INTRODUCTION

-We live in a world surrounded by organisms that can cause diseases.

-Most of us are healthy because we have an immune system.

-Our immune system exhibits memory - offers long-term protection against re-infection. HISTORY

- History of small pox - In humans around 10, 000 BC (in mummies in 3000BC) - Killed 300-500M people in 20th century alone - Care givers - Variolation - Jenner (1796)

- Discovery of microbes - Koch & Pasteur - Vaccination

- The modern era: discovery of immune cells and many features of the immune system IMPACT -Vaccination eradicated small pox from the world in 1979. -Polio basically eradicated (Salk and Sabin). -Major cause of reduction of childhood mortality. -Cancer immunotherapy. -But, there are no vaccines against HIV, HCV, Malaria, TB, etc STATISTICAL PHYSICS IS THE NATURAL LANGUAGE FOR IMMUNOLOGY

- Processes that underlie the immune response involve cooperative events with many participating components that act collectively. Many of the processes are stochastic and often driven far from equilibrium.

- Understanding such emergent behavior is the realm of statistical physics. SYLLABUS

Introduction to the basic elements of immunology

Virus fitness landscapes.

Mounting pathogen-specific responses to unknown pathogens, but not self - Darwinian evolution - Development of a self-tolerant immune repertoire

Kinetic proofreading - how biochemical signaling networks enable pathogen-specific immune cells to respond to foreign pathogens and not self CLASSES OF PATHOGENS

• VIRUSES (Human immunodeficiency virus, influenza) – 300K – 600K people die from influenza/yr – HIV has killed 75M

• BACTERIA (myobacterium tuberculosis, TB) – TB killed 1.6M in 2017

• FUNGI (Candida) – Efficiently dealt with, except for immunocompromised people

• PARASITES (Malaria) – 200M infected and 400K deaths in 2015 MANY ILLUSTRATIONS FOR THE INTRODUCTION ARE TAKEN FROM IMMUNOBIOLOGY JANEWAY, TRAVERS, WALPORT, SHLOMCHIK Garland Publishing IMMUNE SYSTEM OF COMPLEX ORGANISMS IS COMPRISED OF DIVERSE ORGANS AND CELLS

• INNATE IMMUNE SYSTEM - recognizes features that are common to many pathogens - pathogen must evade the innate immune system in order to establish infection

• ADAPTIVE IMMUNE SYSTEM - recognizes features specific to a pathogen - establishes “memory” of a pathogen, and confers protection against re-infection Nature Genetics 38, 431 - 440 (2006) Ng et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells NK Cells

adaptive immunity Innate immunity Phagocytic cells:

Macrophages: -Have receptors that bind to blood components bound to pathogens – promotes phagocytosis. -They also have receptors that bind to repeating patterns of carbohydrates or lipids that are characteristic of microbial surfaces (but, not host cells) – promotes phagocytosis Macrophage actions:

Acidic phagosome break down many pathogens Lysosomes contain antimicrobial agents and fuse with phagosomes

-Can interact with the adaptive immune system DENDRITIC CELLS

. Long-lived, reside in tissues

• receptors recognize common pathogen markers and engulfs them.

• also “eat up” extracellular material including bacteria and viruses without need for receptors.

• Upon engulfment, they mature and lose phagocytic phenotype.

• go to organs where they can interact with cells of the adaptive immune system. THE ROLE OF ADAPTIVE IMMUNITY

-Innate immune system cells recognize conserved and common pathogenic markers -Many bacteria have evolved strategies to evade the innate immune system - protective capsules that allows them to hide their evolutionarily conserved proteins - TB bacteria prevents phagosome-lysosome fusion, and use macrophages as host. -Viruses carry few invariant molecules, and are rarely recognized by macrophages Lymphocytes orchestrate adaptive immunity

optical TEM -Small cells with little cytoplasm -Transcriptionally quite slient (condensed chromatin) -Gowans showed that removal of lymphocytes results in loss of pathogen specific responses T cells and B cells are the two most important lymphocytes • T cells and B cells both express receptors on their surface (called TCR and BCR) • A great diversity of receptors is generated (1011 T cells in a human and a similar number of B cells ~ 1% of all cells) • Most lymphocytes have a distinct receptor (a clone) • Each clone is likely to bind to distinct pathogenic markers; O (107) different clones (>> 20, 000) • Some clones share the same specificity, and lymphocytes exhibit some degeneracy

• Thus, the lymphocyte repertoire enables pathogen-specific responses against a diverse and evolving world of microbes BURNETT’S CLONAL SELECTION THEORY

• Clone with “antibody” specific for a pathogen get activated and carry out effector functions

• Lederberg speculated that lymphocytes displaying antibodies on their surface could mutate to generate even more diversity. B cell receptor 50 kDA Heavy chain 25 kDA light chain -Some residues (framework regions) of the V regions provide structural stablility -These variable loops of the H and L chains are brought together to create 2 identical Ab binding sites. -The 2 heavy chains and 2 light chains are identical giving each protein 2 identical antigen binding sites -Two types of L chains, l and k; a BCR has only one type; no functional differences mediated, but ratio is species-specific THE T CELL RECEPTOR (TCR)

-Two chains of different types connected by a disulfide bond -Have constant and variable regions -Variable region binds a pathogenic marker -Transmembrane domain ends in a short cytoplasmic tail (signaling fn.) -The hinge joint is less flexible than the BCR -There are other differences A great diversity of TCR and BCR are generated

-Genes are inherited as gene segments, each encoding a different part of the receptor

-During development, one member of each set of gene segments is joined to others by a stochastic and irreversible process to form a juxtaposed gene that encodes one chain of the receptor

-The rearranged genes express proteins, and two distinct chains form a unique receptor

-TCR and BCR share this general scheme A great diversity of TCR and BCR variable regions are generated Light chains: -V region (variable) -J region (joining region) -C region -V-J are recombined, followed by C Heavy chains: -V, J, D (diversity), and C regions -D-J, then V-DJ, then VDJ-C recombine

Ubiquitous/specific DNA modifying enzymes mediate DNA recombination (separately for L & H). Non-coding regions adjacent to different types of segments ensure the right type of joining Because DNA recombination is irreversible, if a B cell divides, the daughters have the same DNA HOW DIVERSE IS THE REPERTOIRE Light chains:

For K: 40 types of VL, 5 types of JL; 200 types of K light chains

For l: 30 types of VL and 4 types of JL; 120 types of l light chains 320 types of light chains

Heavy chains: 65 types of VH, 27 types of DH, 6 types of JH ~10, 500 types of heavy chains

So, roughly 3.5 * 106 types of BCR

During recombination, some nucleotides are stochastically added or deleted at junctions - junctional diversity adds significantly to this

Some BCRs do not fold

Usage of types of gene segments is not uniform

Walczak, Callen, Quake (Science, PNAS, etc) HOW DO LYMPHOCYTES MEET PATHOGENS?

WHAT PATHOGENIC MARKERS DO THEY BIND TO, OR “RECOGNIZE”?

HOW DOES THAT LEAD TO IMMUNE RESPONSES? Lymphocytes interact with pathogens and develop in lymphoid organs

B cells mature -Lymphatic vessels drain extracellular fluid (lymph) from tissues to closest (LN) -Lymph contains pathogen, DCs, and macrophages. Lymphocytes enter secondary lymphoid organs from blood by squeezing between cells of capillary walls T cells APCs Von Andrian lab -Lymphocytes interact with APCs and pathogens in LN. -T cell-DC interactions, T cell-B cell interactions, etc. occur. After activation and proliferation, or otherwise, lymphocytes leave LN - Lymph returns them to blood via the thoracic duct. WHAT DO BCR RECOGNIZE?

• BCR recognize proteins, parts of proteins, etc., • They bind to parts of proteins and sugar molecules on bacteria and viruses. -Antigens (epitopes) can bind in grooves of the V regions -Conformational flexibility (induced fit) or picking out a particular conformer, shape complementarity is important. -Non-covalent bonds formed (H-bonds, electrostatic, VDW, hydrophobic, etc). Antibody interacting with Lysozyme

Antibodies can bind to discontinuous parts of surface moieties of proteins. Affinity Maturation – Darwinian evolution in a short time

antibodies Plasma Light zone cell Helper T cell FDC

Memory apoptosis B cell

Activated naïve B cell selection

Receptor mutates

Dark zone Germinal Center Bacterial toxins bacteria viruses B cells principally mount responses principally to free virus particles in blood or extracellular spaces.

They can also mount responses to viruses budding out of infected cells. -Many bacteria multiply in the extracellular spaces, and intracellular pathogens move from cell-to-cell through these spaces. Antibodies protect extracellular spaces and blood. -Some bacteria and all viruses replicate inside cells where antibodies cannot access them. Also, infected cells need to be killed. -T cell mediated or cellular immunity. T cells orchestrate adaptive immunity

T cell

Antigen presenting cell -Intracellular pathogens make proteins that their DNA encode -Proteosome or peptidases cut up these proteins in to short peptide fragments -Proteins coded for by the majorhistocompatibility gene complex (MHC) in chromosome 6 can potentially bind these peptides. - TAP proteins transport peptides to the ER. MHC I, present in excess, binds them here. -These pMHC complexes are transported to the cell surface. T CELLS RECOGNIZE SHORT ANTIGENIC PEPTIDES BOUND TO MHC PROTEINS

UNANUE Antigenic peptide TOWNSEND T CELLS HAVE CO-RECEPTORS

CTLs have CD8, and can bind to peptide-MHCI complexes Bacteria and some parasites are taken up in edosomes; B cells internalize pathogen

- Proteases and peptidases in endosomes degrade proteins to form peptides - MHC II can bind to them - MHC II cannot bind peptides in the ER – protein binds MHC to block peptide binding. This protein – MHC II associated invariant chain also helps target MHC II to vesicles and endosomes. - The acidic environment there cleaves the invariant chain; peptide-MHC class II complex created. - Specialized intracellular vesicles transport pMHC II to cell surface. T CELLS HAVE CO-RECEPTORS

T helper cells have CD4, and can bind to peptide- MHCII complexes MHC class I and II expressed differentially

• CTL attack viruses that live in any nucleated cell – MHC I is present in all nucleated cells.

• RBC do not express MHC I – viruses cannot replicate in RBC (non-nucleated) – but, this allows plasmodium (malaria) live in RBCs.

• Th cells interact with cells that present MHC II - B cells, macrophages, DCs GREAT DIVERSITY OF MHC GENES IN THE POPULATION

CLASS I: HLA-A, HLA-B, HLA-C

CLASS II: HLA-DR, HLA-DP, HLA-DQ

2 Alleles per locus – so, 6 – 12 types.

Great diversity of alleles

Particular combination of MHC alleles called haplotype

Two individuals are highly unlikely to have the exact same set of MHC molecules MHC CLASS I

a: 43 kDa

b2: 12kDa -MHC I bind peptides 8-11 amino acids long -the peptide is elongated; kinking accommodates length variation -invariant sites on MHC bind atoms on the carboxy and amino termini of the peptides -The alleles differ mainly in the peptide binding cleft – so, bind different types of peptides -Major sites of polymorphism in peptide binding region MHC ALLELES CAN BIND DIFFERENT TYPES OF PEPTIDES

- Anchor residues -A given allele can bind diverse peptides - different preference of anchor residues for different alleles

Y: aromatic; V, L, I: hydrophobic MHC CLASS II -MHC II bind peptides 13-17 amino acids long -Ends of peptide unbound -No invariant sites on MHC II -Anchor residues are more permissive for various amino acids -Diagonal orientation of the TCR over the peptide -Va sits on a2 and amino terminal -Vb sits on a1 and carboxy terminal -CDR3 loops of Va and Vb meet over the central amino acids Consequences of antigen recognition by T cells

-Upon antigen recognition, T cells get activated, proliferate, and many clones specific for this pathogen become available. CTL killing (no innate cells needed) -Th1 cells activate macrophages: Leads to killing of internal pathogens. -Tregs, Th17, etc., are other types of CD4 T cells – all Th cells regulate cytokine production -Th2 cells help with B cell responses TOLERANCE OF B CELLS AND T CELLS

Deletion of self-reactive B cells in the bone marrow

Thymic selection of T cells

B cell activation and AM depends upon T cell help. LIFE TIMES OF T CELLS AND B CELLS

Broad distribution of mature B cell life-times (naïve - 3-8 weeks); memory B cells live longer (possibly decades).

T cells live much longer: naïve ~ 4 – 6 years (CD4, CD8); memory ~ 0.4-0.7 years. Memory cells divide faster. Adaptive immunity mounts pathogen-specific responses

distinct receptor T cell Peptide-MHC

virus

Viral protein distinct receptor Host B cell cell Antibodies Adaptive immunity mounts pathogen-specific responses

distinct receptor CTL Peptide-MHC

virus

Viral protein distinct receptor Host B cell cell CTL Antibodies

Infected cell Adaptive immunity mounts pathogen-specific responses

distinct receptor T cell Peptide-MHC

virus

Viral protein distinct receptor Host B cell cell Antibodies

Establishes memory of past infections Adaptive immunity mounts pathogen-specific responses

distinct receptor T cell Peptide-MHC

virus

Viral protein distinct receptor Host B cell cell Antibodies

Establishes memory of past infections – basis for vaccination