Biology of t lymphocytes

Biology of T lymphocyte
Pannipa Kittipongpattana, MD.
13 December 2019
Division of Pediatric Allergy and Immunology
Department of Pediatrics, Faculty of Medicine
King Chulalongkorn Memorial Hospital

Outline
T cells maturation
• Progenitor commitment
• Thymic migration
• Progenitor proliferation
• TCR recombination
• Selection & differentiation
T cells activation & function
• Lymph node migration
• T cells activation
• Effector function

T lymphocyte development
Stage of lymphocyte development (maturation)
1. Commitment of progenitor cells to the B
lymphoid or T lymphoid lineage
2. Proliferation of progenitors
3. Rearrangement (recombination) of antigen
receptor genes
4. Selection events
5. Differentiation of T cells into functionally
and phenotypically distinct

1.Commitment of progenitor cells to the B or T lymphoid lineage
Bone marrow
&
Fetal liver Commitment depends on:
• signals-cell surface receptors
• transcriptional regulators
EBF, E2A & Pax5 B cell lineage
Notch 1 & GATA3 T cell lineage
Induce expression of
• Genes for pre-T cell receptor
• RAG1
• RAG2
Abbas AK, et al. Cellular and Molecular Immunology 9th edition

T cell maturation

Thymus: structure
https://commons.
wikimedia.org/wiki
/File:Thymic_corp
uscle.jpg
Hassall corpuscle
A bilobed organ situates in the anterior
mediastinum
Lobe → lobules → cortex & medulla
• Cortex contains dense collection of T
cells (thymocytes)
• Medulla also contains macrophages
and DCs.
Thymic epithelial cells (TEC) derive from
3rd pharyngeal pouch endoderm
• Cortical TEC (cTEC)
• Medulla TEC (mTEC)
Vascular supply
Diseases affecting thymus
DiGeorge syndrome
thymus development abnormal
Malnutrition → thymic atrophy
→ decrease T cell number (↓CD4/CD8)

Pro
T
Pro
T
CCL25 CCR9IL 7
Pro
T
Pro
T
Pro
T
Pro
T
Pro
T
Pro
T
Pro
T
RAG1,2
Pro
T
TdT
Proliferation
β D-J
recombination
β V-DJ
recombination
Pre
T
DP
T
Weakly binding to
Self Ag + MHC I
Weakly binding to
Self Ag + MHC II
Strongly binding to
Self Ag + MHC
Runx3
GATA3
ThPOK
Lck
CD8
+
CD4
+
Bim
CCL19 CCR7
CCL21 CCR7
CD8
+
CD4
+
???
T
reg
AIRE
α V-J
recombination
Bim
Strongly binding to
Self Ag + MHC
Death by
neglected
Negative
selection
Positive selection
CD8
+
CD4
+
T
reg
Negative
selection
cTEC
mTEC
DC
Cortex
Medulla

Cortex
2. Proliferation of progenitor cells
Proliferation generates a large pool
of progenitors to produce a highly
diverse repertoire of mature,
antigen-specific lymphocytes.
Key cytokine: IL-7
Source of IL-7 in thymus:
cortical thymic epithelium cells
cTEC
IL-7
Mutation of IL-7 receptor α chain → T- B+ NK+
Mutations of common γ chain (receptor of IL-2, IL-4, IL-7, IL-9, IL-
15, IL-21) → X-SCID (T-B+NK-)
Block T cell (IL-7) and NK cell (IL-15) development
Normal B cell development

3. Sequential and ordered rearrangement of antigen receptor genes
TCR complex
Intracellular signal transduction
Binding with peptide-MHC
Structure of the T cell receptor

V(D)J recombination
αβ TCR
T cell from BM
γδ TCR
T cell from fetal liver
TCRs diversity are
generated by
rearrangement of gene
segments region:
- Variable (V)
- Diversity (D)
- Joining (J)

Abbas AK, et al. Cellular and
Molecular Immunology 9th edition
Random joining
= Combination
diversity

1.Synapsis
Two coding segments and their adjacent RSSs are
brought together
2. Cleavage
Double-stranded breaks at RSS-coding sequence
junctions by
recombination-activating genes (RAG1 & RAG2)
Mechanism of V(D)J
recombination

Recognition Signals Sequences (RSSs)
7 912 7 923

Occurs up to 50% in Ig κ locus

3. Hairpin-opening and end-processing
- Artemis
- An endonuclease that opens up the
hairpins at the coding ends
- Terminal deoxynucleotidyl transferase
(TdT)
- adds bases to broken DNA ends
4. Nonhomologous end joining
- Ku70/Ku80: DNA end-binding proteins
- DNA-PK: double strand DNA repair
enzyme
- DNA ligase IV, XRCC4: ligation
Mechanism of V(D)J
recombination

Junctional diversity:
add or cut nucleotide

Diversity of TCR
Diversity of T cell receptors are generated from
1. Rearrangement of V, D and J gene segments
2. Pairing of β (V(D)J) and α (VJ) chains
3. Junctional diversity

Actual number of Ag receptors on T or B cells expressed in
each individual is probably on the order of only 107

β β β
V(D)J
rearrangement
of TCR β chain
V(D)J
rearrangement
of TCR α chain
Pro T cell Pre T cell
immature
T cell
pre TCR
β / pre α
Checkpoints in lymphocyte maturation

Function of pre-TCR
- inhibit the other β chain recombination
(allelic exclusion)
- proliferation of pre T cells
- inhibit pre T α chain transcription
- stimulate of α chain recombination
- expression of CD4, CD8
Double Positive T cell
CD4+ CD8+ TCRαβ

J Allergy Clin Immunol. 2015 Nov;136(5):1186-205.e1-78.
Proliferation
V(D)J Recombination
TCR
complex
Middleton’s 8th edition

4. Selection
Positive selection
● Selects T cells that recognize peptides on self MHC with low affinity
● Self MHC restriction
Negative selection
● Eliminates T cell with TCRs that bind too strongly to self antigen/MHC
complex --> Clonal deletion
● Autoreactive cells are removed by this process
● Self tolerance or Central tolerance

DN
DP
SP
TCR-CD4-CD8- =
Double negative (DN)
TCR+CD4+CD8+ =
Double positive (DP)
TCR+CD4+CD8+ /
TCR+CD4-CD8+ =
Single positive (SP)
MHC class I
MHC class II
5. Differentiation of T cells into functionally and phenotypically distinct

Annu. Rev. Immunol. 2018. 36:579–601
Schematic model of CD4 helper and CD8 cytotoxic T cell differentiation

Experimental & Molecular Medicine (2018) 50,e456;
Schematic diagram of Treg cell development

Stages of T cell maturation

Other T cell subsets
- γδ T and NKT cells
- Recognize a wide variety of antigen, many are not peptides, not
displayed by class I and II MHC molecules on APC
- Antigen receptor of γδ T cells and NKT cells have limited diversity
- Abundant in epithelial tissues (GI tract)
- Mucosa-Associated Invariant T (MAIT) Cells
- 50% of all T cells in the human liver
- limited diversity

γδ T cells
Annu. Rev. Immunol. 2014. 32:121–55.
- Thymic selection/differentiation : none
- Receptors: γδ TCR complex (limit diversity)
○ not recognize MHC-associated peptide
antigens and are not MHC restricted
○ recognize
■ protein or nonprotein antigens that do
not require processing of APCs
■ small phosphorylated molecules,
alkyl amines, or lipids presented by
nonclassical class I MHC–like
molecules
- Effectors: IL-17
- Functions in mice model
○ initiate immune responses to microbes
at epithelia, before the recruitment and
activation of antigen-specific αβ T cells.

Natural Killer T (NKT) Cells
- Thymic selection/differentiation
: CD1d (MHC-I like β2 Microglobulin)
- Subsets: NKT CD4+, NKT DN
- Receptors
○ NKR-P1, CD122
○ TCR [Vα24-JαQ x Vβ11]: recognize
hydrophobic antigen, mostly
glycolipid
- Effectors: IFN γ , IL-4
- Functions in mice model
○ Protect from autoimmunity, tumor,
and infections (Listeria, Toxoplasma,
Mycobacteria, Salmonella,
Plasmodium)
Immunol Today. 2000 Nov;21(11):573-83.

Mucosa-Associated Invariant T (MAIT) Cells
- Thymic selection/differentiation:
MR1 (MHC-I Related molecules)
- Receptors
○ CCR6, CCR9: "gut homing CCR"
○ TCR [Vα7.2-Jα33]: recognize MR1-
Microbial metabolites of B2 or
Folate (yeast, bacteria,
mycobacteria)
○ IL-18R, IL-12R (MR1 independent
activation): response to Monocyte
with TLR-8 (viruses)
- Effectors:
IFN γ , TNF, Granzyme B, Perforin
Nature Reviews Immunology volume 19, pages643–657(2019)

Abbas AK, et al.
Cellular and Molecular Immunology 9th edition
Lymphocyte classes
This table summarizes the major properties of the lymphocytes
of the adaptive immune system. Not included are natural killer
cells and other innate lymphoid cells.
Ig, Immunoglobulin; MHC, major histocompatibility complex.
* The percentages are approximations, based on data from
human peripheral blood and mouse lymphoid organs. In the
liver, almost 50% of the lymphocytes are MAIT cells.
† CD4+ CD8− : CD4− CD8+ ~ 2:1
% within compartment

T cells activation & functions

Migration of naïve T cells to lymph node
• Naive T cells migrate to lymph nodes because of
the presence of homing receptors (L-selectin and
CCR7)
• Peripheral node addressin (CCL19 and
CCL21) expressed on the HEVs of lymph nodes
• CCL21 binds to receptor (CCR7) on naive T cells
and promotes T cell recruitment into the lymph node

Activation of naive and
effector T cells by antigen
Naive T cells move around
and stop when they find the
Ag which they express
specific receptors

Phases of T cell responses

Sequence of events in T cell responses

Signals for T cell activation
- Interaction between specific T cell and
APC
- 1st Signal
- Interaction between TCR and
MHC-peptide complex
- Required for the activation of
antigen-specific T cell
- CD8 cytotoxic (CTL) T cells →
class I MHC molecules that present
on surface of nucleated cells
- CD4+ T cells (Th cells) → class II
MHC molecules that present on APC
(B cells, macrophages, and dendritic
cells) and epithelial cells in thymus
TAP= transporter associated with antigen processing

Major Histocompatibility Complex Molecules
MHC class I molecule MHC class II molecule

The class I MHC pathway
ERAP, Endoplasmic reticulum associated peptidase; ER, endoplasmic reticulum;
β2m, β2-microglobulin; TAP, transporter associated with antigen processing

The class II MHC pathway
CLIP , class II–associated invariant chain peptide; ER , endoplasmic reticulum; I i , invariant chain.

Early tyrosine phosphorylation events in T cell activation
Protein kinases
-Lck: lymphocyte-specific tyrosine kinase
-ZAP-70: ζ-associated protein of 70 kD
Adaptor protein
-LAT: linker for T cell activation

PLCγ 1 : phospholipase C γ1;
MAPK : mitogen activated protein kinase
Early tyrosine phosphorylation events in T cell activation

Structure of tyrosine kinases
Src family kinases:
c-Src, Lyn, Fyn, and Lck
Syk family kinases:
Syk and ZAP-70
Tec family kinases:
Tec, Btk, and Itk

The Ras-MAP kinase pathway in T cell activation

T cell signaling downstream of PLCγ1
DAG , diacylglycerol; IP3 , inositol 1,4,5-trisphosphate; PIP2 , phosphatidylinositol bisphosphate; PKC , protein kinase C.

Activated transcription factors in T cell activation signaling
Abbas AK, et al. Cellular and
Molecular Immunology 9th edition

Peptide-MHC
B7-CD28
Costimulatory Signals for T cell activation
2nd Signal
–B7 (CD80, CD86) is costimulatory
molecule expressed on activated
APC upon stimulation by foreign
antigen, this molecule interacts
with CD28 molecule on activated
T cell
–Resting APC expresses few or no
B7 molecule → no T cell activation

CD28 signaling cascade
in T cell activation

CD28 (appear on naïve and activated T
cell) :B7 → activate T cell response
CTLA-4 (appear on activated
T cell) :B7 → inhibit T cell activation
ICOS:ICOS-ligand → helper T cell-
dependent Ab responses
PD1:PD ligand →inhibit the activation of
effector cells, especially in peripheral
tissues
Costimulators and Inhibitory
receptors

Clinical application of CTLA-4
• CTLA-4-Ig block B7:CD28
• CTLA-4 binds to B7 stronger than
CD28
– Approve: rheumatoid arthritis
– Clinical trials: transplant
rejection, psoriasis, Crohn’s
disease
• Anti-CTLA-4: block CTLA-4
→ T cell has no negative regulation
- T cell activated
- Rx: tumor

Role of CD40 in T cell activation
• CD40L: member of TNF superfamily membrane protein, expressed
primarily on activated T cells
• CD40: member of TNF receptor superfamily, express on APCs
• CD40-CD40L interaction in class switching of B cell

Functional responses of T cell activation
• Changes in surface molecule of T cells
• IL-2 secretion and IL-2 receptor (IL-2R) expression
• Clonal expansion of T cells
• Differentiation of CD4+T cells into TH1, TH2, and TH17 effector
cells
• Differentiation of CD8+T cells into CTL
• Differentiation of memory T cells

Changes in surface molecule of T cells
- CD69 : retain T cells in
lymphoid organ
- IL-2Rα (CD25): response to IL-2
(autocrine growth factor)
- CD40L: help activate APCs
- CTLA-4: inhibit T cell activation

Regulation of IL-2 receptor expression
Structure of IL-2 and its receptor
(CD25)
(CD122) (CD132)

Clonal expansion of T cells
• Clonal expansion: generate large number of cells to eliminate
specific Ag from a small pool of Ag-specific naïve T cells

Differentiation and Functions
of CD4+ Effector T cells

Induction and effector phases
of CD4+ T cell responses
• CD4 + T cells recognize peptides that are
derived from protein antigens and presented
by dendritic cells in peripheral lymphoid
organs.
• The T cells are stimulated to proliferate and
differentiate into effector (and memory)
cells, which enter the circulation and migrate
to sites of infection in peripheral tissues.
• In the tissues, effector T cells recognize the
antigen and respond by secreting cytokines
that recruit more leukocytes and activate
phagocytes to eradicate the infection.

Properties of major subsets of CD4+ helper T cells

Development of Th1 cells
• Driven cytokines: IL-12, IL-18, IFN-γ
• Transcription factors: T-bet, STAT1, STAT4
• TH1 developed → secrete IFN-γ (positive
amplification loop)
• IFN-γ amplifies the reaction and inhibits
differentiation toTh2 and Th17

Effector functions of Th1 cells
• Secrete IFN-γ: activate
macrophages to increase
phagocytosis and killing of
microbes in phagolysosomes
• Th1 –mediated pathological
reactions:
–Delayed-type hypersensitivity
–Granulomatous inflammation
(Mycobact. tuberculosis)

Functions of IFN-γ
• Classic macrophage
activation
• Acts on B cells (in mice)
– To promote switching of Ig
→ IgG subclass
– Inhibit switching to IL-4
dependent isotypes e.g. IgE
• Stimulate expression of MHC
molecules and B7
costimulators on APCs
IL12

Macrophage activation by Th1 cells
Role of CD40-CD40L and IFN-γ

Function of activated macrophages

• Driven cytokines: IL-4
• Transcription factors:
STAT6, GATA-3
• TH2 developed → secrete
IL-4, IL-5, IL-13
• IL-4 amplifies the reaction
and inhibits differentiation
to TH 1 and TH17

Effector functions
of Th2 cells
IL-4
IL-5

Classical and Alternative Macrophage Activation
TH1 TH2

• Driven cytokines: IL-6, IL-1, IL-23, TGF-β
• Microbes: (extracellular) bacteria and
fungus
• Transcription factors: RORγt, STAT3
• TH17 developed → secrete IL-17, IL-22
• IL-21 amplifies the reaction
• TGF-β suppress Th1 and Th2
differentiation

Effector functions of
Th17 cells
• Signature cytokine of TH17 = IL-17
• Main function is
induced neutrophilic inflammation
defense against extracellular bacteria and
fungus
• IL-17 stimulates the production
of antimicrobial substances, including
defensin
• IL-22: produced in epithelial tissues,
especially of the skin and gastrointestinal tract
→ promoting the barrier function
IL-17 IL-22

Role of TH17 cells in host defense
• The principal function of Th17 cells is to destroy extracellular bacteria and
fungi, mainly by inducing neutrophilic inflammation
• Job syndrome (hyper-IgE syndrome) mutations in STAT3 resulting in
defective Th17 development
–increased susceptibility to cutaneous fungal and bacterial infections
–associated with chronic mucocutaneous candidiasis
• Th17 cells contribute to the pathogenesis of many inflammatory diseases
– associated with psoriasis, inflammatory bowel disease, rheumatoid
arthritis, and multiple sclerosis.
• Th17 cells help to maintain the integrity of epithelial barriers, such as in the
intestinal tract.

Leung S., Cellular & Molecular Immunology (2010) 7, 182–189.
Summary
of CD4+

O'Shea JJ, et al. Science. 2010;327(5969):1098-102.

Differentiation and Functions of
CD8+ Effector T cells

Induction and effector phases
of CD8+ T cell responses

Cross-presentation of antigens to CD8 + T cells
(Exogenous Ag is presents on MHC class I)
Differentiation of CD8+T cells

Differentiation of CD8+T cells
Differentiation into CTLs require CD4+ helper T cells
Licensing of APC

Effector functions of CTL
• Cytotoxic T cell (CTL) is an effector T cell that directly kills target cells that are
infected by intracellular microbe
• CTL kill target cells that express peptide (from cytosolic antigen) presented on class I
MHC molecule
• Killing mechanisms:
– Granule exocytosis → release of perforin/granzymes→ target cell apoptosis
– FasL-Fas mediated killing
• Target cells:
– Virus-infected cells, cancer cells
– Phagocytes with intracellular microbes

Steps in CTL-
mediated lysis of
target cells

Granule-dependent mechanism of cell killing
(Perforin/granzyme)

Granule-independent mechanism of cell killing
(Fas/FasL)
• Fas ligand: present on activated CTL
• Fas: present on target cells
• Crosslink of Fas/FasL → target cell apoptosis

Inhibition of CD8+T cells response:
T cell Exhaustion
- In acute infections, CD8 + T cells differentiate
into CTLs that eliminate the infected cells.
- In situations of persistent or chronic antigen
exposure, the response of CD8 + T cells is
suppressed by the expression and
engagement of PD-1 and other inhibitory
receptors = T cell exhaustion
- Chronicity of some viral infections in humans
;HIV, hepatitis C virus

Presentation of extracellular and cytosolic antigens to different subsets of effector T cells

• Effector T cells are short-lived, number
of Ag-specific T cell rapid decline as the
antigen eliminated
•Maintaining homeostasis in the immune
system
•As the level of costimulation and the
amount of growth factors (IL-2) decrease,
the level of anti-apoptotic proteins (Bcl-2
and Bcl-XL) drop → cells that were
produced by activation die and the
generation of newly activated cells
declines, so the pool of antigen-activated
lymphocytes contracts.
Decline of T cell responses

Development of memory T cells
• The mechanisms that determine
whether an individual antigen-
stimulated T cell will become a
short-lived effector cell or the
long-lived memory cell are not
established
• One possibility is that the types
of transcription factors
– T-bet drives differentiate
toward effector cells
– Blimp-1 promotes the
generation of memory cells
Derived from activated T cells or effector T cells (2 models)
Linear
model
Branched
model

Properties of memory T cells
• Express high level of anti-apoptotic protein: Bcl-2 and Bcl-XL, responsible
for prolonged survival
• Larger and more rapid response to Ag than naïve cells
• Slow proliferation and high capacity to self-renew → long life span
• Both CD4+ and CD8+ memory cells exist without need of antigens but need
cytokines
–IL-7: maintenance of memory CD4+ and CD8+ T cells, early lymphocyte
development, survival of naïve T cells
–IL-15: survival of memory CD8+ T cells

Change in proportions of naive and memory T cells with age
The thymus involutes with age
and is virtually undetectable in
postpubertal humans, resulting
in a gradual reduction in the
output of mature T cells.

Biology of t lymphocytes

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