Lecture 10 – Animal models used to understand the immune system

Why are mice good animal models for study of the immune system?

• Immune system is similar to humans

• Extracting cells/molecules for study is quite easy

• Inbred mice are genetically identical

How are transgenic mice made?

Introduce a ‘new gene’ (a transgene) into a mouse genome 

• Microinjection of a plasmid containing a gene into the pro-nucleus of fertilized eggs 

How did early transgenic mice provide a model for type 1 diabetes?

(LCMV glycoprotein)

Rat insulin promoter combined with LCMV glycoprotein

Transgenic mice then infected with LCMV which causes the mouse immune system to be activated

Activated immune system attacks the pancreas due to those cells expressing LCMV glycoprotein

Mouse develops type I diabetes

How were TCR transgenic mice which had only one type of T-cell developed?

Injected ovalbumin into a mouse and then took out the spleen

T-cell hybridoma made from the mouse CD4 T-cells

Isolate TCR a and b genes which encode ovalbumin-specific TCR

Create transgene and produce transgenic mice

These mice only have T-cells specific to ovalbumin

What was the issue with the ovalbumin transgenic mice which only had one type of T-cell?

Wasn’t a true representation of the whole immune system and couldn’t show how these T-cells interacted with other cells.

What was the solution to ovalbumin TCR transgenic mice only having one type of T-cell?

Purify the T-cells and then transfer them into another mouse

This allows you to have just enough of the T-cells to allow easy detection while maintaining the normal T-cell populations 

How were knock-out mouse models made?

Culture multi-potent embryonic stem (ES) cells from mouse embryos

Introduce DNA containing mutations/deletions into the ES cells

Modified ES cells are then reintroduced into the very early embryos 

Why are knock-out models usually better than transgenic ones?

Knock-out models target precesely the same genetic location so only the targeted gene is affected

• In transgenic models the insertion of a gene is done randomly

• This could potentially disrupt other genes/promoters 

How were CD8 T-cell KO mice made?

Target the beta2-microglobulin gene of MHC I molecule

• Causes no positive selection of CD8 T-cells in the thymus

• So vast reduction in CD8 population

How were B-cell KO mice made?

Introduced a STOP codon early in the constant region which causes very short BCRs to be made.

• This reduces surface expression of the BCR

• This prevents B-cell activation

What is the difference between Knock-In mice and Knock-out mice?

Rather than a large insertion/disruption, KI models replace the gene with a subtle knocked in version

• Gene is mutated to delete/alter function 

How were conditional/tissue specific KO/KI models made using Cre/LoxP?

LoxP is placed either side of the target gene in a mouse

• Gene is now ‘floxed’

Cross this mouse with another mouse which expresses Cre

• Cre expression is placed under a promoter to prevent expression until needed

• Cre cuts ‘floxed’ DNA

This allows the KO/KI model to be tissue/cell/drug-specific as Cre will only cut when/where we want Cre to be expressed

What are Bone Marrow (BM) chimera models?

A mice model made from the combination of specially modified bone marrows

• MHC class II KO mouse  

  • All cells are MHC II negative but has B-cells 

• B cell KO mouse 

  • All APCs are MHC II positive  

• Can then combine these two bone marrows to produce a mouse which expresses  

  • Normal DCs and macrophages 

  • But B-cells which are MHC class II negative  

What is the EAE mouse model used to study?

Multiple sclerosis

How can Multiple Sclerosis be induced in a mouse?

Inject nervous tissue/purified preparation in Freund’s adjuvant

Mice then become progressively paralysed

How can arthritis be induced in a mouse?

Purify aggrecan and inject it into a mouse joint.

This will then induce something similar to arthritis in a mouse joint

What are NOD mice used to study?

Type 1 diabetes

What are NOD SCID mice?

NOD mice (Develop type 1 diabetes) which also have no B-cells and no T-cells

When NOD SCID mice (No T/B-cells) are injected with human cells, they were killed by NK and innate cells. 

How did they solve this?

Most innate cells reply upon cytokines for development.

• Common gamma chain was then knocked out which prevents innate cells from developing. 

What is the common gamma chain?

Protein component of several cytokine receptors including IL-2, IL-4, etc…

When NOD SCID mice (No T/B-cells) are injected with human lymphocytes, 4-8 weeks later the humans T/B-cells will attack the mouse cells.

How did they solve this?

Rather than mature human T/B-cells, they injected human CD34+ hematopoietic stem cells

• From these stem cells, functional human T/B-cells will develop

• These developing cells will recognise mouse MHC class I/II without attacking them