IMMUNOLOGY

Part 1

MTAP 2
IMMUNOLOGY
� Study of our immune system
� Study of a host’s reactions when a foreign
substances are introduced into the body
� Study of all aspects of body defenses,
such as antigens, antibodies,
hypersensitivity, graft rejection and
autoimmune diseases.
HISTORY
YEAR SCIENTIST
Edward Jenner, an English countryside
physician, demonstrated that protection from
1798 cowpox could be generated by the transfer of
postural material from a cowpox lesion instead of
a more hazardous smallpox lesion
Elie Metchnikoff demonstrated that certain blood
1888 cells ingest foreign material, a concept now
known as phagocytosis
1894 Jules Bordet discover complement

1897 Robert Kaus discovered precipitins

HISTORY
Emil von Behring had the distinction of being
1901 awarded as the first immunology-related Nobel
Prize for his works on serum therapy
1951 Reed: Vaccine against Yellow Fever
Georges Köhler, César Milstein, and Niels Kaj
1975
Jerne: monoclonal antibodies
1984 Discovery of T-cell receptor gene
Susumu Tonegawa was awarded the Nobel
Prize for his 1978 discovery of the genetic
1987
principles underlying the generation of antibodies
with different specificites
Frazer: Discovery of vaccine against Human
2005
Papilloma Virus (HPV)
NOBEL PRIZE WINNERS
YEAR SCIENTIST RESEARCH
1901 Emil von Behring Serum antitoxins
1905 Robert Koch Cellular immunity in TB
Phagocytosis
Elie Metchnikoff
1908 Antibody formation theory
Paul Ehrlich
(Immunity)
1913 Charles Richet Anaphylaxis
1919 Jules Bordet Complement
1930 Karl Landsteiner Human blood group antigens
Macfarlane Burnet, Discovery of immunologic
1960
Peter Medawar tolerance
Gerald Edelman,
1972 Structure of antibodies
Rodney Porter
1977 Rosalyn Yalow Radioimmunoassay
NOBEL PRIZE WINNERS
YEA
SCIENTIST RESEARCH
R
George Snell, Jean Dausset,
1980 MHC
Baruj Benaceraf
Niels Jerne
Immunoregulation
1984 Georges Koehler
Monoclonal Antibody
Cesar Milstein
1987 Susumu Tonegawa Antibody Diversity
Edward Donnall Thomas,
1991 Transplantation
Joseph Murray
Cytotoxic T cell
Peter Doherty
1996 recognition of virally-
Rolf Zinkernagel
infected cells
Francoise Barre-Sinoussi
2008 HIV
Luc Montagnier
MUST KNOW!
� POXVIRIDAE
⚫ Vaccinia: Cowpox
⚫ Variola major: Smallpox
⚫ Variola minor: Alastrim
� Vaccination came from the Latin word vacca
meaning “cow”
� Chinese: inhaled powder of dried smallpox
lesions (variolation)
� Christopher Columbus
⚫ Old world to New world: Smallpox
⚫ New world to Old world: Syphilis
MUST KNOW!
� Pope Innocent VII: first to receive a blood
transfusion (from 3 children) and caused his
death
� “Typhoid Mary”: Mary Mallon, the chef
who spread typhoid fever
� Dr. Charles Drew of American Red Cross:
blood transfusion and blood preservative
technique
� Yves Lapiere: gel technology
� 1983: HIV discovery
� 1984: T-cell receptor gene discovery
COMPONENTS OF THE IMMUNE
SYSTEM
NATURAL/ ACQUIRED/
INNATE/NONSPECIFIC ADAPTIVE/SPECIFIC
• Ability of the body to • Specific for each
resist infection using only individual pathogen
the normally present body • Not present at birth
functions • Results in increased
• Present at birth response upon repeated
• Standardized response exposure
for all antigens • With memory
• No prior exposure is
required
• Lacks memory
NATURAL IMMUNITY
 NATURAL/ INNATE/ NONSPECIFIC IMMUNITY
FIRST LINE OF DEFENSE SECOND LINE OF DEFENSE
Physical Cellular/Phagocytes
-intact skin and mucous -neutrophils
membranes -monocytes and
-cilia lining of respiratory macrophages
tract -basophils and mast cells
Chemical -eosinophils and NK cells
-lactic acid In sweat Humoral/Fluid Component
-lysozymes in saliva and -complement proteins
tears -APRs
-acidity of the GIT and -defensins
vagina -properdin
Normal microbiota -interferons A and B
-tumor necrosis factor
-betalysins
FIRST LINE OF DEFENSE
� Also known as the EXTERNAL DEFENSE SYSTEM
of the body
� PRIMARY FUNCTION: to prevent the entry of
potential pathogens and hazardous objects into the
body
⚫ Physical Components: credibility is diminished in the
presence of damages (e.g. abrasions, incisions or
ulcers)
⚫ Chemical Components: destroys or prevents
overgrowth of pathogens
⚫ Normal microbiota: mostly commensals; resides on
the surface of the and deep layers of the physical
components which prevent the overgrowth of
pathogens and viruses
SECOND LINE OF DEFENSE
� Included in the INTERNAL DEFENSE system
together with the third line of defense
� PRIMARY FUNCTION: to recognize invading
pathogens
⚫ Cellular Component: acts by phagocytosis and
secretion of substances necessary for destroying
pathogens
⚫ Humoral/ Fluid Components: soluble
substances with a wide range of functions
A. TOLL-LIKE RECEPTORS AND
CELLULAR DEFENSE MECHANISM
� Its binding will cause phagocytosis to occur
� Have highest concentrations on the ff. cells:
⚫ Neutrophils
⚫ Monocytes
⚫ Macrophages
� Examples:
⚫ TLR2 : interacts with Gram positive bacteria
⚫ TLR4 : interacts with Gram negative bacteria
TISSUE MACROPHAGES
� Liver
� CNS
� Bone
� Lung
� Placenta
� Spleen
� Kidney
� Skin
� Tissue
� Synovium
CELLS
� Cells involved are from the myeloid series
⚫ Neutrophils
⚫ Basophils
⚫ Eosinophils
o PRIMARY FXN:
⚫ Monocyte
⚫ Dendritic cells
� BEST ANTIGEN PRESENTOR
� Covered with long membranous extensions

resembling cell dendrites

� Phagocytose and present antigens to T helper

cells
B. PHAGOCYTOSIS
� Concept was credited to Elie Metchnikoff
� Process of engulfing and killing extracellular organisms and
foreign particles
� STEPS: (I.C.E.D)
1. INITIATION
� Results from tissue damage or multiplication of
microorganisms
� Increased surface receptors of phagocytes for
subsequent adherence
2. CHEMOTAXIS
� Migration of phagocytes in a certain direction under the
stimulation of chemotaxins*
� Positive chemotaxis
� Negative chemotaxis
� Random movement: happens in the
absence of chemotactic substances
⚫ JOB’S SYNDROME
� Random Movement: Normal

� Directional/Chemotactic Movement:
Abnormal
⚫ LAZY LEUKOCYTE SYNDROME
� Random Movement: Abnormal

� Directional/Chemotactic Movement:
Abnormal
� BOYDEN-CHAMBER ASSAY
⚫ test for chemotaxis
B. PHAGOCYTOSIS
3. ENGULFMENT
� Process of enclosing the pathogen into a
phagocytic vacuole (phagosome or
phagolysosome)
� Facilitated by amoeboid movement of
phagocytes
� Opsonins: includes antibodies and complement
proteins which interact with bacterial surfaces and
makes them more susceptible to engulfment and
phagocytosis
� Opsonization is downgraded by virulence factors
such as bacterial capsules.
B. PHAGOCYTOSIS
4. DIGESTION
� Facilitated by digestive enzymes enclosed in cell
particles
� As foreign materials or pathogens are digested, the cells
degranulate
� CGD (Chronic Granulomatous Disease)
� Affects neutrophil microbicidal function leading to
inability of the cell to kill ingested organisms
� Characterized by impaired NADPH production
� Normal: Blue formazan formation
� CGD (+): colorless
� G6PD deficiency, MPO deficiency and glutathione
synthetase/reductase deficiency may also show
positive result
B. PHAGOCYTOSIS
4. DIGESTION

� Oxygen dependent killing

❖ Respiratory burst - production of ROS which

are toxic to bacteria.

� Oxygen independent killing

❖ alterations in pH, lysozymes, lactoferrin, and

granular cationic proteins

MUST KNOW!
� Extracellular pathogens: destroyed via
phagocytosis

� Intracellular pathogens: destroyed by NK

cells and T cells via cell killing
[Link]
� Tissue’s reaction to injury
� CARDINAL SIGNS
⚫ Rubor: redness, caused by dilation of blood
vessels and increased blood flow
⚫ Calor: heat, caused by dilation of blood vessels,
increased blood flow and increased interleukin
production
⚫ Tumor: swelling, caused by extravasation and
accumulation of tissue fluids
⚫ Dolor: pain, due to the damage or trauma
caused to nearby nerves
⚫ Functio laesa: loss of tissue function
C. INFLAMMATION
� STAGES
⚫ Vascular Response
1. Mast cells release HISTAMINE
2. Vasodilation occurs
� Causes increased blood flow
� Increased capillary permeability
⚫ Cellular Response
❏ Margination - center to PERIPHERY
❏ Rolling - transient adhesion of WBCs to endothelial cells
❏ Adhesion- firm attachment
❏ Transmigration- migration of WBCs THROUGH
endothelium -> ________
❏ Chemotaxis- unidirectional/targeted movement
❏ Opsonization - coating
❏ Phagocytosis
C. INFLAMMATION
Neutrophils: 1st to migrate (after about 30 minutes); short-
lived; acute inflammation
Monocytes and Macrophages: 2nd to migrate (after about
4 hours) ; long-lived;chronic inflammation
1. ANTIGEN PRESENTING CELLS

⚫ Resolution and Repair

� Initiated by fibroblast proliferation which may result to
total repair, abscess or granuloma formation
 D. ACUTE PHASE REACTANTS (APR)
� Produced primarily by HEPATOCYTES,
� Plasma proteins which increases rapidly by at least 25%
due to infections or injury
PROTEIN RESPONSE N.V. ↑ FUNCTION
TIME (mg/dL)
CRP(MOST SENSITIVE 6-10 hr 0.5 1000x Opsonization, C’ activation
INDICATOR OF INFLAMM)

Serum amyloid A 3.0 1000x Cholesterol removal

a1-antitrypsin 24 hr 200-400 2-5x Protease inhibitor
Fibrinogen 24 hr 110-400 2-5x Clot formation
Haptoglobin 24 hr 40-200 2-10x Binds free Hb
Ceruloplasmin 48-72 hr 20-40 2x Binds Cu and oxidizes iron
C3 48-72 hr 60-140 2x Opsonization and lysis
Mannose-binding 0.15-1.0 C’ activation
protein

� Negative APRs:
D. INTERFERONS
� Glycoprotein family produced by cells which exert a virus-
nonspecific but host-specific antiviral response
1. TYPE 1
⚫ Non-immune; produced in the initial innate response to
viral infection
a) INTERFERON-ALPHA
� also known as leukocyte interferon
� produced by virus-induced leukocyte culture
� Major producers: NK cells
b) INTERFERON-BETA
� a.k.a. fibro/epithelial/ fibroepithelial interferon
� produced by double-stranded fibroblasts
� Major producer: epithelial cells and fibroblasts
D. INTERFERONS
2. TYPE 2
⚫ Immune; produced as a component of the specific
immune response to pathogens and viruses
⚫ INTERFERON-GAMMA
� Also known as immune interferon
� Produced by immunologically-stimulated
lymphocytes
� MAJOR PRODUCER: T-CELLS and _________
� activates _________
E. TUMOR NECROSIS FACTOR
� Exhibit cytotoxic effects against tumor cells and virally infected
cells
� PRINCIPAL MEDIATOR of acute inflamm. response to
gram(-) bacteria. NEGATIVE SIDE EFFECT: _______
1. TNF-ALPHA
⚫ Cachectin
⚫ Produced by macrophages
2. TNF-BETA
⚫ Lymphotoxin
⚫ Produced by CD4+ and CD8+ cells

F. PROPERDIN
• Exerts bactericidal effect in the presence of C’3
and magnesium
G. COMPLEMENT PROTEINS
� Discovery was credited to Jules Bordet
� Functions:
⚫ Immune system activation
⚫ Mediates inflammation(Anaphylatoxin)
⚫ Opsonization
⚫ Cellular lysis

� Order of discovery: C’1,2,3,4,5,6,7,8,9

� Order of activation: C’1,4,2,3,5,6,7,8,9

H. BETALYSINS
� Released by platelets during coagulation
� Heat-stable cationic substances with bactericidal activity
MUST KNOW!
� Cytokines
⚫ General term for the soluble mediators secreted by
cells to regulate the immune system
� Interleukins
⚫ Cytokines secreted by macrophages and other
leukocytes which has a wide range of functions
depending on the secreting and target cell.
� Defensins
⚫ Cysteine-richcationic proteins which acts against
pathogen’s cell wall � lysis
ACQUIRED
IMMUNITY
ACQUIRED/ ADAPTIVE/ NONSPECIFIC
IMMUNITY
THIRD LINE OF DEFENSE
Specialized Lymphocytes
-T lymhocytes
-B lymphocytes and plasma cells
Humoral
-Antibodies
 TYPES OF ACQUIRED IMMUNITY
ACTIVE 1. NATURAL, ACTIVE
-acquired Ag-Ab -INFECTION with pathogens and
production is DONE subsequent production of
by the body antibodies specific to the antigen
2. ARTIFICIAL, ACTIVE
Advantage: -VACCINATION
LONG-TERM a. Live organisms: smallpox
immunity b. Attenuated or weakened
organisms: Bacillus of Calmette
Disadvantage: and Guerin for MTB =
SLOW immune attenuated M. bovis
response c. Dead organisms: cholera,
typhoid
d. Toxins: tetanus
e. Modified virus: poliovirus
MUST KNOW!
Inactivated vaccines: composed of micro-organisms that
have been killed with chemicals and/or heat and are
no longer infectious.
o Whole cell viral: polio, rabies, and hepatitis
o Fractional: made from pieces of bacteria or viruses
o Toxoids: modified toxins (diphtheria and tetanus)
o Subunit: hepatitis B, human papillomavirus,
inactivated influenza, acellular pertussis, anthrax, and
Lyme disease
o Polysaccharide
o Conjugate: Haemophilus influenzae type b,
meningococcal, and pneumococcal
MUST KNOW!
Live, attenuated/weakened antigen: composed of micro-
organisms that have been cultivated under conditions
which disable their ability to induce disease. These
responses are more durable and do not generally require
booster shots.
⚫Viral: measles, mumps, rubella, yellow fever,
vaccinia (smallpox), rotavirus, live attenuated
influenza vaccine (LAIV), varicella, and zoster
(shingles)
⚫Bacterial: oral typhoid and Bacillus Calmette-Guérin
(BCG)
 TYPES OF ACQUIRED IMMUNITY
PASSIVE 3. NATURAL, PASSIVE
-acquired Ag-Ab -transfer of antibodies in vivo
production is NOT -transfer of antibodies through
done by the body ingestion of colostrum*
4. ARTIFICIAL, PASSIVE
Advantage: -IMMUNIZATION or administration
IMMEDIATE immune of immune serum IgG antibodies
response via injection

Disadvantage:
SHORT-TERM
immunity
LYMPHOID ORGANS
1. PRIMARY/CENTRAL LYMPHOID ORGANS
⚫ Maturation site of T and B cells
⚫ Site of ANTIGEN-INDEPENDENT
LYMPHOPOIESIS
a) Bone Marrow
� Bursa of Fabricius
� Maturation site for B cells (If BM is not in choices,
select ________)
b) Thymus
� Maturation site for T cells
� Become hypoplastic as age increases
A. LYMPHOID ORGANS
2. SECONDARY LYMPHOID ORGANS
⚫ Site of proliferation and differentiation of T and B
cells
⚫ Functions:
� Trapping site of pathogens
� Stand by areas of T cells, B cells and phagocytes
� Site of cell-pathogen encounter and phagocytosis
� Site of antibody and lymphokine production
� Site of ANTIGEN-DEPENDENT LYMPHOPOIESIS
A. LYMPHOID ORGANS
SECONDARY LYMPHOID ORGANS
a) Spleen
� Filters antigen found in the blood
� LARGEST secondary lymphoid organ
b) Lymph nodes
� Filters antigen in tissue fluids
c) Tonsils and adenoids
d) MALT, BALT, SALT, GALT
e) Peyer’s patches
f) Appendix
A. CELLULAR COMPONENT
� LYMPHOCYTES are the cells involved in
acquired immunity
� Marker: Terminal deoxynucleotidyl
transferase
⚫ For DNA polymerase immunoperoxidase
⚫ (+) color reaction: NONE
⚫ (+) ALL (lymphoid cells)
⚫ (-) AML (myeloid cells
� 20-40% of circulating WBCs
 T CELLS B CELLS
• Cellular-mediated immunity • Humoral-mediated
• 60-80% immunity
• Long-lived (4-10 yrs) • 20-35%
• Develop in the thymus • Short-lived (3-4days)
• Produce lymphokines • Develop in the BM
(cytokines) • Produce antibodies
• Markers: CD2, CD3, CD4, • Markers: CD19, CD20, CD21,
CD8 CD40, MHC Class II
• Identified by Erythrocyte • Identified by surface Igs
Rosette Assay
• Locations: • Locations:
-medullary, perifollicular -follicular and medullary or
and paracortical region of germinal centers of lymph
lymph nodes nodes
-periarteriolar regions of -primary follicles and red
spleen pulp of spleen
-thoracic duct of the -follicular region of GALT
circulatory system
T LYMPHOCYTES
� Involved in CELLULAR-MEDIATED IMMUNITY
� Most circulating T lymphocytes express 3 of the following
CD markers:
⚫ CD2 – sheep RBC receptor, CLASSICAL T cell RECEPTOR
⚫ CD3 – part of T cell antigen-receptor complex
⚫ CD4 – receptor for MHC class II molecule
⚫ CD8 – receptor for MHC class I molecule

� Helper-inducer T cells: CD4+

� Suppressor-cytotoxic T cells: CD8+
� CD4+: CD8+ normal ratio = 2:1
� In HIV, ratio is : 0.5-1:2
� In AIDS, CD4+ cell count drops to <200/uL (N.V. 500-
1,300/uL)
T LYMPHOCYTE DEVELOPMENT
1. DOUBLE-NEGATIVE THYMOCYTES
⚫ CD2, CD5, CD7 (+)
Unsensitized T
⚫ CD4 (-) AND CD8 (-)
cell
2. DOUBLE-POSITIVE THYMOCYTES -Memory T cell
⚫ CD4 (+) AND CD8 (+)
3. SINGLE-POSITIVE THYMOCYTE (MATURE T CELL)
⚫ CD4 (+) OR CD8 (+)
4. ACTIVATED T LYMPHOCYTE
⚫ Found in the 2° lymphoid organs
⚫ CD25(+) : receptor for IL-2 which causes lymphocyte
proliferation
5. SENSITIZED/SECRETORY T LYMPHOCYTE
⚫ Exposed to antigens
⚫ Secretes lymphokines
TCR
SUBSETS OF T LYMPHOCYTES
1. T-helper/_____ cells (Th)
⚫ 60%
⚫ help B cells to produce antibodies
2. T-suppressor cells (Ts)
⚫ 30%
⚫ suppress B cells NOT to produce antibodies.
3. T-cytotoxic cells (Tc)
⚫ lyse and destroy foreign cells (cancer cells) and
virus-infected cells reject grafts & transplants
4. T-delayed type hypersensitivity (Tdth)
⚫ involved in some delayed allergic reactions
SUBSETS OF T-HELPER CELLS
Th1:
◆ enhance inflammation(____________)
● IL-2:

● IFN-gamma:
○ stimulates B cell production of
___________________________

● IL-12:

● stimulates B cell production of

___________________________
Th2:
◆ suppress inflammation(____________)
● IL-4:

● IL-5:
Source:
Turgeon
B LYMPHOCYTES AND ITS
DEVELOPMENT
� Involved in HUMORAL-MEDIATED IMMUNITY
� Bone-marrow derived and precursor cells for antibody
production
1. PRO-B CELL (PROGENITOR B CELL)
⚫ No antibodies yet
⚫ There is rearrangement of genes coding for the formation of
heavy chains (Ch.14)
2. PRE-B CELL (PRECURSOR B CELL)
⚫ No surface antibodies yet Light Chains:
⚫ Mu chains are present in the cytoplasm Lambda: Ch. 22
⚫ Heavy chains are present on the surface Kappa: Ch. 2
⚫ There is rearrangement of genes coding for light chains
B LYMPHOCYTES AND ITS
DEVELOPMENT
3. IMMATURE B CELL
⚫ Has complete antibodies on the surface
⚫ First antibody on the surface of B cell: monomeric
IgM
⚫ CD21 and CD35 markers are expressed
4. MATURE B CELL
⚫ Increased IgM density
⚫ IgD is expressed
⚫ Cells are releases from the BM and seed in
peripheral lymphoid organs
B LYMPHOCYTES AND ITS
DEVELOPMENT
5. ACTIVATED B CELL
⚫ CD25 � triggered by IL-2 attachment � enhanced
lymphocyte proliferation
� a.k.a lymphocyte capping
6. PLASMA CELL
⚫ Activated B cell
⚫ Surface markers on B cell surface disappear
⚫ WITHOUT surface immunoglobulins but WITH
cytoplasmic immunoglobulins which are later
released as antibodies
B CELL MATURATION
Plasma
Cell

BONE MARROW PERIPHERY

LYMPHOID Pro-B cell Pre-B cell Immature Mature B Activated

STEM B cell cell B cell
CELL

Memory B
cell
CD MARKERS ON T AND B CELLS
ANTIGEN FUNCTION
CD2, CD3,
pan T-cell markers
CD5, CD7
CD19,
B-cell markers
CD20, CD21
CD21 receptor for Epstein-Barr Virus
CD2 sheep RBC receptor
marker for T-helper cells, receptor for MHC
CD4
class II molecule, receptor for HIV
marker for T-cytotoxic cells, receptor for
CD8
MHC class I molecule
CD10 CALLA marker
CD28 Binds B7 (CD80) of B cells
CD33 Myeloid receptor
CD152 a.k.a. CTLA-4; Binds B7 and B7.2 of B cells
CD154 CD40 ligand; Binds CD40 of B cells
 ANTIGEN FUNCTION
CD16, CD56 NK cell markers
CD16 receptor for Fc portion of IgG
Regulator of IgE synthesis;
CD23
triggers release of GM-CSF from monocytes
CD25 receptor for interleukin 2
CD41, CD61 platelet and megakaryocyte markers
Adhesion molecule in most lymphocytes which mediate
CD44
homing to peripheral lymphoid organs
Essential in T and B cell antigen-stimulated proliferation
CD45
Formerly leukocyte common antigen
Decay accelerating factor (DAF) (GPI-linked), decays C3
CD55
and C5 convertases
CD56 Found in NK cells with no known function
Membrane of inhibitory reactive lysis (MIRL), inhibits MAC
CD59
formation
CD71 Glycophorin A, transferrin and erythroid receptor
CD94 Involved in inhibition of Nk cell cytotoxicity
REMEMBER!
� CD4+ T cells recognize antigens ONLY in
the context of MHC class II antigens

� CD8+ T cells recognize them ONLY through

MHC class I antigens.
LABORATORY IDENTIFICATION
1. FLOW CYTOMETRY
⚫ Automated system for cell identification based
on LIGHT SCATTERING as cells flow in single
file through a laser beam
⚫ Forward Light Scatter: SIZE
⚫ Side Light Scatter (90°): CELLULAR
GRANULARITY
2. FLUORESCENCE MICROSCOPY
⚫ Rely on the use of LABELED-MONOCLONAL
ANTBODIES against specific surface antigens
LABORATORY IDENTIFICATION
3. ERYTHROCYTE ROSETTE ASSAY
⚫ For T cells
4. ERYTHROCYTE-ANTIBODY-COMPLEMENT
ROSETTE ASSAY
⚫ For B cells
� Lymphocytes are separated out from whole blood and
then mixed with a suspension of sheep red blood cells
� If 3 or more RBCs attached to a lymphocyte, this is
considered a rosette
� 200 cells are counted and % of cells forming rosettes are
calculated
� False (+)- presence of cold-reacting anti-lymphocyte
antibodies in RA and IM
LABORATORY EVALUATION OF
LYMPHOCYTE FUNCTION
1. LYMPHOCYTE TRANSFORMATION ASSAYS
[Link] are isolated using
FICOLL-HYPAQUE density gradient centrifugation
⚫ Diluted or heparinized blood is
carefully layered on top
of the solution and
the tube is centrifuged
⚫ 3 layers
� Plasma
� Mononuclear layer on top
of density gradient solution
� RBCs and granulocytes
LYMPHOCYTE TRANSFORMATION ASSAYS
2. These are radiolabeled using titrated thymidine
and cultured for several days with a MITOGEN (3–
4 days) or a specific soluble antigen (5–10 days) in
a media enriched with human AB serum
(ROSWELL PARK MEMORIAL INSTITUTE
CULTURE MEDIA)
3. After incubation, the cells are harvested and
transferred to a disk of filter paper.
4. The disks are placed in a scintillation fluid and
radioactive emissions are measured in a liquid
scintillation counter. Lymphocyte analysis may also
be done using flow cell cytometry.
LYMPHOCYTE TRANSFORMATION ASSAYS
� MITOGENS
� substances that initiate DNA synthesis and
mitosis or production of antibodies
� T-cell blastogenesis is induced by:
⚫ Pokeweed mitogens*
⚫ Phytohemagglutinin
⚫ Concanavalin A

� B-cell blastogenesis is induced by:

⚫ Pokeweed mitogens*
⚫ Lipopolysaccharide
⚫ S. aureus-Cowan strain (pure B-cell mitogen)
NK CELLS - BRIDGE
� Part of natural immunity
� 3rd population lymphocytes
� Also known as LARGE GRANULAR
LYMPHOCYTES
� Identified by the presence of CD16 and CD56
& absence of CD3
� Activated by IL-2 and IFN-gamma released by
T helper cells to become LAK cells

� LAK cells: LYMPHOKINE-ACTIVATED KILLER

CELLS
⚫ Part of acquired immunity
MAJOR HISTOCOMPATIBILITY COMPLEX
� Set of genes that control tissue compatibility
� Regulate immune responses and play a role in
graft rejection after organ transplantation
� Genes are located on the short arm of
Chromosome 6
� In humans, it is referred to as Human
Leukocyte Antigen Complex (HLA Complex)
which is encoded by MHC genes
MHC CLASS I molecule:

MHC CLASS II molecule:

 MHC CLASS I MHC CLASS II MHC CLASS III
Genetic HLA-A, B, C HLA-DP C2, C4, TNF
Loci HLA-DQ and Factor B
HLA-DR
Chain alpha-chains alpha-chains Minor MHC
Structure B2-microglobulin Beta-chains antigen
Cell Found on ALL Found on B
Distribution nucleated cells cells and APCs
(Highest in
lymphocytes)
Antigen Cytoplasmically- Extracellularly-
processed derived antigens derived ag
Presents CD8+ cells CD4+ cells
Antigen to
MUST KNOW!
� MHC molecules
⚫ used by APCs to present unsuccessfully destroyed
pathogens to the specialized lymphocytes of the third
line of defense such as T and B cells.
T-cells: _____ to recognize native antigens
B-cells: _____ to recognize native antigens
� Antigen-Presenting Cells
⚫ cells that phagocytose, process, and present
pathogen’s antigens to T and B cells via the MHC
molecules
⚫ Examples of APCs:

NOTE: ALL NUCLEATED CELLS can actually present

antigens in association with MHC CLASS _.
IMPORTANCE OF HLA TYPING
HLA antigens:

1. TISSUE OR ORGAN TRANSPLANTATION

⚫ Recipient and donor or donated organ must be ABO and
HLA matched
� Sibling: 50% chance of being HLA matched; BEST

� Parent: 25% chance of being HLA matched

2. DISEASES ASSOCIATION
⚫ HLA-B27: (90% chance) ankylosing spondylitis
⚫ HLA-DR3: SLE
⚫ HLA-DR4: Rheumatoid arthritis
⚫ HLA-B8:
� Grave’s disease

� Myasthenia gravis

� Addison’s disease
 IMPORTANCE OF HLA TYPING
3. PATERNITY TESTING
⚫ An allele present in the child but not in the mother is
referred to as the obligatory paternal gene (OG)
⚫ Direct exclusion
� If the tested man does not have the possibility of passing
the obligatory paternal gene and the marker for the gene is
absent in the presumed mother BUT the child has it
⚫ Indirect exclusion
� absence of an expected genetic marker in the child when
the parent in question appears to be homozygous (one
allele present) for the gene
� sometimes also known as reverse homozygosity
METHODS OF HLA TYPING
I. LYMPHOTOXICITY ASSAY/
MICROCYTOTOXICITY ASSAY)

⚫ Uses Phase-Contrast Microscope

⚫ Principle:
Complement-dependent
cytotoxicity

⚫ Demonstrates that if the antigen is present on the

lymphocytes, addition of complement will cause
them to become porous and unable to exclude the
added dye.
CLASS I ANTIGENS:
1. Mixture of T and B cells are obtained from the FICOLL-
HYPAQUE CENTRIFUGATION from the potential donors
and recipient
2. Cells are distributed into a series of wells on a microtiter
plate, then antibodies specific for various Class I & Class II
MHC alleles are added to different wells.
3. After incubation, complement is added to the wells and
cytotoxicity is assed by the uptake or exclusion of various
dyes by the cells.
4. If the WBC expresses the MHC allele for which a particular
monoclonal antibody is specific, then the cells will lysed upon
addition of complement, and these dead cells will take up a
dye such as TRYPAN BLUE.
⚫ (+) Color change with TRYPAN BLUE
⚫ (-) Unstained
CLASS II ANTIGENS:

1. Mixture of T and B cells are separated using

NYLON WOOL TECHNIQUE
⚫ B cells adhere to the nylon wool
⚫ These cells are now ready for use and steps
2-4 may be followed.
� (+) Color change with TRYPAN BLUE

� (-) Unstained
 METHODS OF HLA TYPING
II. MIXED LYMPHOCYTE REACTION (MLR)/
MIXED LEUKOCYTE REACTION
⚫ Cellular assay
⚫ Can be used to quantify the degree of class II
MHC compatibility between potential donors and
a recipient.
⚫ Advantage over microcytotoxicity typing:
it gives better indication of the degree of TH cells
activation generated in response to the Class II
MHC antigens of the potential graft
PROCEDURE:
1. Lymphocytes from potential donor that have been x-
irradiated or treated with mitomycin C serve as the
stimulator cells, and lymphocytes from the recipient
serve as responder cells.
2. Proliferation of the recipient T cells, which indicates T
cell activation, is measured by the uptake of
thymidine into cell DNA.

1. The greater the Class II MHC differences between

the donor and recipient cells, the more thymidine
uptake will be observed in an MLR assay.
2. Intense proliferation of the recipient lymphocytes
indicates a poor prognosis for graft survival.
MUST KNOW!
• HLA on RBCs:
• Bennett-Goodspeed (BG) Antigen
• Bga: HLA-B7
• Bgb: HLA-B17
• Bgc: HLA-A28
 B. HUMORAL COMPONENTS
� IMMUNOGEN
⚫ Substance that stimulates antibody production
⚫ Capable of inducing an immune response

� ANTIGEN
⚫ Substance that reacts with a specific antibody but may not
be able to evoke an immune response in the first place
⚫ Capable of combining with an antibody
⚫ MAIN PARTS (of a complete antigen)
� Carrier

� Protein in nature • Hapten

� With high molecular • Non protein in nature
weight • With low molecular weight
� Immunogenic • Antigenic ONLY
ANTIGENIC DETERMINANT
� Also known as EPITOPE
� An antigen must contain at least one epitope
1. Linear Epitope
◆ Made up of amino acids on the same chain
◆ Interact with the paratope based on the primary
structure of antigen
2. Conformational Epitope
◆ Made up of amino acids on different sides of chain,
when they come together become conformational
epitopes
◆ Interact with the paratope based on the 3-D surface
features and shape or tertiary structure of the
antigen
FACTORS AFFECTING IMMUNOGENICITY
� FOREIGNNESS
⚫ ↑ difference in nature = ↑ immune response
� MOLECULAR SIZE
⚫ MW of >10,000 Daltons = POTENT ANTIGEN
⚫ Examples
� Albumin: 40,000 Daltons (good immunogen)

� Hemocyanin: 1,000,000 Dalton (excellent

immunogen)
� CHEMICAL COMPOSITION & COMPLEXITY
⚫ Protein> Polysaccharide>Lipids and Nucleic Acids
FACTORS AFFECTING IMMUNOGENICITY
� ROUTE, DOSAGE AND TIMING
⚫ ↑ Dose = ↑ immune response
⚫ IV and IP> ID> IM and SQ
� GENETIC COMPOSITION
� DEGRADABILITY
⚫ ↑ degradability = ↓ immune response
� STRUCTURAL STABILITY
⚫ ↑ stability = ↑ immune response
� ADJUVANTS
⚫ Substances added to vaccines with less
immunogenic substances
⚫ Added to enhance immune response of the body
ADJUVANTS
1. CFA (Complete Freund’s adjuvant)
⚫
water-in-oil emulsion of M. butyricum
⚫
Stimulates T cell activity = enhaced CMI
2. LPS (Lipopolysaccharides)
⚫ Stimulates B cell activity = enhanced HI
3. Synthetic Muramil Dipeptide
⚫ Stimulates T cell activity = enhanced CMI
4. Alum Adjuvant
⚫
MOST COMMONLY USED adjuvant in vaccination
⚫
enhances phagocytosis
5. Squaline (MF59)
⚫ derived from shark oil, for HIV vaccine
TYPES OF ANTIGEN
� Autoantigen
⚫ derived from the same individual
� Alloantigen/ Homologous antigen
⚫ Derived from different individual of the same
species
� Heteroantigen/ Xenogeneic antigen
⚫ Derived from different species
⚫ Triggers the greatest immune reponse
� Heterophile Antigen
⚫ Found in unrelated plants or animals which cross-
reacts with other antibodies
TYPES OF GRAFTS
� Autograft
⚫ Derived from the same individual
� Isograft/Syngraft
⚫ Derived from different class but identical
individual or twins
� Allograft
⚫ Found on different individual of the same
species
� Heterograft/ Xenograft
⚫ Derived from totally different species
IMMUNOGENECITY OF DIFFERENT
TRANSPLANT TISSUES
� Bone Marrow: MOST IMMUNOGENIC
� Skin
� Islets of Langerhans
� Heart
� Kidney
� Liver
� Bone
� Xenogeneic valve replacements
� Cornea: LEAST IMMUNOGENIC
⚫ Privileged site not reached by the immune system
⚫ Avascular
 CATEGORIES OF GRAFT REJECTION
TYPE TISSUE MECHANISM CAUSE
DAMAGE
Hyperacute Within Humoral Preformed cytotoxic
minutes antibodies to donor
antigens
Accelerated 2-5 days Cellular- Previous
mediated sensitization to
donor antigens
Acute 7-21 days Cellular- Dev’t of allogeneic
mediated reaction to donor
antigens
Chronic > 3 mos Cellular- Disturbance of
mediated host/graft tolerance
ANTIBODY
� [Link].

**subjected to electrophoresis, pH(__):

� Substances produced from antigenic stimulation

� Produced by plasma cells or present on surface
of ______.
� Functions:
⚫ Cell cytotoxicity (intra or extravascular)
⚫ Neutralization(_____)
⚫ Opsonization and complement activation(___)
ANTIBODY STRUCTURE
1. HEAVY CHAIN
⚫ Determines the immunoglobulin class
� Gamma heavy chain: IgG
� Alpha heavy chain: IgA
� Mu heavy chain: IgM
� Delta heavy chain: IgD
� Epsilon heavy chain: IgE
⚫ NOTE:
� IgM and IgE have an extra heavy chain (CH4)
resulting in 5 domains
ANTIBODY STRUCTURE
2. LIGHT CHAIN
⚫ Kappa
� Encoded in Chromosome 2

� 65%

⚫ Lambda
� Encoded in Chromosome 22

� 35%

⚫ Ratio of kappa: lambda = 2:1

ANTIBODY STRUCTURE
3. DISULFIDE BONDS
⚫ H-H and H-L : normal
⚫ L-L : abnormal
� Note: Bence-Jones protein are free monoclonal
immunoglobulin light chains found in the urine of MM px
4. HINGE REGION
⚫ Proline-rich, flexible region of the antibody which
increases the efficiency of antigen-binding and
cross-linking reactions by antibodies
5. DOMAINS
⚫ Sections or regions in an immunoglobulin molecule
⚫ 1 light chain = 2 domains (1 VL + 1CL)
⚫ 1 heavy chain = 4 domains (1 VH + 3CH)
� except IgM and IgE = 5 domains (1 VH + 4CH)
MUST KNOW!
� PARATOPE: site for antigen’s epitope binding

� Isotype
⚫ Heavy chain that determines immunoglobulin class
� Allotype
⚫ Variation
in constant region of both the heavy chain
heavy and light chains
� Idiotype
⚫ Variation in the variable region of both heavy and light
chain
 MUST KNOW!
� Monomer
⚫ Basic immunoglobulin structure
⚫ Composed of 2HC+ 2LC
� IgG, IgD, IgE, Serum IgA and B cell surface IgM

� Dimer
⚫ Consists of 2 monomers
� Secretory IgA : monomers are connected by a

J-chain & has a secretory component

� Pentamer
⚫ Consists of 5 monomers interconnected by a J-chain
� Serum IgM
MUST KNOW!
� J-chain/joining chain
⚫ cysteinerich polypeptide found in dimeric IgA
and pentameric IgM

� Secretory component
⚫ Prevents enzyme degradation of IgA
MUST KNOW!
� In a monomeric antibody, there are:
⚫2 heavy chains
� With 400 amino acid chains
� MW of 50,000 – 77,000 Daltons

⚫2 light chains
� With 200 amino acids
� MW of approx. 25,000 Daltons
THEORIES OF AB DIVERSITY
� EHRLICH’S SIDE-CHAIN THEORY
⚫ Formulated by Paul Ehrlich
⚫ Certain cells had specific surface receptors, once antigen was
introduced, it would select the cell w/ the proper receptor.
Receptors would break off together with the attached antigen,
circulate as antibodies.
� TEMPLATE/INSTRUCTIVE THEORY
⚫ By Felix Haurowitz
⚫ Cells produce a general antibody and when antigen contact
occurs, antigen serves as a template for the general antibody to
be molded as a specifc antibody which then enters the circulation
� CLONAL SELECTION
⚫ The MOST ACCEPTABLE theory by Niels Jerne and MacFarlane
Burnet
⚫ Specific antigen selects the specific cells capable of responding to
it and produces the corresponding antibody
ANTIBODY PORTIONS & FUNCTIONS
� Fab portion:
⚫
Fragment antigen binding
⚫
1LC+1/2 HC
Function: Direct neutralization of antigen
� Fc portion:
⚫
Fragment crystalline
⚫
2 halves of HC
Functions:
1. Responsible for biologic activities of the antibody:
⚫ Activation of complements leading to inflammation,
chemotaxis and lysis
▪ IgG, IgA, IgD: CH2 serves as C1q receptor
▪ IgM, IgE: CH4 serves as C1q receptor
▪ Placental Transfer
▪ Skin fixation
2. Opsonization
IgG
⚫ PREDOMINANT IMMUNOGLOBULIN in humans
⚫ Smallest antibody, coating antibody, clinically significant
antibody which reacts at 37°C
⚫ Longest half-life (23 days)
⚫ Major immunoglobulin class produced in the
secondary immune response
⚫ Monomer
⚫ Consists of four subclasses:
◦ IgG1 (67%): most efficient in crossing the placenta
◦ IgG2 (22%): cannot cross the placenta
◦ IgG3 (7%): largest, most efficient in C’ activation
◦ IgG4 (4%): cannot fix C’
FUNCTIONS:
1. Provides protective immunity to newborns.
2. Fixation of complement
⚫ Two IgGs are needed to fix a complement
⚫ (3>1>2)
3. Opsonization
4. Neutralization of toxins and viruses
⚫ ADCC reactions (CD16: receptor for IgG)
⚫ NK cells + IgG + Viral antigen � Perforin release
5. Participation in agglutination and precipitation
reactions
⚫BEST PRECIPITATING ANTIBODY
IgA
� Migrates in the B region (electrophoresis)
� Associated with ANAPHYLAXIS
� Consists of two subclasses:
⚫ IgA1
⚫ IgA2

� Predominant antibody found in body secretions

⚫ saliva, tears, colostrums, intestinal fluids
� RESPONSIBLE FOR MUCOSAL IMMUNITY

� With secretory component/piece and

a J-chain binding the 2 monomers

**function of secretory component:

IgM
• Known as the MACROGLOBULIN
�in serum:
�on surface of B cell:

• Reacts at rooM temperature

✔ MOST PRIMITIVE
✔ First to appear in phylogeny and the last to leave in
senescence
✔ First antibody to appear on B-cells
✔ Major Ig class produce in primary immune response
✔ Does NOT have a hinge region
✔ Best agglutinating and complement-fixing antibody due to its
multiple binding sites
IgM
• Its heavy chain has an additional domain (CH4)
which serves as the receptor for complement
• Due to its large size, it is confined within
intravascular space and not found in CSF
• Free state = “STAR-LIKE”
• Attached to an antigen = “CRAB-LIKE”
• First effective defense against bacteremia
• Antibody most often formed in response to
stimulus by Gram (-) bacteria
• Consists of two subclasses:
• IgM1
• IgM2
FUNCTIONS:
1. Complement fixation
⚫ BEST COMPLEMENT FIXER
2. Agglutination
⚫ BEST AGGLUTINATING ANTIBODY
3. Opsonization
4. Neutralization of toxins

Examples of IgM:
⚫ Wasserman antibodies,
⚫ Heterophil antibodies
⚫ Rheumatoid factor
⚫ Cold agglutinins and allohemaglutinins
IgD
• Second to appear on B-cell
• Primarily a cell membrane immunoglobulin
found in the surface of MATURE B lymphocytes
in association with IgM
✔Postulated to be anti-idiotypic antibody
✔MOST SENSITIVE TO ENZYME
DEGRADATION

• Function: IMMUNOREGULATION
IgE
✔Least abundant immunoglobulin in serum
✔REAGINIC ANTIBODY
• Heat-labile antibody, originally called reagin
• Binds strongly to a receptor on mast cells and basophils
(via Fcεr) and together with antigen, mediates the
release of histamines and heparin from these cells

• Mediates some type of hypersensitivity reactions

(Type 1 hypersensitivity), allergies and anaphylaxis and
is generally responsible for an individuals immunity to
invading parasites

• Like, IgM it has also an additional domain (CH4)

• Atopy = IgE-mediated allergic reactions
 IMMUNOGLOBULIN PROPERTIES
IgG IgA IgM IgD IgE

Serum: mono
Structure Mono Penta Mono Mono
Sec: dimer
Heavy
γ α μ δ ε
Chain

% Total Ig 70-75 10-15 10 <1 0.002

Serum
23 days 5-6 days 5 days 1-3 days 2-3 days
half-life
150,000 160,000 900,000 180,000 190,000
MW
Daltons Daltons Daltons Daltons Daltons
Sedimentation
coefficient 7S 7S 19S 7S 8S
ANTIBODY/POLYMER
REDUCTION
� Conversion of polymeric antibody into a
monomeric antibody
1. 2-mercaptoethanol (2-ME)

2. Dithiothreitol (DTT)

⚫ They both destroy the J-chain of

antibodies
MONOMER FRAGMENTATION
� Papain
⚫ Fragments a monomer ABOVE OR
EXACTLY at the hinge region
⚫ Produces 3 fragments: 2Fab + 1Fc
� Pepsin
⚫ Fragments a monomer BELOW the hinge
region
⚫ Produces 2 fragments: Fab2 + 1 Fc’
� Fab2 = 2LC + 2halves of HC

� Fc = degraded into smaller fragments

ANTIBODY RESPONSE
� FOUR PHASES
⚫ Lag phase: no antibody detectable
⚫ Log phase: antibody titer rises
logarithmically
⚫ Plateau phase: antibody titer stabilizes
⚫ Decline phase: antibody is catabolized
� TWO TYPES
⚫ Primaryantibody response
⚫ Secondary antibody response
 PRIMARY ANTIBODY
RESPONSE
� Long lag phase: 3-14 days
� Disease usually develops due to the SLOW
antibody production
� FEWER memory B cells and plasma cells are
produced resulting to LOW antibody titer
� IgM
⚫ predominant type of antibody produced
⚫ short-lived immunity
� Antibodies produced have LOW AFFINITY to
corresponding antigens
SECONDARY ANTIBODY
RESPONSE
� Shorter lag phase: hours to few days
� Longer and higher plateau phase
� More gradual decline
� B memory cells: divides QUICKLY to form plasma
cells
� Plasma cells: produce a HIGHER titer of
antibodies
� No sufficient time for disease to develop and the
person becomes IMMUNE IgG
⚫ predominant type of antibody produced
� Antibody produced have HIGHER AFFINITY to
antigen
ANTIGEN-ANTIBODY INTERACTION
� Specificity
⚫ an antibody binds only to a particular and
specific antigen
� Cross Reactivity
⚫ happens when antigenic determinants are
shared by the antigens of different molecules
⚫ Heterophile antibodies: cross-reacting
antibodies
� Affinity
⚫ forceof attraction between a single Fab site
and a univalent antigen
� Avidity
⚫ forceof attraction between multiple Fab sites
and multivalent antigen
ANTIGEN-ANTIBODY INTERACTION
� Immune complex
⚫ multiple noncovalent interaction between
antigen and antibody
⚫ normally cleared by phagocytic cells via the
Fc portion
⚫ if not cleared, it may become deposited in
tissues � immune complex disease

1. Small (Soluble Type)

• either antigen or antibody is in excess
2. Large (Precipitating Type)
• antigen and antibody are in equal amounts
BINDING FORCES
1. Hydrophobic bonds: MAJOR BOND
2. Hydrogen bonds
⚫ formation of hydrogen bridges between appropriate
atoms (O-H-O, N-H-N)
3. Van der Waal’s Force/London Dispersion force
⚫ Nonspecific
⚫ Linkages occur between electrically neutral
molecules when they are so close to each other
4. Electrostatic attraction/force
⚫ Attraction between a (-) with a (+) pole

NOTE: ALL of these produce a NONCOVALENT BOND.

GOODNESS OF FIT
⚫ Complementary matching of determinants &
binding sites
⚫ Create ample opportunities for the simultaneous
formation of several
non-covalent bonds and few opportunities for
disruption of the bond
⚫ Important in determining the binding of an Ab
molecule for a particular Ag
MONOCLONAL ANTIBODIES
⚫ Kohler, Milstein (1975)
◦ won the 1984 Nobel Prize in Physiology & Medicine
for their discovery of somatic cell hybridization - the
technique used to produce monoclonal antibodies
◦ used Sendai virus to fused myeloma cells with with
spleen cells to create an immortal cell line of
hybridoma cells
⚫ Definition:
⚫ Monoclonal antibodies are purified antibodies cloned
from a single cell which exhibit exceptional purity and
specificity and are Able to recognize and bind to a
specific antigen
MONOCLONAL ANTIBODY PRODUCTION
1. Mice are immunized (IP) with an antigen containing the
epitope of choice (from an antigen
2. After 2 to 4 days, splenic B cells are removed and are mixed
with cultured mouse myeloma cell.
3. HGPRT-deficient myeloma cells are fused with the B cells
using polyethylene glycol or PEG (rather than Sendai virus)
producing fused or HYDRIDOMA CELLS. Afterwards, the
normal B cells die leaving only the hybridoma and unfused
myeloma cells.
4. Then, the cell mixture is placed in the Hypoxanthine-
Aminopterin-Thymidine (HAT) medium, unfused myeloma
cells die while hybridoma cells proliferate.
5. Hybridoma cells divide rapidly in HAT medium. Those
producing antibodies specific to the desired epitope will be
grown in mass culture and cloned.
� Whole process: about 3-6 months
� Mice: animal of choice
USES:
� Hormone identification and quantification
� Blood and tissue typing
� Infectious agent identification
� Diagnosis of leukemias and lymphomas via CD
marker identification
� Antigen and autoantibody identification
� Immunotherapy

NOTE: It also has DISADVANTAGES:

⚫ Too specific
⚫ Unable to fix complement
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