GMS 6006 - Fundamentals of Immunology & Microbiology Spring 2006

February 15, 2006February 15, 2006

Introduction to ImmunityIntroduction to ImmunityAntibody Function & Antibody Function & DiversityDiversity

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.

All textbook figures herein identified by Fig. number

© Garland Science

“Immunobiology - The Immune System in Health & Disease”, Janeway et al., 6th edition, 2005

Monday, October 29, 2001

West Nile virus spreads throughout Florida, here to stay

By MIRANDA LEITSINGERAssociated Press Writer

MIAMI (AP) - Chevy could hardly stand on his own. He didn'teat, he wasn't responding to his owners and he was dragging hishind quarters. The 9-year-old horse, a Palomino Appaloosa thathas won medals for sliding stops and spins in equestriancompetitions, could barely walk.

''It was so hard to watch ... to see one reduced to this,'' saidowner Sheila Anderson, of Coral Springs. ''I just cried and cried.''

Chevy, a golden-colored horse with a white mane and tail, wasthe first confirmed case in Broward County of the West Nilevirus. As of last week, 51 of Florida's 67 counties from thePanhandle to the Keys were under a medical alert for theHealth 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 MITCHELLThe New York Times

WASHINGTON - Concern about biological terrorism heightenedWednesday as preliminary tests showed that more than 30workers on Capitol Hill were exposed to anthrax spores from thecontaminated letter sent to the office of the Senate majorityleader. Officials also disclosed that early testing has shown thatthe anthrax samples found in media offices in New York andFlorida were of the same strain.

While there was no evidence that anyone has yet been infectedwith what Attorney General John Ashcroft called a ''virulent,strong, very serious'' form of powdery anthrax that was found ina letter Monday in the office of Sen. Tom Daschle, D-S.D., wordthat more than half the workers in his suite were exposedprompted the closure of all six House and Senate office buildingsfor further screening …...

What disease does this man have?

December 29. 2002

UF scientists: Hold off on smallpox vaccine

By DIANE CHUNSun 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 CHUNSun 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”?

Edward Jenner’s experiment (1796)

material from cowpoxlesion of milkmaid

village boy susceptibleto smallpox

inoculate withcowpox

wait 2 monthsinoculate with

smallpox

child recoversfrom cowpox

child does notdevelop 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

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 erythrocytes (red blood cells) and leukocytes (white blood cells):myeloid cells macrophages monocyteslymphocytes B cells T cells

Immunity

Concept Map of “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′])

Comparison:

Innate Immunity

Adaptive Immunity

“Line of defense”

“first”

“second”

Specificity

general

exquisite

Improved response upon re-exposure

No

Yes

Immunological memory

No

Yes

Cells

Phagocytes NK cells

Lymphocytes (B, T)

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 IMMUNITYInnate 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

Cellsphagocytic monocytes/macrophages dendritic cells (adaptive immunity)

neutrophils (polymorphonuclear leukocytes, PMN)

Fig. 1-4

INNATE IMMUNITYInnate immune response: SUBSURFACE

PROTECTIONCellsProteins complement (C) anti-microbial peptides;

defensins (skin, gut); cryptidins (intestine)

Innate immune responses often trigger INFLAMMATION

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?

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

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

Effector1. Plasma cells produce large amounts of antibody

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

ADAPTIVE IMMUNITY

ImmunizationPassive

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

produces temporary immunity

Activeintentional exposure to Ag to elicit and immune response

produces long-lasting immunity

ADAPTIVE 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

Lymphocytes - lymphoid organsCentral 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

B cell-richB cell-rich T cell-richT 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 (TH): CD4 co-receptor

Fig. 1-5

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

Fig. 1-13

B cells respond to native AgT 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

Lymphocyte ActivationLymphocytes 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 cellsTH, CTL, memory T cells plasma & memory B cells

Questions?Fig. 1-21

Immunity

Concept Map of “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’])

Immunity

Concept Map of “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’])

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

von Behring and Kitasato experiment (1892)

isolate serum from blood

healthy rabbitdiphtheriatoxoid

vaccinated rabbit diphtheria-resistantrabbit

rabbit lives

rabbit dies

x

healthy rabbit

healthy rabbit

virulentdiphtheria

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 STRUCTUREAn antibody molecule is composed of two identical Ig heavy chains (H) and two identical light chainslight 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 chainsfound in the urine of patients with multiple myeloma

Serum protein electrophoresis

multiple myeloma

normal serum

Clinical terms:hypergammaglobulinemiahypogammaglobulinemiaagammaglobulinemia 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 () – IgGalpha () – IgAdelta () – IgDepsilon () – IgE

But what was the Ab specificity of the myeloma proteins?

Monoclonal Antibodies & HybridomasKohler & 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

It is estimated that 108-109 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 segmentscombinatorial diversity (different combinations of gene segments)junctional diversity (additional diversity generated during sloppy recombination)

The problem of antigen receptor diversity

Questions?Fig. 4-2

Classes

Subclasses

Ig isotypes

Fig. 4-18

IgMcomplement fixation (opsonization)

intravascular except during inflammation

primary Ab response

Fig. 4-23,9-27

IgDtrace amount in serum

on surface of mature, naïve B cells

Fig. 7-6

IgG4 subclasses

intravascular & interstitial space

highest concentration in blood

appears late in primary response, predominates in secondary responses

fixes complement

crosses placenta

Fig. 3-4

Fig. 2-18

IgAmonomer in serum

dimer in secretions

mucosal immunity

Fig. 9-20

IgEtrace amount in serum

bound to mast cells via FcR

type I hypersensitivity, allergy

parasite immunity

Fig. 9-33,9-35 Schistosome larva & mast cells

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

Immunity

Concept Map of “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’])

Lymphocyte development &

toleranceT cell recognition of Ag

Immunity

Concept Map of “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’])

Humoral immune response

T cell-mediated immunity

Eric Sobel, M.D., Ph.D.