AMI - Lecture 7 - Autoantibodies in infection and inflammation

explain the following terms; Fc, Fab, hinge, light + heavy chain

• Fc region → immune cells bind here, constant domain

• Fab region → where ag binds, consists of constant + variable region

• hinge → connects Fc + Fab

• heavy chain → determines isotype

What are the different types of ab, explain the different IgG

• IgG is the most prevalent in the serum

  • IgG1, most common

  • IgG2, stiffer hinge region, activates complement and is proteolytic

  • IgG3, long hinge region = flexible, activates complement + proteolytic (better than IgG2).

  • IgG4, binds 2 different ag, no immune response but can stop interactions, anti inflammatory ab, functionally monovalent

• IgD unknown

• IgE role in allergic reaction, asthma, parasites

• IgM is the first Ab produced after infection, pentameric, activation complement

• IgA can be monomeric in the blood, but can also be secretory

explain more about the ab IgA

• produced most of all ab per day

• key antibody involved in mucosal immunity

• IgA is transported to mucosal site (transported across epithelium), then released as secretory IgA

• functions;

  • prevent pathogen entry by binding pathogens + neutralizing

  • IgA coated ag taken up by M cells and delivered to ag presenting cells in Peyer’s patches

  • IgA can be anti inflammatory → promote anti-inflammatory cytokines, prevent overactivation of immune responses

the 3 functions of antibodies

• neutralizes pathogens at mucosal site

• activates complement → MAC

• opsonizes pathogens → coats them with ab so they are more recognizable, will be phagocytosed

activating versus inhibitory fc receptors

• fc receptors are found on the surface of immune cells → bind to fc region of antibodies

• fc receptors can be activating or inhibitory

• activating → lead to phagocytosis, cytokine release

• inhibitory → suppress immune activation to prevent excessive inflammation

• the balance will determine the outcome of the cell

What are autoantibodies

• antibodies produced by the immune system that mistakenly target the body's own tissues, cells, or proteins

• common diseases;

  • Systemic lupus erythematosus (SLE): Anti-dsDNA antibodies.

  • Rheumatoid arthritis: Rheumatoid factor (RF) and anti-CCP antibodies.

  • Type 1 diabetes: Anti-GAD antibodies.

systemic lupus erythematous

• autoimmune disease

• reacts to DNA

• symptom; butterfly rash on face

impaired clearance of apoptotic cells in SLE

• impaired clearance of apoptotic cells

• accumulation secondary necrotic cells (SNEC)

• release of necrotic debris

• autoag activate immune response (BC)

• break of BC tolerance, continuous exposure of autoab in germinal centres leads to production of autoab

NETosis and immune complex formation

• NETosis → process where neutrophils release chromatin and antimicrobial proteins to trap pathogens. Normally DNAse clears NETs to prevent excessive accumulation

• in SLE → defects in DNAse → uncleared NETs → nuclear material exposed to environment such as DNA, histones → these act as autoantigens, trigger immune response → autoantibodies bind to autoantigens, form immune complexes → immune complexes + uncleared NETs result in activation DC and production of interferon alpha → resulting in organ damage

autoab in rheumatoid arthiritis

• chronic auto immune disease

• inflammation of joints → infiltration of neutrophils in synovial fluid → destruction of cartilage and bone

• RF-IgM and anti-CCP-IgG can be used to diagnose

  • RF-IgM → rheumatoid factor → is an antibody against the Fc tail of IgG. (ab against ab).

    • when binding happens immune complexes formed → taken up by neutrophils → induce NETs → tissue damage and inflammation

  • anti-CCP-IgG → anti-cyclic citrullinated peptide (modified forms of self proteins in RA).

increased IgA in RA

• increased IgA autoantibodies in RA correlates with worse disease outcomes

• neutrophils in RA overactivated → NET release mediated with IgA

• in RA overactivated osteoclasts → these break down bone. when IgA activated monocytes were cultured on bone matrix resulted in large hole in bone.

Linear IgA bullous disease (LABD)

• immune system produces IgA autoab against collagen 17 (protein that anchors epidermis to dermis)

• neutrophils recruited to this site → tissue inflammation

• challenges in studying→ mice do have IgA but no fc receptor

• in LABD research → mouse model with knock in human IgA directed against mouse collagen 17

• fluorescent neutrophils injected in ear (very thin, visible under microscope) showed high influx of neutrophil into tissue.

explain monoclonal ab production

• mouse injected with ag to stimulate B cell response (ab against injected ag)

• myeloma cells (cancerous B cells that divide indefinitely) are fused with B cells from mouse (hybridoma)

• these fused cells (hybridomas) grow in drug containing medium only the hybrid cells live

• hybridomas are selected that produce ag specific ab

• this hybridoma is cloned

These ab used in research

hoe are mice derived ab ultimately changed to be used in human therapeutics (3 techniques)

• chimeric ab → 75% human 25% mouse, variabel region (Fab, where ag binds) is from mice, rest of ab from human, very successful in clinic

• production human antibodies (less anti- animal reaction);

  • humanization → graft the complimentary determining region (CDR), these are key ag binding site from mouse, onto human framework

  • HuMab mice → immunoglobulin genes knocked out in mice and replaced with human Ig genes. mice start producing fully human ab

how do ab work in therapeutics (autoimmune disease)

• bind/block cytokines → no signal transduction

  • rituximab → anti CD20 (B surface marker). it depletes B cells, is used to treat B cell malignancies and used in autoimmune disease.

  • Bevacizumab → anti VEGF (vascular endothelial growth factor). mab used in cancer therapy.

  • checkpoint inhibitors → anti PD1, removes the break from immune system, resulting in effective T cells. can cause side effects that immune system starts attacking healthy tissue

  • FcR blocking → used to reduce tissue damage. Prevents immune cells from attacking healthy tissue

explain the 6 ways mab work

• enhancing cancer treatment → increased sensitivity to radiation and chemotherapy

  • herceptin for breast cancer

• blocking growth factor receptors → antagonistic ab, stops tumors from receiving growth signals.

  • EGFR → tumor uses EGF to grow. can block this receptor, tumor can not grow. but sometimes patients have mutation in EGFR then this treatment does not work.

• complement activation → requires at least 2 IgG molecules

  • MAC

• fc receptor mediated effects → several effects;

  • NK cells → also bind to tumor, attract NK cells. KIR receptors on NK cells regulate inhibitory or activating signals, mab increase the activating signals

  • MQs result in phagocytosis, Kupffer cell (specialized MQ in liver) effectively clear circulating tumor cells. (don’t always fully kill tumor leading to gradual death)

• neutrophils → immune repressive in cancer patients.

  • cetuximab (IgG ab) → not very effective

  • cetuximab (IgA ab) → works very well

  • G-CSF → promote pro inflammatory environment, increase numbers at tumor site, neutrophils secrete chemotactic stimuli and attract other immune cells

• trogocytosis → neutrophils mechanically pull of parts of tumor

explain how mab therapy can be improved

• changing isotype (IgG, IgG4)

• improving fc receptor binding

• using ab as drug carriers (ab drug conjugates (ADC))

• IMPORTANT → CD8 T cells do not express fcR → no direct affect of mab therapy that rely on fcR mediated mechanisms

explain BiTEs and DARTs

• BITEs + DARTs → guide T cells directly to tumor cells by binding both CD3 and tumor associated ag

• thus bypass the need for fcR

• BITEs → target CD3 on T cells and CD19 on B cells

• DARTs → works the same but with great specificty and bind multiple targets

both have short half lives → continuous infusion needed

chimeric antigen receptor T cells (CAR T)

• genetically engineered T cells designed to recognize and destroy specific cancer cells

• based on the antigen recognition domain