HOW ARE CELLS STUDIED?

As was indicated at the beginning of this chapter, it is not possible to differentiate between B and T lymphocytes using ordinary microscopy, either in blood or in lymphoid organs.  However, some differences are observed when electron microscopy is used. Nevertheless, there are some methods that allow differentiation and quantification  of lymphocytes, their subpopulations, and even their capability to respond to different antigens. The more frequently used methods are grouped as follows:

1. CELL SURFACE MARKERS AND RECEPTORS. These methods have been traditionally used to differentiate and quantify B and T lymphocyte subpopulations. With the arrival of monoclonal antibodies against different lymphoid populations, traditional methods are gradually being used less. These conventional markers are based on the specific characteristics of some membrane receptors, present in both lymphoid populations. The main markers are: 
   
   

For B lymphocytes:

B lymphocytes have in their membrane surface immunoglobulins that can be detected using anti-porcine-immunoglobulin sera or immunoglobulin.  These anti-immunoglobulin antibodies can be labeled with fluorescent dyes; monoclonal antibodies labeled with fluorescein isocyanate can also be used. All of them are specific against each immunoglobulin isotype. Using a fluorescence microscopy, a positive fluorescent reaction in the membrane can be observed . 

Surface immunoglobulins of a B lymphocyte observed by fluorescence microscopy.

Technique used for the observation of surface immunoglobulins of B lymphocytes. A polyclonal or monoclonal serum (a), labeled with fluorescein isocyanate (b), reacts with porcine immunoglobulins of the membrane. 

For T lymphocytes.

Rosettes formed with erythrocytes. Porcine T lymphocytes, like those of other species, have the characteristic of forming E-rosettes when they bind selectively to sheep red blood cells (a). Lymphocytes can be quantified by acridine orange labeling and afterwards observed with a fluorescence and ordinary light microscope. If T and B lymphocytes are labeled with acridine orange, only T lymphocytes form E-rosettes; B lymphocytes are actually stained but do not form rosettes.  

It is important to note that other rosette types may be formed by porcine cells. In the above picture   rosette formation of porcine erythrocytescan be observed in  macrophages infected by the African Swine Fever virus (b). This phenomenon is known as hemadsorption and must not be confused with E-rosette formation with sheep erythrocytes by porcine T lymphocytes (a).

2. MEMBRANE ANTIGENS

The different porcine lymphocyte populations can be labeled using monoclonal antibodies. The more frequently used methods are:

2.1. Flow cytometry.

A flow cytometer allows "in vitro" characterization and even separation of the different lymphocyte populations by means of monoclonal antibodies labeled with fluoresceine, these antibodies are specific for the surface markers of each targeted population. Nowadays, flow cytometry allows  the evaluation of several cytocromes at the same time. Thus, different cell subpopulations can be studied in the same lymphocyte sample. It is possible to start with whole blood (state of the art cytometers allow working with whole blood) or with the lymphocyte population previously separated  from the blood. The cells, with the specific monoclonal antibody (or antibodies; may be several at the same time) against the marker under study, are forced through a nozzle in a single cell stream that passes through a laser beam. This detects the scattering of light (particle size) and emitted fluorescence (cell type). The obtained measurements indicate each population percentage.  

Picture of one of the flow cytometers used for the study of porcine lymphocyte populations. The basic lecture unit and data processing system can be observed. 

2.2. Immunohistochemistry. Using monoclonal antibodies, labeled with fluoresceine or peroxidase, against the different porcine lymphocytes, it is possible to study these cells in any tissue. It is even possible to study two different populations at the same time. This is achieved using two monoclonal antibodies against two different lymphocyte populations. Each of them is labeled with a different enzyme (double labeling) (peroxidase- alkaline phosphatase). These techniques have  been of great importance in the study of the pathogenic mechanisms in several porcine infectious diseases. Thus, in the case of African Swine Fever Virus (ASFV), the different lymphocyte populations affected, or not, by the virus were studied. It was observed that those macrophages which were infected by the virus, had an altered SLA expression. Nowadays, these techniques are used to know the affected populations in the different viral infections and to study the different pathogenic mechanisms.  

Double labeling study (brown-blue) for the localization of ASFV infection (brown) in cells labeled with a monoclonal antibody against porcine macrophage (blue). No all cells reacting with the monoclonal antibody (blue) are infected, although most of them are.  

3. FUNCTIONAL ASSAYS

These methods are based on the evaluation of the B and T lymphocytes' capability to recognize a specific antigen. The most currently used techniques are:

• Induced lymphocyte proliferation or  blastogenesis

• T lymphocytes (CD 8+) cytotoxic activity

INDUCED LYMPHOCYTE PROLIFERATION.

Induced lymphocyte proliferation. The use of lymphocyte transformation or blastogenesis is nowadays one of the more precise and most frequently used "in vitro" techniques for the study of the specific and non-specific stimulation capability of lymphocytes. This technique is based on the capability of the lymphocytes for responding to an antigen (specific response) which has induced memory lymphocytes, either by vaccination or by natural infection. These lymphocytes, when in repeated contact with the antigen, have a blastogenic transformation. This blastogenic transformation may be induced in a non-specific way, due to the lymphocyte's capability of reacting to different lectins or mitogens.  Lectins induce a non-specific stimulation both in B and T lymphocytes.

Induced lymphocyte proliferation.

Picture of the equipment used for harvesting cells cultured in plaques. It must be noted that cells remain in the filter paper. These filters will  later be analyzed using the liquid scintillation counter or b particle counter. 

Induced lymphocyte proliferation assay implies lymphocyte culture with target antigens and several mitogens, that will be used as a proliferation control (non-specific stimulus). Specific blastogenic transformation is measured by the antigen capability of inducing lymphocyte proliferation. After the incubation period, a radioactive isotope  (tritiated thymidine) is added to the lymphocyte growth medium. If lymphocyte proliferation occurs, thymidine will be incorporated into the newly produced lymphocytes. The cells are then radioactively labeled. Cells and supernatant of each well are forced through a filter using a special cell "Harvester" where only cells remain. These filters will then be analyzed in a liquid scintillation counter or b particles counter. The more tritiated-thymidine uptake, the larger the lymphocyte stimulation.
 

Cytotoxicity study, taking into account the different ways of induction: by CD 8+ lymphocytes, by NK cells and by immunoglobulins  that activate complement. Their evaluation is carried out by measuring Chromium 51 liberation after the destruction of target cells, using a g racioactive particles counter . 

CYTOTOXIC ACTIVITY OF T LYMPHOCYTES (CD 8+)

The cytotoxic activity of T lymphocytes (CD 8+) towards a target cell can be studied measuring the killing ability that a determined number of T lymphocytes have for a certain number of target cells, when both populations are placed together. There are several methods for the evaluation of lysis percentage or target cells killed. However, the one most used is the liberation of Chromium 51 coming from target cells. In this method, target cells (which express certain surface antigens) are labeled with Chromium 51, and then placed together with effector cells (T lymphocytes). After the incubation period, cells are centrifuged and then, chromium 51 in the collected supernatant is measured, using a gamma-particle counter. The more chromium present in the supernatant, the larger the cytotoxic activity.