Primary and Secondary Immune Response

1. When an individual is exposed to an antigenic substance, either by injection, a complex series of events ensues, a. An antigen-presenting cell (usually a macrophage) processes the antigen and presents it to the lymphoid cells of the immune system. (1) For a successful immune response to occur, the processed antigen (specifically, its epitope) must be presented to the lymphocytes in association with a glycoprotein encoded by genes of the major histocompatibility complex (MHC). (2) This requirement for effective cell interaction is called MHC restriction. b. The lymphoid cells recognized that particular epitope and acquire the ability to react with it. c. The result of this sequence of events is the activation of antigenspecific B and T cells, causing them to proliferate and mature. 2. The consequences of the initial interaction between lymphocytes and their homologous epitopes are far-reaching. a. A subsequent exposure to antigen will induce some B lymphocyte (memory cells) to proliferate and differentiate into antibodysecreting plasma cells. (1) These active plasma cells release their specific antibody in large amounts when they contact antigen a second time, a phenomenon known as anamnesis. (2) The secreted antibody reacts specifically with the antigen that originally induced the B cell to proliferate. The potential exists to produce an extremely large (>100,000) variety of different, specifically reactive, antibodies. b. Some T lymphocytes (memory T cells) are induced to differentiate and proliferate to form mature progeny that will be triggered to release biologically active metabolites when they contact antigen a second time. c. Cells Involved In the Immune Response: A. Macrophages:

Macrophages are versatile cells that play many roles. As scavengers, they rid the body of worn-out cells and other debris. They are foremost among the cells that "present" antigen; a crucial role in initiating an immune response. As secretory cells, monocytes and macrophages are vital to the regulation of immune responses and the development of

inflammation; they churn out an amazing array of powerful chemical substances (monokines) including enzymes, complement proteins, and regulatory factors such as interleukin-1. At the same time, they carry receptors for lymphokines that allow them to be "activated" into single-minded pursuit of microbes and tumor cells. After digesting a pathogen, a macrophage will present the antigen (a molecule, most often a protein found on the surface of the pathogen, used by the immune system for identification) of the pathogen to a corresponding helper T cell. The presentation is done by integrating it into the cell membrane and displaying it attached to a MHC class II molecule, indicating to other white blood cells that the macrophage is not a pathogen, despite having antigens on its surface. Eventually the antigen presentation results in the production of antibodies that attach to the antigens of pathogens, making them easier for macrophages to adhere to with their cell membrane and phagocytose. In some cases, pathogens are very resistant to adhesion by the macrophages. Coating an antigen with antibodies could be compared to coating something with Velcro to make it stick to fuzzy surfaces. The antigen presentation on the surface of infected macrophages (in the context of MHC class II) in a lymph node stimulates TH1 (type 1 helper T cells) to proliferate (mainly due to IL-12 secretion from the macrophage). When a B-cell in the lymph node recognizes the same unprocessed surface antigen on the bacterium with its surface bound antibody, the antigen is endocytosed and processed. The processed antigen is then presented in MHCII on the surface of the B-cell. TH1 receptor that has proliferated recognizes the antigen-MHCII complex (with co-stimulatory factors- CD40 and CD40L) and causes the B-cell to produce antibodies that help opsonization of the antigen so that the bacteria can be better cleared by phagocytes. Macrophages provide yet another line of defense against tumor cells and body cells infected with fungus or parasites. Once a T cell has recognized its particular antigen on the surface of an aberrant cell, the T cell becomes an activated effector cell, releasing chemical mediators known as lymphokines that stimulate macrophages into a more aggressive form. These activated or angry macrophages, can then engulf and digest affected cells much more readily. The angry macrophage does not generate a response specific for an antigen, but attacks the cells present in the local area in which it was activated. Macrophages are the major antigen-presenting cells of the body, interacting with antigen as a primary step in the induction of an immune response. Langerhans cells of the skin, dendritic cells, and B lymphocytes can also present antigen. Besides presenting antigen to T and B cells, macrophages release soluble mediators such as the monokine (macrophage-derived mediator with hormone-like effects) interleukin-1 (IL-1), which stimulates T cells to mature and to secrete lymphokines (lymphocytederived mediators with hormone-like effects).

A macrophage of a mouse stretching its arms to engulf two particles, possibly pathogens

Macrophage cell

Function of Macrophage cells: 1. These cells are actively involved in the engulfment and destruction of various substances that enter the body. 2. They also are highly migratory and have the ability to insinuate themselves into the small nooks and crannies of the extracellular matrix of connective tissue. 3. Macrophages ingest some large particular substances by phagocytosis, a process that involves specific attachment and ingestion. Also, they ingest dissolved solutes from their fluid environment by pinocytosis. a. Once a macrophage has adhered specifically to a microorganism, it can ingest and destroy it. In certain instances, cells such as lymphocytes will adhere specifically to macrophages without being ingested. b. Some macrophages, particularly those in the primary alveoli, specialize in the nonspecific phagocytosis of inspired particulate matter such as dust, pollen, and cigarette smoke. c. Specific phagocytosis involves coating microorganisms with specific immunoglobulins or complement factors- a process called opsonizationand then adhesive recognition of bound

immunoglobulins or complement factors by the cell surface proteins of macrophages. d. Ingested bacteria are destroyed by a mechanism that probably is related to low lysosomal pH and the presence of hydrolytic enzymes such as lysozyme. 4. Macrophages not only specifically ingest opsonized microorganisms, they also can potentiate the lymphocyte antibody production by binding certain antigens to their surface and then interacting with lymphocytes to stimulate antibody production. 5. Macrophages have the ability to migrate in a directed fashion up a concentration gradient of dissolved components of bacterial cell walls. This chemotactic behavior probably is responsible for recruiting large numbers of macrophages to areas of tissue destruction or infection.

Lymphocytes: The lymphocyte is a common leukocyte whose heterogenous and complex nature was long misunderstood by the histologists. Recent studies in cellular immunology, however, have revealed two functional classes of lymphocytes : B lymphocyte and T lymphocyte. Both lymphocyte types originate from undifferentiated bone marrow stem cells. a. B lymphocytes leave bone marrow and diffuse throughout the body where they differentiate under poorly understood inductive influences. 1. In birds, B lymphocytes differentiate in an organ known as the bursa of Fabricus and thus are called B cells.

2. Although no structure equivalent to the bursa of Fabricus has been clearly identified in any mammal, even man, the name B cell is considered as misnomer. b. T lymphocytes or T cells leave bone marrow and travel to the thymus where they differentiate.

Figure: Simplified schematic of humoral immunity.

Immune Response
Humoral Immunity (Antibody) Cell-mediated Immunity (Cytotoxicity)

B cell development: a. In mammals, B cells originate from stem cells in the bone marrow. b. During maturation, which also occurs in the bone marrow, the B cell undergoes several gene rearrangements. These establish the B cells antigenic specificity before it travels to secondary lymphoid organs. Later somatic gene recombinations allow the cell line to switch from

one immunoglobulin class to another without a change in antigenic specificity. c. When the B cell moves to the blood and peripheral lymphoid tissues, it carries immunoglobulin in its surface membrane and is ready to interact with antigen.
LYMPHOID TISSUES

Primary
(Responsible for maturation of Agreactive cells)

Secondary
(Sites for Ag contact and response)

Thymus (T-cell maturation)

Bone marrow

Lymph nodes

Spleen

(Expansion of lymphatic system, separate from blood (Similar to lymph circulation. Deep nodes but part of (T-cell maturation) (B-cell maturation) cortex harbors blood circulation. mostly T-cells, Collects bloodsuperficial cortex borne Ags) harbors mostly Bcells)

d. The B cell matures into one of two types of cells. 1. The plasma cells have abundant rough endoplasmic reticulum and actively secretes large amount of the antibody that has been anchored in the parent B cell membrane. 2. The memory B cell is a long-lived cell that is the progenitor responsible for rapid plasma proliferation in the amnestic response. B-cell Function and Characterisatics: 1. B cells comprise approximately 35 percent of the circulating lymphcytes and primarily are responsible for humoral immunity (i.e., production of specific serum immunoglobulins directed against various environmental antigens). 2. B cells function in antibody production in two distinct ways. a. In the first and clearly most simple case, antigens bind directly to the B cells surface; B cells then undergo a clonal proliferation followed by terminal differentiation into plasma cells. Plasma cells are highly specialized for the secretion of immunoglobulins. b. In the second and more complex case, antigens are bound to the surface immunoglobulins of helper T cells. Next, these antigen-antibody complexes are released from helper T cells and bound to macrophages. Finally, the B cells interact specifically with stimulated macrophages and subsequently undergo a clonal proliferation and plasma cell differentiation. 3. Certain B cells persist after initial exposure to an antigen and exist in the form of memory B cells. These cells can undergo a rapid clonal expansion if a person is exposed again, even years later, to the same antigen. 4. B cells are most heavily concentrated in the germinal centers of aggregates of lymphoid tissue. For example, B cells are found in large numbers in the cortical aggregates in lymph nodes and in the white pulp of the spleen. T cell development: Fetal stem cells are destined to become T cells, which enter the thymus and proliferate there. These

immature T cells (called thymocytes while in the thymus for the periphery as mature T cells). a. During T cell development in the thymus, several changes occur. 1. Some type of selection process occurs, which favors the proliferation of thymocytes that are restricted by selfMHC molecules. That is, thymocytes are selected for their ability to recognize antigens associated with molecules of the same MHC type. a. Precursors of both helper T (Th) cells (MHC class II-restricted) and cytotoxic T (Tc) lymphocyte (MHC class I- restricted) are selected. b. Current evidence suggests that cortical thymic epithelial cells, which express both class I and class II MHC glycoproteins are important in this selection event; the mechanism is still unknown. General Characteristics of T cells: a. T cell surface markers: 1. Monoclonal antibody techniques have identified molecules on the T cell membrane that function chiefly as receptors. 2. These surface molecules include: a. Class I and class II MHC molecules b. Thy 1, Ly1, Ly2, 3 and L3T4 in mice. c. CD (cluster of differentiation) antigens (e.g., CD3, CD4, and CD8) in humans. 3. As a thymocyte differentiates toward a particular T cell subtype, it acquires certain CD antigens in its membrane and loses others. Thus, the T cell subsets can be distinguished by their CD markers. 4. CD3, CD2, and CD5 are found on most peripheral blood T cells. a. CD3 is a heteropolymer with at least five polypeptide chains; it appears late in differentiation when the cells are becoming immunocompetent. i. CD3 is associated with the T cell receptor for antigen and it is important in intracellular signaling

to initiate an immune response once the cell has interacted with a homologous epitope. ii. CD3 is not directly involved in antigen recognition, but antibodies against CD3 will block the antigen-specific activation of T cells. b. CD2 (the SRBC receptor) is responsible for resetting of sheep red blood cells (SRBCs) in the E-rosette assay for T cell enumeration. c. CD5 is expressed on all T cells and on a subset of B cells that appear to be predisposed to autoantibody production. 5. CD4 and CD8 are present on different effector T cells and on a subset of B cells that appear to be predisposed to autoantibody production. 6. Antiserum against certain of the membrane markers (e.g., against CD3) is immunosuppressive and has been used to prevent rejection of transplanted tissues. b. The T cell receptor for antigen 1. T cell have an antigen-specific receptor that functions as the antigen-recognition site. This surface component, the T cell receptor (TCR), bears significant structural homology with the Fab portion of an antibody molecule. 2. Structure and Function of TCR: a. The TCR is a heterodimer. i. It consists of two nonidentical polypeptide chains, an chain (about 45kDa) and a chain (about 40 kDa), linked together by disulfide bonds. ii. Both chains of the heterodimer are variable; there may be more variability in the smaller () chain. b. The TCR contains idiotypic determinants similar to those of immunoglobulin molecules. Hypervariability occurs in particular areas of each polypeptide chain in a manner analogous to the complementarity-determining regions (CDRs) of immunoglobulin molecules. c. The TCR heterodimer is noncovalently linked in the T cell membrane to the ,, and chains of the CD3 molecule. d. The TCR-CD3 complex apparently makes contact with both the antigen and a portion of the MHC molecule. Different portions of the hypervariable regions of the and chains interact with:

i. The helical sides of the epitope-binding cleft of the MHC molecule. ii. The epitope lying on the floor of the cleft. e. CD4 or CD8 molecules (depending on the T cell subset) also contact a portion of the MHC molecule. Primary and Secondary Immune Response: a. The primary immune response occurs following the first exposure to antigen and produces a relatively small amount of antibody. b. If a sufficient length of time elapses after the primary antigenic stimulation, the antibody level will decrease markedly. c. However, subsequently exposure to even a small amount of antigen will evoke an anamnestic response (also called booster response, memory response, or secondary immune response). 1. The anamnestic response consists of a rapid proliferation of plasma cells, with the concomitant production of large amounts of specific antibody. 2. The anamnestic response occurs because a large population of memory B and T cells are recruited into the humoral immune response. a. These memory cells are produced during the initial exposure to the antigen. b. The memory cells are precursors of Th cells and plasma cells, and represents another product of the collaboration between T cells and B cells.

Figure: Schematic Diagram of the Development of the Immune Responses

Induction of primary immune responses Induction of a primary immune response begins when an antigen penetrates epithelial surfaces. It will eventually come into contact with macrophages or certain other classes of Antigen Presenting cells (APCs), which include B cells, monocytes, dendritic cells, Langerhans cells and endothelial cells. Antigens, such as bacterial cells, are internalized by endocytosis and "processed" by the APC, then "presented" to immunocompetent lymphocytes to initiate the early steps of the immunological response. Processing by a macrophage (for example) results in attaching antigenic materials to the surface of the membrane in association with MHC II molecules on the surface of the cell. The antigenclass II MHC complex is presented to a T-helper (TH2) cell, which is able to recognize processed antigen associated with a class II MHC molecule on the membrane of the macrophage. This interaction, together with stimulation by Interleukin 1 (IL-1), produced by the macrophage, will activate the TH2 cell. Activation of the TH2 cell causes that cell to begin to produce Interleukin 2 (IL-2), and to express a membrane receptor for IL-2. The

secreted IL-2 autostimulates proliferation of the TH2 cells. Stimulated TH2 cells produce a variety of lymphokines including IL-2, IL-4, IL-6, and gamma Interferon, which mediate various aspects of the immune response. For example, IL-2 binds to IL-2 receptors on other T cells (which have bound the Ag) and stimulates their proliferation, while IL-4 causes B cells to proliferate and differentiate into antibody-secreting plasma cells and memory B cells. IL-4 activates only B cells in the vicinity which themselves have bound the antigen, and not others, so as to sustain the specificity of the immune response. Cross-linked antigens bound to antibody receptors on the surface of a B cell cause internalization of some of the antigen and expression on the B cell membrane together with MHC II molecules. The TH2 cell recognizes the antigen together with the Class II MHC molecules, and secretes the various lymphokines that activate the B cells to become antibody-secreting plasma cells and memory B cells. Even if the antigen cannot cross-link the receptor, it may be endocytosed by the B cell, processed, and returned to the surface in association with MHC II where it can be recognized by specific TH2 cells which will become activated to initiate B cell differentiation and proliferation. In any case, the overall B-cell response leads to antibodymediated immunity (AMI). The antigen receptors on B cell surfaces are thought to be the specific types of antibodies that they are genetically-programmed to produce. Hence, there are thousands of sub-populations of B cells distinguished only by their ability to produce a unique (reactive) type of antibody molecule. A B cell can also react with a homologous antigen on the surface of the macrophage, or with soluble antigens. When a B-cell is bound to Ag, and simultaneously is stimulated by IL-4 produced by a nearby TH2 cell, the B cell is stimulated to grow and divide to form a clone of identical B cells, each capable of producing identical antibody molecules. The activated B cells further differentiate into plasma cells, which synthesize and secrete large amounts of antibody, and into a special form of B cells called memory B cells. The antibodies produced and secreted by the plasma cells will react specifically with the homologous antigen that induced their formation. Many of these reactions lead to host defense and to prevention of reinfection by pathogens. Memory cells play a role in secondary immune responses. Plasma cells are relatively short-lived (about one week) but produce large amounts of antibody during this period. Memory cells, on the other hand, are relatively long-lived and upon subsequent exposure to Ag they become quickly transformed into Ab-producing plasma cells.

Cell-mediated immunity Generation of cell mediated immunity (CMI) begins when (for example) a Tc cell recognizes a processed antigen associated with MHC I on the membrane of a cell (usually an altered self cell, but possibly a transplanted tissue cell or a eukaryotic parasite). Under stimulation by IL-2 produced by TH2 cells the TC cell becomes activated to become a cytotoxic T lymphocyte (CTL) capable of lysing the cell which is showing the new (foreign) antigen on its surface, a primary manifestation of CMI. The interaction between an antigen-presenting macrophage and a TH cell stimulates the macrophage to produce and secrete a cytokine called Interleukin-1 (IL-1) that acts locally on the TH cell. The IL-1 stimulates the TH-cell to differentiate and produce its own cytokines (which in this case might be called lymphokines because they arise from a lymphocyte). These lymphokines have various functions. Interleukin-4 has an immediate effect on nearby B-cells. Interleukin-2 has an immediate effect on T cells as described above. Time is required before a primary immune response is effective as a host defense. Antigens have to be recognized, taken up, digested, processed, and presented by APCs; a few select TH cells must react with Ag and respond; preexisting B or T lymphocytes must encounter the Ag and proliferate and differentiate into effector cells (plasma cells or CTLs). In the case of AMI, antibody level has to build up to an effective physiological concentration to render its host resistant. It may take several days or weeks to reach a level of effective immunity, even though this immunity may persist for many months, or years, or even a lifetime, due to the presence of the antibodies. In natural infections, the inoculum is small, and even though the antigenic stimulus increases during microbial replication, only small amounts of antibody are formed within the first few days, and circulating antibody is not detectable until about a week after infection. Induction of a secondary immune response On re-exposure to microbial antigens (secondary exposure to antigen), there is an accelerated immunological response, the secondary or memory response. Larger amounts of antibodies are formed in only 1-2 days. This is due to the activities of specific memory B cells or memory T cells which were formed during the primary immune response. These memory cells,

when stimulated by homologous Ag, "remember" having previously seen the Ag, and are able to rapidly divide and differentiate into effector cells. Stimulating memory cells to rapidly produce very high (effective) levels of persistent circulating antibodies is the basis for giving "booster"-type vaccinations to humans and pets.

Figure 3. Receptor interactions between B cells, T cells and Antigen Presenting Cells (APC)

Immunoglobulin class switching: a. The IgM-IgG switch. 1. In the primary immune response, the immunoglobulin produced is mainly IgM. Subsequent exposures to the antigen will cause the response to shift to IgG production. 2. This changeover occurs within individual plasma cells to replace IgM-producing plasma cells. 3. The individual plasma cell splices out the constant-region gene complex and places it with a 3, 1, or another constant region gene. 4. The entire light (L) chain gene complex and the variable, diversity, and joining segments of the heavy (H) chain remain intact. Thus, the antigenic specificity of the plasma cell and its immunoglobulins is not changed. Figure 4. Primary and Secondary Immune Responses. Following the first exposure to an b. Class switching to IgA, Ig D, or IgE takes place by similar splicing antigen the immune response (as evidenced by processes. following the concentration of specific antibody
in the serum) develops gradually over a period of days, reaches a low plateau within 2-3 weeks, and usually begins to decline in a relatively short period of time. When the antigen is encountered a second time, a secondory (memory) response causes a rapid rise in the concentration of antibody, reaching a much higher level in the serum, which may persist for a relatively long period of time. This is not to say that a protective level of antibody may not be reached by primary exposure alone, but usually to ensure a high level of protective antibody that persists over a long period of time, it is necessary to have repeated antigenic stimulation of the immune system.

Primary and Secondary Immune Responses

Secondary Immune Response : Primary Immune Response: If a second dose of the same antigen Following the first exposure to a foreign is given days or even years later, an antigen, a lag phase occurs in which no accelerated secondary immune antibody is produced, but activated B response or anamnestic immune cells are differentiating into plasma response (IR) occurs. This lag phase cells. The lag phase can be as short as 2is usually very short (e.g. 3 or 4 3 days, but often is longer, sometimes as days) due to the presence of long as weeks or months. memory cells. The amount of antibody produced is The amount of antibody produced usually relatively low. rises to a high level. Over time, antibody level declines to the Antibody level tends to remain high point where it may be undetectable. for longer. The first antibody produced is manily The main type of antibody produced IgM (although small amounts of IgG are is IgG (although small amounts of usually also produced). IgM are sometimes produced). Note: The crossmatch attempts to prevent a secondary immune response by detecting any antibody present, and then ensuring that only antigen-negative red cells are transfused. It cannot prevent a primary immune response because only autologous red cells or red cells from an identical twin will introduce, no foreign antigens into a person being transfused.

In blood banking, a primary immune response doesn't always cause mainly IgM antibody to be produced. Sometimes only IgG antibody can be detected (e.g., for antibodies in the Duffy or Kidd systems). Similarly, a secondary immune response does not always cause mainly IgG antibody to be produced. Sometimes, only IgM antibody is produced (e.g., for antibodies in the MN or Lewis systems).