Immunology-Exam II

Somatic Hypermutation Theory

"Alternative" hypothesis Explanation to account for the size of the mature antibody repertoire Mutation occurs only in B cells. Since it is not affecting germ-line genes, the changes were not heritable.

iTreg: Effector T Helper Subset

(induced) Similar in function to natural Treg cells originating in the thymus -Arise during activation of T cells in the presence of TGF-B -iTreg cels secrete IL-10 and TGF-B to downregulate inflammation (by inhibiting APCs) and suppress other T-cell subsets Master Regulator: FoxP3 -Induced by TGF-B, shifting activating cells into this subset

MHC Molecules- Class I

-Antigen presentation -Member of the Ig superfamily and possess Ig domains -Smaller 12 kDa B2 protein, larger 45 kDa glycoprotein alpha chain -Interaction with 2 domains -Cytoplasmic anchor section of 30 amino acids The a1 and a2 domains form a cleft region that binds an 8-10 amino acid-long peptide fragment form an antigen -B sheet forms the floor of the cleft -Two alpha helices form the walls Interactions: -Present peptides to CD8+ T cells -Peptides derived from endogenous intracellular proteins -Some amino acids anchor the peptide into the groove -Other amino acids are available to interact with a TCR

MHC Molecules- Class II

-Antigen presentation -Member of the Ig superfamily and possess Ig domains -heterodimeric: 33 kDa a chain, 28 kDa B chain -Interaction with 2 chains -Both chains pass through the plasma membrane A peptide-binding cleft is formed by the pairing of the a1 and B1 domains -Accommodates peptides of 13-19 amino acids in length -Present peptides to CD4+ T cells Interactions: -Usually derived from exogenous extracellular processed antigens -Present antigens to CD4+ T cells -Exhibit anchor amino acid residues that lock them into groove -Extend amino acid residues from groove to interact with TCRs

T-Cell Beginning

-Early T-cell precursor development occurs in the bone marrow -T cell precursors begin their travel through the thymus at the cortex -T cells that survive selection migrate into the medulla

5 Antibody-Diversity Mechanisms- Naive B Cells

1) Multiple Gene segments-which gene segments are put together 2) Heavy chain/light chain combinatorial diversity 3) P nucleotide addition-templated nucleotide addition between joints, resulting from asymmetrical cleaving of hairpin structures 4)Exonuclease trimming-sometimes occurs at junctions, losing nucleotides and changing reading frames 5) Nontemplated N nucleotide addition- Mediated by TdT activity, adding in random nucleotide between joints -Adds diversity -Can lead to frameshift mutations

Immunoglobulin Proteins

2 identical heavy chains, 2 identical light chains Kappa (light)-Human chrom 2 Lambda (light)-Human chrom 22 Heavy Chain genes- human chrom 14 Heavy and light chain combinations in humans = 6210 x (205+165) = 2.3 x 10^6

Ig Structure

4 polypeptide chains, 2 large (heavy) and 2 smaller (light) End of 4 polypeptides are highly variable regions. This is where the antigen binds, allowing for numerous antigen specificities The body of the Ig forms the C or constant region which allows the antibody to perform other cellular functions such as binding to receptors

Superantigens

A special class of T-cell activators Viral/bacterial proteins that bind to specific VB regions of TCRs and a chain of class II MHC molecules -Effectively "short circuits" needed for costimulation -Produces dramatic cytokine secretion by large proportion of inappropriately activated T cells Exogenous: soluble proteins secreted by bacteria Endogenous: cell membrane proteins generated by viral genes integrated into mammalian genomes.

Antigen

A substance that can induce an immune response (mediated primarily by recognition by T and B cells) Usually proteins, may also be polysaccharides, lipids or nucleic acids Origin: Within the body (autoantigen) or externally (viral or bacterial) Specific binding site: epitope

Th17: Effector T Helper Subset

Activation in the presence of Il-1B, IL-6, IL-23, and TGR-Beta form this subset -IL-23 also plays a role in finalizing the subset commitment -IL-17F, IL-21, and IL-22 production may play a role in warding off fungal and extracellular bacterial infections -IL-17A production is associated with chromic inflammatory and autoimmune responses Master regulator: RORyt -Orphan steroid receptor, become activate and differentiates activating T cells into this subset Th17 cells are important in controlling infections by extracellular bacteria and fungi

MHC Class II: Exogenous Processing

Antigenic peptides to CD4+ T cells Class II presentation requires exogenous processing Peptides are generated from internalized antigens in endocytic vesicles -Particles are taken in within endosomes. -Endosomes are fused with lysosome as in MHC late lysosome -Late lysosome becomes acidic, its contents are degraded. Simultaneously, MHC Class II molecules are produced, associated with a protein called Invariant chain (li, CD74) and exported in vesicles from ER to the Golgi Invariant chain Guides transport of Class II MHC molecules to endocytic vesicles -Invariant chain (li, CD74) prevents peptides from binding to the groove too early in the ER -one of the ways the pathway prevents the processing of Class I molecules -li also uses sorting signals in its cytoplasmic tail to direct MHC Class II molecule-containing endocytic compartments.

Immunoglobulin Gene Structure

B cells use recombination of gene segments to create different possible antibodies There are Variable, diversity, joining, and constant region gene segments -D segments are used in antibody heavy chains only Recombination among various V region gene segments generates a diverse repertoire of antibody combining sites.

V(D)J Recombination and RAG

B/T cells have absolute dependence on RAG1/2 expression Gene segments are joined by the RAG1/2 recombinase RAG= Recombination activating gene -Both proteins are needed for recombination -RAG1 is more important-it forms a complex with RSSs stabilized by binding RAG2 -RAG1/2 complex is responsible for recognizing and cutting DNA at the immunoglobulin-encoding region and the RSS V(D)J recombination occurs in a series of well-defined steps -RAG binds to RSSs and cleave dna -Other proteins process the hairpin loops that form after RAG reacts -Products include a recombined coding joint and a leftover signal joint that is later degraded. V(D)J recombination can occur between segments transcribed in either the same or opposite directions -So long as the correct RSSs are lined up, orientation of the actual gene segment is irrelevant

T-Cell Activation Pathways

Begins with tyrosine kinase, Lck -CD4 and CD8 cytoplasmic tails guide Lck to TCR-MHC complex where is phosphorylates ITAMs on CD3 which then become docking sites for ZAP-70. PLCy action activate NF-kB and NFAT -PLCy binds LAT and catalyzes the splitting of PIP2 to soluble IP3 and membrane-DAG -IP3 induces Ca2+ release which binds calcineurin and desphosphorylates NFAT which gones on to enter nucleus. 0DAG also activates PKC which translocated NF-kB to nucleus. Ras-ERK signaling and AP-1 -Ras pathway triggers MAP kinase (MAPK) activation which leads to the MAPK cascade that phosphorylates RAF, MEK, and ERK. -ERK activates transcription factor AP-1 DAG ultimately activates Ras/Erk and PKC which results in activation of the transcription factors, AP-1 and NFkB respectively

Modern Accepted Theory

Both theories, "modified germ-line" and somatic hypermutation are correct. Multiple germ-line segments each encode a part of the antibody variable regions. These segments are rearranged differently in the formation of each naive B cell to produce an extremely diverse primary receptor repertoire. These rearranged genes are acted on after antigen encounter by somatic hypermutation and antigenic selection, resulting in an expanded and exquisitely hone population of antigen-specific B cells.

Lineage Commitment

CD4 or CD8? Instructive Model: -TCR/CD4 and TCR/CD8 coengagement generate unique signals -The signals generated "instruct" the T cells which lineage to fully commit to Stochastic Model: -Positively selected thymocytes randomly downregulate CD4 or CD8 -Only those cells with the "correct" coreceptor receive signals to continue development -Strength of signal and duration of signal from TCR/coreceptor Kinetic Signaling Model: -Cells commit to the CD4 lineage if they receive a continuous signal -Cells commit to CD8 lineage if stimulation signal is interrupted -IL-7 Promotes CD8 differentiation of interrupted thymocytes

Major aB T cell Classes

CD4+ "Helper" T cell -When activated, mostly secrete helper cytokines CD8+ Cytotoxic T cell (CTL) -Killer T cells -Can lyse (kill) infected or transformed cells

Regulatory CD4+ T cells: Peripheral Tolerance

Can be generated naturally in the thymus (nTreg cells) or in the periphery following Ag induction. Self engage Ag-MHC Class II complexes through TCR -Down regulate responses -Helps shut down allergic responses Work via: Dependent mechanisms: as TREG cells express high levels of inhibitory CTLA-4 molecules Independent: rely upon secretion of cytokines (IL-10, TGF-B, IL-35) into the surrounding area, shutting down nearby cells responses. Bystander effect

T-Cell Memory

Can emerge in both CD4+ and CD8+ populations. Generally, longer-lived than effector T cells, and are quiescent -In G0, like naive T cells -Acquire energy from lipids via oxidative phosphorylation Represent roughly 35% of T cells in healthy young adult, and rise to >60% in individuals over 70. Respond to heightened reactivity to a challenge with the same antigen. -This anamnestic (secondary) response is both faster and more robust. Have less stringent requirements for activation than naive cells.

T-Cell Development in the Thymus

Cells migrate to the thymus for further development There they go through a variety of stages -Double negative (DN) cell has no CD4 or CD8 -Double positive cells (DP) is both CD4+CD8+ -Positive/negative selection stages for a cell to become single positive CD4+ or CD8+ Final screening removes autoreactive cells (cells that can bind to self antigens or self peptides) -Release into the peripheral bloodstream -Recombination of TCR gene segments also occurs in the DN stages, yielding either and ab or a yo T cell.

Tcm Cells

Central Memory T cells -Reside in/travel between secondary lymphoid tissues -Live longer/divide more times than Tem cells -Are rapidly reactivated by second Ag exposure -Can differentiate into several subset types depending on cytokine environment

Memory T Cell Subsets

Central memory T cells (Tcm) Effector memory T cells (Tem) Resident memory T cells (Trm) Stem cell memory T cells (Tscm)

Tscm Cells

Circulate to and from secondary lymphoid organs -True stem cells, can self reneew

MHC Molecules-Structure and Function

Class I and II molecules exhibit polymorphism in the peptide-binding region -Allows for massive diversity in the capacity to bind different peptides Several hundred different allelic variants in humans -Up to 6 class I and about 12 class II molecules expressed per person -A given MHC molecule can bind numerous different peptides and some peptides can bind to several MHC molecules

MHC Class I: Endogenous Processing

Class I proteins present "endogenous" antigenic peptide to CD8+ T cells Class I presentation requires cytosolic or endogenous processing Peptides are transported from the cytosol to the RER -Transporter associated with antigen processing (TAP) molecules in the RER membrane move the peptide fragments -MHC Class I molecules synthesized on ribosomes on the RER anchor in the RER membrane after their translation Chaperons aid peptide/MHC class I assembly -Calnexin, ERp57, calreticulin, and tapasin help fold MHC class I and put it in close proximity to TAP (KNOW THESE) -ER aminopeptidase ERAP1 trims long peptides to a suitable size for MHC class I grooves

MHC Alleles in Immune Responsiveness

Class II MHC alleles and antigen presentation -Different capability to present antigens may dictate overall strength of immune response from individual to individual. -Two likely explanations for variability in immune responsiveness 1: Determinant selection model 2:Holes-in-the-repertoire model -Both models appear to be correct

SUMMARY LECTURE 15

Complete activation requires 3 signals -CD4+ T cell differentiation is regulated by polarizing cytokines (signal 3) -Polarizing cytokines are mostly derived from the activated APC -Which polarizing cytokines are produced depends on: which cell has been alerted, what pathogen has alerted it, and tissue context. CD4+ Activation/differentiation have been demonstrated to result in the formation of at least 7 effector varieties -Four main subsets Effector T helper subsets are distinguished by 3 properties: -Distinct polarizing cytokines that induce their generation -A master gene regulator -A signature set of effector cytokines

T-Cell Browsing

Cortical thymic epithelial cells express high levels of mHC Class I and II Developing T cells can "browse" possible self-peptide.MHC complexes These present self-peptides;three possible outcomes with T cells encounter these self-peptide/MHCs -TCRs can't bind; cells die by neglect -TCR bind too strongly; negative selection, apoptosis occurs -TCRs bind just right; positive selection to single-positive stage occurs

T-Cell Activation: Costimulatory Molecules

Costimulatory signals are required for T-cell activation and proliferation Positive costimulatory receptors facilitate activation -CD28: generally involved in initial activation events in T cells, enhances TCR induced proliferation and survival, binds to B7-1 and B7-2 expressed by APCs -ICOS: Expressed on memory and effector T cells, inducible costimulator, binds ICOS-ligand on activated APCs Negative costimulatory (coinhibitory) receptors help turn activation off -CTLA-4: Induced within 24h after activation, peaks 2-3 days post, binds B7-1/B7-2 with higher affinity than CD28 but shuts down pathway -PD-1 (programmed death 1): mediates T-cell tolerance in nonlymphoid tissues -BTLA (B and T lymph attenuator): Downregulates inflammatory and autoimmune responses APCS have characteristic costimulatory properties -Dendritic cells, macrophages, and B cells provide the signals to activate T cells.

Subset Cross Regulation

Cytokines can achieve Th1/Th2 helper subset cross regulation -INF-y from Th1 responses inhibits IgG1/IgE class switching (a common Th2-induced response) -IL-4 from Th2 responses inhibits production of IgG2a -IL-10 from Th2 responses also inhibits Th1 responses by suppressing the production of inflammatory mediators from APCs Master regulators commit T cells to one subset or the other -T-Bet suppresses Th2 pathway gene expression -GATA3 suppresses Th1 pathway gene expression

Thymocyte Beta Selection

DN thymocytes undergo Beta selection, resulting in proliferation/differentiation A successfully produced B chain is paired with the pre-Ta chain -A 33 kDa protein surrogate for real TCRa chain -Allows for formation of a pre-TCR complex (with CD3 proteins) and many early signaling events After beta selection has occurred, thymocytes are at the DP stage of development. -Functional TCRa chain replaces surrogate pre-TCRa -The cell still expresses both CD4 and cd8 (double positive) -Pos/neg selection occurs, yielding mature single positive T cell, CD4+ or CD8+

Thymocyte Commitment: Other Lymphocytes

DP thymocytes may commit to other types of lymphocytes NKT cells: -Express a TCR with an invariant TCRa chain -Interact with CD1 molecules presenting lipid antigens Intraepithelial Lymphocytes (IELs) -Usually CD8+, but also have features of innate immune cells Regulatory T cells (Treg) -CD4+ subset that helps to quench adaptive immunity

Signal 3 Cytokines

Depending on which cytokines are present as the T cell is becoming activated, different outcomes can occur -IL-2 is an example of an autocrine type of cytokine response system. T cells produce the cytokine and the receptor for it. Binding of this ligand induces a very strong prolferation signal during activation stages. -Polarizing cytokines can send the T cell down different subset development pathways

SUMMARY LECTURE 13

Developing T cells arise from multipotent CD4-CD8- precursors that migrate from the bone marrow to the thymus Mature T lymphocytes have a diverse TCR repertoire that is tolerant to self-antigens yet restricted to self-MHC. The fate of a CD4+8+ thymocytes depends on the affinity of its TCR for self-peptide/MHC complexes encountered on stromal cells in the two major thymic microenviornments: the cortex and the medulla. Mechanisms that remove autoreactive T cells during development, central tolerance, are reinforced in the periphery by a variety of mechanism, including the activity of regulatory T cells. Early Thymic emigrant remain multipotent until they encounter the Notch receptor on the surface of Thymic stromal cells -Enocunter allows them to "commit" to a T lineage. Until then they can become NK cells, B cell, Dendritic cells, or other myeloid cells. CD4+ and CD8+ thymocytes that survive positive and negative selection are allowed to migrate from the thymus into the bloodstream and complete their maturation in the periphery.

CD4+ T Helper Cell Functions

Dictate which class of immunoglobulin is expressed by B cells Enhance phagocytic function of innate immune cells like macrophages Differentiate into various effector cells specialized to eliminate specific classes of pathogens

B-cell Receptor Expression-Allelic Exclusion

Each B cells synthesizes only on heavy chain and one light chain Heavy chains are recombined and expressed first -Expression of a functional heavy chain shuts down recombination machinery temporarily -Heavy chain is paired with a surrogate light chain (SLC) to form a pre-BCR If the SLC will pair with the heavy chain, the machinery is started up again -Light chain recombination then takes place Nonproductive arrangements lead to programmed cell death

B-cell Receptor Expression: Receptor Editing

Editing of potentially autoreactive receptors occurs in light chains A functional antibody in an immature B cell may bind to self-antigens Recombination machinery can be turned back on to edit original rearrangement to salvage the rearrangement or at least inactivate it

Tem Cells

Effector Memory T cells -Travel to/between tertiary tissues -Contribute better to first-line defenses -Shift right back into effector functions on second Ag exposure

Holes-In-The-Repertoire Model

Explanation to variable immune responsiveness T cells with TCRs that recognize certain foreign antigens closely resembling self-antigens may be eliminated during thymic development.

Determinant Selection Model

Explanation to variable immune responsiveness MHC Class II molecules differ in ability to bind particular processed antigen peptides.

Helper T Cell Differentiation

Helper T cells may not be irrevocably committed to a lineage -Early in differentiation, Th subpopulations may be able to shift -Fluidity among subsets makes definitive establishment of helper lineages difficult Helper T cells play critical roles in immune health and disease -Leprosy -HIV -EBV

Heavy-Chain Gene Organization

Human Chromosome 14 Vh, Dh, Jh, and Ch segments Humans have at least

Kappa Light-Chain Genes

Human Chromosome 2 V, J, and C segments

Lambda light-Chain Genes

Human Chromosome 22 Pair each J segment with a particular C segment 40% of light chains are lambda type

Th9 Cells

IL-2, TGFB, and IL-4 play roles in polarizing naive T cells to Th9 cells Transcription factors IRF-4 and PU.1 drive Th9 differentiation Th9 cells produce IL-9 in expressing worms and contributes to antitumor responses

Follicular Helper T (Tfh) Cells

IL-6 and IL-21 appear to be the polarizing cytokines Master Regulator: Bcl-6 -Inhibits T-Bet, GATA3, and RORyt expression IL-4 and IL-21 are characteristic secreted cytokines, promoting B-cell differentiation Express CD40L required for cognate B-cell help Express CXR5 that attracts them to B-cell follicle

Th22 Cells

IL-6, IL-23, and TNF polarize naive T cells to Th22 cells by upregulating AHR transcription factor Th22 effector cytokine is IL-22 Distinct from Th17 cells in that Th22 do not express RORyt or make IL-17 Th22 secrete IL-13 and express homing receptors for skin.

T-Cell Differentiation

Initial activation signals 1 and 2 induce: -Upregulation of prosurival genes like Bcl-2 -Transcription of IL-2 and IL-2R genes -Outcome activation and robust proliferation. production of memory cells and effector clonal cells Effector T helper subsets are distinguished by 3 properties: -Distinct polarizing cytokine set that induces expression of a mater gene that regulates the expression of a signature set of effector cytokines produced by that subset. So polarizing cytokines, effector cytokines, and master gene regulators (the three). Differentiation of T helper cell subsets is regulated by polarizing cytokines -Signal 3-what cytokines are nearby when T cell is activated? APCs may bind PAMPS via PRRs inducing cytokine secretion -Different PRRs engaged (via different antigens)=different cytokines produced -Viruses stimulate IL-12 to induce Th1 subsets -Worms stimulate IL-4 to induce Th2 subsets

T-Cell Overview

Intial activation stimulates the upregulation of prosurvival genes (like Bcl-2) that contribute to complete activation, robust proliferation, appropriate differentiation, and formation of memory cells. After the pathogen is cleared, 90-95% of effector cells dies by apoptosis, leaving behind the all-important, antigen-specific memory T cell population.

CD4+ T cell differentiation

Intrinsic and extrinsic metabolic factors are required for specific CD4+ T cell differentiation and function In addition to providing the stimuli needed for T cell activation, cytokines secreted by the antigen-presenting cell during activation can direct CD4+ T cell differentiation into one of at least 7 classes of specialized effectors -Peripheral Treg, Th17, Th2, Th9, Tfh, Th1, Th22. Know basics of all 7 for EXAM

Chromatin Alteration

Involved in the regulation of V(D)J gene recombination The catalytic activity of RAG1/2 occurs in an extraordinarily complex nuclear environment. -RAG1/2 binding is affected by epigenetic modifications on histones associated with the target sequences. -->Histone acetylation or methylation affects accessibility to enzymatic activity in chromatin -Because V,D, and J segments are so spread out along the chromosome, high order chromatin structure must also play a role

Thymocyte Stages Key Molecules

Key molecules in stages: 1: C-kit (CD117)-Receptor for stem cell growth factor 2: CD44-An adhesion molecule 3: CD25-The a chain of the IL-2 receptor

Exogenous Pathways: Peptide Assembly

MHC Class II li is initially degraded by proteolytic activity within endocytic compartments to class II-associated invariant chain (CLIP) li chain prevents MHC class II from interacting with peptide in the ER HLA-DM exchanges CLIP out of the groove for a peptide fragment

Role of MHC Expression

MHC expression can change with changing conditions Genetic regulatory components -Promoters that drive up transcription during times of infection, NLRs are core components transcriptional activators Viral Interference -Viruses like to shut down MHC Class I expression because it targets the cells they're in for destruction Cytokine-mediated signaling -Some cytokines like IFN-a and TNF-a expressed first during infection/disease can drive UP MHC expression, whereas corticosteroids and prostaglandins DOWNregulate MHC expression.

MHC Inheritance

MHC locus encodes three major classes of molecules -Class I MHC genes -Class II MHC genes -Class III MHC genes -->Complement and inflammation proteins Allelic forms of MHC genes are inherited in linked groups called haplotypes -Each individual inherits one haplotype from each parent MHC Alleles are co-dominantly expressed -Both maternal and paternal MHC genes expressed in offspring cells -Gives organism best chance to have some capability of presenting all the possible antigen peptides it encounters -Transplantation somewhat difficult as humans are heterozygous at each locus Class I/II molecules exhibit diversity at individual AND species levels -Individuals express MHC alleles inherited from both parents -Alleles can also differ among individuals creating enormous diversity -This diversity provides flexibility in responding to unexpected environmental changes, now and in the future

MHC Expression Patterns

MHC molecules present both intracellular and extracellular antigens Class I presents intracellular antigen peptides -Includes self proteins -Provides a way for "checking" that cells are self and are generally healthy -Can show which cells have been infected with viruses or are abnormal and display those peptides on class I and activate Tc cells -Expression is found throughout the body, especially on all nucleated cells Class II presents extracellular antigen peptides -More restricted; generally found on cells involved in immune responses -Helps to direct responses against threats-things that shouldn't be in our systems-and display those peptides on class II and activate Th cells -Expression is primarily restricted to antigen-presenting cells, macrophages, B cells, and dendritic cells

MHC Inheritance Organization

MHC region is polygenic Heterozygous individuals express gene products encoded by alleles at each MHC gene locus Each parental chromosome's MHC alleles are codominantly expressed in the offspring MHC polymorphism differences tend to be clustered at amino acid locations within the groove sites

MHC Molecules Role

Major histocompatibility Complex These molecule are encoded by closely associated genes called the MHC locus, or just the MHC (aka HLA genes in humans) Peptide fragments that bind to MHC molecules are generated inside the cell following antigen digestion The antigenic peptide plus MHC molecule is transported to the cell surface, where it can be displayed and "presented" to a T cell whose TCR recognizes that particular peptide-MHC complex.

Thymocyte Review

Mature T lymphocytes have a diverse TCR repertoire that is tolerant to self-antigens yet restricted to self MHC Thymocytes "learn" MHC restriction in the thymus Cells are selected "positively" for their ability to respond to self MHC+ peptide appropriately Cells are selected "negatively" if they respond too strongly (ie bind with too high affinity) and are deleted/inactivated Cells that cannot bind to mHC fail to receive survival signals and die by neglect

Cross-presentation of Exogenous Antigens

Mechanism and functions of cross-presentation -Actual redirection method unclear To prevent redirection of self-antigens into APC pathways, dendritic cells may need "license" -If DC can present foreign antigen to CD4+ helper T cell, it gets license to redirect exogenous Ag into the endogenous pathway -"License" might be back/forth cytokine signal between the APC and helper T cell, a situation right for cross-presentation Dendritic cells appear to be the primary cross-presenting cell type -Exogenous antigens are redirected to endogenous presentation pathway -This allows for their presentation on MHC Class I molcules,p riming CD8+ T-cell receptors -Dendritic cells are the only APC to exhibit this activity in vivo

How/When do Memory Cells Arise

Memory cells may arise very early in immune response -Tcm cells may arise from or prior to Tem cells -Tem cells may be derived from fully differentiated effector cells -They may arise from asymmetrical division of activated T cells -Self-renewing memory stem cell populations may be generated during T cell activation. Memory cells are persistent over the years in the absence of antigen -IL-17 and IL-15 appear important to this homeostatic proliferation

CD4+/CD8+ Memory T Cell Differences

More memory CD8+ T cells than CD4+, but they proliferate more during their responses -Could also be due to difference in life span where CD4+ memory cells are not as long lived

T cell memory: Surface Protein Expression

Naive, effector, and memory T cells display broad differences in surface protein expression. 3 surface makers can differentiate the sets: CD44: increases in response to TCR-mediated activation signals -Expressed on Teff, Tem, Tcm, and Trm CD62L: adhesion protein that regulates homing to secondary lymphoid organs -Expressed on naive, Tscm, Tcm CCR7: Chemokine receptor that regulates homing to secondary lymphoid organs. -Expressed on naive, Tscm, Tcm CD69: C-type lectin that prevents immune cells from leaving tissue -Expressed on Tcm, Trm

Negative Selection-Central Tolerance

Negative selection (central tolerance) ensures self-tolerance Clonal deletion-induction of apoptosis in cells with too strong anti-self singaling/binding Tissue antigens screening -Autoimmune regulator (AIRE) protein induces expression of many tissue-specific proteins in medullary thymic epithelial cells -AIRE binds epigenetic marks on histones to recruit tfs -New T cells can be screened against these antigens safely in the thymus Other mechanism: -Clonal arrest: Autoreactive T cells are prevented from maturing farther -Clonal anergy: Autoreactive T cells are inactivated, not deleted -Clonal editing: Second or third chances at rearranging a non-self reactive TCR alpha gene Clonal deletion remains the best proven and most common method of tolerance induction in the thymus

+/- Selection Occurance

Negative selection can occur at various points in development Positively selected cells must express CCR7 chemokine receptor to move medulla for further development and selection/screening -Occurs in a more finite time/space Medullary region appears to be quite important in removing autoreactive T cells

Trm Cells

Permanent residents of previously infected tissues -Respond upon reinfection -CD8+ Trm found in multiple tissues

Tolerance

Prevention of an immune response against self structures Central tolerance: deletion of lymphocytes before they mature -Takes place in generative lymphoid organs -Limits development of autoreactive T and B cells Peripheral tolerance: either renders self reactive lymphocytes nonresponsive or actively generate inhibiting lymphocytes. -Occurs outside bone marrow and thymus -Regulates autoreactive cells in circulation -Regulatory CD4+ T cells

Antigen-Presenting Cell Types

Professional -Dendritic cells -Macrophages -B cells Nonprofessional -Fibroblasts (skin) -Glial cells (brain) -Pancreatic beta cells -Thymic epithelial cells -Intraepithelial lymphocytes -Vascular endothelial cells

Dryer and Bennett Hypothesis (1965)

Proposed that antibody heavy and light chains are encoded in two separate segments in the germ-line genome In order to form one complete polypeptide encoding gene, in B cell DNA, one V-region must be brought together with a C region. A revolutionary idea that proposed somatic cells undergoing recombination which could account for massive amount of Ab numbers. -"Modified germ-line" hypothesis

Proteasomes

Protease complexes which generate peptides Role in the endogenous pathway, MHC Class I Tagged proteins are fed into proteasomes -Ubiquitin proteins are used to "tag" intracellular proteins for constitutive proteasome degradation -A subtle variant known as an immunoproteasome (found in antigen-presenting cells) cleaves ubiquitinated proteins into fragments that pair better with MHC molecules

Peripheral Mechanisms of Tolerance

Protect against autoreactive thymocytes Some self-antigens are "hidden" because APCs lack the correct costimulatory molecules needed to initiate immune responses Some self-antigens are presented by non-APCs, preventing initiation of autoimmunity Strong self-antigen signaling through the TCR in the absence of co-stimulation may drive the T cells into anergy (nonresponsiveness)

Lymphoid-Specific Proteins

RAG 1/2: Antigen receptor gene recombinase complex. DNA cleavage is mediated by RAG1. Epigenetic targeting is directed by RAG2 -Deficiency: Severe combined immuno deficiency. Terminal deoxyribonucleotidyl Transferase (TdT): Adds non-templated (N) nucleotides to V-D and D-J joints of Ig heavy chain and all joints of TCR chains in a template independent manner. -Deficiency: Reduced N-nucleotide addition at coding joints.

Mechanism of V(D)J Recombination

Recombination is directed by signal sequences Recombination signal sequences (RSSs) flank each antibody gene segment. -Each has a conserved nonamer (9bases) and heptamer (7 bases) sequence -In-between the nonamer/heptamer lies either a 12 or a 23 bp spacer sequence (12 is one turn of the helix, 23 is two turns) -The spacing and arrangement dictates that a 12 bp RSS must pair with a 23 bp RSS for a recombination to occur ("12/23 Rule") Numerous other proteins are required for recombination, including several that are not unique to lymphocytes

Classes of Effectors

SLIDE 14, 16 The four "main" classes of effectors are: 1) T helper 1 (Th1) which targets intracellular and viral pathogens; T-Bet; tissue inflammation 2) Th2, which target helminths (worms); GATA3; allergy 3) Th17 which targets fungal and extracellular bacteria; RORyt; autoimmunity, tissue inflammation 4)Inducible regulatory T cells (iTregs) which have an opposing function in decreasing the inflammatory immune response; FoxP3; inhibits antitumor response Focus on these for EXAM Know MASTER GENE REGULATORS These classes cross regulate each other: -EX: Th17 can cross regulated Treg Th1, Th2, and Th17 effectors serve vital function in eliminating specific classes of pathogens while regulating T cells serve to "soothe inflammation and thus can be considered a special class of effector T cell. Additionally, effectors are inappropriately increased in situations of autoimmunity (Th1 and Th17) and allergies (Th2)

Costimulatory Signals

Signals are required for optimal T-cell activation and proliferation Signal 1: TCR Signaling -Antigen-specific TCR engagement Signal 2: Costimulatory Interaction -Contact with costimulatory ligands Signal 3: Cytokine Signaling -Cytokines directing T-cell differentiation into distinct effector cell types

TCR and BCR Receptors

Similarities: -Both have variable and constant regions -Both proteins can be membrane bound -Both receptors enable the specific recognition of antigen -Use surface molecules to recognize antigens Differences: -B cell receptors can recognize free (whole) antigen -T cell receptors only recognize "pieces" of antigen that are first positioned on the surface of other cells --->These antigen pieces or peptides are held within the binding groove of a cell-surface protein called the MHC molecule

Nonprotein Antigens

Some nonprotein antigens can be recognized by T cells -Presentation not via classic MHC molecules The CD1 and MHC Class I related protein MR1 family of nonclassical class one molecules can be present lipids, lipid-linked molecules, and for MR1 metabolites of vitamin B2 -Five human CD1 genes known; one MR1 gene -Very little polymorphism is displayed -most function similarly to MHC class II molecules

MHC Genotypes

Standardized MHC genotypes to control variability Syngeneic: Identical at all genetic loci Congenic: Genetically identical except at a single genetic region

T-Cell Activation: Overview

Successful T cell-APC interactions organize signaling molecules into an immunological synapse. TCR/MHC-peptide complexes and coreceptors centralize -Central supramolecular activating complex, cSMAC Adhesion molecules/bound ligands peripherally localize -Peripheral supramolecular activating complex, pSMAC

Signals: Memory Cell Commitment

T cell help seems to be required -IL-17, IL-15, and Wnt may play a role for Tscm -IL-2 important in generating effector memory cells (Tem)

T Cell Restriction

T cells are restricted to recognizing peptides presented in the context of self-MHC alleles CD8+/CD4+ T cells can only recognize peptides presented by MHC Class I/II respectively -The MHC haplotype of the presenting cell must match the haplotype of the T cell Two experimental models show self-MHC restriction in T cells -First model: gpigs. Trigger immune response, harvest cells on plate (macros) and from adherence column (T cells). Mix strained antigen-pulse macrophages with antigen primed T cells. Demonstrated that the ability of T cell to respond to antigen is depend on matching MHC -Second model: immunized mice with virus and then harvested spleen cells and inoculated plates with target cells. Lysis and Cr released analyzed. Histocompatibility between T cells and H2 cells. Whole antigens can't be recognized by T cells -Only processed antigen peptide fragments can be recognized

SUMMARY LECTURE 12

T cells must have antigen peptides presented to them for activation Understanding the molecules that perform this presentation is crucial to understanding T-cell activation How intracellular and extracellular antigens are processed for presentation

LECTURE 14 SUMMARY

T cells require 2 (or 3) signals for optimal activation/stimulation Signal 1=Result of antigen-specific TCR engagement (with APC) Signal 2= Delivered via contact with costimulatory ligands (on same APC (in cis)) -Signal 1/2 for optimal activation and proliferation -If T cell recieves signal 1 but not signal 2 the cell is rendered anergic, or unresponsive Signal 3=Provided by cytokines during T-cell differentiation (cis or trans) -Signal 3 is necessary to insure proper generation of effector function -Different cytokines = different responses Successful T-cell-APC interactions organize signaling molecules into an immunological synapse TCR/MHC peptide complexes and coreceptors centralize into a central supramolecular activating complex, cSMAC Adhesion molecules/bound ligands peripherally localize into a peripheral supramolecular activating complex, pSMAC T cell receptor is associated with CD3 molecules -TCR/CD3 is known as "T cell receptor complex" -Complex is made up of alpha and beta chains which facilitate antigen/MHC binding and the CD3 chains mediate signal transduction T cell activation begins with CD4 or CD8 bringing Lck into proximity with TCR/CD3. this initiates a kinase cascade, starting with the phosphorylation of ITAMs on CD3. -Phosphorylated ITAMs become docking sites for ZAP-70, which is phosphorylated and activated by Lck

2 Signal Hypothesis

T cells require antigen presentation as a first signal Other molecular interactions can provide the second required activation signal. Once activated, T cells differentiate into their effector forms -CD8+ T cells go on to become killer T cells -CD4+ T cells differentiate into several subsets

Positive and Negative Selection

T cells undergo positive and negative selection CD4+CD8+ DP thymocytes make up 80% of thymic cells -These cells undergo thymic selection Positive selection: Selects thymocytes bearing receptors capable of binding self-MHC molecules with low affinity, resulting in MHC selection. Negative Selection: Selects against thymocytes bearing high-affinity receptors for self-MHC/peptide complexes, resulting in self tolerance. Most cells (95%) fail positive selection and fail to receive needed survival signals -Die by apoptosis

Self-Tolerance Mechanisms: T-REG

T-REG cells negatively regulate immune responses -They belong to a subset of CD4 T cells characterized by expression of FoxP3 transcription factor T-REG cells function to: -Deplete the local area of stimulating cytokines -Produce inhibiting cytokines -Inhibit APC activity -Directly kill T cells

TCR Genes

TCR genes undergo a process of rearrangement very similar to that of IG genes. TCR expression is also controlled by allelic exclusion -The same machinery doesn't combine Ig genes in T cells, or TCR genes in B cells TCR gene expression is tightly regulated -Many of the same spatial benefits of enhancers and promoters found in B-cell alleles are found in T-cell alleles -Control of gene expression during development is very similar to that found in B cells

T Cell Receptor and Expression

TCR is a heterodimer with an alpha (a) and a beta (B) chain Expressed exclusively on T lymphocytes The beta chain gene setup was discovered first. While looking for the a chain genes and the gamma chain genes were discovered. -A small subset of T cells express a yo TCR rather than an aB TCR, but the two are structurally similar

Th17/Treg Cross-Regulation

TGF-B is a key cytokine for differentiation of both subsets IL-6 is the "switch", allowing RORyt to dominate and induce Th17 subset differentiation. A balance between two subsets is ideal. -Normal state could favor development of suppressive iTreg population to keep inflammation down. -Inflammation from an infection (leading to IL-6 production) would stimulate more antibacterial Th17 differentiation.

Metabolic pathways and T cell subsets

Th1, Th2, and Th17 primarily utilize glycolysis and glutaminolysis for their energy needs and absolutely require GLUT1 iTregs primarily utilize oxidative phosphorylation and are unaffected by GLUT1 deficiency Inhibition of fatty acid catabolism blunts iTreg differentiation and increased AMPK signaling (such as that induced by metformin) increase iTreg differentiation Th17 cells require acetyl coA carboxylase (ACC1), the rate-limiting enzyme in fatty acid synthesis, while iTreg differentiation is unaffected by ACC1 knockout. -The enzyme provides fa ligands that enhance RORyT, the lineage-defining "master" tf for Th17 cells, critical for their activity and IL-17 production -Increased ACC1 activity increases substrates for cholesterol metabolism. Specific oxysterols derived from cholesterol metabolism are potent activating ligands for RORyt, increasing RORyt transcriptional activity, and thus Th17 differentiation.

2 Signal Hypothesis: Clonal Anergy

The result if a costimulatory signal 2 is absent This helps provide tolerance -If only signal 1 is received, the cell is rendered nonresponsive -This might happen if a T cell isn't screened against a peripheral self-antigen during development

SUMMARY LECTURE 9

The story of antibodies and T-cell receptor structures and gene arrangements was one of the largest advances in immunology ever. Once gene arrangements were determined, discovering recombination mechanisms has proves just as crucial By understanding the arrangement and rearrangment of these genes we can understand much more about how B and T lymphocytes operate.

MHC restriction: Thymocytes

Thymocytes "learn" MHC restriction in the thymus Mice experiment Positive selection ensures MHC restriction -TCR that can bid MHC-peptide shifts from T cell to DP to SP -If TCR can bind to an MHC class II molecules, it also binds with the CD4 molecules, selecting the cell to the CD4+ subset -The opposite happens if the TCR binds to an MHC class I molecule, resulting in selection to the CD8+ subset

Thymocyte Receptors

Thymocytes can express either TCRaB or TCRyo receptors -TCRB rearrangements are one of the first to take place and one of the most likely to be productive -TCRaB outcomes are more likely than TCRyo -TCRyo are more common in fetal development

T Cells: Final Maturation Stages

Upregulation of Foxo1 transcription facor -Expression of Klf2, which upregulates sphingosine-1-phosphate (S1P) receptor which is required to help T cells leave the thymus -Foxo1 also upregulates IL-7R (giving survival signals) and CCR7 (a chemokine receptor that helps cells exit and move to the lymph nodes) T cells that have just exited the thymus are recent thymic emigrants (RTEs) -They're not as functionally mature (yet) as older cells-an active area of research

Adjuvants

Used for decades to enhance the effectiveness of vaccines -Usually, the adjuvant is delivered along with the vaccine Now it is understood that adjuvants exert their influence on the innate immune system, by increasing the expression of co-stimulatory ligands and polarizing cytokines by APCs. -These events can shape the consequences of T cell activation. A cytokine or agents that can induce an inflammatory response to enhance the vaccine activity.

Thymocyte Development

When T cells arrive in the thymus, they aren't technically T cells. -They can become NK cells, dendritic cells, B cells, and myeloid cells. A receptor known as NOTCH commits them to the T lineage -GATA-3 transcription factor becomes activated -Notch binding can commit to T lineage in vitro without the thymus being present Thymocytes progress through four DN stages -TCR rearrangement begins in the cortex at the DN2 stage -See table lecture 13 T cell

Combinatorial Diversity

Which heavy genes pair with which light genes -Adds great diversity in products

Selection Paradox

Why don't we delete all cells we positively selection? Affinity model: strength of signal received is critical Support found in the OT-I TCR transgenic mouse system -All TCRs are of one type that can recognize one peptide -MHC class I molecules one thymic epithelial cells have no, low, or high affinity for their peptide -Degree of selection for/against CD8+ SP T cells is determined Altered Peptide model: -Self-peptides produced by thymus epithelial cells are unique and distinct from peptides made by other cells -Thymocytes positively selected by such interactions wouldn't be negatively selected by later interactions -Still under investigation: some evidence that thymus cells process antigens differently from other cells