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The CD34 cell surface antigen is widely expressed in tissues on cells with progenitor-like properties and on mature vascular endothelia. In adult human bone marrow, CD34 marks hematopoietic stem and progenitor cells (HSPCs) starting from the bulk of hematopoietic stem cells with long-term repopulating potential (LT-HSCs) throughout expansion and differentiation of oligopotent and unipotent progenitors. CD34 protein surface expression is typically lost as cells mature into terminal effectors. Because of this expression pattern of HSPCs, CD34 has had a central role in the evaluation or selection of donor graft tissue in HSC transplant (HSCT). Given its clinical importance, it is surprising that the biological functions of CD34 are still poorly understood. This enigma is due, in part, to CD34's context-specific role as both a pro-adhesive and anti-adhesive molecule and its potential functional redundancy with other sialomucins. Moreover, there are also critical differences in the regulation of CD34 expression on HSPCs in humans and experimental mice. In this review, we highlight some of the more well-defined functions of CD34 in HSPCs with a focus on proposed functions most relevant to HSCT biology.

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

Allergic disease is on the rise and yet the underlying cause and risk factors are not fully understood. While lifesaving in many circumstances, the use of antibiotics and the subsequent disruption of the microbiome are positively correlated with the development of allergies. Here, we describe the use of the antibiotic vancomycin in combination with the papain-induced mouse model of allergic disease that allows for the assessment of microbiome perturbations and the impact on allergy development.

Innate lymphoid cells (ILCs) are a recently identified subset of leukocytes that play a central role in pathogen surveillance and resistance, modulation of immune response, and tissue repair. They are remarkably similar to CD4+ T-helper subsets in terms of function and transcription factors required for their development but are distinguished by their lack of antigen-specific receptors. Despite their similarities, the absence of a surface T-cell receptor (TCR) and presence of ILCs and precursors in adult bone marrow has led to speculation that ILCs and T cells develop separately from lineages that branch at the point of precursors within the bone marrow. Considering the common lineage markers and effector cytokine profiles shared between ILCs and T cells, it is surprising that the status of the TCR loci in ILCs was not fully explored at the time of their discovery. Here, we demonstrate that a high proportion of peripheral tissue ILC2s have TCRγ chain gene rearrangements and TCRδ locus deletions. Detailed analyses of these loci show abundant frameshifts and premature stop codons that would encode nonfunctional TCR proteins. Collectively, these data argue that ILC2 can develop from T cells that fail to appropriately rearrange TCR genes, potentially within the thymus.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (Podxl(ΔTie2Cre)). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood–brain barrier (BBB) in Podxl(ΔTie2Cre) mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH(2)) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH(2), LPS-primed Podxl(ΔTie2Cre) mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum—a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens. Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization.

High-grade serous ovarian carcinoma (HGSOC) is an aggressive tumor with a 5-year disease-free survival of roughly 15%, partly because it is usually diagnosed at an advanced stage. Podocalyxin (Podxl) is a highly glycosylated sialomucin normally expressed by vascular endothelia and kidney podocytes. Strikingly, Podxl expression is frequently upregulated by a variety of tumors (including HGSOC) and is consistently associated with poor prognosis. We capitalized on the fact that glycosylation pathways are frequently dysregulated in cancer to develop an antibody, PODO447, that recognizes a tumor-restricted glycoform of Podxl not expressed on normal tissue. While the exact epitope remains to be identified, our results suggest that PODO447 binds an epitope comprising a peptide domain of Podxl in combination with the core 1 O-GalNAc glycan (T-antigen). When coupled to a cytotoxin, a PODO447-antibody-drug conjugate (ADC) effectively kills human tumor cells in vitro and in xenografted mice. While the vast majority of ovarian tumors highly express the Podxl core protein, only a subset of these express the PODO447 epitope. Strikingly, tumors that express a high level of PODO447 epitope tend to be those that lack infiltrating CD8+ T cells and CD20+ B cells: a phenotype that has previously been linked to immune evasion and poorest disease-free survival. Furthermore, we find that PODO447 is a more consistent marker of these immunologically “cold” tumors than a number of other markers, including CA125, mesothelin and folate receptor α. These results highlight the PODO447-epitope as a highly selective diagnostic marker of poor outcome tumors and the PODO447-ADC as a novel strategy for therapeutic intervention. This research was supported by the Canadian Institutes of Health Research (Grant Number: PJT-166180), the School of Biomedical Engineering (The University of British Columbia) postdoctoral fellowship and the Michael Smith Foundation for Health Research (MSFHR) research trainee award.

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

Gut dysbiosis alters the development and severity of atopic disease. We previously demonstrated that nursing dams and newborn mice treated with low-dose vancomycin alters gut microbial diversity with a marked loss of bacteria that produce short-chain fatty acids (including butyrate). Vancomycin-induced gut dysbiosis enhances the TH2 response to lung allergens due to altered dendritic cell trafficking and activation in addition to modifying the behavior of other mature leukocyte lineages. Butyrate supplementation reverses the vancomycin-induced TH2 pro-inflammatory phenotype. Butyrate is known to exert some of its effects on target cells by inhibiting histone deacetylases (HDACs) with consequent effects on gene expression. Consistent with a role for epigenetic skewing of the hematopoietic compartment, we found that engraftment of total bone marrow from dysbiotic mice transferred enhanced TH2 proclivity in normobiotic recipients. Strikingly, we found unique regulatory states (H3K27ac marks) in purified hematopoietic stem and progenitor cells (HSPC) of TH2-skewed recipient mice. Single cell RNA sequence analyses identified a distinct transcriptomic signature in HSPC of dysbiotic mice that was reversed by butyrate supplementation. Together, these data suggest that the gut microbiome alters gene expression in blood progenitor cells with long term consequences on the immune response to peripheral allergens.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair. The transcription factor RORα is essential for ILC2 development but it is also highly expressed by ILC3s where its function remains poorly defined. Previously, we found that Rorasg/sg bone marrow transplant (BMT) mice were protected from intestinal fibrosis in a Salmonella--induced model of Crohn’s disease and that this is due to defective cytokine production by ILC3s. In the current study, whole transcriptome sequencing analysis reveals a striking downregulation of ILC3 signature genes in RORα-deficient ILC3s isolated from Salmonella infected mice. In particular, the expression of genes involved in sensing an inflammatory milieu is attenuated. Moreover, we find that Rorasg/sg ILC3s fail to express IL-17A following ex vivo stimulation with IL-23 and IL-1β. Consistent with these observations, we also find that Rorasg/sg BMT mice are more susceptible to Citrobacter rodentium infection due to attenuated expression of IL-22 and impaired induction of antimicrobial peptides. Collectively, our data suggests that RORα plays a key role in preserving functional ILC3s by modulating their ability to sense environmental cues that stimulate the efficient production of cytokines. These observations also suggest that targeting ILC3 cells could be of therapeutic benefit in fibrosis associated with Crohn’s disease.

Introduction Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. Methods We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. Results Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. Conclusions We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0562-7) contains supplementary material, which is available to authorized users.