Immuno Test 3

• Phases of the humoral immune response

• In one week, a single B cell may give rise to ~5000 antibody secreting cells, which collectively produce > 1 trillion antibody molecules per day

• Antibody responses to protein antigens

• Require T cell assistance

• Involve follicular B cells (lymph nodes)

• We’ll focus on T-dependent responses

• Antibody response to Multivalent antigens

• Repeating determinants: polysaccharides and nucleic acids

  • No T cell help required

  • Rapid and simple

  • Involve marginal zone B cells (spleen) and B1 cells (mucosa)

“Helper T cell” term comes from discovery that

•  T cells stimulate B cells to produce antibodies

Summary of primary and secondary antibody responses

• Lymphocytes are organized with lymph nodes

Antigen delivery to naive follicular B cells

• Small antigens delivered to follicular B cells via conduits

• Larger antigens captured by macrophages in subcapsular sinus or resident DCs in medulla

• Antigen transferred to FDCs for display to B cells

• Antigen generally in its native, folded three-dimensional conformation

• Recognition of the antigen by appropriate B cell receptor initiates B cell activation

Sequence of events in humoral immune responses to T cell-dependent protein antigens

 1. Independent recognition of antigen by Th cells and B cells

2. Activated T cells and B cells migrate toward each other and

interact at interface of T-cell zone and B-cell zone

3. T cell-dependent B cell proliferation and differentiation creates

extrafollicular focus (where B cells proliferate, undergo isotype

switching and differentiate into short-lived plasma cells)

5. Tfh cells and activated B cells migrate back to the follicle to

form a cell cluster termed the germinal center.

4. Some T cells develop into follicular helper T cells

6. The late events in B cell response occur in germinal centers,

including affinity maturation, additional isotype switching,

memory B cell generation, & generation of long-lived plasma cells

• B cells present protein antigen to Th cells

• Antigen recognition is necessary but NOT always sufficient to stimulate significant B cell proliferation and differentiation

=> full response to protein antigen also requires Th cells

B cells present peptide antigens to Th cells via class II MHC

=> B cells are type of APC!

Peptide presented to Th cell is the “same” peptide that activated the naïve CD4+ T cell

Activated Th cells in turn promote B cell response via CD40L and cytokines

• Germinal centers in secondary lymphoid organs Three identifiable regions

• Mantle zone

• Germinal center

  • Light zone

  • Dark zone

Germinal center light zone

• abundant follicular dendritic cells

Germinal center dark zone

 mainly proliferating B cells

Germinal center mantle zone

mainly naive B cells

• Germinal center reaction in a lymph node

• Ig heavy chain isotype switching 

• Driven by Tfh cells in light zone

• Does not affect antibody specificity

• Regulated by Th-produced cytokines activated by microbe type

• Occurs via “class switch recombination” (CSR) of Ig heavy chain

  • Previously formed VDJ exon is placed adjacent to different downstream constant region (Intervening sequence is deleted

• Overview of affinity maturation

• Depends on “somatic hypermutation”

• Early in the immune response (after gene rearrangement)

• As T-dependent humoral responses progress

  • Somatic mutations in Ig V genes => selection of high affinity B cells

• Higher affinity antibodies are more effective at neutralizing and eliminating microbes and toxins

• Both CSR and SHM depend on activation-induced cytidine deaminase (AID)

  • Induces C to U mutations subject to error-prone repair

B cell selection in germinal centers

• Many somatic mutations result in reduction or loss of

antigen-binding ability

• B cells experience alternating rounds of selection in the

light zone and proliferation/mutation in the dark zone

• In light zone, B cells die by apoptosis unless “rescued” by

binding to antigen

• After mutation, B cells undergo form of Darwinian selection

• As more antibody molecules are produced, more of the

antigen is eliminated

• Thus, B cells must express antigen receptors with

increasingly higher affinity to avoid apoptosis

• GCs are sites of significant B cell turnover

• Re-enters dark zone or becomes plasma cell or memory cell

Plasma cells =

• terminally-differentiated B cells committed to Ab production

short lived plasma cells

Generated in early T-dependent responses in extrafollicular foci & in T-independent responses

• Generally found in secondary lymphoid organs and peripheral non-lymphoid tissues

long lived plasma cells

• Generated in T-dependent germinal centers

• Precursors called plasmablasts enter the circulatory system, then move to the bone marrow

where they complete differentiation

• Survive long periods without antigen stimulation due to expression of anti-apoptotic proteins

Antibody production in long-lived plasma cells

• Bone marrow becomes major site of antibody production 2-3 weeks after exposure

• Plasma cells in marrow may continue to secrete antibodies for decades

• Antibodies enter circulation and mucosal secretions (plasma cells do not recirculate)

• Plasma cells are morphologically distinct from the typical B cell

=> Major structural alterations in the ER and other components of secretory pathway

Memory B cells

• Derived from some activated B cells in the germinal center

• May remain in the lymphoid organ of origin or enter circulation

• Responsible for much of enhanced secondary antibody response to protein antigens

• Effective vaccines against microbes induce both efficient affinity maturation and

memory B cells => these only occur if vaccine also activates helper T cells

Effector mechanisms of humoral immunity

• Secreted antibodies eliminate antigens and associated microbes

• Involves collaboration with components of the innate immune system

• Antibodies perform effector functions at locations distant from site of production

• Primary targets include:

• Extracellular bacteria and fungi

• Viruses (pre-infection and post-release)

• Primary basis of vaccine protection

• May contribute to tissue damage in certain immune diseases and transplant rejection

Neutralization of microbes and toxins by antibodies

Antibody binding:

• increases size of antigen or microbe

• blocks ability of microbe to infect host cell

• blocks ability of microbial toxin to harm host cell

Mainly IgA in gut

Mainly IgG in blood

Requires only antigen-binding region (Fab)

=> Any isotype could “work”

Antibody-mediated opsonization

Antibody dependent cell mediated cytotoxicity

• Complement activation depends on a proteolytic cascade

Functions of complements (opsonization and phagocytosis)

• Neutrophils and macrophages

• Last step caused by activating signals

Function of complements (stimulation of inflammatory reactions)

• And activation of endothelial cells

• Degranulation of mast cells releases multiple “vasoactive mediators” (e.g., histamine)

• C3a and C3b = “anaphylatoxins” = can produce anaphylactic shock

Functions of complement (complement-mediated cytolysis)

• MAC related to perforin

• Creates pores in microbial cell membrane 

• Pores allow movement of water into cell

Specialized immunity

• Regional immune systems

• Privileged tissues

Regional immune systems

• Provide protection against microbial challenges encountered at certain locations

• Allow for appropriate balance with nonpathogenic commensal organisms

• Examples:

  • Mucosal epithelial barriers: gastrointestinal, respiratory, urogenital

  • Cutaneous (skin)

• Regional immune systems share certain basic features

  • But each contains own specialized anatomic features, cell types, and molecules

Challenges for GI immunity

1. Large surface area to defend

2. Abundance and diversity of nonpathogenic, commensal microbes

3. Consistent exposure to diverse food antigens

4. Inflammation has negative impact on necessary gi functions

   1. Must limit response to commensal and food antigens

   2. But must still be able to detect small pathogen antigen “signals” amongst background

The gastrointestinal immune system

• Peyer’s patch

  • Organized secondary lympoid tissue

  • Contains B cells, T cells, DCs, Macrophages

  • “Mucosal-associated lymphoid tissue” = MALT

  • “Gut-associated lymphoid tissue” = GAL

The gastrointestinal immune system: Innate Immunity

Barrier aspects

• Mucus secreted by Goblet cells

• Defensins secreted by Paneth cells

• Epithelial sheet sealed by tight junctions

• Basement membrane (ECM)

TLR’s and other PAMP / DAMP receptors on various cells

trigger innate immune response to pathogens

BUT inflammatory response to commensals is limited by

multiple mechanisms:

• TLRs on basolateral surface of epithelial cells

• TLRs in GI tract require high threshold for activation

• Phagocytes secrete IL-10 (inhibitory cytokine)

The gastrointestinal immune system: Adaptive Immunity

Mainly humoral immunity (via IgA)

directed at microbes in the lumen

Dominance of IgA because:

1. B cells in gut region class switch to IgA

2. IgA-expressing B cells home to the gut

Abundant Treg cells act to

control inflammatory reactions

Gut-antigen sampling: the role of M cells

Gut-antigen sampling: intestinal dendritic cells

The gastrointestinal immune system: Humoral Immunity

Major function

=> neutralize microbes in gut lumen via IgA (termed “secretory immunity”)

Transcytosis of IgA

The gastrointestinal immune system: T cell-mediated Immunity

T cells found:

• within epithelia

• throughout lamina propria

• around and within Peyer’s patches

• in draining lymph nodes

Most intraepithelial T cells are CD8+

Most in / around Peyer’s patches or GALTs are CD4+ Th or Tfh or Treg

Regulating immune responses in the gut

Understanding remains incomplete but key factors include:

• Abundant regulatory T cells prevent inflammatory actions against commensals

• Inhibitory cytokines (especially IL-10) from Treg and other cells

• The commensal microbiome influences gut and systemic immune responses

Several inflammatory diseases of the GI tract are related to unregulated responses

to commensal organisms or to food antigens in genetically susceptible individuals

• Immune privilege - protection from immune response

• Occurs in tissues where inflammation carries high risk of organ damage / failure

  • Examples: eye, brain, testes, fetus

• Not well understood but some common contributing mechanisms

  • blood- tissue barrier: tight junctions seal endothelial cells of vasculature

  • Secretion of anti-inflammatory factors and/or signals that inactivate T cells

  • Reduced number of dendritic cells

  • Higher threshold for macrophage activation

  • Reduced vasculature and draining lymphatics

• Immunologic tolerance

• The unresponsiveness to an antigen that is induced by previous exposure to that antigen

• Encountering an antigen can lead to either

1. Lymphocyte activation and an immune response (what we have focused on so far)

2. Lymphocyte inactivation or elimination (which leads to tolerance)

• Which occurs depends on 

  • Affinity of receptor-antigen interaction

  • Conditions of antigen exposure

  • Presence or absence of co-stimulators

• Failure of self tolerance leads to immune reactions against self antigens (autoimmunity)

• Central tolerance to self antigens

• Immune lymphocytes

• generative/primary/central lymphoid organs

• Three possible mechanisms

• Not perfect

peripheral tolerance to self antigens

• Mature lymphocytes

• Peripheral tissues

• Three possible mechanisms

• Backup for central mechanisms

• Maintain unresponsiveness to self antigens expressed

  • Only in peripheral tissues

  • Only in adults

Control tolerance for immature T cells occurs in the thymus

• Occurs for both CD8+ and CD4+ cells that express high-affinity receptor against self antigens

• Most thymocytes that recognize self

  • Negative selection : deletion

• Some CD4+ thymocytes that recognize self

  • Development of regulatory T cells

• Unclear what factors determine outcome

Peripheral tolerance in T cells

• Interaction with activated DC displaying both foreign peptide antigen and B7 costimulator

• Three mechanisms provide T cell tolerance to tissue specific antigens not normally abundant in the thymus

T cell anergy favored by prolonged exposure to self antigen presented by “resting DCs”

=> DCs are not activated by self antigen so don’t express B7 or other costimulators

Regulatory T cells (Tregs) may develop centrally or peripherally

Regulatory T cells (Tregs) have multiple targets

1. T cell activation

2. Effector T cell activity

3. B cell activation

4. NK proliferation

5. IL-10 production inhibits DCs & macrophages

Defects in Treg-mediated suppression of immune response contribute to various autoimmune disease

Antibody avidity is important for B cell tolerance

• Avidity = total strength of interaction between antibody and antigen

• Depends on

1. Affinity of binding to antigen

2. Number of antigen-binding sites in antibody complex (valency)

   1. For example, IgG has 2 binding sites, IgM has 10 binding sites

3. Arrangement and number of antibody binding sites on antigen

   1. Repetitive polymer? Abundant cell surface protein?

As each of these factors increases, so does avidity of antibody or BCR

Central tolerance for immature B cells occurs in bone marrow

• B cell tolerance important for preventing antibody responses to self antigen

• If recombination is successful

  • Non-self reactive B cell

• Low- avidity self antigens => will eventually die

• Peripheral tolerance for mature B cells

• Similar possible outcomes as for T cells

• Self antigens don't trigger innate immune responses

• The immune system protects against five types of pathogens

1. Bacteria

2. Viruses

3. Protozoa

4. Fungi

5. Helminths (worms)

• Immunity to microbes: viruses

• Viruses

  • Are obligatory intracellular microorganisms

  • Use host nucleic acid & protein synthetic machinery to produce more viral particles

  • Debilitate and ultimately kill the infected cell (often through lysis)

  • May indirectly induce tissue damage by triggering host inflammatory response

• Kinetics of innate and adaptive responses to virus infection

• Innate and adaptive protection against viral infection

• “Interferon” derived from ability to interfere with viral infection

• Expression of type 1 IFNs is induced in many cell types through pattern recognition receptors

  • IFNs induce expression of enzymes that block viral replication

• Antibodies:

• Block binding of virus to its receptor

• May also opsonize particles => phagocytosis

• Effective only against extracellular particles

• Prevent cell-to-cell spread and re-infection

• Innate and adaptive eradication of established infection

• Via killing of virus-infected cells

• NKs

  • Activated in inflammatory response

  • Use pattern recognition receptors

• CD8+

  • Massive proliferation during infection

  • Most are specific for just a few viral peptides

• NKs and CTLs use same killing mechanisms

• Viral mechanisms for evading host immunity

1. Alter surface antigens recognized by antibodies or TCRs by one or both of:

   1. Antigenic drift

   2. Antigenic shift

Resulting variation creates strains no longer recognized by immune system

• Antigenic drift

• Results from point mutations that either

  • Accumulate throughout the antigen over time (minor changes add up)

  • Occur in key antigenic site (major change)

• Influenza virus is reasonably well understood example

• Also common in rhinoviruses and HIV

• Antigenic shift

• Genetic recombination of two viral strains

• Less frequent than antigenic drift

  • But more sudden and significant change

• Strains typically found in different hosts

• Simultaneous infection by two strands

  • Allows for reassortment of RNA strands

• Can create new, antigenically distinct virus

• H1N1 influenza virus responsible for 2009 pandemic

Generated by reassortment of swine, avian and

Human viruses in pigs, then passed back to humans

2. Inhibition of antigen processing and MHC class I presentation

Different viruses inhibit different steps in the pathway

Infected cells not recognized or killed by CD8+ T cells

• other mechanisms viruses use to evade the immune system

3. Coding for proteins that

• Act as ligands for Nk cell inhibitory receptors

• Function as decoy signal molecules that compete with cytokines

• Resemble immunosuppressive cytokines such as IL-10

• Bind to and inhibit pro-inflammatory cytokines

  4. Exhaustion of CTLs cells

5. Killing or Inactivation of immune cells (e.g., HIV kills CD4+ T cells)

• Immunity against bacteria, fungi, and parasites

• immunity to extracellular bacteria

• Can survive and reproduce in organ lumens, connective tissues and sometimes blood

• Pathogenic extracellular bacteria promote inflammation and produce toxins

• Innate immune response: complement, phagocytosis, inflammation

• Adaptive immune response: antibody-dependent neutralization & opsonization

• May induce cytokine-mediated, inflammatory damage to host

• Use various immunoevasion and/or immunoresistance strategies

• Immunity to intracellular bacteria

• Survive and even reproduce within phagocytes => inaccessible to Abs

• Innate immune response: phagocytes and NK cells

• Adaptive immune response: CD4+ Th cells activate phagocytes to kill microbes

• May result in macrophage-associated damage

• Resistant bacteria may escape lysosomes or inactivate microbe-killing mechanisms

• Immunity to fungi

• Fungal infections may be endemic or opportunistic

• Compromised immunity most important factor for significant fungal infection

• Fungal infections may be extracellular or intracellular

• Innate immune response: neutrophils and macrophages

• Adaptive immune response: humoral (extracellular), T cell-mediated (intracellular)

• Immunity to parasites

• Protozoa and helminths

• Infections often chronic

• Innate immune response: phagocytosis (protozoa), inflammation (helminths)

• Adaptive immune response: varied humoral and cell-mediated

• Tissue damage varies with parasite and corresponding immune response

• Multiple, effective immunoevasion and immunoresistance strategies

• Features of immunity to microbes

• The immune response is specialized to type of microbe

• Microbial survival and pathogenicity are tied to immunoevasion / immunoresistance

• Defects in immunity are important causes of susceptibility to infections

• Analysis of immune responses can provide info on status of infection

• Impact of vaccines

• Vaccination programs have led to complete or nearly complete eradication of many infectious diseases in developed countries

• Goal is to obtain “heard immunity”

The principle of vaccination

• Administer a harmless form or component of a pathogen

  • Does not cause disease

  • Elicits humoral response to protect against live, pathogenic microbe

• The best vaccines stimulate

• Development of long-lived plasma cells that produce high affinity antibodies

• Development of memory cells

• A strong innate immune response

• Minimal side effects

• Vaccination success depends on properties of target microbe

• Vaccination most effective if the target microbe does not:

  • Establish latency

  • Undergo antigenic variation (i.e., mutation, exhibit different life stages)

  • Interfere with host immune responses

  • Infect other animal hosts

• Live attenuated and killed vaccines

• Live attenuated

  • Genetic or chemical modification

  • Very rarely, the microbe may be reactivated and caused the disease

• Killed or Split

  • Heating, chemicals, or detergents

Subunit & synthetic antigen vaccines

• Used purified, synthesized or conjugated antigens

• Safer than live attenuated vaccines

• Antigen alone typically does not induce a strong response

  • Requires adjuvant to trigger innate immune response

• MRNA/DNA vaccines

• RNA or DNA encodes microbial antigens => induces our cells to express protein fragments

• Viral vector vaccines

• Virus encodes microbial antigens => induces our cells to express protein fragments

• Vaccine hesitancy

• Loss of knowledge / concern over time

• Anecdotal info perceived as significant

• Misinformation

• Mistrust in government / institutions

B cells use B cell receptors to recognize

solube antigens, microbial surface antigens, & host cell surface antigens

FDCs display protein antigen in the native, folded three-dimensional conformation (t/f)

True

T cell independent antigen responses are primary triggered in response to protein antigens (t/f)

false

All mutations in the variable coding region will imporve the affinity of antibody for its antigen (t/f)

False

the most effective somatic hypermutations are clustered in the complementarity determining regions (CDRs) of the ig heacy chain (t/f)

True

which cell also used perforins and granzymes to kill target cells

CTLs

the surface area of the human digestive tract is estimated to be equivalent to the surface area of

a Tenis court

within an epithelial sheet, adjacent cells are sealed to neighboring cells by

tight junctions

IgA is also a critical component of

Breast milk

self antigens generally activate the innate immune system (t/f)

False

IgA has ______ binding sites

4

Actions of NK cells and CTLs can _____ normal tissues

Damage

The “anti-viral state”______

Inhibits virus replication and assembly

immunoevasion or immunoresistance mechanisms are not exhibited by bacteria? (t/f)

false

which immune component would you expect to be least effective against listeria

antibodies