1 2 . MUCOSAL IMMUNITY

12. MUCOSAL IMMUNITY

MUCOSAL IMMUNE SYSTEM

Protect mucous membranes from pathogens

Prevent the development of self-damaging inflammatory immune responses against harmless environmental antigens

Transport of antigens across mucosal surface - M-cells

HUMORAL MUCOSAL RESPONSES

Massive production of IgA (under the influence of TGF-, IL-10, IL-4 in mucosal lymphoid tissues)

Transport of IgA across epithelial cells on the

mucosal surface: Transcytosis; poly-Ig-receptor; secretory component

IgA in mother milk (newborn human does not

have own antibodies; serum contains only small amounts of antibodies obtained transplacentary from the mother)

CELL MEDIATED MUCOSAL RESPONSES

Intraepithelial T-lymphocytes

T-lymphocytes in lamina propria,

Peyer’s plaques CHARACTERISTIC FEATURE:

Mucosal immunization usually results in induction of

„tolerance“ (suppression of TH1, TC; stimulation of TH2 resp.

TH3 - IgA)

Suppressive (anti-inflammatory) cytokines

13. TRANSPLANTATION

TRANSPLANTATION OF TISSUES AND ORGANS

BASIC TERMS

Donor, recipient, “graft“, rejection

Grafts genetically identical with the recipient are syngeneic

Grafts from genetically non-identical donor of the same species are allogeneic

Grafts from a different species donor are xenogeneic

SUPPRESSION OF TRANSPLANTATION

REJECTION REACTION

- Choice of genetically related donors (best - relatives)

- Immunosupression (cyclosporin A; corticosteroids;

antibodies to T-lymphocytes; irradiation)

- Ideal: induction of tolerance

- Xenotransplantation – possibly real; genetically modified

animals (baboons, pigs)

IMMUNE “PRIVILEGED“ ORGANS, TISSUES

Isolated from cells of immune system

- Anterior eye chamber

- Central nervovous system

- (Developing foetus)

Defense system of brain – microglial cells (a form of tissue macrophages)

- Active mechanisms – FasL (destruction of activated T-cells attacking a privileged tissue)

- Privileged sites vs. tissues

- Th2 x Th1. PREVENTION OF INFLAMMATION

BONE MARROW TRANSPLANTATION

- Inborn defects of hematopoiesis, immunodeficiencies

- Radiation, chemical damage

- Leukaemia, lymphoma

PROBLEM:

- Attack “graft vs. host“ (GvHD)

- Necessity of best possible genetic similarity and strong immunosuppression

- Ideal – transplantation of pure stem cells

ALLOREACTIVITY IN VITRO

- Mixed lymphocyte reaction (MLR)

Mutual stimulation of allogeneic lymphocytes (mainly

proliferation of TH, also TC): measurement of the rate of

incorporation of radioactive nucleotides

- Use – selection of potential donors

RATE OF TRANSPLANTATE REJECTION DEPENDS ON:

- Genetic difference between donor and recipient

- Type of tissue

- Activity of recipient immune system

Hyperacute rejection - during minutes to hours

Cause: antibodies (e.g. xeno-);

complement

Acute rejection– during several days

Cause: alloreactive T-lymphocytes (TH1, TC)

ALLOREACTIVITY

Genetic difference in MHC (extremely polymorphic): the cells of

the graft carry complexes MHC-peptide completely different

from the recipient cells. Many T-lymphocytes therefore recognize

graft cells as foreign (as if infected).

Genetic differences in v non-MHC: a similar situation; the

number of the “foreign“ complexes is however much lower

XENOREACTIVITY

Similar to alloreactivity; paradoxically even smaller

(partial inter-species incompatibility of adhesive and

signaling molecules).

Big problem – “natural“ xenoantibodies

MOST COMMON TRANSPLANTATIONS

- Blood transfusion

- Kidney (> 30 000 per year)

- Heart (3 000)

- Liver (5 000)

- Cornea – mostly no imunological problems;

a “privileged“ site

14. ANTI-TUMOUR IMMUNITY

ANTI-TUMOUR IMMUNITY - Tumour-specific antigens (TSA)

- Tumour-associated antigens (TAA)

TSA:

- Complexes of MHC gp with abnormal protein fragments (mutants; abnormal cleavage). (Chemically induced tumors)

- Complexes of MHC gp with fragments of oncogenic viruses (polyoma, SV40, EBV)

- Abnormal forms of glycoproteins

- Idiotopes of myelomas

TUMOUR ASSOCIATED ANTIGENS (TAA)

ONCOFETAL ANTIGENS:

-fetoprotein (AFP)

carcinoembryonal antigen (CEA)

OTHERS

melanoma antigens (MAGE-1, Melan-A)

HER-2/neu (growth factor receptor, epithelial cells) Amplification in breast cancer cells

EPCAM (epithelial cells; metastases of carcinomas)

“Differentiation antigens“ in leukaemia (CALLA - CD10)

Diagnostic, partially therapeutic importance

ANTITUMOUR IMMUNE MECHANISMS

- HYPOTHESIS OF “IMMUNOLOGICAL

SURVEILLANCE” (?)

- Inflammation; macrophages (possible stimulation by BCG

injection, Corynebacterium)

- NK (anomalous expression of MHC I)

- Antibodies, TC

MECHANISMS OF TUMOUR RESISTANCE

- Do not act as APC (absence of costimulatory surface molecules)

- Variability (loss of tumour antigen, immunoselection)

- Paradoxical stimulatory effect of antibodies (“enhancement”)

- Production of factors inactivating generally T-lymphocytes

- “Blocking factors“ (soluble forms of tumour antigens shedded from the cell surface)

(privileged tissue!)

TUMOUR IMMUNOTERAPIES BASED ON ANTIBODIES

- Monoclonal antibodies, resp. immunotoxins against tumour antigens

Problems:

Damage of normal tissue

Immunoselection of the antigen-loss variants

Accessibility (best after surgery; micrometastases);

EPCAM; HER-2/neu; myelomy

- Bispecific antibodies (against tumour antigen x against T, NK)

- Autologous bone marrow transplantation (leukaemia); “purging“ of

leukaemic cells by means of monoclonal antibodies

IMMUNOTHERAPY OF TUMOURS – CELLULAR MECHANISMS

- “Non-specific“ stimulation of inflammation (BCG; bladder

carcinomas)

- Stimulation of LAK, TIL

- Improvement of APC-function (transfection of CD80, cytokines; APC fusion)

- “Tumour vaccines“ (identification of peptides recognized by TIL on melanomas and other tumours, optimal stimulation of TC, TH1)

15. REGULATION OF IMMUNE

RESPONSES

REGULATION OF IMMUNE RESPONSE

Regulation by antigen

- Primary regulator (eliciting, finishing of response, affinity maturation; memory)

- Antigenic competition (for MHCgp) Surface density of the MHC-peptide complexes on APC decisive for TH1 x TH2. Necessity to achieve a threshold density

- Agonistic, semi-agonistic, antagonistic peptides

- Automatic mechanisms of finishing of the response (apoptosis of activated T-cells, short life time of most plasma cells)

REGULATION BY CYTOKINES AND INTERCELLULAR CONTACT

PROBABLY THE MOST IMPORTANT MECHANISM OF REGULATION!

APC – T TH1- M TH1 x TH2

FDC – B TH2 – B

(TH – TC)

Development of various leukocyte subpopulations

REGULATION BY ANTIBODIES

- Competition for antigen between BCR and soluble antibodies

- Crosslinking of BCR and FcR on B-cells by immunocomplexes – negative signal

- Idiotypic network

NEGATIVE REGULATION (SUPRESSION)

1) TH1 x TH2 (cytokines)

2) TREG, TS

Possible mechanisms:

- effect on DC (tolerogenic)

- inhibition of TH

- creation of “cytokine environment“ prefering TH2

- anergic cells competing for IL-2

3) Anergization; clonal elimination (non-professional APC)

ESSENTIAL IMPORTANCE OF

Treg

MECHANISMS OF Treg

DEVELOPMENT AND SELECTION IN THYMUS – ALSO Treg!!!

REGULATORY IMPORTANCE OF DC

NEUROENDOCRINE REGULATION - Neurotransmitters (noradrenaline…) act on leukocytes

- Direct contact of nerve endings and mastocytes

- Many endocrine hormones affect markedly leukocytes (corticosteroids, growth hormone, thyroxine, endorfins)

- Leukocytes produce hormones (endorfins, ACTH, TSH, growth hormone etc.)

- Many cytokines act on neuronal system (IL-1, IL-6, LIF, TNF)

- Clear negative effect of stress on immune system (wound healing, anti-infection immunity, alergy). Activity of fagocytes, NK. Mainly the effects of the released corticosteroids?

IMMUNOLOGIC TOLERANCE

Immunity x tolerance

Inability to react to a certain antigen

Physiologically – tolerance to autoantigens

Experimentally – tolerance induction (instead of immunity) to foreign antigens

Importance – transplantation; autoimmune diseases

FACTORS AFFECTING EXPERIMENTAL TOLEROGENIC RESPONSE

- State of the immune system (immature; weakened by irradiation, immunosupresive drugs)

- Properties of antigen (size of the molecule; monomer x polymer, aggregate)

- Antigen dose (very low and very high doses tend to be tolerogenic)

- Way of application (injection to blood, subcutaneous; adjuvans; peroral)

- Genetic effects (allelic forms of MHC)

EXPLANATION:

Mainly presentation of antigen (type of APC; density of the MHC-peptide complexes; TH1 x TH2; activation of TREG)

Immune paralysis, exhaustion (high antigen doses – saturation of binding sites, activation of all antigen-specific cells at the same time, no memory cells).

MECHANISMS OF SELF-TOLERANCE

- Elimination of autoreactive clones (negative selection)

- Anergization of immature B-cells

- Ignoring of autoantigens present in small amounts or isolated from immune system (“invisible“)

- Anergization of T-cells by contact with non-professional APC (missing costimulatory signal)

- Activity of various types of TREG, TS

BASIC DOGMA OF THE ADAPTIVE RESPONSE:

ANTIBODY RESPONSES (B, Th2) – EFFECTIVE AGAINST EXTRACELLULAR PARASITES

INFLAMMATORY RESPONSES (Th1, Tc) – EFFECTIVE AGAINST INTRACELLULAR PARASITES

MUTUAL COMPETITION Th1 vs. Th2 (REGULATION BY POSITIVE FEEDBACK)

WRONG CHOICE OF THE Th1 vs. Th2 RESPONSE

CAN BE FATAL (LEPROSY…)

Th1 x Th2 („they don’t like each other …“)

IFN vs. IL-4

16. IMMUNOPATOLOGIC

REACTIONS - ALLERGIES

ALERGIES AND OTHER HYPERSENSITIVITIES

Hypersensitivity to harmless antigens; reaction damages the organism

Type I – Alergy, atopy (IgE)

Type II – Hemolytic reactions (complement)

against alloantigens (transfusion reaction;

hemolytic disease of the newborn – Rh)

Type III – Caused by immunocomplexes

Type IV – Delayed type (DTH) and contact hypersensitivity

(TH1 reaction)

MECHANISMS OF ALERGIES (TYPE I)

- Completely analogous to defense IgE reactions against parasites: IgE; Ig-receptors on mastocytes, basophils and eosinophils

- Sensitisation; production of IgE; binding to IgE-R

- After next encounter of allergen - binding to mastocytes coated by IgE – receptor cross-linking

- Consequences:

“degranulation” – release of primary mediators (histamine, enzymes)

stimulation of secondary mediators production (prostanglandins, leukotrienes – arachidonate derivatives)

local inflammation (1. a 2. phase); swelling of mucosa, secretions, itching, pain, smooth muscle contraction; 2nd phase – also other cells participate (typical inflammation)

FACTORS AFFECTING ALLERGIES

- Nature of aergens: unclear why just certain antigens of pollen, house mites, foods, etc. elicit IgE responses

- Genetic predispositions (polymorphism of MHC genes, IL-4, IgE-R)

- Environment (exposition to the allergen; climatic conditions)

- Diet in early childhood (breast feeding?)

- Infections in early childhood (some respiratory viral infections: bronchial hyperreactivity; absence of intestinal parasites in childhood? low exposition to antigens in general? „hygienic hypothesis“)

Incidence of prototypical infectious diseases and immune disorders 1950 to 2000

N Engl J Med, Vol 347, No. 12, 09/2002

HUGE INCREASE OF ALLERGIES AS „CIVILIZATION DISEASES“

Epidemiological studies - hygienic hypothesis:

Early childhood on a (primitive) farm (mycobacteria, LPS?)Intestinal parasitesBreast feedingNon/pasteurized milk “Good” intestinal floraHepatitis ADiesel exhaust particles?

Life style in general (DDR vs. FRG, Turkish immigrants, anthroposophy..)

Allergens that get to blood circulation may cause systemic anaphylaxis (anaphylactic shock)

- insect venoms

- penicillin (alergens are penicilloylated proteins)

TREATMENT OF ALLERGIES

- Profylaxis

- Antialergics (blocking of histamine receptors; corticoids inhibit histamine synthesis; effects on membrane composition – inhibition of degranulation)

- Desensitisation, hyposensitisation (empiric efforts to enhance competing IgG responses instead of IgE). Reliable rational procedures (TH1 x TH2?) do not exist yet.

TYPE II HYPERSENSITIVITY

Transfusion reaction

(A, B, 0; “isohemagglutinins”)

Minor blood groups

Hemolytic disease of newborn:

85% individuals - RhD+

RhD- mother – immunization by RhD+ foetus (delivery)

Next pregnancy – foetus damage

Prevention: passive immunization anti-RhD before the first delivery

TYPE III HYPERSENSITIVITY

Antigen (animal sera, bacterial product, autoantigen) – elicits antibodies

Repeated contact with antigen – massive formation of immunocomplexes

Binding to Fc-receptors of phagocytes, activation of ccomplement, deposition in kidney glomerules

Important component of pathology of pathologies of some infectious and autoimmune diseases

“Serum sickness“ – damage of kidney, blood vessels, joints, skin

Experimental model: Arthus reaction

TYPE IV HYPERSENSITIVITY (DTH)

- Sensitising antigen natural – e.g. mycobacteria experimental – protein given in adjuvans

- Following injection subcutaneous, intradermal: local characteristic reaction after 24-72 hr.

- Mechanism: TH1 – activated macrophages; inflammation

- Tuberculin reaction

CONTACT HYPERSENSITIVITY

- Low-molecular substances (Ni, CrO42-,

components of cosmetics, varnish, dyes). Experimental – DNP, DNFB

- Modification of proteins, stimulation of TH1

- Skin manifestations – essentially DTH

17. IMMUNOPATOLOGIC AUTOIMMUNE REACTIONS

AUTOIMMUNITY Immune system reacts to autoantigen.

Causes:

- Molecular mimikry?

- Exposure of hidden autoantigens and cryptic eptitopes (infection, inflammation)

- Reaction against primary autoantigen intensifies the inflammation, reaction against further autoantigens (cryptic epitopes) develops. “Determinant spreading“

Linkages to MHC polymorphism

AUTOIMUNE DISEASES CAUSED BY AUTOANTIBODIES

Lupus erythematosus autoantibodies to DNA, histones ribonucleoproteins; DR3

Graves disease autoantibodies to TSH-R; mimick TSH → overprodukce of thyroxin; DR3

Myasthenia gravis autoantibodies to AChR; - block effects of ACh; DR3

Hashimoto thyroiditis autoantibodies to thyroid antigens; decreased production of thyroid hormones; DR5

Acute rheumatic fever Antibodies to streptococcal antigens cross-react with surface antigens of heart muscle. The only clear case of antigenic mimikry

Disease Autoantibodies to

Systemic lupus erythematodes Nuclear antigens; blood elements

Rheumatoiod arthritisFc-fragments of immunoglobulins ("rheumatoid factor")

Dermatopolymyositis Extractable nuclear antigens Jo-1, PM/Scl

Sjögren’s disease Extractable nuclear antigens (SS-A, SS-B)

Systemic sclerodermy Extractable nuclear antigens (Scl-70)

Anti/phospholipide syndrom Phospholipids

Some vasculitis Cytoplasmatic antigens of neutrophils

DISEASES CAUSED BY AUTOREACTIVE T-CELLS

Juvenile diabetes mellitus (IDDM)

TC, TH1 against antigens of -cells of pancreatic of Langerhans islets Primary autoantigens – glutamic acid decarboxylase, Hsp60? Elicited after local inflammation following a viral infection? DR3, DR4, (protective DR2)

Multiple sclerosis

TH1 against antigens of myelin (presented by microglia); inflammation; demyelination DR2

Rheumatoid arthritis

TH1 (secondary??) against joint antigens (collagen, Hsp); antibodies to immunoglobulins (“rheumatoid factor”); damage of kidney

DR4

Disease HLA Relative risk*

Ankylosing spondyloarthritis (Bechterev disease)

B27 87.4

Uveitis B27 10

Goodpasture syndrom DR2 15.9

Multiple sclerosis DR2 4.8

Graves-Basedow diseaseDR3 3.7

Systemic lupus erythematodes

DR3 5.8

Myasthenia gravis DR3 2.5

Pemphigus DR4 14.4

Rheumatoid arthritis DR4 4.2

Hashimotova thyreoiditis DR5 3.2

THERAPY OF AUTOIMMUNE DISEASES

Immunosupression (corticosteroids, antibodies to T-lymphocytes, cyclosporin A) Ideal – re-establishing of tolerance by

manipulation of the immune system

18.IMMUNODEFICIENCIES

DEFECTS OF NON-ADAPTIVE (INNATE) PART OF THE IMMUNE SYSTEM

Missing of some complement components (Neisseria; accumulation of immunocomplexes) serious defect – missing of C1 inhibitor

Defects of NADPH oxidase (chronic granulomatous disease)

Chédiak-Higashi syndrome: defect of lysosome-phagosome fusion

Leukocyte adhesion deficiency (LAD): defect of leukocyte integrins (leukocytosis, defective extravasation, no puss formation)

LAD-2 – defect of L-selectin

DEFECTS ANTIGEN PRESENTATION

“Bare lymphocyte syndrome”: missing MHC II (defect of a regulatory transcription factor).

CD4+ T cells are missing

Very rarely – missing MHC I (defect in peptide transport). Very few CD8+ T

DEFECTS OF B-LYMPHOCYTES AND Ig PRODUCTION

X-linked agammaglobulinemia (XLA):

almost no B cells and Ig (O.C.Bruton 1952; the first described inborn immunodeficiency)

Defect in the tyrosine-kinase Btk

X-linked hyper-IgM syndrome

Defect of CD40L (on TH): no affinity maturation, isotype switch. Only large amounts of IgM against TH independent antigens.

Selective Ig (e.g. IgA) deficits

Surprisingly mild consequences; predisposition to respiratory infections, allergies, a risk for transfusion (anti-IgA!)

DEFECTS OF T-CELLS

DiGeorge syndrome – developmental defect, anatomic abnormalities, including missing of thymus.

Nu/nu mice – an important model

Deficiency of adenindeaminase (ADA):

Missing T-cells. Attempts at gene therapy.

Severe combined X-linked immunodeficiency (SCID):

defect in common subunit c of the receptors for cytokines IL-2, -4, -7, -9, -13, -15.

Similar severe diseases in defects of several other signaling molecules.

Autosomal recessive SCID – defect of recombination of the Ig and TCR genes. Missing T and B lymphocytes.

Nu/nu mouse (athymic)

ACQUIRED IMMUNODEFICIENCIES

Temporary or chronic – consequence of some infections, malnutrition, malignancies, stress (injury trauma), irradiation, chemicals (high doses of antibiotics, immunosupressants)

AIDS: infected > 40 000 000 people

HIV – retrovirus (lentivirus); tropism for CD4+ T, monocytes/macrophages

Potentially a number of possible mechanisms of immune system destruction, e.g.:

direct and indirect destruction of TH destruction of APC including FDC affecting TH1 x TH2 loss of memory T-cells

AIDS - therapy:

virostatics – (AZT), HAART

(vaccine development)

looking for immunotherapy (cytokines; IL-16)

rare cases of natural resistance (absence of chemokine receptors)

(successful experimental immunization; HIV-2; chimps)

19. IMMUNOPROPHYLAXIS AND

IMMUNOTERAPY

IMMUNOPROPHYLAXIS TERMS:

Active, passive immunization

Vaccines: microorganisms or their components

Atenuated microorganisms Inactivated microorganisms “Subunit“ vaccines Toxoids

Prophylactic x therapeutic vaccination

Danger of infection or anaphylaxis

Adjuvants (incomplete, complete Freund adjuvans; alum)

The way of administration is important – immunogenic x tolerogenic:

- monomeric x polymeric, aggregated antigen

- parenteral x subcutaneous x peroral

PASSIVE IMMUNIZATION

Natural – maternal antibodies (transplacental, milk)

Prophylactic, therapeutic:

animal antisera to toxins (danger of anaphylaxis after repeated administration)

human IgG (hepatitis, rabies, tetanus; Ig-deficiency); intramuscular, resp. highly purified intravenous

Anti-Rh (prevention of production of own anti-Rh)

ACTIVE IMMUNIZATION

E. Jenner: Vaccinia – against pox (variola) (ERADICATION 1970!)

Atenuated live vaccines (most efficient)

measles, mumps, rubella, polio (peroral), yellow fever, tuberculosis (BCG – only in risk groups, total - 2,5 billion)

Subunit vaccines (in adjuvans)

pertussis (toxoid), hepatitis B (recombinant surface antigen), hemophilus, meningococcus (polysaccharide antigens)

Killed bacteria, inactivated viruses

pertussis, tetanus, cholera, plague, typhoid, influenza, rabies (therapeutic immunization - “outruns“ slow infection!)

NEW TYPES OF VACCINES (UNDER DEVELOPMENT)

Recombinant Vaccinia virus or other vectors (bird poxviruses, adenoviruses, genetically modified Salmonella, BCG and other bacteria)

Futuristic approach – expression in edible plants

“Genetic vacination“ directly by expression plasmid (injection into muscle, “gene gun“)

Better adjuvantsand immunostimulatory substances

Experimentally – carrier KLH, immunostimulators (cytokines, LPS); severe side effects

MARKED SUCCESS – THERAPEUTIC

MONOCLONAL ANTIBODIES:

Discovery 1976, Nobel prize 1984.

Not patented…

>15 years technical difficulties in therapeutic uses

Last cca 10 years – huge progress (humanized mAb), most dynamic field of pharmaceutic industry, sales over 20 billion USD per year…

Cca 30 approved by FDA, other cca 100 under testing

Unconjugated mAbs

Nature Biotechnology 23, 1147 - 1157 (2005)

Trastuzumab (Herceptin)

Humanized Human IgG1

HER2/neu Breast cancer 1998

Rituximab (Rituxan)

Murine-human chimeric

Human IgG1

CD20 Lymphoma 1997

Cetuximab (Erbitux)

Murine-human chimeric

Human IgG1

EGF receptor

Colorectal cancer 2004

Bevacizumab (Avastin)

Murine-human chimeric

Human IgG1

VEGF Colorectal, lung cancers

2004

Alemtuzumab (Campath-1H)

Humanized Human IgG1

CD52 Chronic lymphocytic leukemia

2001

Immunoconjugates

Ibritumomab tiuxetan (Zevalin) together with rituximab

Murine 90Y-radiolabeled murine IgG1

CD20 Lymphoma 2002

Tositumomab and 131I tositumomab (Bexxar)

Murine 131I-radioabeled murine IgG2a

CD20 Lymphoma 2003

Gemtuzumab (Myelotarg)

Human (drug derived from streptomycete)

Human IgG4 conjugated to calicheamicin

CD33 Acute myelogenous leukemia

2000

HOPEFULLY IN FUTURE:

- BETTER VACCINES (HIV, WEAK AND TUMOR ANTIGENS?)

- BETTER IMMUNOSUPPRESSION (AUTOIMMUNE DISEASES, ALLERGIES, TRANSPLANTATION)

- EFFECTIVE IMMUNOTHERAPIES OF TUMOURS (A WEAK SPOT OF IMMUNITY…)