A human immune system mouse model with robust lymph node development

Abstract

Lymph nodes (LNs) facilitate the cellular interactions that orchestrate immune responses. Human immune system (HIS) mice are powerful tools for interrogation of human immunity but lack secondary lymphoid tissue (SLT) as a result of a deficiency in Il2rg-dependent lymphoid tissue inducer cells. To restore LN development, we induced expression of thymic-stromal-cell-derived lymphopoietin (TSLP) in a Balb/c Rag2−/−Il2rg−/−SirpaNOD (BRGS) HIS mouse model. The resulting BRGST HIS mice developed a full array of LNs with compartmentalized human B and T cells. Compared with BRGS HIS mice, BRGST HIS mice have a larger thymus, more mature B cells, and abundant IL-21-producing follicular helper T (TFH) cells, and show enhanced antigen-specific responses. Using BRGST HIS mice, we demonstrated that LN TFH cells are targets of acute HIV infection and represent a reservoir for latent HIV. In summary, BRGST HIS mice reflect the effects of SLT development on human immune responses and provide a model for visualization and interrogation of regulators of immunity.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Characterization of human immune cell reconstitution in BRGST HIS mice.
Fig. 2: Intravital two-photon microscopy of BRGST LN showing human T cell behavior.
Fig. 3: Human thymocyte and TFH cell development in BRGST HIS mice.
Fig. 4: Enhanced humoral responses in BRGST HIS mice.
Fig. 5: Immunoglobulin gene repertoire of human class-switched memory B cells in HIS mice.
Fig. 6: HIV infection and latency in BRGST HIS mice.

References

  1. 1.

    Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V. & Greiner, D. L. Humanized mice for immune system investigation: progress, promise and challenges. Nat. Rev. Immunol. 12, 786–798 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Sugamura, K. et al. The interleukin-2 receptor gamma chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14, 179–205 (1996).

    CAS  Article  Google Scholar 

  3. 3.

    Traggiai, E. et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304, 104–107 (2004).

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Ito, M. et al. NOD/SCID/γc null mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100, 3175–3182 (2002).

    CAS  Article  Google Scholar 

  5. 5.

    Gimeno, R. et al. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2–/– γc–/– mice: functional inactivation of p53 in developing T cells. Blood 104, 3886–3893 (2004).

  6. 6.

    Chappaz, S. & Finke, D. The IL-7 signaling pathway regulates lymph node development independent of peripheral lymphocytes. J. Immunol. 184, 3562–3569 (2010).

    CAS  Article  Google Scholar 

  7. 7.

    van de Pavert, S. A. & Mebius, R. E. New insights into the development of lymphoid tissues. Nat. Rev. Immunol. 10, 664–674 (2010).

    Article  CAS  Google Scholar 

  8. 8.

    Spits, H. & Di Santo, J. P. The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat. Immunol. 12, 21–27 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    Spits, H. et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 13, 145–149 (2013).

    CAS  Article  Google Scholar 

  10. 10.

    Verstraete, K. et al. Structural basis of the proinflammatory signaling complex mediated by TSLP. Nat. Struct. Mol. Biol. 21, 375–382 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Park, L. S. et al. Cloning of the murine thymic stromal lymphopoietin (TSLP) receptor: formation of a functional heteromeric complex requires interleukin 7 receptor. J. Exp. Med. 192, 659–670 (2000).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Chappaz, S., Flueck, L., Farr, A. G., Rolink, A. G. & Finke, D. Increased TSLP availability restores T- and B-cell compartments in adult IL-7 deficient mice. Blood 110, 3862–3870 (2007).

    CAS  Article  Google Scholar 

  13. 13.

    Legrand, N. et al. Functional CD47/signal regulatory protein alpha (SIRPα) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo. Proc. Natl. Acad. Sci. USA 108, 13224–13229 (2011).

    CAS  Article  Google Scholar 

  14. 14.

    Li, Y. et al. A novel Flt3-deficient HIS mouse model with selective enhancement of human DC development. Eur. J. Immunol. 46, 1291–1299 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Lopez-Lastra, S. et al. A functional DC cross talk promotes human ILC homeostasis in humanized mice. Blood Adv. 1, 601–614 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Denton, P. W. et al. IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice. Mucosal Immunol. 5, 555–566 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Okada, T. et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. 3, e150 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Bousso, P. & Robey, E. Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nat. Immunol. 4, 579–585 (2003).

    CAS  Article  Google Scholar 

  19. 19.

    Garcia, Z. et al. Competition for antigen determines the stability of T cell-dendritic cell interactions during clonal expansion. Proc. Natl. Acad. Sci. USA 104, 4553–4558 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    Celli, S., Lemaître, F. & Bousso, P. Real-time manipulation of T cell-dendritic cell interactions in vivo reveals the importance of prolonged contacts for CD4+ T cell activation. Immunity 27, 625–634 (2007).

    CAS  Article  Google Scholar 

  21. 21.

    Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

    CAS  Article  Google Scholar 

  22. 22.

    Halkias, J. et al. Conserved and divergent aspects of human T-cell development and migration in humanized mice. Immunol. Cell Biol. 93, 716–726 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Huntington, N. D. et al. Autonomous and extrinsic regulation of thymopoiesis in human immune system (HIS) mice. Eur. J. Immunol. 41, 2883–2893 (2011).

    CAS  Article  Google Scholar 

  24. 24.

    Jiang, Q., Coffield, V. M., Kondo, M. & Su, L. TSLP is involved in expansion of early thymocyte progenitors. BMC Immunol. 8, 11 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Alves, N. L., Huntington, N. D., Rodewald, H. R. & Di Santo, J. P. Thymic epithelial cells: the multi-tasking framework of the T cell “cradle”. Trends Immunol. 30, 468–474 (2009).

    CAS  Article  Google Scholar 

  26. 26.

    Takahama, Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6, 127–135 (2006).

    CAS  Article  Google Scholar 

  27. 27.

    Crotty, S. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29, 621–663 (2011).

    CAS  Article  Google Scholar 

  28. 28.

    Fazilleau, N., Mark, L., McHeyzer-Williams, L. J. & McHeyzer-Williams, M. G. Follicular helper T cells: lineage and location. Immunity 30, 324–335 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Lang, J. et al. Studies of lymphocyte reconstitution in a humanized mouse model reveal a requirement of T cells for human B cell maturation. J. Immunol. 190, 2090–2101 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Yu, H. et al. A novel humanized mouse model with significant improvement of class-switched, antigen-specific antibody production. Blood 129, 959–969 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tonomura, N., Habiro, K., Shimizu, A., Sykes, M. & Yang, Y. G. Antigen-specific human T-cell responses and T cell-dependent production of human antibodies in a humanized mouse model. Blood 111, 4293–4296 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Becker, P. D. et al. Generation of human antigen-specific monoclonal IgM antibodies using vaccinated “human immune system” mice. PLoS One 5, e13137 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Scheid, J. F. et al. Differential regulation of self-reactivity discriminates between IgG+ human circulating memory B cells and bone marrow plasma cells. Proc. Natl. Acad. Sci. USA 108, 18044–18048 (2011).

    CAS  Article  Google Scholar 

  34. 34.

    Tiller, T. et al. Autoreactivity in human IgG+ memory B cells. Immunity 26, 205–213 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Prigent, J. et al. Scarcity of autoreactive human blood IgA+ memory B cells. Eur. J. Immunol. 46, 2340–2351 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Freed, E. O., Englund, G. & Martin, M. A. Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J. Virol. 69, 3949–3954 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Li, G. et al. Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice. PLoS Pathog. 10, e1004291 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Passaes, C. P. et al. Ultrasensitive HIV-1 p24 assay detects single infected cells and differences in reservoir induction by latency reversal agents. J. Virol. 91, e02296 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    von Andrian, U. H. & Mempel, T. R. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3, 867–878 (2003).

    Article  CAS  Google Scholar 

  40. 40.

    Bousso, P. & Moreau, H. D. Functional immunoimaging: the revolution continues. Nat. Rev. Immunol. 12, 858–864 (2012).

    CAS  Article  Google Scholar 

  41. 41.

    Allam, A. et al. TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1. Sci. Rep. 5, 10443 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Xu, H. et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature 496, 523–527 (2013).

    CAS  Article  Google Scholar 

  43. 43.

    Veiga-Fernandes, H. et al. Tyrosine kinase receptor RET is a key regulator of Peyer’s patch organogenesis. Nature 446, 547–551 (2007).

    CAS  Article  Google Scholar 

  44. 44.

    Banga, R. et al. PD-1+ and follicular helper T cells are responsible for persistent HIV-1 transcription in treated aviremic individuals. Nat. Med. 22, 754–761 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Perreau, M. et al. Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J. Exp. Med. 210, 143–156 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    McGary, C. S. et al. CTLA-4+PD-1 memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques. Immunity 47, 776–788 (2017).

Download references

Acknowledgements

The authors thank the Center for Translational Research and the Animalerie Centrale of Institut Pasteur for collaboration, and N. Court, M. Boussand, O. Fiquet, D. Hardy, M. Tichit and A. Besnard for technical help. This work was supported by the Agence Nationale de la Recherche (ANR) programme RPIB (Im_HIS to J.P.D.), the Laboratoire d’Excellence REVIVE (grant ANR-10-LABX-73 to Y.L. and J.P.D.), the Laboratoire d’Excellence Vaccine Research Institute (grant ANR-10-LABX-77 to T.B. and O.S.), the Laboratoire d’Excellence Milieu Intérieur (grant ANR-10-LABX-69 to H.M.), the Laboratoire d’Excellence IBEID (grant ANR-10-LABX-62 to O.S.), the Agence Nationale de Recherche sur le SIDA et les hepatitis (grant 15465 to O.S.), the European Commission Seventh Framework Programme no. 305578 (PathCO; to J.P.D.), the European Research Council (grant ERC-2013-StG 337146 to A.K. and T.H.; grant ERC-2017-AvG 771167 to P.B.), Gilead Sciences (grant 00397 to G.M.-R. and J.P.D.), the Institut Pasteur (Core grant to J.P.D.; G5 Program to H.M.), and INSERM (Core grant to J.P.D.).

Author information

Affiliations

Authors

Contributions

Y.L. designed and performed experiments, analyzed data and wrote the paper; G.M.-R. designed, performed and analyzed data from in vivo HIV-infection experiments; T.B. performed and analyzed the latent-HIV-reactivation experiments; N.S. analyzed gut ILCs; M.D. performed ELISPOT assays; H.S.-M. performed immunofluorescence studies; A.K., A.I.L., T.H. and H.M. performed single B cell sorting and analysis of antibody gene sequencing data; Z.G. performed intravital imaging; G.J. performed immunohistology; F.B., A.T., H.M., O.S., D.F. and P.B. provided mouse strains, reagents and technological approaches; and J.P.D. designed and supervised the project and wrote the paper, with contributions from all other authors.

Corresponding author

Correspondence to James P. Di Santo.

Ethics declarations

Competing interests

J.P.D. is a stakeholder in AXENIS (founder and member of the executive board).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Mouse ILC and LN anlage development in BRGST mice.

(a) Mouse TSLP concentration in plasma from BRGS and BRGST mice was quantified by ELISA (n = 12 mice/group, bars represent mean, and error bars denote s.e.m.). (b) Representative FACS plot showing the gating strategy for mouse ILCs in lamina propria of small intestines of BRGST mice from 12 mice with similar results. (c) Absolute numbers of ILC subsets in small intestines of Rag2−/−, BRGS and BRGST mice (n = 4 mice/group; bars represent mean, and error bars denote s.e.m., P value is shown, one-way ANOVA with a Turkey test) (d) Representative images of anlagen of inguinal and iliac LNs in non-engrafted BRGS and BRGST mice 1 week after peritoneal injection of Chicago Sky Blue 6B dye from 2 independent experiments with similar results. LN anlagen are indicated by arrow heads. Scale bar = 2 mm. (e) Total number of LN anlagen observed in non-engrafted 8 week-old BRGS and BRGST littermates 1 week after dye injection (n = 5 mice/group, bars represent mean, and error bars denote s.e.m., P value is shown for Two-tailed Mann-Whitney U test). (f) Quantification of different LNs present in non-engrafted 8 week-old BRGS and BRGST littermates (n = 8 mice/group) 1 week after dye injection. To calculate the percentages, the number of mice observed with indicated LN anlage was divided by total number of mice from indicated strain. (g) Numbers of PPs counted on small intestines of BRGST and C57BL/6 newborn pups after VCAM1 whole mount staining (n = 4 mice/group, bars represent mean, and error bars denote s.e.m.).

Supplementary Figure 2 Characterization of human immune cell reconstitution in BRGST HIS mice.

(a) Representative pictures indicated LNs in BRGST HIS mice at 10-14 weeks post fetal liver CD34+ cell transplantation from 3 independent experiments with similar results. Scale bar = 2 mm (b) Kinetics of human cell reconstitution in blood of BRGS and BRGST HIS mice (n = 8 mice/group, centre values represent mean, and error bars denote s.e.m.). (c) Representative flow cytometry analysis of human naïve/memory T cell subsets from 12 HIS mice with similar results, identified by expression of CCR7 and CD45RA among hCD45+CD3+CD4+ splenic T cells.

Supplementary Figure 3 Two-photon microscopy of explanted BRGST LN showing human T cell behavior.

10 million human T cells purified from health donor PBMC were fluorescently labeled and adoptively transferred into 12 week old BRGST HIS mouse. After 24 h, intact inguinal lymph nodes were explanted and subjected to two-photon imaging (a). Representative images of human T cells and corresponding tracks from 8 different movies in 3 different recipients. Scale bar = 20 µm. Right side insets show the migration pattern for single human T cells (n = 3). Scale bar = 10 µm. Graphs show the mean velocity (b) and the straightness index (c) for individual T cells (the measure of centre represents mean). (d) T cells tracks were graphed from a common origin. Data are representative of 8 different movies in 3 different recipients.

Supplementary Figure 4 Mouse thymocyte precursor development in BRGST HIS mice.

(a) Percentages and absolute numbers of Ki67+ cells among human thymocyte subsets in 12 week old BRGS and BRGST HIS mice (n = 5 mice/BRGS, 6 mice/BRGST. bars represent mean, and error bars denote s.e.m., P value is shown for Two-tailed Mann-Whitney U test). (b) Representative images of thymi in 8 week old non-reconstituted BRGS and BRGST mice 1 week after peritoneal injection of Chicago Sky Blue 6B dye. Data from 2 independent experiments with similar results. Scale bar = 2 mm (c) Representative flow cytometry analysis of murine thymocytes gated on indicated populations above each plot (numbers next to outlined areas indicate percentages of cells) from 11 HIS mice with similar results. (d) Absolute numbers of mouse thymocyte subsets in BRGS and BRGST mice (n = 6 mice/BRGS, 5 mice/BRGST, bars represent mean, and error bars denote s.e.m., P value is shown for Two-tailed Mann-Whitney U test).

Supplementary Figure 5 B cell phenotype and antigen-specific immune responses after KLH immunization in HIS mice.

(a) FACS analysis of mature human B cells (gated on hCD45+mCD45CD19+ cells) from HIS mice. Representative analysis from 10 independent mice with similar results. (b) KLH specific IgG ELISA results from sera and (c) IFN-γ ELIspot results from splenocytes of KLH-immunized HIS mice in 2 independent experiments. (n = 4 mice/group, centre values represent mean, and error bars denote s.e.m.).

Supplementary Figure 6 Immunoglobulin gene repertoire of human class-switched memory B cells in HIS mice.

(a) Circular phylograms of representative clonal expansions as shown in B (green and blue clones for BRGS and BRGST, respectively). GL, germline; LN, lymph nodes; Sp, spleen; BM, bone marrow. (b) Circos plots comparing the frequency of VH(DH)JH rearrangements of IgA+/IgG+ memory B-cell antibodies between both groups. (c) Violin plots comparing the number of mutations in the variable genes of heavy (IgH) chain between both groups. The colored bar and the black dot indicate mean and median, respectively. Below each dot plot, pie charts show the distribution of mutation in each group, from 0 (white) to 7 or more (red). The number of sequences analyzed (n) in each group is indicated below the Violon plot. Groups were compared using unpaired two-sided student t-test with Welch’s correction. LN, lymph nodes; Sp, spleen; BM, bone marrow. (d) Graphs show the Bayesian estimation of antigen-driven selection in IgH CDR sequences from both groups as determined with the BASELINe software using a total of 121 and 115 IgL sequences for BRGS and BRGST, respectively (following the groups distribution shown in (c)). Values >0 indicate positive selection.

Supplementary Figure 7 HIV infection in BRGS and BRGST HIS mice.

Longitudinal analysis in the blood of HIV viremia (RNA copies/mL) and of human CD45+ cells, CD4+ and CD8+ T cells, respectively in BRGS (a) and BRGST (b) HIS mice. (n = 6 BRGS mice, n = 12 BRGST mice for acute infection; n = 3 BRGS, n = 4 BRGST mice for HAART treatment).

Supplementary Figure 8 Cell-sorting report.

(a) Representative flow cytometry analysis for T cell enrichment by lineage depletion of pooled HIS mouse splenocytes. Data from 3 independent experiments with similar results. (b) Representative flow cytometry analysis for CD4+ T cell selection from pooled splenocytes and lymph node cells. Data from 3 independent experiments with similar results.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1 and 2

Reporting Summary

Supplementary Video 1

Motility of HIS T cells in popliteal lymph nodes of BRGST HIS mice. Human T cells purified from HIS mice were fluorescently labeled and adoptively transferred into BRGST HIS mice of the same cohort. After 24 h, intact popliteal lymph nodes from the recipients were subjected to intravital two-photon imaging. T cells (green) are shown together with their tracks (white)

Supplementary Video 2

Motility of adult human peripheral blood T cells in lymph nodes of BRGST HIS mice. Human T cells purified from PBMC were fluorescently labeled and adoptively transferred into reconstituted BRGST

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Masse-Ranson, G., Garcia, Z. et al. A human immune system mouse model with robust lymph node development. Nat Methods 15, 623–630 (2018). https://doi.org/10.1038/s41592-018-0071-6

Download citation

Further reading

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing