Regeneration of humoral immunity from pluripotent stem cells by defined transcription factors

Regeneration of humoral immunity from pluripotent stem cells (PSCs) is a crucial aim in translational medicine. However, reconstitution of complete, sustained, and functional B lymphopoiesis from PSCs has not yet been developed. Here, we successfully achieved regenerative B lymphopoiesis in B-cell deficient animals transplanted with PSC-derived hematopoietic progenitors (iHPCs) guided by synergistic expression of Runx1, Hoxa9, and Lhx2. Upon transplantation, the iHPCs immediately gave rise to pro/pre-B cells in recipients' bone marrow, which were able to further differentiate into the entire B cell lineages, including innate B-1a, B-1b, MZ B cells, as well as adaptive FO B cells. In responding to antigen stimuli, the regenerative B cells produced adaptive humoral immune responses, sustained a prolonged antigen-specific antibody production, and formed immune-memory. Particularly, the regenerative B cells in spleen showed developing patterns of immunoglobulin chain-switch and hyper-mutation via a cross-talk with the host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study provides de novo evidence that B lymphopoiesis can be regenerated from PSCs via a HSC-independent approach, which provides insights into treating B-cell related humoral deficiencies using PSCs as unlimited cell resource.

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Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors

Cellular and Molecular Immunology ◽

10.1038/s41423-021-00805-6 ◽

2021 ◽

Author(s):

Qi Zhang ◽

Bingyan Wu ◽

Qitong Weng ◽

Fangxiao Hu ◽

Yunqing Lin ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

B Cell ◽

Pluripotent Stem Cells ◽

De Novo ◽

Immune Memory ◽

B Lymphopoiesis ◽

Marginal Zone B Cells ◽

Humoral Immune

AbstractRegeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

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Conditional Knockout of Sox4 Markedly Affects B Lymphopoiesis in Adult Mice.

Blood ◽

10.1182/blood.v114.22.2524.2524 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2524-2524

Author(s):

Baohua Sun ◽

Saradhi Mallampati ◽

Yun Gong ◽

Donghai Wang ◽

M. James You ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Conditional Knockout ◽

B Lymphopoiesis ◽

Cell Stage ◽

Hematopoietic Stem ◽

Adult Mice

Abstract Abstract 2524 Poster Board II-501 B cells constitute an integral part of the immune system. The development of mature B cells from hematopoietic stem cells is a complex process that is regulated in a hierarchical order by various proteins, particularly transcription factors. Sox4 is a SRY-related HMG box containing transcription factor and is known to be involved in B cell development. However, the role of Sox4 in various stages of B cell development has not been systematically investigated. In this study we used a conditional knockout mouse strain and studied the effect of Sox4 deletion in B lymphopoiesis in adult mice. We crossed the Sox4-floxed mice with different Cre mouse strains that were expected to delete the floxed Sox4 gene at different B cell developmental stages. These Cre strains included Vav-iCre (expressed in hematopoietic stem cell stage, starting from early embryos), MX1-Cre (expression in hematopoietic stem cells, induced by pIpC injection in adults), MB1-Cre (expressed in B cells, starting from early progenitor cells in embryos), and CD21-Cre (expressed in mature B cells). We demonstrated that deletion of Sox4 caused an arrest of B lymphopoiesis at the transition from pre-pro-B cell (fraction A) stage to pro-B cell stage (fraction B): fraction A cells are slightly reduced in number whereas fraction B and later stage cells are nearly absent. The pre-pro-B cells from the Sox4 knockout mice retain a population of AA4.1+ cells, which are considered to be developed into B cells. Deletion of Sox4 in early embryonic stage (Vav-iCre) or in adults (Mx1-Cre) results in a similar phenotype on B lymphopoiesis, except that peritoneal B1 cells appear to be affected with Vav-iCre, but not with Mx1-Cre. MB1-Cre gave rise to similar results as did Vav-iCre, but the arrest was not as dramatic as with Vav-iCre. CD21-Cre produced no significant difference in B cell phenotype. These data suggested that Sox4 is required for early B cell development at the transition from pre-pro-B cells to pro-B cells and is not required for mature B cells. We are currently investigating the transcription program of this transcription factor in B cell development. Disclosures: No relevant conflicts of interest to declare.

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Attempt to Prove the Existence of Abnormal B Lymphocyte As Myeloma-Initiating Cells from B Cell-Derived Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-112759 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1896-1896

Author(s):

Aki Yanagi ◽

Naohiro Tsuyama ◽

Yukari Yanai ◽

Yu Abe ◽

Misaki Sugai ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

B Cell ◽

Stromal Cells ◽

Pluripotent Stem Cells ◽

Research Funding ◽

Chromosomal Translocation ◽

Company Limited ◽

Cd34 Cells ◽

Induced Pluripotent

Abstract We previously established normal B cell-derived induced pluripotent stem cells (BiPSCs; BiPSC13 and MIB2-6). BiPSCs are known to maintain VDJ rearrangement of the IgH gene, and they can be induced by the tet-off system to express activation-induced cytidine deaminase (AID; BiPSC13-AID and MIB2-6-AID) and differentiate into hematopoietic progenitor cells (HPCs) (Scientific Rep, 2017). Using these BiPSCs, we attempted to prove the existence of abnormal B cells, which are thought to be myeloma-initiating cells, originating from mature B cells transformed by reprogramming. We speculated that BiPSCs could develop into myeloma-initiating cells that undergo chromosomal translocation or gain genetic abnormalities during redifferentiation into mature B cells. First, using the comet assay, we confirmed the DNA-damaging effect of AID in BiPSCs-AID. Secondly, we differentiated BiPSC13-AID into CD34+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (mouse embryo cell line: 10T1/2), and we subsequently transplanted the cells into the bone marrow (BM) of immunodeficient NRG mice. The presence of CD34+ cells was still observed in mouse BM 4 months after transplantation; however, no differentiation into B cells was detected. Next, using the CRISPR/Cas9 system, we attempted to make BiPSCs with chromosomal translocation t(11;14); we succeeded in establishing a 293T cell line with t(11;14), then confirmed t(11;14) in MIB2-6-AID, and the clone is now being established. Furthermore, we established BiPSC13-Pax5, which can be induced by the tet-off system to express Pax5, and we then differentiated BiPSC13-Pax5 into CD34+/Pax5+/CD38-/CD43-/CD45- cells by co-culture with stromal cells (10T1/2). We expect that the HPCs or hematopoietic stem cells (HSCs) derived from BiPSCs will further differentiate into B cells due to the expression of Pax5 in the BM of NRG mouse. We also established BiPSC13-AID-p53-/-, in which p53 was deleted using the CRISPR/Cas9 system, and the cells differentiated into HPCs. Interestingly, we detected some CD43+/CD45+ cells among CD34+/CD38- cells after the co-culture of BiPSC13-AID with aorta-gonad-mesonephros-derived stromal cell (AGM-S3) instead of 10T1/2. Therefore, AGM-S3 may promote the differentiation of BiPSCs into cells that are more similar to HSCs. These CD34+ cells differentiated from BiPSCs will be transplanted into the BM of NRG mouse. Disclosures Hanamura: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Fujimoto Pharmaceutical Corporation: Research Funding; Takeda Pharmaceutical Company Limited.: Other: Lecture fee; Bristol-Myers Squibb: Other: Lecture fee, Research Funding; Celgene: Other: Lecture fee.

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Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells

Blood ◽

10.1182/blood-2006-08-041384 ◽

2007 ◽

Vol 109 (9) ◽

pp. 3706-3712 ◽

Cited By ~ 248

Author(s):

Jiang Zhu ◽

Russell Garrett ◽

Younghun Jung ◽

Yi Zhang ◽

Nacksung Kim ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Developmental Process ◽

Bursa Of Fabricius ◽

B Lymphopoiesis ◽

Hematopoietic Stem ◽

Cell Commitment

Abstract Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.

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Homologous and Variant-Specific Memory B-Cell and Antibody Responses after SARS-CoV-2 mRNA Vaccination

10.1101/2021.07.12.21260386 ◽

2021 ◽

Author(s):

Iana H. Haralambieva ◽

Jonathon M. Monroe ◽

Diane E. Grill ◽

Inna G Ovsyannikova ◽

Gregory A. Poland ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Humoral Immunity ◽

Vaccine Dose ◽

Neutralizing Antibody ◽

Memory B Cells ◽

Immune Memory ◽

Antibody Activity ◽

Convenience Sample ◽

Memory B Cell

Abstract. Importance. A better understanding of the immune memory and functional humoral immunity directed at the emerging Variants of Concern (VoC) strains after SARS-CoV-2 vaccination is essential for predicting the longevity of heterotypic protection. Objective. The aim of our study was to characterize functional humoral immunity (including memory B cell response) after COVID-19 mRNA vaccination and to determine/compare the reactivity of COVID-19 vaccine-induced memory B cells to the emerging SARS-CoV-2 Variants of Concern (VoC). Design, setting, participants and interventions. We designed an exploratory longitudinal observational (convenience sample-based) study at Mayo Clinic, Rochester, MN that enrolled and followed naive subjects and recovered COVID-19 subjects from Olmsted County, MN and surrounding areas after COVID-19 vaccination in January-June 2021. The study enrolled 17 relatively healthy subjects, 59% females and 94% White/Non-Hispanic or Latino with median age at enrollment 41 years. The subjects received either the BNT162b2 (Pfizer/BioNtech) or mRNA-1273 (Moderna) vaccine (n=3) and provided a blood sample at baseline, at;3 weeks after their first vaccine dose/before the second dose, and at;2 weeks after the receipt of their second vaccine dose. Main outcomes and measures. Spike-specific humoral and memory B cells responses were assessed over time after vaccination against the original Wuhan-Hu-1/vaccine and against emerging VoC strains/antigens. Results. We observed a robust neutralizing antibody response after COVID-19 mRNA vaccination, but a reduction in the functional antibody activity to several of the emerging SARS-CoV-2 VoC. Consistent with this, we also found differences in the number of isotype-switched/IgG+ MBCs responding to homologous and variant receptor-binding domain/RBDs after vaccination. We found a reduction of MBCs reactive to RBDs of Beta, Gamma and Delta SARS-CoV-2 VoC strains. Conclusion and relevance. In this exploratory study in subjects following receipt of COVID-19 mRNA vaccine, we found differences in antibody titers observed for VoCs after vaccination that are accompanied with, and can partially be explained by, decreased MBC reactivity against the VoCs. This can further attenuate the generated recall humoral immune response upon exposure to these variants.

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PBSCT Similar to BMT Allows Active B-Lymphopoiesis Unless Severe GVHD Occurs.

Blood ◽

10.1182/blood.v106.11.5351.5351 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5351-5351

Author(s):

Julia Winkler ◽

Armin Biller ◽

Natalie Schub ◽

Wolf Rösler ◽

Kerstin Eckart-Schäfer ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

B Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

First Year ◽

B Lymphopoiesis ◽

Hematopoietic Stem ◽

Precursor B Cells ◽

B Cell Precursors

Abstract Long-term engraftment after stem cell transplantation is dependent on pluripotent hematopoietic stem cells capable to multi-lineage reconstitution. The engraftment and prolonged repopulation of B lymphoid progenitor cells is dependent on pluripotent hematopoietic stem cells. We have recently shown that a high number of B-cell progenitors is detectable in the bone marrow from patients both after allogeneic PBSCT and after allogeneic BMT. The percentage of CD19/CD10+ pro-B/pre-B cells showed a high variability and ranged from 0 to 98% of all B-lymphocytes. Interestingly, no difference in numbers of precursor B-lymphocytes between BM recipients and PBSC recipients was found. In micro-satellite analysis, both in PBSC and in BM recipients the B cell precursors were derived exclusively from the donor. To identify predictors of B lymphopoiesis in the bone marrow after stem cells transplantation, a stepwise, multiple regression analysis was performed. The stem cell source, BM or PBSC, the occurrence of GVHD, the conditioning with or without TBI and whether ATG was given, were entered as categorical variables. Time point of BM sampling after transplantation, age of the donor and the recipient, the quantity of stem cell transplanted and the T cell content of the BM sample served as continuous variables. Time elapsed after transplantation (p=0.002) and severe GVHD (aGVHD grade III/IV or extensive cGVHD) (p=0.036) could be identified as parameters with an independent influence on the percentage of B cell precursors. The highest percentage of precursor B cells was found during the first year after transplantation, unless severe GvHD was present, leading to neglible precursor B-cells. This hyperactive B lymphopoiesis in the first year after transplantation contrasts to low circulating B cell counts. Finally, it could be formally demonstrated, that G-CSF mobilized PBSC contain hematopoietic stem cells capable of long-term reconstitution of the B cell compartment.

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Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production

10.1101/2019.12.20.885343 ◽

2019 ◽

Author(s):

Jeremy F. Brooks ◽

Janet M. Davies ◽

James W. Wells ◽

Raymond J. Steptoe

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Antibody Production ◽

Specific Antibody ◽

De Novo ◽

Antigen Expression ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Specific Antibody Production

SummaryPathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund’s adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient’s pre-existing B cell repertoire as well as the repertoire that arose after bone marrow transfer. OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal centre formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

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Methamphetamine Compromises the Adaptive B Cell-Mediated Immunity to Antigenic Challenge in C57BL/6 Mice

Frontiers in Toxicology ◽

10.3389/ftox.2021.629451 ◽

2021 ◽

Vol 3 ◽

Author(s):

Anum N. Mitha ◽

Daniela Chow ◽

Valerie Vaval ◽

Paulina Guerrero ◽

Dormarie E. Rivera-Rodriguez ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Adaptive Immunity ◽

Humoral Immunity ◽

Cell Mediated Immunity ◽

Limited Information ◽

Humoral Immune ◽

Humoral Immune Responses ◽

The Impact

Methamphetamine (METH) is a substance of abuse that causes dysregulation of the innate and adaptive immunity in users. B cells are involved in the humoral component of the adaptive immunity by producing and secreting antibodies (Abs). METH modifies Ab production, although limited information on the impact of this psychostimulant on antigen (Ag)-specific humoral immune responses is available. Since T cell-dependent and T cell-independent Ags are involved in the activation of B lymphocytes, we explored the role of METH on humoral immunity to ovalbumin (OVA; T cell-dependent) and bacterial lipopolysaccharide (LPS; T cell-independent) in C57BL/6 mice. We demonstrated that METH extends the infiltration of B cells into pulmonary and splenic tissues 7 days post-Ag challenge. METH impairs Ab responses in the blood of animals challenged with OVA and LPS. Furthermore, METH diminishes the expression and distribution of IgM on B cell surface, suggesting a possible detrimental impact on users' humoral immunity to infection or autoimmunity.

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