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Home , Allelic exclusion, Junctional diversity

- Lecture 3 Adaptive 2

Generation of Immune Diversity Receptors - 8 Lymphocyte Development- 9 Lymphocyte Activation – 10 Lymphocyte Effector Functions – 11 Regulation of Adaptive Responses - 12

Innate versus Adaptive specificity?

Epitopes recognized by innate receptors have been predetermined by evolution and germ-line encoded

• Based on Ag that ancestors encountered • Limited range of view = TLR4 = LPS, 4 or 5 other things • View is looking back in time

Epitopes recognized by Ig or TCR are determined before they ever encounter Ag

• View is looking forward for thing you might see • Randomness of the future needs to be accounted for as well as the diverse Ag that will be accounted for 15 • It is estimated that each individual has the capacity to recognize 10 epitopes

How do adaptive immune cells generate such diversity? Chromosome rearrangement Junctional diversity Allelic exclusion Switching (B cells)

BCRs and TCRs are similarly organized. Each receptor is composed of two distinct subunit chains (BCR: light chain and heavy chain, TCR: α chain and β chain). The antigen binding surface is formed by the variable region of each chain, which is encoded by recombined V, J, and D (BCR heavy and TCRβ) segments.

VDJ recombination or somatic recombination:

The antigen receptors are encoded by roughly 300 different gene segments - but can be used to generate 1010-15 receptors

DNA chromosomal rearrangement gene segments to produce this significant diversity

Relies on Recombination -Activating (RAG)

RAG deficiency - results in severe combine (SCID) Fig. 8.2

Some genes are inverted before being reannealing

Terminal deoxynucleotide transferase (TdT) - adds or removes nucleotides to exposed ends of DNA before renealing to mediate junctional diversity

http://www.youtube.com/watch?v=QTOBSFJ

WogE Fig.

8.3 Steps in V(D)J recombination:

Antigen receptor gene segments are flanked by a recombination signal sequence (RSS)

In the first phase, recombination activating gene 1 (RAG1) and RAG2 proteins bind to the RSSs

Capture of the second RSS (a process termed synapsis) results in the formation of the paired complex, within which the RAG proteins introduce double strand breaks between the gene segments and the RSSs.

In the second phase, the RAG proteins cooperate with non-homologous end joining (NHEJ) DNA repair factors to rejoin the DNA ends.

Gene segment ends typically undergo non-templated nucleotide addition by terminal deoxynucleotidyl transferase (TdT) and nucleotide loss before being joined to form the coding joint.

RSS ends are typically joined without processing to form the signal joint.

T Receptor (TCR):

Heterodimers composed of ab pairs

- 1 x V and 1x C

CD3 and CD247 for signaling

TCR generation

LC: α on chromosome 14

HC: β is on chromosome 7 Fig. 8.4

LC = VJC chains HC = VDJC chains

Each developing randomly produces a light- heavy chain combination with unique specificity due to the V region at the N-terminus of the protein

V regions combined with limited set of D, J, C

Random combinations of light and heavy chains genetic rearrangement and junctional diversity randomly create TCR chains that vary among, but not within, individual T cells

The theoretical number of possible combinations produced within the body may be estimated to be the product of several possible light chains and several possible heavy chains

Estimated that 1 to 5 million -binding

B-cell receptors or “Ig” are composed of:

2 identical LC (κ or λ)

2 identidal HC

Associated Igα-Igβ for signaling (CD79/a/CD79b) which are required for signaling

IgD>IgM>IgG can all be membrane bound monomers

Gene clusters encoding BCR chains are found on multiple chromosomes

HC V regions account for a significant level of diversity

>9x106 Ag-Binding combinations

Just like the TCR, the different combination of V domains on L and H chains to light and heavy chain results in significant diversity

The choice to use which V, D, J genes is random for each cells

In the end – 1 cells synthesizes an Ig of a single specificity made up of the VL and VH domain

Isotype Switch:

After contact with its specific epitope B cells cease production of lgD, and most of them differentiate into plasma cells that secrete lgM. Some B cells become memory B cells. Express only lgM on their surface.

If reactivated by a new contact with their specific epitope and via interactions with T cells, they can complete rearrangements of their heavy chain genes at the DNA level to juxtapose their VDJ units with other constant genes.

Whatever constant gene is brought adjacent to the VDJ determines what heavy chain gene will be produced.

Some isotype switches may involve the deletion of large tracts of DNA to bring VDJ segments together with distant constant genes.

Reactivations of memory B cells may result in a series of shorter deletions as lgM memory B cells may switch and become lgG memory B cells, then be reactivated again and switch to yet another isotype.

Repeated or constant stimulation by the same epitope drives B cells from lgM expression to other isotypes

The cell's epitope specificity is not altered by isotype switching.

Lymphocyte Development - 9

The thymus is organized into outer or cortical and inner or medullary regions.

Pro- enter the thymus to under several maturational steps.

B cell development: Takes place in the bone marrow: Contains HPSC, stomal cells for instruction, and vasculature enabling cells to come and go. First step is to express Iga and Igb (Signaling complex) Generate and test HC with SLC (pre- receptor If non-functional –cell dies of apoptosis Generation of LC and IgM – tested for functionality Tested for self reactivity on stromal cells of the bone marrow Too strong – receptor editing (reactivates VDJ recombinases) If fails again – delete If successful a IgM positive B cells release to periphery

T cell activation involves the formation of an immune synapse:

Initiated by the TCR recognition of a Ag presented on MHC

Weak interaction of TCR with MHC is stabilized by CD4 or CD8

Generates signal 1 through TCR/CD3

Necessary, but not sufficient to activate a naïve T cell (never activated before - versus a memory cell)

Cell adhesion molecules (CAMs) expressed by the T cells (LFA-1) interact with ICAM-1 on APC form a ring with LFA on outside, then CD4/8 and TCR inside. This helps to initiate appropriate signaling.

Second signal is received from an ACTIVATED APC

Activated APC have recognized non-self and are “pro-inflammatory”

CD86 hi CD40 hi Co-Stim CD80 hi

MHC hi

Secreting

Without these APC derived signals - TCR stimulated T cells become, “anergic” or harder to stimulate into a productive immune response.

CD4 Differentiation:

Cytokines and local factors dictate the shape the CD4 responses takes after activation:

IL-12 from activated DC- Type 1 helper cells (Th1) making IFNgamma and mediating cellular . Helps CD8s and innate immune system remove pathogens

IL-4 from innate cells and other T cells results in a T helper type 2 cells making more IL-4, IL-5, IL-13 which support , , and tissue repair.

Generation of T cell memory:

Activated T cells complete effector responses and undergo activation induced cell death, or upregulate moleucles like CD152 which interact with co-stimulatory molecules to generate regulation.

A small subset of Ag stimulated T cells will upregulate anti-apoptotic proteins such as Bcl2 and avoid death

They will also decrease molecules that direct them into the lymphoid organs. Thus they sit in the blood and peripheral tissues where they can mount a rapid response to their recognized Ag.

Memory cells take less stimulation to respond

Went from a situation where you had a few naïve clones to an expand population of memory cells with the same Ag recognition ready for a rapid response

BCR signaling:

Initiated by clustered BCR - recognition of multiple

epitopes

Both T cells dependent and independent B cell activation

T cell dependent is most

common and relies on co- stimulation via CD154 on activate T cells and CD40 on B cells

T cell independent involves mitogens - that bind structures other then the BCR

Ex. LPS via TLR4 and BCR

Innate APC, B cells, and T cells interact to shape and coordinate immune response to pathogens and after vaccination.

Immunological Memory:

Lymphocyte Effector Functions Upon proper activation via their Ag-receptor, adaptive immune cells undergo clonal expansion (multiple divisions of clone recognizing that Ag and expressing the EXACT same Ag-receptor) and while most will do their function and die, a small percentage will upregulate anti-apoptotic genes to survival.

Become long lasting memory cell that express that Ag-receptor and need less stimulation to initiate immune response.

Immune memory increases both the number of cells that can potentially

recognize an antigen and also decreases the intensity of the stimuli need

to activate them.

Memory increases intensity, duration, and amplitude of adaptive immune

response

Memory is the reason vaccination is possible. Vaccines often use disabled “attenuated live” or killed microbes for vaccines. Still have Ag to stimulate adaptive response - but can not make you sick.

Two effector arms of the

Humoral Immunity: involves fluids or “humors”; and complement

Ancient Greek and Romans; Humors = fluids of the body that define the makeup of the workings of the human body

• Secreted that are targeted to specific epitopes on via variable domain generated epitope binding sites and attached to heavy chains with FC regions that shape the function of bound antibodies

• Once bound – work with complement and innate cells to clear Ag

• There is a T cell-dependent component to humoral immunity, as T cell

help (CD40 ligation via Th cell CD154 and Th cytokines) are required for an effective and diverse humoral response.

Cell-Mediated: Direct cell to cell interaction (or at least localized) for destroying infected or malignant cells.

CTL - Direct killing of cells (infected with intracellular pathogens –virus and bacteria; malignant cells) CD4+ T helpers often directing other cells to destroy pathogens and through the use of cytokines

Humoral Immunity:

Agglutination: Cross-link Ag and entrap them to render them more susceptible to destruction

IgM - 10 binding sites IgA - 4 binding sites IgG – in high concentrations

Also Aided by NETs

Neutralization:

Binding to pathogens or toxins to prevent infection or toxicity

Typically IgG or IgA

Will require prior exposure to pathogen or toxin

Opsonization: ZMAb vs. Ebola

Greek word “Opson” - delicious side dish

IgG1 and IgG3 are highly effective opsonizing antibodies. Generating these isotypes driven by both IL-4 and IFNg. Thus both Th1 and Th2 responses support opsonization.

Antibodies tagging of antigen for destruction by phagocytic cells.

Upon binding to Ag antibodies undergo a conformation change that allows them to interact with FcR via their Fc Recptor

Different classes of FcR bind different Isotypes (Table 11.1)

Opsonization is also Enhanced by C3b

Antibody-dependent Cell-mediated cytotoxicity (ADCC):

“Tagging” of Ag attracts cytolytic cells and enables their use of effector mechanisms

NK cells: (IgG1 - FcgRIII)

Eosinophils:

IgG - FcgRIII IgE - FceRI IgA - FcaR1

Immediate

The Fc portion of IgE can bind to FcR on Mast cells/ without Ag. When Ag cross links Ab on these cells, they respond immediately with release of granules (histamine)

Cell-Mediated Immunity

Delayed Type Hypersensitivity response (DTH):

Once activated - CD4+ T cells leave the lymphoid organs to search the periphery (vasculature, tissues, and lymphatics) looking for APC presenting Ag/MHCII

Activated CD4+ cells then stimulate local tissue - mediated destruction that ensues is not limited by the triggering epitope (IFNg dominated)

Activated destroy not only the infectious agents that initiated the DTH but also other microbes in the immediate vicinity. Associated with tissue injury Cytotoxic T : Once activated - CD8+ T cells

leave the lymphoid organs to search the periphery (vasculature, tissues, and lymphatics) looking for any cells presenting Ag/MHCI

Binds any positive cell and forms a synapse with them. Through it releases perforin and granzyme.

Also express molecules that can directly induce apoptosis of other cells (Fas ligand).

Isotype Switching:

CD4+ helper cell use cytokines to support of isotype switching in B Cells (Table 11.2)

B cells that have prolonged or repeated exposure to Ag undergo isotype switch that places the same epitope binding site on different HC.

Permits the humoral response to initiate diverse functional mechanisms

Complement activation by IgM and IgG IgA is secreted onto epithelial barrier tissues Mast cell and degranulation by IgE

Activated T cells also produce other cytokines that direct the immune response:

Effect on: Sources T cells B cells APC Stromal Deficiency Cells Results in IL-2 Activated CD4+ T Proliferation of Limited Total T cells CTL, Th, and cell responses; Treg (Required for Treg) Interferon-γ Th1, CTL, Inhibits Th2; IgG2a Increased Increased MHC Infections with (IFNγ) Drives CTL and Synthesis CD80-CD86; I; Antiviral intracellular Th1 responses MHCI and II functions; microbes; poor tumor clearance IL-4, -5, -13 Th2 Inhibits Th1 IgG1, IgE, IGA Mucus Poor parasite sythesis production; clearance; tissue repair Lack of allergy

IL-10 Th2 cells and Inhibits T cell Limits their Autoimmunity Treg responses stimulatory capacity IL-17 Th17 cells Secretion of Susceptibility to chemokines, infections defensins, pro- (bacterial, inflammatory fungi); cytokines (IL-6)

Regulation of Adaptive Responses - 12

The innate and adaptive immune system is a dynamic network that can rapidly amplify it’s response to outcompete a developing infection.

Such a system will need regulation to avoid lethal pathology

Also, while the adaptive immune system is educated to not see self, the process is not perfect. Self reactive B and T cells may escape to the periphery where they have the potential to instigate autoimmunity

Cells Intrinsic mechanisms of regulation:

Anergy: State of lymphocyte non-responsiveness.

T cells: Results from Ag/MHC recognition in the absence of co- stimulation.

B cells: Less clear mechanisms; lack of second signals (CD40 on activated T cells; Fig. 10.17)

Prevents self reactive lymphocytes cells from getting started in the absence of appropriate

Upregulation of regulatory molecules:

CD152 (CTLA4) - blocks co-stimulation by competing with CD28 for binding to CD80/CD86 and does so with a greater affinity than CD28.

Provides means for imposing a finite period of activity on each activated T cell.

Activation induced cell death: Due to lack of cytokines driving the response. IL-2 is needed to sustain T cells responses - upregulates the anti-apoptotic protein Bcl2.

Activated T and B cells will also upregulate FasL: Killed by cells expressing Fas, including themselves or other lymphocytes.

B cells need CD40 stimulation via T cell CD154 to sustain response

Cells extrinsic mechanisms of regulation:

Regulatory T cells: CD4+ CD25+ Foxp3+ T cells that control self reactive T cells, including Tumor responses. Also, suppress responses to pathogens

Escape the thymus with strong self reactivity. 10% of T cells.

Multiple suppressive mechanism

Consume or break down factors driving immune response (IL- 2, ATP, amino acids)

Secrete regulatory cytokines (IL-10 and TGF-b)