1Department of Sciences, University College Roosevelt, Middelburg, The Netherlands
2Microvida Laboratory for Medical Microbiology and Immunology, St Elisabeth Hospital, Tilburg, The Netherlands
*Corresponding author: Ger T. Rijkers, Department of Sciences, University College Roosevelt, P.O. Box 94, 4330 AB Middelburg, The Netherlands
Received Date: 11 January, 2021; Accepted Date: 29 January, 2021; Published Date: 02 February, 2021
The painting The Mass of Saint Gregory, by Jheronimus Bosch (Figure 1) is based on a legend from the late middle Ages . This legend deals with the theological question whether during consecration, bread and wine really are transformed into the body and blood, respectively, of Jesus Christ. Pope Gregory the Great, when celebrating mass, prayed to God for assistance because he wanted to convince two pagans attending his mass. The pagans were in doubt about the events which would take place during consecration. According to the legend, God permitted Saint Gregory a view of the resurrected Christ with his wounds. Christ’s blood poured into the chalice and the host (the bread) was infused with the body of Christ. The two onlooking pagans were converted on the spot. There is however another version of the legend in which it is Saint Gregory himself who has doubts on the transformation. In still another version, a woman who baked the bread used as host couldn’t believe that it could be transformed into the body of Christ. Neither the transformation of wine into blood, nor that of bread into human flesh, can readily be explained in strict biological terms, but for a religious belief that also isn’t needed. In biology however, a different type of transformation, involving cells, in particular lymphocytes, is being used in research and for in vitro diagnostic procedures. The medical and biological aspects of that type of transformation, including the history as well as current and future applications, will be discussed in biological terms in this paper.
The first, and for a long time the most common compound used for in vitro lymphocyte transformation is Phytohemagglutinin (PHA). High concentrations of PHA are found in beans (especially Phaseolis vulgaris, red kidney beans; see Figure 2), which, when eaten raw, can be toxic. PHA is composed of 2 subunits, E and L, of which PHA-E can agglutinate erythrocytes (E). PHA-E was used first in medicine for agglutination of red blood cells  and this is why it is called a haemagglutinin. The PHA-L subunit binds to the cell membrane of lymphocytes (see below). An alternative name for (plant) hemagglutinins is lectin, derived from the Latin word legere, meaning to select . The reason for using the term for select is that the capacity of certain plant agglutinins to bind to erythrocytes was dependent on the blood group. Later on the term lectin was used in general, irrespective of blood group specificity.
Phytohemagglutin from the beans of Phaesolus vulgaris (panel a), pokeweed mitogen from the berries of Phytolacca americana (panel b) and Concanavalin A from the beans of Canavalia ensiformis (panel c). Sources and credits Mr. Rasbak, https://commons.wikimedia.org/w/index.php?curid=1677401 (panel a), Ryan Hodnett, https://commons.wikimedia.org/ wiki/File:American_Pokeweed_(Phytolacca_americana)_-_ Kitchener,_Ontario_04.jpg (panel b), and Forest and Kim Starr, https://commons.wikimedia.org/wiki/File:Canavalia_ensiformis_ MHNT.BOT.2013.22.55.jpg (panel c). All sources assessed on June 6, 2020.
Dr. Peter Nowell was the first one to demonstrate that PHA was able to induce proliferation of human lymphocytes . Two versions of this discovery exist. The first one is that Nowell wanted to investigate cell proliferation in leukemia. When he had planned to take blood from a leukemia patient he found out (to his disappointment) that on that particular day the patient had gone into remission. He took the blood anyway, purified the lymphocytes by removal of the erythrocytes with PHA and to his surprise the lymphocytes started to grow and divide in vitro. When he repeated the experiment with his own blood, and that of his technician, he found that also normal lymphocytes could be stimulated to proliferate by PHA. The other version of the accidental discovery is that on a day in the lab he had started to purify lymphocytes from blood by adding PHA, but because he had to rush home for a family event, and because of that had forgotten to centrifuge the blood afterwards. The next morning he saw that the lymphocytes had undergone blastogenesis . Up till the discovery of Nowell, the prevailing paradigm in immunology was that lymphocytes are cells at the end-stage of their lifespan, unable to proliferate or differentiate any further. His paper was eventually published by Cancer Research , but one of the reviewers thought the paper was “interesting . . . but of no conceivable significance to science” . After a lag time of several years, the technique to induce in vitro lymphocyte proliferation by stimulation with PHA became a major laboratory tool in biomedical research and diagnostics (Figure 3). On December 31, 2020 PubMed lists 22,212 papers with the key word phytohemagglutinin [https://pubmed.ncbi.nlm. nih.gov/?term=phytohemagglutinin&sort=date ; see also Figure 1). Soon after the discovery that PHA was a powerful inducer of in vitro lymphocyte transformation, other plant lectins were discovered with properties, the most important being concanavalin A and pokeweed mitogen.
A century ago, James Sumner isolated and crystalized a protein from jackbean, Canavalis ensiformis (Figure 2, panel c) which precipitated by half saturation with ammonium sulphate and was called concanavalin . Upon dialysis, bisphenoid crystals precipitate first (Concanavalin A), precipitates in the form of needles last (Concanavalin B). Concanavalin A (ConA) binds to α-D-mannosyl and α-D-glucosyl sugar moieties. The biological activities of ConA are manifold [8,9]. in vivo, Con A has both proinflammatory as well as anti-inflammatory activity, depending on dosage and route of administration. Both in vitro and in vivo ConA has a vasorelaxation effect. For several tumor cell lines, ConA, because of its ability to induce apoptosis and autophagy, has antiproliferative effects. For selected species, ConA has antibacterial and antifungal activities. In the context of this manuscript, the most relevant biological activity of ConA is its mitogenic property for lymphocytes. This activity is directly related to the carbohydrate specificity of ConA, because the α-D-mannosyl and α-D-glucosyl moieties are (also) present on T cell receptor molecules [8,9]. Because ConA is a tetramer, the binding to T lymphocytes can cause extensive cross-linking of T cell receptors. Unlike other lectins, ConA binds with high affinity to cross-linked dextrans, such as Sephadex G-50. This allowed Mackler et al. to prepare “ConA-free” supernatants of lymphocytes cultured with ConA. These so-called ConA-supernatants contain lymphokines, which by themselves were mitogenic for autologous and allogeneic lymphocytes . Later studies demonstrated that IL-2 was the major cytokine responsible for this activity. Crosslinking of T cell receptors by Con-A induces a rapid, transient increase in the cytoplasmic Ca2+ concentration, one of the first signs of cell activation [11,12] (Figure 4).
The discovery of the biological properties of Pokeweed Mitogen (PWM), like that of PHA as described above, also has been accidental. Drs. Patricia Farnes and Barbara Barker were the pediatricians who discovered in 1961 that a fatal mysterious brain disease in a 3-year old girl was caused by ingestion of berries of pokeweed. When working in the laboratory to study and purify the active compound, now known as pokeweed mitogen, both doctors were exposed accidentally themselves leading to plasmacytosis in peripheral blood [13,14]. This led to the realization that PWM not only is a powerful inducer of T- and B-lymphocyte proliferation, but also of B-lymphocyte differentiation into plasma cells . Pokeweed, Phytolacca americana (Figure 2, Panel b) in its raw form is highly poisonous. When properly cooked it can be eaten as a sallet . Locally, the words sallet and salad are often used intermixed, a dangerous habit because when eaten raw, as when preparing a salad, pokeweed can be lethal. Tony Joe White wrote a song about a poor woman, Poke Salad Annie, who knew the recipe how to safely prepare a meal with pokeweed .
In vitro lymphocyte transformation, induced by either PHA, ConA or PWM, can be measured by the degree of proliferation, i.e. the net increase in number of cells as determined by counting the number of viable cells after a given culture period. The downside of such an approach is that during the culture period cells, especially the ones not taking part in the process, may die. Therefore, the net number of cells after culture actually can be lower than the starting number. For that reason most often 3H thymidine incorporation is used as a proxy for cell proliferation . The method is sensitive, can be semi-automated to a certain degree, but requires substantial laboratory infrastructure, including facilities to work with radioisotopes.
The complex biological nature of plant derived mitogens, and consequently batch to batch variations, have made most of these substances go out of fashion as of the 1990s (Figure 2). Moreover, the specific cellular ligands, being glycosylated surface proteins, have been difficult to characterize. For PHA they include the T cell receptor associated CD3 molecules as well as CD2 [19,20]. The decline of the use of plant lectins for in vitro lymphocyte proliferation was also caused by the availability of better standardized stimuli in the form of monoclonal antibodies directed against cell surface molecules on lymphocytes. Currently, polyclonal in vitro lymphocyte activation is being done with (mixtures of) monoclonal antibodies, e.g. the combination of antiCD3 and anti-CD28 antibodies [21,22].
When assessing the functional capacity of T lymphocytes in vitro, antigen-specific activation would be preferred over polyclonal activation with plant lectins or monoclonal antibodies against T cell receptors. However, in most cases the frequency of antigen-specific lymphocytes in unprimed individuals is too low to be measured in a quantitative manner. As an intermediate between polyclonal activation by mitogens and antigen specific activation, as described above, in vitro lymphocyte transformation can also be induced by so-called superantigens. Bacterial superantigens are produced by Staphylococcus aureus and Streptococcus pyogenes and are the causative agents of the toxic shock syndrome. The reason is that these superantigens can induce a massive cellular immune response and subsequent cytokine storm. Superantigens associate with HLA molecules and T cell receptors, outside of the peptide binding groove, and by crosslinking both molecules can cause activation of up to 20% of all T lymphocytes [23,24]. This effect is further enhanced by their binding to the co-stimulatory molecules CD28 and CD86 [25,26].
Specific antigens, such as peptides from Epstein Barr virus, cytomegalovirus , influenza virus  and yellow fever virus [29,30] can be successfully used for in vitro lymphocyte activation, provided the donors have been previously exposed to or infected by the respective viruses. Tetanus toxoid also is frequently used as an antigen-specific stimulus for lymphocytes from primed individuals .
In vitro lymphocyte transformation induced by either plant lectins, monoclonal antibodies, superantigens or specific antigens is an important component in the assessment of the functionality of the immune system. in vitro lymphocyte transformation tests are also being used for a number of other applications, as will be described below.
The lymphocyte transformation test is useful in vitro tool for diagnosis of a number of hypersensitivity reactions against many different drugs, including antibiotics such as sulfadiazine, acenocoumarol, terbinafine, and beta-lactam antibiotics [32-34]. The LTT also is being used to detect nickel sensitization . The outcome of the in vitro test cannot always be translated directly to the clinical situation, especially because a negative test doesn’t rule out a drug hypersensitivity. Also used for screening or treatment of patients with metal sensitivity before temporomandibular joint replacement surgery .
The immunomodulatory activities of medicines such as cyclosporine, methotrexate, rapamycin, and dexamethasone can be studied using human in vitro lymphocyte activation assays. It has been demonstrated that these substances can cause dose-dependent inhibition of lymphocyte proliferation at concentrations within the therapeutic window . In order to facilitate extrapolation of the obtained data to the clinical situation, the type of assays are performed with defined influenza peptides and lymphocytes from recently vaccinated donors. Using defined protocols, the IC50 of cyclosporine and mycophenolic acid as determined in five individual laboratories was found to be within an acceptable range .
There are many alternatives to 3H thymidine incorporation, including measurement of activation markers on cells and in vitro cytokine production, but few of them are equivalent of cell proliferation. Yet, the in vitro lymphocyte transformation tests, despite the use of standardized protocols, remains rather complicated and technically demanding . A number of technical less demanding alternatives have been tested and developed which will be discussed below.
Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) is a fluorescent dye, capable of covalently binding to intracellular molecules. Upon each cell division, the fluorescent molecules are equally divided between each daughter cell, causing a reduction in fluorescence intensity per cell by approximately 50%. This reduction in fluorescence intensity can be measured by flow cytometry, and as a direct measure of the rate of cell division [38,39]. CFSE is especially effective in T lymphocyte activation studies because of its very low cell toxicity. The technique even can be used for antigen specific T-lymphocyte activation studies.
Another technique that is capable to measure antigen-specific lymphocyte activation in vitro is the so-called Interferon-Gamma Release Assay (IGRA) . IGRA is a whole blood tests used to diagnose both latent as well as active Tuberculosis (TB) infections. White blood cells of TB infected patients release interferon g (IFN-g) upon in vitro (re-)activation with a mixture of specific antigens from Mycobacterium tuberculosis (ESAT-6, CFP-10, and TB7.7). The test includes a tube with PHA as a positive control and a negative control tube. Following incubation at 37 ˚C for 24 hrs, the concentration of IFN-g in the culture supernatant is determined. The test has a high degree of sensitivity and specificity, but cannot distinguish between latent and active TB.
5-bromo-2-deoxyuridine (BrdU) is a non-radioactive thymidine analog which can be used to detect in vitro T-lymphocyte proliferation. Furthermore, activated cells in S-phase of the cell cycle can be detected using flow cytometry with fluorescent labeled anti-BrdU antibodies, which can also be used as an alternative for measure of proliferation. This method has the added advantage that it can be combined with cell surface staining, allowing to distinguish between CD4 and CD8 T-lymphocyte activation .
Ki-67 is a protein that is widely used as a proliferation marker for human tumor cells. Ki-67 is expressed in both interphase and mitotic cells, but is upregulated dramatically during cell cycle progression . It is equally sensitive as CSFE, or the CSFEderivative Oregon Green, and more sensitive than BrdU in detection of in vitro tetanus toxoid specific T-lymphocyte activation. Using a combination of intracellular staining with fluorescent antiKi-67 and cell surface phenotyping, flow cytometric analysis can distinguish between CD8 and CD4 T-lymphocyte activation by PPD or methylisothiazolinone [43,44].
Cyclic voltammetry is a technique that does not rely on the use of any labels for measuring in vitro lymphocyte transformation. Proliferating cells show specific voltametric behavior, which is the current passed through the solution in response to an applied potential. The short duration of 4 hours in combination with the ability to use fresh blood allows for an accurate indication of how cells would respond in their normal environment .
Assessment of the cellular and humoral immune status in health and disease requires a set of diagnostic techniques with high sensitivity and specificity. in vivo techniques (Latin for “within the living”) such as a delayed type hypersensitivity reaction are only applicable for a limited number of indications (e.g. TB diagnostics). In this manuscript the in vitro techniques (Latin for “within the glass”) for measuring lymphocyte transformation have been discussed. Especially in PHA stimulated lymphocytes, chromosomes are easily visible in metaphase, and therefore it also became the standard method in karyotyping for clinical genetic diagnostics . For a (fortunately, limited) period PHA has been used in vivo as a form of treatment of aplastic anemia. The rationale was that “transformed” lymphocytes would thus acquire hematopoietic properties [47,48]. PHA also has been used for treatment of squamous cell carcinoma in a single study of 24 patients . It soon became clear that the use of the lectins PHA, ConA, and PWM, because of their toxicity [13,14] should be restricted to in vitro applications. in vivo immunotherapy of malignant and other diseases requires better defined compounds, and monoclonal antibodies have been demonstrated to be highly effective in that respect.
In the future, techniques for immune status assessment could be developed, based on organoids of lymphoid organs, which would be closer to the in vivo situation. The term “ex vivo” has been proposed for such an approach [50,51].
Saint Gregory’s Mass addresses the transformation which takes place during consecration. It is painted in grisaille, which was common in that period for the closed panels of altar pieces. Exceptional is that the two figures in color, Peter Scheyfve (standing to the left) and his son (kneeling to the right) are not the main character of the painting, because (obviously) that is Saint Gregory himself (Figure 1). By being the patron of the painting, Peter Scheyfve, who was a rich textile merchant from Antwerp , contributed to the transformation of this work by Jheronimus Bosh.
Figure 2: Botanical origin of common lectins used for in vitro lymphocyte transformation.
from the beans of Phaesolus vulgaris (panel a), pokeweed mitogen from the berries of Phytolacca americana (panel
b) and Concanavalin A from the beans of Canavalia ensiformis (panel c). Sources and credits Mr.
Rasbak, https://commons.wikimedia.org/w/index.php?curid=1677401 (panel a), Ryan Hodnett, https://commons.wikimedia.org/wiki/File:American_Pokeweed_(Phytolacca_americana)_-_Kitchener,_Ontario_04.jpg (panel b), and Forest and Kim
Starr, https://commons.wikimedia.org/wiki/File:Canavalia_ensiformis_MHNT.BOT.2013.22.55.jpg (panel c). All sources
assessed on June 6, 2020.
Citation: Rijkers GT, Rietkerken S (2021) Mass of Saint Gregory: Lymphocyte Transformation Tests as a Diagnostic Tool in Clinical Immunology. J Vaccines Immunol 6: 160. DOI: 10.29011/2575-789X.000160