Immunoglobulin light chain determinants on unstimulated and stimulated human blood lymphocytes, assayed by indirect immunofluorescence

Abstract

The occurrence of immunoglobulin determinants on the surface of lymphocytes from human blood was assessed by indirect immunofluorescent staining of living cells after cultivation with phytohaemagglutinin or other stimulants. While antisera to γ, μ or α-determinants only stained a few cells, antisera to light chain determinants stained a larger proportion of the cells. Positive staining was recognized as `ring' staining comprising smaller or larger parts of the cell surface. The specificity of staining was ascertained by several types of controls.

After 48 hours of cultivation, anti-κ serum, applied at dilutions of 1:10–1:16 stained about 35–50 per cent and anti-λ serum about 15–20 per cent of the cells in the PHA cultures but only 3–5 per cent in the cultures incubated without PHA. When the antisera were applied at higher concentrations, positive light chain staining was also seen in the unstimulated cultures. At the highest concentrations, which could be used without increasing the non-specific background, the maximum number of κ-positive cells in the unstimulated cultures was approximately 25 per cent. Antiserum titrations showed that about 5–10 times less antiserum was needed to stain the optimal fraction of PHA treated cells. No increased staining of heavy chain determinants was achieved by increasing antiserum concentrations under the present conditions.

Similar results were obtained with lymphocytes stimulated by 3 days of incubation with concanavalin A, or by 6–7 days of incubation under mixed culture conditions. Lymphocytes of a tuberculin positive donor also gave increased staining for light chain determinants after incubation for 6–7 days with antigen (PPD).

The results indicate that lymphocyte stimulation is accompanied by increased amounts of surface bound immunoglobulins. At the present stage of knowledge, several explanations may account for the fact that light chain determinants are primarily accessible for staining.

The above results were obtained under conditions in which no protein was present in the washings performed during processing for immunofluorescence. In the presence of low concentrations of protein more than 60 per cent of both unstimulated and stimulated cells stained for light chain determinants, while staining for heavy chain determinants remained unchanged and at a low level. It is possible that protein-free washing removed a more loosely adsorbed immunoglobulin fraction passed on from producing to neighbouring non-producing cells.