MCB 150 Exam 1 Review

Soooooo manyyyyyy termmmmmmsssss.

Jenner’s Experiment and what it demonstrates

Jenner injected a boy with cowpox fluid, waited several weeks, and then he injected the boy with smallpox, resulting in no infection. This demonstrated immune memory and forms the basis of vaccines

Four main tasks of the immune system

1. Recognizing an infection

2. Containing it via immune effector functions

3. Regulating itself to limit host damage

4. Generating immune memory

Hematopoiesis

The process of producing diverse immune cells from multipotent stem cells in the bone marrow

Main features of innate vs adaptive immunity

Innate immunity: provides a rapid response (minutes to hours) utilizing macrophages, neutrophils, and dendritic cells. These cells express Pattern Recognition Receptors (PRRs) with predetermined specificities shaped by evolution to recognized conserved microbial features. A single innate cell can express multiple PRRs (in short, it’s fast, uses those types of cells, and uses PRRs)

Adaptive immunity: Takes days to develop, but produces immune memory. It relies on T cells and B cells. Unlike PRRs, we have T cell receptors (TCRs) and B cell receptors (BCRs) that are generated randomly via VDJ recombination, meaning each cell has only one exquisitely unique receptor specificity. Through clonal selection, when a specific T or B cell gets activated, its antigen leads to massive proliferation, which generates many identical cells and memory cells (in short, takes much longer, has TCRs/BCRs, and is very specific)

Main features of PRRs vs Antigen Receptors

PRRs are innate and recognize PAMPs (Pathogen-Associated Molecular Patterns), which are conserved structural motifs shared by whole classes of microbes. BCRs recognize native, intact antigens like proteins, polysaccharides, or lipids in their natural 3D shape floating in extracellular spaces. TCRs recognized processed (or degraded) peptide fragments bound to MHC molecules

Inflammation

When immune cells release cytokines and chemokines upon detecting bacteria, leading to vasodilation, increased vascular permeability, and the recruitment of inflammatory cells to tissues

Clonal selection

When a specific T cell or B cell gets activated, it leads to massive proliferation so you get many identical cells memory cells

What is the purpose of the primary lymphoid organs, secondary lymphoid organs, and lymphatics?

Primary lymphoid organs are where immune cells develop (bone marrow, thymus), secondary lymphoid organs provide an organized environment where cells interact with antigens to start adaptive responses (spleen, lymph nodes), and lymphatics drain fluid, cells, and antigens from the tissues to the lymph nodes

How do dendritic cells link activation of the innate and adaptive immunity?

Dendritic cells engulf pathogens and sense them via PRRs. PRR activation causes them to migrate to lymph nodes, upregulate costimulatory molecules, and present antigen peptides on MHC molecules to activate T cells

What are the effector mechanisms of adaptive immunity?

B cells secrete antibodies (a secreted form of the BCR) that bind directly to antigens to neutralize or block them. Cytotoxic T cells directly kill infected host cells. Helper T cells provide crucial assistance by helping macrophages eliminate bacteria and helping B cells become activated

Vaccine

A substance used to elicit an immune response and establish long-lasting memory against a pathogen without causing a disease

Immune effector functions

The active mechanisms used by immune cells to eliminate pathogens (phagocytosis, cytokine release, or target cell lysis)

Immune memory

The capacity of the adaptive immune system to respond faster and more aggressively upon secondary exposure to a pathogen

Autoimmunity

An inappropriate immune response directed against self-tissue due to a breakdown in self-tolerance

Pattern recognition receptors (PRRs)

Invariant, germline-encoded receptors on innate cells that detect conserved microbial features (PAMPs)

Macrophages

Long-lived phagocytotic tissue-resident cells that engulf pathogens, clean up debris, and trigger inflammation

Neutrophils

Abundant, short-lived innate phagocytes that rapidly migrate to acute infection sites to kill bacteria

Dendritic cells

Antigen-presenting cells that ingest microbes at tissue sites and migrate to lymph nodes to activate naive T cells

T cells

Adaptive lymphocytes that develop in the thymus and execute cell-mediated immune responses (T for thymus)

B cells

Adaptive lymphocytes that develop in the bone marrow and produce antibodies (B for bone marrow)

Antigen receptors

Clonally distributed, highly variable surface receptors (BCRs and TCRs) built via VDJ recombination

B cell receptor (BCR)

Membrane-bound immunoglobin on B cells that binds extracellular antigens directly

T cell receptor (TCR)

Surface receptor on T cells that recognizes peptide-MHC complexes

Antibodies

Secreted forms of the BCR released by effector B cells (plasma cells) into body fluids

Antigen

Any molecule or structure capable of being specifically bound by a BCR, antibody, or TCR

Cytotoxic T cell

T cell that recognizes peptide-MHC Class I complexes and directly kills infected or altered cells

Helper T Cell

T cell that recognizes peptide MHC-Class II complexes and releases cytokines to activate macrophages and B cells

How are epithelial surfaces a barrier to infection?

They utilize mechanical mechanisms (tight junctions, mucus movement), chemical defenses (low pH, lysozyme, antimicrobial peptides), and microbiological elements (normal microbiota) to prevent pathogen attachment, flush microbes away, and directly damage them

How is proteolysis used to regulate complement activation? Give an example with C3.

Complement proteins remain inactive until cleaved by proteases. Cleaving C3 exposes a highly reactive, unstable thioester bond in the C3b fragment that allows it to covalently attach to microbial surfaces

What are the three pathways that activate complement?

Classical Pathway (antibodies binding to a microbe), Lectin Pathway (host lectins binding to specific microbial carbohydrates), and the Alternative Pathway (relies on spontaneous C3 hydrolysis or the amplification of C3b already deposited on a microbe)

What are the ways to generate a C3 convertase?

The Lectin and Classical pathways utilize cleaved C4 and C2 to form the C4b2a convertase. The alternative pathway utilizes C3b and cleaved Factor B to form the C3bBb convertase, which requires stabilization by properdin. Spontaneous C3 hydrolysis also creates a low-level fluid-phase C3(H2O)Bb convertase.

At this point, just draw out the full pathway and memorize it

What are the regulatory proteins that prevent complement activation on host cells?

Host cells utilize membrane proteins (DAF, CR1) and recruit soluble proteins (Factor H, C4BP) that forcefully dissociate C3 convertases. Furthermore, host cofactors like MCP, CR1, Factor H, and C4BP recruit the protease Factor I to permanently degrade C3b and C4b into inactive fragments

What are some ways to generate a C5 convertase?

A C5 convertase is formed when a C3b molecule binds directly to an existing C3 convertase, generating either C4b2a3b for the Classical/Lectin pathways or C3b2Bb for the Alternative pathway

What are the outcomes of complement activation?

Activation drives inflammation via the recruitment of immune cells by C3a and C5a, opsonizes pathogens with C3b to promote phagocytosis, and directly ruptures pathogen membranes via the C5b-initiatied Membrane Attack Complex (MAC)

What is the family of C’ receptors expressed by host cells?

Host immune cells express specific receptors (such as CR1, CR2, CR3, and CR4) that recognize complement-tagged pathogens to heavily stimulate phagocytosis and enhance immune cell responses

How do pathogens evade C’?

Pathogens successfully survive innate immunity by utilizing mechanisms to disrupt pathway initiation, bind and inactive complement proteins, degrade complement components, or deceptively mimic/co-opt host regulatory proteins

Lysozyme

An antimicrobial enzyme secreted at mucosal surfaces that effectively destroys bacterial cell walls by cleaving peptidoglycan, particularly in gram-positive bacteria

Antimicrobial peptides

Small secreted peptides, such as defensins and cathelicidins, that undergo regulated cleavage to form pores that disrupt microbial membranes

Opsonization

The critical process of coating a pathogen with molecules like C3b, marking it for efficient uptake by phagocytic cells

Phagocytosis

The mechanism by which specialized immune cells engulf and destroy invading pathogens

C3

A central complement protein that gets cleaved into C3a and C3b to simultaneously drive inflammation, opsonization, and further downstream complement activation

C3 convertase (C4b2a, C3bBb)

An assembled enzyme complex that cleaves C3 into C3a and C3b, acting as the vital convergence point for all three complement activation pathways

Classical pathway

A complement activation cascade rapidly initiated when C1q binds to the Fc portion of antibodies already attached to a microbial surface

Lectin Pathway

A complement activation cascade triggered when host proteins like mannose-binding lectin (MBL) or ficolins bind to specific carbohydrates on a microbe

Alternative Pathway

A highly self-amplifying complement pathway initiated by the spontaneous hydrolysis of C3 or the continuous deposition of C3b on microbial surfaces

Decay accelerating factor (DAF)

A host cell membrane protein that protects the host by causing the dissociation of C3 convertases

Complement Receptor 1 (CR1)

A versatile host cell receptor that stimulates phagocytosis of C3b-tagged pathogens while also promoting the decay of C3 convertases

Factor H

A selectively binding soluble host protein that blocks C3b from forming convertases and simultaneously recruits Factor I to degrade C3b

C4 binding protein (C4BP)

A soluble host protein that protects host cells by binding C4b to block convertase assembly and recruiting Factor I for C4b degradation

Factor I

A constitutively active soluble serine protease that selectively degrades membrane-associated C3b and C4b when properly recruited by host cofactors

Membrane cofactor of proteolysis (MCP)

A host membrane protein dedicated to recruiting Factor I to efficiently degrade localized C3b and C4b

C5 convertase

An enzyme generated by adding C3b to a C3 convertase that cleaves C5 to trigger deep inflammation and initiate the membrane attack complex (MAC)

C’ receptor

Specialized proteins on immune cells that enable the recognition and subsequent destruction of pathogens coated in complement fragments

What are the traditional and modern methods to make a knockout mouse?

Traditional methods disrupt a gene in embryonic stem cell using homologous recombination before blastocyst injection, while modern methods directly inject CRISPR/Cas9 and guide RNA into embryos to induce sequence deletions or replacements

What does quantitative RT-PCR do?

It measures the precise expression levels of specific genes in a tissue sample (basically measures the amount of mRNA you have)

Immunofluorescence microscopy

An imaging tool that utilizes fluorescently labeled antibodies to visually confirm the presence and localization of specific proteins within a biological sample

Fluorescence in situ hybridization

A molecular visualization technique that uses targeted fluorescent probes to detect specific nucleic acid sequences, such as using 16S rDNA probes to spot intestinal microbiota

Strategies of innate vs adaptive immune recognition

Innate immune recognition relies on germline-encoded PRRs that posses limited diversity and are expressed in a non-clonal fashion on cells like macrophages, dendritic cells, and granulocytes. PRRs recognize PAMPs (Pathogen-associated molecular patterns). PRRs’ activation provides the immune system with immediate information about the nature of the pathogen due to the fact they target specific microbial features.

Adaptive immune recognition relies on somatically generated, clonally expressed antigen receptors with enormous diversity. Adaptive specificities are random, can recognize self or non-self antigens, and they do not inherently provide information about the antigen’s origin or nature

What are pattern recognition receptors (PRRs)?

PRRs recognize PAMPs and provide immediate information about the nature of the pathogen. The specificities of these receptors are genetically predetermined through natural detection to detect conserved, essential products of microbial metabolism (again, PAMPs). Because they are genetically predetermined, they possess limited diveristy

How were PRRs discovered, and what’s the immunologist’s dirty little secret? Mention the Drosophila Toll, C3H/H3J mouse as well.

The Immunologist’s dirty little secret is the observation that purified proteins require the addition of dead bacteria (an adjuvant) to stimulate strong adaptive immune responses. This ultimately led to the discovery of specific receptors that recognize evolutionary conserved, difficult-to-alter microbial components (PAMPs), like lipopolysaccharide (LPS), flagellin, and viral nucleic acids.

In the Drosophila Toll, researchers discovered that mutant fruit flies lacking the Toll gene failed to induce antimicrobial peptides (like Drosomycin) and were highly susceptible to fungal infections. In the C3H/HeJ mouse, this inbred mouse strain was completely unresponsive to LPS. Beutler’s group discovered that a single point mutation in the TLR4 gene was responsible, identifying TLR4 as the mammalian LPS receptor

What are the major PRR families and the cellular compartments in which they function? There’s 5.

1. Cell surface TLRs: Detect extracellular microbes. Specifically, TLR2 recognizes lipopeptides, TLR4 recognizes LPS, and TLR5 recognizes flagellin

2. Endosomal TLRs: Detect nucleic acids from phagocytosed microbes. TLR3 binds dsRNA, TLR7 and TLR8 bind ssRNA, and TLR9 binds DNA containing unmethylated CpG motifs

3. NOD-like Receptors (NLRs): Cytosolic censors. Nod1 and Nod2 detect degraded peptidoglycan fragments from bacterial cell walls

4. RIG-I-like Receptors (RLRs): Cytosolic sensors of viral RNA. RIG-I detects uncapped RNA with a 5’-triphosphate, and MDA-5 detects viral dsRNA

5. cGAS: A cytosolic enzyme that detects ds viral or bacterial DNA

What are some examples and characteristics of PAMPs?

Lipopolysaccharides (LPS), flagellin, and viral nucleic acids. These are evolutionary conserved, difficult-to-alter microbial components

Describe signal transduction by PRRs. What are the key signaling components and transcription factors? If you know nothing else, know cGAS-STING. But in total, there are four.

TLRs: Dimerization recruits the MyD88 adaptor (activation the NF-κB transcription factor to produce pro-inflammatory cytokines like TNF and IL-6) or the TRIF adaptor (used by TLR3 and TLR4 to activate IRFs, including antiviral Type I interferons)

Nod1/2: Utilize the RIP2 adaptor to activate NF-κB and drive inflammatory gene expression

RLRs (RIG-I/MDA-5): Bind the MAVS adaptor on mitochondria to activate both NF-κB and IRF3

cGAS-STING: cGAS binds to cytosolic DNA and synthesizes the second messenger cGAMP. cGAMP binds the STING adaptor on the ER membrane, which activates the TBK1 kinase to phosphorylate IRF3, leading to Type I interferon production

What are the different outcomes of PRR activation (including inflammasomes)?

Certain NLRs, such as NLRP1, NLRP3, NAIP/NLRC4, and Pyrin, assemble into a multiprotein complex called the inflammasome in response to cytosolic PAMPs or the detection of dangerous pathogen activities (e.g., protease activity, potassium efflux, or Rho GTPase manipulation). The inflammasome recruits the ASC adaptor and the enzyme Caspase-1. Active Caspase-1 cleaves the pro-forms of IL-1β and IL-18—which require a prior TLR "priming" signal for their transcription—as well as Gasdermin D. Cleaved Gasdermin D forms structural pores in the host cell membrane, releasing the cytokines and executing a highly inflammatory, lytic cell death known as pyroptosis.

PRRs

Pattern Recognition Receptors that recognize PAMPs that ultimately lead to immune responses from the innate immunity

PAMPs

Pathogen-associated Molecular Patterns that are evolutionarily conserved, difficult-to-alter microbial components like lipopolysaccharide (LPS), flagellin, and viral nucleic acids

TLRs and their ligands

TLR2: lipopeptides

TLR3: dsRNA

TLR4: LPS

TLR5: flagellin

TLR7: ssRNA

TLR8: ssRNA

TLR9: DNA

Nod1/2 and their ligands

They detect degraded peptidoglycan fragments from bacterial cell walls

RLRs and their ligands

RIG-I detects uncapped RNA with a 5’ triphosphate, and MDA detects viral dsRNA

cGAS and its ligand

ds viral or bacterial DNA

Inflammasomes

NAIP/NLRC4, NLRP1, NLRP3, Pyrin

Caspase-1

Caspase-1 cleaves the pro-forms of IL-1β and IL-18—which require a prior TLR "priming" signal for their transcription—as well as Gasdermin D. It basically does pyroptosis

Pyroptosis

A highly inflammatory, lytic cell death

GasderminD

Along with a TLR priming signal for the transcription of IL-1β and IL-18, GasderminD (activated ones at least) make structural pores in the host cell membrane and release cytokines

What were animal models (mice, flies) with pathogens or PAMPs used for?

Utilizing knockout organisms, such as Toll-deficient flies or MyD88/TRIF double-knockout mice, to demonstrate how the absence of PRR signaling severely compromises survival following pathogen challenge (like Salmonella or fungi)

ELISA

Used to directly quantify the concentration of secreted proteins, such as measuring inflammatory cytokines in the blood serum of LPS-challenged mice.

Northern blot

Used to detect and quantify specific mRNA transcripts, historically applied to observe the lack of antimicrobial peptide mRNA in mutant Drosophila.

RT-PCR with Agarose Gel

Used to amplify and visualize the induction of specific target genes (e.g., measuring IL-8 transcription following human TLR4 activation) by assessing band intensity

How do GPCRs facilitate chemotaxis

When a ligand binds to the GPCR, a conformational change enables the receptor to associate with a G protein, causing it to dissociate into $\alpha$ and $\beta\gamma$ subunits. This activates GTPases like Rac, Rho, and Cdc42, which mobilize the actin cytoskeleton to drive cellular movement.

What are the three modes of cytokine action (I know there are four technically but 3 were mentioned in lecture).

Autocrine: A cytokine acts on the same cell that produced it (e.g., IL-2 produced by a T cell acting on itself).

Paracrine: A cytokine acts on nearby cells (e.g., TNF produced by macrophages acting on adjacent endothelium).

Endocrine: A cytokine enters circulation to act on distant cells (e.g., IL-5 traveling to bone marrow). Endocrine is through the blood

The fourth one you were thinking about is juxtacrine. It’s like paracrine, but you have to be physically touching each other

Describe the signaling by the major cytokine receptor families (know the specifics for IL-1, TNF, IL-6, CXCL8, and IL12).

IL-6 and IL-12: Bind to homodimeric or heterodimeric receptors that signal heavily via the JAK/STAT pathway.

IL-1: Utilizes IL-1 family receptors that signal similarly to Toll-like Receptors (TLRs), relying on the MyD88 adaptor to activate the NF-$\kappa$B transcription factor.

TNF: Binds the TNFR family, which signals through TRAFs to activate NF-$\kappa$B, but can also induce cell death.

CXCL8: Binds to the chemokine receptor family, which are GPCRs that signal through G proteins.

What are the downstream effects of major cytokines induced by PRR activation? There are four.

IL-1$\beta$ and TNF-$\alpha$: Locally activate vascular endothelium, increase vascular permeability, and systemically induce fever. TNF-$\alpha$ also mobilizes metabolites and can cause shock systemically.

IL-6: Locally activates lymphocytes and systemically induces fever as well as acute-phase protein production from the liver.

CXCL8: Acts as a chemotactic factor to recruit neutrophils, basophils, and T cells to the site of infection.

IL-12: Activates natural killer (NK) cells and drives the differentiation of CD4 T cells into Th1 cells.

What are leukocyte rolling and extravasation (roles of selectins, integrins, and ICAM)?

Signaling from TNF and IL-1 activates endothelial cells to upregulate adhesion molecules and causes local vessel dilation. Selectins (like E-selectin) on the endothelium mediate weak adhesion by binding to sugars (sialyl-Lewisx) on leukocytes, enabling the cells to "roll" along the vessel wall. Meanwhile, integrins on the leukocyte bind to ICAM on the endothelium; chemokine signaling induces a conformational change in these integrins that vastly increases their affinity for ICAM, locking the cell in place (tight binding) and initiating extravasation (diapedesis) into the tissue

In short, selectins bind to sugars on leukocytes, which enables them to “roll” along the vessel wall. Integrins bind to ICAM, which ultimately leads to extravasation, which is where white blood cells squeeze through intact capillary walls to enter surrounding body tissues!

Acute phase response

Bacterial infection induces macrophages to produce IL-6, which travels to the liver and stimulates hepatocytes to synthesize acute-phase proteins such as C-reactive protein (CRP) and mannose-binding lectin. These proteins act as opsonins to coat bacterial surfaces and can subsequently activate the complement system.

What are the downstream consequences of Type I IFNs?

• Induce cellular resistance to viral replication by generating Mx proteins, 2',5'-linked adenosine oligomers, and PKR.

• Enhance viral sensing by upregulating PRR expression.

• Induce IFIT proteins to suppress viral RNA translation.

• Increase MHC class I expression and antigen presentation.

• Activate dendritic cells, macrophages, and NK cells, and induce chemokines to recruit lymphocytes.

What is the innate immune influence over adaptive immune responses (DCs)?

Pattern Recognition Receptors (PRRs) control dendritic cell (DC) function by triggering their transition from highly endocytic tissue-resident cells to mature cells with high MHC and costimulatory molecule expression. The specific cytokine profiles produced by these activated DCs dictate the trajectory of T cell differentiation (e.g., producing IL-12 drives a Th1 response, while other cytokines drive Th2 or Th17 responses).

Phagocytosis

The process of internalizing bound material (such as bacteria or yeast) into the cell for breakdown.

Phagosome vs phagolysosome

A phagosome is the vesicle containing internalized material; it fuses with a lysosome to form a phagolysosome, where acidification and toxic enzymes break down the microbe.

Chemotaxis

Cellular movement occurring in response to chemical signals, directing cells to sites of infection.

Formylated methionine

A unique amino acid utilized only by bacteria to initiate protein translation; it acts as a potent chemoattractant when bound by host GPCRs.

Cytokine

Small proteins produced by cells that induce specific responses in cells expressing corresponding receptors.

Chemokine

A specific subset of cytokines with chemoattractant properties that recruit cells expressing specific GPCRs to sites of infection.

GPCR (in this class)

G-protein coupled receptors, which initiate chemotaxis and enhance microbial killing upon ligand binding.

NADPH oxidase

An enzyme complex that assembles on the phagosomal membrane, transferring electrons from NADPH to O2 to generate highly reactive toxic oxygen species for microbial killing.

Inflammation

A physiological response to infection characterized locally by heat, pain, redness, and swelling, driven molecularly by blood vessel dilation, vascular permeability, and cell recruitment.

Monocytes

Cells that circulate in the blood and differentiate into macrophages upon migrating into surrounding infected tissue.

JAK/STAT

A primary intracellular signaling pathway utilized by many multi-chain cytokine receptors, leading to gene transcription.

TNF/TNFR

Tumor necrosis factor and its receptor family, which primarily signal via TRAFs to activate NF-$\kappa$B, or to induce cell death.

IL-1

Interleukin-1, a cytokine that signals through MyD88 to induce broad inflammatory responses, similar to TLR signaling.

Selectins

Endothelial adhesion molecules that bind carbohydrates on leukocytes to initiate the weak "rolling" phase of cell recruitment.