A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer’s patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Humanized mice are limited in terms of modeling human immunity, parti cularly with regards to antibody responses.Here we constructed a humanized (THX) mouse by grafting nonγirradiated, genetically myeloablated Kit W41J mutant immunodeficient pups with human cord blood CD34 + cells, followed by 17βestradiol conditioning to promote immune cell differentiation.THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop wellformed lymph nodes and intestinal lymphoid tissue, including Peyer's patches, and human thymic epithelial cells.These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T celldependent and T cellindependent antibody responses, entailing somatic hypermutation, classswitch recombination, and plasma cell and memory B cell differentiation.Upon flagellin or a PfizerBioNTech coronavirus disease 2019 (COVID19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptorbinding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGFβ, IL4 and IFNγ, all at physiological levels.These mice can also develop lupus autoimmunity after pristane injection.By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.
Many of the more than the 1,600 immune response mouse genes are incongruent with their human equivalents, resulting in divergencies or deficiencies of mice as predictors of human immune responses 1 , making availability of a 'humanized' mouse model that faithfully reproduces human immune responses a high priority.The first humanized immune system mice were constructed by injecting human peri pheral blood lym phocytes or (CD34 + ) human hematopoietic stem cells (huHSCs; hu pre fix for human or humanized is used throughout) into severe combined immunodeficiency Prkdc scid (SCID) mice or Rag1/Rag2 knockout (KO) mice [2][3][4][5][6] .Subsequently, huHSC grafting of immuno deficient nonobese diabetic NOD.CgPrkdc scid Il2rg tm1Wjl /Sz or NOD.CgPrkdc scid Il2rg null (NSG) mice 7,8 , in which Il2rg deletion results in defective cytokine signaling in multiple immune cell receptors, furthered the scope of humanized mice 8 .In huNSG mice, the NOD phagocytic cell SIRPα receptor variant crossreacts with human CD47 to induce a 'don't eat me' signal, thereby limiting human cell phagocytosis 5,9 .NSG mice, however, allow for poor Technical Report https://doi.org/10.1038/s41590-024-01880-3blood huCD34 + cells.To make THX mice, we fed huNBSGW mice E2 ad libitum in drinking water starting at 14 to 18 weeks of age.After 4 weeks, THX mice were ready for experimental use or continued on E2 for use at a later time.Female and male THX mice showed comparable blood E2 levels (82.17 ± 10.36 pg ml −1 and 82.75 ± 5.72 pg ml −1 , respectively, mean ± s.e.m.), higher than those in female and male huNBSGW mice (20.94 ± 1.88 and <5 pg ml −1 ) and within women's physiological E2 level (35-500 pg ml −1 ; Extended Data Fig. 1 and Supplementary Table 1).THX and huNBSGW mice sustained human peripheral blood mononuclear cells (huPBMCs) at higher levels (up to 96.1% and 89.3% huCD45 + cells, respectively) than huNSG mice (Fig. 1a,b and Extended Data Fig. 2a).They showed more blood huB cells, huT cells, human dendritic cells (huDCs), human natural killer (huNK) cells and human monocytes, and more huB cells in spleen and LNs than huNSG mice (Fig. 1e and Supplementary Figs. 1 and 2).THX mice displayed higher levels of circu lating huIgM, huIgD, huIgG, huIgA and huIgE, and had a longer lifespan than huNBSGW and huNSG mice (Fig. 1c,d).Their spleens contained a spectrum of huCD45 + lymphoid and myeloid cells, like spleens of humans who died from accidental death 31 (Fig. 1f and Supplementary Tables 2-4a-g).THX mice showed blood huCD45 − CD235a − CD61 + plate lets and, as in other humanized mice, few huCD235a + red blood cells 5 (Supplementary Table 5a,b).THX and huNBSGW mice harbored more BM huCD34 + cells than huNSG mice (Fig. 1g).Thus, female and male THX mice reconstitute human lymphoid and myeloid cells, showed higher levels of huIgM, huIgD, huIgG, huIgA and huIgE than huNBSGW and huNSG mice, and extended survival.

THX mice BCR huV(D)J gene repertoire reflects that of humans
The THX mouse huBCR repertoire mirrored that of humans.Indeed, THX mouse huCD19 + IgM + B cells expressed huV H DJ H Cμ transcripts with probabilistic V H gene usage, that is, reflecting the genomic repre sentation of human V H genes (huIgH locus haploid complement con sists of 36-49 functional V H genes segregated in seven families 32 ), with V3 family genes, particularly V3-V30, as the most frequently utilized, followed by V1 and V4 (Fig. 2a,b).Like humans, THX mouse huIgM + B cells showed preponderant human D3 and J H 3 utilization and dominant V3 to J H 4 combination (Fig. 2c).Their huV H DJ H Cμ transcripts showed a pseudonormal CDR3 length distribution, which peaked at 14 amino acids, mimicking huIgM + B cells in humans (Fig. 2d).Discrete huIgM + B cell clones identified by unique and identi cal huV H DJ H Cμ transcripts showed even greater diversity than in humans (Fig. 2e).THX mouse huIgM + B cells displayed a Vκ gene utilization similar to that of humans 32 , albeit biased to Vκ4, and a JλCλ3 utilization versus human huIgM + B cells JλCλ2 and JλCλ3 (Fig. 2f).Thus, the THX mouse huIgM + BCR repertoire mirrors that of humans, with minor differences in VκJκ and VλJλ gene expression.
Although mutated IgG to ovalbumin have been detected in γirradiated knockin huIL6 Rag2 −/− Il2rg −/− SIRPα h/m mice (RG SKI interleukin (IL)6) 10 , a humanized mouse capable of mounting fully mature antibody responses has yet to be established.Maturation of the antibody response entails B cell somatic hypermutation (SHM), classswitch DNA recombi nation (CSR), differentiation of plasma cells (PCs) making highaffinity antibodies and generation of specific memory B cells (MBCs).The National Institute of Allergy and Infectious Diseases has emphasized the need for a novel and more advanced human immune system mouse model 2 , a recommen dation that has gone essentially unheeded.Gene ration of homo zygous Kit W41J mutant NSG mice has yielded genetically myelo ablated NSGW41 (NOD.CgKit W41J Prkdc scid Il2rg tm1Wjl /WaskJ) and NBSGW (NOD.CgKit W41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ) mice, support ing huHSC engraftment without γradiation 11,12 .Mutated Kit W41J ham pers mouse (mo)HSCs docking onto BM stromal cells and opens up an ample niche for huHSCs docking through binding of mouse stem cell fac tor 11,12 , which is engaged by huHSC cKit.Adult NBSGW and NSGW41 mice grafted intravenously with cord blood huCD34 + cells supported greater huCD45 + lymphoid and myeloid cell reconstitution than γirradiated NSG mice 5,11,12 .Despite their obvious potential, however, NBSGW and NSGW41 mice have not been leveraged to construct an advanced huma nized mouse that faithfully replicates human immune responses [2][3][4][5][6] .

Technical Report
https://doi.org/10.1038/s41590-024-01880-3THX, huNBSGW and NBSGW mice showed distinct and shared gut bacte ria families (Extended Data Fig. 3).Muribaculaceae together with other families contributing to the human gut microbiome 34 made up for the THX mouse microbiome.This shared most bacteria families with huN BSGW mice and substantially differed from that of NBSGW mice, which was dominated by the characteristically 'murine' Rikenellaceae, still

Technical Report
https://doi.org/10.1038/s41590-024-01880-3found in the 'transitional' microbiome of huNBSGW mice, but absent in THX mice.Thus, reflecting the impact of human immune cells and E2, the THX gut microbiome consists of bacteria all found in human gut microbiome and shows little similarity to that of (nongrafted) NBSGW mice.

THX mice huTCRa and huTCRb gene repertoires reflect those of humans
THX mouse spleen huTCRα and huTCRβ repertoire diversity largely reflected the human genomic representation of huVα, huJα, huVβ and huJβ genes 32 (Fig. 3a), and broadly overlapped with huTCRα and huTCRβ gene expression in human blood, including huVβ and huJβ gene pair preferences (Fig. 3b,c).THX mice huVαJα−Cα and huVβDJβ−Cβ CDR3 lengths followed a pseudonormal distri bution, 4 to 21 amino acids, peaking at 11 and 13 amino acids, com parable to huT cells in humans (Fig. 3d).THX mouse huVβDJβ−Cβ transcripts identified discrete huT cell clones of a diversity com parable to humans (Fig. 3e).Thus, THX mouse huT cells express diverse huTCRα and huTCRβ repertoires, with huVα, huJα, huVβ and huJβ gene utilization reflecting huVα, huJα and huVβ, huJβ genomic representation and overlapping with that of huTCRα and huTCRβ in humans.

huB cells from THX mice have full differentiation potential
Naive huIgM + IgD + B cells from THX mice and from humans were cultured in vitro to compare their potential to undergo CSR, PC and memorylike B cell differentiation.Upon culture with T celldependent (CD154, IL2, IL4 and IL21) or T cellindependent (CpG, IL2, IL21, transforming In these, different colors denote different huVκ, huJκ or huVλ, huCλ gene families; color gradients denote individual gene family members-the huIgκ locus comprises 39 functional huVκ genes and 5 huJκ genes, while the huIgλ locus comprises 30 functional huVλ genes segregated into 10 subgroups and 5 functional huJλCλ clusters 32 .

Flagellin-vaccinated THX mice mount a neutralizing response to Salmonella
THX mice vaccinated with purified S. Typhimurium flagellin made antiflagellin huIgM, huIgG and huIgA, a Salmonellaneutralizing response comparable to humans, and survived S. Typhimurium infection, while nonvaccinated THX mice did not (Fig. 6a-d).Their bactericidal antibody response was accompanied by blood and spleen MZ huCD19 + IgM + IgD + CD27 + B cells, flagellinspecific huCD19 + IgG + and huCD19 + IgA + B cells, huCD19 + CD38 + CD138 + CD27 + PBs, CD19 − CD38 + CD138 + CD27 + PCs and specific memory huCD19 + CD27 + B cells, at higher frequencies than similar cells in humans.Flagellinspecific spleen huB cells sorted from THX mice expressed huV H DJ H Cγ and huV H DJ H Cα1 transcripts involving V1, V3 and V4 genes, pseudonormal huIgH CDR3 lengths distribution, peaking at 16 amino acids, and bearing substantial loads of point mutations.huIgM + , huIgG + and huIgA + B cells underwent select clonal expansion and intraclonal diversification, with the three largest huV H DJ H Cα1expressing huB cell clones accounting for a greater proportion of huV H DJ H Cα1huB cells than the three largest huV H DJ H Cγexpressing huB cell clones did of huV H DJ H CγhuB cells (Fig. 6e-i and Extended Data Fig. 7a-e).Also, vaccinated THX mice showed blood incretion of huAPRIL, huBAFF, huTGFβ, human inter feron gamma (huIFNγ), huIL2, huIL4, huIL6, huIL10 and huIL21 at human physiological concentrations (Extended Data Fig. 8 and Supplementary Table 6).Thus, flagellinvaccinated THX mice mount a protective antibody response to Salmonella, entailing SHM/CSR, huB cell clonal selection and intraclonal diversification, huPC and huMBC differentiation, huMZ B cells and blood incretion of antibody responserelated human cytokines.
Fig. 3 | THX mice huTCR cell repertoire and clonality are similar to those in humans.a, huVα and huJα (huTCRa) genomic representation and gene expression in blood and spleen huT cells of healthy humans (n = 3, HS 05, 06, 07) and nonintentionally immunized THX mice (n = 3, THX mice 369, 370, 371) depicted as stacked columns (left).In these, different colors denote different huVα or huJα gene families; color gradients denote individual family members.Heat maps of expressed individual huVα and huJα genes (right).b, huVβ and huJβ (huTCRb) genomic representation and gene expression in huT cells of HS and THX mice as in a, depicted as stacked columns (left).In these, different colors denote different huVβ or huJβ gene families; color gradients denote individual family members.Heat maps of individual huVβ and huJβ genes (right).c, Associated expression of huVβ and huJβ genes in HS and THX mouse huT cell repertoires as in a, depicted by Circos plots.d, CDR3 length distribution (top) and frequency (bottom) in huT cell recombined huVαJαCα (huTCRa) and huVβDJβCβ (huTCRb) transcripts of HS and THX mice as in a.Each dot depicts CDR3 length in an individual cell.e, huT cell clones in HS and THX mice as in a, as identified by unique huVβDJβCβ (including CDR3 as translated amino acid sequence) transcripts and depicted by TreeMaps.Individual rectangle or square (unique color) area reflects huT cell clone size.In THX mice, huVβDJβCβ transcripts identified 5,531, 3,981 and 8,142 discrete huT cell clones in the same order of magnitude as in HS huVβDJβCβ transcripts, which identified 3,437, 11,305 and 4,266 discrete huT cell clones.

Fig. 4 | THX mice mount specific T cell-dependent and T cell-independent class-switched, hypermutated and clonal antibody responses. a-f, THX
(n = 7), huNBSGW (n = 7) and JAX NSG huCD34 (n = 4) mice were injected i.p. with NP 16 CGG (100 μg in 100 μl alum) on day 0, boosted (100 μg in 100 μl PBS) on day 14 and euthanized on day 28.a, Total serum human immunoglobulin and NP 4 specific human antibodies measured by ELISAs.Total human immunoglobulin concentrations expressed as μg eq ml −1 and NP 4 specific human antibodies expressed as relative units (RUs).Fewer than seven data points were derived for human immunoglobulins other than NP 4 specific huIgM, huIgG and huIgG1.b, Left, spleen huIgM + , huIgG + and huIgA + B cells, classswitched memory huCD27 + IgD − B cells and huCD27 + CD38 + PBs/PCs.Right, NPspecific huCD19 + B cells and memory huCD19 + CD27 + IgG + B cells (identified by binding of PElabeled NP 16 ), and MZ huCD19 + IgM + IgD + CD27 + B cells in THX mouse spleen.c, Spleen huB cell intracellular AID and BLIMP1 expression in THX and huNBSGW mice.d, Left, point mutation frequencies (change/base) in spleen huB cell huV H DJ H C H transcripts of THX 406, 407, 408 and 409 mice depicted as scatterplots.Each dot represents a single sequence and the bar depicts the mean with s.e.m.Right, means of total, S and R huV3 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of huV H DJ H C H transcripts depicted as histograms.e, In the SHM pie charts, slices depict proportions of huV H DJ H C H transcripts carrying given numbers of point mutations; slice gray gradients depict increasing numbers of point mutations; the overall mutation frequency is listed below each pie chart.Spectrum of point mutations depicted as donut charts (same mice as in d).f, huV1DJ H Cγ1 B cell clones and intraclonal diversification in THX mice (same mice as in d) depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX 406, 407, 408 and 409 mice, the three largest huV1DJ H Cγ1 clones accounted for 42.9%, 26.6%, 32.0% and 23.4% of huV1DJ H Cγ1 B cells.g-p, THX (n = 7), huNBSGW (n = 7) and JAX NSG huCD34 (n = 4) mice were injected i.p. with DNPCpG (50 μg in 100 μl PBS) on day 0, boosted (50 μg in 100 μl PBS) on day 14 and euthanized on day 28.g, Total serum immunoglobulin concentration (μg eq ml −1 ) and DNP 5 specific human antibodies (RUs) measured by specific ELISAs.Fewer than seven data points were derived for DNPspecific huIgE.h, Spleen huB cells, huMBCs and huPBs/PCs as in b. i, huCD45 + huCD19 + B cells, huCD3 + T cells, huCD11c + DCs, huCD14 + monocytes, memory huCD19 + CD27 + B cells, huCD27 + CD38 + PBs/PCs as well as huIgM + , huIgD + , huIgG + and huIgA + B cells in THX mouse mesenteric LNs and spleen.j, Blood and spleen MZ huCD19 + IgM + IgD + CD27 + B cells (8.1% ± 0.3% and 12.9% ± 0.2% huB cells, respectively) in THX (n = 6) and huNBSGW (n = 6) mice.FACS plots are from one THX and one huNBSGW mouse, each representative of six mice.k, Total and DNP 5 specific huIgM, huIgG and huIgA ASCs in THX mouse spleen and BM, as analyzed by specific ELISPOTs.l, Spleen huB cell intracellular AID and BLIMP1 expression in THX and huNBSGW mice.m, Total human immunoglobulin (μg eq ml −1 ) in the BALF of THX (n = 5) and huNBSGW (n = 5) mice.n, huCD45 + cells, huCD19 + B cells, huCD3 + T cells, and huIgM, huIgD and huIgAproducing cells in THX mouse lamina propria (immunofluorescence; scale bar, 100 μm).Different pseudocolors denote different cells.o,p, Free and bacteriabound huIgD and huIgA in feces of THX (n = 5) and huNBSGW (n = 5) mice measured by specific ELISA (μg eq/g, THX versus huNBSGW mice: huIgD, P < 0.0001; huIgA, P = 0.007, twosided Student's unpaired ttest) and identified by flow cytometry (% total bacterial cells).Flow cytometry plots (b, c, h, i and l) are from one THX, one huNBSGW or one JAX NSG huCD34 mouse, each representative of three mice.huCD45 + cells were pregated in all FACS analyses.ELISPOT images (k) and micrographs (n) are from one THX mouse representative of five mice.In the histograms (a, g, j, m and o), each dot represents an individual mouse and the bar depicts the mean with s.e.m.Statistical significance (a, g, j and m) was assessed by twosided Student's unpaired ttest (NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001).

COVID-19 mRNA-vaccinated THX mice mount an RBD-neutralizing response
THX mice mounted a mature antiviral response.THX mice vaccinated intramuscularly (i.m.) with PfizerBioNTech 162b2 coronavirus disease 2019 (COVID-19) mRNA, according to human vaccination schedule, made huIgM, huIgG and, to a moderate degree, huIgA to SARSCoV2 Spike S1 RBD (37 amino acid core peptide) as well as RBDspecific huASCs, huCD19 + B cells, memory huCD19 + CD27 + B cells and ) huIgG (µg eq ml −1 ) huIgD (µg eq ml −1 ) huIgM ( g eq ml huCD19 + CD27 + CD38 + PBs (Fig. 7a-c).They showed blood incretion of huAPRIL, huBAFF, huTGFβ, huIFNγ, huIL2, huIL4, huIL6, huIL10 and huIL21 at human physiological concentrations (Extended Data Fig. 8 and Supplementary Table 7).THX mice sera with high RBDbinding huIgG titers displayed SARSCoV2neutralizing activity comparable to huIgG1 monoclonal antibodies, as assessed by two different Spike   7f and Extended Data Fig. 9a).In fact, the latter comprised a multitude of huIgM + B cell 'microclones', likely not participants in the antiRBD response.The interplay of SHM and CSR in shaping B cell intraclonal diversification was exemplified by genealogical trees outlining the stepwise evolu tion of two clones, one developing from an unmutated huV353D1 26J H 1CμB cell progenitor, the other from an unmutated huVκ311 Jκ1CκB cell progenitor (Fig. 7g).RBDspecific huB cells were sorted from spleens of additional mRNAvaccinated THX mice, and paired huV H DJ H Cγ and huVκJκ or huVλJλ gene segments were amplified from single huB cells to make 100 recombinant human monoclonal anti bodies.These showed predominant utilization of human V3, V4 and V1, reflecting the human haplotypic representation of these human V H genes, together with human Vκ3, Vκ1 and Vκ2 as well as Vλ1 and Vλ2 genes; as expected, somatic point mutations were more frequent in V H than in Vκ or Vλ gene segments (Extended Data Fig. 9b).Fortyfive of the 100 human monoclonal antibody huB cell clones (27 huIgM, 5 huIgG1 and 13 huIgA1) were selected based on greater RBDbinding activity and characterized for paired huIgH and huIgL genes (Extended Data Fig. 9c).Thus, upon COVID-19 mRNA vaccination, THX mice mount a mature neutralizing antibody response to Spike S1 RBD, entailing SHM/CSR, huB cell select clonal expansion and intraclonal diversification, huPC differentiation, generation of huMBCs and blood incretion of antibody responserelated human cytokines.

THX mice can model SLE autoimmunity
Pristane, a saturated terpenoid alkane with proinflammatory activity, can induce lupuslike autoimmunity in C57BL/6, BALB/c and γirradiated humanized NSG mice 43 .Male and female 18weekold THX mice (gene rated from huNBSGW and huNSGW41 mice) were injected i.p. with pristane or PBS.As early as 3 weeks after pristane injection, THX mice developed a malar rash evocative of the 'butterfly rash' in individuals with systemic lupus erythematosus (SLE), concomitant with rising levels of serum huIgG and huIgA, including antinuclear, antidsDNA, antihistone, antiSm/antiRNP and antiRNA huIgG autoantibodies,

Discussion
Humanized mice have been constructed using BM, fetal liver or umbilical cord blood huCD34 + cells or PBMCs, with cord blood being a highly enriched source of HSCs 5 .In THX mice, cord blood huCD34 + cell engraftment of genetically myeloablated Kit W41J mice enables human cell multilineage development and full immune tolerance.Intracardiac injection would maximize huCD34 + cell dissemination to multifocal BM sites, thereby facilitating huCD45 + cell colonization of peripheral lymphoid organs, such as LNs, gutassociated lymphoid tissue and Peyer's patches.In THX mice, this is promoted by E2 and contrasts with underdeveloped peripheral lymphoid formations in huNBSGW, JAX NSG huCD34 and other humanized mice [2][3][4][5][6] .In these, LN develop ment could be achieved only by supraphysiological expression of transgenic murine thymic stromal cellderived lymphopoietin 44 .Thus, in addition to neonatal grafting of Kit W41J immunodeficient mice by intracardiac injection, the innovative estrogen conditioning is critical to the making of THX mice.An important limitation of humanized mouse models is the failure to mount mature antibody responses [2][3][4][5][6] .E2 support of antibody response maturation is consistent with stronger antibody responses to viral vaccines, such as SARSCoV2 virus, influenza and hepatitis B virus, or bacterial vaccines, such as diphtheria, tetanus and pneumococcus, and greater incidence of autoantibodymediated autoimmunity in female than male mice and humans 21,22,[45][46][47] .Accordingly, E2 promotes differentiation of virtually all immune cells, including B cells, T cells and granulocytes, all of which express ERα and ERβ 14,[20][21][22]25,26 . Althogh more information is needed on E2 impact on HSC differentiation, CD34 + HSCs express ERα and ERβ (encoded by Esr1 and Esr2 genes) and engraft more efficiently in immunodeficient female than male mice [13][14][15]24,48 .
The comparable blood E2 levels in male and female THX mice were higher than in huNBSGW mice, but well within women's E2 physiological range.The critical role of E2 in promoting B cell differen tiation in THX mice likely reflects an intrinsic B cell estrogen activity 16,18,49 , as revealed by the THX mouse mature antibody response to T cellindependent DNPCpG.In THX mice, E2 is critical in promoting development of LNs, Peyer's patches and GCs, supporting differen tiation of huTECs, huT FH cells and huGC B cells, increasing B2:B1 cell ratio and generating huMBCs.E2 conditioning was also important for the appearance of huIgM, huIgD, huIgG and huIgA in BALF and feces, as well as the high baseline levels of huIgD, huIgG and huIgA in nonintentionally immunized THX mice.Additionally, E2 supported differentiation of huMZ B cells, which contribute antibodies that provide the firstline of defense against bloodborne microbial pathogens.Spleen huMZ B cells in THX mice, whether immunized with NPCGG, DNPCpG or Salmonella flagellin, were comparable, as a proportion of B cells, to spleen MZ B cells in humans and mice 50 .As in humans, THX mice huMZ B cells occurred at a greater proportion in circulating blood than spleen.
E2 induces a genetic program, including Ptpn6, Bcl2 and Vcam1 expression, that promotes B cell activation and survival, while dampen ing proapoptotic mediators, such as PD1 (ref.16).The direct impact of estrogen on B cell differentiation was reflected in the ability of huB cells from THX mice to undergo CSR, PC and memorylike B cell differentia tion in vitro as efficiently as B cells from healthy humans, in response to T celldependent and T cellindependent stimuli.Indeed, E2 promotes B cell AID expression and SHM/CSR by upregulating HoxC4, a transcrip tion factor that induces the Aicda promoter to activate this gene [27][28][29] .E2 also downregulates miR26a, a most abundant microRNA in B cells   C/EBPβ, leading to increased RNA polymerase II recruitment 14 .High estrogen:androgen ratios support differentiation of classswitched MBCs and PCs, as in human aromatase transgenic male mice 52 .By con trast, progesterone (P4), the most important progestogen, precursor of testosterone and potent agonist of nuclear progesterone receptor, exerts a negative activity on B cell proliferation, differentiation and Aicda expression, thereby dampening SHM/CSR 53,54 .P4 impact on B cells can reduce antibodymediated defense and promote disease, such as in P4treated female mice infected with influenza virus 55 .Like P4, testosterone would exert a negative impact on immune cell activi ties, thereby contributing to weaker antibody responses to bacterial and viral vaccines in men than women 21,22,[45][46][47] .THX mouse human antibody responses to T celldependent and T cellindependent conjugated haptens, Salmonella flagellin and viral SARSCoV2 Spike S1 RBD peptide, entailed SHM/CSR mediating intra clonal diversification of selectively expanded huIgG + and huIgA + B cell clones, whose sizes accounted for major proportions of their respective huIgG + and huIgA + B cell repertoires.This contrasted with the, gener ally, multitude of huIgM + B cells with virtually no clonal expansion, possibly progenitors of expanded classswitched and somatically hypermutated huIgG + and huIgA + B cell clones.In COVID19 mRNA or flagellinvaccinated THX mice, the lower level of circulating antiRBD or antiflagellin huIgA than huIgG was incongruous with the compa rable huIgA + and huIgG + B cell clonal expansions, huIgA and huIgG mutational loads and huIgA and huIgG ASC numbers.It, however, is consistent with the lower level of antiRBD huIgA than huIgG in blood and saliva of COVID19 mRNAvaccinated humans 56,57 as well as the lower level of antiflagellin huIgA than huIgG in humans infected with Salmonella 58 .The predominant V3, V4 and V1 gene utiliza tion by the classswitched antibodies in COVID19 mRNAvaccinated THX mice is evocative of similar V gene utilization by the classswitched antibody response in COVID19 mRNAvaccinated humans 59 .The muta tional load of greater than 10 −2 changes per base in huB cell huV H DJ H Cγ transcripts in COVID19 mRNA and RBD-KLHvaccinated THX mice is also evocative of the heavy mutational load of COVID19 mRNA vaccineinduced huIgG response in humans [59][60][61] , possibly reflecting the high immunogenicity of Spike S1 RBD 62,63 .
Humanized mice generally lack thymic huMHCs, resulting in huT cells selected on mouse MHC, a shortcoming corrected by graft ing human thymus fragments, as in BLT mice 5 .THX mouse mature antibody responses induced by NP 16 CGG, Salmonella flagellin and Pfizer COVID19 mRNA were presumably dependent on CD4 + T cells educated on huTECs or other human cells expressing MHC class II [40][41][42] , such as huB cells and huDCs, also present in THX mouse thymus.But how could THX mice populate their thymus with huTECs, which supposedly emerge from nonhematopoietic CD34 − progenitors?In fact, epithelial cells can differentiate from CD34 + stem cells, including cord blood CD34 + cells [64][65][66] , possibly giving rise to huTECs.Interest ingly, TECs express ERα and ERβ, consistent with an E2 role in promot ing their differentiation 67 .
THX mouse maturation of antibody response involved blood incretion of huAPRIL and huBAFF at human physiological concentra tions.APRIL supports B cell proliferation, CSR and PC differentiation, while BAFF supports immature B cell survival, B cell differentiation and antibody production 68 .Flagellinvaccinated and Pfizer COVID 19 mRNAvaccinated THX mice displayed comparable concentra tions of blood huAPRIL.The former, however, showed higher levels of circulating huBAFF, likely reflecting flagellin induction of this B cell cytokine 69 .In THX mice, huAPRIL and huBAFF occurred together with huTGFβ, huIFNγ, huIL2, huIL4, huIL6 and huIL10, all at human physiological levels and, possibly, as promoted by ERα signaling 14,19,20,49 .THX mouse physiological levels of human B cell growth factors and cytokines contrast with the generally dysregulated levels of knockin or transgenic growth factors and cytokines in other immunized mice 5 , as exemplified by the supraphysiological expression of human granulocytemacrophage colonystimulating factor (huGMCSF) and huIL3 in huNSGSGM3 mice, huGMCSF, huIL3 and huIL6 in huMIS TRG(6) mice or huBAFF (TNFS13B) in huBAFFKI mice 70 .
A shortcoming of humanized mice has been the lack of GCs, con tributing to impaired antibody responses 5 .In THX mice, E2 supports differentiation of huT FH cells, which make cytokines, such as IL4, IL6, IL10 and IL21, and critically promote GC huB cell differentiation, GC formation, BCR affinity maturation and generation of PCs and MBCs 71,72 .E2 promotes expansion of T FH cells via PPARγ, thereby supporting the classswitched antibody response 49,73 .In activated huPBMCs, E2 increases not only PD1 + CXCR5 + T FH but also ICOS + T FH cells, both important for GC formation 49,72 .In addition, E2 enhances expression of CXCR4 and CXCR5, which are central to GC dark and light zone organization as well as T cell homing by modulating expression of T cell chemokine receptors, such as CCR5 (refs.49,74).Finally, E2 increases CD4 + T cell CD154 expression 22 and upregulates EZH2 histone methyltransferase, which helps T FH cell differentiation 75 .
Another shortcoming of humanized mice is poor develop ment of human myeloid cells, particularly neutrophils 5 .Expression of huGMCSF and huIL3 in γradiation myeloablated humanized NSGSGM3 and MISTRG mice as well as additional expression of human granulocyte colonystimulating factor (hGCSF), as in humanized MISTRGGR mice, has partially corrected human myeloid cell under representation 5,76 .Neutrophils express both ERα and ERβ 20,25,26 , and estrogen has been shown to increase neutrophils in women's peripheral blood and in mouse blood, BM and spleen 25 .THX mice reconstituted human neutrophils, to almost onefourth of spleen huCD45 + cells, a proportion comparable to neutrophils in spleen of humans 31 .Finally, human platelets in THX mice accounted for approximately onethird of total platelets, possibly also as a result of direct E2 impact on megakaryocytes, which express ERα and ERβ and whose maturation is boosted by estrogen 77 .
The THX mouse gut microbiome, which consisted of Muribaculaceae and other bacterial families found in humans, profoundly differed from NBSGW mice microbiome, which was dominated by the exquisite 'murine' Rikenellaceae.By contrast, it shared bacte ria, including the dominant Muribaculaceae, with huNBSGW mice, which, possibly reflecting the lack of E2 conditioning, also harbored remnants of Rikenellaceae, not found in THX mice.The humanlike gut microbiome together with free and bacteriabound fecal huIgD and huIgA, likely induced by microbial stimulation of gut lymphoid cells' TLRs 38,39,78 , suggests that THX mice are suited to model human intestinal mucosa antibody responses.Nevertheless, further investi gation is needed to elucidate the mechanisms underpinning E2 con tribution to shaping the THX mouse microbiome in gut and airways and, possibly, the potential E2 contribution to support huILCs and peripheral resident T cells, both important in mucosal homoeostasis and defense.
Lupus murine models, such as MRL/lpr and genetically modified Sle1, Sle2 and Sle3 mice, all share a nonhuman immune system, mediat ing an autoantibody response that does not faithfully reproduce that of individuals with SLE.Estrogen plays a role in accelerating mouse lupus autoimmunity and may play a role in the development of human lupus 16,17,19,23,43,79 .E2 enhances antidsDNA antibody production in lupus huB cells and ERα accelerates lupus development in autoimmune (NZBxNZW)F1 mice in a B cellintrinsic fashion 17,20,79 .Consistent with B cell clonal expansion in individuals with lupus, Lupus THX mice expanded and intraclonally diversified select huIgG + and huIgA + B cells and made classswitched autoantibodies to cell nuclear components, eventually leading to lupuslike symptoms and immunopathology.By overcoming limitations posed by the differences between mouse and human lupus 43 , Lupus THX mice would lend themselves to testing novel therapeutic approaches with immediate translatability to individuals with lupus.They would also provide a first proofofconcept of THX mice modeling human disease.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.© The Author(s) 2024 https://doi.org/10.1038/s41590-024-01880-3
huCD34 + HSCs to be used for construction of THX, huNBSGW, huNSGW41 and huNSG mice were isolated from human umbilical cord blood collected immediately after cesarean section from fullterm, nor mally developed male and female newborns (in approximately equal numbers) upon informed consent from healthy puerperae (18-45 years old with no infectious disease or history of cancer) of different ages, races and ethnic backgrounds (Supplementary Table 9; Department of Obstetrics and Gynecology, The University of Texas Long School of Medicine).CD34 + cells were purified using EasySep Human CD34 Positive Selection Kit II (17856, STEMCELL Technologies) according to the manufacturer's instructions, yielding at least 99% huCD34 + cell preparations.Freshly purified huCD34 + cells were resuspended in PBS supplemented with 2% FBS for immediate grafting or frozen in 10% dimethylsulfoxide, 72% FBS, 18% RPMI medium and kept in liquid nitrogen for later grafting.
huNSG mice (Supplementary Table 10) were constructed by myelo ablative conditioning of NSG mice neonates (within 48 h of birth) with (1 Gy) γradiation, followed by intracardiac (left ventricle) injection with purified cord blood huCD34 + cells (1.5 × 10 5 freshly isolated or frozenthawed huCD34 + cells in 50 μl PBS supplemented with 2.0% FBS) using a 27gauge needle.huNBSGW (Supplementary Table 11) and huNSGW41 mice were constructed by grafting nonγirradiated, genetically myeloablated NBSGW and NSGW41 mice neonates (within 48 h of birth) intracardially (left ventricle) with cord blood huCD34 + cells.THX mice were generated by feeding huNBSGW or huNSGW41 mice E2 (3301, SigmaAldrich; 1.5 μM in drinking water resulting in a dose of 6.1 × 10 −4 mg per kg body weight per day) ad libitum starting at 14-18 weeks of age (18 weeks in most cases) and continuing thereafter.After 4 weeks of E2 conditioning, huNBSGW or NSGW41 mice (referred to as THX mice; Supplementary Table 12) were ready for experiments or continued E2 for later use.E2 conditioning of huNBSGW or huNSGW41 mice did not start before 14 weeks of age, as estrogen (albeit at high dose) might inhibit early thymus development by T cell proliferation.
Most THX mice were constructed using NBSGW mice as only a dozen NSGW41 mice were acquired in 2019 from The Jackson Labora tory before the sale of such mice was discontinued.NSGW41based THX mice were used in the human antibody response to NPCGG (n = 3), in the lupus studies as part of the healthy THX controls (n = 4 of 12) as well as for generation of Lupus THX mice (n = 5) as described in 'Lupus THX mice, human autoantibodies, immunopathology and mortality'.huCD45 + cells in blood, spleen and BM of humanized mice were identi fied by flow cytometry using APCantihuCD45 monoclonal antibody (304011, BioLegend; 1:100 dilution) and Pacific BlueantimoCD45 monoclonal antibody (103125, BioLegend; 1:100 dilution).Generally, THX and huNBSGW mice displayed up to 96.1% and 89.3% huCD45 + cells in circulating blood, respectively.huNSG and JAX NSG huCD34 mice displayed, at peak, approximately 45% and 20% huCD45 + cells, respectively.Circulating huCD45 + mononuclear cell numbers (cells per ml of blood) were measured by complete blood count analysis, in which blood was collected in EDTAcoated microtubes and analyzed using a XT2000iV or XE5000 blood analyzer (Sysmex).THX, huNBSGW and huNSG mice used in all experiments were 20 to 24 weeks of age, unless indicated otherwise.JAX NSG huCD34 mice were 23 weeks of age.Mice used in all experiments were housed in a pathogenfree barrier animal vivarium facility at The University of Texas Health Science Center at San Antonio and were free of infection or disease.Housing rooms were maintained at a 14h light/10h dark cycle and controlled temperature of approximately 22-23 °C with 40-60% humidity.Food (Teklad LM485 Sterilizable Mouse/Rat Diet, 7912, Inotiv) and water were sterilized.

Estrogen
Serum estradiol concentrations in nonintentionally immunized THX and huNBSGW mice (18-24 weeks old) were measured using Cayman Estradiol ELISA Kit (501890, Cayman Chemical), according to the manufacturer's instructions, and compared to mice and human physiological range [80][81][82][83][84][85][86] .This platform uses an estradiol acetylcho linesterase conjugate (estradiol acetylcholinesterase Tracer) in an inhi bition/competition assay, measuring serum estradiol concentration by OD at 414 nm.High OD readings reflect low estradiol concentrations, while low OD readings reflect high concentrations.Sera were collected from equal numbers of male and female mice, with female mice sera collected generally during proestrus, metestrus and diestrus.
Flow cytometry analysis and sorting were performed using singlecell suspensions.Cells were gated by forward and side scatter ing to exclude dead cells and debris (Supplementary Fig. 1a-c).Cell analysis was performed on pregated huCD45 + cells using a BD LSRII or FACS Celesta flow cytometer (BD Biosciences) with FACSDiva software v9.4 (BD Biosciences).Data were acquired and analyzed using FlowJo v10.9 (Tree Star).
To assess human immune lymphoid and myeloid cell reconstitu tion in THX mice, singlecell suspensions of splenic white cells from nonintentionally immunized THX mice (20-24 weeks old) were incubated for 30 min at 4 °C with a 50 μl cocktail of metal conjugated antihuman monoclonal antibodies (Supplementary Table 2) from the MaxPar Direct Immune Profiling Assay, 30 Marker Kit (201325, Fluidigm), followed by washing for 10 min at room temperature.Cell via bility was measured by DNA intercalation (CellID Intercalator103Rh).Labeled cells were analyzed by Helios mass cytometer (CyTOF software v6.7,Fluidigm) using a flow rate of 0.045 ml min −1 .Human immune lymphoid and myeloid cell population frequencies, qualitycontrol metrics and data plot displays were acquired using Maxpar Pathsetter software v3.0 (401018, Fluidigm).
Bacteriabound huIgD and huIgA in THX and huNBSGW mice were detected as we described 38,78 .Briefly, feces (10 mg) were suspended in 100 μl PBS, homogenized and centrifuged at 400g for 5 min to remove large particles.Supernatant was centrifuged at 8,000g for 10 min, then analyzed for free huIgD and huIgA by ELISA.To detect bacteriabound huIgD and huIgA, the bacterial pellet was resuspended in 1 ml PBS containing 1.0% (wt/vol) BSA.After fixation with 7.2% formaldehyde for 10 min at room temperature, bacteria were washed with PBS, stained with FITCantihuIgD (clone IA62, 348205, BioLegend; 1:100 dilution) or APCantihuIgA (clone IS118E10, 130113427, Miltenyi Biotec; 1:50 dilution) monoclonal antibodies on ice for 30 min, washed again then resuspended in PBS containing 0.2 μg ml −1 DAPI for flow cytometry analysis.All events revealed by DAPI were considered as bacteria.
Human immune cells were isolated from humanized mouse blood, BM, thymus, spleen, LNs and/or Peyer's patches, and suspended in ACK Lysis Buffer (BP10548E, Lonza) to lyse erythrocytes.Peripheral blood (approximately 250 μl) was collected from the submandibular vein into microtubes containing heparin (H19, Fisher Scientific; 25 μl, 1,000 units per ml).After quenching with FBSRPMI and centrifuga tion, erythrocytefree cells were resuspended in FBSRPMI for further preparation or analysis.
To identify individual huB and huT cell clones and analyze huB or huT cell clonal diversity, huB cell V H DJ H Cμ or huT cell VβDJβCβ tran scripts (up to 250,000 sequences) of healthy humans and THX mice were analyzed by Illumina MiSeq amplicon sequencing and segregated based on the same huV H or huVβ gene segment, the same and unique huIgH or huTCRβ CDR3 together with the same huJ H or huVβ sequence [87][88][89][90][91] .Each discrete clone was depicted as an individual rectangle or square (unique color), whose area reflects huB or huT cell clone size, as inferred from the sum of identical huV H DJ H Cμ or huVβDJβCβ transcripts (TreeMaps, Microsoft Excel v16.83 and IMGT/HighVQUEST statistic data).

THX mice huB cell SHM/CSR, clonality and intraclonal diversification
To analyze SHM in the NP 16 CGGinduced antibody response, RNA (2 μg) was extracted from THX mice total and sorted NP 16 specific huB cells using the RNeasy Mini Kit (74104, Qiagen), and cDNA was synthesized using the SuperScript III FirstStrand Synthesis System (18080051, Invitrogen) with oligodT primer.Rearranged huV1DJ H Cγ, huV3DJ H Cγ, huV1DJ H Cα1 and huV3DJ H Cα1 cDNA was amplified using a huV1 or huV3 leaderspecific forward primer together with a nested huCγ or huCαspecific reverse primer tagged with Illumina overhang adaptors (Supplementary Table 16) and Phusion highfidelity DNA polymerase (M0530S, New England BioLabs)-amplification of huIgH V1 and V3 genes was chosen as these families include gene members of high sequence similarity to mouse V172 (V186.2/V3gene), the gene encoding the most efficient 'NPbinding' mouse IgH V segment (https:// www.imgt.org/ligmdb/view?id=J00239/) 36,92 .PCR amplification conditions were 98 °C for 10 s, 60 °C for 45 s and 72 °C for 1 min for 30 cycles.The cDNA amplicons were further amplified and sequenced as described in 'huBCR IgM + B cell and huTCR repertoires and huIgM + B and T cell clonality'.Somatic point mutations in recombined transcripts were analyzed using IMGT/HighVQUEST v1.9.2 (https:// www.imgt.org/HighVQUEST/login.action/)and corrected for poly merase and sequencing error rates (0.008) to calculate the frequency of somatic point mutations.To analyze huB cell clonality and SHM in the DNPCpG, S. Typhimurium flagellin, Pfizer COVID19 mRNA and RBD-KLHinduced antibody responses, THX mouse huB cell V H DJ H Cμ, V H DJ H Cγ, V H DJ H Cα, VκJκCκ or VλJλCλ transcripts were reverse tran scribed, amplified and sequenced as described in 'huBCR IgM + B cell and huTCR repertoires and huIgM + B and T cell clonality', then analyzed for point mutations as described above.
B cell clonal diversity in immunized THX mice was analyzed as described in 'huBCR IgM + B cell and huTCR repertoires and huIgM + B and T cell clonality'.To analyze intraclonal diversification, shared and unique point mutations in huV H DJ H C H transcripts within each huB cell clone were used to construct genealogical trees (phylogenetic maps), revealing sequential multistep accumulation of point mutations from unmutated progenitors, and allowing for detailed intraclonal diversi fication analysis.Genealogical trees were constructed by uploading FASTA files of all segregated huV H DJ H C H transcripts onto PHYLOViZ Online v2.0 (http://www.phyloviz.net/),which uses a JAVA implementa tion of the Feil's goeBURST algorithm rules for visualization of multiple phylogenetic inference trees.
To quantify AICDA, PRDM1, huV H DJ H Cμ, huV H DJ H Cγ1, huV H DJ H Cα1 and huV H DJ H Cε transcript expression in huB cells from THX mice in vitro and ex vivo and huB cells from humans in vitro, RNA extraction and cDNA synthesis were performed as described above.Transcript expression was analyzed by SYBR Green dye (IQ SYBR Green Supermix, 115010139, BioRad) incorporation in PCR reactions involving specific forward and reverse primers (Supplementary Table 16).Reactions were performed in an iCycler (BioRad) realtime qPCR system under the following amplification cycles: 95 °C for 15 s, 40 cycles at 94 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s-data acquisition was performed during this 72 °C extension step (BioRad CFX Manager Software v3.1).Melting curve analysis was performed from 72 to 95 °C.The 2 −ΔCt method (2 −ΔCt = 2 [Ct(HPRT1)Ct(target gene)] ) was used to determine levels of transcripts, and data were normalized to levels of human HPRT1.

THX mice neutralizing response to Salmonella and in vivo protection
THX mice (20-24 weeks old) were injected i.p. with S. Typhimurium flagellin (CVD1925 FliC, University of Maryland School of Medicine Center for Vaccine Development, 50 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (50 μg in 100 μl PBS or 100 μl PBS alone) on day 14 and euthanized on day 28 (ref.39).
Total human immunoglobulin and flagellinspecific human anti bodies were analyzed by specific ELISAs, as described in 'ASCs and titration of human antibodies'.Bactericidal activity of flagellininduced antibodies in sera from flagellinvaccinated and nonvaccinated THX mice was measured by in vitro killing of S. Typhimurium 39 .S. Typhimu rium IR715, a virulent nalidixic acidresistant derivative of wildtype isolate ATCC 14028 (provided by M. Raffatellu, University of California, San Diego) was grown in LB broth (BP14262, Fisher Scientific) overnight at 37 °C.Logphase cultures were prepared by diluting overnight cul tures to an OD 600 of 0.05 in fresh LB medium and incubating them at 37 °C, with shaking at 250 rpm until an OD 600 of 0.7 or 0.8 was attained.Stock cultures were prepared by diluting 500 μl of logphase cultures in 500 μl of 50% sterile filtered glycerol (G331, Fisher Scientific) then https://doi.org/10.1038/s41590-024-01880-3further diluted in PBS to a cell density of approximately 10 4 CFUs per ml.Sera from flagellinvaccinated THX mice, nonvaccinated THX mice and healthy humans were serially twofold diluted in PBS in roundbottom 96well plates.Diluted sera (50 μl) or PBS (50 μl, negative control) were mixed with 25 μl babyrabbit complement (CL3441, CEDARLANE, 25% final concentration) and incubated with 25 μl diluted S. Typhimurium (250 CFUs).Each sample mixture was shaken (115 rpm) at 37 °C for 1 h and then struck onto LBagar plates.These were incubated at 37 °C overnight, after which CFUs were enumerated.To assess the protective response induced by flagellin vaccination in vivo, flagellinvaccinated and nonvaccinated THX mice were infected orally with S. Typhimurium (1 × 10 5 CFUs) by gavage on day 21.The effective dose of bacteria given to mice was verified by plating dilutions of S. Typhimurium on LBagar plates supplemented with nalidixic acid (N887825G, SigmaAldrich, 0.05 mg ml −1 ).Mice were monitored for 10 days, and Kaplan-Meier sur vival plots were generated (GraphPad Prism v10.0.3).For cell sorting, flagellinspecific spleen huB cells from flagellinvaccinated THX mice underwent singlecell FACS after staining with AF647flagellin (synthe sized using iLink Andy Fluor 647 Antibody Labeling Kit, L038, ABP Bio sciences).V H DJ H C H transcripts from sorted huB cells were analyzed for SHM/CSR, B cell clonality and intraclonal diversification, as described in 'huB cell SHM/CSR, clonality and intraclonal diversification'.

THX mice neutralizing antibody response to COVID-19 mRNA or RBD-KLH
THX mice (20-24 weeks old) were injected i.m. with PfizerBioNTech 162b2 COVID19 vaccine (Pfizer COVID19 mRNA, 5 μg in 50 μl PBS) or nil (50 μl PBS) on day 0, boosted (5 μg in 50 μl PBS or 50 μl PBS alone) on day 21, according to the human vaccination schedule, and euthanized on day 28.'Discarded' vials of Pfizer COVID19 mRNA vaccine were obtained from The University of Texas Health Science Center at San Antonio vacci nation facility within 6 h of opening and contained less than one full vac cine dose, thereby not diverting any amount of vaccine from humans for the purpose of this study.THX mice were injected i.p. with SARSCoV2 Spike S1 RBD (47 amino acid peptide containing the core 37 amino acids: FRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNG, custom synthesized by ABI scientific) conjugated to KLH (RBD-KLH, 50 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (50 μg in 100 μl PBS or 100 μl PBS alone) on day 21 and euthanized on day 28.
Total human immunoglobulin and RBDspecific human antibodies or ASCs were analyzed by specific ELISAs or ELISPOTs, as described in 'ASCs and titration of human antibodies'.The SARSCoV2 neutraliza tion power of antibodies induced by COVID19 mRNA vaccine in THX mice was measured using two different platforms: SARSCoV2 Neutral izing Antibody Detection ELISA Kit (502070, Cayman Chemical) and SeroFlash SARSCoV2 Neutralizing Antibody Assay Fast Kit (D100896, EpigenTek), according to the manufacturer's instructions.Sera from COVID19 mRNAvaccinated THX mice were serially twofold diluted in PBSTween 20 in 96well plates precoated with SARSCoV2 Spike S1 RBD peptide (EpigenTek platform), or a recombinant rabbit Fctagged SARSCoV2 Spike S1 RBD peptide bound to an antirabbit Fcspecific antibody (Cayman platform), followed by addition of recombinant Histagged ACE2 protein to each well.These platforms use a horserad ish peroxidase (HRP)conjugated antiHis antibody in an inhibition/ competition assay to measure serum neutralizing human antibody concentration by OD reading at 450 nm.High OD readings reflect a low concentration of neutralizing antibodies, while low OD readings reflect a high concentration.SARSCoV2neutralizing human mono clonal antibodies were provided as a positive control by EpigenTek and Cayman.Extensive controls performed by both Cayman Chemical and EpigenTek have validated measurements of their RBD compe tition assays with actual virus neutralization in COVID19positive and COVID19negative human sera (https://www.caymanchem.com/product/502070/sarscov2neutralizingantibodydetectionelisakit; www.epigentek.com/docs/D1008.pdf).
Sequencing and cloning of original paired heavychain V H DJ H C H and lightchain VκJκCκ or VλJλCλ gene segments for construction of human antibodyproducing cell microcultures was performed by The Univer sity of Texas MD Anderson Cancer Center Recombinant Antibody Pro duction Core.Briefly, RBDspecific spleen huB cells of three COVID19 mRNAvaccinated THX mice underwent singlecell FACS using bioti nylated RBD peptide (47 amino acids) and FITCstreptavidin (405201, BioLegend).huV H DJ H C H and lightchain huVκJκCκ or huVλJλCλ gene segments from sorted huB cells were amplified as cDNAs by singlecell RT-PCR and then sequenced.The single B cell huIgH constant region and huIGκ or huIGλ constant regions were determined.The amplified huV H DJ H and huVκJκ or huVλJλ cDNAs were sequenced and cloned into pcDNA3.4vectors that included the coding sequence for either human heavychain (γ1) or lightchain (κ or λ) constant regions to transfect ExpiCHO cells (A29127, Thermo Fisher).Transfected ExpiCHO cells were cultured in ExpiCHO Expression Medium (A2910001, Thermo Fisher) in 100 singlecell microcultures to produce recombinant human monoclonal antibodies.After 5 days, media were collected and analyzed for RBDspecific recombinant human antibodies by specific ELISA.

Human cytokines
To measure circulating human cytokines, sera were collected from flagellinvaccinated and COVID19 mRNAvaccinated THX mice and ana lyzed for huAPRIL, huBAFF, huIFNγ, huIL2, huIL4, huIL6, huIL10 and huIL21 by Luminex Human Discovery Assay 8Plex (LXSAHM08, R&D Systems).Analysis of huTGFβ1 was performed by TGFβ Premixed Mag netic Luminex Performance Assay (FCSTM17, R&D Systems).Samples and reagents were prepared according to the manufacturer's instruc tions.Briefly, sera were diluted at a 1:2.5 (Luminex Human Discovery Assay) or 1:15 (TGFβ Luminex Performance Assay) ratio in Calibrator Diluent RD652 or Calibrator Diluent RD650, respectively.Next, 50 μl working standards and 50 μl diluted sera were each mixed with 50 μl Human Magnetic Premixed Microparticle Cocktail (colorcoded magnetic beads coated with analytespecific capture antibodies) and incubated in 96well microplates at room temperature for 2 h with shaking at 800 rpm.After washing plates with 100 μl per well of wash buffer using a Luminex microplate magnet, human cytokines were detected by addition of 50 μl Human Premixed BiotinAntibody cocktail (biotinylated detection monoclonal antibodies specific for analytes of interest) followed by reaction with 50 μl streptavidin-phycoerythrin and measurement using a duallaser flowbased detection Luminex FLEXMAP 3D analyzer (Luminex).One laser classifies the beads and determines the analyte that is being detected.The second laser deter mines the magnitude of the PEderived signal, which is proportional to the amount of analyte bound.Cytokine concentrations were calculated using Belysa Immunoassay Curve Fitting Software (40-122, Millipore Sigma) and compared to human physiological range [93][94][95][96][97][98] .

Intestinal microbiota
Microbial DNA was extracted from feces of nonintentionally immu nized THX, huNBSGW and NBSGW mice (22 weeks old) using QuickDNA Fecal/Soil Microbe Microprep Kit (Zymo Research) according to the manufacturer's instructions.To analyze gut bacterial microbiome com position, microbial DNA was tagged and sequenced using the Illumina MiSeq platform.Briefly, the V3-V4 hypervariable region of the bacteria 16S rRNA gene was amplified by PCR using tagged bact341F primer 5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGG CWGCAG3′, bact850R primer 5′GTCTCGTGGGCTCGGAGATGTGTA TAAGAGACAGGACTACHVGGGTATCTAATCC3′ and Phusion highfidelity DNA polymerase (M0530S, New England BioLabs).Multi plexing indices and Illumina sequencing adaptors were then added to the amplicons by limitedcycle amplification using the Nextera XT Index Kit (Illumina).Libraries were normalized, pooled and sequenced using the Illumina MiSeq platform.Sequencing and quality assessment were performed by The University of Texas Health Science Center at San Antonio Genome Sequencing Facility.Bacterial taxonomy was assigned using the Ribosomal Database Project (RDP) classifier v2.14 (http://rdp.cme.msu.edu/classifier/).Principle component analysis of gut bacterial composition in THX, huNBSGW and NBSGW mice was performed by ClustVis v1.0 (biit.cs.ut.ee/clustvis/), which uses clustering algorithms to construct plots visualizing similarities and/or differences between groups of samples.

Extended Data
Extended Data Fig. 10 | RBD-KLH-vaccinated THX mice mount a classswitched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.a, b, THX mice were injected i.p. with RBDKLH (100 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (100 μg in 100 μl PBS or 100 μl PBS alone) on day 21 and euthanized on day 28.Total serum human immunoglobulin and RBDspecific huIgM, huIgG and huIgA antibodies in RBDKLHimmunized (n=6) and nonimmunized (n=6) THX mice measured by specific ELISAs (total human immunoglobulin concentrations expressed as μg eq ml −1 and RBD specific human antibody titers as OD readings at different dilutions or RUs).In the histograms, each dot represents an individual mouse and the bar depicts the mean with s.e.m.

Fig. 2 |
Fig. 2 | THX mice huBCR repertoire and clonality are similar to those in humans.a, huIgH V H , D and J H gene genomic representation and expression in blood and spleen huIgM + B cells of healthy humans (n = 3, HS 01, 02, 03) and nonintentionally immunized THX mice (n = 3, THX 365, 366, 367), depicted as stacked columns.In these, different colors denote different huV H , huD or huJ H gene families; color gradients denote individual family members-the huIgH locus haploid complement consists of 36-49 functional huV H genes segregated into 7 families 32 .b, Heat map of individual huV H family members in huIgM + B cells of HS and THX mice as in a. c, Associated expression of huV H and huJ H genes in huIgM + B cell repertoire of HS and THX mice as in a, depicted by Circos plots.Outermost Circos plot tracks mark the boundaries of each huV H or huJ H region subfamily.d, huIgH CDR3 (translated amino acid sequence) length distribution (left) and frequency (right) in huIgM + B cell recombined huV H DJ H Cμ transcripts of HS and THX mice as in a-the somatically generated IgH CDR3 is the most polymorphic BCR region and provides the main structural correlate for antigen binding.In the violin plots, the upper and lower edges of the box plot indicate the 75th and 25th percentiles, respectively, and the middle line indicates the median.Each dot depicts CDR3 length in an individual huB cell.e, huB cell clones in HS and THX mice as in a, as identified by unique huV H DJ H Cμ (including CDR3 as translated amino acid sequence) transcripts and depicted by TreeMaps.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX mice, huV H DJ H Cμ transcripts identified 521,859, 23,052 and 20,045 discrete huB cell clones in the same order of magnitude as in HS huV H DJ H Cμ transcripts, which identified 11,115, 9,016 and 11,570 huB cell clones.f, huIgK chain (Vκ and Jκ) and huIgL chain (Vλ and JλCλ) gene genomic representation and expression in huIgM + B cells of HS and THX mice as in a, depicted as stacked columns.In these, different colors denote different huVκ, huJκ or huVλ, huCλ gene families; color gradients denote individual gene family members-the huIgκ locus comprises 39 functional huVκ genes and 5 huJκ genes, while the huIgλ locus comprises 30 functional huVλ genes segregated into 10 subgroups and 5 functional huJλCλ clusters32 .

Fig. 7 |
Fig. 7 | THX mice vaccinated with Pfizer COVID-19 mRNA mount a mature neutralizing antibody response to SARS-CoV-2 Spike S1 RBD.a-g, THX mice were injected i.m. with PfizerBioNTech 162b2 COVID19 mRNA vaccine (5 μg in 50 μl PBS) or nil (50 μl PBS) on day 0, boosted (5 μg in 50 μl PBS or 50 μl PBS) on day 21 and euthanized on day 28.a, Serum RBDspecific human antibodies in COVID19 mRNAvaccinated (n = 8) and nonvaccinated (nil, n = 8) THX mice by specific ELISA (titers expressed as optical density (OD) readings at different dilutions).b, Total and RBDspecific spleen huCD45 + cells, huB cells, huMBCs and huPBs/PCs in COVID19 mRNAvaccinated THX mice, as identified by binding of labeled SARSCoV2 Spike S1 RBD.c, Total and RBDspecific huIgM, huIgG and huIgA ASCs in spleen and BM of COVID19 mRNAvaccinated THX mice, as analyzed by specific ELISPOTs.Data in b and c are from one THX mouse representative of three THX mice.d, Dosedependent neutralizing antibody activity of sera from COVID19 mRNAvaccinated THX mice (n = 4), as analyzed by EpigenTek and Cayman SARSCoV2neutralizing antibody detection platforms.SARSCoV2neutralizing humAb4 and humAb15 were provided as positive control by EpigenTek and Cayman.e, Point mutation frequencies (changes per base) in spleen huB cell huV H DJ H C H (huV H DJ H Cμ: 0.8 ± 0.01 × 10 −2 , huV H DJ H Cγ: 1.5 ± 0.2 × 10 −2 , huV H DJ H Cα1: 1.2 ± 0.1 × 10 −2 ) transcripts of COVID19 mRNA vaccinated THX mice (n = 3, THX 477, 478, 479) depicted as scatterplots and pie charts.Each dot represents a single sequence and bars depict the mean with s.e.m. f, huV H DJ H C H B cell clones and intraclonal diversification in THX mice as in e, depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX 477, 478 and 479 mice, the three largest huV H DJ H Cμ, huV H DJ H Cγ and huV H DJ H Cα1 clones accounted for 4.7%, 3.9% and 2.8% of huV H DJ H Cμ huB cells, 22.0%, 16.1% and 36.9% of huV H DJ H Cγ huB cells and 42.8%, 40.7% and 22.5% of huV H DJ H Cα1 huB cells.g, Interplay of SHM and CSR shapes B cell stepwise intraclonal diversification in COVID19 mRNAvaccinated THX mice, as exemplified by genealogical trees outlining the evolution of two huB cell clones, one tracked from its huV353D126J H 1Cμ heavy chain huB cell progenitor, the other from its huVκ311Jκ1Cκ lightchain huB cell progenitor.

Fig. 8 |
Fig. 8 | THX mice can develop human autoantibodies and model SLE.a-g, Lupus THX mice were generated by injecting THX mice (n = 11) once i.p. with 500 μl pristane.THX mice (n = 12) injected with 500 μl PBS served as healthy controls.a, Left, malar rash in a (huNSGW41derived) Lupus THX mouse (3 weeks after pristane injection).Middle, serum antinuclear IgGs (scale bar, 20 μm) and kidney immunopathology (H&E and antihuIgG immunofluorescence; scale bar, 100 μm) in Lupus THX and THX mice (12 weeks after pristane or PBS injection).Micrographs are from one Lupus THX and one THX mouse, each representative of 3 mice.Right, survival of Lupus THX (n = 11) and THX (n = 12) mice through 12 weeks after pristane or PBS injection (Kaplan-Meier curves, P = 0.04, logrank Mantel-Cox test).b, Total serum huIgM, huIgG and huIgA (μg eq ml −1 ) as well as antidsDNA, antihistone, antiRNP and antiRNA huIgG (RUs) in Lupus THX (n = 7) and THX (n = 7) mice measured by specific ELISAs.c, Mesenteric LN huIgM + IgD + , huIgM + , huIgG + and huIgA + B cells as well as spleen and BM huCD27 + CD38 + PBs/ PCs in Lupus THX and THX mice.Data are from one Lupus THX and one THX mouse, each representative of three mice (3 Lupus THX mice were euthanized when showing obvious signs of disease; 3 euthanized healthy THX mice entered into the study in addition to the 12 followed in the survival study).d, Numbers of point mutations in recombined huV H DJ H Cγ and huV H DJ H Cα1 transcripts in Lupus THX mice (n = 3, Lupus THX 715, 716, 717) huB cells depicted as scatterplots.Each dot represents a single sequence and bars depict the mean with s.e.m. e, Spleen huB cell intracellular AID and BLIMP1 expression in Lupus THX (n = 3) and THX (n = 3) mice.f,g, huV3DJ H Cγ and huV3DJ H Cα1 huB cell clones and intraclonal diversification (f) as well as huVβDJβCβ huT cell clones (g) in Lupus THX mice (n = 2, Lupus THX 715, 717), as depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB or huT cell clone size.Analyses in b-g were performed at 6 weeks after pristane or PBS injection.huCD45 + cells were pregated in all FACS analyses.In the histograms (b and e), each dot represents an individual mouse and bars depict the mean with s.e.m.Statistical significance (b and e) was assessed by twosided Student's unpaired ttest (NS, not significant; **P < 0.01, ***P < 0.001).

Fig. 2 |
THX mice huCD45 + cell reconstitution and THX mice but not huNBSGW mice develop Peyer's patches, containing huB cells, huMZ B cells, huGC B cells, huMBCs, huPBs/PCs and huT cells.a, Identification of circulating huCD45 + PBMCs in nonintentionally immunized THX mice (n = 7) by flow cytometry.huCD45 + cells account for 92−96% of total (human plus mouse) CD45 + cells in blood of THX mice.b, THX (n = 6 of the 7 as in Fig. huCD45 + cells were pregated in all FACS analyses.Captions on top of FACS plots indicate pregating markers.(Bottom row) Quantification of huMZ B cells, classswitched huB cells, huGC B cells, huMBCs and huPBs/PCs in Peyer's patches of THX and huNBSGW mice.Each dot represents an individual mouse, the bar depicts the mean with s.e.m.Statistical significance was assessed by twosided Student's unpaired ttest (NS, not significant; ***P < 0.001).Extended Data Fig. 3 | Gut microbiome composition in NBSGW, huNBSGW and THX mice.Left, bacterial families identified in gut microbiome of nonintentionally immunized (nonhuHSCgrafted, nonE2conditioned) NBSGW (n = 6), (huHSCgrafted, nonE2conditioned) huNBSGW (n = 6) and (huHSCgrafted, E2conditioned) THX mice (n = 6 including the 3 mice as in Fig. 2a-f) by highthroughput 16 s rRNA gene MiSeq amplicon sequencing.In histograms, different colors denote different bacterial families, depicted as stacked columns.Each column depicts microbiome composition in an individual mouse.THX, huNBSGW and NBSGW mice developed distinct gut bacterial microbiomes (THX, 8; huNBSGW, 78; NBSGW, 6 families).THX mice gut was colonized by Lactobacillaceae, Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae bacterial families (phylum: Firmicutes), Muribaculaceae (Bacteroidetes), Akkermansiaceae (Verrucomicrobia) and Enterobacteriaceae (Pseudomonadota).NBSGW mice gut harbored predominately (up to virtually 80%) Rikenellaceae (Bacteroidetes), which are characteristic of mouse gut microbiome and were not found in THX mice.Rikenellaceae contributed moderately to gut microbiome of 3 out of 6 huNBSGW mice, suggesting a human pseudonormalization of the mouse microbiome by human immune system elements' development and differentiation.Disappearance of Rikenellaceae in THX mice suggested an important impact of E2 conditioning on further 'humanization' of these mice gut microbiome.Right, principal component analysis (PCA) of gut bacterial composition in the same nonintentionally immunized NBSGW (blue), huNBSGW (green) and THX (red) mice.Each dot depicts an individual mouse; colors denote NBSGW, huNBSGW and THX mice.THX and huNBSGW mice, both hosting bacterial families contributing to gut microbiota in healthy humans, fully segregate from NBSGW mice, which host predominately 'murine' Rikenellaceae.Extended Data Fig. 4 | NP 16 -CGG-immunized THX mice mount a T-dependent class-switched antibody response to NP entailing select B cell oligoclonal expansion and SHM-mediated intraclonal diversification.a, b, Spleen huB cell huV3DJ H Cγ and huV3DJ H Cα1 transcripts in NP 16 CGGimmunized THX mice (n = 3, same mice as in Fig. 4d-f) were analyzed for SHM, B cell clonal expansion and intraclonal diversification.(a) In the SHM pie charts, slices depict proportions of transcripts carrying given numbers of pointmutations; slice gray gradients depict increasing numbers of pointmutations; the overall mutation frequency (change/base) is listed below each pie chart.Spectrum of pointmutations depicted as donut charts.Means of total, S and R huV3 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of recombined huV3DJ H Cγ and huV3DJ H Cα1 transcripts depicted as histograms.(b) huV3DJ H Cγ1 and huV3DJ H Cα1 huB cell clones and intraclonal diversification, as depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX 406, 407, 408 and 409 mice, the 3 largest huV3DJ H Cγ1 huB cell clones accounted for 3.5%, 6.9%, 8.3% and 4.6% of huV3DJ H Cγ1 huB cells, while the 3 largest huV3DJ H Cα1 huB cell clones accounted for 22.6%, 31.2% and 12.5% of huV3DJ H Cα1 huB cells in THX 406, 407 and 408 mice.Select huIgG + B cell clones expressed V3 with V330 overutilization (over 24% of V3DJ H Cγ1 transcripts).Intraclonal diversification is depicted for each of the three largest clones as a genealogical tree constructed based on shared and unique point mutations in recombined huV3DJ H Cγ1 and huV3DJ H Cα1 transcripts.Extended Data Fig. 5 | DNP-CpG-immunized THX mice mount a T-independent class-switched antibody response to DNP entailing select B cell oligoclonal expansion and SHM-mediated intraclonal diversification.a, Spleen huB cell huV H DJ H Cγ transcripts in a DNPCpGimmunized THX mouse (n = 1, THX 425 as in Fig. 4g) were analyzed for SHM.Left, huV H mutation frequency (change/base) in recombined huV H DJ H Cγ transcripts, as depicted by scatter plots.Each dot depicts a single sequence and bar depicts mean with s.e.m.Middle, in the SHM pie charts, slices depict proportions of transcripts carrying given numbers of pointmutations; slice gray gradients depict increasing numbers of pointmutations; the overall mutation frequency (change/ base) is listed below each pie chart.Spectrum of pointmutations depicted as donut charts.Right, means of total, S and R huV H , huV1, huV3, huV4 mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of recombined huV H DJ H Cγ transcripts depicted as histograms.b, huV H DJ H Cμ and huV H DJ H Cγ huB cell clones and intraclonal diversification in a nonintentionally immunized THX mouse (n = 1, THX 437) and DNPCpGimmunized THX mouse 425 as in (a), as depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In the DNPCpGimmunized mouse (THX 425), the 3 largest huV1DJ H Cμ, huV3DJ H Cμ and huV4DJ H Cμ huB cell clones accounted for 7.2%, 7.7% and 4.5% of huV1DJ H Cμ, huV3DJ H Cμ and huV4DJ H Cμ huB cells, while only accounting for 2.1%, 1.4% and 1.4% of similar huB cells in the nonintentionally immunized THX mouse (THX 437).In the same DNPCpGimmunized THX mouse, the 3 largest huV1DJ H Cγ, huV3DJ H Cγ and huV4DJ H Cγ huB cell clones accounted for 22.3%, 16.8% and 29.4% of huV1DJ H Cγ, huV3DJ H Cγ and huV4DJ H Cγ huB cells.Extended Data Fig. 9 | THX mice vaccinated with Pfizer-BioNTech 162b2 COVID-19 mRNA mount a class-switched and somatically hypermutated antibody response to SARS-CoV-2 Spike S1 RBD.a, Spleen huB cell huV H DJ H Cμ, huV H DJ H Cγ and huV H DJ H Cα1 transcripts in COVID19 mRNAvaccinated THX mice (n=3, same mice as in Fig. 7e, f) were analyzed for CDR3 length, R:S mutations and huB cell clonal size.Left, CDR3 length distribution in recombined huV H DJ H C H transcripts.Colors denote different antibody isotypes; color gradients denote different THX mice.Middle, huV H DJ H Cμ, huV H DJ H Cγ and huV H DJ H Cα1 huB cell clonal size depicted as scatter plots.Bars depict the mean with s.e.m.Right, means of total, S and R V H mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 depicted as histograms.R:S data are from one THX mouse representative of 3 THX mice.b-c, Spleen RBDspecific huB cell huV H DJ H C H , huVκJκ and huVλJλ transcripts in 3 additional COVID19 mRNA vaccinated THX mice were reverse transcribed and amplified by RTPCR.Paired huV H DJ H and huVκJκ or huVλJλ gene segments from 100 single cells were used to make recombinant human monoclonal antibodies.(b) Left, huV H , huVκ or huVλ gene family member expression in 100 recombinant human monoclonal antibodies, as depicted by pie charts.Colors depict different huV H , huVκ or huVλ gene families; color gradients denote individual gene family members.The 100 human monoclonal antibodies showed predominant utilization of V3, V4 and V1, together with Vκ3, Vκ1 and Vκ2 as well as Vλ1 and Vλ2 genes.Middle, mutation frequency (change/base) of recombined huIgH V H DJ H and huIg VκJκ or VλJλ regions in recombinant human monoclonal antibodies, as depicted by scatter plots.Each dot represents a single sequence and the bar depicts the mean with s.e.m.Right, CDR3 length distribution in paired V H DJ H and human immunoglobulin VκJκ or VλJλ human monoclonal antibodies.huIgH CDR3 lengths varied between 5 and 25 amino acids, peaking at 12, 13 and 15 amino acids; Statistical significance was assessed by twosided Student's unpaired ttest (NS, not significant; **P<0.01,***P<0.001).c-f, Spleen huB cell huV H DJ H C H transcripts in RBDKLHimmunized THX mice (n=3 of the 6 as in a-b, THX 488, 490, 492) were analyzed for CDR3 length, clonal expansion and intraclonal diversification.(c) CDR3 length distribution in huV H DJ H C H transcripts.Colors denote different antibody isotypes; color gradients denote different THX mice.(d) huV H mutation frequency (change/base) in huV H DJ H Cγ (2.7+0.08x10 2 , mean+s.e.m.) transcripts depicted as scatter plots (left) and pie charts (middle).Each dot represents a single sequence and the bar depicts the mean with s.e.m.Right, means of total, S and R huV H mutation frequencies in FR1, CDR1, FR2, CDR2 and FR3 of huV H DJ H Cμ and huV H DJ H Cγ transcripts depicted as histograms.R:S data are from one THX mouse representative of 3 THX mice.(e) huV H DJ H Cμ and huV H DJ H Cγ huB cell clonal size and diversity in RBDKLHimmunized THX mice (THX 488, 490, 492) depicted as scatter plots.Bars depict the mean with s.e.m.(f) huV H DJ H Cμ and huV H DJ H Cγ huB cell clones and intraclonal diversification, as depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX mice 488, 490 and 492, the 20 largest huV H DJ H Cμ and huV H DJ H Cγ huB cell clones accounted for about onetenth of huV H DJ H Cμ huB cells and onefourth of huV H DJ H Cγ huB cells.

and Jβ genes paired expression in huT cells from humans TCRβ: Vβ and Jβ genes paired expression in huT cells from THX mice
S1 RBD-ACE2 platforms (Cayman Chemical and EpigenTek; Fig.7d).In vaccinated THX mice, huB cell huV H DJ H C H transcripts displayed huIgH CDR3 lengths peaking at 13 and 17 amino acids and substantial loads of V gene somatic point mutations, greater in huV H DJ H Cγ and huV H DJ H Cα1 than in huV H DJ H Cμ transcripts, all with high R:S mutation ratios (Fig.7eand Extended Data Fig.9a).huIgG + , huIgA + and huIgM + B cells underwent select clonal expansion and intraclonal diversification, with the three largest huV H DJ H Cγ and huV H DJ H Cα1expressing huB cell clones accounting for a greater proportion of huV H DJ H CγhuB and huV H DJ H Cα1huB cells than the three largest huV H DJ H Cμexpressing huB cell clones did of huV H DJ H CμhuB cells (Fig.

-vaccinated THX mice mount a mature antibody response to RBD
THX mice mounted a mature antibody response to SARSCoV2 Spike S1 RBD, as elicited by RBD (47 amino acid peptide containing a core of 37 amino acids) conjugated to keyhole limpet hemocyanin (KLH; i.p. priming and boost).RBD-KLHinjected THX mice, not nonvaccinated controls, made specific huIgM, huIgG and, to a lesser extent, huIgA anti bodies to RBD (37 amino acid core peptide) (Extended Data Fig.10a,b).Their huB cell huV H DJ H Cμ and huV H DJ H Cγ transcripts displayed het erogeneous huIgH CDR3 lengths and heavy loads of somatic point mutations with high R:S mutation ratios (Extended Data Fig.10c,d)-huV H DJ H CγhuB cells underwent greater select clonal expansion and intraclonal diversification than huV H DJ H CμhuB cells, which comprised a multitude of 'microclones', reflecting moderate to no clonal expansion (Extended Data Fig.10e,f).Thus, upon vaccination with SARSCoV2 Spike S1 RBD-KLH, THX mice mount a specific mature antibody response to RBD, involving SHM/CSR, huB cell clonal selection and intraclonal diversification.
Each dot represents a single sequence and bars depict the mean with s.e.m. h, huV H DJ H Cγ and huV H DJ H Cα1 huB cell clones and intraclonal diversification in THX mice as in g, depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX 450, 451 and 452 mice, the three largest huV H DJ H Cγ and huV H DJ H Cα1 clones accounted for 18.6%, 9.2% and 18.4% of huV H DJ H Cγ huB cells and 19.6%, 33.4% and 57.9% of huV H DJ 5CFUs, day 21; Kaplan-Meier curves, P = 0.0026, logrank Mantel-Cox test).e, Flagellinspecific huCD19 + B cells, huIgG + B cells, huIgA + B cells and classswitched memory huCD19 + CD27 + B cells in flagellinvaccinated THX mouse spleen and healthy human blood, as identified by binding of Andy Fluor 647 (AF647)labeled flagellin (AF647 alone as negative control); H Cα1 huB cells.i, Evolutive lineage of a huB cell clone that underwent SHM and CSR in a flagellinvaccinated THX mouse (THX 450).huCD45 + cells were pregated in all FACS analyses.In the histograms (a and f), each dot represents an individual mouse and bars depict the mean with s.e.m.
mutation frequencies (changes per base) in spleen huB cell huV H DJ H C H (huV H DJ H Cμ: 0.8 ± 0.01 × 10 −2 , huV H DJ H Cγ: 1.5 ± 0.2 × 10 −2 , huV H DJ H Cα1: 1.2 ± 0.1 × 10 −2 ) transcripts of COVID19 mRNA vaccinated THX mice (n = 3, THX 477, 478, 479) depicted as scatterplots and pie charts.Each dot represents a single sequence and bars depict the mean with s.e.m. f, huV H DJ H C H B cell clones and intraclonal diversification in THX mice as in e, depicted by TreeMaps and phylogenetic trees.Individual rectangle or square (unique color) area reflects huB cell clone size.In THX 477, 478 and 479 mice, the three largest huV H DJ H Cμ, huV H DJ H Cγ and huV H DJ H Cα1 clones accounted for 4.7%, 3.9% and 2.8% of huV H DJ H Cμ huB cells, 22.0%, 16.1% and 36.9% of huV H DJ H Cγ huB cells and 42.8%, 40.7% and 22.5% of huV H DJ H Cα1 huB cells.g, Interplay of SHM and CSR shapes B cell stepwise intraclonal diversification in COVID19 mRNAvaccinated THX mice, as exemplified by genealogical trees outlining the evolution of two huB cell clones, one tracked from its huV353D126J H 1Cμ heavy chain huB cell progenitor, the other from its huVκ311Jκ1Cκ lightchain huB cell progenitor.