I&I B3/4 tut.

Site of T cell development

thymus

a) T and b) B cell development occurs primarily before/after/during puberty

a) before puberty; thymus begins to atrophy after

b) continuously produced throughout life

Development of T cells from double negative (DN) to single positive (SP)

1. Common lymphoid progenitors from bone marrow seed the thymus

2. T cell lineage commitment via Notch signaling (= DN1)

3. cells begin to rearrange DJ (1st) and VDJ (2nd) genes at TCR-β locus (= DN2)

4. Pre-TCR formed from TCR-β and Pre-Tα (= DN3)

5. Presence of pre-TCR indicates that a productive TCR-β gene rearrangement has taken place

6. Signals from pre -TCR lead to clonal proliferation (= DN4)

7. upregulate both CD4 and CD8 (= DP)

8. TCR-α VJ genes rearrange

9. TCR-α replaces Pre-Tα

10. selection (= SP)

Pair the corresponding MHC (1, 2) and CD (4, 8) molecules

MHC I:CD8

MHC II:CD4

What are the selection processes DP thymocytes are subject to?

Positive selection: eliminates those that don’t bind to self-MHC on cortical thymic epithelial cells

negative selection: eliminates thymocytes bearing very high affinity receptors for self-MHC

How is it determined whether a T cell will become CD4 or CD8 SP?

Kinetic signaling: on the basis of which CD produces a continuous signal (indicating specific binding) to a peptide on MHC

negative selection of SP thymocytes (3 steps)

1. SP thymocytes migrate back towards thymic medulla

2. SP thymocytes exposed to a array of self peptides including tissue specific ones by AIRE⁺ medullary thymic epithelial cells and Medullary DCs

3. SP thymocytes with TCRs reactive to self antigen eliminated by apoptosis

At what stages are thymocytes in the lymph a) cortex and b) medulla?

a) DN2→ DP

b) DN1, SP

(__) control thymocyte migration through the thymus

BONUS: name some key ones

chemokines; CCR7, CCR9 and S1PR

nTreg

CD4 + T cell subset that inhibits adaptive immune responses • Driven by expression of FOXP3 transcription factor • IL-2 and intermediate affinity TCR stimulation by self-peptide/MHC in thymus promotes nTreg development • Self reactive TCRs so promote regulation of self antigen

TRUE OR FALSE: all mature cells originating from lymphoid progenitor undergo positive selection and negative selection

FALSE: B cells don’t have positive selection because they recognise unprocessed native antigen

How do B cells differ from T cells in terms of the site of a) of gene rearrangement, b) of maturation?

a) bone marrow

b) periphery (spleen)

B cell development

1. Receive survival signals via IL-7/IL-7R

2. STAT5→ series of pathways including RAG→ PAX5 transcription factor confirms B cell lineage

3. Heavy chain D-J recombination (early pro-B)

4. Heavy chain V-DJ recombination (pro-B)

5. Production of CD19, Igα and Igβ

6. Pre-B cell receptor is expressed→ promote proliferation of productive clones, inhibits further rearrangement of H chain locus on other chromosome (pre-B)

7. Ig heavy chain complexed with Surrogate Light Chain (allelic exclusion)

8. light chain rearrangement of κ

9. light chain rearrangement of λ

10. express IgM on surface (immature)

11. tested against self antigen (survival= mature)

What happens to strongly autoreactive B cells in bone marrow?

Apoptosis or receptor editing; RAG genes re-expressed→ Light chain genes undergo additional rounds of rearrangement

TRUE OR FALSE: B cells are mature after being tested with self-antigen in the bone marrow

FALSE: They are transitional B cells because they have not been tested against peripheral antigens in the spleen

Describe the selection process of a) T1 and b) T2 B cells in the spleen

a) negative selection for self-reactivity in the PALS

b) positive selection to ensure they have not evaded negative selection by losing expression of a BCR entirely in the follicles

Mature follicular B cells migrate to (__) via (__)

Mature follicular B cells migrate to secondary lymphoid follicles via expression of the chemokine receptor CXCR5

What surface molecules are expressed by mature B cells?

IgM, IgD

B1 cells develop from what?

Develop from fetal liver

What are the functions of a) marginal zone, b) B1, c) B2/follicular B cells?

a) defend against blood-borne antigens

b) protect fetus from common microbial antigens.

c) Highly specific adaptive responses

TRUE OR FALSE: adults don’t have B1 cells

FALSE, BUT have them in very low amount

The five cardinal signs of inflammation and their underlying causes

redness: increased vasodilation/ blood flow to deliver immune components

swelling: increased vascular permeability delivers plasma proteins and fluid also enters tissue

pain: Production of inflammatory mediators activate nociceptors to make aware of injury thus protect tissue

heat/fever: see redness

loss of function: protects injured tissue

What is the fundamental difference between inflammation and infection?

inflammation can occur without the presence of microbes in response to tissue damage

Roles of the lipid mediators (2; 3 + 1)

prostaglandin E2: vasodilation, sensitizes nociceptors, acts in hypothalamus to cause fever

leukotriene B4: neutrophil chemoattractant/activator

Tissue mast cell activated by a)… to produce b)… causing c)…

a) C3a

b) release histamine from granules

c) vasodilation and permeability

Tissue macrophage cell activated by a)… to produce b)… (4) causing c)…

a) phagocytosis of bacteria

b) IL-1β & TNFα, IL-6 and chemokines

c) a response to be mounted against inflammation

Myeloid cells primed to produce pro-IL-1 (inactive precursor) by PRR signals • A 2nd signal (e.g. extracellular ATP, ROS, or uric acid crystals) induces formation of the NLRP3 inflammasome. • Inflammasomes are multiprotein complexes assembled around an intracellular danger sensor • Activate an inflammatory cascade • Convert caspase-1 (a protease) into its active form • Active caspase-1 cleaves pro-IL-1, pro-IL-18 into active forms that can be secreted - both highly inflammatory cytokines • Allows tight regulation of IL-1 cytokine production and rapid production of large quantities when required

Acute phase proteins from liver: • C-reactive protein (opsonin, complement activator) • Serum amyloid A (pro-inflammatory functions) • Fibrinogen – (coagulation)

rank the following from quickest to slowest recruited following inflammation: lymphocyte, monocyte, neutrophil

neutrophil

monocyte (→ macrophage)

lymphocyte

TRUE OR FALSE: cell composition of response recruited following inflammation depends on the stimulus

TRUE

neutrophil netosis

Expulsion of nuclear material in the form of neutrophil extracellular traps (NETs), which prevent dissemination of and kill pathogens (using degranulation)

Process of phagocytosis (5 steps)

1. opsonization of pathogen attracts phagocyte

2. binding of opsonin and receptors on phagocyte

3. internalisation into phagosome

4. phagolysosome formed

5. internalised matter processed

6. processed matter either used by cell, or expelled by exocytosis if waste

What mechanisms are used to kill ingested microbes in the phagolysosome? (6)

• low pH

• Toxic O₂-derived products

• Toxic NO derived products

• Antimicrobial peptides

• Enzymes

• Competitors

Macrophages functions (4)

• phagocytose pathogens

• clear dead neutrophils (efferocytosis) and debris

• produce inflammatory mediators

• coordinate repair/resolution

NK cells defence against virally infected cells (5 steps)

1. respond to inflammatory cytokines IL-12 + IL-15 + IL-18

2. irregular MHC I on ailing cell failing to stimulate negative signal

3. release perforin (pore forming) and granzymes (induce apoptosis)

4. produce IFNγ

5. activate macrophage

Eosinophils defend against…

large parasites too large to phagocytose

Type 2 inflammatory signals ↓ Eosinophil recruitment and activation ↓ Degranulation onto parasite surface 3 key granule components: • Major basic protein (MBP) • Eosinophil peroxidase (EPO) • Eosinophil cationic protein (ECP) → damage to parasite (potential collateral tissue damage too)

DCs present Ag to naïve T cells where?

paracortex of lymph node

3 signals for T cell activation by DCs:

• MHC-peptide antigen – TCR

• Costimulation CD80/86 – CD28

• Cytokines (dictate differentiation)

leukocyte extravasation steps and mediators (5 steps)

1. Tethering/rolling: Selectins (upregulated on tissue) + Carbohydrate ligands (on leukocyte)

2. Leukocyte activation: Cytokines, chemotactic factors

3. Adhesion: Integrins (on leukocyte) + Ig superfamily CAMs (upregulated on tissue)

4. Trans-endothelial migration: Vasoactive factors, degradative enzymes

5. Chemotaxis (migration to target): Chemotactic factors & receptors

integrins structure

Basement membrane: a) what is it, b) what is it made of, and c) what does it do?

a) specialised extracellular matrix underlying endothelial and epithelial cells

b) Collagens, Laminin, perlecan proteoglycan

c) Bind cell surface integrins

TRUE OR FALSE: leukocytes produce proteinases that can degrade all components of ECM

TRUE

What are proteinases produced by (during inflammation)?

connective tissue cells and leukocytes

How do proteinases function during inflammation?

locally remodel basementmembrane ECM to facilitate leukocyte passage

4 major proteinase classes

serine

matrix

cysteine

aspartic

fMet-Leu-Phe chemotactic factor a) origin and b) nature

a) processed bacteria

b) peptide

C5a chemotactic factor a) origin and b) nature

a) complement

b) peptide

LTB4 chemotactic factor a) origin and b) nature

a) plasma membrane

b) lipid

chemokine chemotactic factor a) origin and b) nature

a) host gene expression

b) protein

Which chemotactic factors are specific to a) myeloid leukocytes only, b) lymphoid leukocytes only, c) all leukocytes?

a) fMet-Leu-Phe, LTB4, C5a

b) none

c) chemokine

a) Necrosis vs b) apoptosis

a) caused by cell lysis releasing the (damaged) contents of the cell→ DAMPS

b) organelles bleb into/ are contained within apoptotic bodies→ homeostasis maintained

efferocytosis

phagocytic clearance of apoptotic cells by macrophage

What changes occur in macrophage phenotype following efferocytosis? (2)

• downregulate proinflammatory cytokines

• upregulate cytokines (IL-10, TGF-β) and programmes involved in repair and reduction of inflammation

What is the Lipid mediator profile and effects like during a) early (2) and b) end of (4) inflammation?

a) prostaglandins and leukotrienes→ inflammation / leukocyte recruitment

b) lipoxins, resolvins, protectins, maresins→ limit further recruitmen, promote efferocytosis and resolution

scar formation is mediated by…

fibroblast activation and ECM/collagen deposition

What determines whether tissue will be regenerated/repaired or scarred? (3)

Nature/extent of injury

regenerative capacity

persistence of injury/infection

What if you have excessive TGFβ?

extensive scarring leading to eventual reduction of tissue function

What causes persistent/ chronic inflammation? (5)

• Persistent infection

• Persistent tissue injury

• Presence of foreign material that cannot be removed

• Continued DAMP generation/ necrosis

• Defective resolution

How are tissue destruction and repair involved in chronic inflammation?

persistent inflammation leads to tissue remodelling and fibrosis

Granuloma formation: a) when and b) how does it occur?

a) persistent stimulus from a pathogen that is not eliminated

b) recruited macrophages and T1 cells surround the pathogen to create a physical barrier to prevent the infection from spreading

sequence of systemic inflammation (6 steps)

1. excessive/widespread cytokine activation

2. systemic inflammatory response

3. endothelial dysfunction

4. vasodilation/permeability

5. coagulation abnormalities

6. organ dysfunction

Massive influx of inflammatory cells and production of pro-inflammatory mediators in airways • Pro-inflammatory mediators: IL-6, IL-1, TNF → alveolar-capillary barrier damage → ↑ vascular permeability → protein-rich oedema in lung → impaired gas exchange → ± subsequent fibrosis → ARDS

E.g. in chronic HCV infection in liver • Chronic inflammation • Macrophage / fibroblast activation • Collagen / ECM deposition • Altered tissue architecture • Loss of function

Failure to eliminate the trigger → persistent inflammation → chronic inflammation / granuloma Failure to resolve/repair appropriately → fibrosis/remodelling Excessive inflammatory response → collateral tissue damage / systemic inflammation

corticosteroids: a) effects, b) receptor, c) benefit, d) detriment

a) decrease inflammatory cytokine/chemokine production, adhesion molecules on endothelium & inflammatory-cell activation/recruitment

b) glucocorticoid receptor (regulates gene transcription)

c) suppress inflammation at multiple levels

d) lack of specificity produces adverse effects

Non-steroidal anti-inflammatory drugs (NSAIDs): a) target, b) effect, c) benefit, d) detriment

a) cyclooxygenase (inhibition)

b) reduced prostaglandin

c) suppress inflammatory symptoms

d) lowers host defence which inhibits tissue repair/regeneration