Biology of T lymphocytes.pdf

Biology of T lymphocyte
Tharida Khongcharoensombat, MD
Division of Pediatric Allergy and Immunology
Department of Pediatrics, Faculty of Medicine
King Chulalongkorn Memorial Hospital
22 April 2022

Outline
• T cell maturation
• Progenitor commitment
• Thymic migration
• Progenitor proliferation
• TCR recombination
• Selection and differentiation
• T cell activation and function
• T cells activation
• Effector function

Lymphocyte development
• The process by which lymphocyte progenitors in the thymus and bone marrow differentiate into
mature lymphocytes that populate peripheral lymphoid tissues
• Occur in the generative lymphoid organs (BM, thymus).
• Commitment of progenitor cells to the B lymphoid or T lymphoid lineage.
• Proliferation of progenitors at specific early stages of development
• Rearrangement of antigen receptor genes
• Selection events
• Preserve cells that have produced functional antigen receptor proteins
• Eliminate potentially dangerous cells that strongly recognize self antigens.
• Differentiation of B and T cells into functionally and phenotypically distinct subpopulations.
• B cells develop into follicular, marginal zone, and B-1 cells
• T cells develop into CD4 + and CD8 + αβ T lymphocytes, natural killer T (NKT) cells, MAIT cells,
and γδ T cells. Abbas AK. et al. Cellular and Molecular immunology 9th edition

Abbas AK. et al. Cellular and Molecular immunology 9th edition

Role of the Thymus in T Cell Maturation
• Site of maturation
• GA 7-8 weeks
• While in the cortex, thymocytes first
express γδ and αβ TCRs
• Double positive in cortex
• Single positive in medulla
The thymic environment provides stimuli that are required for the proliferation and maturation of thymocytes.

• Naive T cell migration out of the blood through the HEVs into the lymph node parenchyma
involves the adhesion molecules L-selectin and LFA-1 and the chemokine receptor CCR7.
• Mechanism of egress of lymphocytes from lymphoid organs.
• Gradient of sphingosine-1 phosphate in the blood stream

1. Commitment to the B and T Cell Lineages
• Commitment to different lineages is driven by
• Cell surface receptors
• Transcription factors
T cell
• Notch-1 and GATA-3 transcription
• Pre-TCR
• RAG1, RAG2
B cell
• EBF, E2A, and Pax-5
• Pre-BCR, BCR
• RAG1, RAG2

2. Proliferation of Progenitors
• Proliferate first in response to cytokines and later in response to
signals generated by a preantigen receptor that select cells that have
successfully rearranged the first set of antigen receptor genes.
• Proliferation ensures that a large enough pool of progenitor cells will be
generated to eventually produce a highly diverse repertoire of mature,
antigen-specific lymphocytes.
• IL-7
• Required for proliferation of T cell progenitors (not B cell)
• Produced by stromal cells in the bone marrow and by cortical thymic
epithelium cells

IL-7: T cell development
IL-15: NK cell development

3. Rearrangement of antigen receptor gene
• This process is responsible for the generation of a diverse adaptive
immune repertoire.
• Small number of genes can give rise to a vast number of distinct Ig and
TCR molecules, each capable of binding to a different antigen
• Antigen independent
• Antigen receptor genes are randomly recombined and nucleotide
sequence variations are introduced at one of the joints, resulting in the
production of a large number of variable region-encoding exons.

Organization of T Cell Receptor Gene Loci
• In the TCR β and δ proteins, the V domain is encoded by the V,
D, and J gene segments, and in the TCR α and γ proteins, the
V domain is encoded by the V and J gene segments.

TCR diversity are generated by
the recombination of different gene
segment.
• Variable (V)
• Diversity (D)
• Joining (J)
The actual number of antigen receptors on B or T cells expressed in each individual
at any one point in time is probably on the order of only 10 7 or 10 8

V(D)J recombination
• Tremendous diversity of antigen receptors
• Each cell and its clonal progeny produce a distinct antigen receptor

Recognition Signals that drive V(D)J recombination
• Recombination signal sequences (RSS)
12/23 rule
Recombination occurs between two segments only if
• One of the segments is flanked by a 12-nucleotide spacer
• The other is flanked by a 23-nucleotide spacer
Occur 50% in Ig κ locus

Mechanism of V(D)J Recombination is mediated by the coordinated
activities of several enzymes.
1. Synapsis
• Two coding segments and their adjacent RSSs are brought together
2. Cleavage
• V(D)J recombinase (RAG1, RAG2 )
• RAG1/RAG2 complex is required for V(D)J recombination, but
only RAG1 possesses catalytic activity.
• Creates double-stranded breaks at RSS-coding sequence
junctions, forming a covalent hairpin
• Hold the hairpin ends and the blunt ends together
RAG genes are expressed only in developing B and T cells
RAG1 or RAG2 mutations are a cause of SCID (T-B-NK+)

3. Hairpin opening and end processing
• Artemis; an endonuclease
• Opens up the hairpins at the coding ends
• In the absence of Artemis, hairpins cannot
be opened and mature T and B cells
cannot be generated
• Terminal deoxynucleotidyl transferase (TdT)
• Adds nucleotides to broken DNA ends
Mutations in ARTEMIS are a rare cause of SCID. (T-B-NK+)

4. Joining
• Nonhomologous end joining
• KU70 and KU80 are DNA end-binding proteins
that bind to the breaks and recruit the catalytic
subunit of DNA-dependent protein kinase
(DNA-PK), a DNA repair enzyme
• DNA ligase IV, XRCC4: ligation
Mutations in affecting DNA-PK result in a failure to
produce mature B and T lymphocytes, causing SCID (T-B-NK+)

Generation of Diversity TCR
• Combinatorial diversity
• Recombination of V(D)J
• Junctional diversity
• The largest contribution to the
diversity of antigen receptors
• Addition or removal of
nucleotides at the junctions of
the V-D, D-J or V-J segments
• Pairing of β (VDJ), α (VJ) chains

Junctional diversity
• P nucleotides
• N nucleotides: randomly
add up to 20 non-template
encoded nucleotides
TdT
Nucleotides may be added to or removed from the coding ends
to generate new sequences that are not present in the germline.

4. Maturation
Stage of T cell maturation
1. Double-negative (DN) thymocytes
• The majority (>90%) of the DN thymocytes:
αβ T cell
• Pre-TCR
2. Double-Positive Thymocytes
• Thymocytes that fail to rearrangement of the
TCR α chain gene will die by apoptosis.
• The expression of RAG genes and further
TCR gene recombination cease after this
stage of maturation.
3. Single positive T cells

Stage of T cell maturation: Pre-TCR
• Productively rearranged the TCR β chain gene
• Proliferation of Pre-T cells
• Stimulation of alpha chain recombination
• Drive the transition from the double-negative to the
double-positive stage of thymocyte development
4. Maturation
Lack of any component of the pre-TCR complex or associated
signaling molecules (i.e., the TCR β chain, pre-Tα, CD3, ζ, or LCK)
• Results in a block in the maturation of T cells at the double-negative stage
• CD3ε mutations in humans result in SCID. (T-B+NK+)

4. Maturation
Stage of T cell maturation
1. Double-negative (DN) thymocytes
• The majority (>90%) of the DN
thymocytes: αβ T cell
• Pre-TCR
2. Double-Positive Thymocytes
• Thymocytes that fail to rearrangement of
the TCR α chain gene will die by
apoptosis.
• The expression of RAG genes and
further TCR gene recombination cease
after this stage of maturation.
3. Single positive T cells

5. Selection process
Checkpoints - developing cells are tested and continue to mature
1. Successful production of one of the polypeptide chain
• Expression of the pre-antigen receptor; Pre-TCRs in T cells
2. Assembly of a complete receptor
• Requires the second chain and thus assembly of a complete receptor
• At this immature stage, cells that express useful antigen receptors
may be preserved
• Potentially harmful cells that strongly recognize self structures may
be eliminated or induced to alter their antigen receptors

Rearrangement of
TCR β chain
Rearrangement
of TCR α chain
The presence of multiple checkpoints ensures that
only cells with useful receptors complete their maturation.

Positive selection
• TCRs bind with low avidity (i.e., weakly) to self peptide–self MHC
complexes are stimulated to survive and to differentiate either into CD4 + T
cells or CD8 + T cells
• MHC Restriction: Class I MHC - CD8, Class II MHC – CD4
• Do not recognize self MHC molecules are permitted to die by a default
pathway of apoptosis -> called death by neglect
• Mature T cells whose precursors were positively selected by self MHC
molecules in the thymus are able to recognize foreign peptide antigens
displayed by the same self MHC molecules on antigen-presenting cells in
peripheral tissues

Negative selection
• Eliminates or alters developing
lymphocytes whose antigen receptors bind
strongly to self antigens
• T cell - clonal deletion
• B cell - receptor edition - clonal deletion
• Maintaining tolerance to many self antigens
-> central tolerance
TCR signaling may induce
expression of a
pro-apoptotic protein
“BIM”
Weak – positive selection
Strong – negative selection
Intermediate – Treg

• AIRE (autoimmune regulator) that induces low-level
expression of many self antigens that are normally expressed
only in specific peripheral organs (tissue-restricted antigens).
• Their AIRE-dependent expression in the thymus makes these
tissue-specific antigens available for presentation to immature T
cells, facilitating the deletion (negative selection) of these cells
A mutation in the gene that encodes AIRE results in
an autoimmune polyendocrine syndrome (APS)/APECED

6. Differentiation
• Mature single-positive thymocytes
leave the thymus to populate
peripheral lymphoid tissues.
T cells develop into
• CD4 + and CD8 + αβ T lymphocytes
• γδ T cells
• Natural killer T (NKT) cells
• MAIT cells

• Less than 5% of all T cells express this form of TCR
• 10% of human intestinal intraepithelial T cells
• Diversity is limited
• Only a few of the available V, D, and J segments are used in mature γδ T cells
• Little or no junctional diversity
• Do not recognize MHC-associated peptide antigens and are not MHC restricted
• Recognize
• Small phosphorylated molecules, alkyl amines, or lipids that are commonly found in
mycobacteria and other microbes and that may be presented by nonclassical class I
MHC–like molecules.
• Protein or nonprotein antigens that do not require processing or any particular type of
APCs for their presentation
6. Differentiation: γδ T Lymphocytes

Function
• Serve as an early defense against a
limited number of commonly
encountered microbes at epithelial
barriers.
• Before the recruitment and activation
of antigen-specific αβ T cells
• IL-17
6. Differentiation: γδ T Lymphocytes
γδ Tcells have several innate cell-like features
In psoriasis, γδ T cells
: earliest IL-17–producing cells in lesions
Bonneville M, et al. Nature Reviews Immunology 2010;10(7):467–78.

• Expresses markers such as CD56 that are found on NK cells
• Limited diversity: invariant NKT cells (iNKT cells)
• All NKT cell TCRs recognize lipids that are bound to class I MHC–like
molecules called CD1 molecules
• Function
• IL-4, IFN-γ
• Produce antibodies against lipid antigens
• Mediate innate and adaptive immune responses
• Protect from autoimmunity, tumor, infection (Listeria, Toxoplasma,
Mycoplasma, Salmonella, Plasmodium)
6. Differentiation: NKT cell

• IFNγ promotes DC activation and
maturation
• Augmentation of CD4+and CD8+T
cell responses.
• Help in B cell activation through
CD40–CD40L interactions, improves
antibody titers and B cell memory
responses.
• Production of IL-2 induces regulatory
T cell proliferation
Figure 2. iNKT regulation of NK, T cell, and B cell activation.
Juno JA, et al. PLOS Pathogens 2012;8(8):e1002838.

• Most MAIT cells are CD8+ and can be activated either by
• MR1-restricted presentation of microbial riboflavin derivatives
• Directly by cytokines, including IL-12 and IL-18
• The effector functions of MAIT cells
• IFN-γ and TNF
• Cytotoxicity against infected cells: Granzyme, perforin
• MAIT cells are found in the blood, the gastrointestinal tract, and the liver
• 50% of all T cells in the human liver
• Gut barrier function
6. Differentiation:
Mucosa-Associated Invariant T (MAIT) Cells

TCR-dependent activation
• IL-17A (RORγt)
TCR-Independent activation
• IFN-γ (PLZF)
Hinks TSC, et al. Front Immunol. 2020 May 27;11:1014.

Clinical implication: SCID
Proliferation
TCR complex
V(D)J
recombination
J Clin Immunol (2020) 40:66-81
Rebecca H. Buckley, Middleton’s 9th edition

Antigen processing and presentation pathways
Exogenous
(Endocytic, MHCII) pathway
Endogenous
(Cytosolic, MHC I) pathway

Class I MHC pathway
• Microbial protein
• Proteasome
• TAP - Tapasin
• ERAP
• Peptide-Class I MHC-CD8+ T cell
Peptides preferentially bind Class I MHC
• Newly synthesized class I MHC are
attached to the luminal aspect of the
TAP molecules
• Class II MHC in ER are blocked by
invariant chain

Class II MHC pathway

T cell activation
• Occurs mainly in peripheral lymphoid organs
• IL-2
• Effector T cells: remain in Lymphoid organ or migrate to sites of infection
Sequence event in T cell response

Signal for T lymphocyte activation
First signal: Peptide-MHC-TCR
• Class I MHC on surface of
nucleated cells - CD8+ T cell – TCR
• Class II MHC on APC (B cells,
macrophages, DCs) and epithelial
cell in thymus - CD4+T cell - TCR
T cell activation
Recognition of Antigen

• TCR - MHC-peptide ligands
• Clustering of coreceptors
(CD4, CD8)
• Phosphorylation of ITAM tyrosine
residues in CD3 and ζ proteins
• CD4 and CD8 are T cell
coreceptors
• Bind to nonpolymorphic regions
of MHC molecules
• Facilitate signaling by the TCR
complex during T cell activation
T cell activation
Costimulation
Inhibition
Adhesion

Mutation in CD3 complex T-B+NK+ SCID
Tangye SG, et al. J Clin Immunol. 2020 Jan;40(1):24-64.

Activation of Tyrosine Kinases and a Lipid Kinase During T Cell Activation
• Within seconds of TCR ligation, Lck phosphorylates the ITAMs of the CD3 and
ζ chains
T cell activation
1. TCR-MHC complex
2. Phosphorylation of ITAM

3.
4.
Early tyrosine phosphorylation
events in T cell activation.
Tyrosine kinase
Src: Lyn, Fyn, Lck
Syk: ZAP-70, Syk
Tec: Tec, Btk, Itk

ZAP-70 deficiency Combine immunodeficiency
,generally less profound than SCID

• PLCγ1 (which is phosphorylated by ZAP-70 and Itk) are activated.
• Active PLCγ1 hydrolyzes membrane PIP2 to generate IP3, DAG
• IP3: stimulates an increase in cytosolic calcium
• DAG: activates the enzyme PKC

• IP3 causes depletion of endoplasmic reticulum calcium, which is sensed by STIM1
• STIM1 activated CRAC channel Influx of extracellular calcium
• Cytosolic free calcium act as a signaling molecule
• Calcium – calmodulin complexes activate several enzymes including calcineurin,
important transcription factor activation

1.
2.
3.

Mutations in ORAI gene
Combined immunodeficiency disease

• Recessive STIM1 and ORAI1 LoF mutations resulting in insufficient SOCE cause
CRAC channelopathies characterized by severe combined immunodeficiency
• Recurrent and chronic infection, Autoimmunity, Muscular hypotonia, Ectodermal
dysplasia, Anhidrosis, Mydriasis
• Dominant STIM1 and ORAT1 GoF mutations inducing excessive ca2+ entry
• Tubular aggregate myopathy (TAM), Stromorken syndrome (STRMK)
• Progressive muscle weakness and myalgia predominantly affecting the lower
limbs
• Miosis, ichthyosis, short stature, hyposplenism, thrombocytopenia, dyslexia
Silva-Rojas R, et al. Front Physiol. 2020 Nov 4;11:604941.

Silva-Rojas R, et al. Front Physiol. 2020 Nov 4;11:604941.

Signal for T lymphocyte activation
Second signal: Co-stimulation
Co-stimulator: activation / Inhibition
• CD28 - B7-1 (CD80) and B7-2 (CD86)
• CD28 expressed on more than
• 90% of CD4 + T cells
• 50% of CD8 + T cells
T cell activation

• In the absence of costimulation
• T cells fail to respond or enter a state
of prolonged unresponsiveness
• Microbe products (TLR), IFN-γ
• Enhance expression of costimulators
• APC -> activated naïve T cell
• Activated CD4+ T cells -> enhance
the expression of B7 costimulators
(pathway dependent on CD40)
• Mature dendritic cells are the most
potent stimulators of naive T cells.
Resting – few or no costimulators
Activated APC express
costimulators

CD28 signals + antigen recognition promote the survival,
proliferation and differentiation of the antigen-specific T cells

Other costimulators
• ICOS (inducible costimulator, CD278)
• ICOS-L (CD275)
• Expressed on dendritic cells, B cells
• Function
• T cell–dependent antibody responses
• Development and activation of T
follicular helper cells - essential for the
formation of germinal centers
• Generation of high-affinity B cells

Mutations in ICOS Combined immunodeficiency disease
(generally less profound than SCID)

Coinhibitors
• Expressed following T cell activation and
function to limit immune responses.
• CTLA-4: competitive inhibitor of CD28 by
binding more strongly to B7 molecules
• PD-1: delivers inhibitory signals that block
signaling by the antigen receptor and by CD28

Defective function of Tregs Autoimmunity

Clinical implication of CTLA-4
• CTLA-4 has a higher affinity for B7 than
does CD28.
• CTLA-4-Ig approved therapy for
• Rheumatoid arthritis
• Transplant rejection
• Immunotherapy of tumors
• Mechanism
• Reducing inhibition
• Enhancing T cell activation
• Enabling the cancer-bearing individual
to mount more effective antitumor
immune responses

Role of CD40 in T cell activation
• CD40L: activated T cell, CD40: B cell, macrophages, DCs
• CD40L-CD40 interaction:
• T cell licensing
• Germinal center reaction
• Induced B cell express enzyme AID

CD40L deficiency
• Lack of signaling between activated T cells and B cells
• Failure of B cells to upregulate CD80 and CD86
• Failure of B cell to become IgD-CD27+ memory B cell
• Fail isotype switching, produce only IgM
• Failure of monocyte to production of proinflammatory and anti-
infectious cytokines

CD40L deficiency
• Flow cytometry
• Normal number B cell, Normal or low number T cell
• Low memory B cell (CD27), Low memory T cell
• Low Treg >> underlie autoimmune manifestation
• Defective Th1 response
• Low IFN-g production, failure of APC to synthesize cytokine ; esp IL-12 -> defective
cellular immune response, increase OI infection
• T cell function : normal
• Immunoglobulin level
• IgM : normally or elevated (especially during infectious episode) (some : low IgM)
• Markedly low IgG, A
• IgM isotypes (natural isohemagglutinin, antipolysaccharide, anti non-typable H
influenza antibodies): produced normally BUT Absent IgG

Functional response of T cell activation
• Changes in the expression of various surface molecule
• IL-2 Secretion and IL-2 Receptor Expression
• Clonal expansion of T cell
• Differentiation of CD4+ T cell to Th1, Th2, Th17
• Differentiation of CD8+ T cell to CTL
• Differentiation memory T cell

• CD69: decreased S1PR1 activated T cell retained in lymphoid organ
• IL-2Rα: IL2
• CD40L: help activated APCs
• CTLA-4: Inhibit T cell activation

Regulation of IL-2 expression
• IL-2 is a growth, survival, and differentiation factor for T lymphocytes
• The high-affinity IL-2 receptor (IL-2R) is transiently expressed on activation of naive and
effector T cells; regulatory T cells always express this receptor.

Biologic function of IL-2
• IL-2 stimulates
• Survival
• Proliferation
• Differentiation of
antigen-activated T
cells.
• IL-2
• Required for the
survival and
function of
regulatory T cells

Clonal expansion of T cell
• Signal from
• Antigen receptor
• Costimulators
• Autocrine growth factor: IL2
• To generate a large number of
antigen-specific clones to eliminate
specific antigen from a small pool
of antigen-specific naïve T cells

Differentiation of CD4+ T cell to Th1, Th2, Th17
Subsets of CD4 + Effector T Cells
CXCR3, CCR5
CCR3, CCR4, CCR8
CCR6 – CCL20

• Cytokines drive development of CD4+ T cell subsets
• Stimuli other than cytokines influence the pattern of helper T
cell differentiation
• Affinity of the T cell receptor (TCR) for antigen
• Amount of antigen
• Nature of the APC
• Host genetic
General features of T cell subset differentiation

Development of Th1 cells
• Driven cytokines
• IL-12, IL-18, IFN-γ
• T-BET, STAT1, and STAT4 IFN-γ
• TH1 developed
• Secrete IFN-γ
• Inhibit differentiation of Th2 and
Th17

IFN-γ Function
• Activate macrophages to increase phagocytosis and
killing of microbes in phagolysosomes
• Promotes the differentiation of CD4+ T cells to the Th1
subset.
• Stimulates expression MHC molecules and B7
costimulators on APCs
• Produce cytokines that amplify the host response.
• TNF
• IL-1
• IL-12
• Th1-mediated pathology
• Delayed-type hypersensitivity
• Granulomatous inflammation

• Th1 cells activate macrophages
through contact-mediated signals
delivered by CD40L-CD40
interactions and by IFN-γ
• Classically activated macrophages
also called M1 macrophage.
• CD40 activate NF-kB, AP-1
Th1-Mediated classical
macrophage activation and
killing of phagocytosed microbes

Mendelian Susceptibility to Mycobacterial Disease
Bhattad SJc. Mendelian Susceptibility to Mycobacterial Disease: A Clinical and Laboratory Approach. 2019;1:4.

• IL-4 (others: IL-25, IL-33, TSLP by damaged
epithelial, ILC2)
• STAT6, GATA3
• TH2 developed
• Secrete IL-4, IL-5, IL-13
• Inhibit differentiation of Th1 and Th17

• IL-4 (IL-13)
• IgE
• Mucosal barrier
• M2 macrophage
• IL-5: Eosinophil
Effector function of
Th2 cells

M2 Macrophage
• Terminate inflammation
• Promote tissue repair
and fibrosis
• Scar formation (platelet-
derived GF, IL-13, TGF-
B, FGF)
Classical and alternative
macrophage activation

• IL-6, IL-1, IL-23, TGF-β
• RORγt and STAT3
• TH17 developed
• Secrete IL-17, IL-22
• IL-21: amplifies the reaction
• TGF-β suppress differentiation of Th1 and Th2
IL-21

IL-17
• Recruitment of Neutrophil (monocyte)
• IL-8, TNF
• G-CSF >> enhance neutrophil
generation
• Increase production of antimicrobial
peptides (defensins)
IL-22
• Maintain epithelial integrity: repair, AMP
Effector function of
Th17 cells

Roles of Th17 Cells in Host Defense

Differentiation of CD8+ T cell to CTL
3 transcription
factors for CTL
differentiation are
• T-BET
• Eomesodermin
• BLIMP-1
Cytokines
• IL-2, IL-12, IL-15

Killing mechanism
• Release of perforin/granzymes >>
target cell apoptosis
• Fas-FasL mediated killing
Target cells
• Virus-infected cells, cancer cells
• Phagocytes with intracellular
microbes
Effector function of CTL

Granule-dependent mechanism of killing
(Perforin- granzyme)
• Perforin/granzymes
• Lethal hit to the
target cell
• Death occurs during
the following 2-6 hr

• FasL is expressed on activated CTLs
• Fas on the surface of target cells
• Then induces apoptosis
Granule-independent mechanism of killing
(FAS/FAS-L)

• The effector response start but is
shut down ≠ Tolerance
• Persistent or chronic antigen
exposure
• The response of CD8 + T cells
is suppressed by the
expression and engagement
of PD-1 (programmed cell
death protein-1) and other
inhibitory receptors.
T cell exhausted

Differentiation memory T cell
Properties of memory T cells
• Increase level of anti-apoptotic
proteins: BCL-2, BCL-X
• Response more rapidly
• Greater number
• Migrate to peripheral tissue
• Self-renew
• Exist without need of antigens but
need cytokines
• IL-7: maintenance of CD4+,
CD8+ T cells, early lymphocyte
development, survival of naïve T
cell
• IL-15: survival of memory CD8+ T
cell

Change in proportions of naive and memory T cells with age

Biology of T lymphocytes.pdf

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