Plasma Cells

Plasma cells play a critical role in the adaptive immune response. The purpose of the following experimental outline is to demonstrate, observe and study the nature of plasma cells, by producing and utilizing the splenocyte suspensions from the spleen of a previously immunized mouse with sheep erythrocytes and another mouse with saline. We used a hemolytic plaque assay, the Cunningham-Szenberg Plaque Assay, to detect and quantify the number of plaque-forming lymphoid cells, plasma cells secreting IgM antibodies against the foreign sheep erythrocytes with added complement, as a percent of total splenocytes. In addition, the characteristic features of plasma cells were observed and identified in fixed histological (tissue) slides derived from nasal polyps containing well-defined plasma cells.

Using the Cunningham-Szenberg Plaque Assay and the hematocytometer to determine the concentration of cells in a suspension, the detection, visualization and quantification of the number of plaque forming cells was performed. These plaque forming cells secrete IgM in a primary response against sheep erythrocytes and were present only in the immunized mouse at a calculated level of PFC/million splenocytes. The examination of fixed pathological slides of nasal polyps allowed the visualization and characterization of plasma cells and an understanding of their features. Plasma cells have abundant cytoplasm, a large and irregular nucleus with prominent nucleoli, clear staining next to the nucleus with abundant rER, Golgi apparatus and mitochondria. These characteristics enable plasma cells to carry out their function of secreting large amounts of antibody.

In the procedure, large single chambered slides, that approximately take 0.15 mL of reaction mixture. We injected 0.1 ml. The chambers were sealed with elastic tap, rather than dipping in a paraffin wax bath which is not always available. During counting, the spleen cell suspension was mixed thoroughly and well in order to distribute the splenocytes approximately equally throughout the sample and only a small sample of the spleen suspension for hemacytometer counting. The concentration of this sample was approximately the same as the total cell solution concentration. Due to high concentration of suspensions, small hematocytometer squares should be used in the triple-ruled area.

Plasma Cells: B Lymphocyte Effector Cells and Antibody Producing Cells

Humoral immuniy, the antibody-mediated specific immunity made in a humoral immune response, part of the adaptive immune response, is mediated significantly by plasma cells, the effector function cells of B lymphocytes. Plasma cells are terminally differentiated B lymphocytes that arise from naive B cells and are found in splenic red pulp, the medulla of lymph nodes, blood, lymph, and in the bone marrow. Moreover, plasma cells are the main antibody-secreting cells within the body and are essential mediators of immunity. Plasma cells secreted antibodies play essential roles in the neutralization, opsonization and destruction of extracellular and intracellular pathogens. Intracellular pathogens, like viruses, must spread from cell to cell through the extracellular spaces in order to infect more cells, while the majority of bacteria multiply extracellulary. Antibodies can neutralize pathogens by binding to specific cell entrance molecules found on the pathogen and thereby blocking their subsequent entrance into cells. Moreover, neutralization can provide protection from bacterial toxins, by inactivating these molecules and mediating their destruction and clearance from the body. Antibodies can coat pathogens surface and enhance the process of phagocytosis, which is known as opsonization. In addition, antibodies can activate classical complement system, which recruits phagocytes and also activate the terminal components of complement, which can lyse certain microorganisms directly through the formation of pores in their membranes. Moreover, antibodies of different isotypes operate in distinct locations within the body and provide distict effector functions where they are found (1).

Naive B cells arise from hematopoietic stem cells found in the bone marrow, which differentiate initially to a common lymphoid progenitor. Maturation proceeds within the bone marrow until maturation is complete. Once B cells are fully matured, they enter the bloodstream through which they migrate to the peripheral organs, such as the lymph nodes and spleen. The peripheral organs are the sites of B, as well as T, lymphocyte activation by antigen. Extracellular fluid as lymph, is drained through the lymph nodes and into the thoracic duct by the lymphatics, which returns the lymph to the bloodstream by emptying into the left subclavian vein. B lymphocytes circulating in the bloodstream enter the peripheral organs and are eventually carried by lymph to the thoracic duct where they re-enter into the blood. All antigen specific naive B lymphocytes (and T) continually circulate between the lymph and blood, always passing through peripheral lymphoid organs and tissues in order to meet antigen. If no antigen is encountered while travelling through the peripheral lyphoid tissues, this process continues repeatedly until stimulation with antigen halts the cells to proliferate and mature. However, in the presence of foreign antigen, as with an infection, antigens from sites of infection reach lymph nodes via lymphatics (antigens from blood enter the spleen) and lymphocytes that recognize the foreign antigen become trapped in the lymphoid tissue where they proliferate and differentiate into effector cells capable of combating the foreign invader(s). Naive B cells circulate, enter the lymphoid tissues, encounter their specific antigen (APCs, e.g. macrophages present it from engulfing it from the infected site), bind to the antigen with their surface antigen-specific immunoglobulin, transmit signals to the interior of the cell and internalize the Ig and bound antigen, process the antigen and present the antigen to armed helper CD4+ T cells in a MHC class II-T cell receptor restricted fashin and become arrested in the T cell zones of the lymphoid tissues. T cells begin secreting specific B-cell stimulatory cytokines IL-4, -5, -6, at the site of contact and also synthesize CD40 ligand (on the T cell) that interacts with CD40 on the B cell. Both processes synergize to stimulate the proliferation and differentiation of B cells into antibody-secreting plasma cells (also memory cells, and some microbial antigens can activate B cells directly without T-cell help). However, B cells and helper T cells must recognize molecularly identical epitopes in order to interact. The antigen specificity of the T cell and B cell therefore, must be identical (1).

The T cells that were activated and produced the mediatory cytokines were T helper cells. These Th2 cells provide help to the B cells and are involved in stimulating B cells to become antibody secreting plasma cells, as well as memory cells (animal is now primed to meet the antigen in the future, with an antibody response prepared). The activated B cells proliferate in the specialized environment provided by the germinal centers. Germinal centers are formed when activated B cells enter lymphoid follicles. These are areas of B cell interactions with antigen presenting cells, follicular dendritic cells, which present antigen to the B cells and allow for the further survival, affinity maturation, selection and somatic hypermutation of the B cells. Thereafter, the subsequent B cells form two distinct populations after leaving the germinal center. A population migrates into the red pulp of the spleen, the bone marrow and the medullary cords of lymph nodes forming plasma cells. The rest form memory B cells that do not secrete antibody, but can be rapidly activated upon subsequent challenge with the same antigen (1).

Plasma cells initially always produce IgM as the first antibody in a humoral response. These IgM are usually produced before B cells have undergone somatic hypermutation and therefore have low affinity. Increases in affinity occur during subsequent responses to the antigen, through, for example repeated immunizations with antigen. During the end of the primary response, IgG appears. Later phases of responses are dominated by other isotypes besides IgM. B cells can produce antibodies, all specific for the same antigen, that provide protective functions for each body compartment due to Ig isotypes (1).

References:

1. Janeway, C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12

2. Kissinger R, and Myl A.D. 1984. Improvements to the Plaque Assay for Antibody Secreting Cells. J. of Immun. Meth. 66:377-382

3. Wilson S., Munson A., and Meade J. 1999. Assessment of the Functional Integrity of the Humoral Immune Response: The Plaque-Forming Cell Assay and the Enzyme-Linked Immunosorbent Assay. Methods 19:2-7

4. Cunningham, A.J. 1965. Nature (London) 207, 1106

5. Cunningham A.J. and Szenberg A. 1968. Immuniligy 14: 599

6. Jerne N. and Nordin A. 1963. Science 140: 405

7. Delves P. and Roitt I. 1999. Encyclopedia of Immunology, Academic Press Inc., 2nd ed., San Diego, USA page 238.

8. Cruse, J and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965