Transgenic expression of human signal regulatory protein alpha (SIRPα) in BALB/c Rag2 null /Jak3 null immunodeficient mice improves human lymphoma xenograft via reduction of phagocytosis

Transgenic expression of human signal regulatory protein alpha (SIRPα) in BALB/c Rag2 null /Jak3 null immunodeficient mice improves human lymphoma xenograft via reduction of phagocytosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transgenic expression of human signal regulatory protein alpha (SIRPα) in BALB/c Rag2 null /Jak3 null immunodeficient mice improves human lymphoma xenograft via reduction of phagocytosis Jutatip Panaampon, Tatsuya Ogawa, Akira Shiota, Seiji Okada This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8510586/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 4 You are reading this latest preprint version Abstract The immunodeficient mouse model provides a platform for assessing therapeutic options in preclinical cancer research by allowing the establishment of human xenografts. We previously developed BALB/c Rag2nullJak3null (BRJ), an immunodeficient mouse model valuable for cancer research. However, susceptibility to xenograft acceptance can be enhanced by the signal regulatory protein alpha (SIRP)-CD47 “don‘t eat me” signal. In this study, we created BALB/c human-SIRP BAC transgenic mice and crossed them with BRJ mice. The resulting human SIRP transgenic BRJ (BRJ-S) mice showed significantly increased engraftment of human B-cell lymphoma compared to BRJ mice. Additionally, we demonstrated improved human tumor engraftment in BRJ-S mice by reducing phagocytosis. In summary, hSIRP-transgenic BRJ mice (BRJ-S) serve as a promising immunodeficient model with a greater capacity for human lymphoma engraftment. BRJ-S provides an advantageous and permissive model for xenografting and for studying cancer in research and drug development. SIRPα phagocytosis immunodeficient mice xenotransplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Xenotransplantation of human tumors into an immunodeficient mouse model is a well-established method for studying human tumor development. After the discovery of Nude mice, the xenograft model became more popular, and lymphocyte-depleted mice, such as SCID mice and Rag-1 or Rag-2 knockout mice, were introduced. However, depleting T and B lymphocytes alone is not sufficient for human cell xenografts. The introduction of NOD/Scid mice improved the success of human cell xenografts, and NK cell activity plays a key role in xenograft rejection. Therefore, mice lacking lymphocytes and NK cells on a NOD and BALB/c background, such as NOD/Scid commonγ null mice (NSG and NOG mice) (Ito et al. 2002 ; Shultz et al. 2005 ), BALB/c Rag-1/2 null commonγ null (BALB-RG) mice (Traggiai et al. 2004 ), and BALB/c Rag-2 null /Jak3 null (BRJ) mice (Ono et al. 2011 ), have been used as standard models for human xenotransplantation. However, C57BL/6 background immunodeficient mice do not accept human cells (Okada et al. 2019 ; Ono et al. 2011 ). Signal regulatory protein alpha (SIRPα) is one of the immune checkpoint proteins and is also known as cluster of differentiation 72 alpha (CD152a) or Src homology 2 (SH2) domain-containing phosphatase substrate-1 (Matozaki et al. 2009 ). Its expression is primarily found on the surface of myeloid cells, including monocyte-macrophage lineages, granulocytes, dendritic cells, and hematopoietic stem cells. SIRPα interacts with CD47, a well-known overexpressed surface protein on malignant cells, to regulate immune responses, particularly phagocytosis (Barclay and Brown 2006 ; Matozaki et al. 2009 ). SIRPα is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC), probably by inhibiting macrophage-mediated phagocytosis of tumor cells and suppressing antitumor immunity (Koga et al.). The interaction between SIRPα and CD47 on macrophages and target cells plays a crucial role in preventing phagocytosis, known as the “Don’t eat me signal” (Barclay and Brown 2006 ; Barclay and Van den Berg 2014 ). Human cells in mouse xenografts are effectively eliminated by mouse macrophages when there is an SIRPα-CD47 mismatch. Takenaka K et al. showed that macrophages from NOD- and BALB/c-background mice have a high affinity for human CD47, whereas those from C57BL/6-background mice do not (Takenaka et al. 2007 ). This group also generated human SIRPα knock-in C57BL/6 Rag-2null commonγnull mice (Jinnouchi et al. 2020 ) and demonstrated that these mice can accept both normal and malignant human hematopoietic cells. In this study, we created BRJ mice expressing a human SIRPα transgene (BRJ-S) and examined their ability to support human lymphoma xenografts. We show that hSIRPα expression notably improves human tumor engraftment, mainly by decreasing macrophage phagocytosis. Our findings establish BRJ-S mice as a robust and versatile platform for preclinical cancer research and therapy development. Materials and Methods Cell cultures and media Primary effusion lymphoma (PEL) cell lines BCBL-1 (obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA), GTO (Goto et al. 2013 ) (established in our Labo and registered at JCRB cell bank, Osaka Japan: JCRB4057), and TY-1 (kindly provided by Dr. H. Katano, National Institute of Infectious Diseases, Tokyo, Japan) were used in this study. In addition, the human diffuse large B-cell lymphoma (DLBCL) cell line, OCI-Ly3, was included. All cell lines were maintained in RPMI-1640 medium (Fujifilm Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France), penicillin G (100 IU/mL, Meiji Seica Pharm, Tokyo Japan), and streptomycin (100 U/mL, Fujifilm Wako). Cultures were incubated at 37°C under humidified conditions with 5% CO₂. Flowcytometry BALB/c and C57/BL6N mice were purchased from Japan SLC (Hamamatsu, Japan). Mouse bone marrow (BM), spleen, and peritoneal macrophages were stained with anti-human/mouse CD11b (M1/70)-BV421, anti-human CD172a/b (SIRPα/β)(SE5A5)-PE, anti-mouse CD172a (SIRPα)(P84)-APC, anti-mouse CD49b (DX5)-APC (pan-NK marker); anti-mouse CD122 (IL-2Rβ)-PE, anti-mouse CD19-FITC, and anti-mouse CD3-PE (BioLegend, San Diego, CA), and analyzed with FACS Celesta (BD Bioscience, San Jose, CA). The collected data were analyzed with FlowJo Version 10.4 software (Tree Star, San Jose, CA). BAC Transgenic Construct A mouse SIRP BAC clone, RP23-389N11, was identified from the RPCI-23 Female C57BL/6J murine BAC Library by searching the murine BAC end sequence database at the National Center for Biotechnology Information (NCBI). Analysis of the BAC end sequences indicates that this clone encompasses the complete 19.3-kb mouse SIRPα genomic region, along with upstream (5‘) and downstream (3’) flanking genomic sequences (Fig. 1 A). A human SIRP BAC clone (RP11-636L22) was identified from the RPCI-11 human BAC Library using BLAST search in the GenBank database at NCBI. Sequence analysis of this 172-kb BAC revealed that it contains the entire 45.9-kb human SIRPα genomic locus. To create a chimeric human-mouse SIRP BAC transgenic construct, the full-length coding exons and an intervening intron of the human SIRPα gene were inserted into the corresponding mouse SIRPα genomic locus via BAC recombineering. Transfer of the human SIRPα sequence from the human BAC clone to the mouse BAC clone was performed using the Red/ET Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany) (Muyrers et al. 2000 ). Briefly, a rpsL-neo counterselection cassette flanked by homologous sequences matching an intron of the human SIRPα gene was amplified by PCR and integrated into the human BAC through Red/ET recombination. The human SIRPα gene fragment was subsequently subcloned into the pDNR-1r plasmid (Takara Bio USA, Mountain View, CA) using Red/ET recombination, with the gene's terminal sequences serving as homology arms. This subcloned fragment was engineered to contain 40-nucleotide sequences derived from the orthologous mouse SIRPα untranslated regions immediately adjacent to the start codon and the stop codon, respectively. The modified human SIRPα fragment was then transferred to the mouse SIRPα gene. Finally, the remaining rpsL-neo cassette within the intron was removed thr ough rpsL counterselection. All BAC modifications were confirmed by sequencing. Preparation of BAC DNA for microinjection Human-SIRP BAC transgenic constructs were purified for pronuclear microinjection using a modified protocol based on that of Abe et al. (Abe et al. 2004 ). The BAC transgenic constructs were extracted from 250 ml of the E. coli culture with the Nucleobond Plasmid Purification kit (MACHEREY-NAGEL, Düren, Germany). Purified BAC transgenic DNA (10 µg) was linearized overnight with PI-SceI endonuclease (New England Biolabs, Ipswich, MA), which cleaves the unique site within the BACe3.6 vector backbone. The linearized BAC DNA was separated by pulsed-field gel electrophoresis (PFGE) and recovered from the gels through electroelution. After dialysis against TE buffer containing 0.1 mM EDTA, aliquots were reanalyzed by PFGE to verify DNA integrity and size. The DNA concentration was adjusted to 1 ng/µl for microinjection. The BAC DNA solution aliquots were stored at 4°C until use for microinjection. Generation of Human SIRPα BAC Transgenic Mice Human SIRP BAC transgenic mice were generated by injecting BALB/cA mouse embryos (Charles River Laboratory Japan Inc., Yokohama, Japan) into the pronucleus. The transgenic founders and germ-line transmission of the BAC transgenic construct were assessed using Southern blot analysis of PstI-digested tail DNA, probed with a [32P]-labeled human SIRP gene probe. Out of 197 offspring screened, four mice (2.0%) tested positive for the human SIRPα BAC transgene. No significant phenotypic abnormalities were noted when transgene-positive mice were compared to transgene-negative littermates. Multiple transgenic founder lines were subsequently bred with BALB/cA mice, and after several generations, mice exhibiting stable, high-level expression of human SIRPα were selected and maintained as founder lines. Generation and confirmation of Human SIRPα transgenic in BALB/c Rag2 null /Jak3 null Immunodeficient Mice (BRJ-S) BALB/c Rag2 null /Jak3 null SIRPα transgenic mice (BRJ-S mice) were generated by crossing BALB/c Rag2 null /Jak3 null (BRJ) mice with SIRPα transgenic mice. PCR confirmed the presence of the human SIRP transgene, Rag2 null , and Jak3 null . The PCR primers are as follows: human SIRPα: hSIRPaBIF6;5'-GAGGACAAGTTGGAAAAGATGGT, hSIRPaBIR6; 5'-GGAGGTCTCATTACTGCTTC (610bp), Rag-2 5’-CCAACGCTATGTCCTGATAGCGGT-3’ RG2-2 5’-TTAATTCAACCAGGCTTCTCACTT-3’, RG2-3 5’-GCCTGCTTATTGTCTCCTGGTATG-3’ (Wilde type allele: 973 bp, Mutant allele 1107 bp), Jak3; Oligo1:5’-CCAGACCAGCAGAGGGACTT-3’, Oligo2: 5’-GAACCT GCGTGCAATCCATCTTG-3’, Oligo3: 5’-ACCCAGGTACTCCATGCCCT–3’ (Wild type allele: 400 bp, Mutant allele: 700 bp). Preparation of mouse macrophages Peritoneal macrophages were obtained from 10-week-old BRJ mice. To elicit macrophage recruitment, mice were injected intraperitoneally with 2 mL of 4% (w/v) thioglycolate broth (BD Diagnostic Systems, Sparks, MD, USA). Three days later, peritoneal cells were collected by peritoneal lavage with 10 mL of cold phosphate-buffered saline (PBS) (Goto et al.). Flow cytometric analysis confirmed that more than 95% of the recovered cells expressed the macrophage marker CD11b. CD47 protein binding assay Recombinant Human CD47-Fc Chimera and Recombinant Mouse CD47-Fc Chimera were purchased from BioLegend (San Diego, CA, USA), and conjugated with HiLyte Fluor™ 647 by HiLyte Fluor™ 647Labeling Kit - NH 2 (Dojindo, Kumamoto, Japan). Isolated mouse peritoneal macrophage FC receptors were blocked with Purified anti-mouse CD16/32 Antibody (Biolegend), and incubated with HiLyte Fluor™ 647 conjugated Recombinant Human CD47-Fc Chimera or Recombinant mouse CD47-Fc Chimera, and analyzed with FACS Celesta. Phagocytosis assay Macrophage phagocytic capacity was assessed by flow cytometry based on a previously established protocol (Panaampon et al. 2022 ). Briefly, macrophages were seeded into 96-well plates in 10% FBS/RPMI-1640. CFSE-labeled lymphoma cells were subsequently added at the indicated effector-to-target (E:T) ratios and co-cultured for 2 hours at 37°C in a humidified atmosphere containing 5% CO 2 . Following incubation, cells were harvested and stained with anti-CD11b-APC antibody. Samples were analyzed using a FACS Celesta flow cytometer. Phagocytosis was quantified as the proportion of CD11b⁺CFSE⁺ cells among total CD11b⁺ macrophages. Western Blot Bone marrow cells, splenocytes, peritoneal macrophages, and human peripheral mononuclear cells (PBMCs) were collected. Twenty µg protein was loaded onto 10% or 12% SDS-PAGE and subsequently transferred onto a PVDF membrane. The antibodies used were anti-γ-tubulin (C-20) and anti-human SIRP (SE7C2) (Santa Cruz Biotechnology, Inc.). The protein signal was detected using the ImageQuant Biomolecular Imager (GE Life Sciences, Uppsala, Sweden). Xenograft of Lymphoma Cell Lines BRJ (Ono et al. 2011 ) and BRJ-S mice were maintained at the Center for Animal Resources and Development (CARD), Kumamoto University, under specific pathogen-free conditions in accordance with institutional animal care guidelines. Animals had unrestricted access to food and water throughout the study. All experimental procedures were reviewed and approved by the Animal Ethics Committee of Kumamoto University (approval number A2019-041). Subcutaneous xenograft; For subcutaneous tumor establishment, GTO or OCI-Ly3 cells (1 × 10 7 cells per mouse) were injected into both flanks of 10- to 12-week-old female BRJ and BRJ-S mice (n = 6 per group). Tumor growth was monitored longitudinally, and mice were euthanized at the experimental endpoint for excision and measurement of tumor mass. Intraperitoneal xenograft; For the intraperitoneal model, GTO cells (1 × 10 7 cells per mouse) were administered intraperitoneally into 10- to 12-week-old female BRJ and BRJ-S mice (n = 5 per group). Body weight was recorded regularly as an indicator of disease progression. At day 35 following tumor inoculation, mice were sacrificed, and ascitic fluid was collected and quantified. Statistical analysis Data are expressed as mean ± standard error (SE) or standard deviation (SD), as specified in the figure legends. Statistical comparisons were performed using Student’s paired t -test with GraphPad Prism software (version 6; GraphPad Software, San Diego, CA, USA). A P value < 0.05 was considered statistically significant. Results Generation of Human SIRPα BAC Transgenic Mice. Since SIRP-CD47 signaling is crucial for the rejection and acceptance of xenografts, we created transgenic mice that express human SIRP. The BAC transgenic method is a valuable tool for transgene expression because it depends on copy number and is independent of integration position (Heintz 2000 ; Masuda et al. 2018 ; Moritoki et al. 2018 ). We selected a mouse SIRPα BAC clone that includes over 100 kb of upstream regulatory sequences, the complete SIRPα structural gene, and downstream genomic regions for this purpose. Using mouse-specific genomic elements to maintain natural expression patterns, we precisely replaced the endogenous mouse SIRPα coding region within the BAC with the human SIRPα gene through BAC recombineering. This approach produced a chimeric human–mouse BAC transgenic construct that drives strong expression of human SIRPα in myeloid-lineage cells. After recombineering in Escherichia coli, the modified BAC was linearized, purified (Fig. 1 B), and injected into pronucleus-stage mouse embryos to create independent transgenic lines. All four founder mice successfully transmitted the human SIRPα BAC transgene to their F1 offspring. As shown in Fig. 1 C, individual lines carried either one or three copies of the SIRPα BAC transgene. The 22 − 1 line, which contained three copies of the construct, was chosen for all subsequent experiments. Generation and Phenotypic expression of human SIRPα in BALB/c Rag-2 null Jak3 null (BRJ-S) immunodeficient mice The human SIRPα BAC transgenic mice were crossed with BRJ mice for 10 generations and established human SIRPα-expressing Rag-2/Jak3 double knock-out mice (BRJ-S). As shown in Fig. 2 A, the wild-type BALB/c mouse has T and B lymphocytes and NK cells, whereas these cells are absent in BRJ and BRJ-S mice. We isolated cells from bone marrow, spleen and peritoneal macrophages to confirm human (h) SIRPα expression. As shown in Fig. 2 B, hSIRPα was positive in BRJ-S mice by western blotting. Mouse (m) SIRPα was expressed on CD11b + cells of wild-type BALB/c, BRJ, and BRJ-S, whereas human SIRP was expressed on CD11b + cells of bone marrow, spleen, and peritoneal macrophages of only BRJ-S mice (Fig. 2 C). The CD11b + population co-expresses both hSIRPa and mSIRPα (Fig. 2 D). CD11b + of BRJ-S binds to recombinant hCD47 To test the binding of mouse CD11b + cells with recombinant human and mouse CD47-Fc protein, we incubated peritoneal macrophages with HiLyte Fluor™647-conjugated recombinant human or mouse CD47, and assessed binding by fluorescent intensity of FACS. As shown in Fig. 3 A, the binding between CD11b + -hCD47 is strongest in BRJ-S. In contrast, the binding of recombinant mouse CD47 to peritoneal macrophages BRJ-S, BRJ, and wild-type C57/BL6, showed no significant difference among the three types of mice. In vitro tumor cell phagocytosis is weaker in human SIRPα transgenic BALB/c Rag-2 null Jak3 null (BRJ-S) immunodeficient mice The SIRPα-CD47 interaction is well known as the "Don’t eat me” signal. The binding of human SIRPα to mouse macrophages and of human CD47 to human tumor cells inhibits phagocytosis by mouse macrophages, suggesting that BRJ-S mice would have a higher capacity to accept human tumor xenografts than BRJ immunodeficient mice. To test this hypothesis, we xenografted multiple human lymphoma cells into BRJ-S and BRJ mice. As shown in Fig. 2 B, macrophages isolated from BRJ-S showed a lower percentage of phagocytosis than that of BRJ for all four lymphoma cell lines; OCI-Ly3, BCBL-1, GTO, and TY-1. The results suggest that BRJ-S mice exhibit higher efficacy against human lymphoma xenografts than BRJ mice. BRJ-S mice provide a higher capacity for human lymphoma xenograft than BRJ mice To confirm the suitability of the BRJ-S xenograft model, we transplanted OCI-Ly3 and GTO cells into BRJ-S and BRJ mice, respectively. Furthermore, we monitored tumor growth in these two mouse strains. Tumor volume of OCI-Ly3 was measured, and tumor weight was determined at the end of the experiment. As shown in Fig. 4 A-C, OCI-Ly3 grew faster in BRJ-S mice compared with BRJ, resulting in a larger size, volume, and weight than in BRJ mice. In correlation with Fig. 4 D- 4 H, GTO, a PEL cell line, which forms a tumor mass by subcutaneous (s.c.) and forms ascites by intraperitoneal (i.p.) transplantation, was transplanted into BRJ-S and BRJ mice. GTO cells showed more rapid growth in BRJ-S mice than in BRJ mice, as measured by tumor volume, tumor size, and tumor weight (Fig. 4 D-F). Moreover, we intraperitoneally injected GTO into BRJ-S and BRJ mice to directly represent the clinical manifestation of PEL. As shown in Fig. 4 G, GTO forms higher ascites volume in BRJ-S than BRJ. The percentage of weight increase in BRJ-S mice was also more than the weight increase in BRJ mice (Fig. 4 H). Taken together, the results demonstrate that BRJ-S is a superior mouse model for human lymphoma xenograft than BRJ. Discussion In this study, we generated human SIRPα-expressing BAC transgenic mice by the standard method (Mogi et al. 2025 ), then established BALB/c human SIRPα-expressing Rag-2/Jak3 double deficient (BRJ-S) mice, and showed BRJ-S mice had superior engraftment efficiency of lymphoma cells. Severe immunodeficient mice lacking B and T lymphocytes and NK cells have become widely used hosts for xenotransplanting human cells because they exhibit diminished rejection of both normal and malignant human cells. In addition, the genetic background of the mice has been observed to influence engraftment efficiency. In contrast, NOD and BALB/c background immunodeficient mice accept human cells, C57/BL6 background immunodeficient mice cannot support human cell engraftment. Recently, the CD47-SIRPα axis, also known as the “Don’t eat me” signal, has been shown to play an essential role in macrophage phagocytosis. Takenaga et al. showed that NOD SIRPα efficiently binds to human CD47 but not C57/BL6 SIRPα by polymorphism. This group established C57/BL6 Rag-2 null /IL2R null human SIRPα knock-in (BRGS human ) mice and showed efficient reconstitution of both human normal and malignant hematopoietic cells. The advent of NOD/Scid mice enabled the efficient creation of humanized mice; however, the mechanism by which human hematopoietic stem cells engraft easily in NOD/Scid mice remained unclear. The SIRPα immunoglobulin variable region is known to be highly polymorphic in mice. It has been shown that NOD SIRPα has a strong affinity for human CD47, while BALB/c SIRPα has a moderate affinity, and C57/BL6 SIRPα has no affinity. Currently, NOD/Scid background mice are further modified and have been used as recipients of humanized mice, such as NOD/Shi-SCID IL-2R null (NOG) mice, NOD/LtSz-SCID IL-2R null (NSG) mice, and NOD/Scid Jak3 null (NOJ) mice (Ito et al. 2002 ; Okada et al. 2008 ; Okada et al. 2019 ; Shultz et al. 2005 ). However, these NOD/Scid mice have several limitations as recipients for humanized mice and patient-derived xenografts; they are difficult to breed and exhibit poor recovery from DNA damage induced by irradiation and cytotoxic drug treatment. In this context, human SIRPα transgenic mice and BALB/c or C57BL/6 knock-in mice are expected to be easier to handle. In particular, the BALB/c strain is gentle by nature, making it more manageable in experiments than the C57/BL6 strain. Previously, we established BALB/c Rag-2null/Jak3null (BRJ) mice, which demonstrated appropriate homing efficiency of both hematopoietic stem cells and peripheral blood mononuclear cells. We also showed that the NOD genetic background most effectively induces lymphomatous effusion when transplanted into the peritoneal cavity. In this study, we further demonstrated that human SIRPα transgenic BRJ mice had improved transplantation efficiency. The CD47-SIRPα signal is essential for maintaining homeostasis in normal hematopoiesis. Additionally, tumor cells are known to upregulate CD47 expression to inhibit macrophage phagocytosis through the “Don’t eat me” signal mediated by the CD47-SIRPα interaction. This mechanism helps tumor cells evade the immune system, and blocking the CD47-SIRPα signal with anti-CD47 or anti-SIRPα antibodies allows macrophages to recognize and destroy tumor cells. We have previously shown that primary effusion lymphoma and cholangiocarcinoma highly express CD47 on their surfaces, and anti-CD47 antibody treatment suppressed tumor growth in xenograft mouse models. Currently, the anti-CD47 antibody is in clinical trials for hematological malignancies and solid tumors. Therefore, the CD47-SIRPα signal plays a crucial role in supporting tumor cell survival, and human SIRPα transgenic immunodeficient mice are valuable for establishing patient-derived xenograft models. Human SIRPα knock-in mice were developed on a C57BL/6 background. Although transplantation efficiencies may be similar across NOG, NSG, and NOJ mice, C57BL/6 mice are generally known for their high aggression levels, which makes them more difficult to handle than less aggressive BALB/c mice (An et al. 2011 ; Brinks et al. 2007 ; Tsuchimine et al. 2020 ). The behavioral traits of NOD-derived strains (including NOD/Scid mice and NOG/NSG/NOJ mice) have not been fully characterized; however, our experience suggests they display greater aggression compared to BALB/c mice. Additionally, the breeding efficiency of the C57BL/6 strain is usually lower than that of BALB/c mice (Tsuchimine et al. 2020 ; Weber et al. 2013 ), and the reproductive success of NOD/Scid background mice is limited; therefore, efficient production often requires advanced breeding techniques (Goto et al. 2021 ; Kim et al. 2025 ; Kumagai et al. 2011 ). This evidence suggests that BALB/c background immunodeficient mice, such as BRJ-S mice, are relatively easier to handle in experimental settings. Conclusion In conclusion, we successfully established BRJ-S, BALB/c human SIRPα-transgenic Rag2 −/− Jak3 −/− (BRJ-S) mice, and showed that BRJ-S mice can accept human lymphoma cells more than BRJ mice. Thus, BRJ-S mice offer a promising immunodeficient mouse model for cancer research. Declarations Acknowledgments We thank Ms. S. Fujikawa for technical assistance and Ms. M. Teramoto for secretarial assistance. Availability of data and materials The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request. Funding This research was funded by the Strategic Core Technology Advancement Program from the Ministry of Economy, Trade, and Industry, Japan. Authors’ contributions Conceptualization, A.S. and S.O.; methodology, T.O. and J.P.; validation, J.P. and S.O., formal analysis, J.P. and T.O. ; investigation, J.P. and T.O. ; resources, A.S. and S.O.; data curation, J.P.; writing original draft preparation, J.P.; writing review and editing, S.O.; visualization, J.P. and T.O.; supervision, A.S. and S.O.; project administration, A.S. and S.O.; funding acquisition, S.O. All authors have read and agreed to the published version of the manuscript. Ethical consideration: All experimental procedures and protocols were conducted in accordance with the ARRIVE guidelines and approved by the Kumamoto University Committee of Animal Care (A2021-053, A2023-153). Consent to publish Not applicable. Competing interests Tatsuya Ogawa and Akira Shiota are employees of the Institute of Immunology, Co., Ltd. Jutatip Panaampon and Seiji Okada declared no conflicts of interest. References Abe K, Hazama M, Katoh H, Yamamura K and Suzuki M (2004) Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus. Exp Anim 53:311–320. https://doi.org/10.1538/expanim.53.311 An XL, Zou JX, Wu RY, Yang Y, Tai FD, Zeng SY, Jia R, Zhang X, Liu EQ and Broders H (2011) Strain and sex differences in anxiety-like and social behaviors in C57BL/6J and BALB/cJ mice. 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EMBO Rep 1:239–243. https://doi.org/10.1093/embo-reports/kvd049 Okada S, Harada H, Ito T, Saito T and Suzu S (2008) Early development of human hematopoietic and acquired immune systems in new born NOD/Scid/Jak3(null) mice intrahepatic engrafted with cord blood-derived CD34 (+) cells. Int J Hematol 88:476–482. https://doi.org/10.1007/s12185-008-0215-z Okada S, Vaeteewoottacharn K and Kariya R (2019) Application of Highly Immunocompromised Mice for the Establishment of Patient-Derived Xenograft (PDX) Models. Cells 8:https://doi.org/10.3390/cells8080889 Ono A, Hattori S, Kariya R, Iwanaga S, Taura M, Harada H, Suzu S and Okada S (2011) Comparative study of human hematopoietic cell engraftment into BALB/c and C57BL/6 strain of rag-2/jak3 double-deficient mice. J Biomed Biotechnol 2011:539748. https://doi.org/10.1155/2011/539748 Panaampon J, Kariya R and Okada S (2022) Efficacy and mechanism of the anti-CD38 monoclonal antibody Daratumumab against primary effusion lymphoma. Cancer Immunol Immunother 71:1017–1031. https://doi.org/10.1007/s00262-021-03054-8 Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL and Handgretinger R (2005) Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174:6477–6489. https://doi.org/10.4049/jimmunol.174.10.6477 Takenaka K, Prasolava TK, Wang JC, Mortin-Toth SM, Khalouei S, Gan OI, Dick JE and Danska JS (2007) Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat Immunol 8:1313–1323. https://doi.org/ni1527 [pii] /ni1527 Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A and Manz MG (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304:104–107. https://doi.org/10.1126/science.1093933 Tsuchimine S, Matsuno H, O'Hashi K, Chiba S, Yoshimura A, Kunugi H and Sohya K (2020) Comparison of physiological and behavioral responses to chronic restraint stress between C57BL/6J and BALB/c mice. Biochem Biophys Res Commun https://doi.org/10.1016/j.bbrc.2020.02.073 Weber EM, Algers B, Wurbel H, Hultgren J and Olsson IA (2013) Influence of strain and parity on the risk of litter loss in laboratory mice. Reprod Domest Anim 48:292–296. https://doi.org/10.1111/j.1439-0531.2012.02147.x Additional Declarations Competing interest reported. Tatsuya Ogawa and Akira Shiota are employees of the Institute of Immunology, Co., Ltd. Jutatip Panaampon and Seiji Okada declared no conflicts of interest. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 Jan, 2026 Editor assigned by journal 06 Jan, 2026 Submission checks completed at journal 05 Jan, 2026 First submitted to journal 04 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8510586","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570255424,"identity":"ca3b47fc-5597-4264-a8e6-40b54f095402","order_by":0,"name":"Jutatip Panaampon","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Jutatip","middleName":"","lastName":"Panaampon","suffix":""},{"id":570255425,"identity":"7aeba16d-9de3-44a4-ae80-14139e6748ca","order_by":1,"name":"Tatsuya Ogawa","email":"","orcid":"","institution":"Institute of Immunology","correspondingAuthor":false,"prefix":"","firstName":"Tatsuya","middleName":"","lastName":"Ogawa","suffix":""},{"id":570255432,"identity":"34e230a4-7c41-4511-8b5e-cff221d3ae09","order_by":2,"name":"Akira Shiota","email":"","orcid":"","institution":"Institute of Immunology","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Shiota","suffix":""},{"id":570255435,"identity":"fd59610b-6f60-4e15-a8c2-28d164e13f52","order_by":3,"name":"Seiji Okada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYDACHgYGCQYGGwNMYQJa0qBaEojXchhdCx7Az3P44Y0ff84bG1w7/HTDxx82DAYHmB9+YJC5g1OLZG+bsWVv220zg9tpZjdnJKQBtbAZSzDwPMOpxeA8g5kEb8NtG4PbCWa3eRIO1284wGAGdO9hPFrYv0n++XMOqCX9G0gL0Bb2b/i1nO0xk+ZhOwB0WI4ZVAsPflske84UW8u2JRtL3s4puzkjLY1B8jBPsUQCHr/w86RvvPnmj51h3+30bTc+2Ngw8B1v3/jhYw/uEMMCmIE4secAKVrA4AfpWkbBKBgFo2DYAgC3R1ZZkb3CmwAAAABJRU5ErkJggg==","orcid":"","institution":"Kumamoto University","correspondingAuthor":true,"prefix":"","firstName":"Seiji","middleName":"","lastName":"Okada","suffix":""}],"badges":[],"createdAt":"2026-01-04 06:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8510586/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8510586/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100949666,"identity":"925dab91-e5a7-4e4c-9c3a-349c5a0fa572","added_by":"auto","created_at":"2026-01-23 07:04:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHuman SIRPa\u003c/strong\u003e \u003cstrong\u003etransgenic construction.\u003c/strong\u003e (A) Schematic diagram of the transgene construction. Structure of human SIRPa and BAC clones. Each BAC clone contains the full-length of coding sequence, and 5’- and 3’-flanking sequence. The coding region of mouse SIRPa gene (middle) was then replaced by the human SIRPa gene sequence (top) by four times of Red/ET recombination. (B) Purified SIRPa transgenic constructs. PFGE shows that the purified human SIRPa BAC transgenic construct is almost 200 kb in size. M indicates Low Range PFG Marker (New England Biolabs). (C) Southern blot analysis of SIRPa BAC transgenic lines. SIRPa BAC transgenic was detected as a 1.4 kb PstI fragment hybridizing with a human SIRPa probe. The copy number of an integrated transgene in each line was determined as compared with copy control signal intensity.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8510586/v1/b6abba741a5f0703cd84168c.png"},{"id":100951163,"identity":"04b50360-7375-41c8-a2a2-d6181695c8ca","added_by":"auto","created_at":"2026-01-23 07:10:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":54935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic expression of human SIRPa in BALB/c Rag-2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e Jak3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e (BRJ) immunodeficient mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)\u0026nbsp;\u0026nbsp; Splenocytes of BRJ and BRJ-S mice were isolated and the immunodeficiency condition was determined compared with wild-type BALB/c immunocompetent mice. (B) Cell lysate was obtained from bone marrow, spleen, and peritoneal macrophages of BRJ and BRJ-S mice. Western bloting was performed to detect hSIRPa. γ- tubulin was used as housekeeping protein. (C) and (D) Cells were isolated from bone marrow, spleen, and peritoneal macrophages of BRJ and BRJ-S mice. CD11b\u003csup\u003e+\u003c/sup\u003e cells were gated and hSIRPa was positive in BRJ-S, while mSIRPa was positive in wild-type BALB/c, BRJ, and BRJ-S mice.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8510586/v1/e8aac93288f69438e0868293.png"},{"id":100905420,"identity":"6ada5cb4-c7f9-4ffa-963d-847712cce343","added_by":"auto","created_at":"2026-01-22 15:44:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":85454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD11b\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells of BRJ-S \u0026nbsp;specifically bind to human CD47 and provides superior human tumor xenograft compared with BRJ \u003c/strong\u003e(A) CD11b\u003csup\u003e+\u003c/sup\u003e of BRJ-S binds to recombinant hCD47. CD11b\u003csup\u003e+\u003c/sup\u003e cells were incubated with recombinant hCD47-AF647, the binding was assessed by MFI by FACS. (B) Peritoneal macrophages from BRJ-S and BRJ mice were plated into a well. CFSE-labeled hematologic malignant cells (OCI-Ly3, and PELs (BCBL-1, GTO, and TY-1)) were added to the wells of macrophages at the indicated ratios and incubated for 2 hours. Phagocytic cells were defined as CD11b\u003csup\u003e+\u003c/sup\u003eCFSE\u003csup\u003e+\u003c/sup\u003e cells. Data are presented as the % phagocytosis (CD11b\u003csup\u003e+\u003c/sup\u003eCFSE\u003csup\u003e+\u003c/sup\u003e cells). The data are presented as mean values ± the standard error (SE). * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001, **** P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8510586/v1/e42150cdaafd1d505ed02d9f.png"},{"id":100950345,"identity":"20df20ec-ffc8-47b0-b32b-39590561e859","added_by":"auto","created_at":"2026-01-23 07:07:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158155,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBRJ-S is high susceptible and exhibits higher potential\u003c/strong\u003e \u003cstrong\u003efor the xenograft of human B cell malignancies. \u003c/strong\u003e(A-C) 1 × 10\u003csup\u003e7 \u003c/sup\u003eOCI-Ly3 cells were subcutaneously injected into BRJ-S and BRJ mice. (A) Tumor volumes were monitored. (B) The tumor size and morphology. (C) Tumor weights were measured at the end of the experiment. (D-H) 1 × 10\u003csup\u003e7 \u003c/sup\u003eGTO cells were xenografted into BRJ-s and BRJ mice. (D-F) GTO cells were subcutaneously injected into BRJ-S and BRJ mice. (D) Tumor volumes were monitored. (E) The tumor size and morphology. (F) Tumor weights were measured at the end of the experiment. (G and H) GTO cells were intraperitoneally injected into BRJ-s and BRJ mice. (G) Ascites volume was measured at day 35 after the tumor xenograft. (F) The percentage increase in weight was monitored. The data are presented as mean values ± standard deviation (SD). * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001, **** P\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8510586/v1/5f7388472efe2871ae9e82de.png"},{"id":100953047,"identity":"5ccc2c40-57a9-4287-b6a3-a1ce72d5eeed","added_by":"auto","created_at":"2026-01-23 07:19:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1284273,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8510586/v1/f4d4cd01-6b31-4b01-a11a-cf440390bcce.pdf"}],"financialInterests":"Competing interest reported. Tatsuya Ogawa and Akira Shiota are employees of the Institute of Immunology, Co., Ltd. Jutatip Panaampon and Seiji Okada declared no conflicts of interest.","formattedTitle":"Transgenic expression of human signal regulatory protein alpha (SIRPα) in BALB/c Rag2 null /Jak3 null immunodeficient mice improves human lymphoma xenograft via reduction of phagocytosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eXenotransplantation of human tumors into an immunodeficient mouse model is a well-established method for studying human tumor development. After the discovery of Nude mice, the xenograft model became more popular, and lymphocyte-depleted mice, such as SCID mice and Rag-1 or Rag-2 knockout mice, were introduced. However, depleting T and B lymphocytes alone is not sufficient for human cell xenografts. The introduction of NOD/Scid mice improved the success of human cell xenografts, and NK cell activity plays a key role in xenograft rejection. Therefore, mice lacking lymphocytes and NK cells on a NOD and BALB/c background, such as NOD/Scid commonγ\u003csup\u003enull\u003c/sup\u003e mice (NSG and NOG mice) (Ito et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Shultz et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), BALB/c Rag-1/2\u003csup\u003enull\u003c/sup\u003e commonγ\u003csup\u003enull\u003c/sup\u003e (BALB-RG) mice (Traggiai et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and BALB/c Rag-2\u003csup\u003enull\u003c/sup\u003e/Jak3\u003csup\u003enull\u003c/sup\u003e (BRJ) mice (Ono et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), have been used as standard models for human xenotransplantation. However, C57BL/6 background immunodeficient mice do not accept human cells (Okada et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ono et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSignal regulatory protein alpha (SIRPα) is one of the immune checkpoint proteins and is also known as cluster of differentiation 72 alpha (CD152a) or Src homology 2 (SH2) domain-containing phosphatase substrate-1 (Matozaki et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Its expression is primarily found on the surface of myeloid cells, including monocyte-macrophage lineages, granulocytes, dendritic cells, and hematopoietic stem cells. SIRPα interacts with CD47, a well-known overexpressed surface protein on malignant cells, to regulate immune responses, particularly phagocytosis (Barclay and Brown \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Matozaki et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). SIRPα is associated with poor prognosis in esophageal squamous cell carcinoma (ESCC), probably by inhibiting macrophage-mediated phagocytosis of tumor cells and suppressing antitumor immunity (Koga et al.). The interaction between SIRPα and CD47 on macrophages and target cells plays a crucial role in preventing phagocytosis, known as the \u0026ldquo;Don\u0026rsquo;t eat me signal\u0026rdquo; (Barclay and Brown \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Barclay and Van den Berg \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Human cells in mouse xenografts are effectively eliminated by mouse macrophages when there is an SIRPα-CD47 mismatch. Takenaka K et al. showed that macrophages from NOD- and BALB/c-background mice have a high affinity for human CD47, whereas those from C57BL/6-background mice do not (Takenaka et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This group also generated human SIRPα knock-in C57BL/6 Rag-2null commonγnull mice (Jinnouchi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and demonstrated that these mice can accept both normal and malignant human hematopoietic cells.\u003c/p\u003e \u003cp\u003eIn this study, we created BRJ mice expressing a human SIRPα transgene (BRJ-S) and examined their ability to support human lymphoma xenografts. We show that hSIRPα expression notably improves human tumor engraftment, mainly by decreasing macrophage phagocytosis. Our findings establish BRJ-S mice as a robust and versatile platform for preclinical cancer research and therapy development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures and media\u003c/h2\u003e \u003cp\u003ePrimary effusion lymphoma (PEL) cell lines BCBL-1 (obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD, USA), GTO (Goto et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) (established in our Labo and registered at JCRB cell bank, Osaka Japan: JCRB4057), and TY-1 (kindly provided by Dr. H. Katano, National Institute of Infectious Diseases, Tokyo, Japan) were used in this study. In addition, the human diffuse large B-cell lymphoma (DLBCL) cell line, OCI-Ly3, was included. All cell lines were maintained in RPMI-1640 medium (Fujifilm Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaill\u0026eacute;, France), penicillin G (100 IU/mL, Meiji Seica Pharm, Tokyo Japan), and streptomycin (100 U/mL, Fujifilm Wako). Cultures were incubated at 37\u0026deg;C under humidified conditions with 5% CO₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlowcytometry\u003c/h3\u003e\n\u003cp\u003eBALB/c and C57/BL6N mice were purchased from Japan SLC (Hamamatsu, Japan). Mouse bone marrow (BM), spleen, and peritoneal macrophages were stained with anti-human/mouse CD11b (M1/70)-BV421, anti-human CD172a/b (SIRPα/β)(SE5A5)-PE, anti-mouse CD172a (SIRPα)(P84)-APC, anti-mouse CD49b (DX5)-APC (pan-NK marker); anti-mouse CD122 (IL-2Rβ)-PE, anti-mouse CD19-FITC, and anti-mouse CD3-PE (BioLegend, San Diego, CA), and analyzed with FACS Celesta (BD Bioscience, San Jose, CA). The collected data were analyzed with FlowJo Version 10.4 software (Tree Star, San Jose, CA).\u003c/p\u003e\n\u003ch3\u003eBAC Transgenic Construct\u003c/h3\u003e\n\u003cp\u003eA mouse SIRP BAC clone, RP23-389N11, was identified from the RPCI-23 Female C57BL/6J murine BAC Library by searching the murine BAC end sequence database at the National Center for Biotechnology Information (NCBI). Analysis of the BAC end sequences indicates that this clone encompasses the complete 19.3-kb mouse SIRPα genomic region, along with upstream (5\u0026lsquo;) and downstream (3\u0026rsquo;) flanking genomic sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A human SIRP BAC clone (RP11-636L22) was identified from the RPCI-11 human BAC Library using BLAST search in the GenBank database at NCBI. Sequence analysis of this 172-kb BAC revealed that it contains the entire 45.9-kb human SIRPα genomic locus. To create a chimeric human-mouse SIRP BAC transgenic construct, the full-length coding exons and an intervening intron of the human SIRPα gene were inserted into the corresponding mouse SIRPα genomic locus via BAC recombineering. Transfer of the human SIRPα sequence from the human BAC clone to the mouse BAC clone was performed using the Red/ET Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany) (Muyrers et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Briefly, a rpsL-neo counterselection cassette flanked by homologous sequences matching an intron of the human SIRPα gene was amplified by PCR and integrated into the human BAC through Red/ET recombination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe human \u003cem\u003eSIRPα\u003c/em\u003e gene fragment was subsequently subcloned into the pDNR-1r plasmid (Takara Bio USA, Mountain View, CA) using Red/ET recombination, with the gene's terminal sequences serving as homology arms. This subcloned fragment was engineered to contain 40-nucleotide sequences derived from the orthologous mouse \u003cem\u003eSIRPα\u003c/em\u003e untranslated regions immediately adjacent to the start codon and the stop codon, respectively. The modified human \u003cem\u003eSIRPα\u003c/em\u003e fragment was then transferred to the mouse SIRPα gene. Finally, the remaining rpsL-neo cassette within the intron was removed thr\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eough\u003c/span\u003e rpsL counterselection. All BAC modifications were confirmed by sequencing.\u003c/p\u003e\n\u003ch3\u003ePreparation of BAC DNA for microinjection\u003c/h3\u003e\n\u003cp\u003eHuman-SIRP BAC transgenic constructs were purified for pronuclear microinjection using a modified protocol based on that of Abe et al. (Abe et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The BAC transgenic constructs were extracted from 250 ml of the E. coli culture with the Nucleobond Plasmid Purification kit (MACHEREY-NAGEL, D\u0026uuml;ren, Germany). Purified BAC transgenic DNA (10 \u0026micro;g) was linearized overnight with PI-SceI endonuclease (New England Biolabs, Ipswich, MA), which cleaves the unique site within the BACe3.6 vector backbone. The linearized BAC DNA was separated by pulsed-field gel electrophoresis (PFGE) and recovered from the gels through electroelution. After dialysis against TE buffer containing 0.1 mM EDTA, aliquots were reanalyzed by PFGE to verify DNA integrity and size. The DNA concentration was adjusted to 1 ng/\u0026micro;l for microinjection. The BAC DNA solution aliquots were stored at 4\u0026deg;C until use for microinjection.\u003c/p\u003e\n\u003ch3\u003eGeneration of Human SIRPα BAC Transgenic Mice\u003c/h3\u003e\n\u003cp\u003eHuman SIRP BAC transgenic mice were generated by injecting BALB/cA mouse embryos (Charles River Laboratory Japan Inc., Yokohama, Japan) into the pronucleus. The transgenic founders and germ-line transmission of the BAC transgenic construct were assessed using Southern blot analysis of PstI-digested tail DNA, probed with a [32P]-labeled human SIRP gene probe. Out of 197 offspring screened, four mice (2.0%) tested positive for the human SIRPα BAC transgene. No significant phenotypic abnormalities were noted when transgene-positive mice were compared to transgene-negative littermates. Multiple transgenic founder lines were subsequently bred with BALB/cA mice, and after several generations, mice exhibiting stable, high-level expression of human SIRPα were selected and maintained as founder lines.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and confirmation of Human SIRPα transgenic in BALB/c Rag2\u003csup\u003enull\u003c/sup\u003e/Jak3\u003csup\u003enull\u003c/sup\u003e Immunodeficient Mice (BRJ-S)\u003c/h2\u003e \u003cp\u003eBALB/c Rag2\u003csup\u003enull\u003c/sup\u003e/Jak3\u003csup\u003enull\u003c/sup\u003e SIRPα transgenic mice (BRJ-S mice) were generated by crossing BALB/c Rag2\u003csup\u003enull\u003c/sup\u003e/Jak3\u003csup\u003enull\u003c/sup\u003e (BRJ) mice with SIRPα transgenic mice. PCR confirmed the presence of the human SIRP transgene, Rag2\u003csup\u003enull\u003c/sup\u003e, and Jak3\u003csup\u003enull\u003c/sup\u003e. The PCR primers are as follows: human SIRPα: hSIRPaBIF6;5'-GAGGACAAGTTGGAAAAGATGGT, hSIRPaBIR6; 5'-GGAGGTCTCATTACTGCTTC (610bp), Rag-2 5\u0026rsquo;-CCAACGCTATGTCCTGATAGCGGT-3\u0026rsquo; RG2-2 5\u0026rsquo;-TTAATTCAACCAGGCTTCTCACTT-3\u0026rsquo;, RG2-3 5\u0026rsquo;-GCCTGCTTATTGTCTCCTGGTATG-3\u0026rsquo; (Wilde type allele: 973 bp, Mutant allele 1107 bp), Jak3; Oligo1:5\u0026rsquo;-CCAGACCAGCAGAGGGACTT-3\u0026rsquo;, Oligo2: 5\u0026rsquo;-GAACCT GCGTGCAATCCATCTTG-3\u0026rsquo;, Oligo3: 5\u0026rsquo;-ACCCAGGTACTCCATGCCCT\u0026ndash;3\u0026rsquo; (Wild type allele: 400 bp, Mutant allele: 700 bp).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of mouse macrophages\u003c/h3\u003e\n\u003cp\u003ePeritoneal macrophages were obtained from 10-week-old BRJ mice. To elicit macrophage recruitment, mice were injected intraperitoneally with 2 mL of 4% (w/v) thioglycolate broth (BD Diagnostic Systems, Sparks, MD, USA). Three days later, peritoneal cells were collected by peritoneal lavage with 10 mL of cold phosphate-buffered saline (PBS) (Goto et al.). Flow cytometric analysis confirmed that more than 95% of the recovered cells expressed the macrophage marker CD11b.\u003c/p\u003e\n\u003ch3\u003eCD47 protein binding assay\u003c/h3\u003e\n\u003cp\u003eRecombinant Human CD47-Fc Chimera and Recombinant Mouse CD47-Fc Chimera were purchased from BioLegend (San Diego, CA, USA), and conjugated with HiLyte Fluor\u0026trade; 647 by HiLyte Fluor\u0026trade; 647Labeling Kit - NH\u003csub\u003e2\u003c/sub\u003e (Dojindo, Kumamoto, Japan). Isolated mouse peritoneal macrophage FC receptors were blocked with Purified anti-mouse CD16/32 Antibody (Biolegend), and incubated with HiLyte Fluor\u0026trade; 647 conjugated Recombinant Human CD47-Fc Chimera or Recombinant mouse CD47-Fc Chimera, and analyzed with FACS Celesta.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhagocytosis assay\u003c/h2\u003e \u003cp\u003eMacrophage phagocytic capacity was assessed by flow cytometry based on a previously established protocol (Panaampon et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, macrophages were seeded into 96-well plates in 10% FBS/RPMI-1640. CFSE-labeled lymphoma cells were subsequently added at the indicated effector-to-target (E:T) ratios and co-cultured for 2 hours at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Following incubation, cells were harvested and stained with anti-CD11b-APC antibody. Samples were analyzed using a FACS Celesta flow cytometer. Phagocytosis was quantified as the proportion of CD11b⁺CFSE⁺ cells among total CD11b⁺ macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eBone marrow cells, splenocytes, peritoneal macrophages, and human peripheral mononuclear cells (PBMCs) were collected. Twenty \u0026micro;g protein was loaded onto 10% or 12% SDS-PAGE and subsequently transferred onto a PVDF membrane. The antibodies used were anti-γ-tubulin (C-20) and anti-human SIRP (SE7C2) (Santa Cruz Biotechnology, Inc.). The protein signal was detected using the ImageQuant Biomolecular Imager (GE Life Sciences, Uppsala, Sweden).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eXenograft of Lymphoma Cell Lines\u003c/h2\u003e \u003cp\u003eBRJ (Ono et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and BRJ-S mice were maintained at the Center for Animal Resources and Development (CARD), Kumamoto University, under specific pathogen-free conditions in accordance with institutional animal care guidelines. Animals had unrestricted access to food and water throughout the study. All experimental procedures were reviewed and approved by the Animal Ethics Committee of Kumamoto University (approval number A2019-041).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSubcutaneous xenograft;\u003c/em\u003e For subcutaneous tumor establishment, GTO or OCI-Ly3 cells (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells per mouse) were injected into both flanks of 10- to 12-week-old female BRJ and BRJ-S mice (n\u0026thinsp;=\u0026thinsp;6 per group). Tumor growth was monitored longitudinally, and mice were euthanized at the experimental endpoint for excision and measurement of tumor mass.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIntraperitoneal xenograft;\u003c/em\u003e For the intraperitoneal model, GTO cells (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells per mouse) were administered intraperitoneally into 10- to 12-week-old female BRJ and BRJ-S mice (n\u0026thinsp;=\u0026thinsp;5 per group). Body weight was recorded regularly as an indicator of disease progression. At day 35 following tumor inoculation, mice were sacrificed, and ascitic fluid was collected and quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) or standard deviation (SD), as specified in the figure legends. Statistical comparisons were performed using Student\u0026rsquo;s paired \u003cem\u003et\u003c/em\u003e-test with GraphPad Prism software (version 6; GraphPad Software, San Diego, CA, USA). A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGeneration of Human SIRPα BAC Transgenic Mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince SIRP-CD47 signaling is crucial for the rejection and acceptance of xenografts, we created transgenic mice that express human SIRP. The BAC transgenic method is a valuable tool for transgene expression because it depends on copy number and is independent of integration position (Heintz \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Masuda et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Moritoki et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We selected a mouse SIRPα BAC clone that includes over 100 kb of upstream regulatory sequences, the complete SIRPα structural gene, and downstream genomic regions for this purpose. Using mouse-specific genomic elements to maintain natural expression patterns, we precisely replaced the endogenous mouse SIRPα coding region within the BAC with the human SIRPα gene through BAC recombineering. This approach produced a chimeric human\u0026ndash;mouse BAC transgenic construct that drives strong expression of human SIRPα in myeloid-lineage cells. After recombineering in Escherichia coli, the modified BAC was linearized, purified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and injected into pronucleus-stage mouse embryos to create independent transgenic lines. All four founder mice successfully transmitted the human SIRPα BAC transgene to their F1 offspring. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, individual lines carried either one or three copies of the SIRPα BAC transgene. The 22\u0026thinsp;\u0026minus;\u0026thinsp;1 line, which contained three copies of the construct, was chosen for all subsequent experiments.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and Phenotypic expression of human SIRPα in BALB/c Rag-2\u003csup\u003enull\u003c/sup\u003e Jak3\u003csup\u003enull\u003c/sup\u003e (BRJ-S) immunodeficient mice\u003c/h2\u003e \u003cp\u003eThe human SIRPα BAC transgenic mice were crossed with BRJ mice for 10 generations and established human SIRPα-expressing Rag-2/Jak3 double knock-out mice (BRJ-S). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the wild-type BALB/c mouse has T and B lymphocytes and NK cells, whereas these cells are absent in BRJ and BRJ-S mice. We isolated cells from bone marrow, spleen and peritoneal macrophages to confirm human (h) SIRPα expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, hSIRPα was positive in BRJ-S mice by western blotting. Mouse (m) SIRPα was expressed on CD11b\u003csup\u003e+\u003c/sup\u003e cells of wild-type BALB/c, BRJ, and BRJ-S, whereas human SIRP was expressed on CD11b\u003csup\u003e+\u003c/sup\u003e cells of bone marrow, spleen, and peritoneal macrophages of only BRJ-S mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The CD11b\u0026thinsp;+\u0026thinsp;population co-expresses both hSIRPa and mSIRPα (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCD11b\u003csup\u003e+\u003c/sup\u003e of BRJ-S binds to recombinant hCD47\u003c/h2\u003e \u003cp\u003eTo test the binding of mouse CD11b\u003csup\u003e+\u003c/sup\u003e cells with recombinant human and mouse CD47-Fc protein, we incubated peritoneal macrophages with HiLyte Fluor\u0026trade;647-conjugated recombinant human or mouse CD47, and assessed binding by fluorescent intensity of FACS. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the binding between CD11b\u003csup\u003e+\u003c/sup\u003e-hCD47 is strongest in BRJ-S. In contrast, the binding of recombinant mouse CD47 to peritoneal macrophages BRJ-S, BRJ, and wild-type C57/BL6, showed no significant difference among the three types of mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003etumor cell phagocytosis is weaker in human SIRPα transgenic BALB/c Rag-2\u003c/b\u003e\u003csup\u003e\u003cb\u003enull\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eJak3\u003c/b\u003e\u003csup\u003e\u003cb\u003enull\u003c/b\u003e\u003c/sup\u003e \u003cb\u003e(BRJ-S) immunodeficient mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe SIRPα-CD47 interaction is well known as the \"Don\u0026rsquo;t eat me\u0026rdquo; signal. The binding of human SIRPα to mouse macrophages and of human CD47 to human tumor cells inhibits phagocytosis by mouse macrophages, suggesting that BRJ-S mice would have a higher capacity to accept human tumor xenografts than BRJ immunodeficient mice. To test this hypothesis, we xenografted multiple human lymphoma cells into BRJ-S and BRJ mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, macrophages isolated from BRJ-S showed a lower percentage of phagocytosis than that of BRJ for all four lymphoma cell lines; OCI-Ly3, BCBL-1, GTO, and TY-1. The results suggest that BRJ-S mice exhibit higher efficacy against human lymphoma xenografts than BRJ mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBRJ-S mice provide a higher capacity for human lymphoma xenograft than BRJ mice\u003c/h2\u003e \u003cp\u003eTo confirm the suitability of the BRJ-S xenograft model, we transplanted OCI-Ly3 and GTO cells into BRJ-S and BRJ mice, respectively. Furthermore, we monitored tumor growth in these two mouse strains. Tumor volume of OCI-Ly3 was measured, and tumor weight was determined at the end of the experiment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, OCI-Ly3 grew faster in BRJ-S mice compared with BRJ, resulting in a larger size, volume, and weight than in BRJ mice. In correlation with Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, GTO, a PEL cell line, which forms a tumor mass by subcutaneous (s.c.) and forms ascites by intraperitoneal (i.p.) transplantation, was transplanted into BRJ-S and BRJ mice. GTO cells showed more rapid growth in BRJ-S mice than in BRJ mice, as measured by tumor volume, tumor size, and tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Moreover, we intraperitoneally injected GTO into BRJ-S and BRJ mice to directly represent the clinical manifestation of PEL. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, GTO forms higher ascites volume in BRJ-S than BRJ. The percentage of weight increase in BRJ-S mice was also more than the weight increase in BRJ mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Taken together, the results demonstrate that BRJ-S is a superior mouse model for human lymphoma xenograft than BRJ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we generated human SIRPα-expressing BAC transgenic mice by the standard method (Mogi et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), then established BALB/c human SIRPα-expressing Rag-2/Jak3 double deficient (BRJ-S) mice, and showed BRJ-S mice had superior engraftment efficiency of lymphoma cells. Severe immunodeficient mice lacking B and T lymphocytes and NK cells have become widely used hosts for xenotransplanting human cells because they exhibit diminished rejection of both normal and malignant human cells. In addition, the genetic background of the mice has been observed to influence engraftment efficiency. In contrast, NOD and BALB/c background immunodeficient mice accept human cells, C57/BL6 background immunodeficient mice cannot support human cell engraftment. Recently, the CD47-SIRPα axis, also known as the \u0026ldquo;Don\u0026rsquo;t eat me\u0026rdquo; signal, has been shown to play an essential role in macrophage phagocytosis. Takenaga et al. showed that NOD SIRPα efficiently binds to human CD47 but not C57/BL6 SIRPα by polymorphism. This group established C57/BL6 Rag-2\u003csup\u003enull\u003c/sup\u003e/IL2R\u003csup\u003enull\u003c/sup\u003e human SIRPα knock-in (BRGS\u003csup\u003ehuman\u003c/sup\u003e) mice and showed efficient reconstitution of both human normal and malignant hematopoietic cells.\u003c/p\u003e \u003cp\u003eThe advent of NOD/Scid mice enabled the efficient creation of humanized mice; however, the mechanism by which human hematopoietic stem cells engraft easily in NOD/Scid mice remained unclear. The SIRPα immunoglobulin variable region is known to be highly polymorphic in mice. It has been shown that NOD SIRPα has a strong affinity for human CD47, while BALB/c SIRPα has a moderate affinity, and C57/BL6 SIRPα has no affinity. Currently, NOD/Scid background mice are further modified and have been used as recipients of humanized mice, such as NOD/Shi-SCID IL-2R\u003csup\u003enull\u003c/sup\u003e (NOG) mice, NOD/LtSz-SCID IL-2R\u003csup\u003enull\u003c/sup\u003e (NSG) mice, and NOD/Scid Jak3\u003csup\u003enull\u003c/sup\u003e (NOJ) mice (Ito et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Okada et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Okada et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shultz et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, these NOD/Scid mice have several limitations as recipients for humanized mice and patient-derived xenografts; they are difficult to breed and exhibit poor recovery from DNA damage induced by irradiation and cytotoxic drug treatment. In this context, human SIRPα transgenic mice and BALB/c or C57BL/6 knock-in mice are expected to be easier to handle. In particular, the BALB/c strain is gentle by nature, making it more manageable in experiments than the C57/BL6 strain. Previously, we established BALB/c Rag-2null/Jak3null (BRJ) mice, which demonstrated appropriate homing efficiency of both hematopoietic stem cells and peripheral blood mononuclear cells. We also showed that the NOD genetic background most effectively induces lymphomatous effusion when transplanted into the peritoneal cavity. In this study, we further demonstrated that human SIRPα transgenic BRJ mice had improved transplantation efficiency.\u003c/p\u003e \u003cp\u003eThe CD47-SIRPα signal is essential for maintaining homeostasis in normal hematopoiesis. Additionally, tumor cells are known to upregulate CD47 expression to inhibit macrophage phagocytosis through the \u0026ldquo;Don\u0026rsquo;t eat me\u0026rdquo; signal mediated by the CD47-SIRPα interaction. This mechanism helps tumor cells evade the immune system, and blocking the CD47-SIRPα signal with anti-CD47 or anti-SIRPα antibodies allows macrophages to recognize and destroy tumor cells. We have previously shown that primary effusion lymphoma and cholangiocarcinoma highly express CD47 on their surfaces, and anti-CD47 antibody treatment suppressed tumor growth in xenograft mouse models. Currently, the anti-CD47 antibody is in clinical trials for hematological malignancies and solid tumors. Therefore, the CD47-SIRPα signal plays a crucial role in supporting tumor cell survival, and human SIRPα transgenic immunodeficient mice are valuable for establishing patient-derived xenograft models.\u003c/p\u003e \u003cp\u003eHuman SIRPα knock-in mice were developed on a C57BL/6 background. Although transplantation efficiencies may be similar across NOG, NSG, and NOJ mice, C57BL/6 mice are generally known for their high aggression levels, which makes them more difficult to handle than less aggressive BALB/c mice (An et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Brinks et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Tsuchimine et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The behavioral traits of NOD-derived strains (including NOD/Scid mice and NOG/NSG/NOJ mice) have not been fully characterized; however, our experience suggests they display greater aggression compared to BALB/c mice. Additionally, the breeding efficiency of the C57BL/6 strain is usually lower than that of BALB/c mice (Tsuchimine et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Weber et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and the reproductive success of NOD/Scid background mice is limited; therefore, efficient production often requires advanced breeding techniques (Goto et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kumagai et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This evidence suggests that BALB/c background immunodeficient mice, such as BRJ-S mice, are relatively easier to handle in experimental settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we successfully established BRJ-S, BALB/c human SIRPα-transgenic Rag2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003eJak3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e (BRJ-S) mice, and showed that BRJ-S mice can accept human lymphoma cells more than BRJ mice. Thus, BRJ-S mice offer a promising immunodeficient mouse model for cancer research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. S. Fujikawa for technical assistance and Ms. M. Teramoto for secretarial assistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Strategic Core Technology Advancement Program from the Ministry of Economy, Trade, and Industry, Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Conceptualization, A.S. and S.O.; methodology, T.O. and J.P.; validation, J.P. and S.O., formal analysis, J.P. and T.O. ; investigation, J.P. and T.O. ; resources, A.S. and S.O.; data curation, J.P.; writing original draft preparation, J.P.; writing review and editing, S.O.; visualization, J.P. and T.O.; supervision, A.S. and S.O.; project administration, A.S. and S.O.; funding acquisition, S.O. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical consideration:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All experimental procedures and protocols were conducted in accordance with the ARRIVE guidelines and approved by the Kumamoto University Committee of Animal Care (A2021-053, A2023-153).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTatsuya Ogawa and Akira Shiota are employees of the Institute of Immunology, Co., Ltd. Jutatip Panaampon and Seiji Okada declared no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbe K, Hazama M, Katoh H, Yamamura K and Suzuki M (2004) Establishment of an efficient BAC transgenesis protocol and its application to functional characterization of the mouse Brachyury locus. Exp Anim 53:311\u0026ndash;320. https://doi.org/10.1538/expanim.53.311\u003c/li\u003e\n \u003cli\u003eAn XL, Zou JX, Wu RY, Yang Y, Tai FD, Zeng SY, Jia R, Zhang X, Liu EQ and Broders H (2011) Strain and sex differences in anxiety-like and social behaviors in C57BL/6J and BALB/cJ mice. Exp Anim 60:111\u0026ndash;123. https://doi.org/10.1538/expanim.60.111\u003c/li\u003e\n \u003cli\u003eBarclay AN and Brown MH (2006) The SIRP family of receptors and immune regulation. 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We previously developed BALB/c Rag2nullJak3null (BRJ), an immunodeficient mouse model valuable for cancer research. However, susceptibility to xenograft acceptance can be enhanced by the signal regulatory protein alpha (SIRP)-CD47 “don‘t eat me” signal. In this study, we created BALB/c human-SIRP BAC transgenic mice and crossed them with BRJ mice. The resulting human SIRP transgenic BRJ (BRJ-S) mice showed significantly increased engraftment of human B-cell lymphoma compared to BRJ mice. Additionally, we demonstrated improved human tumor engraftment in BRJ-S mice by reducing phagocytosis. In summary, hSIRP-transgenic BRJ mice (BRJ-S) serve as a promising immunodeficient model with a greater capacity for human lymphoma engraftment. BRJ-S provides an advantageous and permissive model for xenografting and for studying cancer in research and drug development.\u003c/p\u003e","manuscriptTitle":"Transgenic expression of human signal regulatory protein alpha (SIRPα) in BALB/c Rag2 null /Jak3 null immunodeficient mice improves human lymphoma xenograft via reduction of phagocytosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 15:44:12","doi":"10.21203/rs.3.rs-8510586/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T14:01:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-06T13:45:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T14:38:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Transgenic Research","date":"2026-01-04T05:56:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"transgenic-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trag","sideBox":"Learn more about [Transgenic Research](http://link.springer.com/journal/11248)","snPcode":"11248","submissionUrl":"https://submission.nature.com/new-submission/11248/3","title":"Transgenic Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0758ae77-a25f-44f1-a1c1-0771789fd185","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T08:10:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 15:44:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8510586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8510586","identity":"rs-8510586","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}