One‐step CRISPR /Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice
2018; Springer Nature; Volume: 37; Issue: 18 Linguagem: Inglês
10.15252/embj.201899243
ISSN1460-2075
AutoresYing‐Cing Lin, Simone Pecetta, Jon M. Steichen, Sven Kratochvil, Eleonora Melzi, Johan Arnold, Stephanie K. Dougan, Lin Wu, Kathrin H. Kirsch, Usha Nair, William R. Schief, Facundo D. Batista,
Tópico(s)RNA and protein synthesis mechanisms
ResumoResource7 August 2018Open Access Transparent process One-step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock-in mice Ying-Cing Lin Ying-Cing Lin Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Simone Pecetta Simone Pecetta Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Jon M Steichen Jon M Steichen Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Sven Kratochvil Sven Kratochvil Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Eleonora Melzi Eleonora Melzi orcid.org/0000-0002-1520-735X Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Johan Arnold Johan Arnold Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Stephanie K Dougan Stephanie K Dougan Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Lin Wu Lin Wu Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Kathrin H Kirsch Kathrin H Kirsch Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Usha Nair Usha Nair Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author William R Schief William R Schief Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Facundo D Batista Corresponding Author Facundo D Batista [email protected] orcid.org/0000-0002-1130-9463 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Ying-Cing Lin Ying-Cing Lin Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Simone Pecetta Simone Pecetta Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Jon M Steichen Jon M Steichen Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Sven Kratochvil Sven Kratochvil Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Eleonora Melzi Eleonora Melzi orcid.org/0000-0002-1520-735X Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Johan Arnold Johan Arnold Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Stephanie K Dougan Stephanie K Dougan Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Lin Wu Lin Wu Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA Search for more papers by this author Kathrin H Kirsch Kathrin H Kirsch Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Usha Nair Usha Nair Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author William R Schief William R Schief Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Facundo D Batista Corresponding Author Facundo D Batista [email protected] orcid.org/0000-0002-1130-9463 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA Search for more papers by this author Author Information Ying-Cing Lin1,‡, Simone Pecetta1,‡, Jon M Steichen2, Sven Kratochvil1, Eleonora Melzi1, Johan Arnold1, Stephanie K Dougan3, Lin Wu4, Kathrin H Kirsch1, Usha Nair1, William R Schief1,2 and Facundo D Batista *,1 1Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA 2Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA 3Dana-Farber Cancer Institute, Boston, MA, USA 4Genome Modification Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 857 268 7071; E-mail: [email protected] The EMBO Journal (2018)37:e99243https://doi.org/10.15252/embj.201899243 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Here, we describe a one-step, in vivo CRISPR/Cas9 nuclease-mediated strategy to generate knock-in mice. We produced knock-in (KI) mice wherein a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. To validate our knock-in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B-cell development and performing single B-cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock-in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases. Synopsis We describe an in vivo, CRISPR/Cas9 nuclease-mediated strategy to generate knock-in (KI) mice. A large 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. We optimized this strategy to generate two independent heavy chain KI mouse models in about 3 weeks with a high frequency of homologous recombination (30–50%). Via a CRISPR/Cas9-nuclease-mediated strategy, a 1.9-kb DNA fragment bearing a pre-arranged human B-cell receptor heavy chain was recombined into the native murine immunoglobulin locus. The frequency of recombination is 30–50%. This strategy enabled us to generate knock-in mice in about 3 weeks. Introduction B lymphocytes are central to humoral immunity because of their ability to produce antibodies in response to infection and immunization. Antibodies not only confer protection from infections, but also represent the basis of most of the currently licensed human vaccines. Despite the great importance of antibodies for public health, the precise molecular mechanisms that drive their specificity, affinity, class switching, and durability are still under investigation. A more detailed understanding of the cellular and molecular mechanisms that drive B-cell function is critical for the development of treatments for B-cell dyscrasias and autoimmune diseases, and for the advancement of next-generation vaccines, particularly against challenging targets such as influenza and HIV (Pappas et al, 2014; Dosenovic et al, 2015; Jardine et al, 2015; Sanders et al, 2015; Briney et al, 2016; Escolano et al, 2016; Joyce et al, 2016; Kallewaard et al, 2016; Lee et al, 2016; Tian et al, 2016). Antigen binding to pre-clustered B-cell receptors (BCRs) on the B-cell membrane triggers a complex signaling cascade leading to B-cell activation. This includes extensive reorganization of the actin cytoskeleton, which is involved in BCR signaling-induced Ca2+ flux (Lanzavecchia, 1985; Wienands et al, 1996; Aman & Ravichandran, 2000; Batista & Neuberger, 2000; Hao & August, 2005; Lillemeier et al, 2006; Pierce & Liu, 2010; Treanor et al, 2010, 2011; Yang & Reth, 2010; Mattila et al, 2013; Tolar & Spillane, 2014; Spillane & Tolar, 2017). These events are followed by antigen internalization and endosomal trafficking, both of which are pre-requisites for antigen presentation to T cells, which drives the development of naïve B cells to germinal centers (GC) and differentiation into long-lived memory B cells (MBCs) and plasma cells (PCs) (Dal Porto et al, 2002; Shih et al, 2002; Okada et al, 2005; O'Connor et al, 2006; Paus et al, 2006; Qi et al, 2008; Gatto et al, 2009; Pereira et al, 2009; Schwickert et al, 2011; Taylor et al, 2012). In the past, mouse models expressing pre-arranged BCRs having defined specificity for particular model antigens have proven to be extremely valuable in studying different aspects of B-cell function. This was initially accomplished by expressing BCRs at non-native loci. Indeed, the studies of such mice have been extremely valuable in advancing our knowledge of concepts such as B-cell tolerance and allelic exclusion (Goodnow et al, 1988, 1989). More recently, knock-in (KI) mouse models expressing pre-arranged BCRs at their native loci have been generated (Verkoczy et al, 2017). These animal models have been instrumental in furthering our understanding of fundamental B-cell attributes such as negative selection, receptor editing, anergy, class switch recombination, and somatic hypermutation, which were not possible to study when BCRs were recombined at random sites in the genome (Taki et al, 1993; Pelanda et al, 1997). However, all the BCR KI models described thus far have been generated by using gene modification in embryonic stem (ES) cells or by somatic cell nuclear transfer (SCNT) (Dougan et al, 2012). A major drawback of these methods is the long time required to generate a BCR KI mouse or highly specialized technical skills and equipment needed in the case of SCNT. The ES gene modification approach not only requires the generation of correct ES clones, but also all of the founder mice produced by this method are chimeras. These chimeric founders require further crossing for the complete germline transmission of insertions or deletions in order to obtain a stable line. To alleviate this hurdle, we have developed a rapid one-step CRISPR/Cas9 nuclease-mediated KI strategy. In this method, Cas9 nuclease is directed by two guide RNAs to introduce double-stranded breaks within specific regions of the native murine immunoglobulin heavy chain locus of fertilized oocytes. Subsequently, large DNA fragments comprising pre-arranged immunoglobulin heavy (IgH) chains serve as templates for HDR giving rise to BCR KI mice within a matter of weeks rather than months. While the CRISPR/Cas9 technology is routinely used to generate mice bearing gene deletions and targeted insertions of short DNA fragments, our method allows for the rapid and reliable generation of KI mice with large DNA insertions, which was hitherto considered a challenge (Verkoczy et al, 2017). Using our methodology, we have generated and validated two such human IgH KI mouse models, which we report here. These KI mouse models express Ig V(D)J sequences encoding predicted germline sequences of HIV-1 broadly neutralizing antibodies (bnAbs) specific for the HIV-1 envelope V3 loop and surrounding spike glycans, PGT121, and BG18 (Julien et al, 2013; Freund et al, 2017). We envision that KI mouse models bearing specific immunoglobulin heavy or light chains will be valuable tools in testing the efficacy of particular immunogens in eliciting desired immune responses. As such, they offer the potential to drive vaccine development against HIV and other infectious diseases. Results One-step CRISPR generation of KI mice expressing human heavy chain Our goal was to generate KI mice in a rapid and reproducible manner. To this end, we designed a strategy that relies on Cas9 nuclease to introduce double-stranded breaks specifically at the D4-J4 region of the native IgH locus of fertilized oocytes (Fig 1A). The introduction of double-stranded DNA breaks can be repaired by either NHEJ or HDR (Maruyama et al, 2015). To drive integration of PGT121 via HDR in addition to the concomitant deletion of the native, murine D-J region, we introduced double-stranded breaks using not one, but two sgRNAs. As the substrate of homologous repair, we generated a donor plasmid bearing 5′ (3.9 kb) and 3′ (2.6 kb) homology arms, a mouse VHJ558 promoter and leader region (1.5 kb), and a pre-assembled BCR (0.4 kb) expressing PGT121-gH (hereafter referred to as PGT121 for simplicity), the predicted germline VDJ sequence of the heavy chain of a monoclonal antibody, PGT121 (Fig 1A and B; described in Materials and Methods). This sequence, previously described as PGT121 GLCDR3rev1, has been fully germline-reverted except at two positions in the D gene (Steichen et al, 2016), and differs at eight CDR3 positions from the less-reverted sequence (PGT121CDR3rev4) used in a previously described PGT121 germline-reverted heavy chain KI mouse (Fig EV1) (Escolano et al, 2016; Steichen et al, 2016). Figure 1. One-step CRISPR zygote injection to generate mice carrying PGT121 heavy chain in the mouse IgH locus Schematic depicting CRISPR/Cas9 injection. A circular plasmid bearing germline PGT121-gH VDJ sequences, two guide RNAs, and Cas9 protein were injected into zygotes and implanted into pseudopregnant mice. Cas9-induced double-stranded breaks in the genome of zygotes are used to insert germline PGT121 VDJ sequences flanked by homologous arms on each side of the cut site via HDR. After 3 weeks, F0 founder mice are born, some of which bear the human bnAbs germline precursor. Strategy for insertion of PGT121 rearranged VDJ into mouse IgH locus. Targeting DNA donor with 5′ (3.9 kb) and 3′ (2.6 kb) homology arms to the C57BL/6 WT mouse IgH locus, murine VHJ558 promoter, leader, and the human PGT121 heavy chain VDJ sequences are located between two homology arms. CRISPR/Cas9-mediated HDR leads to the insertion of the promoter and PGT121 sequences into the C57BL/6 mouse genome. P: murine VHJ558 promoter; HDR: homology-directed repair; bnAbs: broadly neutralizing antibodies. sgRNA targeting sites are indicated in red. Three distinct fragments of genomic DNA were amplified by PCR, and in vitro digestion assay was performed with each of the sgRNAs to validate the efficiency of Cas9-mediated cleavage. Analysis sgRNA off-target effects in unrelated genes. Amplicons corresponding to Aakt, Map3K10, and Nop9 were generated by PCR by using gene-specific primers. In vitro digestion assay was performed to measure the Cas9-directed cleavage efficiency. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Alignment of inferred germline PGT121 heavy chain sequences with full PGT121 bnAb heavy chain sequencesSequences were aligned in Jalview using ClustalO and post-processed in BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html). Relevant publications, from which these sequences originate, are listed: (i) GL-CDR3rev1 Steichen—PMID: 27617678, (ii) GL-CDR3rev4 Escolano—PMID: 27610569. Download figure Download PowerPoint The specificity of CRISPR/Cas9-mediated genome editing is dependent on the sgRNA. Accordingly, we used the CRISPR DESIGN database (http://crispr.mit.edu/) to identify potential candidate protospacers, including 20 nucleotides complementary to the target sequence upstream of a protospacer adjacent motif (PAM) sequence (NGG). To avoid the cleavage of the homologous recombination arms of the DNA donor by Cas9 upon oocyte injection, we designed sgRNAs that only target sequences within the wild-type IgH locus but are not present within the homology arms of our donor plasmid. In an attempt to select for highly specific sgRNAs, which can potentially render this process more efficient in the mouse embryo, we first designed and examined the ability of 11 different sgRNAs to cleave a PCR amplicon containing the wild-type genomic DNA target in an in vitro assay (Appendix Table S1). As shown in Fig 1C, we identified three sgRNAs (sgRNAs 1, 4, and 6) that guide Cas9 to cleave the genomic DNA target around the D4 region and three other guide RNAs (sgRNAs 7, 8, and 10) capable of targeting Cas9 to the J1-4 regions. We chose sgRNA1 and sgRNA8 because they appeared to be the two most efficient candidates and confirmed that they did not exhibit any off-target effects on three selected amplicons from unrelated genes (Fig 1D and Appendix Table S2). After the injection of the two sgRNAs, Cas9 protein and plasmid DNA containing PGT121 germline sequence into fertilized oocytes, and subsequent implantation into pseudopregnant females, we obtained F0 founder mice potentially carrying our KI heavy chain. As a first step to ascertain which of these founder mice is carrying the PGT121 insertion, we designed a screening protocol with three, independent TaqMan probes for genotyping. The first probe, Ighm-1 WT, is targeted to the WT C57Bl/6 mouse IgH D4-J1-4 region; testing positive for this probe indicates that the WT locus is intact (WT mouse). The second probe, HuIghV-4 Tg, is directed to the introduced PGT121 sequence and detects the integration of our PGT121 DNA. The third probe, KI-P, is targeted to the junction region between the 5′ arm and VHJ558 promoter, and testing positive to this probe indicates the correct site of insertion of our PGT121 DNA (Figs 2A and EV2A). Figure 2. Characterization of PGT121 KI mice Schematic of the TaqMan probes and their targeting sites within the WT IgH and PGT121 IgH. T: TaqMan probe. Schematic showing the annealing sites of primers used to validate PGT121 KI animals. Fo.1F and Fo.2F primers were targeted at promoter region and PGT121 region, respectively, and combined with Re.1R primer targeted to the genomic region after homologous 3′ Arm. KI alleles are predicted to result in the amplification of a Fo.1 fragment (3.3 kb) and Fo.2 fragment (2.8 kb). Genomic DNA was extracted from the F0 founders born after CRISPR injection or from a C57BL/6 (WT) mouse. Long-range PCR was performed to detect the insertion of the PGT121 VDJ sequences at the correct genomic locus. Table showing the frequency of the different genotypes of mice generated after CRISPR injection with plasmid donors containing long or short homology arms. # of HDR occurrence indicates the integration of the PGT121 heavy chain in the mouse IgH locus. # of Cas9-mediated D4-J4 deletions indicates the efficiency of our sgRNA-directed Cas9 double-stranded breaks. HC: heavy chain. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. TransnetYX probes design and KI mice named 3 TaqMan probes, Ighm-1 WT, HuIghV-4 Tg, and KI-P designed for genotyping. Schematic showing nomenclatures of WT and PGT121 KI mice according to genotyping results. Download figure Download PowerPoint In our initial experiment, after microinjecting 400 fertilized oocytes with sgRNA, Cas9 protein, and plasmid DNA containing PGT121 germline sequence and subsequently implanting them into pseudopregnant females, 15 pups were born. As determined from our screening protocol, out of these 15 pups, we found eleven founders that carried no deletions or insertions (WT+/+), three founders that carried deletions of the D4 to J1–4 segment in both alleles with no insertion of PGT121 (WT−/−), and lastly one founder in which the D4 to J segment was replaced with a monoallelic insertion of PGT121 (PGT121+/WT; Figs 2C and EV2B). Taken together, we observed that Cas9-driven deletion occurred at 26.7%, while the frequency of homologous recombination was only 6.7%. To validate whether the inserted IgH germline sequence (PGT121) was at the right genomic locus, we performed long-range PCR in the PGT121 mouse by amplifying the genomic DNA fragments using specific primers (Appendix Table S3). The two forward primers, Fo.1F and Fo.2F, were targeted at the promoter and PGT121 regions, respectively, and the reverse primer, Re.1R, was targeted at the region after the homologous 3′ arm. We found amplicons of the correct size only from genomic DNA amplified from the PGT121 KI mice, but not from WT mice, indicating that the PGT121 fragment had been inserted in the correct genomic locus (Fig 2B). We performed another CRISPR injection (200 fertilized oocytes) with short arms (3.9 kb) of plasmid DNA and we observed that the frequency of Cas9-driven D4-J1-4 deletion was 35.7%, while the frequency of homologous recombination was 0% (Fig 2C). We hypothesized that the low efficiency of HDR was likely to be a result of our relatively short homology arms. Accordingly, to increase the efficiency of this process, we extended each of the homology arms to 5 kb (Fig EV3). This enhancement in arm length coincided with an increase in HDR observed in the founders. Indeed, 50 pups were born following the injection of this plasmid, among which there were 14 PGT121+/WT mice, one PGT121+/− mouse, and one WT−/− and 34 WT+/+ mice. It appears that the extended homology arms increased our HDR efficiency to 30%, while the rate of deletion remained constant (32%). This higher rate of recombination was confirmed by performing three independent injections, from which the average frequency of HDR reached 40% with a similar rate of genomic DNA cleavage (Fig 2C). Click here to expand this figure. Figure EV3. Schematics of elongated DNA donor armsThe original arms of DNA donor are 3.9 kb and 2.6 kb in length. We extended both homology arms to 5 kb for later microinjections. Download figure Download PowerPoint We further crossed several of our positive heavy chain KI F0 founders with WT mice and followed the frequency of germline transmission of the KI heavy chain. Six out of 10 F0 mice revealed the Mendelian transmission of the heavy chain, while three out of ten F0 founders exhibited non-Mendelian transmission, indicating likely mosaicism, and one of our founders most likely has more than one insertion of our KI heavy chain (Fig EV4). Click here to expand this figure. Figure EV4. Genetic tree of KI mice depicting the F0, F1, and F2 generationsSix out of ten F0 mice revealed the Mendelian transmission of the heavy chain. Three out of ten F0 founders exhibited non-Mendelian transmission. One of our founders has at least more than one insertion of our KI heavy chain. White triangles—F0 mouse; black hexagon—F1 homozygous KI; gray hexagon—F1 heterozygous KI; white hexagon—F1 WT; the number of mice is shown next to the triangles. The percentages of animals of each genotype detected are shown below each of the hexagons. Download figure Download PowerPoint Collectively, our results demonstrate that we can reliably generate heavy chain KI mice in vivo by CRISPR/Cas9 within a short period of few weeks. This represents a significant step forward towards the rapid and reproducible generation of IgH KI mice. Characterization of B lymphocytes in the PGT121 KI mouse To examine the impact of PGT121 germline heavy chain insertion on B-lymphocyte development, we analyzed the bone marrow progenitors of 8- to 10-week-old WT and PGT121+/WT mice by flow cytometry, using the Hardy classification system (Hardy et al, 1991). When compared to WT mice, we observed a similar frequency of immature, defined as early (B220+CD43+) and late (B220+CD43−), B cells in the bone marrow of PGT121+/WT mice (Fig 3A). However, when we examined the B cell subpopulations in the early immature compartment, we observed a significant reduction in the (CD24+BP-1+) B cell fraction C (Fig 3B). In contrast, the late immature B cell subpopulations from the bone marrow were not affected (Fig 3C). These results suggested that the expression of the PGT121 germline heavy chain might compromise the ability of KI B cells to pass tolerance checkpoints in the bone marrow (positive selection, stage C), possibly leading to the elimination or BCR editing of these cells. Figure 3. Characterization of B-lymphocyte development in the bone marrow of PGT121+/WT mice Bone marrow cells from WT and PGT121+/WT mice were analyzed by flow cytometry using the gating strategy shown on the left. B-cell progenitors (B220+) were divided into immature (CD43+) and mature (CD43−) cells on the basis of CD43 expression. Data quantified in the panels on the right show the percentage of live cells in the indicated gates (WT: n = 9, PGT121+/WT: n = 8, mean ± SEM). Early (CD43+) B-cell progenitors were subdivided according to CD24 and BP-1 expression into Hardy populations A (CD24−BP-1−), B (CD24+BP-1−), and C (CD24+BP-1+). Data quantified in the panels on the right show the percentage of CD43+ cells in the indicated gates (WT: n = 6, PGT121+/WT: n = 4, mean ± SEM). Late (CD43−) B-cell progenitors were subdivided according to IgM and IgD expression into Hardy populations D (IgM−IgD−), E (IgM+IgDint), and F (IgM+IgD+). The data quantified in the panels on the right show the percentage of CD43− cells in the indicated gates (WT: n = 6, PGT121+/WT: n = 4, mean ± SEM). Data information: For all flow cytometry experiments, data are from one out of three representative experiments with three or more animals in each group, and each dot represents an individual mouse. Student's t-test, ns P > 0.05, *P < 0.05. Download figure Download PowerPoint To further characterize the progression of transitional B cells toward mature B lymphocytes, we analyzed peripheral B cells from blood, spleen, and peritoneal cavity by flow cytometry. Consistent with defects we observed in the bone marrow, we found a significant diminution in the number of B cells in the blood (Fig 4A). In contrast, we observed comparable numbers of B and T cells between these KI and WT mice, indicating that spleen cellularity was largely unaltered in PGT121+/WT mice (Fig 4B and C). However, we noticed a slightly decreased proportion of T1 cells (CD21loCD24hi) within the B220+ B-cell compartment. Furthermore, PGT121+/WT B cells retained their maturation potential, as we found similar amounts of both mature follicular (CD21hiCD24lo), marginal zone B cells (CD21hiCD24hiCD23−), and T2 cells (CD21hiCD24hiCD23+) in WT and PGT121+/WT mice (Fig 4D and E). We also analyzed the lymphocyte populations in the peritoneal cavity of these animals. Here, we observed that WT and PGT121+/WT mice had comparable numbers of B2 (CD5−CD11b−) and B1a (CD5+CD11b+) but a slight diminution of B1b (CD5−CD11b+) cells (Fig 4F). Together, these results suggest that the establishment of mature lymphocyte populations is not affected in the PGT121+/WT mouse. Figure 4. Characterization of B- and T-lymphocyte development in peripheral blood and spleen of PGT121+/WT mice A. B cells from blood of WT and PGT121+/WT mice were analyzed by flow cytometry. Identification and quantification of B-cell population (B220+IgD+). Data quantified in the panels on the right show the percentage of live cells in the indicated gates (WT: n = 3, PGT121+/WT: n = 14, mean ± SEM). B–E. Spleens from WT and PGT121+/WT mice were analyzed by flow cytometry. (B, C) Identification and quantification of B cells (B220+TCRβ−) and T cells (B220−TCRβ+). T cells were subdivided into CD4 (CD4+CD8−) and CD8 (CD4−CD8+) T cells. Quantification—right. (D, E) B cells were divided on the basis of CD21, CD23, and CD24 expression into T0/T1 cells (CD21−CD24hi), follicular B cells (CD21loCD24lo), T2 cells (CD21hiCD24hiCD23−), marginal zone B (MZB) cells (CD21hiCD24hiCD23+), and quantified (right panels) (n = 5/group, mean ± SEM). F. Peritoneal lavage was performed on WT and PGT121+/WT mice, and the exudate was analyzed by flow cytometry. B cells (IgM+) were divided into B2 (CD11b−CD5−), B1b (CD11b+CD5−), and B1a (CD11b+CD5+). Data quantified in the panels on the right show the percentage of IgM+ cells in the indicated gates (WT: n = 8, PGT121+/WT: n = 4, mean ± SEM). G. Confocal images spleen cryosections showing the organization of the spleen and the localization of T cells, macrophages, and B cells. Spleen was collected from WT and PGT121+/WT mice. Sections were stained with B220 (blue, B cells), CD169 (red, metallophilic macrophages), TCRβ (green, T cells), and F4/80 (white, red pulp macrophages). In the insets, sequential sections were stained for B220 (blue), CD169 (white), IgM (green), and CD23 (red) to identify follicular (Fo, IgMlowCD23high) and marginal zone (MZ, IgMhighCD23−) B cells. TC: T cells, BC: B cells, Fo: follicular B cells, MZ: marginal zone. Data information: For all flow cytometry experiments (A–F), data are from one out of three representative experiment with three or more animals in each group, and each dot represents an individual mouse. Student's t-test, ns P > 0.05, *P < 0.05. Download figure Download PowerPoint To understand whether the histological organization of lymphoid organs was altered in the PGT121 KI mouse line, we assessed the anatomical structure of the spleen by confocal microscopy. We observed a normal compartmentalization of the splenic tissue, comprised of white pulp and red pulp area in normal proportion, the latter populated by F4/80+ macrophages (Fig 4G). The cellular localization appeared normal in the white pulp, with central T cells (TCRβ) surrounded by B cell (B220+) organized into follicles delimited by metallophilic marginal zone macrophages (CD169+). Inside the follicles, B cells presented a
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