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.

We studied the role of CD28 in T-cell biology and T cell–mediated pathology using a novel mouse anti–mouse CD28 antibody, E18, which recognizes an epitope close to the B7 binding site. In vitro, this antibody completely blocked binding of B7 molecules to CD28 expressed on mouse thymocytes but enhanced anti-CD3–induced proliferation of peripheral T cells. Injections of E18 monoclonal antibody into normal BALB/c mice in vivo, however, led to a reversible reduction in Treg cell frequencies among CD4⁺ cells, both in the thymus and in secondary lymphoid organs, suggesting that E18 acted as an inhibitor of CD28 signaling under these conditions. Antagonistic activity of E18 in vivo was further implied by suppressed responses of conventional CD4⁺ T cells to stimulation with the superantigen staphylococcal enterotoxin B and in a model of acute graft-versus-host disease. In contrast to healthy mice, intact monoclonal antibody E18, but not its nonstimulatory Fab fragment, increased the frequencies of Treg cells among CD4⁺ T cells in these pro-inflammatory settings allowing for efficacious protection from acute graft-versus-host disease. Thus, the agonistic signal generated by conventional, ie, nonsuperagonistic, anti-CD28 antibodies is important for their immunotherapeutic potential in vivo.

Genetic deficiency for acid sphingomyelinase or its pharmacological inhibition has been shown to increase Foxp3⁺ regulatory T-cell frequencies among CD4⁺ T cells in mice. We now investigated whether pharmacological targeting of the acid sphingomyelinase, which catalyzes the cleavage of sphingomyelin to ceramide and phosphorylcholine, also allows to manipulate relative CD4⁺ Foxp3⁺ regulatory T-cell frequencies in humans. Pharmacological acid sphingomyelinase inhibition with antidepressants like sertraline, but not those without an inhibitory effect on acid sphingomyelinase activity like citalopram, increased the frequency of Foxp3⁺ regulatory T cell among human CD4⁺ T cells in vitro. In an observational prospective clinical study with patients suffering from major depression, we observed that acid sphingomyelinase-inhibiting antidepressants induced a stronger relative increase in the frequency of CD4⁺ Foxp3⁺ regulatory T cells in peripheral blood than acid sphingomyelinase-non- or weakly inhibiting antidepressants. This was particularly true for CD45RA⁻ CD25^high effector CD4⁺ Foxp3⁺ regulatory T cells. Mechanistically, our data indicate that the positive effect of acid sphingomyelinase inhibition on CD4⁺ Foxp3⁺ regulatory T cells required CD28 co-stimulation, suggesting that enhanced CD28 co-stimulation was the driver of the observed increase in the frequency of Foxp3⁺ regulatory T cells among human CD4⁺ T cells. In summary, the widely induced pharmacological inhibition of acid sphingomyelinase activity in patients leads to an increase in Foxp3⁺ regulatory T-cell frequencies among CD4⁺ T cells in humans both in vivo and in vitro.

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 CD25^high 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.

Myocardial infarction (MI) induces the generation of proinflammatory Ly6C^high monocytes in the spleen and the recruitment of these cells to the myocardium. CD4⁺ Foxp3⁺ CD25⁺ T-cells (Tregs) promote the healing process after myocardial infarction by engendering a pro-healing differentiation state in myocardial monocyte-derived macrophages. We aimed to study the effects of CD4⁺ T-cells on splenic myelopoiesis and monocyte differentiation. We instigated MI in mice and found that MI-induced splenic myelopoiesis is abrogated in CD4⁺ T-cell deficient animals. Conventional CD4⁺ T-cells promoted myelopoiesis in vitro by cell–cell-contact and paracrine mechanisms, including interferon-gamma (IFN-γ) signalling. Depletion of regulatory T-cells enhanced myelopoiesis in vivo, as evidenced by increases in progenitor cell numbers and proliferative activity in the spleen 5 days after MI. The frequency of CD4⁺ T-cells-producing factors that promote myelopoiesis increased within the spleen of Treg-depleted mice. Moreover, depletion of Tregs caused a proinflammatory bias in splenic Ly6C^high monocytes, which showed predominantly upregulated expression of IFN-γ responsive genes after MI. Our results indicate that conventional CD4⁺ T-cells promote and Tregs attenuate splenic myelopoiesis and proinflammatory differentiation of monocytes.

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.