Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses

doi:10.1016/j.imlet.2014.11.013

Immunology Letters

Volume 163, Issue 1, January 2015, Pages 84-95

https://doi.org/10.1016/j.imlet.2014.11.013 Get rights and content

Highlights

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Human megakaryocyte progenitors (MKp) are MHC Class II expressing professional APC.
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Human MKp express cytokines and mediators augmenting Th17 and Th1/Th17 response.
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MKp augment Th17 response against Candida albicans, serious opportunistic pathogen.
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Unlike MKp, mature MK and platelets express only Class I and do not augment Th17.
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MKp probably play immunologic role in conditions with extramedullary hematopoiesis.

Abstract

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34⁺CD117⁺CD41⁺CD151⁺ megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFβ, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC.

Introduction

Development of the megakaryocyte (MK) lineage in hematopoietic differentiation is considered to be exclusively geared for production of platelets. However, megakaryocyte progenitors (MKp) may have other functions that remain to be defined. In the case of reactive extramedullary hematopoiesis that occurs in various infections, autoimmune inflammatory diseases or graft-versus host disease, megakaryopoiesis accompanies myelopoiesis in the vicinity of T and B cells in peripheral lymphoid organs [1]. Whether the extramedullary MKp play a role in the immune responses under these conditions or are there solely to produce platelets, is not clear. In this regard, we recently showed that cells resembling MKp and bipotent megakaryocyte-erythroid progenitors (MEP) are markedly expanded in spleens of lupus-prone mice and they can act as professional APC that efficiently present nuclear autoantigens to selectively induce a Th17 response, without requiring Th17-polarizing culture conditions [2]. An expanded population of similar MKp-like cells was also found in the peripheral blood of lupus patients, but their functional activity could not be characterized due to the rarity of such cells in the periphery. To assess the immune potential of this human population in the present study, we derived MKp in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC), using culture conditions akin to bone marrow niche or organs that support extramedullary hematopoiesis [3]. We found that the MKp were professional APCs that expressed MHC class II, in marked contrast to platelets, which express only MHC class I similar to stromal cells capable of presenting antigen only to CD8 T cells [4], [5]. Indeed, surface MHC Class II expression diminished as the MKp matured. Moreover, in contrast to platelets, which suppress Th17 responses [6], we found that the MKp produced mediators that augmented Th17, Th1 and potent Th1/Th17 responses even in non-cognate interactions under non-polarizing conditions.

Section snippets

Materials and methods

Unless otherwise specified, reagents were obtained from Sigma-Aldrich (St. Louis, MO), cytokines from Peprotech (Rocky Hill, NJ), and antibodies from BD Biosciences (San Jose, CA).

Efficient generation of MKp from CD34⁺HSPC of normal donors

In lupus-prone mouse strains, splenocytes with MKp phenotype that express CD41, CD151 and CD117, are potent APC for inducing pathogenic Th17 cells in response to nuclear autoantigens [2]. In order to assess the functionality of the human counterpart of this recently reported murine population, CD34⁺ HSPC from nine normal adult donors were separately cultured for MKp differentiation in serum-free medium with SCF, Tpo, IL-3, IL-6, and IL-11 at 5% O₂ for 4–6 days. Consistent with previous

Discussion

We have established that human MKp derived from mobilized peripheral blood stem cells of normal individuals have the properties of professional APC and they can enhance Th17 or Th17/Th1 responses, including those to pathogens. Using a previously described method to mimic conditions in vivo, CD34⁺ HSPCs cultured in hypoxic conditions (5% O₂) in media supplemented with Tpo, SCF, IL-3, IL-6, and IL-11 expanded and committed to the MK lineage giving rise to CD34⁺CD117⁺CD41⁺CD151⁺ MKp with a stable

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by funding from the National Institutes of Health (NIAID, ARRA – R01AI41985 to S.K.D, and NIH/NCI training grant T32CA09560 to A.C.S. under W.M.M.) and a John N. Nicholson fellowship to A.C.S.

We thank Drs. Christian Stehlik and Lucia Maria V De Almeida and Teresa DeLuca for technical advice and assistance.

Ariel Finkielsztein, PhD has a licentiate degree in biology from the University of Buenos Aires, a Master of Science in Genetics and Molecular Biology from the University of Alberta (1999), and a PhD in developmental genetics from the University of Western Ontario (2009). He has done postdoctoral training at the University of Chicago (Pathology), the University of Illinois at Chicago (Multiple Sclerosis and Stem Cells), and Northwestern University (CD4T-Th17 and CD4T-IFNalpha regulation by

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The fungus Candida albicans can cause mucosal infections including oropharyngeal candidiasis (OPC) in immunocompromised patients. In humans, an increased risk of fungal infections correlates with thrombocytopenia. However, our understanding of platelets and megakaryocytes (Mks) in mucosal fungal infections is almost entirely unknown. When megakaryocyte- and platelet-depleted mice were infected with OPC, the tongue showed higher fungal burden, due to decreased neutrophil accumulation. Protection depended on a distinct population of oral-resident Mks. Interleukin-17, important in antifungal immunity, was required since mice lacking the IL-17 receptor had decreased circulating platelets and their oral Mks did not expand during OPC. The secretion of the peptide toxin candidalysin activated human Mks to release platelets with antifungal capacity. Infection with a candidalysin-deficient strain resulted in decreased expansion of tongue Mks during OPC. This is the first time that a distinct megakaryocyte population was identified in the oral mucosa which is critical for immunity against fungal infection.
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Protein arginine methyltransferase 1 in the generation of immune megakaryocytes: A perspective review
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Citation Excerpt :
Whether CD53+CD41+ Mks can recapitulate all the biological functions of immune Mks, as defined by scRNA-seq analysis, requires further investigation. Both MHC classes I and II are involved in antigen presentation in Mks (42, 56). MHC class I is expressed in mature Mks (42).
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2022, Blood Advances
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MKs express Toll-like receptors6-9 and other immune receptors10-12 and produce inflammatory cytokines and chemokines.13-15 Early MK progenitors express major histocompatibility complex (MHC) class II.16 Mature MKs cross-present antigens to CD8+ T cells via MHC I17 to CD4+ T cells via MHC II18 and exhibit antiviral potency.19
Neutrophils transit through megakaryocytes in a process termed emperipolesis, but it is unknown whether this interaction is a single type of cell-in-cell interaction or a set of distinct processes. Using a murine in vitro model, we characterized emperipolesis by live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome to enter the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or the perinuclear endoplasmic reticulum. These findings reveal that megakaryocyte emperipolesis reflects at least 2 distinct processes differing in transit time and morphology, fast and slow emperipolesis, suggesting divergent physiologic functions.
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Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41⁺ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche–related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1⁺ and CD53⁺ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8–associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53⁺ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

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Alaina Schlinker, PhD obtained BS in chemical engineering, University of Southern California, and PhD in Chemical and Biological Engineering, McCormick School of Engineering, Northwestern University (2014). Research work involves studies on adhesion molecules essential for initiation of proplatelet formation in megakaryocytes, and different ways to develop in vitro-derived platelet production from hematopoietic stem cells to generate a transfusable product.

Li Zhang, MD, PhD is research assistant professor of Medicine-Rheumatology at Northwestern University School of Medicine, Chicago. She received MD from Suzhou University School of Medicine, Suzhou, China (1984), Master of Science in Immunology from Dong Nan University School of Medicine, Nanjing, China (1990), and Ph.D. in immunology from Saga University School of Medicine, Saga, Japan (2000). She did post-doctoral research at Northwestern University School of Medicine, Chicago. Her research interests include nucleosomal histone peptide epitopes for tolerance therapy of lupus and pathogenic circulating T_FH cells and APC in lupus patients and SLE mouse model.

William M. Miller, PhD is professor of chemical and biological engineering at Northwestern University. He obtained BS from Lehigh University, MS from MIT, and PhD from the University of California, Berkeley. His research is focused on expansion and controlled differentiation of hematopoietic stem and progenitor cells, culture surfaces that mimic aspects of the in vivo bone marrow niche, mechanisms that regulate hematopoietic cell differentiation, and bioreactor systems for hematopoietic cell and platelet production. He is a Fellow of AAAS and AIMBE, Editor of the Biochemical Engineering Journal, and served on the Scientific Advisory Boards of the Australian Stem Cell Center and the Stem Cell Network of Canada.

Syamal K. Datta, MD is professor of medicine and microbiology-immunology at Northwestern University School of Medicine, Chicago. He has held Solovy Arthritis Research Society Professorship; received MERIT award from NIH; is member of Association of American Physicians, and Faculty of 1000. After graduating from Calcutta Medical College, India in 1967, he did post-doctoral research at Tufts University, Boston. In 1980s Datta group showed that pathogenic anti-DNA autoantibodies are encoded by genes of normal subjects. They also identified nucleosomal histone peptide epitopes for tolerance therapy of lupus, defined role of Cbl-b in hyperexpression of CD40L and COX-2 in lupus, and MKp as a new category of APC.

¹: These authors contributed equally to this work.

View full text

Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses

Highlights

Abstract

Introduction

Section snippets

Materials and methods

Efficient generation of MKp from CD34+HSPC of normal donors

Discussion

Conflict of interest

Acknowledgments

J Proteomics

Methods

Blood

Immunity

Blood

Clin Immunol

Blood

Blood

Blood

Trends Immunol

Semin Hematol

Extramedullary hematopoiesis: a new look at the underlying stem cell niche, theories of development, and occurrence in animals

Vet Pathol

Megakaryocyte progenitors are the main APCs inducing Th17 response to lupus autoantigens and foreign antigens

J Immunol

Three-stage ex vivo expansion of high-ploidy megakaryocytic cells: toward large-scale platelet production

Tissue Eng Part A

Platelets present antigen in the context of MHC class I

J Immunol

An unexpected role for platelets in blocking Th17 differentiation

J Clin Invest

Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-β producing CD8+ Treg cells are associated with immunological remission of lupus

J Immunol

Efficient generation of MKp from CD34⁺HSPC of normal donors

Regulatory T cell (Treg) subsets return in patients with refractory lupus following stem cell transplantation, and TGF-β producing CD8⁺ Treg cells are associated with immunological remission of lupus