THE LYMPHOID SYSTEM T – lymphocytes

Lymphocytes  Effector cells that serve a regulatory role

 The key cell involved in the adaptive immunity B – lymphocytes
 Represent 20-40% of the circulating WBC  Produce antibody
 Typical lymphocyte is between 8-12 um in diameter  Both types recirculate continuously from the
with a large rounded nucleus that may be somewhat bloodstream to the secondary lymphoid organs
indented and back in an attempt to increase contact with
 Nuclear chromatin is dense and stains deeply foreign antigens
 Sparse cytoplasm, few organelles, no specific
granules; consists of a narrow ring surrounding the
nucleus
 Unique because they arise from a hematopoietic stem cell
lymphoid organs (basis of
two main classes of
lymphocytes)
 Primary lymphoid organs:
- Bone marrow (B-cells)
- Thymus (T-cells)
 Secondary lymphoid organs PRIMARY LYMPHOID ORGANS
- main contact with foreign antigens takes place BONE MARROW
- Spleen  Considered as the largest tissue of the body
> serves as a filtering mechanism for  Total weight 0f 1300 to 1500 g in adult
antigens in the blood stream  Fills the core of all long bones
-Lymph nodes  Main source of hematopoietic stem cells which
> filter fluid from the tissues develop into erythrocytes, granulocytes, monocytes,
- Appendix platelets, and lymphocytes
- Tonsils  Functions as the center for antigen-independent
- other mucosal-associate lymphopoiesis
lymphoid tissue (MALT) - Lymphocyte stem cells are released and travel
to primary lymphoid organs for further
maturation
- One subset goes to the thymus (T cells); B cell
maturation takes place in the BM in humans;
together they produce approximately 109 mature
lymphocytes each day
THYMUS
 T-cells develop their identifying characteristics

 A small, flat, bilobed organ found in the thorax

or chest cavity right below the thyroid gland
and overlying the heart
 Weighs an average of 30 g at birth, reaches
about 35 g at puberty, and then gradually
atrophies (though diminishes in size, it is still
capable of producing T lymphocytes throughout
Mucosal surfaces in RT and Alimentary tract a person’s lifespan)
- additional means of contracting antigens as they  Each lobe is divided into lobules filled with
enter the body epithelial cells that play a central role in the
differentiation process
Lymphocytes are segregated within the secondary o Surface antigens are acquired as
organs according to their particular functions: lymphocytes travel from the cortex

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 (outer) to medulla (inner) (2-3
weeks)
o Mature T lymphocytes are then
released form the medulla

 Progenitor T- cells appear in the fetus as early

as 8 weeks in the gestational period
 Differentiation of lymphocytes appear to take
place very early in fetal development and is
essential to acquisition of immunocompetence
by the time the infant is born

SECONDARY LYMPHOID ORGANS

 After differentiation, mature T and B lymphocytes are
released form BM and thymus and migrate to
secondary lymphoid organs and become part of a
recirculating pool the red pulp and then exists by way of the
 each lymphocyte spends most of its lifespan in solid splenic vein
tissue, entering the circulation only periodically to go 2. White pulp
from one secondary organ to another - contains the lymphoid tissue, arranged
 Lymphocytes in these organs travel through the around arterioles in a periarteriolar lymphoid
thoracic duct sheath (sheath contains small lymphocytes,
large lymphocytes, macrophages, plasma cells,
and granulocytes)
- Lymphocytes enter and leave this area
by means of the many capillary branches that
connect to the arterioles
-T cells are found close to the central
arterioles, while B cells are typically organized
into clusters called follicles
 Primary follicles – contain B cells that
are not yet stimulated by antigen
 Comprises approximately 20%of the
total weight of the spleen
LYMPH NODES
 Located along lymphatic ducts
 Serve as central collecting points for lymph fluids
 1-2% of total lymphocyte pool recirculates every hour from adjacent tissues
 This is where lymphopoiesis occurs but is strictly  Numerous near joints and where the arms and legs
dependent on antigenic stimulation, while formation in join the body
BM is antigen-dependent  Nodes range in size from 1mm to 25 mm in diameter
 Most naive or resting lymhphocytes die within a few  Main function is filtration
days after leaving the primary lymphoid organs unless - Lymph fluid flows slowly through spaces
activated by the presence of a foreign antigens (sinuses), which are lined by macrophages,
 Sites of antigen-dependent lymphopoiesis creating an ideal location for phagocytosis to
- This second process gives rise to long-lived memory take place
cells and shorter-lived effector cell responsible for the  Organized into an outer cortex and an inner medulla
generation of the immune response  Lymphocytes and any foreign antigens present enter
SPLEEN nodes via afferent lymphatic vessels
 The largest secondary lymphoid organ
 Approximately 12 cm in length and weighs 150 g in
adult
 Located in the upper left quadrant of the abdomen just
below the diaphragm,
surrounded by a thin
connective tissue
capsule
 Characterized by a large
discriminating filter
- Major function is to filter out old and
damaged cells and foreign antigens from
the blood
 Divided into two types of splenic tissue:
1. Red pulp
- makes up more than one half of the
total volume
- Destruction of old rbcs
- Blood flows from the arterioles into
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 Small number are also able to enter the nodes from the - are small masses of macrophages and
bloodstream by means of specialized venules (high lymphoid tissue found in the mucous
endothelial venules) located in paracortical areas membrane lining of the oral and pharyngeal
 Subcapsular sinus cavities
- Underneath the outer capsule - Function is to respond to pathogen
- Lined with macrophages entering the respiratory and
- Phagocytosis and antigen presenting take place alimentary tract
 Cortex MALT (Mucosal-associated lymphoid tissue)
- Contains aggregation of B cells in primary 3. Appendix
follicles similar to those found in the spleen 4. Epidermis
(mature, resting B cells not exposed to - contains a number of intraepidermal
antigen) lymphocytes (most are T cells), uniquely
- Small number of T cells and specialized positioned to combat any antigens that enter
cells (follicular dendritic cells) are also through the skin (cutaneous-associated lymphoid
located tissue)
- Follicular dendritic cells
 Only found in lymphoid follicles *Note: within each of these secondary organs, T and B cells
and have long cytoplasmic are segregated and perform specialized function:
processes that radiate out like B cells – differentiate into memory cells and plasma
tentacles cells and are responsible for humoral immunity
 Exhibit a large number of T cells- play a role in cell-mediated immunity,
receptors for antibody and producing sensitized lymphocytes that secrete cytokines or
complement
 Thought to be responsible for the
regulation of activities of memory
B cells
 Secondary follicles
- Consist of antigen-stimulated proliferating
B cells
- Interior is known as germinal center (blast
transformation of the B cells occurs here)
- Plasma cells (secrete antibody) and
memory cells are present
 Paracortex
- region between follicles and medulla
- T - lymphocytes are mainly located and are
in close proximity to antigen-presenting cells
called interdigitating cells
 Medulla
- Less densely populated but contains some
T cell (in addition to B cells), macrophages,
and small number of plasma cells
Lymph nodes...... lymphokines
 Particulate antigens are removed as the fluid travels
across the node from the cortex to medulla Surface markers on Lymphocytes
 Transit time through a lymph node is approximately  Proteins that appear on cell surfaces and used as
18 hours markers to differentiate T cells and B cells
 Fluid and lymphocytes exit by way of the efferent  Proteins are also used to distinguish the developmental
lymph vessel stages of the two types of cells according to when
- Form larger duct that eventually connects these proteins appear
with the thoracic duct and the venous system  Such proteins or antigens are detected by monoclonal
 If contact with antigen takes place, lymphocyte antibodies
traffic is shut down for about 24 hours in an  MONOCLONAL ANTIBODIES
attempt to immobilize the antigen  Extremely specific
- Lymph nodes become enlarged, and  Kohler and Milstein (1975)
within several days, germinal centers are  Made by cloning a single antibody-
visible (lymphadenopathy) producing cell
OTHER SECONDARY LYMPHOID ORGANS  Uses myeloma cells
MALT (Mucosal-associated lymphoid tissue)  Produces hybridoma
 Found in the gastrointestinal, respiratory, and  Clusters of Differentiation (CD)
urogenital tracts  International Workshops on Human
 Macrophages and lymphocytes are localized at some Leukocyte Antigens (1982)
of the main ports of entry for foreign organisms  Came about because the exact nature of
1. Payer’s patches proteins identified by various antibodies
- Represent a specialized type of MALT was not known
- Located at the lower ileum of the intestinal  Acts as reference in standardizing names of
tract membrane proteins found on all human
2. Tonsils white blood cells

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 during the immune response
Stages in B Cell Differentiation  At this stage, there is evidence that self-
1. Pro-B Cells antigens give a negative signal to immune B
 Derived from lymphoid-myeloid precursor cells, resulting in arrested maturation and cell
that remains in the BM for maturation death
 Part of the B-cell antigen-independent phase - Immature b cells that tightly bind
of development self-antigens through cross-linking of
 First recognizable cell in the B cell lineage surface IgM molecules are eliminated or
 Distinctive markers include: inactivated
- CD19 - Thus, many B cells capable of producing
- CD45R antibody to self-antigens are deleted from the
- Terminal deoxyribonucleotide transferase BM by the process of programmed cell death
(TdT) (apoptosis); it is estimated that 90% of B
- Recombinase activating gene cells die in this manner without leaving the
(RAG)– 1 and 2 enzymes BM
 RAG- and RAG-2 4. Mature B Cells
- cleave the DNA at certain possible  Exhibit IgD, in addition to IgM, on their surface;
recombination sites Surface IgM increases in density
 TdT - Both have the same specificity for a particular
- Helps to join the pieces back together by antigen or group antigens
incorporating additional nucleotides in the - Provide the primary activating signal to B cells
joining areas when contact with antigen takes place
 Also associated are MHC class II products
 CD19 - These are glycoproteins embedded in the cell
- Acts as a coreceptor that helps to regulate B membrane and rare recognized by T-helper cells,
cell development and activation which aid in antibody production
 CD45R  Mature B cells are released from the BM and seed
- Largest form of CD45 (membrane glycoprotein peripheral lymphoid organs
found on all hematopoietic cells  Unless contact with an antigen, life span is only a
- A tyrosine-specific phosphatase that is involved few days; if stimulated, it undergoes transformation
in signaling in B cell activation to a blast stage, which eventually forms memory
2. Pre-B Cells cells and antibody secreting plasma cells (antigen-
 Development begins when synthesis of part dependent phase)
of the antibody molecule (heavy chains)  B-1 cells
occurs  Have IgM but litlle or no IgD
- The first heavy chains synthesize are the µ  Express a surface marker, CD5, which is
chains (class IgM) normally found on T cells
- The µ chains accumulate in the cytoplasm;  Appear to arise before the major group of B cells
some on the cell surface and these are (B-2 cells)
accompanied by an unusual light chain  Capable of editing the gene coding for variable
molecule (surrogate light chain) regions of both heavy and light chains, giving
- The combination of the two heavy chains them the ability to respond to more than I
with the surrogate light chains forms the pre- antigen
B cell receptor, which adheres to the bone  Most respond to carbohydrate rather than protein
marrow stromal cell membranes and antigens
transmits a signal to prevent rearrangement of  Tend to produce only antibody of the IgM class
any other heavy chains genes (the only B-  Formed during fetal life and are usually found in
cells that survive and proceed to further the peritoneal cavity
differentiation)  Major source for antibody production in the
- Typically divide several times before further infant and young child
differentiation occurs and lasts for 5. Activated B Cells
approximately 2 days  Exhibit other identifying markers that include
3. Immature B Cells CD25 (acts as receptor for interleukin-2, a
 Recognized by the appearance of complete growth factor produced by T cells)
IgM molecule on the cell surface (indicates  Additional factors that appear at this time are
that rearrangement of the genetic sequence specific for other growth factors produced by T
coding for light chains on either chromosome cells and for other immunoglobulin classes
2 or 22 has taken place)  When activated in this manner (contact with
 µ chains are no longer detectable in the antigen), they transform to become blasts that
cytoplasm will give rise to both plasma cells and memory
 Other surface proteins include receptors for cells
complement components :  Within these germinal centers blasts can give
Ex. CD21 - acts as a receptor for a rise to as many as 104 cells in 3 to 4 days
breakdown product of C3 (C3d) 6. Plasma Cells
- Receptors enhance the likelihood of  Large, spherical, or ellipsoidal cells between
contact between B cells and antigen 10 to 20 um in size and are characterized by
because antigen frequently becomes the presence of abundant cytoplasmic
coated with complement fragments immunoglobulins and little to no surface

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 immunoglobulins (TCR) is expressed on the cell surface,
 Nucleus is eccentric or oval with heavily positive selection begins
clumped chromatin that stains darkly  This receptor must be able to
 Abundant ER and a clear well-defined Golgi recognize both foreign antigens
zone and self-MHC antigens
 Represents the most fully differentiated  Any thymocytes that are unable to
lymphocyte; main function is antibody recognize self-MHC antigens die
production without leaving the thymus (this
 Not normally found in blood but in germinal selection process eliminates more
centers than 90% of the double-positive
 After production of antibody for several days, thymocytes in the cortex)
they die without further proliferation  Selection appears to be influenced
by the number of TCR receptors
 T Cell Differentiation expressed on the T cell
 T-lymphocytes - Very low TCR levels – do
 60-80% of circulating T lymphocytes in not survive
peripheral blood  A second selection process takes place
 Precursors are pro-thymocytes among surviving double-positive T cells
- Bone marrow to thymus (negative selection)
- CD44 and TdT  T cells capable of reacting with
self-antigens are destroyed
 3 main stages of development:
 Antigen presenting cells are
 Double-negative stage
responsible (macrophages and
 Double-positive stage
dendritic cells)
 Mature lymphocytes
 T cells that bind with high affinity
 Double-negative stage
to self-antigens undergo death
 Early thymocytes (lack CD4 and CD8
(apoptosis)
markers)
 Process takes place in the cortico-
 Surface markers: CD2, CD5, CD7, medullary junction
CD45R  Mature T cells
- CD2 : “Rosetting”
 Survivors of negative selection that exhibit
 Rearrangement of genes that code for only 1 type of marker
antigen receptor (CD3) begins at this
 Selection may depend on with which MHC
stage
protein the cell interacts and exposure to
- Complex that serves as T cell antigen
certain cytokines
receptor
- CD4+ T cells recognize antigen along with
- Consists of 8 nocovalently associated
MHC class II protein (T-helper or Inducer
chains (6 are common to all T cells; 2 chains
cells; 2/3 of peripheral T cells)
(alpha and beta) contain variable regions that
- CD8+ T cells interact with antigen and MHC
recognize specific antigens
class I proteins (Cytotoxic T cells; 1/3 of
- Under control of RAG enzymes
peripheral T cells)
- Rearrangement of beta chains occurs
 Differ greatly in function
first
 Released from the thymus and
 Some thymocytes (5% or less) rearrange seed peripheral lymphoid organs
and express two other chains (gamma  Resting T cells have a lifespan of
and delta) up to several years in these
 Proceed a different developmental peripheral organs
pathway
 Remain negative for CD4 and  Antigen Activation of T Cells
CD8  T cells are transformed into large activated
 As mature T cells, they appear to cells characterized by polyribosome-filled
represent the dominant T cell cytoplasm when antigen activation occurs
population in the skin, intestinal - Express receptors for IL-2
epithelium, and pulmonary  T lymphoblasts differentiate into functionally
epithelium active small lymphocytes that produce
 Function is not absolutely clear; cytokines and T memory cells
new evidence indicates that they  Cytokines
may act like natural killer (NK)
 Assiting B cells in the
cells to mediate tumor-cell lysis
commencement of antibody
 Capable of recognizing stress
production
proteins (MICA and MICB) that
 Killing of tumor and other target
are found only on tumor and
cells
virally-infected cells
 Double-positive stage  Rejection of grafts
 Thymocytes express both CD4 and  Stimulation of hematopoiesis in
CD8 markers the BM
 Proliferate and begin to rearrange genes  Initiation of delayed
coding for the alpha chain hypersensitivity allergic reactions
 When CD3-ąß receptor complex
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 MHC class I proteins (expressed on healthy
cells)
 If NK cells react with MHC proteins, then
inhibition of natural killing occurs
 KIRs – killer cell inhibitory receptors
- specific
receptors on
NK cells
responsible
for binding
- different
types of
KIRs
expressed on
NK cells
T Cells B Cells  MECHANISM
 develop in the thymus  develop in the bone marrow OF
 found in blood ( 60-80% of  found in bone marrow, spleen,
circulating lymphocytes), lymph nodes
thoracic duct fluid, lymph  identified by surface
nodes immunoglobulins
 identified by rosette  end product of activation is
formation with SRBCs antibody
 end product of activation  antigens include CD19, CD20,
are cytokines CD40, MHC class II
 antigens include CD2, locate in cortical region of
CD3, CD4, CD8 lymph nodes
 locate in paracortical
region of lymph nodes
CYTOTOXICITY cont.
 Diseased and cancerous cells tend to
lose their ability to produce MHC
proteins
Natural Killer Cells  NK cells are triggered by a lack of MHC
 Small percentage of lymphocytes that do not antigens (recognition of “missing self”)
express the markers of either T cells or B cells  This lack of inhibition appears to be
 15 um in diameter (larger than T and B cells) combined with an activating signal
 Kidney shaped nuclei; condensed chromatin; switched on by the presence of proteins
prominent nucleoli produced by cells under stress (MICA
 Higher cytoplasmic-nuclear ratio and MICB)
 Azurophilic granules in cytoplasm  NKG2D – receptor on NK cells that
 5-15% of circulating lymphoid pool; found binds MICA or MICB and sends a signal
mainly in spleen and peripheral blood to destroy the cell
 Have the ability to mediate cytolytic reactions
 If an inhibitory signal is not received,
and kill target cells without prior exposure to
NK cells release:
them (nonspecific response)
1. Perforins – pore-forming proteins that
 No surface antigens unique to them
polymerize in the presence of Ca++ and forms
 Express a specific combination of antigens
channels in the target cell membrane
 CD16 – receptor for crytallizable 2. Granzymes – packets of serine esterase
portion (nonspecific) end of enzymes that may enter through the channels
immunoglobulin molecule (IgG) and mediate cell lysis
 CD56
 CD94  ANTIBODY-DEPENDENT CELL
 Lack CD3, CD4, and CD8 CYTOTOXICITY
 Arise from a common progenitor cells - Another method of destroying target
 Recent evidence shows that in the absence of a cells by NK cells
thymus, fetal thymocytes develop into NK cells  Ability to recognize and lyse antibody-coated
instead of CD4 or CD8 thymocytes cells
 Capable of recognizing any foreign cell and - Binding occurs through CD16 (receptor
destroying it without regard to MHC restriction for IgG)
 First line of defense against virally infected and - Exhibited also by macrophages,
tumor cells monocytes, and neutrophils
 Play complementary role to that of CD8 Laboratory Identification of Lymphocytes
 MECHANISM OF CYTOTOXICITY  Identification of lymphocytes as either T cells or
- How NK cells tell the difference B cells may be useful in the diagnosis of:
between normal and abnormal cells 1. Lymphoproliferative malignancies
 Balance between activating and inhibitory
2. Immunodeficiency diseases
signals that enable NK cells to distinguish
healthy cells from infected or cancerous cells 3. Unexplained infectious diseases
 The inhibitory signal is based on recognition of 4. Monitoring of transplants
5. Acquired Immunodeficiency Syndrome
6
 (AIDS) Cytometry
 Most methods are based on separation of  Also known as fluorescence-activated cell sorter
mononuclear cells from whole blood and and  An automated system for identifying cells based
detection of specific cell surface markers on the scattering of light as cells flow in a single
1. Density gradient centrifugation with Ficoll-
file through a laser beam
Hypaque
 Scattering is read on both a forward and a side
- one of the most frequently used methods
direction
- available commercially
 The amount of forward light scatter (LS) is a
- medium has specific gravity between 1.077 and
measure of cell size, while the side scatter is a
1.114 depending on manufacturer
measure of granularity
- diluted defibrinated blood or heparinized blood
is layered on top of the solution, and the tube is
centrifuged Other Methods
 Once a lymphocyte population has been  The rosette technique
obtained, segregation into subsets can be  Lymphocytes are separated out from
accomplished by: whole blood and then mixed with a
1. Fluorescence Microscopy suspension of SRBCs
2. Flow Cytometry  If three or more red blood cells are
- both techniques rely on the use of attached to a lymphocyte, this considered
labeled monoclonal antibodies against a rosette
specific surface antigens  200 cells are counted (counting chamber)
- Some of the more common antigens and percent forming rosettes are
tested are: calculated
- T cells – CD2, CD3, CD4, CD7, and  Monoclonal antibodies with an enzyme label
CD8  ELISA
- B cells – CD19, CD20, CD22, and  uses antibodies to specific CD antigens
surface immunoglobulins which are reacted with the sample
Fluorescence Microscopy  After washing a second antibody with an
 Either direct or indirect enzyme tag is reacted with the specimen
 A. Direct Immunofluorescence  Second wash is performed and substrate
 Use of monoclonal antibodies to which a is added
fluorescent tage is attached  Color change will take place if antigen is
 Commonly used dyes: present
– fluorescein and phycoerythrin
(490 nm)
– Rhodamine (545 nm)

 B. Indirect immunofluorescence
 Uses unlabeled antibody that first combines
with the antigen by itself
 Then an immunoglobulin that is complexed
to a dye is added
 This will react only with cells previously
coated with antibody
 Fluorescence is read manually

Flow
Cell