GMS 6006 - Fundamentals of Immunology & Microbiology Spring 2006 February 15, 2006 Introduction to Immunity Antibody Function & Diversity Wayne T. McCormack, Ph.D. R1-102, 392-7413, mccormac@pathology.ufl.edu Introduction: The goal of these two lectures is to provide an overview of the immune system and how it operates.

Antibody Gene Rearrangement Why in the world are we considering antibodies now? I thought this section was about molecular biology . . . For further reading: “Immunobiology - The Immune System in Health & Disease”, Janeway et al ., 6th edition, 2005. For the basics: GMS 6006 – Fundamentals of Immunology & Microbiology For the whole story: GMS 6140 “Principles of Immunology” = GMS 6031/6032/6033 immunology advanced modules September 30, 2005 Wayne T. McCormack, Ph.D. PLEASE REVIEW!

“ Immunobiology - The Immune System in Health & Disease”, Janeway et al ., 6th edition, 2005 All textbook figures herein identified by Fig. number © Garland Science

Monday, October 29, 2001 West Nile virus spreads throughout Florida, here to stay By MIRANDA LEITSINGER Associated Press Writer MIAMI (AP) - Chevy could hardly stand on his own. He didn't eat, he wasn't responding to his owners and he was dragging his hind quarters. The 9-year-old horse, a Palomino Appaloosa that has won medals for sliding stops and spins in equestrian competitions, could barely walk. ''It was so hard to watch ... to see one reduced to this,'' said owner Sheila Anderson, of Coral Springs. ''I just cried and cried.'' Chevy, a golden-colored horse with a white mane and tail, was the first confirmed case in Broward County of the West Nile virus. As of last week, 51 of Florida's 67 counties from the Panhandle to the Keys were under a medical alert for the Health experts expected the number to keep increasing …...

Thursday, October 18, 2001 More than 30 on Capitol Hill test positive for anthrax By TODD S. PURDUM and ALISON MITCHELL The New York Times WASHINGTON - Concern about biological terrorism heightened Wednesday as preliminary tests showed that more than 30 workers on Capitol Hill were exposed to anthrax spores from the contaminated letter sent to the office of the Senate majority leader. Officials also disclosed that early testing has shown that the anthrax samples found in media offices in New York and Florida were of the same strain. While there was no evidence that anyone has yet been infected with what Attorney General John Ashcroft called a ''virulent, strong, very serious'' form of powdery anthrax that was found in a letter Monday in the office of Sen. Tom Daschle, D-S.D., word that more than half the workers in his suite were exposed prompted the closure of all six House and Senate office buildings for further screening …...

What disease does this man have?

December 29. 2002 UF scientists: Hold off on smallpox vaccine By DIANE CHUN Sun Staff Writer The UF researchers say mass vaccinations present more risks than benefits. Beginning in January, county and state public health officials will begin to carry out the Bush administration's plan to vaccinate 10.5 million medical personnel and emergency responders against smallpox. Should the federal government decide to proceed with the final stage of those plans, scheduled for sometime in 2004, as many as 10 million Floridians might be eligible for voluntary vaccination against smallpox. The question will then become: Should they get vaccinated? ……

February 28. 2003 3 have reaction to smallpox shot By DIANE CHUN Sun Staff Writer Officials with the state Department of Health reported that three additional health care workers have apparently had adverse reactions to the smallpox vaccine they received as part of Operation Vaccination. Thursday's announcement came slightly more than a week after the focus of the state's effort shifted from health department workers to groups drawn from hospitals throughout Florida. A total of 1,186 people had received the vaccine as of Monday. One earlier reaction had been reported. ……

Are there any other medical conditions in which something “foreign” resides in your body? Is an immune response always beneficial? c.2000 G.Caviness, BioGrafix

What does “immunity” mean to you? What are the key features of an “immune response”? How old is the concept of “immunity”?

What does “immunity” mean to you?

What are the key features of an “immune response”?

How old is the concept of “immunity”?

Edward Jenner’s experiment (1796) material from cowpox lesion of milkmaid village boy susceptible to smallpox inoculate with cowpox wait 2 months inoculate with smallpox child recovers from cowpox child does not develop smallpox

GOALS OF THE IMMUNE SYSTEM Distinguish between “self” and “non-self” Separate/exclude self from non-self Remove non-self if self is penetrated

Distinguish between “self” and “non-self”

Separate/exclude self from non-self

Remove non-self if self is penetrated

Spaces of the Body That Need to Be Monitored and Defended Major classes of pathogens: bacteria viruses protozoa, fungi, parasites Fig. 10-4

Cells of the Immune System Hematopoietic stem cells in bone marrow give rise to e rythrocytes (red blood cells) and leukocytes (white blood cells): myeloid cells macrophages monocytes lymphocytes B cells T cells

Hematopoietic stem cells in bone marrow give rise to e rythrocytes (red blood cells) and leukocytes (white blood cells): myeloid cells macrophages monocytes lymphocytes B cells T cells

Concept Map of “Immunity” Immunity Innate (phagocytes) Adaptive (lymphocytes) Surface (skin, mucous membrane) Subsurface Humoral (Ab) Cellular (CMI) Systemic (IgM, IgG) Mucosal (IgA) Cells (PMN; Macrophages; NK) Proteins (Complement [C ′ ])

What happened that time you cut your finger? What did it feel like & look like? Did it get infected? What was that yucky stuff that leaked out? Did you have to get a tetanus shot? The next time you got a splinter, did the same thing happen?

What happened that time you cut your finger? What did it feel like & look like?

Did it get infected?

What was that yucky stuff that leaked out?

Did you have to get a tetanus shot?

The next time you got a splinter, did the same thing happen?

INNATE IMMUNITY Innate immune response: nonspecific, no memory, same intensity with each response SURFACE PROTECTION anatomic barriers (skin, mucous membranes) chemical & enzymatic barriers (stomach acidity, lysozyme in tears) mechanical: flow & motility (cilia) commensal organisms

Innate immune response:

nonspecific, no memory, same intensity with each response

SURFACE PROTECTION

anatomic barriers (skin, mucous membranes)

chemical & enzymatic barriers (stomach acidity, lysozyme in tears)

mechanical: flow & motility (cilia)

commensal organisms

INNATE IMMUNITY Innate immune response: SUBSURFACE PROTECTION Cells phagocytic monocytes/macrophages dendritic cells (adaptive immunity) neutrophils (polymorphonuclear leukocytes, PMN) Fig. 1-4

Innate immune response:

SUBSURFACE PROTECTION

Cells

phagocytic

monocytes/macrophages

dendritic cells (adaptive immunity)

neutrophils (polymorphonuclear leukocytes, PMN)

INNATE IMMUNITY Innate immune response: SUBSURFACE PROTECTION Cells Proteins complement (C  ) anti-microbial peptides; defensins (skin, gut); cryptidins (intestine) Innate immune responses often trigger INFLAMMATION

Innate immune response:

SUBSURFACE PROTECTION

Cells

Proteins

complement (C  )

anti-microbial peptides; defensins (skin, gut); cryptidins (intestine)

Many bacteria activate phagocytosis and trigger inflammation

Principles of Innate and Adaptive Immunity The innate immune system provides first line of defense against many microorganisms, and is critical for the control of some bacterial pathogens. It also plays a role in initiating the adaptive response, and controls the infection until the adaptive response takes effect in 4-7 days. The adaptive immune response recognizes pathogens not seen by the innate response, and provides additional effector functions, as well as an improved secondary response upon re-exposure to the same pathogen. Some phagocytic cells ( i.e. macrophages, dendritic cells) act as “antigen-presenting cells” to help promote the specific immune response. Questions?

The innate immune system provides first line of defense against many microorganisms, and is critical for the control of some bacterial pathogens. It also plays a role in initiating the adaptive response, and controls the infection until the adaptive response takes effect in 4-7 days.

The adaptive immune response recognizes pathogens not seen by the innate response, and provides additional effector functions, as well as an improved secondary response upon re-exposure to the same pathogen.

Some phagocytic cells ( i.e. macrophages, dendritic cells) act as “antigen-presenting cells” to help promote the specific immune response.

also called the specific or acquired immune response occurs throughout lifetime as an adaptive response to infection often confers life-long immunity (immunological memory) enhanced secondary (anamnestic) responses mediated by lymphocytes (B and T cells) ADAPTIVE IMMUNITY

also called the specific or acquired immune response

occurs throughout lifetime as an adaptive response to infection

often confers life-long immunity (immunological memory)

enhanced secondary (anamnestic) responses

mediated by lymphocytes (B and T cells)

The immune response can be divided into 3 phases: Recognition 1. discrimination between various pathogens (bacteria, viruses, fungi, parasites) 2. discrimination between foreign and self antigen Activation 1. lymphocyte activation requires 2 signals: antigen recognition & co-stimulation 2. clonal proliferation and differentiation into effector cells Effector 1. Plasma cells produce large amounts of antibody 2. Cytotoxic T cells (CTL) kill virus-infected cells ADAPTIVE IMMUNITY

The immune response can be divided into 3 phases:

Recognition

1. discrimination between various pathogens

(bacteria, viruses, fungi, parasites)

2. discrimination between foreign and self antigen

Activation

1. lymphocyte activation requires 2 signals:

antigen recognition & co-stimulation

2. clonal proliferation and differentiation into effector cells

Effector

1. Plasma cells produce large amounts of antibody

2. Cytotoxic T cells (CTL) kill virus-infected cells

Immunization Passive injection/infusion of Ab (“pre-packaged” immunity) produces temporary immunity Active intentional exposure to Ag to elicit and immune response produces long-lasting immunity ADAPTIVE IMMUNITY

Immunization

Passive

injection/infusion of Ab (“pre-packaged” immunity)

produces temporary immunity

Active

intentional exposure to Ag to elicit and immune response

produces long-lasting immunity

Antigens & Immunogens Immunogen : substance that elicits (stimulates) an immune response Antigen : substance that can be recognized by the immune system Unfortunately the terms are often used interchangeably Immunogenicity: protein > CHO > NA & lipids >40 kD +++ ~10 kD + <2 kD +/- very small nonimmunogenic molecules (haptens) can be made immunogenic by coupling to a larger molecule (carrier), e.g. DNP-BSA

Immunogen : substance that elicits (stimulates) an immune response

Antigen : substance that can be recognized by the immune system

Unfortunately the terms are often used interchangeably

Immunogenicity: protein > CHO > NA & lipids >40 kD +++ ~10 kD + <2 kD +/-

very small nonimmunogenic molecules (haptens) can be made immunogenic by coupling to a larger molecule (carrier), e.g. DNP-BSA

Lymphocytes - lymphoid organs Central lymphoid organs sites of lymphocyte development bone marrow and thymus Peripheral lymphoid organs where adaptive immune responses occur spleen, lymph nodes, Peyer’s patches organized to trap Ag and facilitate interaction of Ag-presenting cells and T cells, and of T and B cells Fig. 1-7

Central lymphoid organs

sites of lymphocyte development

bone marrow and thymus

Peripheral lymphoid organs

where adaptive immune responses occur

spleen, lymph nodes, Peyer’s patches

organized to trap Ag and facilitate interaction of Ag-presenting cells and T cells, and of T and B cells

B cell-rich T cell-rich Lymph node Fig. 1-8

Spleen Peyer’s patches Fig. 1-9,1-10

Lymphocytes B cells : (humoral immunity) surface receptor for antigen is membrane-bound immunoglobulin (Ig), or antibody differentiate into plasma cells , which secrete large amounts of antibody T cells : (cell-mediated immunity) surface receptor for antigen is called the T cell receptor (TCR) two major classes: cytotoxic T lymphocytes (CTL): CD8 co-receptor helper T cells (T H ): CD4 co-receptor Fig. 1-5

B cells : (humoral immunity)

surface receptor for antigen is membrane-bound immunoglobulin (Ig), or antibody

differentiate into plasma cells , which secrete large amounts of antibody

T cells : (cell-mediated immunity)

surface receptor for antigen is called the T cell receptor (TCR)

two major classes:

cytotoxic T lymphocytes (CTL): CD8 co-receptor

helper T cells (T H ): CD4 co-receptor

Lymphocytes are activated by antigen & Ag-specific cells proliferate in a clonal fashion Fig. 1-13 B cells respond to native Ag T cells respond to “processed & presented” Ag

Burnet’s Clonal Selection Hypothesis Lymphocytes are activated by Ag & Ag-specific cells proliferate in a clonal fashion each lymphocyte bears a unique receptor self-reactive lymphocytes are removed from the repertoire specific interaction with Ag  activation differentiated effector cells express same receptor

Lymphocytes are activated by Ag & Ag-specific cells proliferate in a clonal fashion

each lymphocyte bears a unique receptor

self-reactive lymphocytes are removed from the repertoire

specific interaction with Ag  activation

differentiated effector cells express same receptor

Lymphocyte Activation Lymphocytes proliferate and differentiate in response to (1) antigen and (2) signals from other cells in peripheral lymphoid tissues T cells : B cells : (1) Ag via TCR (1) Ag via surface Ig (2) costimulatory receptors on (2) costimulatory receptors on professional APC helper T cells T H , CTL, memory T cells  plasma & memory B cells Questions? Fig. 1-21 Friday

Lymphocytes proliferate and differentiate in response to

(1) antigen and (2) signals from other cells in peripheral lymphoid tissues

T cells : B cells :

(1) Ag via TCR (1) Ag via surface Ig

(2) costimulatory receptors on (2) costimulatory receptors on

professional APC helper T cells

T H , CTL, memory T cells  plasma & memory B cells

Concept Map of “Immunity” Immunity Innate (phagocytes) Adaptive (lymphocytes) Surface (skin, mucous membrane) Subsurface Humoral (Ab) Cellular (CMI) Systemic (IgM, IgG) Mucosal (IgA) Cells (PMN; Macrophages; NK) Proteins (Complement [C’])

Concept Map of “Immunity” Immunity second half Innate (phagocytes) Adaptive (lymphocytes) Surface (skin, mucous membrane) Subsurface Humoral (Ab) Cellular (CMI) Systemic (IgM, IgG) Mucosal (IgA) Cells (PMN; Macrophages; NK) Proteins (Complement [C’])

Immunity to disease results from host production of protective substances Pasteur had hypothesized that immunity resulted from depletion of key nutrients, the accepted mechanism for immunity Emil von Behring demonstrated that diphtheria microbes produced a toxic, lethal substance detectable in culture supernatants This “toxin” could be inactivated by heating, and the inactivated “toxoid” was protective by vaccination Hypothesis: Transfer of serum from protected animals will protect passively immunized normal animals Early History

Immunity to disease results from host production of protective substances

Pasteur had hypothesized that immunity resulted from depletion of key nutrients, the accepted mechanism for immunity

Emil von Behring demonstrated that diphtheria microbes produced a toxic, lethal substance detectable in culture supernatants

This “toxin” could be inactivated by heating, and the inactivated “toxoid” was protective by vaccination

Hypothesis: Transfer of serum from protected animals will protect passively immunized normal animals

von Behring and Kitasato experiment (1892) isolate serum from blood healthy rabbit diphtheria toxoid vaccinated rabbit diphtheria-resistant rabbit rabbit lives rabbit dies x healthy rabbit healthy rabbit virulent diphtheria

Metchnikoff’s “anti-korperen” Microorganisms are engulfed & digested by phagocytic cells (macrophages) Substances in the blood, called or anti-korperen ( antibodies ) enhanced phagocytosis

Antibody Structure: The Puzzle solved (mostly) by Porter, Edelman, Nisonoff in 1959-1964 Experimental approach : Break Ab molecules into smaller fragments Reducing agent (break S-S bonds) 2 heavy chains (55 kD) + 2 light chains (25 kD) Fab: fragment antigen-binding Fc: fragment crystalizable

ANTIBODY STRUCTURE An antibody molecule is composed of two identical Ig heavy chains (H) and two identical light chains (L), each with a variable region (V) & constant region (C). Amino acid sequences were determined from myeloma proteins. Fig. 1-17,1-16

Bence-Jones proteins (myeloma proteins) = Ig light chains found in the urine of patients with multiple myeloma Serum protein electrophoresis multiple myeloma normal serum Clinical terms: hypergammaglobulinemia hypogammaglobulinemia agammaglobulinemia monoclonal gammopathy

Amino acid sequences of Bence-Jones proteins XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX -----------XX-------------XX----------XXXX--------------------------------------------------- ------------X------------XX----------XX-XXX-------------------------------------------------- ------XXX-XX-------------X-X-----------XXXX-------------------------------------------------- XXXXX ---XX-----X ^ XX-----X-----XXXX---------XXXXX-------------------------------------------------- XXX --XXXX-X--- ^ ------XX----X-XX----XXX---XX-XX-------------------------------------------------- XX------XXXX—X----XX----XX-X----XXX---XXXX-------------------------------------------------- VARIABLE REGION CONSTANT REGION ***** ***** ****** *hypervariable regions *complementarity-determining regions Light chains: kappa (  ) and lambda (  ) Heavy chains: mu (  ) – IgM gamma (  ) – IgG alpha (  ) – IgA delta (  ) – IgD epsilon (  ) – IgE But what was the Ab specificity of the myeloma proteins?

Amino acid sequences of Bence-Jones proteins

XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

-----------XX-------------XX----------XXXX---------------------------------------------------

------------X------------XX----------XX-XXX--------------------------------------------------

------XXX-XX-------------X-X-----------XXXX--------------------------------------------------

XXXXX

---XX-----X ^ XX-----X-----XXXX---------XXXXX--------------------------------------------------

XXX

--XXXX-X--- ^ ------XX----X-XX----XXX---XX-XX--------------------------------------------------

XX------XXXX—X----XX----XX-X----XXX---XXXX--------------------------------------------------

VARIABLE REGION CONSTANT REGION

***** ***** ******

*hypervariable regions

*complementarity-determining regions

Light chains: kappa (  ) and lambda (  )

Heavy chains: mu (  ) – IgM

gamma (  ) – IgG

alpha (  ) – IgA

delta (  ) – IgD

epsilon (  ) – IgE

But what was the Ab specificity of the myeloma proteins?

Monoclonal Antibodies & Hybridomas Kohler & Milstein (1974) Fig. A-14

Fig. 3-5,3-7

Fig. 3-8,3-9

Fig. 3-10

The problem of antigen receptor diversity Each lymphocyte has a unique receptor Ig for B cells, TCR for T cells How are antigen receptors with a virtually infinite range of specificities encoded by a finite number of genes? Germline vs . somatic diversity Tonegawa discovered in 1976 that antibody genes are encoded as gene segments , and must undergo gene rearrangement during lymphocyte development. During the next ~10 years it was discovered that the V regions of all germline Ig and TCR genes are encoded by families of gene segments. Fig. 1-18

The problem of antigen receptor diversity It is estimated that 10 8 -10 9 different receptor specificities may be present in an individual at any one time. Receptor diversity is generated by: germline diversity of the V region gene segments combinatorial diversity (different combinations of gene segments) junctional diversity (additional diversity generated during sloppy recombination) Questions? Fig. 4-2

It is estimated that 10 8 -10 9 different receptor specificities may be present in an individual at any one time.

Receptor diversity is generated by:

germline diversity of the V region gene segments

combinatorial diversity (different combinations of gene segments)

junctional diversity (additional diversity generated during sloppy recombination)

Classes Subclasses Ig isotypes Fig. 4-18

IgM complement fixation (opsonization) intravascular except during inflammation primary Ab response Fig. 4-23,9-27

IgM

complement fixation (opsonization)

intravascular except during inflammation

primary Ab response

IgD trace amount in serum on surface of mature, naïve B cells Fig. 7-6

IgD

trace amount in serum

on surface of mature, naïve B cells

IgG 4 subclasses intravascular & interstitial space highest concentration in blood appears late in primary response, predominates in secondary responses fixes complement crosses placenta Fig. 3-4

IgG

4 subclasses

intravascular & interstitial space

highest concentration in blood

appears late in primary response, predominates in secondary responses

fixes complement

crosses placenta

Fig. 2-18

IgA monomer in serum dimer in secretions mucosal immunity Fig. 9-20

IgA

monomer in serum

dimer in secretions

mucosal immunity

IgE trace amount in serum bound to mast cells via Fc  R type I hypersensitivity, allergy parasite immunity Fig. 9-33,9-35 Schistosome larva & mast cells

IgE

trace amount in serum

bound to mast cells via Fc  R

type I hypersensitivity, allergy

parasite immunity

DISTRIBUTION OF Ig ISOTYPES Fig. 9-22

Fig. 4-17

FUNCTIONS OF Ig ISOTYPES Fig. 9-1

Fig. 9-24,9-25

Fig. 9-31,9-32

Course of Typical Acute Infection Fig. 10-1

Kinetics of Antibody Response Fig. 1-20

The affinity as well as the amount of antibody increases with repeated immunization Questions? Fig. 10-31

Concept Map of “Immunity” Immunity Lymphocyte development & tolerance T cell recognition of Ag Friday Innate (phagocytes) Adaptive (lymphocytes) Surface (skin, mucous membrane) Subsurface Humoral (Ab) Cellular (CMI) Systemic (IgM, IgG) Mucosal (IgA) Cells (PMN; Macrophages; NK) Proteins (Complement [C’])

Concept Map of “Immunity” Immunity Humoral immune response T cell-mediated immunity Monday Eric Sobel, M.D., Ph.D. Innate (phagocytes) Adaptive (lymphocytes) Surface (skin, mucous membrane) Subsurface Humoral (Ab) Cellular (CMI) Systemic (IgM, IgG) Mucosal (IgA) Cells (PMN; Macrophages; NK) Proteins (Complement [C’])