Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow.

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

Zfp318 regulates IgD expression by abrogating transcription termination within the Ighm/Ighd locus.

The protein Zfp318 is expressed during the transition of naive B cells from an immature to mature state. To evaluate its role in mature B cell functions, a conditional gene deficiency in Zfp318 was created and deleted in bone marrow lineages via Vav-Cre. B cell development was minimally altered in the absence of the protein, although transitional 2 (T2) B cell populations were depressed in the absence of Zfp318. Intriguingly, the analysis of IgM and IgD expression by maturing and mature naive B cells demonstrated an elevated level of IgM gene products and a virtual loss of IgD products. Transcriptome analysis of Zfp318-deficient B cells revealed that only two gene products showed altered expression in the absence of Zfp318 (Ighd and Sva), demonstrating a remarkable specificity of Zfp318 action. In the absence of Zfp318, Ighm/Ighd transcripts, which would normally encode IgM and IgD from heterogeneous nuclear RNA transcripts via alternative splicing, lack intron and exon sequences from the IgD (Ighd)-encoding region. This finding indicates that Zfp318, in a novel manner, functions by repressing recognition of the transcriptional termination site at the 3' end of the terminal IgM-encoding exon, allowing for synthesis of the complete Ighm/Ighd heterogeneous nuclear RNA.

Fatal autoimmunity results from the conditional deletion of Snai2 and Snai3.

Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as 3 weeks of age and die by 30 days after birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. Additionally, transplantation of Snai2/Snai3 double deficient bone marrow into Snai2 sufficient Rag2(-/-) recipients resulted in autoantibody generation. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance.

Bone marrow-induced Mef2c deficiency delays B-cell development and alters the expression of key B-cell regulatory proteins.

The Mef2 family transcriptional regulator Mef2c (myocyte enhancer factor 2c) is highly expressed in maturing bone marrow and peripheral mature B-cells. To evaluate the role of this transcription factor in B-cell development, we generated a B-cell-specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B-cell numbers in bone marrow and spleen. This phenotype was evident in all B-cell subsets; however, as the animals mature, the deficit in the peripheral mature B-cell compartments was overcome. The absence of Mef2c in mature B-cells led to unique CD23+ and CD23- subsets that were evident in Mef2c knockout primary samples as well as Mef2c-deficient cultured, differentiated B-cells. Genome-wide expression analysis of immature and mature B-cells lacking Mef2c indicated altered expression for a number of key regulatory proteins for B-cell function including Ciita, CD23, Cr1/Cr2 and Tnfsf4. Chromatin immunoprecipitation analysis confirmed Mef2c binding to the promoters of these genes indicating a direct link between the presence (or absence) of Mef2c and altered transcriptional control in mature B-cells.

Jchain-DTR Mice Allow for Diphtheria Toxin-Mediated Depletion of Antibody-Secreting Cells and Evaluation of Their Differentiation Kinetics.

Antibody-secreting cells (ASCs) are generated following B cell activation and constitutively secrete antibodies. As such, ASCs are key mediators of humoral immunity whether it be in the context of pathogen exposure, vaccination or even homeostatic clearance of cellular debris. Therefore, understanding basic tenants of ASC biology such as their differentiation kinetics following B cell stimulation is of importance. Towards that aim, we developed a mouse model which expresses simian HBEGF (a.k.a., diphtheria toxin receptor (DTR)) under the control of the endogenous Jchain locus (or J-DTR). ASCs from these mice expressed high levels of cell surface DTR and were acutely depleted following diphtheria toxin treatment. Furthermore, proof-of-principle experiments demonstrated the ability to use these mice to track ASC reconstitution following depletion in 3 distinct organs. Overall, J-DTR mice provide a new and highly effective genetic tool allowing for the study of ASC biology in a wide range of potential applications.

Retro-orbital CD45 antibody labeling to evaluate antibody-secreting cell trafficking in mice.

Antibody-secreting cells (ASCs) are critical regulators of the humoral immune response. However, differences between tissue resident populations versus those that have recently migrated to their final anatomic destination are poorly understood. Here, we present a protocol for using retro-orbital (r.o.) CD45 antibody labeling to identify tissue resident versus recently immigrated ASCs in mice. We describe steps for r.o. injection of antibodies, animal euthanasia, and tissue harvesting. We then detail tissue processing, cell counting, and cell staining for flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Pioli et al. (2023).1.

Thymus antibody-secreting cells possess an interferon gene signature and are preferentially expanded in young female mice.

Antibody-secreting cells (ASCs) are key contributors to humoral immunity through immunoglobulin production and the potential to be long-lived. ASC persistence has been recognized in the autoimmune thymus (THY); however, only recently has this population been appreciated in healthy THY tissue. We showed that the young female THY was skewed toward higher production of ASCs relative to males. However, these differences disappeared with age. In both sexes, THY ASCs included Ki-67+ plasmablasts which required CD154(CD40L) signals for their propagation. Single cell RNA-sequencing revealed that THY ASCs were enriched for an interferon responsive transcriptional signature relative to those from bone marrow and spleen. Flow cytometry confirmed that THY ASCs had increased levels of Toll-like receptor 7 as well as CD69 and major histocompatibility complex class II. Overall, we identified fundamental aspects of THY ASC biology which may be leveraged for future in depth studies of this population in both health and disease.

Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions.

Background: Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that ASCs regulate multiple aspects of biology through the secretion of various cytokines. In this regard, ICFC is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Methods: Paraformaldehyde plus saponin or the eBioscience Foxp3/Transcription Factor Staining Buffer Set were used to evaluate the non-specific intracellular retention of phycoerythrin-containing antibody conjugates by ASCs. Results: We showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of ASC intracellular protein expression compared with other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within ASCs. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between ASCs and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen ASCs expressed toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1ß. Conclusion: Increasing the number of centrifugation steps performed on ASCs post-fixation leads to inappropriate retention of phycoerythrin-containing antibody conjugates during ICFC.

Do haematopoietic stem cells age?

Genetic defects that accumulate in haematopoietic stem cells (HSCs) are thought to be responsible for age-related changes in haematopoiesis that include a decline in lymphopoiesis and skewing towards the myeloid lineage. This HSC-centric view is based largely on studies showing that HSCs from aged mice exhibit these lineage biases following transplantation into irradiated young recipient mice. In this Opinion article, we make the case that the reliance on this approach has led to inaccurate conclusions regarding the effects of ageing on blood-forming stem cells; we suggest instead that changes in the environment contribute to haematopoietic system ageing. We propose that a complete understanding of how ageing affects haematopoiesis depends on the analysis of blood cell production in unperturbed mice. We describe how this can be achieved using in situ fate mapping. This approach indicates that changes in downstream progenitors, in addition to any HSC defects, may explain the reduced lymphopoiesis and sustained myelopoiesis that occur during ageing.

Plasma Cells, the Next Generation: Beyond Antibody Secretion.

Plasma cells (PCs) represent the terminal differentiation step of mature B lymphocytes. These cells are most recognizable for their extended lifespan as well as their ability to secrete large amounts of antibodies (Abs) thus positioning this cell type as a key component of humoral immunity. However, it is now appreciated that PCs can have far reaching effects on pathologic as well as non-pathologic processes independent of Ab secretion. This is highlighted by recent studies showing that PCs function as key regulators of processes such as hematopoiesis as well as neuro-inflammation. In part, PCs accomplish this by integrating extrinsic signals from their environment which dictate their downstream functionality. Here we summarize the current understanding of PC biology focusing on their ever-growing functional repertoire independent of Ab production. Furthermore, we discuss potential applications of PC immunotherapy and its implementation for translational benefit.

Lymphoid-Biased Hematopoietic Stem Cells Are Maintained with Age and Efficiently Generate Lymphoid Progeny.

Current models propose that reductions in the number of lymphoid-biased hematopoietic stem cells (Ly-HSCs) underlie age-related declines in lymphopoiesis. We show that Ly-HSCs do not decline in number with age. Old Ly-HSCs exhibit changes in gene expression and a myeloid-biased genetic profile, but we demonstrate that they retain normal lymphoid potential when removed from the old in vivo environment. Additional studies showing that interleukin-1 inhibits Ly-HSC lymphoid potential provide support for the hypothesis that increased production of inflammatory cytokines during aging underlies declines in lymphocyte production. These results indicate that current models proposing that lymphopoiesis declines with age due to loss of Ly-HSCs require revision and provide an additional perspective on why lymphocyte development in the elderly is attenuated.

Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs.

T lymphocytes are essential contributors to the adaptive immune system and consist of multiple lineages that serve various effector and regulatory roles. As such, precise control of gene expression is essential to the proper development and function of these cells. Previously, we identified Snai2 and Snai3 as being essential regulators of immune tolerance partly due to the impaired function of CD4(+) regulatory T cells in Snai2/3 conditional double knockout mice. Here we extend those previous findings using a bone marrow transplantation model to provide an environmentally unbiased view of the molecular changes imparted onto various T lymphocyte populations once Snai2 and Snai3 are deleted. The data presented here demonstrate that Snai2 and Snai3 transcriptionally regulate the cellular fitness and functionality of not only CD4(+) regulatory T cells but effector CD8(α+) and CD4(+) conventional T cells as well. This is achieved through the modulation of gene sets unique to each cell type and includes transcriptional targets relevant to the survival and function of each T cell lineage. As such, Snai2 and Snai3 are essential regulators of T cell immunobiology.

MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors.

Snail transcription factors in hematopoietic cell development: a model of functional redundancy.

Coordinated gene expression is crucial in facilitating proper lymphoid cell development and function. The precise patterns of gene expression during B- and T-cell development are regulated through a complex interplay between a multitude of transcriptional regulators, both activators and repressors. We have recently identified the Snail family of transcription factors as playing significant and overlapping roles in lymphoid cell development, in that deletion of both SNAI2 and SNAI3 was required to fully impact the generation of mature T and B cells. Analyses using compound heterozygote animals further demonstrated that SNAI2 and SNAI3 were partially haplosufficient and relatively equivalent in their ability to preserve B-cell generation in the bone marrow. In this review, we summarize studies elucidating the role of the Snail family in hematopoiesis, with a focus on lymphoid cell development. Using the Snail family as an example, we discuss the concepts of functional redundancy and strategies employed to assay transcription factor families for intramember compensation.

Deletion of Snai2 and Snai3 results in impaired physical development compounded by lymphocyte deficiency.

The Snail family of transcriptional regulators consists of three highly conserved members. These proteins regulate (repress) transcription via the recruitment of histone deacetylases to target gene promoters that possess the appropriate E-box binding sequences. Murine Snai1 is required for mouse development while Snai2 deficient animals survive with some anomalies. Less is known about the third member of the family, Snai3. To investigate the function of Snai3, we generated a conditional knockin mouse. Utilizing Cre-mediated deletion to facilitate the ablation of Snai3 in T cells or the entire animal, we found little to no effect of the loss of Snai3 in the entire animal or in T cell lineages. This finding provided the hypothesis that absence of Snai3 was mitigated, in part, by the presence of Snai2. To test this hypothesis we created Snai2/Snai3 double deficient mice. The developmental consequences of lacking both of these proteins was manifested in stunted growth, a paucity of offspring including a dramatic deficiency of female mice, and impaired immune cell development within the lymphoid lineages.

Sequential proteolytic processing of an interferon-alpha receptor subunit by TNF-alpha converting enzyme and presenilins.

It is well established that interferons trigger tyrosine-kinase-dependent signaling via JAK kinases and STAT transcription factors. However, we have observed both IFNaR2 receptor cleavage and functional activity of the liberated intracellular domain (ICD), suggesting that interferon-alpha (IFN-alpha) can also signal via regulated intramembrane proteolysis (RIP), an evolutionarily conserved mechanism of receptor-mediated signaling. Sequential cleavage of the receptor ectodomain and transmembrane domain is a hallmark of the most common class of RIP. To investigate the mechanisms of IFNaR2 RIP signaling, we examined IFNaR2 cleavage by TNF-alpha converting enzyme (TACE) and presenilin proteases. We tracked the fate of epitope-tagged and fusion variants of IFNaR2 in cells expressing wild-type, mutant, or null versions of TACE and presenilins 1 and 2. Cleavage and subcellular location were determined by immunoblot, fluoresence microscopy, and reporter assays. We found that both TACE and presenilin 1/2 cleave IFNaR2, in a sequential manner that allows the ICD to move to the nucleus. TACE cleavage was induced by IFN-alpha but was not consistently required for the anti-proliferative effects of IFN-alpha. In conclusion, IFNaR2 is cleaved by TACE and Presenilin 1/2, suggesting that interferons signal by both kinase and RIP-mediated pathways.