2010-11-02

Cells of the immune system

Immunological response
All blood cells arise from a type of cell called the
hematopoietic stem cell
- pluripotent = able to differentiate in various ways
- the number of cells are maintained at stable levels

• hematopoietic stem cell Lymphoid progenitor cell

Åsa Håkansson
Division of Applied Nutrition and Food Chemistry
differentiates into
Myeloid stem cell
Department of Food Technology, Engineering and Nutrition
Lund Institute of Technology, Lund University 2010

Cells of the immune system

• Lymphoid progenitor cells - B cells
- T cells
- NK cells
• Myeloid stem cells - erythrocytes (red blood cells)
- neutrophils
- eosinophils
- basophils
- monocytes
- mast cells
- platelets

• Progenitor commitment depends on the aquisition of

responsiveness to particular growth factors and cytokines.

Components of immunity-
Components of immunity
innate immunity
Immunity Innate immunity: 4 types of defensive barriers
- Anatomic
- Physiologic
Nonspecific components Specific components - Phagocytic
- Inflammatory
= =
Innate immunity Adative immunity

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Components of immunity- Components of immunity-

innate immunity innate immunity
Anatomic barrier: Skin • Phagocytic barrier:
Mucous membranes -Phagocytosis is the uptake by a cell of
-mucus material from it´s environment, including
Normal flora whole pathogenic microorgasims.
-Conducted by specialized cells such as neutrophils and
Physiologic barriers: Temperature tissue macrophages. The internalized microbes are
pH destroyed by production of microbicidal substances.
Soluble factors • Inflammatory barrier: The reaction is initiated by a
- e.g. lysozyme, complement complex series of events aiming at protection

Components of immunity- Components of immunity-

adaptive immunity adaptive immunity
Four characteristic attributes:
• Antigenic specificity An effective immune response involves two major groups
• Diversity of cells:
-Lymphocytes (B cells and T cells)
• Immunologic memory
• Self/nonself recognition
-Antigen-presenting cells (Macrophages, B cells and
dendritic cells)

Components of immunity- Components of immunity-

adaptive immunity
B cells:
• Mature in the bone marrow, where they express antigen-
receptors (antibodies).
• When a naive B cell first meets the antigen that matches its
membrane-bound antibodies, the binding causes the cell to
divide and differentiate into memory B cells and effector B
cells=plasma cells.
adaptive immunity
B cells:
Memory B cells: Longer lifespan than naive cells.
Express the same membrane-bound antibodies
as their parent naive B cell.
Plasma cells: Do not express membrane-bound antibodies.
Instead, they produce antibodies in a form that can
be secreted.

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Components of immunity-
adaptive immunity
T cells:
• Arise in the bone marrow but migrate to the thymus gland to
mature.
• During maturation, T cells come to express a unique antigen-
binding T cell receptor on their membrane.
• The T cell receptors can recognize only antigen that is bound
to cell membrane proteins called major histocompatibility
complex (MHC).
Two major types of MHC molecules
-Class I MHC: expressed by nearly all nucleated cells
-Class II MHC: expressed only by antigen-presenting cells
Components of immunity-
adaptive immunity
TH cells:
• Express a membrane glycoprotein, called CD4.
• After a TH cell recognizes and interacts with an
antigen-MHC
antigen MHC class II molecule complex, the cell
is activated - It becomes an effector cell that
secretes cytokines.
- cytokines = proteins that regulate the intensity
and duration of the immune response by exerting
effects on lymphocytes and other immune cells e.g.
macrophages.
Components of immunity-
adaptive immunity
Treg cells:
• Regulatory T cells come in many forms and can express either
CD4 or CD8.
• Regulatory T cells are defined by expression of the forkhead
family transcription factor FOXP3 (forkhead box p3).
• Regulatory T cells suppress immune responses of other cells
and prevent pathological self-reactivity, i.e. autoimmune
disease.
2010-11-02
Components of immunity-
adaptive immunity
T cells:
• There are three subpopulations of T cells:
-T helper cells (TH cells)
-TT cytotoxic
t t i cells
ll (TC cells)
ll )
-T regulatory cells (Treg cells)
Components of immunity-
adaptive immunity
TC cells:
• Express a membrane glycoprotein, called CD8.
• Under the influence of TH – derived cytokines, a TC cell that
recognizes an antigen-MHC class I molecule complex
proliferates and differentiates into an effector cell.
• In contrast to TH cells, TC cells generally do not secrete many
cytokines. Instead they exhibits cytotoxic activity against, for
exemple, virus-infected cells and cancer cells.
Components of immunity-
adaptive immunity
The specificity of each T and B lymphocyte is determined before
its contact with an antigen.
• As a B cell matures in the bone marrow, its specificity is created by
random rearrangements of a series of gene segments that encode
the antibody molecule. As a result, each mature B cell possesses a
single functional gene encoding the antibody. All 105 antibodies on
a given B cell have identical specificity, given each B cell, and the
clone of daughter cells to which it gives rise, a distinct specificity for
a single epitope on an antigen.
• The random gene rearrangement during B cell maturation in the
bone marrow generates an enormous number of different antigenic
specificities. The resulting B cell population exhibits more than 108
different specificities.
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Components of immunity- Components of immunity-

adaptive immunity
• As the B cell maturation, the process of T cell maturation
includes random rearrangements of a series of gene
segments, that encodes the cells antigen-binding
receptor.
• Each T cell expresses about 105 receptors, and all
receptors on a cell and its daughter cells have identical
specificity for an antigen. The random rearrangements is
capable of generating 109 unique specificities.
adaptive immunity
The enormous diversity in the mature B and T cell
populations are later reduced by a selection process
in the bone marrow (for B cells) and in the thymus
((for T cells),
), that eliminates the cells whose
membrane-bound antibodies/receptors recognize
self-components.
-This process is referred to as negative selection
or ”central tolerance” to self antigens.

Components of immunity- Components of immunity-

adaptive immunity adaptive immunity
Antigen-presenting cells:
• Includes macrophages, B cells and dendritic cells.
• Distinguished by two properties
- express class II MHC molecules on their membranes
- are able to deliver a co-stimulatory signal that is necessary
for TH cell activation.
• Antigen-presenting cells first internalize antigen, for example
through phagocytosis, and then display a part of the antigen,
bound to a class II MHC molecule, on their membrane.
• The TH cell regognizes and interacts with the antigen-class II
MHC molecule complex on the membrane of the antigen-
presenting cell. An additional co-stimulatory signal is then
produced by the antigen-presenting cell, leading to activation
of the TH cell

Components of immunity Immune responses

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Humoral response Cell-mediated response

• The humoral response = interaction of B cells with antigen and their • The cell-mediated response = effector T cells generated
subsequent proliferation and differentiation into antibody-secreting in response to antigen are responsible.
plasma cells.

• Antibodies function as effectors of the humoral rsponse by binding • Both activated TH and TC cells can serve as effector
to antigen and neutralizing it or facilitating its elimination. When an cells.
g is coated with antibodies,, it can be eliminated in several
antigen
ways:
• Cytokines produced by TH cells can activate phagocytic
- Antibodies can crosslink several antigens, forming clusters that can
cells, enabling them to phagocytose and kill
be ingested by phagocytic cells. microorganisms.
- Binding of antibodies to antigen on a microorganism can activate
the complement system, resulting in lysis of the foreign organism.
- Antibodies can also neutralize toxins or viral particles by coating • TC cells participate in the response by killing altered
them, which prevents them from binding to host cells. self-cells, virus-infected cells and tumor cells.

Immune responses- Immune responses-

generation of the humoral immune response generation of the humoral immune response
1. Mature antigen-commited B cells are seeded out from the bone marrow
to circulate in the blood, the lymph and lymphoid organs.
2. Interaction of a mature B cell with antigen triggers its activation,
proliferation and differentiation. This process begins when antigen cross-
links membrane-bound antibodies on a B cell.
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3. Some of the bound antigen is internalized by endocytosis.
endocytosis
4. After processing the antigen, B cells presents the resulting antigenic
peptides, combined with class II MHC, on its membrane.
5. A TH cell specific for the presented antigen-MHC complex then binds to
the complex. As a result of this interaction, the TH cell secretes cytokines,
that stimulate various stages of B cell division and differentiation.
6. The activated B cell undergoes a series of cell divisions over about 5 days,
differentiating into a population of both antibody-secreting plasma cells
and memory cells.

Immune responses-
generation of the cellular-mediated immune response
1. As in the humoral response, a clonally expanded population
of antigen-specific activated TH cells is required.
2. TH cell activation is initiated by interaction of the receptor
with a processed antigen bound to a class II MHC molecule,
on the surface of the antigen-presenting cell. The activating
signal
i l leads
l d to proliferation
lif i and d differentiation
diff i i into
i effector
ff
and memory cells.
3. Cytokines secreted by these TH cells help to activate other T
effector cells.
4. The cytokines also regulate the proliferation and
differentiation of non-specific effector cells such as natural
killer cells and activated macrophages.

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Immune response The crypts of Leiberkuhn, also

known as intestinal glands
secrete various enzymes, and the
• Primary immune response = The initial encounter of a basal portion of the crypt
naive lymphocyte with an antigen. contains multipotent stem cells.
Goblet cells produces mucus and
the cells increase in number from
the duodenum to the iluem.
• Secondary immune response = A later contact of the host Paneth cells are found at the
with the antigen will induce a more rapid and heightened base of the crypts of Leiberkuhn.
secondary response. These cells secrete anti-bacterial
proteins: lyzosyme, an enzyme
that digests bacterial cell wall and
- The population of memory cells accounts for the defensins, small peptides that
rapidity and intensity that distinguishes a make bacterial plasma
membranes permeable.
secondary response from the primary response.

Anatomy of the large intestine

GALT-
Gut-Associated Lymphid Tissue
• The digestive tract's immune system is often referred to as gut-
associated lymphoid tissue (GALT).
• About 70% of the body's immune system is found in the digestive
tract. The GALT is made up of several types of lymphoid tissue that
store immune cells, such as T and B cells, dendritic cells and
p g
macrophages.
• These tissues range from less organized clusters of lymphoid cells to
organized structures such as peyer´s patches (small intestine),
mesenteric lymph nodes (large intestine) and follicles (small and
large intestine).
• Populations of immune cells can also be found within the epithelial
cell layer. These include dendritic cells, that sample antigens directly
from the lumen and intraepithelial lymphocytes (IEL), which consist
mostly of T cells.

GALT-
Gut-Associated Lymphid Tissue
• The epithelial cells play an important role in promoting
the immune response by delivering foreign antigens from
the lumen of the digestive tract to the underlying
lymphoid tissue.
• This antigen transport is carried out by specialized cells,
called M cells (microfold cells).
• Antigens transported across the mucous membrane by M
cells can activate antigen presenting cells, resulting in T
cell activation.

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GALT-
Gut-Associated Lymphid Tissue
• Activated B cells differentiate into plasma cells,
which secrete the IgA class of antibodies.
• These
h antibodies
b d are transported d across the
h
epithelial cells and released as secretory IgA into the
lumen, where they can interact with antigens.
Microflora and the immune system
• Intestinal epithelial cells are capable of detecting bacterial antigens and
initiating, participating in and regulating both innate and adaptive immune
responses.
• Through molecules expressed on the epithelial cell surface such as major
histocompatibility complex I and II molecules, and Toll-like receptors
(TLRs) (pattern recognition receptors (PRR)), signals from bacteria can be
transduced to adjacent immune cells in lamina propria, such as
macrophages, dendritic cells and lymphocytes.
• In healthy adults, TLRs are expressed in most tissues. Interaction of TLRs
and bacterial molecular patterns (pathogen-associated molecular patterns
(PAMPs)) results in activation of a complex intracellular signalling cascade,
up-regulation of inflammatory genes and production of pro-inflammatory
cytokines and recruitment of cells.
• It also stimulates expression, upon antigen presenting cells, of co-
stimulatory molecules required to induce an adaptive immune response.
• The expression level of TLRs on the gut epithelium is particularly
sophisticated so as to avoid over-stimulation.
2010-11-02
Microflora and the immune system
• There is a complex relationship between the intestinal immune
system and the bacterial flora in the intestine
• It is extremely important for the intestinal epithelial cells and the
mucosal immune system, to distinguish between pathogenic and
non-pathogenic bacteria.
• The intestinal bacterial flora can significantly alter the response and
the interaction can occur in different ways
ways.
- M cells have the capacity to capture soluble antigens, apoptotic
epithelial cells or bacteria from the luminal compartment, and
transport them to lymphoid cells.
- Both pathogenic and non-pathogenic bacteria can enter into the
mucosal tissue through lamina propria dendritic cells, which
extend their dendrites through epithelial cell tight junctions.
- Also the intraepithelial lymphocytes located in the epithelium
might recognize microbial antigens.
Microflora and the immune system
• Whilst TLR responses are critical for survival and defence against invading
pathogens, inappropriate signalling in response to alterations in the local
bacterial environment can be detrimental.
• Human intestinal epithelial cells normally express TLR3 and TLR5, whilst
TLR2 and TLR4 are barely detectable .
• The two most studied TLRs are TLR4 and TLR2
- TLR4 is known as the LPS receptor (Gram-negative bacteria)
- TLR2 recognizes bacterial lipoproteins and peptidoglycans
(Gram-positive bacteria).
• TLR3 is responsible for recognizing double-stranded RNA while TLR5
controls responses to bacterial flagellin.
• In health, TLR signalling protects the intestinal epithelial barrier and
confers commensal tolerance. In disease, aberrant TLR signalling may
stimulate diverse inflammatory responses leading to acute and chronic
intestinal inflammation.
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Microflora and the immune system
Probiotics and the immune system
• Indirectly, by changing the composition of the intestinal
microflora, the probiotic bacteria will be able to modify the
stimulation of the pattern recognition receptors (for example,
an increased amount of LPS in the lumen will stimulate an
inflammatoryy reaction).
)
• Indirectly, by minimizing bacterial translocation, the
immunsystem will be less affected by Gram-negative bacteria.
• By stimulating the production of mucus, the epithelial cells
will be protected from bacterial stimulation.
Flow cytometry-
an immunological method
2010-11-02
Probiotics and the immune system
Through bacterial-epithelial cell interactions, probiotics are able to affect
both the innate and adaptive immune system:
• When presented by antigen-presenting cells, the probiotic bacteria will be able to
modify the activity signal in T cells, leading to production of different cytokine
profiles (changing the production of pro-inflammatory cytokines to production of
anti-inflammatory cytokines).
• The cytokine pattern produced by macrophages my also be changed
changed.
• Up and down-regulation of membrane receptors can affect the signal transduction
(making it stronger or weaker).
• Some strains have the ability to promote the differentiation of B cells into plasma
cells and increase the production of secretory immunoglobulin A.
• Through the activation signal, the probiotic bacteria may also increase the number
of different subsets of T cells, e.g. regulatory T-cells.
Flow cytometry-
an immunological method
Flow cytometry is a widely used method for analyzing expression of cell
surface and intracellular molecules, characterizing and defining different cells.
• Cell suspensions from blood, cell cultures or tissue samples are incubated with
fluorescent-labelled antibodies. The flow cytometer performs the analysis by
passing thousands of cells per second through a laser beam, and capturing the
light that emerges from each cell as it passes through.
• Each suspended cell, passing through the beam, scatters the light and
fluorescence chemicals may be excited into emitting light.
• A number of detectors are aimed at the point where the stream passes through
the light beam. The combination of scattered and fluorescence light is picked up
by the detectors and by analysing fluctuations in brightness at each detector, it is
possible to deduce various facts about the physical and chemical structure of each
individual cell.
• The data gathered can be analysed statistically by flow cytometry software to
report cellular characteristics such as size, complexity, phenotype and health.
ELISA-
a method to detect cytokines
• Enzyme-linked immunosorbent assay (ELISA), involves at least one antibody with
specificity for a particular antigen.
• The sample with an unknown amount of antigen is immobilized on a solid support
(usually a polystyrene microtiter plate) either non-specifically (via adsorption to
the surface) or specifically (via capture by another antibody specific to the same
antigen, in a "sandwich" ELISA).
• After the antigen is immobilized the detection antibody is added, forming a
complex with the antigen. The detection antibody can be covalently linked to an
enzyme, or can itself be detected by a secondary antibody which is linked to an
enzyme through bioconjugation.
• Between each step the plate is typically washed with a mild detergent solution to
remove any proteins or antibodies that are not specifically bound.
• After the final wash step the plate is developed by adding an enzymatic substrate
to produce a visible signal, which indicates the quantity of antigen in the sample.
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ELISA- Dextran sulphate sodium (DSS)-

a method to detect cytokines inducerad acute colitis
To verify the model:
• 4% DSS in drinking water for 7 days
• The animals were fed standard chow

Disease Activity Index (DAI)

• Body weight
• Stool consistency
• Blood in faeces

Results:
DAI for normal control 0.0 (0.0-0.3)
DAI colitis animals 2.8 (2.3-3.0) ***

Dextran sulphate sodium (DSS)- Dextran sulphate sodium (DSS)-

inducerad acute colitis inducerad acute colitis
Resultat: Results:
Shorter colon Heavier colon Increased weight of caecum
Colon wet weight

0,35
Caecum ratio wet weight / dry weight

0 30
0,30 7
***
Tissue weight (g)

0,25 6

***

0,20 5
Weight (g)

0,15 4

0,10 3
Normal group Colitis group

Groups
2
Normal group Colitis group

Groups

Dextran sulphate sodium (DSS)- Dextran sulphate sodium (DSS)-

inducerad acute colitis inducerad acute colitis
Results (qPCR):
Results: The amount of laktobacilli 12

Normal mucosa Mucosa with inflammation decreased but the amount of 11

Akkermansia (Gram-negative 10
**

bakteria) and the total amount

Bacterial population copy/g

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of bacteria on the mucosa *

increased. 8

Also, the incidence of 6

Enterobacteriaceae increased
5
and a positive correlation obac
illus
obac
illus
erm
ansi
a
erm
ansi
a
l ba
cter
ia
l ba
cter
ia
Lact Lact Akk Akk Tota Tota
NC DSS NC DSS NC DSS
BetweenEnterobacteriaceae and
DAI was found.

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Dextran sulphate sodium (DSS)- Dextran sulphate sodium (DSS)-

inducerad acute colitis
T-RFLP analysed by PCA (Principal Component Analysis)
Results:
The composition of the microflora differed between the groups.
inducerad acute colitis
By using flow cytometry, different populations of and expression
on e.g. lymfocytes, macrofages and dendritic cells can be
analysed in tissues and blood.

Results (mesenteric lymph nodes):

Increased population of CD11b+/CD11c+ (Macrofages)
Increased population of CD11c+/TRL4+ (Dendritic cells)
Increased population of CD4+/CD25+/FoxP3+ (Regulatory T-cells)

Increased concentration of cytokines/chemokines in serum:

IL-6, IL-17 och KC.

Dextran sulphate sodium (DSS)-

Thank you for your attention
inducerad acute colitis
Results: When combining the findings from flow cytometry
analysis with the results from the microflora analysis (T-RFLP) by
using PCA, distinct differences between the groups were found.

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