Innate Immunity and Pathogenic Bacteria

• Innate immunity combats new, previously unencountered bacteria.

T Cell-Mediated Immunity

• Neutrophils are the first responders, guided by chemoattractants, to infection sites.
• Complement activation occurs via the lectin or alternative pathway.
• C3b is activated to opsonize bacteria, facilitating neutrophil recognition.
• The MAC attack complex lyses bacteria by creating holes in their membranes.
• Opsonization involves neutrophils possessing receptors that recognize opsonized bacteria.
• Complement component C5a attracts neutrophils to the infection.
• Macrophages recognize bacteria using opsonic receptors and PRRs, leading to phagocytosis and inflammatory cytokine release, activating neutrophils.

Pathogenic Bacteria

• Pathogenic bacteria must circumvent the immune system to cause disease.
• The immune system's inability to clear bacteria defines pathogenicity.
• Dendritic cells (DCs) detect hidden bacterial antigens, activate T cells.

Immature Dendritic Cells

• Immature DCs reside in secondary lymphatic tissues and are specialized for antigen uptake.
• They exhibit high endocytic activity, capturing and processing antigens into small peptides.
• Immature DCs utilize receptors similar to macrophages, like PRRs.

Antigen Detection

• DCs determine if an antigen is associated with a pathogen through PRR signaling.
• Self/non-self discrimination is a theory on how the immune system identifies what to attack.
• DC maturation is triggered by danger signals.
• Histones leaking from cells indicate danger.
• DCs internalize bacteria, shutting down antigen uptake mechanisms and migrating to T-cell zones with antigen presentation.
• Naive T cells in secondary lymph nodes express CCR7, attracting migrating DCs.
• Mature DCs upregulate MHC I/II, CD80, and BB6 locate to the T cell zone to present antigen to T cells while diminishing phagocytosis.

Mature Dendritic Cells

• Mature DCs specialize in presenting antigens in the T cell zone of secondary lymphoid tissue.

Activation of Naive CD4+ T Cells

• Activation requires interaction with a mature DC presenting antigen via MHC II (signal 1).
• Costimulation is necessary (CD80/86; B7-1/B7-2), as CD28 on naive T cells binds to costimulatory molecules, providing a second signal.
• Without costimulation, T cells become inactive or die.
• Naive T cells are mature, having undergone thymic selection, and are ready to respond to antigen, but have yet to encounter any.

Three-Signal Hypothesis for T Cell Activation

• TCR-antigen specific activation signal
• Not all T cells will recognize an immature DC
• Only those with a TCR recognizing the antigen presented by the DC will be activated.
• If the TCR binds to antigen, a signal is provided, initiating the activation process.
• Signals 2 and 3 required, or the T cell becomes anergic
• Co-stimulatory signal: CD28 on T cells binds to B7 (CD80/86) on mature DCs.
• This provides the primary costimulatory signal for T cell activation.
• Leads to IL-2 production, enhancing T cell proliferation.
• ICOS on T cells interacts with ICOSL on DCs, aiding T cell survival and function.
• CTLA-4, a negative regulator, binds B7 with higher affinity than CD28, inhibiting T cell activation.
• CD40L on activated T cells binds to CD40 on DCs and B cells, enhancing B7 expression and immune response

Cytokine Signaling

• Cytokines determine T cell response and effector T cell differentiation.
• TCR signaling and costimulation (CD28-B7) upregulate IL-2 production, driving T cell proliferation.
• Some pathogens evade the immune response by surviving inside macrophages.
• Intracellular bacteria induces DCs to secrete IL-12/18/IFN gamma, promoting Th1 differentiation and IFN gamma and TNF production.
• Extracellular bacteria and fungi induce DCs to secrete IL-6 and TGF beta, promoting Th17 differentiation and IL-17 production.
• Viral infections induce IL-12 and IFN gamma secretion, promoting Th1 and CD8+ T cell differentiation. Resulting in CD8+ T cells killing infected cells.
• Parasitic infections trigger Th2 responses.
• IL-4 encourages Th2 differentiation and IL-4/5/13 production.

Professional Antigen-Presenting Cells (APCs)

• APCs include dendritic cells (DCs), macrophages, and B lymphocytes.
• Mature DCs migrate to T cell zones to interact with naive T cells.
• Macrophages present antigens to CD4+ T cells, aiding the effector phase, and localize to T cell zones of lymph nodes following activation.
• B lymphocytes present antigen to helper T cells, participating in humoral immunity and producing antibodies, which are essential for T cell-dependent antibody responses.

Key Features of Professional APCs

• MHC expression: MHC I (expressed by all nucleated cells, presenting endo antigen to CD8+) and MHC II (specialized for exo antigen to CD4+, expressed by DCs, macrophages, and B cells).
• Costimulatory signals: B7.1 (CD80) and B7.2 (CD86) are expressed by professional APCs upon activation, interacting with CD28 on naive T cells for full T cell activation.
• Activation of naive T cells.

Lymph Node Function in T Cell Activation

• Naive T cells express CCR7, recognizing CCL19/21 in secondary T cell zones.
• The signaling directs naive T cells to migrate and localize, efficiently scanning DCs.
• DCs also express CCR7 to present antigen to naive T cells.
• DCs are surrounded by naive CD4+ T cells, scanning for matching antigens.
• T cells recognizing the antigen bind to the MHC peptide complex and receive costimulation. This leads to activation and proliferation.

T Cell/DC Interactions in the T Cell Zone

• Immune synapse: Mature DCs show off their antigen presenting to the latching T cells
• Important for signal integration.
• Also called supramolecular activation complex (SMAC)
• Middle (cSMAC): contains TCR, costimulatory molecules, and coreceptors, stabilizing interaction.
• Outside (pSMAC): contains adhesion molecules (LFA1), forming a tight interaction

Experiment

• Lipid bilayers prepared with MHC and ICAM allow T cell adhesion.
• T cell adhesion occurs via ICAM-LFA-1 interaction.
• MHC-TCR interaction initiates signal 1.
• MHC-TCR complexes accumulate centrally (cSMAC).
• ICAM-LFA-1 interaction shifts to the periphery (pSMAC).
• The T cell forms a mature immune synapse with TCR concentrated centrally and adhesion molecules around it.
• Signaling and downstream activation are optimized by the reorganization.

Th1 T Cells

• Th1 T cells combat intracellular pathogens and activate macrophages.
• IL-12 and IFN gamma are good at inducing Th1 differentiation.
• These cells produce TNF and IFN gamma.

Effector T Cells

• Naive T cells activated in lymph nodes differentiate into effector T cells.
• Changes in homing parameters alters how they find infection sites.
• They transition from naive to effector T cells, decreasing L-selectin, increasing PSGL-1, and increasing VLA-4.
• They change chemokine receptors and decrease CCR7, increase CCR3/5 expression to be attracted to inflammatory sites.
• Effector T cells no longer require costimulation.
• They secrete cytokines like IFN gamma to activate macrophages, and TNF to promote inflammation.
• CD40L on T cells binds CD40 on macrophages, activating macrophage fusion, and lysosome.
• CD40L and IFN gamma are central to macrophage activation.

Activation of Naive CD8+ T Cells

• This happens in the T cell zone of secondary lymphoid tissue via licensed mature DCs.
• The cells engage with self cells in the periphery, searching for infected or sick cells.

Key points

• Cytotoxic cells target the cell not the pathogen
• CD4 recognizes antigen presented by DC on MHCII
• The CD4 binds to CD40 on the DC which provides a signal back to the DC
• This process enhances the activation of the CD8 by increasng costimulatory molecules and IL12
• CD8 recognizes antigen on MHC1 presented by DC (previously licensed by CD4) with cytokines.
• The CD8+ cells activate and differentiate into to cytotoxic T lymphocytes.

CD8+ Cytotoxic T Lymphocyte

• Survey intracellular infections and induce apoptosis.
• Similar to NK cells but antigen-specific, targeting cells presenting specific antigen on MHC1.
• All nucleated cells express MHC1, presenting peptides from inside the cell including pathogens. If infected, T cells recognize as evidence of infection.
• CD8+ T cells forms tight immune synapse w infected cells
• alr an effector cell so no need for costimulatory signals*
• Perforin and granzyme pathway
  • Perforin pathway forms pore in cell membrane and granzyme enter activating caspases
• Fas-FasL (Receptor mediated apoptosis)
  • CD8 T cells do this repeatedly and selectively

Humoral Immunity

• Could induce immunity to something by taking its blood
  • Bottom: cells, when transferred, associated with B cells
  • Top: fluid, where antibodies are, passive/humoral immunity
• Humoral immunity = B cell mediated immunity

Naive B cell

• Bear cell surface IgM, does not secrete antibody
• Mature cell that hasn't encountered what it recognizes yet
• Surface BCR: IgM+, IgD+
  • Same v region with 2 diff constant regions
  • mRNA splicing of long mRNA transcript
  • Isotype of BCR determined by heavy chain constant region expressed
• Naive B cell is the ONLY scenario where 2 isotypes are present at the same time

Plasmablast

• Differentiated b cells begin to secrete antibodies
• Not yet lost the capacity to proliferate and still bear cell surface BCRs

Plasma Cells

• Differentiated that can no longer divide
• Little to no cell surface immunoglobulin bc mRNA manipulation
• Rapidly secrete large numbers of antibody molecules
  • Removes transmembrane region and make soluble BCR
  • mRNA splicing to remove terminal transmembrane region

Activating a B cell with specific antigen

• IgM starting point
  • Pentameric
  • Low affinity binding to antigen
• End point: tailored Ab response
  • Class switch recombination (CSR)
    • Change Ab isotype
      • Manipulating part of the gene that encodes constant regions
  • Somatic hypermutation (SHM)
    • Targeted random mutations in heavy chain variable region
      • Direct mutation in the VDJ region
    • Combined with selection -> improved affinity = affinity maturation
      • Low -> high affinity to the same antigen
    • Activation induced by AID
    • C to U conversion
      • G:U mismatch
      • diff DNA repair path to generate diversity uracil DNA glycosylase path
        • cuts out U
        • error prone path to fill in gaps
        • mismatch repair path larger gaps filled in error prone path
          • Lesion w mismatch cut out and filled in randomly
            • Significant and random mutations mutations introduced by AID are processed by DNA repair mechanism for diversity can result in dsDNA break for recombination can result in random mutations - works on ssDNA NOT dsDNA - likes the AGCT motif but may work on other sequences

Class switch recomb

• Process of one isotype to another
• Permanent genetic change that occurs in the heavy chain gene of B cell
• Enable to produce diff types of antibodies wo expressing multiple isotypes
• Irreversible genomic recomb at switch regions on the heavy chain
  • Switch region are open and accessible due to upstream promoter generating sterile transcripts
  • Once DNA unwound through transcription
    • AID act on both strands and introduce dsbreak on ssDNA
  • Just like RAG, once dsbreak, everything in between cut out
• NOT mRNA splicing
• Choice of which constant region to switch to influenced by cytokines and TFs they activate
  • Cytokines determine which switch regions are accessible
  • Important for tailoring the response to engage different effector mechanisms
  • A given B cell clone can switch more than once
    • Unidirectional as long as its going downstream
• IL4 -> IgE, IL5 -> IgA

Problem of B cell receptors

• RAG mediated VDJ recomb
• Baseline low affinity binding
  • WHY: IgM is the first immunoglobulin produced (typically low affinity)
    • RAG mediated VDJ recomb generate baseline diversity of BCR that bind to wide array of antigens wo tuning of binding affinity t cells undergo thymic selection eliminating cells with weak or excessively strong binding but b cells don't
  • Therefore generally low and non specific
    • “Raw material” for further improvement
  • For effective effector function, antibody needs to bind strongly to target bc low affinity antibodies aren't good at triggering immune response
• After V(D)J recombination, BCR affinity is initially low and requires fine-tuning through somatic hypermutation to improve binding strength and ensure an effective immune response. to enhance affinity, SHM
• Occurs primarily in the variable region responsible for binding site (Fab) B cells suppress standard DNa repair pathways and use error prone repair path to process mutation by AID
  • Error prone repair lead to mutations AID induced mutations may cause adult leukemia
    • however usually mutations only in the BCR gene

Somatic Hypermutation

• Targeted to the variable region of the heavy chain gene
• ssDNA - opened up by transcription of the gene
• Mutation hot spots in the CDR coding regions
  • Based on presence of sequences preferred by AID
• Of target SHM can result in cancer

Affinity maturation

• Process of actually increasing antibody affinity for the target antigen via SHM success of producing better antibody response depends on B cell antigen and context in which the B cell is activated diff types of antigen and/or activation stimuli variably drive two processes

Activating a B cell

relies on 3 primary signals

• antigen specific signal through BCR if antigen high conc or strong affinity, cluster the BCR, providing potent activating signal to B cell
• PRRs amplifiers activation signals
• Int w T cells via CD40/40L activated T cells in with B cells, helping them activate and start antibody production cytokine influence
  • surrounding cytokine influence type of antibodies a B cell produce

thymus= t cells thymus dependent antigen vs thymus independent antigens (more specialized and can activate b cells themselves)

TI-1 antigen

• antigen acts as TLRL
  • can be protein or carb physically linked to TLRL
• stimulates naive B cells by binding to BCR and activating TLR when b cell encounter these antigens, upregulate TLR expression, making them
  • 1000x more sensitive to TLRL -> B cell activated by much lower conc of antigen high conc of TLRL-> polyclonal activation (activate B cells non specifically), proliferate and produce antibodies clonal activation occurs when BCR binds to antigen
• activation antigen specific -does not involve T cell help and lack selection mechanism leading to low affinity antibody response, IgM ex/ LPS, flagellin

TI-2 antigens

• Activate B cells through extensive BCR crosslinking
  • Repetitive molecules featuring multiple epitopes that crosslink BCR results in strong activation signal to the B cell -> proliferation and antibody causing B cell to activate wo T cell: purely BCR signaling
• Initial antibody predominantly IgM w minimal class switch (IgG may be present)
  • Bc mechanism BCR crosslinking, no affinity selection present

Thymus independent activation

• Doesn't require int w T cells and can occur outside 2nd lymphoid tissue
• B-1 cells and MZ B cells are involved, specialized to respond to Tl antigens, producing IgM
  • TI1 require BCR engagement and TLR signaling
  • T12 leads to clonal activation where only B cells that recognize antigen via their BCR become highly sensitive to TLR signals result in IgM production w minimal class switch and low affinity antibodies activation through BCR hypercrosslinking independent of TLRs mostly IgM production B1 cells found in cavities, produce IgM w low affinity
    • First line of defense MZ B cells
      • encounter antigens early before getting into T cell zone and involved in both TI and TD activation

Thymus Dependent activation

• Involvement of T cells required specific for linked antigen
  • Activation occurs in secondary lymphoid, particularly lymph nodes
• B cells in follicles (CXCR5) interact w T cells that migrated to T cell zone (CCR7)
• CCR7 responds to CCL19/21
  • CXCR5 follow CXCL13 int lead to class switching, SHM and affinity maturation follicular DC

Lymph Node

• Do not activate T cells or process antigen to present on MHC
• Dont express co stimulation
• Display unprocessed antigens on surfaces using complement receptors
• Antigen on surface helps naive B cells in follicles to encounter and recognize specific antigen
• Facilitates high affinity antibody response by T and B cells specific for same antigen Naive B cells are all in a follicle bc all express CXCR5 Naive T cells are in the t cell zone bc they express CCR7 Ag enter the lymph node through lymphatic system and transported to the subcapsular sinus If Ag small and soluble, diffuse freely When Ag large
• subcapsular sinus macrophages capture the antigen and transport it across into follicles Within the follicle, B cells constantly scanning for antigens that match their BCR
• When bound BCR signal, but alone usually not enough to activate B cell After initial antigen binding, B cell upregulates CCR7 and EBI2 allowing it to migrate to the border of the T cell zone in search of help from T cells T cells activated when int w DC -> clonal expansion Activated T cell express CXCR5 while maintaining CCR7 enabling them to move back towards border Border region where recently activated b and t cells goes to Once B cells migrated to the border, they act as APCs selective antigen presentation crucial for T cell activation

B cell as an APC

• Potent APC to CD4 T cells but have limited range of antigens they can present
  • Antigen uptake occurs primarily through BCR binding to antigens
• Linked recognition
• B cell must bind antigen via BCR and present it to T cells
  • T cell must recognize the same antigen or a protein physically associated with the antigen via its TCR T cell help is essential for full activation of B cells

Linked Recognition

• B cells must bind antigen via BCR and present it to T cells.
• T cells engage with the same or a physically associated antigen via the T cell receptor (TCR).
• T cell help is essential for B cell activation.

Activation Requirements

• B cells' antigen specificity stems from the BCR. Resulting antibodies recognize and bind the same antigen the BCR initially bound.
• What remains constant is antigen specificity.
• All that is related to the antibody is molecules important to get b cells and going, through CD40/40L

Fate choices

• T cells determine the target
• At the border of the follicles, b and t cell forms strong immune synapse where signals are coming in and fully activating the b cells -> proliferation

Some differentiate into short lived plasma blasts - low affinity IgM

Memory cells

• No class switch/SHM but may run into the same one again
• Mostly IgM OR goes back into the middle of the follicle has to downregulate CCR7, EBI2 and only express CXCR5 once goes back, committed to make germinal centre once class switch induced, induced prior to formation of the germinal centre formation class switch dependent on the cytokine available what kind of induced inflammation -> diff cytokine -> influence isotype it'll switch into fate choice controlled by balance of transcriptional regulators t follicular helper cell express BCL6 plasma cell, express BLIMP1

In T cell DEPENDENT responses

• Occurs AFTER B and T int
• Before terminal centre formation
• Second co signal needed for full activation
  • CD40 in T dependent response TLR in T independent response drives proliferation, class switching, differentiation into antibody secreting plasma cells

T cell fate decisions

• T follicular helper cells (Tfh) decreased CCR7, CXCR5+ => migrate towards B cell follicle provides essential T cell help to GC B cells secrete IL4 and 21 engage in ongoing int w B cells via CD40/40L signaling for B cell activation, class switching and affinity maturation Tfh - BCI6 (supports Tfh differentiation and GC function)
• Enters the germinal center
  • Helps b cell undergo SHM and affinity maturation
    • Promotes class switching Support selection of high affinity B cell clones
      • wo Tfh, germinal center response impaired-> defective antibody production downregulate CCR7 and keep CXCR5 so goes to the middle of the follicle controlled by counter expression of BCL6 and BLIMP1 BCL6 expressed by both germinal centre and Tfh
    • Teff - BLIMP

Germinal center

• Source of large high affinity class switched b cell responses
  • Structured to generate high affinity class switch response
    • B cells no longer express IgD

Dark Zone

• Where B cells proliferate.
  • Express Bcl6 here.
  • SHM occurs here - mediated by AID.
    • AID-related random heavy and light chain variable region

Light Zone

• contains FDCs which present antigen to B cells and most Tfh.
• This is the furthest zone from the T cell zone.
  • B cells compete for antigen and Tfh help.

Cyclic Re-Entry

Is dependent on CXCR4/12. B cells bind with antigen. These receive a signal, entering the dark zone to either mutate further or become a plasma/memory cell. B cells upregulate CXCR4 and downregulate CXCR12 to go to the dark zone or vice versa.

B cell output is dependent on survival signals and diff choices

• 1. Recommitment to GC
  • Cycle back to the dark zone for one round of affinity maturation.
• 2. Plasma cells
• Terminally-differentiated to produce antibodies.
• Capable of relocating to the body.
• 3. Memory B cells
• Undifferentiated .
• Capable of responding rapidly upon re-exposure with a better version that have been switched.

B cell mechanism

• Require Ab production
• Antibody structure determines its effecter functions
• Cytokine production is important for its mechanism like IL10 - Inhibitory cytokine, and IL6 Inflammatory Cytokine
• agglutination and neutralization are the only things antibody can do all by itself
• 1. Agglutination*
• Physical clumping
• Dependent on affinity binding
  • FAb region involved! FAb region involved!
• 2. Neutralization*
• Physical blocks
• Dependent on high affinity binding
  • FAb region involved!
• 3. Complement Activation*
• FC region based
• Trigger Classical Complement cascade
• Leads to opsonization, inflammation and lysis.
• C1q binds to Ab, activating the cascade
• Require multiple Ag for activation.
• 4. FC Receptor-Mediated Function*
• Opsonization
• Phagocytes use their FCgammaR to recognize the Ab once made.
  • Ab then coat the pathogen
• Phagocytes then bind to them
• Enhances Destroying the pathogen via FCgammaR Ab-Dependent Cell Cytoxicity (ADCC) - NK Cells use FCgammaR3 (CD16) to detect the opsonized cells, inducing apoptosis FcER and Cell Degranulation
• IgE and FCER = * Only receptor to bind Antibody without Ag*.
• Releases histamine if allergen crosslinks

Fc region receptors

FcgammaRI (CD64)

• Phagocytosis
• Cell activation
• Activating respiratory burst
• ADCC
• IgE

FcgammatR3 (CD16)

• Receptor on NK cells - mediating ADCC
• cell activation for microbial killing
• and cytokine production
• recognizes IgG
• uses ITAMs on gamma chain

FCERI - Degranulation

• Granulocytes, mast
• Recongnizes IgE Uses ITAMS for activation Trigger histamine release for mast cells

Switching Ab isotopes

• Usually associated with T cell help
• depends of the isotopes and generated cytokines
• is what is needed to fight the threat
  • influences effector response
  • removes the transmembrane region - surface bound BCR but soluble Ab

Plasma change to make soluble BCR

full differential with no surface BCR

IgM

• First is made in TD response
• Most common in TI response
• Low affinity
• High avidity
• Large, in the blood
• Monomer creates a pentamer overcome poor affinity
• Good at activating complement

IgD

• • Rare, higher in airway compnent
• Higher conc

IgG

• Smaller and high affinity
  • Found in circulation and extracellular space*
• Went though Terminal center to have high affinity

IgE

• Binds to FCER1 Central to mast cell surveillance of epithelia binds antibody high affinity to cells without circulates .
• Important for parasite and allergy.

IgA

• A monomer that circulates
• Is dimeric *mucosal barrier
• pglR transports it
• produced near mucoidal layers and binds mucoidal surfaces
• Combined with a chain to make a dimer
• plays a part In maternal milk

all engage with Fc receptor

Immune dynamics and memory

• If the virus isn't in check there an initiated response with the adaptive

Th1 and Th2 cell responses

• conditions described mostly derived from experiments when took cells out and on dish* Naive T cell signal 1 and 2 for proliferate What that cell needs to decide for direction not like cytokine at all.
• not made much in and does happen.
• Cytokines prioritize an response against diff types of pathogens.* HOWEVER If want to push hard for TH 1 you can blocking the Ab on IL4 so the IL4 is not the system. pathways back and support themselves. TF Beta a. suppresses Immune responses signals bias to that fate then enforcing a strict.

Th1 IL 12 and 18 IF Ny Itbet Ifg Ny, TNF enhances APC enhances Tc activation protect intracellular pathogens

Th 3 Ig4 Gata 3 Ig 4/5/ 13 Extra cellular bacteria

TGFb //16 //23 and RORyt // a 17 /22 fugal and extracellular

Reg Cell *treg

foxp //3 beta //2

bacterial infection inhibits any inflammation

Tfh cell and helps with germination enter

Bcl6 //6 //21 //4//24

• T bet is the Transciptioned Regulator

• In order to promote a response you have to eliminate on the others Both actively balances

T cell types

• Ensure response

• • TH 1 is by Nkt and Nki
• Helping by cd8
• activates though cd 40 and

TH1

• pathogen and y Cd8 T DC Macro TIR switch

• T 2 Extra* ++1 I 4 MASt BASOPHILS IGE switch

Th17 vs Treg //1 7

• Treg - INhibitory* Beta Fox
• *TR 1 7- In I B beta
• if Il 6 present can // 17/ ++8/ // 1 g CD 1* EFFector does not the home to Second

• the expression goes with the ++ to mad cam 1

B response is plasma cell

• If you dont want it
• go though the *
• **Downregulates If * the of for and *
• Contraction reduction to
• lack the by ***
• Is The *
• role Re initaiatlize * , for tissue stays
• the Second is the*
• all *** #is and ##*

Immune impriatning

• The old stuff
• Involves what *

the to with

is When **

• emerge is //12 *** the I'the/

Peripheral to to for ***

• is

• Cd //8 is THE

to ( DCs) by