Eliana Ribechini

Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1lowCD11bhighLy‐6ChighSSClowMo‐MDSC and Gr‐1highCD11blowPMN‐MDSC populations with suppressive potential, whereas Gr‐1highCD11bhigh neutrophils and Gr‐1lowCD11bhighSSClow eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8+ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.

CCL17-expressing dendritic cells drive atherosclerosis by restraining regulatory T cell homeostasis in mice

Immune mechanisms are known to control the pathogenesis of atherosclerosis. However, the exact role of DCs, which are essential for priming of immune responses, remains elusive. We have shown here that the DC-derived chemokine CCL17 is present in advanced human and mouse atherosclerosis and that CCL17+ DCs accumulate in atherosclerotic lesions. In atherosclerosis-prone mice, Ccl17 deficiency entailed a reduction of atherosclerosis, which was dependent on Tregs. Expression of CCL17 by DCs limited the expansion of Tregs by restricting their maintenance and precipitated atherosclerosis in a mechanism conferred by T cells. Conversely, a blocking antibody specific for CCL17 expanded Tregs and reduced atheroprogression. Our data identify DC-derived CCL17 as a central regulator of Treg homeostasis, implicate DCs and their effector functions in atherogenesis, and suggest that CCL17 might be a target for vascular therapy.

Subsets, expansion and activation of myeloid-derived suppressor cells

Tumor cells and microorganisms manipulate the immune system to minimize any counter response in order to survive. Myeloid-derived suppressor cells (MDSC) in the mouse represent activated Gr-1+ CD11b+ myeloid precursor cells. Activation may occur through endogenous or exogenous factors leading to the suppression of immune responses. Under steady state conditions the same precursors differentiate into dendritic cells, macrophages and neutrophils. Their linkage to tumor progression and several suppression mechanisms employing the arginine metabolism are well documented, but knowledge of their role in chronic infections, autoimmune diseases and graft-versus-host reactions is just emerging. Several factors have been described to promote MDSC expansion and activation in bone marrow, spleen and tumor sites. New evidence suggests that the Gr-1 antibody itself may differentially trigger myelopoiesis under steady state conditions or induce apoptosis in inflammatory situations after binding to a common epitope expressed on Ly-6C and Ly-6G molecules, respectively. Moreover, two subsets of neutrophil- and monocyte-related MDSC have been described in tumor-bearing and healthy mice. In the present review, we summarize some early work leading to recent findings on these two MDSC subsets, the factors supporting MDSC expansion and activation, as well as novel insights on Gr-1 antibody functions.

Gr-1 antibody induces STAT signaling, macrophage marker expression and abrogation of myeloid-derived suppressor cell activity in BM cells

The Gr‐1 (RB6‐8C5) Ab binds with high affinity to mouse Ly‐6G molecules and to a lower extent to Ly‐6C and has been widely used for cell depletion in infected or tumor‐bearing mice. Here we found that Gr‐1 treatment of BM cells in vitro and in vivo showed no depleting effects. The epitope recognized by the Gr‐1 Ab overlapped with Ly‐6G (1A8 Ab) but not Ly‐6C (ER‐MP20 Ab). In vitro the Gr‐1 Ab transmitted signals via STAT‐1, STAT‐3 and STAT‐5 into BM cells, similar to GM‐CSF. In healthy mice injected with the Gr‐1 Ab, the Ab remained attached to the surface of myeloid cells for at least four days. Gr‐1 Ab induced myeloid cell expansion, upregulation of macrophage markers, but not the DC marker CD11c. Suppressor activity of two distinct Gr‐1high and Gr‐1low expressing BM‐myeloid‐derived suppressor cell subsets was transiently ablated by Gr‐1 Ab injection. Depleting effects of Gr‐1 Ab could only be observed on inflammatory Ly‐6CintLy‐6Ghigh neutrophils from the peritoneal cavity, which occurred via apoptosis and was associated with the absence of Mcl‐1 expression. Together, Gr‐1 Ab induces signals leading to myelopoiesis and affects myeloid‐derived suppressor cell activity, suggesting functional roles for Ly‐6C/G molecules in macrophage differentiation and neutrophil apoptosis.

Exogenous TNFR2 activation protects from acute GvHD via host T reg cell expansion.

Activation of TNFR2 with a novel agonist expands T reg cells in vivo and protects allo-HCT recipients from acute GvHD while sparing antilymphoma and antiinfectious properties of transplanted donor T cells.

Sialoadhesin promotes neuroinflammation-related disease progression in two mouse models of CLN disease

CLN diseases are mostly fatal lysosomal storage diseases that lead to neurodegeneration in the CNS. We have previously shown that CD8+ T‐lymphocytes contribute to axonal perturbation and neuron loss in the CNS of Ppt1−/− mice, a model of CLN1 disease. We now investigated the role of the inflammation‐related cell adhesion molecule sialoadhesin (Sn) in Ppt1−/− and Cln3−/− mice, a model of the most frequent form, CLN3 disease. Microglia/macrophages in the CNS of both models showed an upregulation of Sn and markers for proinflammatory M1 polarization and antigen presentation. Sn+ microglia/macrophages associated with SMI32+ axonal spheroids and CD8+ T‐lymphocytes. To analyze their pathogenic impact, we crossbred both models with Sn‐deficient mice and scored axonal degeneration and neuronal integrity using immunohistochemistry, electron microscopy and optical coherence tomography. Degenerative alterations in the retinotectal pathway of Ppt1−/−Sn−/− and Cln3−/−Sn−/− mice were significantly reduced. Ppt1−/−Sn−/− mice also showed a substantially improved clinical phenotype and extended lifespan, attenuated numbers of M1‐polarized microglia/macrophages and reduced expression levels of proinflammatory cytokines. This was accompanied by an increased frequency of CD8+CD122+ T‐lymphocytes in the CNS of Ppt1−/−Sn−/− mice, the regulatory phenotype of which was demonstrated by impaired survival of CD8+CD122− effector T‐lymphocytes in co‐culture experiments. We show for the first time that increased Sn expression on microglia/macrophages contributes to neural perturbation in two distinct models of CLN disease. Our data also indicate that a rarely described CD8+CD122+ T‐cell population can regulate the corresponding diseases. These studies provide insights into CLN pathogenesis and may guide in designing immuno‐regulatory treatment strategies. GLIA 2016;64:792–809

Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells.

The ability of CD4+Foxp3+ regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7+CCR5− lymph node-seeking to a CCR7−CCR5+ inflammation-seeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression.

Novel GM-CSF signals via IFN-γR/IRF-1 and AKT/mTOR license monocytes for suppressor function

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls proliferation and survival of myeloid cells including monocytes. Here, we describe a time-dependent licensing process driven by GM-CSF in murine Ly6Chigh and human CD14+ monocytes that disables their inflammatory functions and promotes their conversion into suppressor cells. This 2-step licensing of monocytes requires activation of the AKT/mTOR/mTORC1 signaling cascade by GM-CSF followed by signaling through the interferon-γ receptor (IFN-γR)/interferon regulatory factor-1 (IRF-1) pathway. Only licensing-dependent adaptations in Toll-like receptor/inflammasome, IFN-γR, and phosphatidylinositol 3-kinase/AKT/mTOR signaling lead to stabilized expression of inducible nitric oxide synthase by mouse and indoleamine 2,3-dioxygenase (IDO) by human monocytes, which accounts for their suppressor activity. This study suggests various myeloid cells with characteristics similar to those described for monocytic myeloid-derived suppressor cells, Mreg, or suppressor macrophages may arise from licensed monocytes. Markers of GM-CSF-driven monocyte licensing, including p-Akt, p-mTOR, and p-S6, distinguish inflammatory monocytes from potentially suppressive monocytes in peripheral blood of patients with high-grade glioma.

The 30-kDa and 38-kDa antigens from Mycobacterium tuberculosis induce partial maturation of human dendritic cells shifting CD4+ T cell responses towards IL-4 production

BackgroundMycobacterium tuberculosis (Mtb) infections are still a major cause of death among all infectious diseases. Although 99% of individuals infected with Mtb develop a CD4+ Th1 and CD8+ T cell mediated immunity as measured by tuberculin skin test, this results only in partial protection and Mtb vaccines are not effective. Deviation of immune responses by pathogens towards a Th2 profile is a common mechanism of immune evasion, typically leading to the persistence of the microbes.ResultsHere we tested the stimulatory capacity of selective Mtb antigens on human monocyte-derived dendritic cell (DC) maturation and cytokine production. DC maturation markers CD80, CD86 and CD83 were readily upregulated by H37Ra- and H37Rv-associated antigens, the 30-kDa (from Ag85 B complex) and 38-KDa Mtb antigens only partially induced these markers. All Mtb antigens induced variable levels of IL-6 and low levels of IL-10, there was no release of IL-12p70 detectable. Substantial IL-12p40 production was restricted to LPS or H37Ra and H37Rv preparations. Although the proliferation levels of primary T cell responses were comparable using all the differentially stimulated DC, the 30-kDa and 38-kDa antigens showed a bias towards IL-4 secretion of polarized CD4+ T cells after secondary stimulation as compared to H37Ra and H37Rv preparations.ConclusionTogether our data indicate that 30-kDa and 38-kDa Mtb antigens induced only partial DC maturation shifting immune responses towards a Th2 profile.

RelB+ Steady-State Migratory Dendritic Cells Control the Peripheral Pool of the Natural Foxp3+ Regulatory T Cells

Thymus-derived natural Foxp3+ CD4+ regulatory T cells (nTregs) play a key role in maintaining immune tolerance and preventing autoimmune disease. Several studies indicate that dendritic cells (DCs) are critically involved in the maintenance and proliferation of nTregs. However, the mechanisms how DCs manage to keep the peripheral pool at constant levels remain poorly understood. Here, we describe that the NF-κB/Rel family transcription factor RelB controls the frequencies of steady-state migratory DCs (ssmDCs) in peripheral lymph nodes and their numbers control peripheral nTreg homeostasis. DC-specific RelB depletion was investigated in CD11c-Cre × RelBfl/fl mice (RelBDCko), which showed normal frequencies of resident DCs in lymph nodes and spleen while the subsets of CD103− Langerin− dermal DCs (dDCs) and Langerhans cells but not CD103+ Langerin+ dDC of the ssmDCs in skin-draining lymph nodes were increased. Enhanced frequencies and proliferation rates were also observed for nTregs and a small population of CD4+ CD44high CD25low memory-like T cells (Tml). Interestingly, only the Tml but not DCs showed an increase in IL-2-producing capacity in lymph nodes of RelBDCko mice. Blocking of IL-2 in vivo reduced the frequency of nTregs but increased the Tml frequencies, followed by a recovery of nTregs. Taken together, by employing RelBDCko mice with increased frequencies of ssmDCs our data indicate a critical role for specific ssmDC subsets for the peripheral nTreg and IL-2+ Tml frequencies during homeostasis.