Humanized animal models

Question 1

Why do we humanise mice?

Answer 1

• humans can‘t serve as experimental subject
• mice have different immunological requirements
• some pathogens don‘t infect murine cells
• better evaluation of new drugs

Question 2

Which features do mice need to be humanized?

Answer 2

• no rejection of human xenografts:
  
  • differentiated cells
  • hematopoetic stem cells (HSC)
  • tissue
• direct expression of transgenic human genes

Question 3

SCID mouse

Answer 3

• SCID = severe combined immunodeficiency
• no T and B cells (leaky = spontaneous generation of mouse lymphocytes)
• high NK-cell activity
• engraftment of human PBMC, fetal hematopoetic tissue and HSCs

Question 4

What does Prkdc-scid mean?

Answer 4

CB17 mice with mutations in protein kinase, DNA and catalytic polypeptide

Question 5

RAG1/2-/- mice

Answer 5

• targeted mutation of the recombination-activating gene (RAG) 1 & 2
• immunodeficienct (no T and B cells) -> not leaky
• high NK cell activity
• limited engraftment of human HSCs

Question 6

NOD-SCID mice

Answer 6

• non-obese diabetic SCID
• no T and B cells + reduced innate immune response (still leaky)
• combination with other immunodeficienct mice improves engraftment of hPBMCs and hHSCs

Question 7

IL-2Rγ-/- mice

Answer 7

• Interleukin-2 receptor (common) gamma-chain deficiency
• IL-2Rγ is component of receptor for many ILs -> indispensable for signalling
• four different mutations (truncation or deletion of γ-chain
• low T and B cells, no NK cell activity (not leaky)
• combination with other immunodeficienct mice improves engraftment of hPBMCs and hHSCs

Question 8

Overview

Answer 8

Question 9

Name 3 general approaches of engraftment human immune systems and name (dis-)advantages)

Answer 9

1. NOD/SCID-hu BLT (BLT) = thymus and liver tissue implanted under renal capsule + injection of autologous fetal liver HSC
   - more complete human immune system
   - hT cells are HLA-restricted
   - development of wasting syndrome
2. Hu-PBL-SCID = injection of hPBMCs
   - easy access to clinically relevant samples
   - mainly CD3+ T cell engraftment
   - GVHD leads to short window
3. Hu-SRC-SCID = injection of hHSCs
   - mice develop a complete human immune system
   - hT cells are educated on murine thymus -> H2-restricted

Question 10

How can we improve humanized immune system mice models?

Answer 10

• sublethal γ-radiation or pretreatment (beware that newborns are radiosensitive)
• injection should direct human tissue/HSCs to supportive niche (intravenous, intraoeritoneal, intrahepatic or into bone marrow)

Question 11

Why are SCID mice extremely radiosensitive?

Answer 11

SCID defect causes defect in DNA repair

Question 12

Describe human hematopoiesis in „empty“ mice

Answer 12

• pluripotent HSC
  
  • long-term self renewing stem cells
  • repopulation possible
  • sustain long-term hematopoiesis
• was early in vivo model to study human HSC function

Question 13

Describe human hematopoiesis in SCID mice

Answer 13

• SCID-hu-mice: engraftment of human fetal tissue (thymus & liver cells) into SCID
• Hu-SRC-SCID mice: engraftment of human HSCs

Question 14

Describe human hematopoiesis in NOD-SCID-IL-2Rγ-/- mice

Answer 14

• transplantation of CD34+ blood cells
• mice develop human dendritic cells and lymphocytes
• experiments with IL-2Rγ-/- mice hint to a treatment for Fanconi anemia, when coupled with retrovirus-mediated transfer of FANCA-cDNA —> long-term reconstruction of stem cell compartment was archived

Question 15

What would be an important advancement for immunological research?

Answer 15

Establishment of a functional human immune system with primary and secondary immune response

Question 16

Next steps in mouse humanisation (future outlook)?

Answer 16

• further reduction of host innate immunity
• transgenic expression of human HLA
• transgenic expression of human cytokines
• development of a human lymphoid architecture