Elsevier

Molecular Immunology

Volume 126, October 2020, Pages 46-55
Molecular Immunology

CD40-miRNA axis controls prospective cell fate determinants during B cell differentiation

https://doi.org/10.1016/j.molimm.2020.07.007Get rights and content

Highlights

  • CD40 signal alters miRNA levels in naïve primary B-lymphocytes.

  • miRNA-target genes set shows significant enrichment in CD40 signaled B cells.

  • Candidate miRNA-targets shows differential regulation in CD40 signaled B cells.

  • Transfection of mimics for differentially regulated miRNA leads to alteration in levels of miRNA-targets.

  • miRNAs effect key CD40 signaled events by regulating expression of genes that contribute to eventual B cell differentiation.

Abstract

Immunological memory is a critical characteristic of a successful long-term adaptive immune response. During the initial phases of antigen:B lymphocyte interactions, B cells participate in the germinal center reaction, in which T-B cell interactions take place. CD154 on T cells acts as a ligand that binds to the CD40 receptor on B cells and facilitates the differentiation of B cells to memory B cells. However, cell fate determinants controlled by CD40 signal for cellular differentiation are unclear. In this study, we explored miRNA and miRNA-targets as cell fate determinants in CD40 signaled B cells. We selected candidate miRNAs based on their involvement in the regulation of B cell development, activation, and differentiation. We found that CD40 signal reduced transcript levels of miR150-5p, 17-5p, 146a-5p, 26a-5p and increased levels of miR292a-5p. Gene set enrichment analyses of previously submitted microarray data revealed accordant changes in levels of gene targets of these miRNA. Gene ontology analysis of miRNA-targets showed enrichment of genes participating in pathways such as DNA damage response, RNA/protein metabolism, and cell cycle regulation. Subsequently, studies on candidate miRNA-targets showed a CD40 signal driven differential regulation of Ccnd2, Pten, Traf6, c-Myb, and Btla. Further, 'gain of function' studies using mimics of the downregulated miRNAs, confirmed a predicted reduction in miRNA responsive targets; such as reduction of Ccnd2 levels in mimic treated groups of miR146a-5p, 26a-5p, and 17-5p. In conclusion, our study reveals that CD40 signal modulates levels of selected miRNAs as well as their cognate targets, whose enriched participation in diverse processes may help delineate downstream cell fate decisions.

Introduction

Currently all prophylactic vaccines are dependent on the formation of an adaptive immune memory. Immunological memory involves a wide spectrum of cell types including long-lived plasma cells, memory B cells, and memory T cells. Each of these cell types contributes diverse functions upon re-exposure to antigenic challenge. Long lived plasma B cells reside in the bone marrow and continue to secrete immunoglobulins into systemic circulation against the cognate antigen (Brynjolfsson et al., 2018); immediately subsequent immune insult is often cleared successfully. Memory B cells retain the B cell receptor on their surface. Upon re-exposure to the original antigenic challenge at an indeterminate time, they differentiate into plasma cells that secrete high-affinity antibodies to clear the target antigen (Engels and Wienands, 2018). Typically, this secondary response is faster in onset and higher in amplitude, thereby improving the effectiveness of clearing. Quite clearly, memory B cells acquire distinguishing features as compared to their parent ‘naïve B cells’. These new features lead to phenotypic outcomes: high rate of antigen-dependent division, high-affinity class-switched antibody as B cell receptor, and the ability to participate in immune response for subsequent antigenic exposures (Kurosaki et al., 2015).

Acquisition of these specific characteristics is dependent upon a coordinated differentiation of B cells that take place in a micro-anatomically specialized site, the germinal center (Nossal et al., 1968; Weisel et al., 2016). Different durations of antigen/non-antigen mediated interactions occur between CD4+ follicular T helper cells and naive B cells in the germinal center (Tangye et al., 2015). As a result, B cells receive several signals, some of which contribute towards memory B cell formation. One such signal is mediated by the CD154 ligand on T cells interacting with the CD40 receptor on B cell. In ex-vivo and in-vivo studies, the agonist, α-CD40, has been shown to bind to the CD40 receptor and mimics CD40 signal to B cells. α-CD40 co-stimulus at the time of primary antigenic challenge has shown to enhance the secondary immune response in the murine model (Raman et al., 2003). In the absence of T cells, just the administration of agonistic α-CD40 (along with antigen) facilitated differentiation of naïve B cells to a memory B cell phenotype (Taylor et al., 2012). Earlier work from our group reported that α-CD40 signaling positions naïve B-lymphocytes in an intermediary stage, between naïve and differentiated Plasma cells (Upadhyay et al., 2014). Despite the generalized awareness and acceptance of CD40 signal as a key event in memory B cell formation, there is an incomplete understanding of B cell-intrinsic molecular players downstream of CD40 signal.

miRNA are non-coding RNA molecules, contributing to cellular physiology by interfering translation of genes or affecting mRNA stability (O’Brien et al., 2018). miRNAs regulate various aspects of B cell development, activation, and differentiation. For example, miR 17-92/Pten axis regulates B cell selection during immature stages of B cell development (Benhamou et al., 2016). miR 17-92 cluster also contributes to the expression of S1pr1, thereby control the homing of plasma cells to bone marrow (Xu et al., 2015). Similarly, miR150 targets c-Myb, which controls plasma cell homing to bone marrow (Xiao et al., 2007 and Good-Jacobson et al., 2015). The depletion of miR 146a in B cells leads to a spontaneous germinal center formation (Cho et al., 2018). Furthermore, conditionally knock out of Dicer (a component of miRNA biogenesis) in B cells blocks the formation of memory B cells by failure in formation of germinal center (Xu et al., 2012). These studies highlight the existence of additional miRNAs, as key regulators in mounting B lymphocyte-mediated immune response. Therefore, we decided to explore CD40 signal-dependent changes in miRNA and miRNA-targets as prospective cell fate determinants of B cell differentiation. In the present study, we focused on CD40 mediated events at early stages in stimulated IgD+ naïve murine B cells. We have sought to identify miRNAs altered in a CD40 signal-dependent manner in B cells and to elucidate their role in cellular differentiation/memory outcome by analyzing cognate miRNA-target genes.

Section snippets

Isolation and stimulation of IgD+ B cells

For the present study, Balb/c (Jackson lab, USA), 2–6 month old male mice were procured from the CSIR-CCMB animal house. The required permissions for animal usage and approvals of experiment procedures were obtained from the Institutional Animal Ethics Committee of CSIR-CCMB for all experiments. Mice were euthanized using a CO2 gas chamber. Splenectomies were performed in sterile conditions. Single cell suspensions were generated from whole Spleen organs by dispersal, using sterile frosted

CD40 signal differentially alters miRNA levels

To identify miRNAs regulated by CD40 signaling, we analyzed relative levels of candidate miRNAs between B lymphocytes stimulated with α-CD40+LPS vs LPS alone. Candidate miRNA were selected based on their reported involvement in B cell development, activation, and differentiation e.g. miR17-5p and 150-5p regulate pro to pre-B cell development (Coffre and Koralov, 2017). Furthermore, we selected miRNA targeting signal transduction pathways e.g. miR146a-5p targeting Traf6 as a downstream component

Discussion

Memory B cells form a crucial component of the adaptive immune response. During primary immunological challenges, the process of germinal center reaction selects for memory B cells. In the germinal center reaction, B cells recognize and present antigen to the cognate T cells; in return, they receive input in the form of T cell signaling. The extent, duration, and quality of T cell help (interaction between T-B cells) decide the nature of B cell fate, including memory (Shinnakasu et al., 2016;

Conclusion

We report that CD40 signaling coordinates initial phases of B cell differentiation by regulating specific miRNAs. Exploring the effects of CD40 signal on the targets of these regulated miRNAs revealed that a CD40-miRNA axis might control a large pool of genes with diverse functionality in cellular and metabolic processes to aid B cells in adapting to the germinal center reaction. We are intrigued whether these linkages suggests coordinated changes at a transcriptome level between miRNAs and

CRediT authorship contribution statement

Satyajeet Salunkhe: Conceptualization, Methodology, Visualization, Investigation, Formal analysis, Software, Validation, Data curation, Writing - original draft, Writing - review & editing. Tushar Vaidya: Conceptualization, Methodology, Supervision, Formal analysis, Writing - original draft, Writing - review & editing, Resources, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Satyajeet Salunkhe was supported by Junior and Senior Research Fellowships from Council of Scientific and Industrial Research (CSIR), Government of India. This research was funded through support to Dr Tushar Vaidya by Council of Scientific and Industrial Research (CSIR), Government of India through CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India. We are thankful to Kaneez-eRabab Khan, V Devi Prasad, Pradyumna Paranjpe, and other members of Dr Tushar Vaidya lab for

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