Heth R. Turnquist

Ischemic heart disease is a highly prevalent and debilitating condition throughout the world. Following an ischemic injury, the immune response can become overly active and spread damage to nearby healthy tissue. Because the heart lacks regenerative capacity, preventing excessive damage is critical. Existing immunosuppressive therapeutics provide indiscriminate suppression of inflammatory and repair functions of infiltrating immune cells. Regulatory T cells (Treg) are the body's highly sophisticated system for regulating immunologic activity. These cells can provide powerful immune suppression while amplifying repair pathways through direct stimulation of local cells. Our research group has previously developed a polymeric delivery system comprised of poly(lactic‐co‐glycolic acid) (PLGA) degradable microparticles (MP) loaded with the Treg‐attracting protein, CCL22. In our previous work, we demonstrated CCL22MP attracts Treg to the injection site and can suppress disadvantageous inflammation. Here we combine our Treg‐attracting MP technology with H ydrogels to E nhance or A ttract R egulatory T cells ( HEART ). Topically applied HEART significantly reduced infarct size and improved ejection fraction after permanent‐ligation MI, and in a cardiac transplant ischemia model reduced graft fibrosis while significantly increasing intragraft Foxp3 ⁺ Treg at 30 days. Efficacy tracked with injury severity, identifying severe ischemic injury as a setting where this therapeutic strategy may be most beneficial.

Skeletal muscle injuries are a common consequence of physical activity, repetitive movements, and trauma. Regulatory T cells (Treg) act as critical mediators of immune repair response after injury. Thus, treatments effectively targeting Treg may accelerate injury resolution. CCL22 is a chemokine that recruits CCR4-expressing cells, particularly Treg, to sites of inflammation or immune regulation, such as tumor microenvironments. When an immunomodulatory, sustained release formulation of polymeric microparticles (MP) delivering CCL22 (CCL22MP), was administered after cardiotoxin (CTx)-mediated muscle injury, significantly improved limb function was observed on days 3 and 5 post injury. Histologic evaluation of the injured limbs showed reduced injury and enhanced myofiber cross-sectional area in CCL22MP treated limbs. Analysis of the local immune populations revealed augmented Treg concentrations, as well as altered infiltrating myeloid populations towards pro-repair subsets. These findings reveal that amplifying local Treg to damaged areas improves outcomes, thus offering a translationally promising approach after muscle injury.

Extracellular vesicles (EV) represent a conserved and highly efficient mechanism for cell-to-cell communication. Matrix-bound nanovesicles (MBV) are a recently identified type of EV embedded within the extracellular matrix with potent local and systemic immunomodulatory effects on myeloid cells. These effects are durable and last beyond the predicted life span of differentiated myeloid cells such as macrophages. The present study investigated MBV-directed epigenetic modification in myeloid precursors as a potential explanation for their prolonged immunomodulatory effects. Flow cytometry and ATAC sequencing studies show that MBV are internalized by myeloid cell progenitors in the bone marrow and in macrophages after terminal differentiation. This internalization is coincident with epigenetic changes that are associated with modulation of macrophage responses to inflammatory stimuli. Furthermore, MBV treatment differentially alters chromatin accessibility as a function of cell differentiation state (i.e., myeloid progenitor versus macrophage). The present study shows the epigenetic effects of MBV on myeloid cells, representing a potential avenue to exploit the therapeutic potential of biologic scaffold materials.

Skeletal muscle injuries are a common consequence of physical activity, repetitive movements, and trauma. Regulatory T cells (Treg) have recently been identified as critical mediators of immune repair response after injury, and treatments effectively targeting Treg may accelerate injury resolution. CCL22 is a chemokine that recruits CCR4-expressing cells, particularly Treg, to sites of inflammation or immune regulation, such as tumor microenvironments. When a sustained release formulation of polymeric microparticles (MP) delivering CCL22 (CCL22MP), was administered after cardiotoxin (CTx)-mediated muscle injury, significantly improved limb function was observed on days 3 and 5 post injury. Histologic evaluation of the injured limbs showed reduced area of injury in CCL22MP treated limbs. Analysis of the local immune populations revealed augmented Treg concentrations, as well as increased myeloid derived suppressor cell and neutrophil frequency. These findings reveal that amplifying local Treg to damaged areas improves outcomes, thus offering a translationally promising approach after muscle injury.

Mechanisms that mediate allograft tolerance differ between organs. We have previously shown that Foxp3 ⁺ T cell–enriched bronchus-associated lymphoid tissue (BALT) is induced in tolerant murine lung allografts and that these Foxp3 ⁺ cells suppress alloimmune responses locally and systemically. Here, we demonstrated that Foxp3 ⁺ cells that reside in tolerant lung allografts differed phenotypically and transcriptionally from those in the periphery and were clonally expanded. Using a mouse lung retransplant model, we showed that recipient Foxp3 ⁺ cells were continuously recruited to the BALT within tolerant allografts. We identified distinguishing features of graft-resident and newly recruited Foxp3 ⁺ cells and showed that graft-infiltrating Foxp3 ⁺ cells acquired transcriptional profiles resembling those of graft-resident Foxp3 ⁺ cells over time. Allografts underwent combined antibody-mediated rejection and acute cellular rejection when recruitment of recipient Foxp3 ⁺ cells was prevented. Finally, we showed that local administration of IL-33 could expand and activate allograft-resident Foxp3 ⁺ cells, providing a platform for the design of tolerogenic therapies for lung transplant recipients. Our findings establish graft-resident Foxp3 ⁺ cells as critical orchestrators of lung transplant tolerance and highlight the need to develop lung-specific immunosuppression.

Chronic rejection (CR) after organ transplantation is alloimmune injury manifested by graft vascular remodeling and fibrosis that is resistant to immunosuppression. Single-cell RNA-Seq analysis of MHC class II–mismatched (MHCII-mismatched) heart transplants developing chronic rejection identified graft IL-33 as a stimulator of tissue repair pathways in infiltrating macrophages and Tregs. Using IL-33–deficient donor mice, we show that graft fibroblast–derived IL-33 potently induced amphiregulin (Areg) expression by recipient Tregs. The assessment of clinical samples also confirmed increased expression of Areg by intragraft Tregs also during rejection. Areg is an EGF secreted by multiple immune cells to shape immunomodulation and tissue repair. In particular, Areg is proposed to play a major role in Treg-mediated muscle, epithelium, and nerve repair. Assessment of recipient mice with Treg-specific deletion of Areg surprisingly uncovered that Treg secretion of Areg contributed to CR. Specifically, heart transplants from recipients with Areg-deficient Tregs showed less fibrosis, vasculopathy, and vessel-associated fibrotic niches populated by recipient T cells. Mechanistically, we show that Treg-secreted Areg functioned to increase fibroblast proliferation. In total, these studies identify how a dysregulated repair response involving interactions between IL-33 ⁺ fibroblasts in the allograft and recipient Tregs contributed to the progression of CR.

Background: Troponin I is a blood biomarker of cardiac injury and levels measured using a high‐sensitivity assay after pediatric heart transplantation (HT) have not been described. We sought to assess the association between high‐sensitivity troponin I (hsTnI) and N‐terminal pro‐B‐type natriuretic peptide (NTproBNP) with treated acute rejection (AR) and graft loss in pediatric heart transplant (HT) recipients.Methods: Serum was collected and banked from pediatric HT recipients prior to cardiac catheterization. Patients with samples drawn within 365 days post‐HT were included and followed for up to 5 years. Generalized linear mixed‐effect models examined the association between hsTnI and treated AR using a random intercept per patient. Cox proportional hazards models tested the association between maximal hsTnI and NT‐proBNP and death/graft loss.Results: HsTnI and NTproBNP values decline in the weeks following HT, after which these biomarkers stabilize. HsTnI was higher in AR versus no AR (6.2 vs. 3.5 ng/L, p < 0.001); doubling of hsTnI increased the odds of AR by 33% (p = 0.004). HsTnI showed moderate discrimination for AR with an AUC of 0.811 (95% CI 0.76, 0.87) and a NPV of 96.4% (95% CI 93.0, 98.1). Elevation in NT‐proBNP was not associated with AR. In multivariable Cox modeling, a doubling of maximal NT‐proBNP was associated with graft loss (HR 8.96, p = 0.014).Conclusions: In this pediatric HT cohort, HsTnI was moderately discriminative for AR and higher maximal NT‐proBNP was associated with graft loss. HsTnI may add value in pediatric HT non‐invasive AR surveillance, and elevated NTproBNP could suggest an increased risk of graft loss.