The McGill Physiology Virtual Lab

Immunology Laboratory

Basic Concepts

When a foreign antigen is introduced into an animal, the animal will respond immunologically to it. Innate (natural) responses occur as many times the foreign agent is encountered and lacks immunological memory, whereas acquired (adaptive) responses improve on repeated exposure to the antigen. The immunological response may be of two different types: cell-mediated or humoral.

Organization of the Immune System

The cells involved in the immune response are effectively organized into tissues and organs. The major lymphoid organs are classified into either primary or secondary.
 Primary lymphoid organs (thymus and bone marrow) are the major sites of lymphocyte development (lymphopoiesis). Their function is to produce a large repertoire of reactive cells (they acquire their repertoire of specific antigen receptors) and to eliminate self-reacting cells (cells with receptors for autoantigens are mostly eliminated).
Secondary lymphoid organs (spleen, lymph nodes, mucosal associated lymphoid tissue) provide the environment for the proliferation and maturation of cells involved in the adaptive immune response, for filtering and trapping antigens. They also provide the environment for cell-cell interaction and cytokine-cell interaction.

Cells of the Immune System
Immune responses are mediated by a number of cells and by the soluble molecules they secrete.

Origin of cells of the immune system: all the cells shown above arise from the hemapoietic stem cell. Platelets are released into the circulation. Granulocytes and monocytes pass from the circulation into the tissues. Mast cells are evidenced in all tissues. B cells mature in the primary lymphoid organs (bone marrow...). T cells mature in the thymus (primary lymphoid organs). Lymphocytes leave primary lymphoid organs and recirculate through secondary lymphoid tissues. Interdigitating and dendritic cells act as antigen-presenting cells in secondary lymphoid tissues.
Function of the cells of the immune system:
The innate responses use phagocytic cells (neutrophils, monocytes, and macrophages), cells which release inflammatory mediators (basophils, mast cells, and eosinophils), and natural killer cells. The molecular components of the innate responses comprise complement, acute-phase proteins, and cytokines such as the interferons.
Acquired responses involve the proliferation of antigen-specific B and T cells, occurring when their surface receptors bind to the antigen. Specialized cells (antigen-presenting cells) display the antigen to T lymphocytes and collaborate with them in the response to the antigen.
B cells secrete immunoglobulins (antigen specific antibodies) responsible for eliminating extracellular microorganisms or foreign agents. Innate and acquired responses usually work together.
Major Histocompatibility Complex (MHC): this gene complex was first identified when it was observed that histocompatibility depended on the donor and recipient sharing the same haplotype. The molecules which determine graft rejection are a limited group: Class I and Class II of cell-surface structures. The molecules which present antigens to T cells are mostly encoded within the MHC. All nucleated cells of the body express MHC Class I. By contrast, MHC Class II molecules are used by antigen presenting cells to present antigens to helper T cells, so cells expressing Class II are smaller in numbers than those expressing Class I.
Antigen presenting cells, in a lymphoid organ, include macrophages, dendritic cells and B-cells.
Mononuclear phagocytes: most important group of long-lived phagocytic cells. These cells are bone marrow-derived and their function is to engulf particles, internalize them and destroy them.
Dendritic cells which are found in the T-cell areas of lymph node and spleen are the most effective cells for the initial activation of naive T-cells.
B-cells: bone marrow-derived. Each B cell is genetically programmed to encode a surface receptor specific for a particular antigen. Upon recognition of the antigen, B cells multiply and differentiate into plasma cells, which produce large amounts of the receptor molecule in a soluble form that can be secreted (antibody).
The germinal centre: with the initiation of the acquired immune response, germinal centres form in the secondary lymphoid tissues, where all the antigen-specific and antigen-presenting cells can interact.
Effector cells: T-cells and B lymphocytes.
T cells: there are several types of T cells with a variety of functions. They originate from bone marrow stem cells and require further differentiation in the thymus where they migrate. T cell maturation requires a number of cell interactions:
T cells express in an orderly fashion certain markers or cell surface proteins. The nomenclature used to refer to cell surface molecules, characterized on the basis of their reactivity to monoclonal antibodies, follows the CD (for "cluster of differentiation") numbering. The CD4+ T cells, cytokine-secreting helper cells can be divided into two major types:
  • Type I helper T cells TH1 which secrete Interleukin (IL) 2 and interferon (IFN) g.
  • Type II  helper T cells TH2 which secrete IL4 and 5.

The production of cytokines by TH1 facilitates cell-mediated immunity (activation of macrophages and T-cell mediated cytotoxicity). TH2 cells help B cells produce antibodies. Class II MHC on antigen presenting cells interact with CD4 on T cells. Peptides which bind to MHC Class II come from proteins which have been internalized by the cell and then degraded.

CD8+ T cells play a role in the elimination of virally infected cells. The infected cell marks itself as a target for the cytotoxic T cell by displaying peptides derived from intracellular viral protein on its surface. Viral proteins are bound to peptide-binding regions of class I MHC molecules. Peptides which bind to MHC Class I molecules come from proteins synthesized within the cell (which are broken down) and transported to the endoplasmic reticulum.

B cells: the earliest cells which develop are called B1 cells; they express CD5 cell-surface molecule and are the source of "natural antibodies", which are IgM antibodies and are frequently polyreactive (recognize different antigens, pathogens and autoantigens). Natural antibodies have a relative low affinity. Most B cells lack the CD5 molecule, they develop later and are called B2 cells. Mature B2 cells coexpress IgM and IgD antibodies on their cell surface. The genes encoding B-cell receptors undergo a process of somatic hypermutation; the final stages of differentiation of B2 cells into antibody secreting plasma cells occurs within the germinal centres of secondary lymphoid tissues.
To elicit a strong antibody response, B cells require:
  • Antigen
  • T cells for direct contact (usually TH2 cells)
  • Soluble cytokines (e.g. IL4 + IL13, INF-γ or IL10)
  • Certain adhesion molecules
Clonal selection involves the proliferation of cells which recognize a specific antigen: each B cell is programmed to make just one antibody specificity, located on its surface as an antigen receptor. Antigen binds to only those B cells with the appropriate surface receptor; these cells are stimulated to proliferate and mature into antibody producing cells.
Functions of Antibody
The primary function of the antibody is to bind the antigen; the antibody directs specificity towards the antigen. By doing do, it also arms the killer cells and activates complement, processes that eliminate foreign organisms, which the antibody by itself cannot do. Of the different classes of antibodies, two will be discussed briefly:
IgM class: is the predominant antibody (it is the first to appear) in primary immune responses and is associated with the immune responses to antigenically complex, blood -borne agents. Once bound to the antigen, it is a powerful activator of the classical pathway complement: a single molecule of bound IgM is able to initiate the cascade because of the adjacent positioning of the Fc regions.
IgG class: is the most important class of immunoglobulin in secondary immune responses

The exposure to foreign antigen yields a biphasic response. The first phase is associated with production of IgM, followed by production of IgG. The second phase is characterized by a reduction of IgM followed by an increase of IgG. The antigen will select and expand a clone of effector B cells which will develop into plasma cells and produce antibodies.

Complement system
The complement system consists of about 20+ serum molecules (some of them being proteases) constituting nearly 10% of the total serum proteins and forming one of the major defence systems of the body. The complement system is activated by the Ag-Ab complex. The principle functions are the initiation of:
chemotactic factors: polymorphs and macrophages have specific receptors for small complement fragments generated during complement activation. The fragments diffuse away from the site of activation and stimulate chemotaxis, the way chemokines do.
vasodilation factors: some of the protein fragments of the complement system (C5a) causes degranulation of mast cells and basophils with release of histamine and other vasoactive mediators. Consequently, there are indirect effects on blood vessels, vasodilation and increased permeability of capillaries.
factors that increase phagocytosis: phagocytic cells carrying receptors for the complement components are then able to bind to the foreign particle (opsonization), and this triggers phagocytosis and cell activation.
membrane attack complex: the final step in complement activation brings about the assembly of a membrane attack complex, which can insert itself into lipid bilayers, causing the lysis of the foreign body.
The lytic pathway:

Click here to continue with the topic of Elisa