23 Introduction to Immunopathology

Functions of the Immune System

Recognize, respond to and destroy invading microbes

Destroy non-self

Surveillance of new/foreign Ags on tumor cells

Diseases of the Immune System

Hypersensitivity reactions

Auto-immune diseases

Immunodeficiency disorders
Tissue transplantation reactions

Graft vs. host and rejection

Cells of the Immune System
Lymphoid: T cells (60-70%)

CD4 60%; CD8 30%

CD4 has Th1 and Th2
TCR rearrangement(thymus)
CD4 binds to MHC class II
CD8 to MHC class I
B cells (10-20%)
IgM as antigen receptor;
CD 21 for complement and EBV

Other Lymphoid cells:

1. Antigen presenting cells: macrophage,
dendritic cells and Langerhans cells
2. Natural killer cells: CD 16 and 56
Large granular lymphocytes in smears

Nonlymphoid cells: neutrophils, eosinophils,

basophils and mast cells

Humoral factors:
1. Ig
2. Complement
3. Cytokines

Major Histocompatibility Complex

Properties of epitopes of antigen

Chemically complex
Foreign

High molecular weight

Properties of innate and adaptive immune system

Innate: no memory, no specificity

Adaptive: with memory and specificity

Immunogenicity of molecules

Protein
+4

Polysaccharide
+2
Lipid
+1

Nucleic Acid
+1

Activators of lymphocytes

Monoclonal

Oligoclonal

Polyclonal
Mitogens

T cells:

B cells:

Antigens
Superantigens
Mitogens

Fate of nave B cells

Endocytosis of Ag

Presentation of Ag fragment/Class II MHC

Activated helper T cells secrete cytokines

Clonal expansion IgM, IgG plasma cells and

memory cells
B cell enumeration and assessment of function

Flow cytometry for enumeration

Total serum antibody for function and enumeration

Reverse hemolytic plaque assay for polyclonal

antibody synthesis
Reverse Hemolytic Plaque Assay

Pokeweed mitogen added to mononuclear cells

B cells differentiate into Ig secreting plasma cells

Cells incubated with protein A sensitized RBCs to

which rabbit IgG antibodies that target human IgG Fc

regions are bound, and complement
RBCs are lyzed resulting to a plaque

Cell lineages and subsets

CD4+ T cells secrete cytokines needed for all immune

response
CD8+ T cells destroy infected cells

Activated CD4+ T cells (Thp)

Clonally expand to form Th0 intermediate cells that

differentiate into Th1 and Th2 cells

Th1 secrete type 1 cytokines for immune responses

involving macrophages, T cells and NK cells

Th2 secrete type 2 cytokines for immune responses

involving B cells
Enumeration and assessing function of T cells

Flow cytometry for CD4: CD8 ratio

Cell proliferation through concanavalin and

phytohemmaglutinin by measuring the amount of 3H
thymidine that is incorporated during DNA replication

Cytotoxicity by incubating activated CD8+ T cells with

radiolabelled 51Cr target cells. Cr are released and
counted by gamma counter when targets cells are
lyzed by T cells

Cytokine secretion by CD4+ T cells added with IL-2 IgG

antibody
Deficiencies of Complement proteins
C1 INH
Hereditary angioneurotic edema
C2 and C4
Immune complex disorders
C3
Encapsulated bacterial infections
C5,6,7,9
Recurrent infections with Neisseria spp
GPI Linkages
P.N.H.
(DAF,CD59)

Concanavalin A
Phytohemagglutinin
Pokeweed Mitogen
Lipopolysaccharide
Pokeweed Mitogen

Active molecules in phagosomes

Reactive oxygen
intermediates

Lactoferrin

Activated macrophages secrete cytokines

IL-1 and TNF: fever and inflammation

IL-6: stimulate hepatocytes to secrete C reactive

protein which acts as opsonin for recognition leading
to phagocytosis

IL-12: stimulate NK cells to secrete interferon

Lysosome
Defensins
Nitric oxide
rainwater@mymelody.com || 1st semester, AY 2011-2012

Evaluating Complement Pathways

CH50 and AH50 measures overall activities of classical

and alternate pathways
CH50: incubate patients serum with sheep rbcs

labelled by IgM osmotic lysis if complement is

present
Primary Immunodeficiencies
Defects in the components of cellular and humoral

immunity

B cell: X-linked Agammaglobulinemia, Selective IgA

deficiency, Immunoglobulin deficiency with increased
IgM

T cell: DiGeorges syndrome

T and B cell:Combined Immunodeficiency Disorders,

Wiskott Aldrich syndrome

Phagocytic Disorders: Chronic granulomatous disease,

Chediak Higashi syndrome, Leukocyte adhesion
deficiency
Complement Disorders:C1 inhibitor deficiency;C1,2 or

4 def; C5-9 def; Decay Accelerating Factor defect

Secondary Immunodeficiency Disorders

Viral infections

Drugs
Malnutrition

Aging
Abnormal production of immunological effectors like

monoclonal gammopathy

Schematic representation of Immunoglobulin G molecule

Summary of Lab exams for immunodeficiency disorders

Humoral deficiencies: electrophoresis of proteins;

nephelometry, ELISA, RIA to quantitate specific Igs;
Flow Cytometry

Complement Deficiencies: CH50 and AH50, total

activity by ELISA

C3 and C4: enzyme immunoassay, RIA

PNH: Hams or Sucrose Hemolysis; Flow Cytometry

C1 INH Def: ELISA, nephelometry, RIA

Lab exams for Cellular Immunity Deficiencies and Phagocytic
deficiencies

CMI: WBC count, Flow Cytometry, CD4:CD8, skin

test(e.g. Candida)

Phagocytic: Nitroblue tetrazolium test, Migration

tests in vitro, Flow cytometry for cell surface adhesion
molecule CD 18

Cellular interactions in the immune system

rainwater@mymelody.com || 1st semester, AY 2011-2012

Functional Classes of Cytokines

Area of Activity Cytokine
Innate
IL-1, TNF, IL-6. ILimmunity
12, IFNy

Lymphocyte
growth,
differentiation,
and activation
Inflammatory
cell activation

IL-2, IL-4, IL-12, IL15, TGF

Chemokinesis

IL-8 superfamily
members

Hematopoiesis

IFNy, IL-5, TNF,

TNF

Action
Protect against
viral infection,
recruit leukocytes,
stimulate acute
inflammatory
response
NK, B cell, and T
cell growth factors,
down-regulate
immune response
Induce acute
inflammation,
activate
macrophages,
eosinophils,
neutrophils and
endothelial cells
Released from T
cells and other cells
as homing signals
for B and T cells
Stimulate
progenitor and
stem cells

Colony-stimulating
factors (CSF-GM,
CSF-G), stem cell
factor
When grouped according to major activity, many cytokines
share similar and apparently overlapping activities.

Laboratory evaluation of the cell mediated immunity

Presentation of possible immunodeficiency:

frequent bacterial infections
severe systemic reactions to a virus
fungal/ protozoal infections
systemic reactions after vaccination
family history of recurrent infections
exposure to HIV
Basic Scanning Immunology Studies

Complete blood count/differential

Lymphocyte subpopulation analysis (numbers and

percentages of T and B cells) by flow cytometry

Lymphocyte activation in vitro to mitogens and

microbial activators
Serum immunoglobulins, including immunoglobulin

subclasses if evidence of clinical infections with

encapsulted bacteria In some cases, immunoglobulin
levels are normal but heterogenous nonbinding
antibodies are produce, thus, additional studies are
needed
Primary Investigation

History and physical examination

CBC/differential
X-rays

Quantitative immunoglobulins
Review previous culture results

Pulmonary function tests

Secondary Investigation

Titers for vaccines administered(tetanus, diphtheria,

pneumococcus)
IgG subclass analysis

Lymphocyte enumeration panel(CD3,4,8,16,56)

Complement levels(CH50, C3 and 4)

Skin testing

Mononuclear cell proliferation studies

NBT

Model of the molecular interactions involved in antigen

recognition

Tertiary Investigation
Enzyme studies(adenosine deaminase, purine

nucleoside phosphorylase)

NK cell cytotoxic studies

Phagocytic studies
Histology and immunochemistry or Flow cytometry of

lymph nodes and lymph organs

Cytokine production

Advanced complement studies

Molecular biology
Causes of skin test anergy

Lack of appropriate antigenic history when panel does

not include ubiquitous activators

Primary immunodeficiency

Viral infections

Malnutrition

Granulomatous diseases

Neoplasia

rainwater@mymelody.com || 1st semester, AY 2011-2012

Infections associated with cellular immune defects

rainwater@mymelody.com || 1st semester, AY 2011-2012