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Though myocardial infarction (MI) in pigs is a well-established translational large animal model, it has not yet been widely used for immunotherapy studies, and a comprehensive description of the immune response to MI in this species is lacking. We induced MI in Landrace pigs by balloon occlusion of the left anterior descending artery over 90 min. Within 14 days, the necrotic myocardium was progressively replaced by scar tissue with involvement of myofibroblasts. We characterized the immune response in the heart ex vivo by (immuno)histology, flow cytometry, and RNA sequencing of myocardial tissue on days 3, 7, and 14 after MI. Besides a clear predominance of myeloid cells among heart-infiltrating leukocytes, we detected activated T cells and an increasing proportion of CD4 + Foxp3 + regulatory T cells (T reg ), especially in the infarct core-findings that closely mirror what has been observed in mice and humans after MI. Transcriptome data indicated inflammatory activity that was persistent but markedly changing in character over time and linked to extracellular matrix biology. Analysis of lymphocytes in heart-draining lymph nodes revealed significantly higher proliferation rates of T helper cell subsets, including T reg on day 7 after MI, compared to sham controls. Elevated frequencies of myeloid progenitors in the spleen suggest that it might be a site of emergency myelopoiesis after MI in pigs, as previously shown in mice. We thus provide a first description of the immune response to MI in pigs, and our results can aid future research using the species for preclinical immunotherapy studies., (© 2024. The Author(s).)

Regulatory T cells (Tregs) maintain immune homeostasis by regulating the activation of other immune cells. Preclinical studies show that the infusion of Tregs can promote immunological tolerance to allografts and prevent or cure multiple autoimmune diseases. However, Treg therapy is limited by high numbers of cells required to induce tolerance. In this study, we aimed at improving the in vitro expansion of sort purified mouse Tregs using the CD28 Superagonist (CD28-SA) D665 and comparing it to the conventional expansion using anti-CD3/anti-CD28 Dynabeads®. CD28-SA-stimulated Tregs expanded more than Dynabead®-stimulated Tregs while maintaining their phenotype by expressing the same level of CD4, CD25 and Foxp3. CD28-SA-expanded Tregs produced comparable amounts of IL-10 and TGFβ while showing a slightly superior suppressive capacity compared to Dynabead®-stimulated Tregs. Thus, stimulating murine Tregs with the CD28-SA is a promising alternative since it maintains their suppressive capacity without altering their phenotype and yields a higher fold expansion within 14 days., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Acid sphingomyelinase (ASM) is the rate-limiting enzyme cleaving sphingomyelin into ceramide and phosphorylcholin. CD4+ Foxp3+ regulatory T (Treg) cells depend on CD28 signaling for their survival and function, a receptor that activates the ASM. Both, basal and CD28-induced ASM activities are higher in Treg cells than in conventional CD4+ T (Tconv) cells. In ASM-deficient (Smpd1-/-) as compared to wt mice, membranes of T cells contain 7-10-fold more sphingomyelin and two- to three-fold more ceramide, and are in a state of higher order than membranes of T cells from wt mice, which may facilitate their activation. Indeed, the frequency of Treg cells among CD4+ T cells in ASM-deficient mice and their suppressive activity in vitro are increased. Moreover, in vitro stimulation of ASM-deficient T cells in the presence of TGF-β and IL-2 leads to higher numbers of induced Treg cells. Pharmacological inhibition of the ASM with a clinically used tricyclic antidepressant such as amitriptyline in mice or in tissue culture of murine or human T cells induces higher frequencies of Treg cells among CD4+ T cells within a few days. This fast alteration of the balance between T cell populations in vitro is due to the elevated cell death of Tconv cells and protection of the CD25high Treg cells by IL-2. Together, these findings suggest that ASM-inhibiting antidepressants, including a fraction of the serotonin re-uptake inhibitors (SSRIs), are moderately immunosuppressive and should be considered for the therapy of inflammatory and autoimmune disorders.

CD4 + Foxp3 + regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4 + T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1 -/- ; Asm -/- ) mice, as compared with wt mice, the frequency of Tregs among CD4 + T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8 + T cells in brains of Asm -/- mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4 + T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Nonalcoholic fatty liver disease is increasing in prevalence. It can be subdivided into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). Five to twenty percent of cases progress from NAFL to NASH. Increased hepatic Th17 cells and IL-17 expression were observed in NASH mice and patients, respectively. We analyzed CD4(+) effector T cells and regulatory T cells (Tregs) from peripheral blood and livers of NAFL and NASH patients. A total of 51 NAFL patients, 30 NASH patients, 31 nonalcoholic fatty liver disease patients (without histology), and 43 healthy controls were included. FACS analysis was performed on PBMCs and intrahepatic lymphocytes. Compared with healthy controls, a lower frequency of resting Tregs (rTregs; CD4(+)CD45RA(+)CD25(++)) and higher frequencies of IFN-γ(+) and/or IL-4(+) cells were detected among CD4(+) T cells of peripheral blood in NASH, and to a lesser degree in NAFL. In hepatic tissue, NAFL to NASH progression was marked by an increase in IL-17(+) cells among intrahepatic CD4(+) T cells. To define immunological parameters in peripheral blood to distinguish NAFL from NASH, we calculated different ratios. Th17/rTreg and Th2/rTreg ratios were significantly increased in NASH versus NAFL. The relevance of our findings for NASH pathogenesis was highlighted by the normalization of all of the changes 1 y after bariatric surgery. In conclusion, our data indicate that NAFL patients show changes in their immune cell profile compared with healthy controls. NAFL to NASH progression is marked by an increased frequency of IL-17(+) cells among intrahepatic CD4(+) T cells and higher Th17/rTreg and Th2/rTreg ratios in peripheral blood., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Targeting regulatory T cells (Treg cells) with interleukin-2 (IL-2) constitutes a novel therapeutic approach for autoimmunity. As anti-cancer therapy with IL-2 has revealed substantial toxicities a mutated human IL-2 molecule, termed AIC284 (formerly BAY 50-4798), has been developed to reduce these side effects. To assess whether AIC284 is efficacious in autoimmunity, we studied its therapeutic potential in an animal model for Multiple Sclerosis. Treatment of Lewis rats with AIC284 increased Treg cell numbers and protected the rats from Experimental Autoimmune Encephalomyelitis (EAE). AIC284 might, thus, also efficiently prevent progression of autoimmune diseases in humans., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide-specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL-10(-) Foxp3(-) CTLA-4(+) CD25(low) Egr2(+) T cells could be converted into suppressive IL-10(+) Foxp3(-) CTLA-4(+) CD25(high) Egr2(+) cells resembling type-1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF-β during anergy induction favored Foxp3(+) Treg-cell induction, while TGF-β had little effect when added to the second stimulation. Expression of both CD28 and CTLA-4 molecules on anergic T cells was required to allow their conversion into Tr1-like cells. Suppressor activity was enabled via CD28-mediated CD25 upregulation, acting as an IL-2 sink, together with a CTLA-4-mediated inhibition of NFATc1/α activation to shut down IL-2-mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1-like cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

While the detrimental role of non-regulatory T cells in ischemic stroke is meanwhile unequivocally recognized, there are controversies about the properties of regulatory T cells (Treg). The aim of this study was to elucidate the role of Treg by applying superagonistic anti-CD28 antibody expansion of Treg. Stroke outcome, thrombus formation, and brain-infiltrating cells were determined on day 1 after transient middle cerebral artery occlusion. Antibody-mediated expansion of Treg enhanced stroke size and worsened functional outcome. Mechanistically, Treg increased thrombus formation in the cerebral microvasculature. These findings confirm that Treg promote thrombo-inflammatory lesion growth during the acute stage of ischemic stroke.

Rationale: An exaggerated or persistent inflammatory activation after myocardial infarction (MI) leads to maladaptive healing and subsequent remodeling of the left ventricle. Foxp3(+) CD4(+) regulatory T cells (Treg cells) contribute to inflammation resolution. Therefore, Treg cells might influence cardiac healing post-MI., Objective: Our aim was to study the functional role of Treg cells in wound healing post-MI in a mouse model of permanent left coronary artery ligation., Methods and Results: Using a model of genetic Treg-cell ablation (Foxp3(DTR) mice), we depleted the Treg-cell compartment before MI induction, resulting in aggravated cardiac inflammation and deteriorated clinical outcome. Mechanistically, Treg-cell depletion was associated with M1-like macrophage polarization, characterized by decreased expression of inflammation-resolving and healing-promoting factors. The phenotype of exacerbated cardiac inflammation and outcome in Treg-cell-ablated mice could be confirmed in a mouse model of anti-CD25 monoclonal antibody-mediated depletion. In contrast, therapeutic Treg-cell activation by superagonistic anti-CD28 monoclonal antibody administration 2 days after MI led to improved healing and survival. Compared with control animals, CD28-SA-treated mice showed increased collagen de novo expression within the scar, correlating with decreased rates of left ventricular ruptures. Therapeutic Treg-cell activation induced an M2-like macrophage differentiation within the healing myocardium, associated with myofibroblast activation and increased expression of monocyte/macrophage-derived proteins fostering wound healing., Conclusions: Our data indicate that Treg cells beneficially influence wound healing after MI by modulating monocyte/macrophage differentiation. Moreover, therapeutic activation of Treg cells constitutes a novel approach to improve healing post-MI., (© 2014 American Heart Association, Inc.)

The mouse killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor known to be expressed on a subset of NK cells and antigen-experienced CD8 T cells. Here, we have characterized expression of KLRG1 on CD4+ T cells from normal mice. While a polyclonal TCR repertoire suggests thymic origin of KLRG1+ CD4+ cells, KLRG1 expression was found to be restricted to peripheral CD4+ T cells. Based on phenotypic analyses, a minority of KLRG1+ CD4+ cells are effector/memory cells with a proliferative history. The majority of KLRG1+ CD4+ cells are, however, bona fide Treg cells that depend on IL-2 and/or CD28 and express both FoxP3 and high levels of intracellular CD152. KLRG1-expressing Treg are contained within the CD38+ subset but are only partially overlapping with the CD25+ CD4+ Treg subset. In functional assays, KLRG1+ CD4+ cells were anergic to TCR stimulation with respect to proliferation, and sorted KLRG1+ CD25+ CD4+ cells were equal or superior to KLRG1+ CD25- CD4+ cells, which were more potent than KLRG1- CD25+ CD4+ cells in suppressing responder cell proliferation. Together, our results demonstrate that KLRG1 expression defines novel and distinctive subsets of senescent effector/memory and potent regulatory CD4+ T cells.

Recruitment of naturally occurring CD4+ CD25+ regulatory T (T(reg)) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T(reg) cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T(reg) cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T(reg) cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T(reg) cells may play an additional role within the target tissue. In addition, the blood-brain barrier remained largely intact after JJ316 treatment, the secretion of T(H)2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-gamma. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T(reg) cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T(reg) cells in controlling autoimmunity in the CNS.

There is strong evidence that a quantitative and/or functional deficiency in CD4+CD25+ regulatory T cells (T(reg) cells) plays a key role in the pathogenesis of many human autoimmune diseases. Therefore, targeting regulatory T cells with novel forms of immunotherapy should provide a means for successfully battling autoimmunity in humans. We have recently shown that superagonistic monoclonal antibodies with specificity for CD28 (CD28 superagonists) are capable of activating and preferentially expanding T(reg) cells over conventional T cells in vitro and, importantly, also in vivo. Moreover, therapeutic application of CD28 superagonists elicited profound therapeutic effects in various animal models of autoimmunity, including experimental autoimmune encephalomyelitis (EAE) and adjuvant arthritis (AA) of the Lewis rat. Adoptive transfer experiments with T(reg) cells from CD28 superagonist-treated rats proved that protection from EAE is, indeed, mediated by CD28 superagonist-activated T(reg) cells. Therefore, effective targeting of CD4+CD25+ regulatory T cells makes CD28 superagonists a promising novel tool for the treatment of human autoimmune diseases.