Lymphocyte circulation

9.4 Lymphocyte circulation

Lymphocytes do not circulate in a random manner athere is a functional division of lymphocytes into the so-called recirculating and non-recirculating pools. A full knowledge of the parameters affecting the passage of cells from one pool to the other during the immune response is essen- tial. Cells show set migration patterns during ontogeny, moving, for example, from the central to the peripheral lymphoid organs. The traffic pattern of lymphocytes may be followed using radioisotope- or fluorescein-labelled cells as detailed below.

9.4.1 Ontogenic migration patterns

Dividing cells in the primary lymphoid organs may be labelled by an intraorgan infusion of 3 H- thymidine or 131 I-iododeoxyuridine.

Caution: It is essential to minimize the effect of ‘spill-over’ of the labelled material into the peripheral tissues, and so the animal is ‘flooded’ by an intravenous injection of non-radioactive DNA analogue.

Isotopically labelled cells in the peripheral lymphoid organs may then be identified by auto- radiography of tissue sections as described in Section 8.2.3. Please refer to the following websites for comprehensive details on radioisotope health and safety procedures as well as useful informa- tion regarding detection and half-life: http://www.practicingsafescience.org http://www.hse.gov.uk

9.4.2 Lymphocyte ‘homing’

If lymphocytes are removed from an animal, isotopically labelled in vitro and returned to a syn- geneic recipient, they show definite migration patterns, localizing within different organs at dif- ferent times.

MATERIALS AND EQUIPMENT Inbred mice

Sodium 51 chromate (Na 5l CrO 4 )

Gamma spectrometer

9.4LYMPHOCYTE CIRCULATION

METHOD

1 Prepare a lymph-node suspension from two to four donor mice (Section 6.3).

2 Count and adjust the suspension to 10 8 viable lymphocytes/ml.

3 Incubate 1 ml of cells with 11.1 × 10 5 Bq Na 5l CrO 4 for 30 min at 37°C in tissue culture medium buffered with HEPES (20 m M ) and containing 5% fetal bovine serum.

4 Wash the cells five times by centrifugation and resuspend in 2 ml of medium.

5 Inject 5 × 10 6 cells intravenously into each of 12 recipients, and retain an aliquot of 5 × 10 6 cells for g counting.

6 Kill three recipient mice at 4, 24, 48 and 72 h postinjection.

7 Remove the thymus, spleen, mesenteric lymph nodes and liver from each recipient

and count the amount of isotope in each organ. ( 51 Cr is a high-energy g emitter and therefore whole-organ counting is possible.) Count also the aliquot of 5 × 10 6 original cells.

8 Calculate the amount of radioactivity in each organ as a percentage of the original counts injected.

9 Plot a graph of percentage radioactivity against time for each organ.

TECHNICAL NOTES • Indium-111 is an alternative label for cells. • The technique may also use fluorescently-labelled cells and confocal microscopy to identify the

trafficking of cells to other organs (see Section 9.4.4).

Interpretation of results The percentage radioactivity per organ is an estimate of the proportion of lymphocytes localizing

in that organ. The ratio of counts localizing in the spleen relative to the liver is a good estimate of the viab- ility of the original lymphocyte suspension. If the suspension has a high viability the index is high, and vice versa. The ratio of spleen : liver localization also changes if cells are coated, but not necessarily killed, with anti-membrane antibodies.

Lymphocyte suspensions with high viability pass from the blood and localize predominantly in the spleen. Eventually cells leave the spleen and enter the lymph nodes, as shown by the change in radioactivity of each organ with time. No re-injected cells were detected in the thymus; the thymus is virtually excluded from the recirculation pathway of immunocompetent lympho- cytes once they have left the thymic cortex. The mature T lymphocytes in the thymic medulla seem to be a long-term resident population, members of which recirculate only rarely. These data were obtained with thoracic-duct lymphocytes which have almost 100% viability. It is necessary to combine dead-cell removal (Section 6.4) with the method outlined in the text to obtain com- parable results.

9.4.3 Intraorgan distribution of cells

It is necessary to use a different isotope as high-energy γ emissions cannot be efficiently captured by a photographic emulsion.

C H A P T E R 9: Lymphocyte function

MATERIALS AND EQUIPMENT Inbred mice Tritiated uridine Materials for autoradiography (see Section 8.2.3)

METHOD

1 Prepare a lymph-node suspension from two or three donor mice.

2 Count cells and adjust to 5 × 10 7 cells/ml in tissue culture medium buffered with HEPES (20 m M ) and containing 5% fetal bovine serum.

3 Add 3 H-uridine to a final concentration of 92.5 × 10 4 Bq/ml and incubate at 37°C for 30 min.

4 Wash the cells three times by centrifugation and inject 1 × 10 7 cells intravenously into each of four recipients.

5 Kill recipients at 0.5, 4, 8 and 24 h postinjection.

6 Remove the spleen and mesenteric lymph nodes from each recipient and fix for histological sectioning.

7 Prepare sections of each organ and dip in photographic emulsion for autoradiography (see Section 8.2.3).

8 After development of the autoradiographs, stain the tissue sections in haematoxylin and eosin.

Labelled cells may be identified by the presence of black grains of silver over their nucleus and cytoplasm. Examine the slides at low power and determine the change in distribution of labelled cells within the spleen and lymph node with time.

This technique can be used to determine the differential localization of any pure cell-line populations.

9.4.4 Fluorescent label for in vivo studies

Radioisotopes and autoradiography may be avoided by using intensely fluorescing dyes that bind to the cytoplasmic proteins (e.g. carboxy-fluorescein diacetate) or DNA (e.g. the bis benzimide H33342) of viable cells. These dyes do not impair the migration or localization pattern of cells and are diluted only at cell division (small lymphocytes divide only rarely). Moreover, they can be visualized under the fluorescent microscope in viable cell suspensions or histological sections, either frozen or after formaldehyde fixation.

MATERIALS AND EQUIPMENT Inbred mice H33342 dye

METHOD

1 Prepare a stock solution of the dye at 600 µg/ml in distilled water at 4°C.

2 Prepare a lymph-node suspension from two to three donor mice as in Section 6.3.

3 Count cells and adjust to 5 × 10 7 cells/ml in tissue culture medium buffered with HEPES (20 m M ) and containing 5% fetal bovine serum.

Continued on p. 276

9.4LYMPHOCYTE CIRCULATION

4 Dilute the stock solution of the dye 1 : 100 into the cell suspension, final dye concentration

6 µg/ml.

5 Incubate for 15 min at 37°C in a water bath.

6 Dilute in tissue culture medium and wash twice by centrifugation, then use as part of in vivo assays as appropriate. Cells may be visualized by confocal microscopy of sections from target organs.

9.4.5 T–B lymphocyte cooperation

In the intact immune response the production of antibody by B cells depends not only on an interaction with antigen-presenting cells but also on an interaction with T cells, i.e. cooperation. Cooperation involving T and B cells cannot be easily demonstrated using thymus and bursa cells from the chicken as they are essentially immunoincompetent cells, and the experiment would require the use of relatively rare inbred chickens. In the mouse the bone marrow behaves as though it were a source of B cells devoid of T cells and it is therefore operationally equivalent to the avian bursa.

MATERIALS AND EQUIPMENT 6-month-old inbred mice for X-irradiation

3–4-week-old inbred mice as thymus and bone marrow donors Sheep erythrocytes X-ray machine or g source Materials for haemolytic plaques (see Section 9.1)

We will use X-irradiated (immunosuppressed) mice as a suitable vehicle to examine the response of T and B cells, separately and together, to sheep erythrocytes.

Protocol. Group

Number of mice*

Cells for transfer

No. ml i.v. Sheep-cell challenge

A 3 Bone marrow

0.1 10 8 i.p.

B 3 Thymocytes

0.1 10 8 i.p.

10 8 i.p. * For experimental purposes it is better to use five or more mice per group.

C 3 Bone marrow + thymocytes

i.p., intraperitoneally; i.v., intravenously.

METHOD

1 Give nine mice 8 Gy of irradiation.

2 Remove the femurs from six untreated donor mice.

3 Cut each end off the bones and ‘blow out’ the marrow with tissue culture medium using a hypodermic syringe with an 18-gauge needle.

4 Disperse the cells gently with a Pasteur pipette.

Continued

C H A P T E R 9: Lymphocyte function

5 Count the cells and adjust to 2 × 10 8 /ml.

6 Remove the thymus from each of four donors and tease the cells into medium.

7 Wash the cells twice by centrifugation (150 g for 10 min at 4°C), count and adjust the cells to 10 9 /ml. Reconstitute and challenge the X-irradiated recipient mice as shown in the Protocol.

8 After 8 days assay the recipient spleens for direct haemolytic plaques as described in Section 9.1.2.

9 Calculate and tabulate the number of plaque-forming cells per spleen for each group of mice.

TECHNICAL NOTES • No control group is included to show that the X-irradiation was successful in suppressing the

immune response of the recipient mice. • Bone marrow is a relatively poor source of B cells. In the regime described you will obtain a maximum of 4.0–6.0 × 10 3 plaques per spleen. More satisfactory results may be obtained using

‘B’ spleens, prepared either by reconstitution of X-irradiated, thymectomized recipients, nude mice (T-lymphocyte deficient) (see Appendix B) or, more conveniently, by anti-Thy-1 treatment

of normal spleen (Sections 6.3 and 11.10.2). Using 2 × 10 7 ‘B’ spleen cells + 10 8 thymus cells

you can expect at least 3 × 10 4 plaques per spleen.