Francesco Liotta

Phenotypic and functional features of human Th17 cells

T helper (Th) 17 cells represent a novel subset of CD4+ T cells that are protective against extracellular microbes, but are responsible for autoimmune disorders in mice. However, their properties in humans are only partially known. We demonstrate the presence of Th17 cells, some of which produce both interleukin (IL)-17 and interferon (IFN)-γ (Th17/Th1), in the gut of patients with Crohns disease. Both Th17 and Th17/Th1 clones showed selective expression of IL-23R, CCR6, and the transcription factor RORγt, and they exhibited similar functional features, such as the ability to help B cells, low cytotoxicity, and poor susceptibility to regulation by autologous regulatory T cells. Interestingly, these subsets also expressed the Th1-transcription factor T-bet, and stimulation of these cells in the presence of IL-12 down-regulated the expression of RORγt and the production of IL-17, but induced IFN-γ. These effects were partially inhibited in presence of IL-23. Similar receptor expression and functional capabilities were observed in freshly derived IL-17–producing peripheral blood and tonsillar CD4+ T cells. The demonstration of selective markers for human Th17 cells may help us to understand their pathogenic role. Moreover, the identification of a subset of cells sharing features of both Th1 and Th17, which can arise from the modulation of Th17 cells by IL-12, may raise new issues concerning developmental and/or functional relationships between Th17 and Th1.

Role for Interferon‐γ in the Immunomodulatory Activity of Human Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA‐unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell‐activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact‐dependent and required the presence of interferon (IFN)‐γ produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN‐γ. The suppressive effect of IFN‐γ was related to its ability to stimulate the production by MSCs of indoleamine 2,3‐dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft‐versus‐host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell– derived IFN‐γ.

Human interleukin 17–producing cells originate from a CD161+CD4+ T cell precursor

We demonstrate that CD161 is a highly up-regulated gene in human interleukin (IL) 17 T helper cell (Th17) clones and that all IL-17–producing cells are contained in the CD161+ fraction of CD4+ T cells present in the circulation or in inflamed tissues, although they are not CD1-restricted natural killer T cells. More importantly, we show that all IL-17–producing cells originate from CD161+ naive CD4+ T cells of umbilical cord blood, as well as of the postnatal thymus, in response to the combined activity of IL-1β and IL-23. These findings implicate CD161 as a novel surface marker for human Th17 cells and demonstrate the exclusive origin of these cells from a CD161+CD4+ T cell progenitor.

Isolation and Characterization of Multipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys

Regenerative medicine represents a critical clinical goal for patients with ESRD, but the identification of renal adult multipotent progenitor cells has remained elusive. It is demonstrated that in human adult kidneys, a subset of parietal epithelial cells (PEC) in the Bowmans capsule exhibit coexpression of the stem cell markers CD24 and CD133 and of the stem cell-specific transcription factors Oct-4 and BmI-1, in the absence of lineage-specific markers. This CD24+CD133+ PEC population, which could be purified from cultured capsulated glomeruli, revealed self-renewal potential and a high cloning efficiency. Under appropriate culture conditions, individual clones of CD24+CD133+ PEC could be induced to generate mature, functional, tubular cells with phenotypic features of proximal and/or distal tubules, osteogenic cells, adipocytes, and cells that exhibited phenotypic and functional features of neuronal cells. The injection of CD24+CD133+ PEC but not of CD24-CD133- renal cells into SCID mice that had acute renal failure resulted in the regeneration of tubular structures of different portions of the nephron. More important, treatment of acute renal failure with CD24+CD133+ PEC significantly ameliorated the morphologic and functional kidney damage. This study demonstrates the existence and provides the characterization of a population of resident multipotent progenitor cells in adult human glomeruli, potentially opening new avenues for the development of regenerative medicine in patients who have renal diseases.

Phenotype, Localization, and Mechanism of Suppression of CD4 CD25 Human Thymocytes

Phenotypic markers, localization, functional activities, and mechanisms of action in vitro of CD4+CD25+ T cells, purified from postnatal human thymuses, were investigated. These cells showed poor or no proliferation in mixed lymphocyte culture (MLC), and suppressed in a dose-dependent fashion the proliferative response to allogeneic stimulation of CD4+CD25− thymocytes. Virtually all CD4+CD25+ thymocytes constitutively expressed cytoplasmic T lymphocyte antigen (CTLA)-4, surface tumor necrosis factor type 2 receptor (TNFR2), and CCR8. They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells. After polyclonal activation, CD4+CD25+ thymocytes did not produce the cytokines interleukin (IL)-2, IL-4, IL-5, IL-13, interferon γ, and only a very few produced IL-10, but all they expressed on their surface CTLA-4 and the majority of them also transforming growth factor (TGF)-β1. The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) α-chain (CD25) expression in target cells. Such a suppressive activity was partially inhibited by either anti–CTLA-4 or anti–TGF-β1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R α-chain and, therefore, their responsiveness to IL-2. These data demonstrate that CD4+CD25+ human thymocytes represent a population of regulatory cells that migrate in response to the chemokine CCL1/I-309 and exert their suppressive function via the inhibition of IL-2R α-chain in target T cells, induced by the combined activity of CTLA-4 and membrane TGF-β1.

Toll-Like Receptors 3 and 4 Are Expressed by Human Bone Marrow-Derived Mesenchymal Stem Cells and Can Inhibit Their T-Cell Modulatory Activity by Impairing Notch Signaling

Bone marrow (BM)‐derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM‐derived MSCs expressed high levels of Toll‐like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor κB (NF‐κB) activity, as well as the production of interleukin (IL)‐6, IL‐8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged‐1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti‐Jagged‐1 neutralizing antibody and N[N‐(3,5‐difluorophenacetyl‐l‐alanyl)]‐S‐phenylglycine t‐butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T‐cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T‐cell response in the course of dangerous infections, such as those sustained by double‐stranded RNA viruses or Gram‐negative bacteria, respectively.

CD14+CD34low Cells With Stem Cell Phenotypic and Functional Features Are the Major Source of Circulating Endothelial Progenitors

Endothelial progenitor cells (EPCs) seem to be a promising tool for cell therapy of acute myocardial infarction, but their nature is still unclear. We show here that EPCs obtainable from peripheral blood (PB) derive from the adhesion-related selection in culture of a subset of CD14+ cells, which, when assessed by the highly-sensitive antibody-conjugated magnetofluorescent liposomes (ACMFL) technique, were found to express CD34. These CD14+CD34low cells represented a variable proportion at individual level of CD14+ cells, ranging from 0.6% to 8.5% of all peripheral-blood leukocytes, and constituted the dominant population among circulating KDR+ cells. By using the ACMFL technique, virtually all CD14+ cells present in the bone marrow were found to be CD14+CD34low double-positive cells. EPCs, as well as purified circulating CD14+CD34low cells, exhibited high expression of embryonic stem cell (SC) markers Nanog and Oct-4, which were downregulated in a STAT3-independent manner when they differentiated into endothelial cells (ECs). Moreover, circulating CD14+CD34low cells, but not CD14+CD34− cells, proliferated in response to SC growth factors, and exhibited clonogenicity and multipotency, as shown by their ability to differentiate not only into ECs, but also into osteoblasts, adipocytes, or neural cells. The results of this study may reconcile apparently contradictory data of the literature, showing the generation of PB-derived EPCs from either CD34+ or CD14+ cells. We suggest that the use of this previously unrecognized population of circulating CD14+CD34low cells, which exhibit both phenotypic and functional features of SCs, may be useful in improving cell-based therapies of vascular and tissue damage.

Th17 cells: new players in asthma pathogenesis

To cite this article: Cosmi L, Liotta F, Maggi E, Romagnani S, Annunziato F. Th17 cells: new players in asthma pathogenesis. Allergy 2011; 66: 989–998.

Identification of a novel subset of human circulating memory CD4+ T cells that produce both IL-17A and IL-4

BACKGROUND IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. OBJECTIVE Study of the relationship between human IL-17A-producing CD4(+) T(H) cells (T(H)17) and IL-4-producing CD4(+) T(H) (T(H)2) cells. METHODS T-cell clones generated from the CCR6(+)CD161(+) fraction of human circulating CD4(+) T cells, which contains virtually all T(H)17 cells, as well as circulating CD4(+) T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. RESULTS A small proportion of CCR6(+)CD161(+)CD4(+) T-cell clones showed the ability to produce both IL-17A and IL-4 (T(H)17/T(H)2). T(H)17/T(H)2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few T(H)17/T(H)2 cells were found among circulating CD4(+) T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. T(H)17/T(H)2 cells could not be derived from naive umbilical cord blood CD4(+) T cells under any experimental condition. However, when circulating memory CCR6(+)CD161(+)CD4(+) T cells were cloned under appropriate polarizing conditions, T(H)17/T(H)2 clones originated in the presence of IL-4, suggesting that an IL-4-rich microenvironment may induce the shifting of memory T(H)17 cells into T(H)17/T(H)2 cells. CONCLUSION Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of T(H)17/T(H)2 cells may play some role in the pathogenesis of this disease.

CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL‐23 receptor (IL‐23R), CCR6, CD161, and the transcription factor retinoic acid‐related orphan receptor C (RORC), and originate from a CD161+CD4+ naïve T‐cell precursor in response to the combined activity of IL‐1β and IL‐23. We show here that not only CD4+TCRαβ+, but also CD8+TCRαβ+, CD4−CD8− TCRαβ+, and CD4−CD8− TCRγδ+ circulating lymphocytes that produce IL‐17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8+ and CD4−CD8− umbilical cord blood T cells that already express RORC and IL‐23R mRNA and that can be induced to differentiate into IL‐17‐producing cells in the presence of IL‐1β and IL‐23. Finally, we provide evidence that umbilical cord blood naïve CD4+CD161− T cells, upon lentivirus‐mediated transduction with RORC2 can acquire the ability to express IL‐23R, IL‐1RI, and CD161, as well as to produce IL‐17. Taken together, these data allow to conclude that T‐cell subsets able to produce IL‐17, as well as precursors of IL‐17‐producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2‐dependent.