Specific Immunity

Third line of defense or adaptive immunity or specific immunity

 Adaptive immunity or acquired immunity

- cellular component- lymphocytes

- Humoral mediated immunity- antibodies

 Acquired immunity are of two types:

-active

- passive

Acquired immunity
 Active immunity

- can be acquired by natural exposure in response to an infection,

-or through intentional injection of an antigen (vaccination)

 Passive immunity

- natural = antibodies from the mother

- artificial= preformed antibodies from other sources (man or other animals

 Reticuloendothelial System
 The system consists of a network of reticular fibers which supports the phagocytic cells.

 In addition, phagocytes may process antigens for specific immune response.

 The Lymphocyte Family
 T Cells
- T helper cell
- Cytotoxic T cells
- Suppressor T cells
- Delayed Hypersensitivity T cells
 B Cells
- plasma cells
- memory cells

T cells
- longer life span
- 80%
- rosette formation
- thymus
- cell mediated immunity

B cells
- shorter life span
- 20%
- not capable of forming rosette
- Bursa of Fabricius (Bursal equivalents)
- humoral immunity
 Th Cells
 Helper T cells use their specific receptors to recognize antigen. They also have molecules on their surface (CD4)
which recognize special “self” molecules called major histocompatibility complex (MHC) Class II, found on

antigen-presenting cells (APC).

Tc Cells
 Cytotoxic T cells (CTL) kill infected cells and tumor cells. They have molecules on their surface (CD8) which
recognize “self” molecules called major histocompatibility complex (MHC) Class I, formed on all nucleated cells.
 Antigen found in infected cells are bound by MHC Class I and brought to the surface where they are recognized
by cytotoxic T cells. Other CD8 positive cells (suppressor T) help to regulate the immune response.

 LGL
 have killer inhibitory response and can recognize MHC Class 1
 can also kill antibody coated B cells
 Cell-mediated immunity
 CMI is moderated by the link bet. T lymphocyte and phagocytic cells (i.e., monocytes-macrophages)
 T lymphocyte responds to antigens presented by other cells in the context of major histocompatibility complex
(MHC) proteins.
 T lymphocytes recognizes antigens present on the surface of an antigen-presenting cell (APC), the macrophage
 Lymphocytes are immunologically active through various types of direct cell-to-cell contact and by the
production of soluble factors.
 Nonspecific soluble factors are called cytokines.
 Some mediators that act between leukocytes are called interleukins
 Under some conditions, the activities of the CMI may not be beneficial
 Suppression of the normal adaptive immune response by drugs or other means is necessary in conditions or
procedures such as organ transplantation, hypersensitivity, and autoimmune disorders,
 T cells
 Key component of CMI
 When T cells recognize foreign antigens, they do not secrete antibodies but instead differentiate into several
types of specialized T cells and into long-lived memory cells.
 The different classes of T cells are responsible for attacking foreign antigens and regulating the immune
response

Types of T cells
 Helper T (Th)
 Delayed hypersensitivity (Td) cells
 Suppressor T (Ts) cells
 Cytotoxic T (Tc) cells

Helper T (Th)
 The cause of the pathogenicity of the AIDS virus was due to destruction of the Th cells, specifically the CD4
surface antigens.
 Certain Th cells present T-dependent antigens to B cells
 Some other Th cells help other T cells to respond to antigens
 Delayed hypersensitivity T (Td) cells
 Associated with certain allergic reactions, such as poison ivy, and with rejection of transplanted tissues.
 Td cells are also impt. in the body’s defense against cancer.
 Early investigators found that transfer of these cells bet. animals also transferred immunity to tuberculosis
 In response to antigenic stimulation, Td cells produce substances called lymphokines which recruit defensive
cells like macrophages.

Suppressor T (Ts) cells

 They inhibit the conversion of B cells into plasma cells which is a way of turning off the immune system when
the Ag is no longer present.
 They also suppress the activity of other T cells so that immune responses are not developed against self-antigens
 Both Th and Ts are called regulatory T cells
 Cytotoxic T (Tc) cells
 Destroy target cells such as cancer cells and transplanted tissue, upon contact.
 Protection against viral infections and intracellular bacterial infections is the most impt.function of Tc cells.
 Tc cells cause the lysis of the target cells by coming into close contact with them and releasing a protein called
perforin. Perforin forms a pore in the target cell membrane
 Also plays a role in the defense ag. Cancer and diseases caused by protozoans and helminths

Other soluble immune response cytomediators

 Cytokines-are polypeptide products of activated cells that control a variety of cellular responses.
 Cytokines produced by leukocytes that act on other leukocytes are also referred to as interleukines.
 cytokines have a variety of roles in host defense.

Cytokines
 In innate immunity, cytokines mediate early inflammatory reactions to microbial organisms and stimulate
adaptive immune response,
 In adaptive immunity, cytokines stimulate proliferation and differentiation of antigen-stimulated lymphocytes
and activate specialized effector cells (e.g., macrophages)
 Interleukines (Ils)
 Ils include molecules that are made by and act on lymphocytes
 Ils have widely overlapping functions. These molecules modulate inflammation and immunity by regulating
growth, mobility and differentiation of lymphoid cells

Il-1
 source: macrophage
 target: T cell, macrophage, endothelium
 effect: lymphocyte activation, cell adhesion, fever, inflammation, increase Il-2 receptor

Il-2
 source: T cells
 target: T cells, NK cells
 effects: proliferation and activation of CTL, NK cells, and macrophage

IL-4
 source: T cells
 target: T, B cells
 effects: proliferation of B cells, isotype switch to IgE, IgG1

IL-5
 Source: T cells
 Targets: B cells
 Effects: Proliferation and activation of B cells, isotype with to IgA

IL-6
 Source: macrophage, T, B cells
 Targets: T, B cells
 Effects: T cell proliferation, B cells activation, induce plasma phase protein from liver

Interferon
 A group of cytokines discovered in virally infected cultured cells.
 One of the body’s natural defensive responses to foreign components (e.g., microbes, tumors, Ag)
 IFNs are the most broadly active physiologic regulators, enhancing the expression of specific genes, inhibiting
cell proliferation, and augmenting immune effectors cells.
 IFNs act as antiviral agents, immunomodulators, and antineoplastic agents.
 Source: T cells, NK cells (gamma)

EC and fibroblast (alpha and beta)

 Targets: Tissue cells, WBC, macrophage, Th-1 cells, NK cells

 Effects: Type I IFN mediate the early innate I.R. to viral infections. Consists of two groups of
proteins-IFN-alpha and IFN-beta
 IFN gamma is the principal macrophage-activating cytokine and serves a critical function in innate
immunity and CMI. Stimulates expression of MHC I and MHC II molecules. and costimulates APCs

Tumor necrosis factor

 Principal mediator of the acute inflammatory response to gram-neg. bacteria and other infectious microbes.
 TNF is responsible for many of the systemic complications of severe infections
 The principal physiologic functions of TNF:

- stimulate recruitment of neutrophils and monocytes to site of infection

- activate these cells to eradicate microbes

TNF- tumor necrosis factor

 Source: macrophage (alpha); lymphocyte (alpha and beta)
 Target: tissue cells, macrophage, granulocytes
 Effects: activation of Tc, granulocytes, and macrophage

Inc. WBC-endothelial adhesion

Inc. MHC-I activation

Inc. acute phase protein

MIF—migration inhibitory factor

 First cytokine activitiy to be described
 Source: T cells
 Target: macrophage
 Effects: Keeps macrophage in area by inhibiting movement, which may cause retention and accumulation of
phagocytes at sites of inflammation.

Macrophage activation factor

 Activates macrophages to improve phagocytic activity

Lymphotoxin
 Destroys nonlymphocytic target cells in vitro; function in vivo uncertain

Hematopoetic stimulators-stem cell factor (c-kit ligand)

 Stem cell factor is a cytokine that interacts with a tyrosine kinase membrane receptor, the protein product of
the cellular oncogene c-kit.
 The cytokine that interacts with this receptor is called c-ligand, or stem cell factor because it acts on immature
stem cells.
 SCF is needed to make bone marrow stem cells responsive to colony-stimulating factor.
 SCF play a role in sustaining the viaability and proliferative capacity of immature Tcells in the thymus and masst
cells in mucosal tissues.
 Acute -phase proteins
 Acute-phase response is an innate body defense. This response is a nonspecific indicator of an inflammatory
process.
 Acute-phase proteins or acute-phase reactants are glycoproteins associated with acute phase response.
 These proteins rise in response to tissue injury(e.g., inflammation, infection, malignant neoplasia, various
disorders, trauma, surgical procedures,drug response ).

Epitope
 The part of antigen that binds with the antibody binding site (paratope).
 Hapten
 A small molecule which can bind with the antibody binding site, but is not immunogenic unless attached to a
larger molecular backbone.

Difference of Immunogen, Antigen, Epitope and Hapten

Immunogen
 a stimulus that produces a humoral or cell-meditated immune response.

Antigen
 Any substance that binds specifically to an antibody or a T-cell receptor.

Epitope
 The portion of an antigen that is recognized and bound by an Ab or TCR/MHC complex (aka antigenic
determinant)

Hapten
 A low molecular weight molecule that can be made immunogenic by conjugation to a suitable carrier.

Tolerogen
 When a potential antigenic molecule is encountered early in the development of specific B or T cells, the cells
die and an immune response will not occur the next time this molecule is seen. The molecule is then called
tolerogen, and the host said is to be tolerant to it. The immune system considers it to be ‘’self.’’
 Immunogenicity

The immunogenicity of an antigen depends on many factors:

Foreignness
 The immune response is greater the more different an antigen is from ‘’self’’’.
 A person will not normally respond to his or her own antigens, or to those of an identical twin, but will react to
proteins which are from different persons, plants or micoorganisms.
 The antigens on our own tissues which result in graft rejection are called major histocompatibility complex
antigens.
 Some molecules of microorganisms are similar but to human tissue antigens, so that an immune response to the
microorganisms can cause immune injury to the host.
 Chemical complexity
 A good molecule is more immunogenic than a simple one.
 Good Immunogens are:
 Proteins (>20 amino acids)
 Lipoproteins (endotoxin)
 Glycoproteins (blood groups)
 Nucleic Acids, in combination with basic proteins
 Small peptides (e.g insulin) can be weak immunogens.
 Pure lipids or pure polysaccharide are not good immunogens.
 Some polymeric antigens can trigger an IgM B cell response .
 Some polymetric can trigger an IgM B cell response without T cell help. They are called T-independent antigens.

Molecular size
 a larger molecule usually more immunogenic than small one.
 Immunogenic molecules are usually >10 kiloDaltons (kD) in size
 Size usually correlates with complexity and the number of antigenic determinants.
 Simple, repetitive molecules like sugar are poor immunogens.
 Larger molecules are more easily phagocytosed.
 Molecules must be degradable by macrophages for presentation.

Antigen dose
 The higher dose of antigen, the greater the immune response
 Repeated exposure to an antigen leads to activation of large numbers of immunocompetent cells.
 This activation allows a faster response each time the antigen is encountered.
 This is called the anamestic or secondary immune response.

Route of immunization
 The immune response will be different depending on the route of immunization :
 Subcutaneous or Intramuscular – good response in draining nodes.
 Intravenous – general (Systemic) response, particularly the spleen.
 Intranasal, Intestinal – mucosal response

 Adjuvants

Route of Immunization
 Intravenous and intraperitoneal routes are stronger than stimuli than subcutaneous and intramuscular routes.
 Subsequent exposure to the same antigen produces a memory response or anamnestic response.

Antigen-Antibody Reaction: Specific and Cross-Reactivity

 The ability of a particular antibody to combine with a particular antigen is referred to as its specificity. This
property resides on the Fab molecule called the combining site.
 When some of the determinants on the surface of apparently unrelated molecules a portion of the Ab directed
against one type of antigen will also react with the other type of antigen. This is called cross-reactivity.
 Cross-reactivity occurs between bacteria that possess the same cell wall polysaccharide as mammalian RBC.
 Intestinal bacteria as well as other substances found in the environment, possess A-like or B-like antigens similar
to A and B erythrocyte antigens.
 Cross-reacting antibodies of this type are termed heterophile antibodies.
 Heterophile antigens are heteroantigens that exist in unrelated plants or animals but are either identical or
closely related in structure so that antibody to one will cross-react with antigen to the other.
 Ex. Human blood group A and B antigens which are related to bacterial polysaccharides. Anti-A antibody is found
in person with blood type B and O but also formed after exposure to pneumococci or other related bacterias.

Antibody Affinity
 Is the initial force of attraction that exists between a single Fab site on an antibody molecule and a single
epitope or determinant site on the corresponding antigen.

Antibody Avidity
 The functional combining strength of an antibody with its antigen.

Immune Complexes
 The concovalent combination of antigen with its respective specific antibody is called this IC.
 An IC maybe of the small (soluble) or large (precipitating) type, depending on the nature and proportion of
antigen and antibody.
 Under conditions of Ag or Ab excess, soluble complexes tend to predominate.
 If equivalent amounts of Ag and Ab are present, a precipitate may form.
 However, all Ag-Ab complexes will not precipitate, even at equivalence.
 Immune complexes once formed is usually removed by phagocytic cells through the interaction of the Fc of the
antibody with complement and cell surface receptors.
 Under normal circumstances, this process does not lead to pathologic consequences.
 It is only in unusual circumstances that the IC persists as a soluble complex in the circulation and, escapes
phagocytosis, and is deposited in endothelial or vascular structures-where it causes inflammatory damage, the
principal characteristics of immune complex disease- or in organs (e.g., kidney), or inhibits useful immunity (e.g.,
tumors, parasites).
 Detection of IC and identification of associated Ag are impt. to the clinical diagnosis of IC disorders.
 Major Histocompatibility Complex
 Human tissue cell have molecules on the plasma membrane called MHC
 These cell surface proteins are called HLA (Human Leukocyte Antigen) in humans.
 They are important in antigen presentation and the regulation of the immune response.
 Differences in HLA antigens among different individuals are the cause of graft rejection reactions.
 The two MHC classes responsible for these reactions are called MHC I MHC class II.
 MHC Class I
 Consists of a single chain anchored into the cell membrane and stabilized by ß2 microglobulin.
 The molecule forms a groove which holds antigen peptides for presentation to cytotoxic T cells.
 Present on all nucleated cells. Cytotoxic CD8 cells ‘’see’’ antigen presented by MHC I.
 Class I HLA are known as HLA-A, HLA-B, HLA-C.

MHC Class II
 Are made up of two chains, which also form a groove to hold and present antigenic peptides.
 Class II molecules present antigen to helper T cells.
 The groove on this allows to present larger peptides than class I molecules
 Present only certain cells of the immune system i.e., antigen presenting cells – macrophages, B cells, dendritic
cells. Helper CD4 ‘’see’’ antigen presented by MHC II.
 Class II HLA are known as HLA-DP, HLA-DQ, HLA-DR.

MHC Genomic Organization

 The genomic organization of MHC is important to determine compatibility between individuals (transplantation
compatibility):
 On chromosome 6
 Co-dominant alleles, one from each parent, both expressed.
 The total set of alleles on each chromosome is called haplotype.
 Each individuals has 2 haplotypes (e.g. HLA-A1, B2, C3; A1, B4, C2)
 Both alleles make up the genotype (e.g. HLA A1, B2, B4, C2, C3)
 Differentiate bet MHC Class I and MHC Class II
 Cellular distribution
 Structure
 Classes
 Size of peptides bound
 Nature of peptide binding cleft
 Interaction with T cells

Antigen Presenting Cells (APC) –

 Different dangers – different process

Exogenous
 Outside the cell. E.g. bacteria
 Taken in, processed and presented cell surface by MHC II molecules.

Endogenous
 Synthesized within the cell. E.g. virus, fungi and tumor antigens
 Processed in cytoplasm and presented cell surface by MHC Class I molecules.

HLA System and Disease:

 The positive association between diseases and HLA- alleles can divided into:
o Those ass. With class I HLA-Ag
 Ex. Ankylosing spondilitits +HLA-B27
 Psoriasis +HLA-CW6
 Myesthenia Gravis +HLA-B8
o Those ass. With class II Ag
 Ex. Rheumatoid Arthritis +HLA-DR4
 Autoimmune thyroiditis +HLA-DR3

MCH (HLA) Testing

 HLA Typing is important in transplantation. The closer the match, the greater the likelihood of successful
transplant.
 2 methods are:
 Serologic – using antibodies against the different HLA antigens to determine the donor and recipient types.
 Mixed Lymphocyte Reaction – using the recipient’s lymphocytes to determine if they react against the
donor’s class II (HLA-D) antigens.

HLA Typing or Tissue typing

 Serologic Techniques:
 Utilize standardized antisera that are specific for particular HLAs
 Lymphocytes from the person being tested are incubated with a selected specific antiserum .
 Complement and dye such as typan blue, are then added.if the Ab in the antiserum have reacted specifically
with the lymphocyte, the cell is damaged.
 The cell will take up the dye (undamaged cells will not)., thus indicating that the lymphocyte possesses a
certain antigen.
 Serological Typing
 Lymphocytes are HLA-typed by crossmatching to panel reactivate antibodies (PRA) using the complement-
dependent cytotoxicity (CDC) test.

Mixed Lymphocyte Reaction

 To determine identity of class II HLA antigens between potential donor and recipient lymphocytes from the
donor are irradiated or treated with mitomycin C to prevent cell division and then added to cells from the
recipient.
 If the class II antigens on the two cell populations are different, the recipient cells will divide rapidly and take up
large quantities of radioactive nucleotides into the newly synthesized nuclear DNA.
 The amount of radiaoactive nucleotides uptake is roughly proportionate to the MHC class II differences between
the donor and recipient lymphocytes.
 Immunoglobulin determinants
 An antigenic determinant is the specific determinant group or molecular configuration against which immune
response is directed.
 Since they are proteins, immunoglubulins can be used as antigens when given to mammals of different species
and produce antiimmunoglobulins.
 When these antiglobulins are analyzed, 3 principal antigenic determinants can be recognized.
 Isotype determinant
 Dominant type found on Ig of all animals of a species
 Heavy chain, constant region structures associated with the different classes and subclasses are termed isotypic
variants.
 Determinants in this category include those specific for each Ig class, such as gamma(γ) for IgG, mu (μ) for IgM,
and alpha (α) for IgA , as well as subclass –specific determinants Κ and λ

Allotype determinants
 2nd principal determinant is found on the immunoglobulins of some, but not all, animals of a species.
 Antibodies to these types (alloantibodies) may be produced by injecting Ig of one animal into another member
of the same species.
 The allotypic determinants are genetically determined variations representing the presence of allelic genes at a
single locus within a species.
 In humans, 5 sets have been found: Gm,Km,Mm,Am, and Hv.

Idiotype determinants
 A result of unique structures on light and heavy chains , individual determinants characteristic of each antibody
are called idiotypes.
 The idiotype determinants are located in the variable part of the antibody associated with the hypervariable
regions that form the antigen-binding site.

Molecular Basis of Antigen-Antibody Reactions:

 Bonding of Ag to an Ab results from the formatioon of multiple, reversible, intermolecular attractions bet. An Ag
and amiinoacids of the binding site.
 Types of bonding
 Hydrophobic bonding
 Major bonds formed bet. Ag and Ab
 Many of the nonpolar side chains of proteins are hydrophobic
 When Ag and Ab come together, these side chains interact and exclude water molecules from the area
of interaction which results in a gain of energy and forms an energetically stable complex
 Hydrogen bonds
 results from the formation of hydrogen bridges bet. O-H-O,N-H-N, and O-H-N.
 Van der Waals forces
 nonspecific attractive forces generated by the interaction bet electron clouds and hydrophobic bonds.
 Electrostatic forces
 result from the attraction of oppositely charged amino acids located on the side of two a.a. residues.

Detection of Ag-Ab Reactions

 Agglutination- process whereby particulate Ag (e.g., cells) aggregate to form larger complexes in the
presence of a specific Ab
 Precipitation – combine soluble Ag with soluble Ab. to produce insoluble complexes that are visible
 Hemolysis testing involves the rxn of Ag and Ab with a cellular indicator (e.g., lysed RBCs.)
 ELISA measures immune complexes formed in an in vitro system

Monoclonal Antibodies
 Are purified antibodies cloned from a single cell.
  These antibodies exhibit exceptional purity and specificity and are able to recognize and bind to a specific
antigen.

Uses of monoclonal antibodies

 Identifying and quantifying hormones
 Typing tissue and blood
 Identifying infectious agents
 Identifying clusters of differentiation for the classification of leukemias and lymphomas and follow-up therapy
 Identifying tumor antigens and autoantibodies
 Delivering immunotherapy