Erythrocyte E rosette

The most widely used rosetting technique in human immunology is the interaction between the sheep erythrocyte and human lymphocytes of the thymic or T cell lineage. This interaction, first appreciated by the laboratories of Brain, Coombs and Wybran, was a major step in the capacity to identify, separate and characterize the T and B cell lineages of lymphocytes in normal physiology and disease. The assay is performed by the incubation of a population of mononuclear cells obtained by a preliminary processing of blood, usually by differential density cen-trifugation of peripheral blood or of organ suspensions, with sufficient sheep erythrocytes to have a ratio of 50:1 or 100:1 erythrocytes to mononuclear cells. The suspension is incubated briefly at 37°C then centrifuged at 200 g and a further incubation for 1-2 hours or overnight at 4°C. The cells are gently suspended and counted in a hemocytometer.

The rosettes formed are more stable in a high protein solution such as 10% human AB serum or 25% bovine serum albumin. The prior treatment of the sheep erythrocytes with neuraminidase also increases the stability. Kaplan found that prior treatment of the sheep erythrocytes with neuraminidase also increases the stability. Kaplan found that prior treatment of the sheep erythrocytes with a sulfhydryl reagent such as 2-amino-ethyisothiouronium (AET) obviates the differences between batches of sheep erythrocytes and enhances the stability of the rosette. Recently Ocklind suggested the use of 1% w/v of polyethylene glycol 10 000 molecular weight in the medium allows for the detection of 97% of T cells. These modifications have proved very useful, particularly when the method is used to isolate T cells in preparative techniques.

The molecule on the surface of T cells responsible for this interaction is the 50 kDa glycoprotein CD2, present on more than 95% of thymocytes and all peripheral T cells. It binds to a protein present on sheep erythrocytes designated TllTS, which is the homolog of the human protein, lymphocyte function-associated antigen 3 (LFA-3). This latter protein is present on human erythrocytes and accounts for the observation that human erythrocytes can form rosettes with human thymocytes. The utility of the sheep erythrocyte over the human in this assay and its reaction with peripheral T cells is due to a three-to five-fold higher ligand density on its surface. The CD2 molecule is a T cell activation molecule involved in one alternate pathway. Thus T cells isolated by this technique may under certain circumstances be activated by the procedure. This phenomenon must be kept in mind when designing experiments or making conclusions about the in vivo state of such populations.

In other species E rosette formation is less successful. Limited use of the technique occurs with sheep E and primate T cells. The cat T cell forms rosettes with guinea pig and gerbil erythrocytes.

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