Describe how NK cells circulate in the bloodstream and are able to enter inflamed tissues to participate in the innate
immune response.

Describe the types of cytokines that activate and recruit NK cells, and describe how NK cells can
influence the activity of tissue macrophages through their release of IFN-γ

• NK cells circulate through the body in the bloodstream but can be rapidly recruited to enter tissues. NK cells play an important role in innate immunity and can contribute to inflammation. As components of innate immunity, NK cells are rapid responders to infection, and function in a more general manner, not in an antigen-specific manner as do the B and T lymphocytes.
• Natural killer (NK) cells are a critical component of the innate immune system, tasked with identifying and eliminating infected or abnormal cells. These cells circulate throughout the bloodstream, constantly surveying for signs of distress. The process by which NK cells enter inflamed tissues involves a complex interplay of adhesion molecules and chemokines.
• When tissues become inflamed, various signals are released, including chemokines such as CXCL8 (IL-8) and CXCL10 (IP-10), which act as chemoattractants. NK cells express receptors that recognize these chemokines, allowing them to follow the gradient towards the site of inflammation. Additionally, adhesion molecules such as integrins and selectins are upregulated on the surface of endothelial cells lining blood vessels in inflamed tissues. NK cells express corresponding ligands for these adhesion molecules, facilitating their extravasation from the bloodstream into the tissue.
• Once inside inflamed tissues, NK cells interact with other immune cells, including tissue macrophages. NK cells are activated by cytokines such as interleukin-12 (IL-12), interleukin-15 (IL-15), and type I interferons (IFNs), which are typically produced by dendritic cells, macrophages, and other immune cells in response to infection or inflammation. Upon activation, NK cells release cytokines such as interferon-gamma (IFN-γ), which plays a crucial role in shaping the immune response.
• IFN-γ produced by NK cells can influence the activity of tissue macrophages in several ways. Firstly, IFN-γ enhances the antimicrobial activity of macrophages by stimulating the production of reactive oxygen species (ROS) and nitric oxide (NO), which are toxic to pathogens. Secondly, IFN-γ promotes the upregulation of major histocompatibility complex (MHC) molecules on the surface of infected cells, facilitating their recognition and elimination by cytotoxic T cells. Lastly, IFN-γ can modulate the polarization of macrophages towards a pro-inflammatory phenotype, enhancing their ability to engulf and destroy

Describe the relationship between virus infection, interferon production, and NK cell activation.

1. Virus Infection: When a virus infects host cells, it triggers various intracellular signaling pathways that are recognized by the host's immune system as foreign. Infected cells may display viral antigens on their surface, signaling danger to the immune system.
2. Interferon Production: In response to virus infection, infected cells and nearby immune cells release signaling molecules called interferons. These interferons, particularly type I interferons (such as interferon-alpha and interferon-beta), serve as alarm signals to neighboring cells, alerting them to the presence of viral infection. This triggers an antiviral state in neighboring cells, making them more resistant to viral replication and spread.
3. NK Cell Activation: Interferons, along with other cytokines like interleukin-12 (IL-12) and interleukin-15 (IL-15), play a crucial role in activating natural killer (NK) cells. NK cells are a type of lymphocyte that forms a critical part of the innate immune response. Upon activation, NK cells become cytotoxic and start to target and kill virus-infected cells. They do so by recognizing and engaging with infected cells that display abnormal or stressed markers, such as reduced expression of MHC class I molecules, which are often downregulated by viruses to evade detection by cytotoxic T cells. The relationship between virus infection, interferon production, and NK cell activation is intricate and crucial for mounting an effective immune response against viral pathogens.
4. Effector Functions: Once activated, NK cells exert their effector functions. They release cytotoxic molecules such as perforin and granzymes, which induce apoptosis (programmed cell death) in the infected cells. NK cells also produce cytokines like interferon-gamma (IFN-γ), which further amplify the immune response by activating macrophages, enhancing antigen presentation, and promoting inflammation.
5. Feedback Loop: The production of type I interferons by infected cells not only activates NK cells but also creates a positive feedback loop. IFN-γ produced by activated NK cells can further stimulate infected cells to produce more type I interferons, reinforcing the antiviral state of neighboring cells and enhancing overall antiviral immunity.

So, the relationship between virus infection, interferon production, and NK cell activation is intertwined: virus infection triggers the production of interferons, which in turn activate NK cells to target and eliminate virus-infected cells, thus helping to control the spread of the virus within the host.

In summary, virus infection leads to the production of interferons, which not only establish an antiviral state in infected and neighboring cells but also play a pivotal role in activating NK cells. Activated NK cells then contribute to the immune response by directly targeting and eliminating virus-infected cells, thus helping to contain the spread of the infection and limit its severity. This coordinated response between virus-infected cells, interferon production, and NK cell activation forms an essential aspect of the innate immune defense against viral pathogens.

Describe the types of ligands that NK cell activating receptors bind to (stress-induced proteins such as MIC).
Describe the types of ligands that NK cell inhibitory receptors bind to (classic MHC-I or HLA-E).

• Stressed genes : they will express MIC on their surface
• Cells that are not stressed do a poor job of producing and stimulating NK cells

NK cells express a variety of receptors that allow them to interact with target cells, either activating their cytotoxic functions or inhibiting them. These receptors recognize specific ligands on the surface of target cells, influencing NK cell activity. Here's a breakdown of the types of ligands recognized by NK cell activating and inhibitory receptors:

1. NK Cell Activating Receptors Ligands: a. Stress-Induced Proteins: Activating receptors on NK cells, such as NKG2D (natural killer group 2 member D), recognize stress-induced proteins that are upregulated on the surface of target cells during cellular stress or transformation. Examples of ligands for NKG2D include MICA (MHC class I chain-related protein A) and MICB (MHC class I chain-related protein B). These ligands are often expressed on virus-infected cells, tumor cells, or cells undergoing cellular stress, marking them for NK cell recognition and activation. b. Viral Proteins: Some activating receptors on NK cells can directly recognize viral proteins expressed on infected cells. For example, the NKp30 receptor can bind to viral hemagglutinins, while NKp44 and NKp46 can recognize viral hemagglutinin-neuraminidase proteins. These interactions trigger NK cell activation and cytotoxicity against virus-infected cells.
2. NK Cell Inhibitory Receptors Ligands: a. Classical MHC-I Molecules: The most well-characterized inhibitory receptors on NK cells are the killer cell immunoglobulin-like receptors (KIRs) and the CD94/NKG2A heterodimer. These receptors primarily recognize classical MHC class I molecules (HLA-A, HLA-B, and HLA-C) on the surface of healthy cells. When these receptors engage with MHC class I molecules, they deliver inhibitory signals to the NK cell, preventing it from initiating cytotoxicity against the target cell. This mechanism serves as a "self-recognition" system, ensuring that NK cells spare healthy cells expressing normal levels of MHC class I molecules. b. HLA-E Molecule: Another ligand for the inhibitory receptor CD94/NKG2A is the non-classical MHC class I molecule HLA-E. HLA-E presents peptides derived from the leader sequences of other MHC class.

In summary, NK cell activating receptors bind to stress-induced ligands such as MICA, MICB, and ULBP, triggering NK cell activation and cytotoxicity. In contrast, NK cell inhibitory receptors bind to self-MHC class I molecules (classic MHC-I) or HLA-E, delivering inhibitory signals that prevent NK cell activation and killing of healthy cells. This balance between activating and inhibitory signals helps regulate NK cell activity and ensure proper immune surveillance without causing harm to healthy tissues.

Explain the fundamental difference between direct and indirect assessment of MHC-I expression on a target cell by
NK cells. How do the mechanisms differ from one another? Understand the role of HLA-E.

Direct and indirect assessment of MHC class I (MHC-I) expression by natural killer (NK) cells represent distinct mechanisms by which NK cells evaluate the "self" status of target cells. These mechanisms involve the recognition of MHC-I molecules on target cells, which serve as ligands for inhibitory receptors on NK cells. The fundamental difference lies in how MHC-I molecules are detected and interpreted by NK cells:

1. Direct Assessment: In direct assessment, NK cells directly interact with classical MHC-I molecules (HLA-A, HLA-B, and HLA-C) displayed on the surface of target cells. Classical MHC-I molecules present peptides derived from endogenous proteins to T cells, contributing to antigen presentation and immune surveillance. NK cells possess inhibitory receptors, such as killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A, that recognize specific epitopes on classical MHC-I molecules. When these inhibitory receptors engage with MHC-I molecules on target cells, they deliver inhibitory signals to the NK cell, preventing it from initiating cytotoxicity. This mechanism ensures that healthy cells expressing normal levels of MHC-I molecules are spared from NK cell-mediated killing, thereby maintaining self-tolerance.
2. Indirect Assessment: In indirect assessment, NK cells monitor the levels of a non-classical MHC-I molecule called HLA-E on the surface of target cells. HLA-E presents peptides derived from the leader sequences of other MHC-I molecules to CD94/NKG2A heterodimers on NK cells. These peptides are typically derived from nascent MHC-I molecules in the endoplasmic reticulum and reflect the overall intracellular MHC-I pool. Under normal conditions, abundant MHC-I molecules are processed and loaded with peptides, leading to the presentation of a diverse repertoire of peptides on HLA-E. Engagement of CD94/NKG2A with peptide-presenting HLA-E delivers inhibitory signals to NK cells, preventing them from attacking cells with sufficient MHC-I expression. However, decreased MHC-I expression results in reduced peptide presentation on HLA-E, leading to decreased engagement of CD94/NKG2A and, consequently, reduced inhibition of NK cell activity.

In summary, the fundamental difference between direct and indirect assessment of MHC-I expression lies in the receptors and ligands involved, as well as the mechanism by which MHC-I expression is.

The role of HLA-E in this context is crucial for immune surveillance. By presenting peptides derived from the leader sequences of other MHC-I molecules, HLA-E acts as a sensor of MHC-I synthesis and trafficking within the cell. The recognition of HLA-E by the CD94/NKG2A inhibitory receptor provides a mechanism for NK cells to indirectly assess the overall integrity of the MHC-I antigen presentation pathway in target cells. If HLA-E presents appropriate peptides, indicating proper MHC-I expression and function, NK cell cytotoxicity is inhibited, allowing healthy cells to be spared from attack. However, if MHC-I expression is compromised or inhibited, leading to reduced HLA-E presentation of MHC-I-derived peptides, NK cell cytotoxicity may be unleashed against abnormal or infected cells lacking MHC-I expression, thus contributing to immune surveillance and elimination of target cells.

Explain how the balance of signaling delivered through the two types of NK cell receptors (activating/inhibitory) dictate
NK activity (to kill or not to kill).

The balance of signaling through activating and inhibitory receptors on natural killer (NK) cells plays a crucial role in determining NK cell activity, specifically whether to initiate cytotoxicity against target cells or spare them from destruction. This balance is often referred to as the "missing self" or "induced self" recognition model and is essential for maintaining immune tolerance while enabling effective immune responses against infected or abnormal cells. Here's how it works:

1. Activating Receptors: Activating receptors on NK cells recognize ligands expressed on the surface of target cells, typically induced during stress, infection, or transformation. When activating receptors engage their ligands, they trigger signaling pathways within the NK cell that promote cytotoxicity and cytokine production. These pathways can involve phosphorylation cascades, calcium influx, and cytoskeletal rearrangements, leading to the release of cytotoxic granules containing perforin and granzymes and the secretion of pro-inflammatory cytokines such as interferon-gamma (IFN-γ). Examples of activating receptors include NKG2D, NKp30, NKp44, and NKp46. These receptors recognize stress-induced ligands, viral proteins, or other markers of cellular distress.
2. Inhibitory Receptors: Inhibitory receptors on NK cells recognize self-antigens presented by MHC class I molecules on the surface of healthy cells. When inhibitory receptors engage with their ligands, which are typically classical MHC-I molecules (HLA-A, HLA-B, and HLA-C), they deliver inhibitory signals that counteract the activating signals from activating receptors. This inhibition prevents NK cell activation and cytotoxicity against normal, healthy cells expressing normal levels of MHC-I molecules. Examples of inhibitory receptors include killer cell immunoglobulin-like receptors (KIRs), CD94/NKG2A, and the Ly49 family of receptors.
3. Balance of Signaling: The balance of signaling between activating and inhibitory receptors ultimately determines NK cell activity. If activating signals dominate and outweigh inhibitory signals, NK cells become activated and initiate cytotoxicity against target cells expressing stress-induced or infected markers. Conversely, if inhibitory signals predominate and suppress activating signals, NK cell activity is inhibited, and healthy cells expressing normal levels of MHC class I molecules are spared from destruction. This delicate balance ensures that NK cells selectively target and eliminate aberrant or infected cells while sparing healthy cells, thus contributing to immune surveillance and homeostasis.

In summary, the interplay between activating and inhibitory signals received by NK cells dictates their activity, determining whether NK cells will kill or spare target cells. This balance ensures effective immune surveillance and helps maintain tissue integrity and function.

Explain why virus-infected cells are particularly susceptible to killing by NK cells (upregulation of stress molecules and
decreased expression of MHC-I).

Virus-infected cells are particularly susceptible to killing by natural killer (NK) cells due to two main factors: the upregulation of stress molecules and the decreased expression of major histocompatibility complex class I (MHC-I) molecules. These changes in infected cells make them more visible to NK cells and reduce the inhibitory signals that normally prevent NK cell activation. Here's how these mechanisms contribute to the susceptibility of virus-infected cells to NK cell killing:

1. Upregulation of Stress Molecules: Virus infection triggers intracellular stress responses within infected cells. As a result, infected cells often upregulate stress-induced ligands on their surface, which serve as targets for activating receptors on NK cells. These stress molecules, such as MICA (MHC class I chain-related protein A) and MICB (MHC class I chain-related protein B), are recognized by activating receptors like NKG2D on NK cells. The upregulation of stress molecules on virus-infected cells acts as a danger signal, alerting NK cells to the presence of infection. Engagement of activating receptors with stress molecules activates NK cells and triggers their cytotoxic response against the infected cells.
2. Decreased Expression of MHC-I Molecules: MHC-I molecules play a crucial role in presenting viral antigens to cytotoxic T cells, initiating adaptive immune responses against viral infections. However, many viruses have evolved strategies to evade immune detection by downregulating the expression of MHC-I molecules on infected cells. Decreased expression of MHC-I molecules reduces the ability of infected cells to present viral antigens to cytotoxic T cells, allowing viruses to evade adaptive immune surveillance. However, this downregulation of MHC-I expression also renders virus-infected cells more susceptible to NK cell-mediated killing. NK cells possess inhibitory receptors, such as KIRs and CD94/NKG2A, that recognize MHC-I molecules on target cells. When MHC-I expression is decreased or absent, inhibitory signaling through these receptors is diminished, leading to NK cell activation and cytotoxicity against the infected cells.

In summary, virus-infected cells are particularly susceptible to killing by NK cells due to the upregulation of stress molecules, such as MICA and MICB, which trigger activating signals on NK cells, and the decreased expression of MHC-I molecules, which reduces inhibitory signaling and enhances NK cell activation. This dual mechanism of recognition allows NK cells to efficiently detect and eliminate virus-infected cells, contributing to the early control of viral infections and the maintenance of immune surveillance.

Explain the role of antibody and Fc receptors in NK cell-mediated ADCC (antibody-dependent cellular cytotoxicity).

Antibody-dependent cellular cytotoxicity (ADCC) is a vital mechanism of the immune response mediated by natural killer (NK) cells in conjunction with antibodies and Fc receptors. This process is crucial for the elimination of antibody-coated target cells, such as virus-infected cells or tumor cells, and plays a significant role in both innate and adaptive immunity. Here's how the components interact in ADCC:

1. Antibody Production: ADCC begins with the production of antibodies by B cells in response to a specific antigen, such as viral proteins or tumor-associated antigens. These antibodies can be of the IgG class, particularly IgG1 and IgG3 subclasses, which have a high affinity for Fc receptors on NK cells. Upon encountering the antigen, B cells differentiate into plasma cells, which secrete antibodies into the bloodstream.
2. Coating of Target Cells with Antibodies: In ADCC, antibodies bind to specific antigens expressed on the surface of target cells, such as viral-infected cells or tumor cells. This binding results in the coating or "opsonization" of target cells with antibodies, forming immune complexes.
3. Recognition of Antibody-Coated Cells by NK Cells: NK cells express Fc receptors, primarily FcγRIIIA (CD16), on their surface. These Fc receptors specifically recognize the Fc portion of antibodies bound to target cells. When NK cells encounter antibody-coated target cells, the Fc receptors on NK cells bind to the Fc portion of the antibodies, forming a bridge between the NK cell and the target cell.
4. Activation of NK Cells: Engagement of Fc receptors on NK cells with antibody-coated target cells triggers intracellular signaling pathways within the NK cell, leading to NK cell activation. This activation results in the release of cytotoxic granules containing perforin and granzymes, as well as the secretion of pro-inflammatory cytokines such as interferon-gamma (IFN-γ).
5. Cytotoxicity Against Target Cells: Activated NK cells exert their cytotoxic functions against antibody-coated target cells through several mechanisms. Perforin forms pores in the target cell membrane, allowing entry of granzymes, which induce apoptosis (programmed cell death) in the target cell. Additionally, NK cells may also release cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) upon target cell recognition. These cytokines further enhance the immune response by promoting inflammation, activating macrophages, and modulating adaptive immune responses.

THIS IS WHERE TOP HAT BEGINS

THIS IS WHERE TOP HAT BEGINS

True/False: The nude mouse (lacks a thymus) does not produce NK cells.

False

*NK Cells do not go to the thymus

SCID mice do not express a functional form of the VDJ recombinase enzyme (RAG); therefore, SCID mice do not produce functional B or T lymphocytes (fact). True/False: SCID mice do not produce functional NK cells.

False

Which two (2) of the following statements about NK cells are true?

a.NK cells are unable to kill extracellular bacteria.

b.NK cells function as components of adaptive immunity.

c.NK cells display a high degree of antigen specificity.

d.NK cells are unable present peptides to Tc cells.

e.NK cells are derived from hematopoietic stem cells that reside in the bone marrow.

a.NK cells are unable to kill extracellular bacteria.

e.NK cells are derived from hematopoietic stem cells that reside in the bone marrow.

In a future lecture we will discuss how NK cells play an important role in identifying and killing cancer cells that arise in the body. Which two (2) of the following phenotypes displayed by cancer cells do you think play a role in their recognition and elimination by NK cells?

a.Their ability to divide in an uncontrolled manner.

b.Their ability to move into tissues (metastasis).

c.Their expression of MIC proteins on their surface.

d. Their ability to evade CTLs by decreasing expression of MHC-I on their surface.

e. Their ability to mutate their DNA at a higher than normal rate.

c.Their expression of MIC proteins on their surface.

d. Their ability to evade CTLs by decreasing expression of MHC-I on their surface.

Which one of the following statements about NK cells is false?

a.NK cells are lymphocytes and function as cellular components of innate immunity.

b.NK cells are more likely to kill a "stressed" cell than they are to kill a "non-stressed" cell.

c.NK cells develop within a primary lymphoid tissue.

d.NK cells possess antigen-specific surface receptors (different NK cells bind to different non-self antigens).

e.NK cells are capable of killing virus-infected cells by participating in the process of ADCC.

d.NK cells possess antigen-specific surface receptors (different NK cells bind to different non-self antigens).

Which complex (MHC-1 or MHC-2) is more likely to be bound by a NK cell inhibitory receptor?

a.) MHC-1

b.) MHC-2

a.) MHC-1

NK cells and CTLs are both highly effective at identifying and killing virus-infected cells (fact). True/False: NK cells and CTLs use similar strategies/mechanisms for differentiating between infected cells (which they kill) and uninfected cells (which they do not kill).

False

Individual X has a genetic condition that results in the inability to produce functional NK cells. Which one of the following types of microbes do you think individual X is most susceptible to due to this condition?

a.Worm infections

b.Virus infections

c.Bacteria infections

d.Fungal infections

e.Unicellular parasite infections.

b.Virus infections

One population of cells is infected with a virus called HCMV and a second population of cells is infected with Zika virus. Infections are conducted so that only 50% of the cells become infected. Six hours after infection the cells are analyzed by FLOW cytometry using a monoclonal antibody that bind to a virus antigen (HCMV or ZIKA) which will bind to virus-infected cells, and a monoclonal antibody that binds to the MIC A protein. Which virus-infected cells (HCMV or Zika) appear to be more susceptible to killing by NK cells?

a.)HCMV

b.)Zika

b.) Zika