IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 29. Immunological Methods

ISOLATION OF LYMPHOCYTE POPULATIONS

Many immunological studies, both in vivo and in vitro, require specific lymphocyte populations. These are typically obtained from experimental animals, primarily from the Thymus, Spleen, and peripheral Lymph Nodes. Certain specialized investigations may necessitate the isolation of Cells from other anatomical sites, such as Peyer's patches. Recirculating cells can be harvested by cannulating the Thoracic duct and collecting the cells over several hours. In humans, peripheral Blood lymphocytes are the easiest to isolate, while spleen, tonsil, and lymph node cells can also be obtained surgically. However, surgically sourced material often contains infectious agents or tumor cells, depending on the underlying pathology that necessitated the Procedure. It should be borne in mind that the cellular populations found in these various Tissues differ significantly both in the degree of lymphocyte maturity and in the relative proportions of different Cell types.

The thymus is a source of a relatively pure T-cell population, although the constituent lymphocytes vary in their maturation stage. When working with lymphocytes from other Organs and tissues, it is frequently necessary to isolate distinct subpopulations for functional analysis. The fluorescence-activated cell sorter (FACS) described above, which separates lymphocytes based on their surface markers, yields only a limited number of cells due to the relatively low speed of flow cytometry and sorting. There are, however, several other techniques that allow lymphocytes and their individual subpopulations to be isolated directly from the entire volume of a sample: density gradient centrifugation, rosetting, panning, and magnetic Separation.

Density gradient isolation relies on the fact that lymphocytes have a lower buoyant density than erythrocytes and granulocytes (Fig. 29.19). This method allows the recovery of the majority of blood lymphocytes. Rosetting and panning are employed to isolate specific subpopulations (Figs. 29.20 and 29.21). Panning is essentially a form of Affinity Chromatography adapted for lymphocytes. A similar principle underlies magnetic separation using microbeads coated with specific Antibodies (e.g., anti-CD4). When mixed with a cell suspension, the beads bind those cells that are recognized by the Immobilized Antibodies. These cells can subsequently be eluted from the beads or isolated by applying a magnetic field.

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Fig. 29.19. Lymphocytes can be isolated from whole blood using a Ficoll density gradient. To achieve this, blood is defibrinated by shaking with Glass beads, and the resulting clot is removed. The blood is then diluted with culture medium and layered over a Ficoll solution occupying the lower half of a centrifuge tube. The density of Ficoll is higher than that of lymphocytes, but lower than that of erythrocytes and granulocytes (such as neutrophils). Following centrifugation, erythrocytes and polymorphonuclear neutrophils (PMNs) pass through the Ficoll layer and pellet at the bottom of the tube, whereas lymphocytes remain at the interface between the suspension medium and the Ficoll. The lymphocyte preparation is subsequently freed from macrophages and residual PMNs by adding iron filings; these are phagocytosed, enabling the phagocytes that have ingested them to be removed using a strong magnet. Macrophages can also be removed alternatively by placing The Cell suspension in a plastic petri dish, where the macrophages adhere to the plastic surface, leaving only lymphocytes in suspension.

Fig. 29.20. The rosetting technique is based on the property of certain lymphocyte subpopulations to bear receptors that bind erythrocytes. Human T cells possess receptors for sheep red Blood Cells (SRBC) — specifically, the CD2 molecule (1). Mouse T cells express very few such receptors, making it impossible to isolate murine T lymphocytes using this approach. When mixed together, T cells and erythrocytes form "rosettes", which can be separated from non-rosetting B cells via Ficoll density gradient centrifugation. Variations of this method also exist to isolate cells bearing other receptors (2). For instance, Tγ lymphocytes (T cells bearing receptors for the Fc fragment of IgG, Fcγ) can be identified and isolated by rosetting with bovine erythrocytes sensitized with a subagglutinating dose of antibodies against these red blood cells. The photomicrograph (3) shows a lymphocyte that has formed a rosette with erythrocytes. (Micrograph kindly provided by Dr. R. M. Lydyard).

Fig. 29.21. Cell populations can be fractionated on antibody-sensitized solid Supports. Antibodies applied to the support bind non-covalently to the plastic surface (much like in an immunoabsorbent assay), after which a cell suspension is added. Antigen-positive cells (Ag⁺) bind to the antibodies, whereas antigen-negative cells (Ag⁺) can be removed by gentle washing. Bound cells can sometimes be detached from the support by altering culture conditions or by enzymatic Treatment. Binding of cells to immobilized antibodies frequently induces functional changes; for instance, the cross-linking of surface molecules by the plastic-bound antigen may trigger cell activation. This method is generally best suited for depleting specific subpopulations rather than purifying them from a total lymphocyte pool. Examples of this approach include the separation of T helper (Th) and cytotoxic T (Tc) cell populations using anti-CD4 or anti-CD8 antibodies, and the separation of T cells from B lymphocytes using anti-Ig antibodies (which bind to B-cell surface IMMUNOGLOBULINS). In the reverse setup (sensitizing the support with an antigen), antigen-binding cells can be separated from non-binders.

Another approach — typically used to deplete unwanted cell populations — relies on The Use of antibodies and Complement. If specific antibodies (e.g., anti-CDS) and subsequently complement are added to a cell mixture, the Cells of the corresponding subpopulation will undergo lysis. Naturally, this method requires antibodies capable of fixing complement, and the target cells must express a sufficient density of surface antigen molecules to engage a lytic dose of complement.

Antigen-specific T-cell lines maintained in continuous culture can also serve as a reliable source of defined lymphocyte populations (Fig. 29.22). Generating such lines eliminates The Need for frequent isolation of primary cultures from animal organs and tissues.

Fig. 29.22. One of the possible Procedures for establishing T-cell lines. Mice are primed with an antigen, typically administered subcutaneously into the footpad of the hind paw. One week later, the draining lymph nodes (in this case, popliteal and inguinal) are harvested, and the cells derived from them are cultured in the presence of the antigen on a feeder layer of syngeneic cells (i.e., from mice of the same inbred strain), such as normal lymphocytes or splenocytes. After 4 days, lymphoblasts are isolated from the culture and induced to proliferate using IL-2. Once the cell number has increased sufficiently, their MHC and antigen Specificity are verified via a lymphocyte transformation assay. The resulting line is then maintained by alternating culture cycles on an antigen-stimulated feeder layer and in an IL-2-containing medium.