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From Bench to Cageside:Immunology of Humanizing Mice

Zachary T Freeman

Goals

• Understand relevant immunology related to humanized mice

• Understand three main components that contribute to formation of humanized mice

• Clinical management of severely immunodeficientmice

Goals




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Dranoff, G Nat. Rev. Cancer, 2004

Innate Lymphoid Cells (ILCs) Are a Novel Cell Type for Immune Responses

Shortman K, et al Nat Rev Imm2002

Innate Lymphoid Cells (ILCs) Are a Novel Cell Type for Immune Responses

Spits H, Nat Rev Imm, 2013

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Humoral Immunity Cellular Immunity

Kumar et al. Robbins Basic Pathology, 8 edn. 2007, pp. 109, Copyright Elsevier.

B Cells Produced Antibodies as the Main Mediator of Humoral Immunity

Cooper MD, Nat Rev Imm, 2015

Zou W, Nat Rev Imm, 2010

CD4 T Cells Coordinate and Modulate Cellular Immune Responses

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Restifo NP, Nat Rev Imm, 2012

CD8 T Cells Mediate Effector Portion of Cellular Immunity

Goals




Crotty S, Nat Rev Imm, 2015

T and B cell Interactions are Important for Protective Immunity

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https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology/humanized-mice/cd34

What is an NSG mouse?

https://www.jax.org/strain/005557

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ

Non Obese Diabetic Mice Have Altered Innate and Adaptive Immunophenotype

Calis JJA, Trends in Imm, 2014

• T cell mediated islet destruction/diabetes

• Defective NK cell function

• Defective macrophage cytokine production

• Altered immunoregulatory functions of antigen presenting cells

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Antigen Specificity Defines B and T cells


B and T cell Receptors Share Common Features


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Lymphoid Specific General

Villartay, JP, Nat Rev Imm, 2003

Deletion of Prkdc Leads to Defect in DNA Repair Mechanisms in All Cells




IL-2 Receptor Gamma is Essential B, T, NK and DC Function

Rochman Y, Nat Rev Imm, 2009

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Ionizing Radiation Has Variable Effects on Immune Cells

Finkelstein SE, et al Frontiers in Oncology, 2012


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Origins of “Humanizing” Component

• Peripheral blood mononuclear cells (PBMC/PBL)

• CD34+ hematopoietic stem cells (HSCs)

• Bone marrow, liver, thymus (BLT mice)

CD34+ Cells Serve as Total Precursor to Immune System

Sharpless NE et al, Nat Rev Mol Cell Bio, 2007

Shultz LD, Nat Rev Imm, 2012

Unknown Cross talk between Mouse and Human Cytokines

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• S: Human Stem Cell Factor (SCF) gene

• maintains normal basal hematopoiesis.

• GM: Human Granulocyte/Macrophage-colony stimulating factor 2 (GM-CSF)

• Innate cell numbers and function

• 3: Human interleukin-3 (IL-3)

• multipotent hematopoietic stem cells differentiate into myeloid progenitor cells

• myeloid lineage to proliferate

Human Cytokines Enhance Innate Cell Engraftment in NSG-SGM3 Mice

Shultz LD, Nat Rev Imm, 2012

Human Cytokines Enhance Innate Cell Engraftment in Humanized Mice

Goals




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Immunology Problems Underlying Clinical Disease in Humanized Mice

• Increased susceptibility to commensal/opportunisitcorganisms• Cornybacterium bovis• Klebsiella oxytoca• Pasturella pneumonia• Pseudomonas sp• Staphlococcus sp• Pneumocystis murina

• Graft Versus Host Disease• Limited resources for the immune system• Risk of anthropozoonosis

Human Immune Cells Do No Recognize Murine Immune Cells

Kobayashi KS, Nature Reviews Immunology, 2012

Human Immune Cells Do No Recognize Murine Immune Cells

Bleakley M, Nature Reviews Cancer, 2004

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Limited Host Resources Alter Immune Cell Expansion and Homeostasis

Badovinac VP, Immunity 2007

Unknown Risk of Anthropozoonosis?

• Human immune cells host to many infectious pathogens

• HIV, EBV, CMV, HTLV

• Unknown risk of infection for severely immunodeficient mouse

• Unclear if murine immune system responds to these organisms

Management of Humanized Mice

• Barrier housing• Air locks/showers• Designated clothing• PPE• PAPRs

• Individually ventilated cages• HEPA filtered air• Automated water

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• Vary levels of “sterilization”• Cages• Food• Bedding• Laminar flow hoods

• Food/water practices• Acidification of water• Prophylactic antibiotics not recommended


• Infectious Agent Surveillance• Sentinel selection

• Age?• Immune status?• Direct vs. indirect

• Agent exclusion• Testing methodology/frequency

• Limited information about efficacy of treatment• Rederivation to clean up infections


https://www.jax.org/news-and-insights/jax-blog/2015/april/top-5-tips-to-take-care-of-your-nsg-mice

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Basic immunology of humanizing mice is important for clinical management and use of the model.

Questions?

Acknowledgements

• Jessica Xu DVM

• ULAM faculty and residents

References• 1.Badovinac, V.P., Haring, J.S. & Harty, J.T. Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the

CD8(+) T cell response to infection. Immunity 26, 827-841 (2007).• 2.Bleakley, M. & Riddell, S.R. Molecules and mechanisms of the graft-versus-leukaemia effect. Nat Rev Cancer 4, 371-380 (2004).• 3.Calis, J.J. & Rosenberg, B.R. Characterizing immune repertoires by high throughput sequencing: strategies and applications. Trends

Immunol 35, 581-590 (2014).• 4.Cooper, M.D. The early history of B cells. Nat Rev Immunol 15, 191-197 (2015).• 5.Crotty, S. A brief history of T cell help to B cells. Nat Rev Immunol 15, 185-189 (2015).• 6.de Villartay, J.P., Fischer, A. & Durandy, A. The mechanisms of immune diversification and their disorders. Nat Rev Immunol 3, 962-

972 (2003).• 7.Dranoff, G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer 4, 11-22 (2004).• 8. Finkelstein, S.E. & Fishman, M. Clinical opportunities in combining immunotherapy with radiation therapy. Front Oncol 2, 169

(2012).• 9. Kobayashi, K.S. & van den Elsen, P.J. NLRC5: a key regulator of MHC class I-dependent immune responses. Nat Rev Immunol 12,

813-820 (2012).• 10. Restifo, N.P., Dudley, M.E. & Rosenberg, S.A. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev

Immunol 12, 269-281 (2012).• 11. Rochman, Y., Spolski, R. & Leonard, W.J. New insights into the regulation of T cells by gamma(c) family cytokines. Nat Rev

Immunol 9, 480-490 (2009).• 12. Sharpless, N.E. & DePinho, R.A. How stem cells age and why this makes us grow old. Nat Rev Mol Cell Biol 8, 703-713 (2007).• 13. Shortman, K. & Liu, Y.J. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2, 151-161 (2002).• 14. Spits, H. et al. Innate lymphoid cells--a proposal for uniform nomenclature. Nat Rev Immunol 13, 145-149 (2013).• 15. Walsh, N.C. et al. Humanized Mouse Models of Clinical Disease. Annu Rev Pathol 12, 187-215 (2017).• 16. Zou, W. & Restifo, N.P. T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol 10, 248-256 (2010).• 17. Kumar et al. Robbins Basic Pathology, 8 edn. 2007, pp. 109, Copyright Elsevier.• https://www.jax.org/strain/005557• https://www.jax.org/news-and-insights/jax-blog/2015/april/top-5-tips-to-take-care-of-your-nsg-mice• https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology/humanized-mice/cd34

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