Specificity and Heterogeneity of Trained Immunity in Hematopoietic Stem and Progenitor Cells

When innate immune cells and hematopoietic stem and progenitor cells (HSPCs) are exposed to pathogenic agents, they develop heightened responses to subsequent infection through a process called trained immunity. After exposure to a pathogen, bone marrow derived macrophages (BMDMs) generated from trained HSPCs are capable of enhanced pathogen clearance and cytokine production, while exhibiting persistent metabolic rewiring. Because some models of HSPC trained immunity are dependent on interferon gamma (IFNγ) signaling, and our lab has described extensive changes in HSC self-renewal and differentiation upon IFNγ exposure, we hypothesized that persistent IFNγ signaling induced by chronic infection results in reprogramming of HSPCs, causing improved non-specific immunity. To test our hypothesis, we generated a chimeric mouse model of trained immunity by transplanting control or M. avium-exposed HSPCs into naïve recipient mice. Mice that received M. avium trained HSPCs had decreased bacterial load, less splenomegaly, and fewer granulomas upon subsequent M. avium infection, indicating improved immunity. Furthermore, BMDMs generated from mice trained with a single dose of recombinant IFNγ (rIFNγ) exhibited increased pathogen clearance and metabolic profiles ex vivo. To test if rIFNγ training was sufficient to induce HSPC trained immunity in vivo, we isolated HSPCs from rIFNγ-exposed mice and challenged the recipients 4 months later. These in vivo experiments demonstrated that rIFNγ training was insufficient to induce substantial protection against subsequent M. avium challenge, but still induced BMDM metabolic rewiring four months post HSPC training. Collectively, our studies indicate that there are degrees of training that occur upon IFNγ exposure, likely related to the concentration and duration of the primary stimulus. To assess the specificity of cross protection of HSPC trained immunity, we utilized our chimeric mouse model and tested two different training and infection pathogens: M. avium and influenza. When we challenged M. avium-trained HSPC recipients with influenza, we found that although there was mildly decreased lung histopathology and increased production of IFNγ and TNFα, mice succumbed to infection like untrained controls. When we swapped the order of pathogens, we observed that mice receiving influenza-trained HSPCs produced BMDMs with increased killing capability and systemically higher IL-6 and RANTES levels, but these features were insufficient to significantly reduce bacterial CFU counts upon M. avium challenge. These transplant experiments indicate that trained immunity encoded in HSPCs is pathogen specific. To dissect the mechanism of M. avium-induced trained immunity in HSPCs, we performed RNAseq analysis on M. avium-trained HSPCs post-transplant and cross referenced it with RNAseq and WGBS data on primary M. avium-exposed HSPCs. These studies showed consistent differences in cellular signaling, metabolism, immunity, and antigen processing and presentation in the trained HSPCs, indicating that epigenetic and transcriptional reprogramming induced by M. avium exposure is durable following transplant and secondary challenge. To ascertain whether transcriptional changes are homogeneous throughout the HSC compartment, we completed scRNA-seq on naïve and M. avium-exposed hematopoietic cells. We found that genes upregulated upon M. avium exposure in HSCs, including Batf2 and Cxcl9, were induced in a subset of HSPCs, indicating that there is a heterogeneous response to training within the HSPC pool. Strikingly, the trained immunity signature was maintained in neutrophils and macrophages but lost in mature B cells, indicating specific propagation of genetic signatures induced by training in certain lineages. Finally, we found an emerging population of HSCs with B cell gene signatures upon M. avium exposure. Emergence of this HSPC subpopulation may suggest the development of a cell that acts as a direct intermediate between HSC and B cells following training. Our work shows that trained immunity induced by M. avium and persistent IFNγ signaling is pathogen-specific and heterogeneous among primitive HSPCs. Emergence of specific responder cell populations within the HSPC pool may be responsible for enhanced protection against specific infection stimuli, whereas the presence of non-responders may insure long term health of the HSPC pool. Disclosures No relevant conflicts of interest to declare.

Arachidonic acid-regulated calcium signaling in T cells from patients with rheumatoid arthritis promotes synovial inflammation

Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are two distinct autoimmune diseases that manifest with chronic synovial inflammation. Here, we show that CD4+ T cells from patients with RA and PsA have increased expression of the pore-forming calcium channel component ORAI3, thereby increasing the activity of the arachidonic acid-regulated calcium-selective (ARC) channel and making T cells sensitive to arachidonic acid. A similar increase does not occur in T cells from patients with systemic lupus erythematosus. Increased ORAI3 transcription in RA and PsA T cells is caused by reduced IKAROS expression, a transcriptional repressor of the ORAI3 promoter. Stimulation of the ARC channel with arachidonic acid induces not only a calcium influx, but also the phosphorylation of components of the T cell receptor signaling cascade. In a human synovium chimeric mouse model, silencing ORAI3 expression in adoptively transferred T cells from patients with RA attenuates tissue inflammation, while adoptive transfer of T cells from healthy individuals with reduced expression of IKAROS induces synovitis. We propose that increased ARC activity due to reduced IKAROS expression makes T cells more responsive and contributes to chronic inflammation in RA and PsA.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model

ABSTRACTInfection with hepatitis C virus (HCV) remains to be a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, currently there is no approved therapeutic targeting this potentially druggable biomarker. Here, we investigated the therapeutic potential of the lectibody Avaren-Fc (AvFc), a HMG-binding lectin-Fc fusion protein. In vitro assays showed AvFc’s capacity to neutralize cell culture-derived HCV in a genotype independent manner with IC50 values in the low nanomolar range. A histidine buffer-based AvFc formulation was developed for in vivo studies using the PXB human liver chimeric mouse model. Systemic administration of AvFc was well tolerated; after 11 consecutive doses every other day at 25 mg/kg, there were no significant changes in body or liver weights, nor any impact noted in blood human albumin levels or serum alanine aminotransferase activity. Gross necropsy and liver pathology further confirmed the lack of discernible toxicity. This treatment regimen successfully prevented genotype 1a HCV infection in all animals, while an AvFc mutant lacking HMG binding activity failed to block the infection. These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection. In particular, AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Using a barcoded AAV capsid library to select for novel clinically relevant gene therapy vectors

ABSTRACT:While gene transfer using recombinant adeno-associated viral (rAAV) vectors have shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet derived cells into functional β-cells in animal models, we constructed two highly complex barcoded replication competent capsid shuffled libraries and selected for high transducing variants on primary human islets. We describe a chimeric capsid (AAV-KP1) that penetrated and transduced primary human islet cells and human embryonic stem cell derived β-cells with up to 10-fold higher efficiency compared to previously studied best in class AAV vectors. Remarkably, this chimeric capsid was also able to transduce both mouse and human hepatocytes at very high levels in a humanized-chimeric mouse model, thus providing a versatile vector which has the potential to be used in both preclinical testing and human clinical trials for both liver-based diseases and diabetes.