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Cellular and Molecular Immunology > 5. Investigating the human immune system > Flashcards

5. Investigating the human immune system Flashcards

1. Discuss the knowledge we gain through human immunology and animal models. 2. The range of techniques to investigate the human immune responses. 3. The main methods to investigate human antigen-specific T cell responses. 4. In vivo human challenge models and how they advance medicine and immunology.

1
Q

What was the birth point of immunology?

A

Edward Jenner and his vaccination for smallpox. This was the first demonstration that vaccination could protect against infectious diseases.

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2
Q

How did Edward Jenner create the first vaccination?

A
  1. He observed that diary maids that had been in contact with cow pox were protected from small pox.
  2. He proves this by taking material from cox pox lesions and putting them into a small boy.
  3. The boy develops a mild illness and recovers quickly.
  4. 6 weeks later, he injects the boy with small pox. He did this up to 20 times.
  5. The boy didn’t develop small pox.
  6. This showed injection with an attenuated pathogen can provide protection.
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3
Q

How is immunological research in humans (in vitro/ex vivo) used?

A
  1. This involves taking calls or tissues from patients to under stand the mechanisms of disease.
  2. Insights into the functions of immune cells and some mechanistic studies.
  3. It has been boosted by major technological advances in the last decades
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4
Q

How is immunological research in animal models used?

A
  1. These are important for basic and mechanistic understanding of the immune system.
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5
Q

How is immunological research in human challenge studies used?

A

To accelerate vaccine development against pathogens of global impact

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6
Q

What was an important human in vitro studies that gave us mechanistic insights?

A

A study from 1985 that showed how B cells and T cells interact.

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7
Q

1985 B cell/T cell study: What model antigen was used?

A

An inactivated tetanus toxin called tetanus toxoid.

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8
Q

1985 B cell/T cell study: What cell populations were used?

A
  1. B and T cell populations from a person who had been vaccinated using tetanus toxoid.
  2. The B and T cell clones are specific to the tetanus toxoid.
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9
Q

1985 B cell/T cell study: How did they determine that T cells were aiding B cell function?

A
  1. The T and B cells specific for tetanus toxoid are co-cultured with the native tetanus toxoid antigen.
  2. T cell proliferation is measured and an increasing T cell population was observed despite not being able to recognise the native antigen.
  3. The B cells efficiently present the antigen on MHC to the T cells.
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10
Q

How do T and B cells recognise antigens?

A

T cells: fragments of antigens presented on MHC
B cells: native antigens

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11
Q

1985 B cell/T cell study: What is important about T and B cell specificity?

A

The T and B cells must recognise the same antigen to interact with each other.

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12
Q

1985 B cell/T cell study: What is the role of the BCR in antigen presentation to T cells?

A

The BCR enables the uptake and presentation of very small amounts of antigens to specific CD4+ T cells.

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13
Q

1985 B cell/T cell study: How does antigen presentation occur?

A
  1. The T and B cell clones must come from the same person - suggests recognition of self.
  2. When a MHC1 inhibitor is introduced antigen presentation still occurs.
  3. This shows that antigen presentation to T cells is MHC2 restricted therefore it is CD4 T cells interacting and aiding B cells.
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14
Q

What is the B cells MHC2 presentation pathway?

A
  1. The antigen binds with the BCR by the epitope.
  2. The whole pathogen is internalised, degraded and some peptide fragments (T cell epitopes) enters the MHC2 pathway.
  3. Peptides from the pathogen proteasome are presented to the T cell which activates the B cell.
  4. The activated T cell produces cytokines to aid the B cell.
  5. The activated B cell produces antibodies specific to the pathogen b cell epitope.
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15
Q

Why are animal models important?

A
  1. They are often used for immunology studies and to determine how the immune system works.
  2. The immune cells work together within tissues so studying them in isolation is not always accurate.
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16
Q

What are some major immunological discoveries that animal models supported?

A
  1. Discovery of phagocytes and their role.
  2. Discovery of immunological tolerance.
  3. Discovery of major histocompatibility genes and the MHC complex’s role in mediating the detection and killing of virally infected cells.
  4. Discovery of dendritic cells.
  5. Discovery of cancer immunotherapy by inhibition of negative immune regulation.
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17
Q

What are the problems with animal models?

A

Some therapies or discoveries failed to translate into humans due to:
1. Limited life spans
2. Living in sterile conditions with limited exposure to microorganisms
3. Genetically identical due to inbreeding.
4. Ultimately, they don’t represent human complexity and heterogeneity.

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18
Q

Why are animal models used?

A

To have an environment where different components of the immune system and body can interact without the ethical problems of doing the same studies on humans.

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19
Q

What is an example of differences in genetic diversity between humans and animal models?

A
  1. Just for HLA class 1 humans have 6 different genes with almost every person having a unique combination of gene variants.
  2. The mice animal models have 3 MHC genes with 1 or few variants.
  3. As the HLA are fundamental for T cell studies, having limited version effects the results and the ability to present the antigens needed.
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20
Q

Technology used to study human immunity: Flow cytometry and CyTOF

A

Use to identify different cell populations through the markers they express.

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21
Q

Technology used to study human immunity: ATAC-seq

A

To study epigenetics. This tells us what genes are expressed and what factors are causing the expression.

22
Q

Technology used to study human immunity: RNA-seq

A

To study the transcriptome to see what genes are expressed.
Single cell RNA-seq is used to understand heterogeneity.

23
Q

Technology used to study human immunity: Mass spectrometry

A

To measure metabolites

24
Q

Technology used to study human immunity: ELISA or Luminex

A

Used to measure plasma cytokines and proteins

25
Q

Technology used to study human immunity: MRI

A

Tissue imaging to look at whole tissue structures

26
Q

Technology used to study human immunity: Clinical data

A

Clinical metadata is used to correlate disease state with all the other information about cell populations, expression, proteins, etc

27
Q

What are the 4 main methods used to study human T cell responses?

A
  1. High dimensional flow cytometry
  2. CyTOF
  3. Peptide-HLA tetramers
  4. Enzyme-linked Immunospot (ELISPOT)
28
Q

How is Flow cytometry used to study immune cells?

A
  1. Fluorescently labelled antibodies are used to identify specific markers on T cells and we can measure and classify T cell populations.
  2. Linage markers can tell us what the type of cell is.
  3. Viability markers are used to exclude cells and prevent dead cells affecting the results.
  4. Tissue homing markers are used to show where cells are migrating to.
  5. Activation, differentiation and cytotoxicity markers show us what the function of the cell could be.
29
Q

How is flow cytometry data visualised?

A
  1. You can measure around 18-30 parameters in one sample.
  2. This gives multidimensional data that needs to be visualised in 2 dimensions and preserve the complexity of the data.
  3. A computer algorithm groups cells together by similar gene expression patterns into clusters.
  4. Closer cells = more genetically similar
  5. You can layer different colour over the data to visualise different cell types of subset of cells expressing the marker you are measuring.
30
Q

What are some flow cytometry data visualisation programs?

A

t-SNE = t distributed stochastic non-linear embedding
UMAP = Uniform Manifold Approximation and Projection

31
Q

How are Peptide-HLA tetramers used to identify antigen specific T cells?

A
  1. The HLA fragment presents the peptide of interest.
  2. It binds to the specific TCR and stays bound.
  3. It can bind multiple TCRs on the same T cell.
  4. Flow cytometry is used to see what the antigen-specific T cells are or what they are doing.
32
Q

What are peptide-HLA tetramers?

A
  1. They are purified MHC molecules, either class 1 or 2, depending on the T cell you are studying.
  2. They present the peptide of interest
  3. They are bound to a fluorescently labelled steptavidin to visualise and tetramise them.
33
Q

Why are peptide-HLA tetramers used?

A
  1. To identify antigen specific population of T cells
  2. They bind more stably in the tetramer form, so they bind for longer.
34
Q

What is CyTOF?

A

Cytometry by time-of-flight.
It is a form of mass spectrometry.

35
Q

How is CyTOF used with peptide-HLA tetramers?

A
  1. The antibodies are tagged with heavy metal isotypes that are not present in nature.
  2. They antibodies bind antigen specific cells.
  3. The cells pass one at a time through a nebuliser and then argon plasma to ionise them.
  4. The cells are quantified using time of flight mass spectrometry and flow cytometry to identify the heavy metal labels.
  5. You end up with a flow cytometry-like plot.
36
Q

What is the advantage of CyTOF?

A
  1. The metal labels don’t have spill over.
  2. Fluorescent labels can spill over into other channels so you need to be careful about what colours you put next to each other.
37
Q

Why is CyTOF now a bit redundant?

A

Flow cytometry technology has caught up so there is no real advantage of doing CyTOF over it.

38
Q

When using CyTOF what can be used as a positive control?

A

Common antigens where there would be a small population of memory cells but would not increase due to no infection. This could be Flu or EBV peptides.

39
Q

When using CyTOF what can be used as a negative control?

A

healthy patients

40
Q

What is intracellular cytokines staining?

A
  1. Fluorescently label antibodies to specific cytokines.
  2. Use a Golgi inhibitor to prevent cytokines from leaving the cell.
  3. This means we can identify the cells producing the cytokines.
41
Q

What is Enzyme-linked immune absorbent spot assay (ELISpot)?

A
  1. You plate out cells on a plate with catcher antibodies that are specific for the antigen of interest.
  2. Similar to a sandwich ELISA you use a detection antibody with either fluorescences or a linked enzyme.
  3. Every spot that appears on the plate is a cell producing the cytokine of interest.
  4. You always need a positive control.
42
Q

How do you find out a T cell epitope of interest when you don’t know it?

A
  1. Use a synthetic peptide library
  2. Use short overlapping fragments that spans the sequence of interest to determine which binds.
43
Q

How was ELISpot used to quantify HBV-specific T cells?

A
  1. Antibodies to many different HBV antigens/epitopes were produced.
  2. ELISpot was used to quantify the different populations of T cells to the different HBV proteins.
  3. The patients who didn’t experience flare-ups had higher HBV-specific T cell levels while they were still on treatment.
  4. These T cells mainly targeted the polymerase and core proteins, suggesting these were the most effective antigens and T cells.
44
Q

Why was ELISpot used to quantify HBV-specific T cells?

A
  1. HBV has very antiviral treatment but it needs to be taken for life.
  2. Some patients that stopped taking the antivirals experienced flare ups and others didn’t
  3. It was thought that differences in T cell population while on treatment and during initial infection could be the cause.
45
Q

What are controlled human infection models?

A
  1. Intentional exposure of a person to an attenuated pathogen
  2. The people must be healthy consenting adults.
  3. Undergo strict ethical approvals by ethics committees.
  4. They are carefully controlled and safe.
46
Q

What are controlled human infection models used for?

A
  1. To allow researchers to understand the onset and development of the disease the pathogen causes
  2. To test the effectiveness of drugs and vaccines
47
Q

Examples of some diseases that have used controlled human infection models

A
  1. Yellow fever
  2. Malaria
  3. Dengue
  4. Influenza
  5. Cholera
  6. Typhoid
  7. COVID-19
48
Q

What was the first controlled human infection model?

A
  1. Done in Cuba during the Spanish-American war.
  2. To study Yellow fever.
  3. They took a volunteer with yellow fever and exposed him to mosquitoes and then exposed the mosquitoes to a healthy volunteer who then developed yellow fever.
  4. this showed mosquitoes transmitted yellow fever.
  5. This lead to the eradication of yellow fever in Havana in 90 days by killing mosquitoes.
49
Q

What are the ethical consideration of human challenge models?

A
  1. The volunteers need to understand what they are signing up for
  2. They must be able to freely consent - no prisoners or children
  3. They must receive compensation like money.
50
Q

How are human challenge models being used today for Dengue vaccines?

A
  1. Dengue is closely related to yellow fever but has no treatment or vaccines.
  2. Due to human challenge models a vaccine is in phase 3 trials.
  3. when exposed to an attenuated Dengue the control group developed viremia but the treated group didn’t.

Cellular and Molecular Immunology (17 decks)

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