Antigen-binding lymphocytes

8.2.2 Antigen-binding lymphocytes

A basic technique is described below by which antigen-binding lymphocytes can be visualized. Applicability of this approach is limited principally by the experimental design to ensure that non-specific binding is avoided. It is difficult in this type of experiment to include a satisfactory specificity control. The approach adopted here is to show that the number of antigen-binding lymphocytes increases after immunization.

Sheep erythrocyte rosettes

A rosette inhibition experiment with anti-mouse immunoglobulin is described at the same time as determining the number of antigen-binding lymphocytes.

Preparation in advance

1 Immunize a mouse 7 days before the experiment with 10 8 sheep erythrocytes (RBC) given intraperitoneally.

2 The anti-mouse immunoglobulin and normal rabbit serum must be absorbed with mouse liver and red blood cells, as well as sheep RBC to be sure they are free of anti-species activity.

MATERIALS AND EQUIPMENT Sheep erythrocytes (SRBC) Normal and SRBC-immunized mice Tissue culture medium Rabbit anti-mouse immunoglobulin serum (anti-Ig) Normal rabbit serum (NRS) Nylon wool 2-ml syringe barrels 2-ml plastic round-bottomed tubes

8.2LYMPHOCYTE SURFACE MEMBRANE

METHOD

1 Kill both the control and immunized mice and prepare single-cell suspensions from the spleens.

2 Remove phagocytic cells.

3 Wash the cells three times by centrifugation (150 g for 10 min at 4°C).

4 Count the number of viable lymphocytes/ml and adjust to 3 × 10 7 /ml.

5 Label tubes and add 0.1 ml aliquots of the lymphocyte suspensions as shown in the Protocol.

Protocol. Tube

Lymphocytes from:

Antiserum incubation before rosetting

1 Immune spleen

None

2 Normal spleen

None

3 Immune spleen

Anti-mouse immunoglobulin

4 Immune spleen

NRS

6 Add 0.1 ml of the appropriate sera to tubes 3 and 4. (Use the optimal dilution as determined for immunofluorescence; see Section 8.2.1.) Alternatively a full titration curve of rosette inhibition may be carried out.

7 Incubate tubes 3 and 4 at 4°C for 30 min.

8 Add 0.1 ml SRBC suspension (2.4 × 10 8 SRBC/ml) to tubes 1 and 2. Mix well.

9 Centrifuge tubes 1 and 2 at 150 g for 10 min at 4°C.

10 Add 0.3 ml of 0.5% acridine orange solution to tubes 1 and 2 if UV microscope is available (otherwise use tissue culture medium) and resuspend the cells on a vertical rotor turning at 8–10 r.p.m. for 5 min. (Alternatively, you may resuspend the cells using a Pasteur pipette. This, however, reduces the number of rosettes because of the shear forces generated.)

11 Repeat the addition of SRBC, centrifugation and resuspension for the cells pretreated with antisera (i.e. tubes 3 and 4). This time, however, add only 0.2 ml of acridine orange solution (or tissue culture medium).

12 Count the number of rosettes in each suspension using a haemocytometer and a

microscope. Sample each tube four times and count the rosettes. If you are using a microscope with UV light, live cells may be seen at the centre of the rosette by their green fluorescence (dead cells are deep red), thus confirming the cell group as a rosette rather than an aggregate of SRBC. If a UV microscope is not used then 0.1% toluidine blue may be used to visualize nucleated cells. Do not count rosettes with more than one lymphocyte, or clumped red cells without lymphocytes. This is shown in Fig. 8.2.

Calculation and evaluation of results

1 Calculate the number of rosettes/ml of suspension and from this the number of rosettes per

10 6 lymphocytes.

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C H A P T E R 8: Lymphocyte structure

Fig. 8.2 Mouse lymphocytes showing antigen-specific immunocytoadherence with sheep erythrocytes.

(a) Antigen-specific lymphocytes will bind sheep erythrocytes to their surface to form rosettes; two are shown in this field. (b) At 4°C the erythrocytes bind as a single layer. (c) The nucleus of the lymphocyte at the centre of this rosette has been stained with acridine orange and is visualized under UV and tungsten light. (d) The morphology of the rosette-forming cell may be seen after Giemsa staining of a cytocentrifuge preparation.

A rosette may be arbitrarily defined as a single lymphocyte binding five or more erythrocytes. If rosettes are incubated at 37°C, in the absence of metabolic inhibition, erythrocyte ‘caps’ will form in an analogous manner to that shown for anti-immunoglobulin.

2 Compare the number of rosettes per 10 6 lymphocytes from the normal and immune animals (tubes 1 and 2) and calculate the factor of immunization.

3 You should find that all the rosettes are blocked by the anti-immunoglobulin serum.