Case Studies Flashcards
What is WAS?
Wiskott-Aldrich Symdrome
caused by mutations in the Wiskott-Aldrich syndrome protein (WASP)
–> nucleation-promoting factors for the ubiquitously expressed Arp2/3 complex, which drives the generation of branched actin filaments
Immune cells (and other cells from the hematopoietic lineage) in patients with WAS have an
impaired ability to reorganize their cytoskeleton
For example, T cells are morphologically
abnormal in patients with WAS, losing their surface microvilli and appearing ‘bald’
The inability of T cells to reorganize their actin cytoskeleton has profound effects on their
function, and thus on the immune system as a whole.
Based on your knowledge of how T cell responses are initiated, how would an impaired ability to reorganize the actin cytoskeleton inhibit T cell activation. What are the implications for impaired proliferative ability for the generation of a T cell
response? (WAS)
Poor formation of the immunological synapse:
–> will prevent T cells from clustering receptors together on the cell surface and will impact the signal strength
–> limit T cell activation and proliferation which will then impact the subsequent functions of T cells, limiting cell-cell contacts which are important for
both CD4 and CD8 T cell function
Rearrangement of the cytoskeleton is also important for regulation of the signalling
scaffolding
–> during signalling events; the correct molecules may not be brought into contact with the synapse and therefore limit the generation of a signalling cascade and impact the generation of an effective immune response
impairment in cytoskeleton rearrangement will also
impact phagocytosis
–> impacts antigen presentation and impairs the
ability of APCs to induce T cell activation
Why is cell migration and lymph node organization important for the generation of
immune responses? What impact would impaired T cell migration have on your
ability to respond to infection? (WAS)
Lymph node organisation helps cells to find each other and allow for communication between the correct cells at the correct time
Impacting this organisation will inhibit:
–> the development of immune responses by preventing these interactions from occurring in a timely and efficient manner
Migration is impacted, meaning:
–> T cells can’t efficiently circulate between lymph nodes
–> T cells will also struggle to migrate to the B cell follicles preventing T helper cells from communicating with B cells
–> This will in turn prevent class switching and
generation of a robust antibody response as B cells will lack T cell stimulation
What effect would a defect in WASP have on the ability of T cells to direct immune
effector responses?
- T cells migration would be impaired hindering their ability to direct other cells
- Inability to rearrange cytoskeleton would impact the ability of the T cells to communicate effectively with other cells via cell-cell contact
- a lack of T reg function might prevent dampening of immune responses leading to
uncontrolled cellular responses and an increase in autoimmunity
-inability to communicate with B cells, so a lack of isotype switching = inability to effectively produce the correct antobodies to the infection
Why are patients with WAS more susceptible to viral infections?
- Without the ability to rearrange the cytoskeleton there will be an impairment in contact dependant cytotoxicity in addition to an impairment in CD8 T cell activation and proliferation resulting in an increases susceptibility to intracellular pathogens
- The cytoskeletal defects will also impact process such as phagocytosis which will impact clearance of infected cells and the ability to present antigens to T cells,
limiting T cell stimulation further - NK cells will also be impacted by this as they will
not be able to form a good synapse with target cells to release perforin and granzyme
If WAS leads to impaired immune responses, can you think why patients with WAS
have higher incidences of allergy and immune-complex mediated diseases?
Impairment in T reg function that will inhibit the regulation of abhorrent immune responses
A lack or T regs to control immune responses against allergens will result in an increase in allergy related diseases
–> This could be due to multiple factors and
intrinsic deficiencies within the Treg compartment
A lack of WASP also seems to prevent T cell death upon reactivation and may also lead to activation of T cells that should be killed resulting in an autoimmune response
Immune complexes will cross link receptors which can increase the signalling received to help activate cells even when they cannot form a proper immune
synapse
–> can lead to the formation of allergy and autoimmune responses
What is EBV?
The Epstein–Barr virus
- one of the most common viruses in humans
- belongs to the herpesvirus family and causes acute infectious mononucleosis
- Most people become infected with EBV before
the age of five and about 90 percent of adults have evidence of previous infection and have gained
adaptive immunity - During childhood, these infections usually cause no symptoms or are indistinguishable from other mild illnesses
- during adolescence infection causes acute
infectious mononucleosis in about 50 percent of cases - Symptoms include fever (usually lasting for about two weeks), sore throat and swollen glands - usually
located around the back of the neck but also throughout the body, including substantial spleen
enlargement - exhaustion/fatigue
- Most symptoms disappear after about 2–3
weeks and most people are able to resume their normal activities within 2–3 months; however, in some
cases fatigue may last for up to 2 years
Cytotoxic CD8+ T cells contribute to immune protection against EBV. How are intracellular
antigens processed and presented to CD8+ T-cells at the cell surface? (EBV)
- cytosolic protein digested in immunoproteasomes
- the peptides, 8-10 amino acids in length, are transported from the cytoplasm into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) proteins. TAP forms a channel in the ER membrane through which peptides can pass. These are loaded onto newly synthesised MHC class I molecules.
- movement through ER, endocytosed (enclosed in vesicle), the subsequently through the GA
- vesicle travels to cell membrane where antigen-MHC I complex is presented to a CD8+ T cell
What do the following
terms mean: MHC restriction; MHC anchor residues; MHC binding motifs; polygenicity; allele
and haplotype? (EBV)
MHC restriction:
- CD4 T cells only recognise peptides presented by MHC class II molecules
- CD8 T cells only recognise peptides presented by MHC class I molecules
MHC anchor residues:
- Certain residues on a peptide that have strong binding in pockets in the MHC molecule peptide binding domains
MHC binding motifs:
- Class I molecules have fewer, more specific pockets. As they only bind viral peptides.
- Class II molecules has more, less specific pockets. As they can bind a wide range peptides from many different organisms.
polygenicity:
- a complex genetic trait or phenotype that is influenced by multiple genes, each contributing a small effect to the overall phenotype
- no single gene has a large or dominant effect on the trait; instead, the trait is determined by the combined action of many genes, often interacting with each other and with environmental factors
- eg height, weight, susceptibility to disease
allele:
- a variant form of a gene, which can result in different phenotypic traits or characteristics
haplotype:
- a specific combination of alleles located on the same chromosome, which are inherited together as a unit because they are physically close to each other on the chromosome and are passed down as a unit through generations
What is clonal expansion? (EBV)
- APCs will present antigen to T cells which will induced a signalling cascade. The T cell will then begin to
divide multiple times, generating a larger population stemming from a single T cell clone - These cells can then polarize and migrate into the periphery to perform their effector function, or go on to generate a pool of memory T cells to response to the same epitope should it be encountered in the future
Methods for studying T cell expansion:
Which techniques did researchers use prior to the Callan et al. paper to measure the expansion of pathogen-specific CD8+ T cells? (EBV)
- One method used to measure the expansion of pathogen-specific CD8 T cells was the use of an ELISPOT assay, a paper in 1995 (Miyahira et al) says it is an assay based in the detection of IFN-gamma secretion by single cells after their stimulation with antigen
- The secretion is visualised as spots revealed by using enzyme labelled anti-IFN-gamma monoclonal antibodies
–> It was determined that the assay detects 80-95% if these CD8 T cells.
Modern method: Flow Cytometry
1. Cell labelling - T cells of interest are isolated from a sample and labelled with fluorescent dyes that bind to specific cell surface markers
- These markers may include CD3, a common marker for T cells, as well as markers specific to certain T cell subsets or activation states
- Flow cytometry analysis - The labelled cells are suspended in a fluid stream and passed through a laser beam, causing them to emit fluorescence
- Detectors measure the intensity of fluorescence emitted by each cell, providing information about the expression levels of the labelled markers.
How did Callan et al. measure the antigen-specificity of CD8+ T cells during infection? What are
the main advantages of this approach? Are there any drawbacks? (EBV)
They used tetramers of MHC molecules complexed to EBV epitopes. These can bind stably and specifically to desired MHC-peptide-specific T cells.
Advantages: Any desired T cell can be measured
Drawbacks: synthesis of the MHC-tetramers seems to be a hard process. Require biotinylation which involves folding, use of protease inhibitors and purification. Yield is 80%.
How did Callan et al. test the specificity of their tetramers?
- They used a fluorescent probe to label their synthesised tetrameric complexes and subsequently tested them to see which CD8 T cells that recognised specific sequences bound to them.
Why did Callan et al. measure HLA-DR, CD38, CD62L, CD45RO and CD57? (EBV)
- These are all different markers indicating levels of CD8 T cell activation. This allowed the researchers to measure the levels of T cells that were activated and antigen specific against EBV
The authors find that about 40% of all CD8+ T-cells in the blood of some patients are specific for
one epitope. What are the advantages/disadvantages of such a huge expansion? (EBV)
- As soon as a T cell recognises a presented antigen, it will undergo clonal expanion. This means that the immune system can respond quickly and effectively.
But if that epitope were to undergo a mutation on reinfection, that large number of T cells would be ineffective - Potential for large expansion to target a poorly accessible antigen which might hamper immunity.
