells.