CYTOTOXICITY TEST, NEUTRALIZATION,

MEASUREMENT OF CELL MEDIATED IMMUNE RESPONSE

CYTOTOXICITY TEST
- Secondary binding test (like Precipitation, Agglutination and Complement Fixation)
- Detect cell destruction as a result of Ag-Ab reaction in presence of complement
- Complement may cause damage to the cell
- If there is no Ag-Ab reaction the complement cannot fix to the cell. Thus the three (Ag,
Ab and Complement) must be present in the test.
- Dye is added- only damaged cells will take up the dye
(Trypan Blue and Eosin Y)
Uses
HLA typing
Transplant surgery
Mediated Application
1. In transplant surgery
- Detects the closest match for transplant
2. Identification of HLA antigen associated with diseases
HLA DR4 Rheumatoid Arthritis
HLA B8 Graves disease (goiter), Hashimoto Thyroiditis (autoimmune disease that
starts as hyperthyroidism and after a time present as hypothyroidism), Sarcoidosis
(enlarged lymph nodes)
HLA B27 Ankylosing Spondilitis arthiritis in the backbone ( 85% of px infected with
A.S. Presents with HLA B27), Graves disease, Reiters disease ( present with
symptoms of arthritis, conjunctivitis and urethritis)
A13-B17 Psoriasis

HLA TYPING BY LYMPHOTOXICITY

Ficoll - Hypaque method

- Used for isolation of mononuclear cells from blood
- Ficoll is a high molecular weight polysaccharide
that induces RBC agglutination
Procedure
Heparinized blood + Ficoll Hypaque solution
2 bands are formed in Ficoll Hypaque
upper: 60-85% lymphocytes
15-40% monocytes
lower: mostly mature granulocytes
Upper band is carefully removed with a Pasteur
pipette washed with HBSS (Hanks Balanced Salt
solution)

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HLA TYPING BY LYMPHOTOXICITY
- 96 well microtiter tray
- Each well contains 1uL of Ab to react with HLA Ag
Procedure
1) Add IuL of washed cell is added to each well (2000 lymphocytes
Incubate at RT for 1hr (for Ag-Ab reaction)
2) Add rabbit complement
Incubate at 37C for 1-2hrs (allow complement to bind to Ag-Ab reaction)
3) Add the dye ( Eosin Y or Trypan blue)
4) Formalin (to fix cells/ preserve morphology)
5) Examine under phase contrast microscope

If Abs reacted with Ag on a lymphocyte, the

complement is fixed and damages the cell; dye
can then enter
A (+) result (cell death) indicates that person
has the particular HLA being tested for

NEUTRALIZATION TEST
- Secondary binding test
- Ag-Ab reaction in which specific antiserum is able to diminish/abolish some biologic
property of Ag but not its antigenicity:
Toxicity
Infectivity
Enzymatic activity
- An important mechanism in host defense against
o Extracellular viruses
o Most infective bacteria
o Microbial toxins
Antitoxin
- specific toxin neutralizing Ab
- Protective Ab that inactivates reaction produced by microorganism
Uses
1) To identify its corresponding toxin
2) To identify certain viruses isolated from patients (w/ a stock of known virus Abs in
patients serum can be Identified)
Toxoids
- Toxin treated with formaldehyde (process of attenuation) to make toxin harmless while
maintaining its antigenic activity
Uses
1) Immunization to induce formation of antitoxins (e.g. DPT)

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Antistreptolysin O Test (ASOT)
- Measures the titer of Ab against the Streptolysin O in patients infected with
Streptococcus pyogenes (Group A streptococci)
- 2 hemolysins produced by S. pyogenes
a) SLS not immunogenic ; oxygen stable
b) SLO antigenic ; oxygen labile (has toxic effects on the heart and the kidney)
- 2-3% of patients with S. pyogenes infection develop Rheumatic fever (heart)
- Some may develop Acute Glomerulonephritis (kidney)
- This test involves inhibition of hemolysin by Streptolysin O with the antitoxin, ASO
(+) no hemolysis
(-) hemolysis
ASOT
- Reciprocal of highest dilution showing no hemolysis
- Each unit of titer is called Todd Unit
- Healthy person may have ASO titer as high as 200 IU/mL / 125 IU /mL
o A titer of >200 IU/mL = significant / suggestive of disease
o A fourfold increase in between paired sera (2-3 weeks apart) is indicative or
current /recent infection

1 2 3 4 5 6 7 8 9 10 11 12 13 14
1:10 1:100 1:500
Serum 0.8 0.2 1 0.8 0.6 0.4 0.3 1 0.8 0.6 0.4 0.2 0.0 0.0
B saline 0.2 0.8 0 0.2 0.4 0.6 0.7 0 0.2 0.4 0.6 0.8 1.5 1.0
Shake tube gently + mix
Strep O 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0 0.5
RGT
Shake gently, incubate at 37C for 15 mins
O cells 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Shake gently, incubate at 37c for 45 mins

Todd 12 50 100 125 166 250 333 500 625 833 1250 2500
Units

Buffer used Phosphate buffer saline

After 2nd incubation, tubes are centrifuged and titer of ASO is read

INSERT FORMULA FOR DILUTION HERE

1st incubation: if ASO is present in serum, neutralization of SLO occurs

2nd incubation: any free SLO can attach and lyse RBC

A titer of 166 IU/mL or 166 Todd Units is used since 200 is not part of the serial dilution
Tube 13: (RBC Control) : should show no hemolysis (+)
Tube 14: (SLO Control) : should produce complete hemolysis (-)

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MEASUREMENT OF CELL MEDIATED IMMUNE RESPONSE
- Measure reaction mediated by cells
- Participation of Ab and Complement is not required
- In some bacterial infection (Tuberculosis) and viral infections it is primarily the CMI that
imparts resistance & keeps record
- CMI is important in defending against fungi, parasites, tumor and rejection of organ
transplant
- CMI plays an important role in ALLOGRAFT REJECTION (Host graft rejection)

Cells involved in CMI reaction

1) T lymphocytes
a) Helper T cells (CD4 + T cells)
- Antigen recognition and secretion of cytokines
b) Cytotoxic T cells (CD8 + T cells)
- Kills infected cells
2) Monocytes & Macrophages
- Present Ag to T cells (and B cells) & secretion of cytokines (known as
monokines IL12 : activates T helper cells and NK cell to secrete interferon
gamma) that activates T helper and NK cells
3) Large granular lymphocytes / NK cells
- Inactivates pathogen
4) Killer cells of ADCC ( Ab Dependent Cellular Cytotoxicity)
o Neutrophil
o Monocyte and Macrophage
o NK Cell
*** all three has Fcr

CMI response in 2 phases

a) RECOGNITION
- Host cells recognize Ag / donor cells as foreign and they proliferate
b) DESTRUCTION
- Host cells respond by attacking foreign cells
Example:
o in Graft vs Host disease = cells in graft recognize foreign Ags in host cells and
destroy the host cells
o In Host vs Graft = host cells recognize foreign Antigens (host cells are active w/c
attack transplanted organs and destroys it)

CMI is more difficult to measure than Humoral immunity

CMI can be measured by:

a) Ability of T cell to secrete cytokines
b) Proliferating capacity of T cells they proliferate when they recognize foreign Ag or cells
c) The cytotoxic ability of T cells respond by attacking foreign cells
d) Enumeration of T cells, macrophage and NK cells
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TESTS FOR CMI

1) IN VIVO for lymphoid cell competence

A) Skin test for the presence of delayed type hypersensitivity

(Old name: INTRADERMAL SKIN TEST)

- Most persons respond with delayed type reactions to skin test Ag because
of past exposures to these Antigens
- Delayed type Hypersensitivity is mediated by T CELLS, not Ab
- It is dependent upon the release of lymphokines from activated T cells that
attract & hold inflammatory mononuclear cells at the skin test area
- Macrophage and Monocyte Chemotactic Factors lymphokine
- Main cells involved are: CD4 (helper) T cell and Macrophages

Procedure:
1) Intradermal injection of Ag into a suspected sensitized host
Ag will be recognized by CD4T helper cells which will secret the lymphokine:
MMCF Macrophage & Monocyte Chemotactic Factor, which when release
attracts monocyte and macrophage
2) A reaction at site of injury occurs as a red bump/lesion (w/in 24-48 hrs 72 hrs
max)
Lesion: firm, indurated (hard), non-edematous, erythema, red swelling
Histology: shows mononuclear cells, basophils and few neutrophil
*** if it is Ab mediated: shows neutrophil infiltration and edema

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1.A.1 TUBERCULIN TEST (to check if patient was exposed to TB bacilli)
- classic example of Delayed Type Hypersensitivity reaction
- First Ag used = OT (Old Tuberculin) a crude filtrate of broth culture of M. tuberculosis
which was first demonstrated by Robert Koch
-Ag used now: PPD (Purified protein derivative) of tuberculin, prepared by precipitiation
of broth culture of M. tuberculosis.

METHODS OF APPLYING PPD

1) Von Pirquet test
- Ag rubbed into a scarified skin (scratches/shallow cuts on skin)
2) Tine test
- Ag is rubbed into multiple punctures in skin
3) Vollmer Patch Test
- Ag is impregnated in paper and taped to skin
- Patient friendly
4) Mantoux test
- Not accurate & reliable method of skin test
- Intradermal injection of 1ml PPD in forearm of px
- Read after 48-72 hours
- (+) area of indurated at site of injection >6mm surrounded by erythema
- Means that patient has been sensitized by vaccination (BCG), presence of
primary TB infection
- Serves as a screening test
- A (+) result requires additional testing through:
o Chest X-ray and CT scan for lung lesion
o Inoculation of bacterium
Medium of choice: Leuwenstein Jensen medium
- A (-) result shows no erythema or lesion is <6mm
o No TB infections / patient is in a state of anergy (state of
unresponsiveness)

ANERGY - inability to react to a battery of common Skin Ag can be due to

a) Technical error in skin testing (usually done by Nurses)
b) Immunologic deficiency (Inefficient T cell, does not release MMCF)
c) Infections that suppress Cell Mediated Immune response
- Influenza, mumps, measles, typhoid, military TB, disseminated mycotic
infection, leprematous leprosy, scarlet fever)

1.A.2 Coccidioides / SPHERULIN SKIN TEST

- Same procedure with the Tuberculin test but differs in the Ag used
- Used to diagnose Coccidioidomycosis
- Ag used: Mycelial extract of Spherule
- (+) test = induration on skin is produced by patients with Coccidioidomycosis,
which indicates past or present infection with C. immitis

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 B) Skin test for the ability to Develop Delayed Type Hypersensitivity
Old term: (Contact Hypersensitivity) / Contact Dermatitis test
- Detects the ability of patient to mount a CMI response to a new Ag to which
he has not been previously exposed
- Normal person readily develop reactivity to new Ag applied
Antigens used:
Simple chemicals
o Dinitrochlorobenzene (DCNB) confirms anergy in patients
o Lethal formaldehyde
Plant materials
o Posion ivy
o Poison oak
Topical drugs
o Sulfonamides
o Neomycin
o Some cosmetics
o Soap

- When some Ag is applied to same area 1-2 weeks later, they respond with a
delayed type skin reaction
- Immunocompromised patient with immunocompetent CMI produce a (-)
result
PROCEDURE
1) High dosage of DNCB is directly applied to skin (volar aspect of forearm)
2) 1-2 weeks later, a lesser dose is applied to the same area

(+) result = FLARE (erythema of skin that spreads outward), vesicular reaction,
itching, eczema, necrosis within 12 48 hours

Induration rarely occurs since test does is not applied intradermally

Intensity of the resulting contact dermatitis provides a rough estimate of
patients ability to amount a CMI response

C) GRAFT REJECTION
- Done only in animal study
- Transplantation of Organ tissue between non -identical strains of mice
result (+) to Graft rejection
Example is the use of Skin Grafts
- If a skin graft from an unrelated donor is applied, it is recognized as non-self
and rejected by the recipient within 1-2 weeks
- The transplant is attacked by T cells that directly lyse the grafted cells and by
macrophage activated by T cells
- The main immune cells involved are CD8 Cytotoxic T cells) and macrophages
cells

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2) IN VITRO
Measures
- Proliferation of Lymphocytes in response to Ag
- Production of lymphokines

A. Lymphocytes Blast Transformation

- In vitro tests for CMI hypersensitivity (uses Ficoll Hypaque)
- Ag is recognized by receptors on B & T cells which then undergoes blast
transformation
- Lymphocytes increase in size, nucleolus enlarged, RER and microtubules
become prominent, increase rate of DNA synthesis; mitosis ensues
- Mitogens: substance that can trigger mitosis of cells

Non- specific Mitogens that Activate Human Lymphocytes

Mitogens Biologic source Relative Specificity
*Phytohemagglutinin (PHA) Kidney Source T cells
*Concanavalin A (Con A) Jack Bean T cells, B cells
Antithymocyte globulin (ATG) Heterologous antisera T cells
Staph. Protein A (SpA, SAC) S. aureus (Cowan 1) B cells
*Pokeweed mitogen (PWM) Phytolacca Americana B cells, T cells
Streptolysis (SLS) S. pyogenes Probably T cells
Endotoxin (LPS) Gram (-) bacteria B cells
(*) MITOLECTIN

PROCEDURE
1) Add suspension of purified lymphocyte (by Ficoll Hypaque)
2) Mix with Mitogen
3) Cultivate for 48-96 hours
4) 12 hours before end of incubation period (cells are actively dividing)
5) Add the indicator, 3H Thymidine, a radiolabeled nucleotide (only actively dividing cells
take up stain)
6) Harvest (using automated harvester) and wash cells
7) Transfer to a tube containing scintillating fluid
8) Measure radioactivity of 3H Thymidine as CPM (counts per minute) using Scintillation
Spectrophotometer

- Non dividing cells do not take up Thymidine, while dividing cells take up
thymidine which means that they are actively synthesizing DNA
- Number of myeloblast formed is proportional to uptake of thymidine &
functional competence of lymphocyte which determines immune
responsiveness of patient
- Normal control must be run simultaneously with the patient lymphocyte in
lab

- To obtain the simulation index: =
>1 = normal <1 = not normal
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B) Mixed Lymphocyte Culture / MIC

- primary clinical use:

selection of a compatible donor for organ transplant
- cellular approach of tissue matching
- performed together with HLA typing
* HLA typing alone may not be satisfactory since not all allo Ag important for
transplant have been Identified
- detects cellular recognition of foreign class II Ag present on other cell
- The stimulator cells are treated with Mitomycin C & Radiaton (blocks DNA synthesis
of cells so it cannot proliferate)

PROCEDURE

lymphocyte from 2 individuals when mixed together stimulate each other to undergo
blast transformation and proliferate

STRAIN A Mix with STRAIN B

(5-6 days incubation)
Responder cells Treated with Mitomycin C
or irradiated (stimulator
cells)
Recognize foreign Class II
Ags on Stimulator cells

Proliferation

- Proliferation is measured by uptake of 3H - Thymidine

- Increased CPM result is not good, and indicates that donor is not compatible with
recipient.
- However, if patient needs immediate transplant and most donors produce CPM
responses, choose the donor with the least amount of CPM response
- The greater the amount of DNA synthesized in the responder cells, the more foreign are
the Class II Ags of the stimulator cells, this indicates unsatisfactory match of the Graft
and is likely to be rejected
- A lack of MLC indicates compatibility between donor and recipient

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GRAFT VS HOST DISEASE
- Occurs when donors Cytotoxic T cells recognize foreign cells and destroy recipients
cells
- Symptoms: maculopapular rash, jaundice, hepatosplenomegaly, diarrhea
- May leas in overwhelming infection and death
- Anticipated by doing MLC, donor cells are the responders, while recipient cells are the
stimulator cells

HOST VS GRAFT DISEASE

- Occurs when ___ cells recognize foreign Ags on Donor cells and respond by
proliferation

e.g When kidney recipients lymphocytes respond by proliferation to the donors

lymphocytes

In Bone Marrow transplant, the proliferation of the donor cells in response to the
recipient cells indicate poor prognosis, an example of Graft vs Host.

When to do BM transplant
a) Severe Combines Immunodeficiency disease (SCID)
b) Aplastic anemia
c) Leukemia

SEROLOGY MIXED LYMPHOCYTE MOLECULAR ASSAYS

Gold standard
Detects HLA Ag of donor and
recipient
Low resolution typing
Identify molecular ___ that
are actually expressed on the
cell and involved in minor
rejection of mismatched graft
CULTURE
Cellular approach PCR (method of choice)
Detects foreign Class II Ags Identify genotype
found on cells
Not applicable for cadaver Cannot see CPM
organ transplant
Originally used for Class II
typing
Rarely used
Involves culture of
lymphocytes of 2 individuals

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 C) MIGRATION INHIBITION TEST
- Macrophages, when packed into a capillary tube by centrifugation, will begin to
migrate out after a few hours
- The test measures the ability of the T cells to release the lymphokine: Macrophage
Migration Inhibition Factor (MMIF), to inhibit migration of macrophages out of the
capillary tube
- In vivo, this lymphokine inhibits the migration of macrophages and that they are
retained at the site of Ag response
- Considered as in vitro test of CMI hypersensitivity

I. DIRECT METHOD II. INDIRECT METHOD

- Lymphocyte collected by Ficoll-
Hypaque and Ag are added to
capillary tubes containing guinea pig
macrophages (target cells)
(+) reaction: no migration of
macrophages
(-) reaction: migration of
macrophages
- Purified lymphocyte suspension + Ag
or Mitogen (PHA,
Phytohemmagglutinin)
- incubate for 24 48 hours
- remove supernatant liquid
(Concentrated by Lyophilization)
- assay for MIF activity (Guinea Pig
macrophages in capillary tube)
(+) result: No migration of
macrophages
(-) result: Migration of macrophages

D) ENUMERATION OF T CELL SUBSETS

- First separate lymphocyte using Ficoll-Hypaque method
- Identify T cells by
a) Using mouse anti-T4 or anti-T8 monoclonal Abs
- Monoclonal Abs may be conjugated with peroxidase or fluorescin
isothiocyanate (FITC)
o Peroxidase stained cells = reddish brown stain at periphery under LM
o FITC stained cell = fluoresce under fluorescent microscope
b) Performing E rosette test
- Dependent on CD2 which is present in all T cells
Procedure
- Washed SRBC + washed lymphocyte (50:1 or 100:1 ration)
- Refrigerated for at least 16 hours
- Cells are then examined in a hemacytometer to count rosetting cells
- Lymphocytes that bind at least 3 SRC are generally considered T cells
** E rosette forming cell in normal blood is about 65-75% of the circulating
lymphocyte
E) MONOCYTE MACROPHAGE ASSAY
- monoclonal Abs tagged with a fluorescent dye

F) NATURAL KILLER CELL ASSAY

- monoclonal Abs tagged with fluorescent dye

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