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.
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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.).
J.P.D. is a stakeholder in AXENIS (founder and member of the executive board).
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Integrated supplementary information
(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.).
(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.
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.
(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+mCD45−CD19+ 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.
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).
(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 Figures 1–8 and Supplementary Tables 1 and 2
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)
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
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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
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