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Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

Laboratory Investigationvolume 99pages93106 (2019) | Download Citation


The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4+ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4+ Tregs and splenic CD8+ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4+ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8+ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.

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  1. 1.

    Doulatov S, Notta F, Laurenti E, Dick JE. Hematopoiesis: a human perspective. Cell Stem Cell. 2012;10:120–36.

  2. 2.

    Dzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol. 2008;9:129–36.

  3. 3.

    Plas DR, Rathmell JC, Thompson CB. Homeostatic control of lymphocyte survival: potential origins and implications. Nat Immunol. 2002;3:515–21.

  4. 4.

    Rathmell JC, Thompson CB. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell. 2002;109:Suppl:S97–107.

  5. 5.

    Zhao E, Xu H, Wang L, et al. Bone marrow and the control of immunity. Cell Mol Immunol. 2012;9:11–19.

  6. 6.

    Bronte V, Pittet MJ. The spleen in local and systemic regulation of immunity. Immunity. 2013;39:806–18.

  7. 7.

    Hensel JA, Khattar V, Ashton R, et al. Location of tumor affects local and distant immune cell type and number. Immun Inflamm Dis. 2017;5:85–94.

  8. 8.

    Juul S, Pliskin JS, Fineberg HV. Variation and information inwhite blood cell differential counts. Med Decis Making. 1984;4:69–80.

  9. 9.

    Sellers RS, Clifford CB, Treuting PM, et al. Immunological variation between inbred laboratory mouse strains: points to consider in phenotyping genetically immunomodified mice. Vet Pathol. 2012;49:32–43.

  10. 10.

    Bogue MA, Grubb SC. The Mouse Phenome Project. Genetica. 2004;122:71–4.

  11. 11.

    Grubb SC, Churchill GA, Bogue MA. A collaborative database of inbred mouse strain characteristics. Bioinformatics. 2004;20:2857–9.

  12. 12.

    Chen J, Harrison DE. Quantitative trait loci regulating relative lymphocyte proportions in mouse peripheral blood. Blood. 2002;99:561–6.

  13. 13.

    Chen J, Flurkey K, Harrison DE. A reduced peripheral blood CD4(+) lymphocyte proportion is a consistent ageing phenotype. Mech Ageing Dev. 2002;123:145–53.

  14. 14.

    Peters LL, Cheever EM, Ellis HR, et al. Large-scale, high-throughput screening for coagulation and hematologic phenotypes in mice. Physiol Genomics. 2002;11:185–93.

  15. 15.

    Petkova SB, Yuan R, Tsaih SW, et al. Genetic influence on immune phenotype revealed strain-specific variations in peripheral blood lineages. Physiol Genomics. 2008;34:304–14.

  16. 16.

    van de Geijn GJ, van Rees V, van Pul-Bom N, et al. Leukoflow: multiparameter extended white blood cell differentiation for routine analysis by flow cytometry. Cytometry A. 2011;79:694–706.

  17. 17.

    Zitvogel L, Pitt JM, Daillere R, et al. Mouse models in oncoimmunology. Nat Rev Cancer. 2016;16:759–73.

  18. 18.

    Sawant A, Schafer CC, Ponnazhagan S, et al. The dual targeting of immunosuppressive cells and oxidants promotes effector and memory T-cell functions against lung cancer. Oncoimmunology. 2014;3:e27401.

  19. 19.

    Sawant AC, Adhikari P, Narra SR, et al. Neutrophil to lymphocyte ratio predicts short- and long-term mortality following revascularization therapy for ST elevation myocardial infarction. Cardiol J. 2014;21:500–8.

  20. 20.

    Schafer CC, Wang Y, Hough KP, et al. Indoleamine 2,3-dioxygenase regulates anti-tumor immunity in lung cancer by metabolic reprogramming of immune cells in the tumor microenvironment. Oncotarget. 2016;7:75407–24.

  21. 21.

    Larson-Casey JL, Deshane JS, Ryan AJ, et al. Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity. 2016;44:582–96.

  22. 22.

    Sawant A, Deshane J, Jules J, et al. Myeloid-derived suppressor cells function as novel osteoclast progenitors enhancing bone loss in breast cancer. Cancer Res. 2013;73:672–82.

  23. 23.

    Levy S, Feduska JM, Sawant A, et al. Immature myeloid cells are critical for enhancing bone fracture healing through angiogenic cascade. Bone. 2016;93:113–24.

  24. 24.

    Guidance Development Review C, Working Group for Clinical Studies of Cancer I, Working Group for Effector Cell T. et al. 2015 Guidance on cancer immunotherapy development in early-phase clinical studies. Cancer Sci. 2015;106:1761–71.

  25. 25.

    Elderman M, van Beek A, Brandsma E, et al. Sex impacts Th1 cells, Tregs, and DCs in both intestinal and systemic immunity in a mouse strain and location-dependent manner. Biol Sex Differ. 2016;7:21.

  26. 26.

    Klein SL, Flanagan KL. Sex differences in immune responses. Nat Rev Immunol. 2016;16:626–38.

  27. 27.

    Pinchuk LM, Filipov NM. Differential effects of age on circulating and splenic leukocyte populations in C57BL/6 and BALB/c male mice. Immun Ageing. 2008;5:1.

  28. 28.

    Sivan A, Corrales L, Hubert N, et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 2015;350:1084–9.

  29. 29.

    Li QX, Feuer G, Ouyang X, et al. Experimental animal modeling for immuno-oncology. Pharmacol Ther. 2017;173:34–46.

  30. 30.

    Abdullah M, Chai PS, Chong MY, et al. Gender effect on in vitro lymphocyte subset levels of healthy individuals. Cell Immunol. 2012;272:214–9.

  31. 31.

    Chen Z, Huang A, Sun J, et al. Inference of immune cell composition on the expression profiles of mouse tissue. Sci Rep. 2017;7:40508.

  32. 32.

    Hahne F, Khodabakhshi AH, Bashashati A, et al. Per-channel basis normalization methods for flow cytometry data. Cytometry A. 2010;77:121–31.

  33. 33.

    Finak G, Langweiler M, Jaimes M, et al. Standardizing flow cytometry immunophenotyping analysis from the Human ImmunoPhenotyping Consortium. Sci Rep. 2016;6:20686.

  34. 34.

    Bashashati A, Brinkman RR. A survey of flow cytometry data analysis methods. Adv Bioinformatics. 2009;2009:584603.

  35. 35.

    Ostrand-Rosenberg S. Animal models of tumor immunity, immunotherapy and cancer vaccines. Curr Opin Immunol. 2004;16:143–50.

  36. 36.

    Clapp NK, Tyndall RL, Otten JA. Differences in tumor types and organ susceptibility in BALB-c and RF mice following dimethylnitrosamine and diethylnitrosamine. Cancer Res. 1971;31:196–8.

  37. 37.

    Ullrich RL. Tumor induction in BALB/c female mice after fission neutron or gamma irradiation. Radiat Res. 1983;93:506–15.

  38. 38.

    Mekada K, Abe K, Murakami A, et al. Genetic differences among C57BL/6 substrains. Exp Anim. 2009;58:141–9.

  39. 39.

    Jiang LI, Nadeau JH. 129/Sv mice—a model system for studying germ cell biology and testicular cancer. Mamm Genome. 2001;12:89–94.

  40. 40.

    Clapcote SJ, Roder JC. Deletion polymorphism of Disc1 is common to all 129 mouse substrains: implications for gene-targeting studies of brain function. Genetics. 2006;173:2407–10.

  41. 41.

    Rodgers RJ, Augar R, Berryman N, et al. Atypical anxiolytic-like response to naloxone in benzodiazepine-resistant 129S2/SvHsd mice: role of opioid receptor subtypes. Psychopharmacology. 2006;187:345–55.

  42. 42.

    Owen JA, Punt J, Stranford SA et al. Kuby immunology. New York: W.H. Freeman; 2013.

  43. 43.

    Wei S, Kryczek I, Zou W. Regulatory T-cell compartmentalization and trafficking. Blood. 2006;108:426–31.

  44. 44.

    Vivier E, Tomasello E, Baratin M, et al. Functions of natural killer cells. Nat Immunol. 2008;9:503–10.

  45. 45.

    Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009;9:162–74.

  46. 46.

    Merad M, Sathe P, Helft J, et al. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol. 2013;31:563–604.

  47. 47.

    McKenna K, Beignon AS, Bhardwaj N. Plasmacytoid dendritic cells: linking innate and adaptive immunity. J Virol. 2005;79:17–27.

  48. 48.

    Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013;13:159–75.

  49. 49.

    Stein JV, Nombela-Arrieta C. Chemokine control of lymphocyte trafficking: a general overview. Immunology. 2005;116:1–12.

  50. 50.

    Ngiow SF, Young A, Jacquelot N, et al. A threshold level of intratumor CD8+ T-cell PD1 expression dictates therapeutic response to anti-PD1. Cancer Res. 2015;75:3800–11.

  51. 51.

    Kamphorst AO, Pillai RN, Yang S, et al. Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients. Proc Natl Acad Sci USA. 2017;114:4993–8.

  52. 52.

    Hackstein H, Wachtendorf A, Kranz S, et al. Heterogeneity of respiratory dendritic cell subsets and lymphocyte populations in inbred mouse strains. Respir Res. 2012;13:94.

  53. 53.

    Velasquez-Lopera MM, Correa LA, Garcia LF. Human spleen contains different subsets of dendritic cells and regulatory T lymphocytes. Clin Exp Immunol. 2008;154:107–14.

  54. 54.

    Langeveld M, Gamadia LE, ten Berge IJ. T-lymphocyte subset distribution in human spleen. Eur J Clin Invest. 2006;36:250–6.

  55. 55.

    Asselin-Paturel C, Brizard G, Pin JJ, et al. Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody. J Immunol. 2003;171:6466–77.

  56. 56.

    Watanabe H, Numata K, Ito T, et al. Innate immune response in Th1- and Th2-dominant mouse strains. Shock. 2004;22:460–6.

  57. 57.

    Rivera J, Tessarollo L. Genetic background and the dilemma of translating mouse studies to humans. Immunity. 2008;28:1–4.

  58. 58.

    Gueders MM, Paulissen G, Crahay C, et al. Mouse models of asthma: a comparison between C57BL/6 and BALB/c strains regarding bronchial responsiveness, inflammation, and cytokine production. Inflamm Res. 2009;58:845–54.

  59. 59.

    Schulte S, Sukhova GK, Libby P. Genetically programmed biases in Th1 and Th2 immune responses modulate atherogenesis. Am J Pathol. 2008;172:1500–8.

  60. 60.

    Lechner MG, Karimi SS, Barry-Holson K, et al. Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy. J Immunother. 2013;36:477–89.

  61. 61.

    Jovicic N, Jeftic I, Jovanovic I, et al. Differential immunometabolic phenotype in Th1 and Th2 dominant mouse strains in response to high-fat feeding. PLoS ONE. 2015;10:e0134089.

  62. 62.

    Florez-Vargas O, Brass A, Karystianis G, et al. Bias in the reporting of sex and age in biomedical research on mouse models. eLife. 2016;5:e13615.

  63. 63.

    Ito C, Okuyama-Dobashi K, Miyasaka T, et al. CD8+ T cells mediate female-dominant IL-4 production and airway inflammation in allergic asthma. PLoS ONE. 2015;10:e0140808.

  64. 64.

    Lloyd CM, Hawrylowicz CM. Regulatory T cells in asthma. Immunity. 2009;31:438–49.

  65. 65.

    Rothermel AL, Gilbert KM, Weigle WO. Differential abilities of Th1 and Th2 to induce polyclonal B cell proliferation. Cell Immunol. 1991;135:1–15.

  66. 66.

    Bryan MA, Guyach SE, Norris KA. Specific humoral immunity versus polyclonal B cell activation in Trypanosoma cruzi infection of susceptible and resistant mice. PLoS Negl Trop Dis. 2010;4:e733.

  67. 67.

    Morbach H, Eichhorn EM, Liese JG, et al. Reference values for B cell subpopulations from infancy to adulthood. Clin Exp Immunol. 2010;162:271–9.

  68. 68.

    Pyzik M, Kielczewska A, Vidal SM. NK cell receptors and their MHC class I ligands in host response to cytomegalovirus: insights from the mouse genome. Semin Immunol. 2008;20:331–42.

  69. 69.

    Lee SH, Girard S, Macina D, et al. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat Genet. 2001;28:42–45.

  70. 70.

    Ouzounova M, Lee E, Piranlioglu R, et al. Monocytic and granulocytic myeloid derived suppressor cells differentially regulate spatiotemporal tumour plasticity during metastatic cascade. Nat Commun. 2017;8:14979.

  71. 71.

    Ortiz ML, Kumar V, Martner A, et al. Immature myeloid cells directly contribute to skin tumor development by recruiting IL-17-producing CD4+ T cells. J Exp Med. 2015;212:351–67.

  72. 72.

    Schmid M, Zimara N, Wege AK, et al. Myeloid-derived suppressor cell functionality and interaction with Leishmania major parasites differ in C57BL/6 and BALB/c mice. Eur J Immunol. 2014;44:3295–306.

  73. 73.

    Van Ginderachter JA, Beschin A, De Baetselier P, et al. Myeloid-derived suppressor cells in parasitic infections. Eur J Immunol. 2010;40:2976–85.

  74. 74.

    Santos JL, Andrade AA, Dias AA, et al. Differential sensitivity of C57BL/6 (M-1) and BALB/c (M-2) macrophages to the stimuli of IFN-gamma/LPS for the production of NO: correlation with iNOS mRNA and protein expression. J Interferon Cytokine Res. 2006;26:682–8.

  75. 75.

    Depke M, Breitbach K, Dinh Hoang Dang K, et al. Bone marrow-derived macrophages from BALB/c and C57BL/6 mice fundamentally differ in their respiratory chain complex proteins, lysosomal enzymes and components of antioxidant stress systems. J Proteomics. 2014;103:72–86.

  76. 76.

    Bertolini TB, de Souza AI, Gembre AF, et al. Genetic background affects the expansion of macrophage subsets in the lungs of Mycobacterium tuberculosis-infected hosts. Immunology. 2016;148:102–13.

  77. 77.

    Billard E, Cazevieille C, Dornand J, et al. High susceptibility of human dendritic cells to invasion by the intracellular pathogens Brucella suis, B. abortus, and B. melitensis. Infect Immun. 2005;73:8418–24.

  78. 78.

    Pina A, de Araujo EF, Felonato M, et al. Myeloid dendritic cells (DCs) of mice susceptible to paracoccidioidomycosis suppress T cell responses whereas myeloid and plasmacytoid DCs from resistant mice induce effector and regulatory T cells. Infect Immun. 2013;81:1064–77.

  79. 79.

    Lande R, Gilliet M. Plasmacytoid dendritic cells: key players in the initiation and regulation of immune responses. Ann NY Acad Sci. 2010;1183:89–103.

  80. 80.

    Zhang H, Gregorio JD, Iwahori T, et al. A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes. Proc Natl Acad Sci USA. 2017;114:1988–93.

  81. 81.

    Sousa LM, Carneiro MB, Resende ME, et al. Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice. Parasite Immunol. 2014;36:13–31.

  82. 82.

    Chen L, Watanabe T, Watanabe H, et al. Neutrophil depletion exacerbates experimental Chagas’ disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions. Eur J Immunol. 2001;31:265–75.

  83. 83.

    Leynaert B, Sunyer J, Garcia-Esteban R, et al. Gender differences in prevalence, diagnosis and incidence of allergic and non-allergic asthma: a population-based cohort. Thorax. 2012;67:625–31.

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Financial support from the National Institute of Health research grants CA184770 and AR060948, and the Department of Defense grant PR141945, is greatly acknowledged. We sincerely thank ENVIGO for providing mice for the study.

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  1. These authors contributed equally: Jonathan A. Hensel, Vinayak Khattar


  1. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    • Jonathan A. Hensel
    • , Vinayak Khattar
    • , Reading Ashton
    •  & Selvarangan Ponnazhagan


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Correspondence to Selvarangan Ponnazhagan.

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