Post-midterm

Where does signal 3 of T cells occur?

Lymph node

When do T cells become effector T cells?

When they have received all three signals

They then go to site of infection to exert their function

What are the three signals for T cell activation?

1) pMHC:TCR

2) B7:CD28 (B7.1/CD80;B7.2;CD86)

3) Cytokines

What are the effector functions (in general) of CD4+ and CD8+ T cells?

CD4+: activation of macrophrages, B cells, other cells

CD8+: killing of infected target cells, macrophage activation

*memory CD4+ and CD8+ T cells

How do T cells leave to get to the site of infection?

Efferent lymphatic vessels → Thoracic duct → Blood stream (to site of infection)

Where do T cells exert cell-mediated immunity?

At the site of infection

What happens to T cells when the infection is cleared?

Downregulation of T cell activity and Immunological memory

Briefly describe signal 3 of T cells.

Differentiation - cytokines directing T cell differentiation into distinct effector cell types

*all three signals result in intracellular signaling, leading to change (activation, proliferation, survival, differentiation)

Activated T cells proliferate in response to what?

IL-2 cytokine

What can activated T cells differentiate into?

CD4+ helper cells: T_H0 into T_H1 or T_H2 or T_FH or T_H17 or others

CD8+: cytotoxic T lymphocyte (CTL)

Regulatory T cells

*T_H0 are naive T cells, have not received all three signals

How can effector T cells be classified?

• The TFs they express

• Profile of cytokines they make

• Main function in adaptive immunity

Once activated, an effector T cell does not require what to act?

Co-stimulation

To kill an infected cell, it does not require signal 2 again. It will only kill if their TCR is specific to the MHC and peptide

*on nucleated cell therefore MHC I

C

What are the subtypes of CD4 T cells?

• T_H1

• T_H2

• T_H17

• T_FH

• T_reg

*different effector cells have different functions, different cytokines secreted and different function

*can impact functionality of other immune cells and clears different microorganisms

What are polarizing cytokines?

Responsible for guiding a T cell toward one of several different effector fates (fate-specifying cytokines)

It is what signal 3 cytokines are referred as

*APCs bind PAMPs via PRRs → cytokine secretion; different PRRs engaged means different cytokines produced

*effector cytokines are produced by differentiated T cells

How does signal 3 exert its effect?

Polarizing cytokines bind their receptor and induce signaling, which occurs through STAT proteins (when phosphorylated act as TFs to go in nucleus and bind to promoters for specific gene)

When specific gene is transcribed, another TF known as master transcriptional regulator is produced (determines functionality of cell)

The MTR binds to response elements on promoter regions of genes coding for specific effector cytokines, which determines activity of activated and differentiated T cell subtype

B

The STAT proteins are waiting to get phosphorylated, which then leads to expression of master transcriptional regulators

What are the general characteristics of Fas ligand and CD40 ligand?

• Expressed on surface of effector T cells (not naive)

  • included in genes that are induced following the three signals

• Crucial for effector T cell function

• Transmembrane ligands part of TNF family (cell to cell interaction)

What is Fas ligand?

• Expressed on surface of effector CD8+ and T_H1

  • used by CD8+ to exert cytotoxic effect

• Binds Fas on surface of infected cells in periphery (site of infection)

What is CD40 ligand?

• Expressed by T_H1, T_H2, T_H17, T_FH

• Binds CD40 on B cells and innate immune cells

• Used to activate these target cells

• Allows DC licensing and expression of more co-stimulatory moleculaes

  • CD40 signaling leads to DC licensing and expression of more co-stim molecules

What are the signals for effector CTL generation from naive CD8+ precursors?

1) TCR binds peptide presented by APC on MHC I

2) Co-stimulatory signal transmitted by CD28-B7 (CD80/86) interaction between T cell and APC

3) Provided mainly by IL-2 and other cytokines to some extent (IL-12) inducing proliferation and differentiation into CTL form

What are the specific considerations for CD8+ T cells?

• Requires more co-stimulation

• IL-2 can be autocrine and paracrine from T_H1 or T_H17

• Requires help of effector CD4+ cells

How can CD8+ cells be activated simply but rarely, and majority of the time?

Simple: by activated DCs that have high co-stim activity (in some viral infections, DCs become infected and sufficiently activated)

Majority: extra help with CD4 effected T cells and license DCs to cross present

What is sequential activation?

• APC becomes further licensed following interaction with a CD4+ cell

  • signal APC receives through CD40 signaling

• Interacts with CD8+ independently

• IL-2 produced by CD8+ cells alone induces proliferation (autocrine)

(1 - CD4+ gets activated, 2 - give permission through CD40 ligand binding, 3 - CD8+ activation)

What is simultaneous activation?

• APC interacts with both CD4+ and CD8+ cells at the same time (CD4 interaction takes long or CD8 was close by)

• First thing that happens is CD4+ activation (must be effector to give CD40 ligand binding to CD40 licensing)

  • CD40 (DC) - CD40L (T cell)

• As CD4+ produces IL-2, it gives some to CD8+ through paracrine

  • IL-2 still secreted by CD8+ (autocrine) to induce proliferation of CD8+

What needs to happen to CD4+ T cells in order for CD8+ T cells activation to occur?

• Find its p:MHC II match

• All three signals received

• CD40L gets expressed and binds CD40 on APC

What needs to happen to DCs to allow them to present antigen to both CD4+ and CD8+ T cells?

• Encounters a PAMP

• Travels to lymph node (secondary lymphoid organ) as mature DC

• Present antigen on MHC II to CD4+

• Gets licensed through CD40 binding

• Presents exogenous antigen on MHC I to CD8+ (cross presentation) (increased expression of CD80/86)

What does CD4+ cells secrete and express when activated?

IL-2 (increased production when APC is licensed by activated CD4+)

CD40L

What 4-1BBL and CD70?

An additional molecule that activates CD8+ T cells and binds to 4-1BB (co stim signals for CD8)

CD70 binds to CD27 to provide costimulatory molecules along with B7

What helps generate memory CD8+ T cells?

CD4+ T cells (due to cytokines)

C & D

What are cytotoxic T lymphocytes?

• When naive CD8+ cells get activated and differentiates

• Leaves lymph node to travel to site of infection

• CTLs can kill infected cells in the periphery

  • interaction of TCR (& CD8 co-receptor) with pMHC I

How do CTLs target infected cells?

*all nucleated cells express MHC I

1) initial interaction via nonspecific adhesion molecules - if pMHC is not a match CTL moves on

2) When CTL recognizes pMHC (infected or cancerous cell), the infected cell dies

How can CTLs induce apoptosis?

• Fas-FasL

• Granules

• Also secrete cytokines to direct immune response

Explain Fas-FasL interaction.

Effector CTL expresses FasL; Infected cells express Fas (after TCR and MHC I interaction)

Signaling cascade involves cleavage of pro-caspases into caspases to lead to apoptosis of infected cell (8→3&7)

Explain granule mediated killing.

1) CTL first makes contact to target cell via nonspecific adhesion

2) Specific recognition via (CTL) TCR:pMHC (triggers intracellular signaing); reorganization of cytoskeleton and cytplasmic contents

3) Granules released at point of cell contact

What granules are used in granule-mediated killing? What are their functions?

Perforin: aids in delivering content of granules to cytoplasm of target cell through forming pores

Granzymes/Granzyme B: serine proteases, which activate apoptosis once in the cytoplasm of target cell

• Granzyme B: initiates signaling through pro-caspases cleavage into caspases (3), leads to DNA fragmentation and cell death (blebbing where cytoskeleton breaks and membrane bugles indicating apoptosis)

Explain granzyme/perforin-mediate cytolysis.

When stimulated CTLs release granule contents:

• Perforin is pore-forming; granzymes are serine proteases

• Perforin punches holes in membranes and granzyme enters cytoplasm of target cell to induce apoptosis

Which mechanism does CTL use?

*both converge on various caspase 3 activation leading to apoptosis)

Perforin/Granzyme is fast-acting and CTLs primarily use this

Fas-FasL is slow-acting

Reports show that CTL lytic action is enhanced when both mechanisms operate simultaneously

C

• CD8+ T cells in trachea 8 days post infection

• Influenza has gone down after 8 days

• Conclusion:

  • CD8+ responsible for reducing virus titer

  • Takes time (8 days) for adaptive immune response to exert function

(*** is significant change)

B

Which cytokines do CTLs secrete? What are their function?

IFNgamma (Type II IFN)

• Increase MHC I expression in neighbouring cells (increases chance of CTLs finding match)

• Activates macrophages and stimulates productive of chemokines that can recruit additional macrophages and CD8+ cells to site of infections

**type I IFN are potent antiviral effects (PRR activation effects); type II has role agaisnt intracellular pathogens, secreted mainly by T cells

What secretes polarizing cytokines?

Secreted by dendritic cell and neighbouring innnate and adaptive immune cells

(fate specifying)

T_H1 responses are involved in the response to which infections?

Viruses and Intracellular pathogens (bacteria and parasites)

CTLs are involved too

T_H2 responses are involved in the response to which infections?

Parasites/helminths and other extracellular pathogens

*facilitates tissue repair

Dysregulated TH2 response are involved in allergies or asthma

T_H17 responses are involved in the response to which infections?

Extracellular bacteria and fungi (and autoimmunity)

T_FH responses are involved in what?

Activating B cells in the lymph node

In response to all types of pathogens

Based on what type of infection, the ___ response is dominated by ___

Cell-mediated; one of the T cell subsets

What are the key effector molecules and cytokines for CTLs?

• Perforin

• Granzyme B

• Fas ligand

• IFNgamma (Type II IFN)

(p:MHC I)

What are the key effector molecules and cytokines for T_H1 cells?

• IFNgamma

• CD40 ligand

• Fas ligand

(p:MHC II)

What are the key effector molecules and cytokines for T_H2 cells?

• IL-4

• IL-5

• IL-13

• CD40 ligand

(p:MHC II)

What are the key effector molecules and cytokines for T_H17 cells?

• IL-17

• IL-22

• CD40 ligand

(p:MHC II)

What are the key effector molecules and cytokines for T_reg cells?

• IL-10

• TGF-B (Beta)

(p:MHC II)

What is cross-regulation? Give some examples.

During response to an infection, there will be a combination of different T cells but one will dominate

• IL-4 (secreted by T_H2) inhibits T_H1 differentiation

• IFNgamma (secreted by T_H1) inhibits T_H2 proliferation

• IL-4 or IFNgamma inhibits T_H17 differentiation

These effector cytokines can help reinforce the predominant subtype they are part of

Explain T_H1/T_H2 cross-regulation.

Master transcriptional regulators commit T cells to one subset or the other

T-Bet (master transcriptional regulator for T_H1) suppresses T_H2 pathway gene expression

• positively regulate IFNgamma and negatively regulates IL-4 and IL-5

GATA3 (MTR of T_H2) suppresses T_H1 pathway gene expression

• negatively regulate IFNgamma and positively regulates IL-4 and IL-5

Explain the decision point of T_H17/T_reg.

• TGF-B is a key cytokine for differentiation of both subsets

• IL-6 is the switch which induces T_H17 subset differentiation instead

• A balance between the two is beneficial:

  • Normal state can favour development of iT_reg population to keep inflammation down

  • Infection (leading to IL-6 production) would stimulate a more antibacterial T_H17 differentiation

Which T cells leave the lymph node and migrate to the site of infection? Which ones stay in the lymph and activated B cells?

Leave: T_H1, T_H2, T_H17

Stay: T_FH

Different ___ molecules are expressed by naive vs effector T cells since they ___

Cell surface molecules; travel to different locations in the body

*effector T cells express molecules that target them to specific tissues (skin vs gut) depending on the site of infection

How is the travel of effector T cells partly determined by?

The travel is partly determined by which lymph node they get activated

Ex: if they are activated in a lymph node that drains the skin, the effector T cells will up-regulate homing that send them to the skin)

Which polarizing cytokines/signal 3 are used for T_H1?

IFNgamma

IL-12

Which effector cytokines are secreted by T_H1?

IFNgamma

What is the master transcriptional regulatory of T_H1?

T-bet

What is the function of T_H1 in cellular immunity?

• Macrophage activation

• Activation and differentiation of naive cytotoxic T lymphocyte precursors into effector CTL (through CD40L binding)

Which transcription factors are needed for T_H1?

STAT 1 and STAT 4

(signal 3 cytokines phosphorylate them)

Why would BALB/c mice with anti-IL-4 survive?

a) BALB/c mice would normally induce a TH2 response and with anti-IL-4 antibody, that would inhibit differentiation of naive T cells to TH2 and instead develop a TH1 response

b) Anti-IL-4 antibodies would lead naive T cells to differentiate into TH17 which are important to clear intracellular parasites

c) BALB/c mice would normally induce a TH1 response and with anti-IL-4 antibody, that would inhibit differentiation of naive T cells to TH1 and instead develop a TH2 response

a) BALB/c mice would normally induce a TH2 response and with anti-IL-4 antibody, that would inhibit differentiation of naive T cells to TH2 and instead develop a TH1 response

*TH1 to be dominant to clear intracellular infections

What type of response are T_H1 cells?

Type I response

How can T_H1 cells activate macrophages?

• Some pathogens persist in macrophages (inhibit fusion of phagosome and lysosome; prevent acidication so lysosomal proteases cannot activate)

• The T_H1 cell recognizes pMHC II on the surface and uses CD40L to bind to CD40

• Secretes IFNgamma

• Boosts macrophage antimicrobial activity and production of TNFa from macrophage

What is an activated M1 macrophage (classically activated macrophage)?

• Function induced in the context of TH1 and have a boost to their antimicrobial mechanisms (ROS)

• TNFa is secrete which provides autocrine signaling as survival signaml to macrophage

• CD40L binding activates macrophages and increases expression of IL-12 (part of signal 3 for TH1)

• TNFa and IFNgamma increase expression of MHC I and II, CD40, B7 molecules, and IL-12 which further activates TH1 cells (feed forward loop)

  • increases chance of TH1 and CTLs to find match

*IFNgamma from effector CTLs can also activate M1 macrophages

How can TH1 kill infected macrophages?

• For chronically infected macrophages

• TH1 cells recognize pMHCII on infected macrophage

• FasL on TH1 cell binds Fas on infected cells to trigger apoptosis

• Bacteria released can be phagocytosed by freshly recruited macrophages

How can TH1 cells help CD8+ cells?

• TH1 can secrete IL-2 and stimulate CD8+ proliferation and differentiation

• Occurs in lymph node

• CTLs can recognize via pMHC I and kill infected macrophages

How can TH1 stimulate increased differentiation of monocytes in bone marrow?

• TH1 secrete IL-3 and GM-CSF which circulate in the blood and act on precursors in the bone marrow (endocrine)

How can TH1 change expression of adhesion molecules on neighbouring endothelium to recruit more macrophages?

TH1 cells secrete cytokines that induce these changes

How can TH1 recruit macrophages by chemotaxis?

TH1 cells secrete chemokine CCL2 that attract macrophages to the site of infection

C

What type of response do TH2 cells have?

Type 2 response

What is the third signal for TH2?

IL-4

What are the effector cytokines for TH2?

IL-4, IL-5, IL-13

What is the master transcriptional regulator for TH2?

GATA-3

What do TH2 cells target/activate when responding to their pathogens?

In response to helminths, they activate eosinophils, mast cells, basophils, and macrophages

Which transcription factors are needed for T_H2?

STAT 6

What is the weep and sweep method used for?

Sometimes the helminth is not cleared (chronic infection), so the worm burden is reduced through weep and sweep

Which antibodies do TH2 cells recruit?

Recruits IgE antibodies, as they are important for immune responses against helminths

How can TH2 promote cell turnover and mucous production?

• IL-13 can increase mucus production by goblet cells and increase turnover of epithelial tissue

  • parasites have a harder adhering to gut and colonizing

• The weep in weep and sweep response

How can TH2 enhance worm expulsion?

• IL-13 can stimulate smooth muscle cells to contract

  • can lead to worm expulsion (physical removal of worm)

• The sweep in weep and sweep response

How can TH2 recruit and activate M2 macrophages?

M2 macrophage: alternatively activated macrophage

• IL-4 and IL-13 important for M2 macrophage

• M2 macrophages aid tissue repair and participate in worm killing and expulsion (increase smooth muscle contraction)

• M2 mac can form granulomas to entrap worms

• M2 mac release toxic mediators directly onto the worm by antibody-dependent cell-mediated cytotoxicity (ADCC)

  • killing of antibody coated target cells by cell with Fc receptors (receptors bind to antibodies) - mediated by NK

How can TH2 play a role in eosinophil activation?

• IL-5 activates, recruits, and enhances eosinophil differentiation

• Eosinophil granules contain major basic protein (MBP) which can kill parasites

• IL-4 and IL-13 lead to IgE (opsonin) generation

  • bind antigens on parasites, eosinophils express receptors that recognize the Fc portion of IgE

  • eosinophils can then specifically target pathogen and degranulate to kill it

How can TH2 play a role in mast cell activation?

• Cytokines activate mast cells, which contain granules of histamine and other molecules

  • increases vascular permeability

  • increases intestinal motility

  • increases recruitment of inflammatory cells

• IgE Abs coat parasite, mast cells express receptors that recognize the Fc portion of IgE

How are basophils involved in TH2 effector functions?

• Secrete IL-4 and IL-13

• Activate goblet cells

• Allow vasodilation

• Binds to IgE and releases histamines

Explain the immune response to allergies.

*Type 2 response

• Allergens enter via mucosal tissues and induce TH2 response

  • IL-4 and IL-13 induce IgE generation

• Allergies are initiated by an interaction between an IgE Ab and an Ag

  • IgE Abs bind to mast cells or basophils and induce degranulation

    • Granule contents released include histamine, proteases, chemokines (inflammatory)

    • Act on surrounding tissues and cells to cause symptoms

• Free circulating IgE is usually low in concentration in blood serum

• Healthy individuals only make IgE in response to parasites

B

*IgE part of humoral immunity

What is signal 3 of TH17 cells?

• TGF-B

• IL-6

• IL-23

What effector cytokine is secreted by TH17?

IL-17 and IL-22

What is the master transcriptional regulator for TH17?

RORgamma T

What cells does TH17 interact with? What does it respond to?

• Enhances neutrophil responses and mucosal immunity

  • respond to extracellular bacteria and fungi

• Pro-inflammatory response and involved autoimmune disorders (from dysregulation)

  • psoriasis, inflammatory bowel disease, asthma, arthritis, MS

What transcription factor is used for TH17?

STAT 3

What type of response do TH17 cells have?

Type 3 response

How can TH17 induce production of antimicrobial peptides?

• IL-17 and IL-22 induce epithelial cells to produce antimicrobial peptides

• Can contribute to killing/slowing replication of bacteria/fungi

How can TH17 increase epithelial turnover?

• IL-22 increases division and shedding of epithelial cells

• Hinders bacterial/fungal growth

How can TH17 induce other cells to produce G-CSF?

• IL-17 acts on stromal and myeloid cells

  • Secretes G-CSF (cytokine)

• G-CSF enters circulation (endocrine) and targets bone marrow precursors to differentiate intro neutrophils