Daniela Sauma

CD73 and CD39 ectonucleotidases in T cell differentiation: Beyond immunosuppression

Extracellular ATP is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. However, extracellular adenosine acts as an immunoregulatory signal that modulates the function of several cellular components of the adaptive and innate immune response. Consequently, the balance between ATP and adenosine concentration is crucial in immune homeostasis. CD39 and CD73 are two ectonucleotidases that cooperate in the generation of extracellular adenosine through ATP hydrolysis, thus tilting the balance towards immunosuppressive microenvironments. Extracellular adenosine can prevent activation, proliferation, cytokine production and cytotoxicity in T cells through the stimulation of the A2A receptor; however, recent evidence has shown that adenosine may also affect other processes in T‐cell biology. In this review, we discuss evidence that supports a role of CD73 and CD39 ectonucleotidases in controlling naive T‐cell homeostasis and memory cell survival through adenosine production. Finally, we propose a novel hypothesis of a possible role of these ectonucleotidases and autocrine adenosine signaling in controlling T‐cell differentiation.

Retinoic Acid as a Modulator of T Cell Immunity.

Vitamin A, a generic designation for an array of organic molecules that includes retinal, retinol and retinoic acid, is an essential nutrient needed in a wide array of aspects including the proper functioning of the visual system, maintenance of cell function and differentiation, epithelial surface integrity, erythrocyte production, reproduction, and normal immune function. Vitamin A deficiency is one of the most common micronutrient deficiencies worldwide and is associated with defects in adaptive immunity. Reports from epidemiological studies, clinical trials and experimental studies have clearly demonstrated that vitamin A plays a central role in immunity and that its deficiency is the cause of broad immune alterations including decreased humoral and cellular responses, inadequate immune regulation, weak response to vaccines and poor lymphoid organ development. In this review, we will examine the role of vitamin A in immunity and focus on several aspects of T cell biology such as T helper cell differentiation, function and homing, as well as lymphoid organ development. Further, we will provide an overview of the effects of vitamin A deficiency in the adaptive immune responses and how retinoic acid, through its effect on T cells can fine-tune the balance between tolerance and immunity.

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection.

CD4+CD25+Foxp3+ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3+ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.

Transplant tolerance: new insights and strategies for long-term allograft acceptance.

One of the greatest advances in medicine during the past century is the introduction of organ transplantation. This therapeutic strategy designed to treat organ failure and organ dysfunction allows to prolong the survival of many patients that are faced with no other treatment option. Today, organ transplantation between genetically dissimilar individuals (allogeneic grafting) is a procedure widely used as a therapeutic alternative in cases of organ failure, hematological disease treatment, and some malignancies. Despite the potential of organ transplantation, the administration of immunosuppressive drugs required for allograft acceptance induces severe immunosuppression in transplanted patients, which leads to serious side effects such as infection with opportunistic pathogens and the occurrence of neoplasias, in addition to the known intrinsic toxicity of these drugs. To solve this setback in allotransplantation, researchers have focused on manipulating the immune response in order to create a state of tolerance rather than unspecific immunosuppression. Here, we describe the different treatments and some of the novel immunotherapeutic strategies undertaken to induce transplantation tolerance.

Transforming Growth Factor-β and All-Trans Retinoic Acid Generate Ex Vivo Transgenic Regulatory T Cells With Intestinal Homing Receptors

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) mediate immunologic self-tolerance and suppress immune responses. In the gut, a subset of dendritic cells is specialized to induce Treg in a transforming growth factor-beta (TGF-beta)- and retinoic acid (RA)-dependent manner. The aim of this study was to establish if RA synergizing with TGF-beta induced antigen specific CD4(+) CD25(high) Foxp3(+) Treg portraying gut homing receptors. Splenic CD4(+)CD25(-) Foxp3(-) naïve T cells from DO11.10 mice were cocultured with splenic CD11c(+) dendritic cells from Balb/c mice in the presence of TGF-beta, RA, and low levels of an antigenic peptide. After 5 days of culture, cells were analyzed for the expression of Foxp3 and the gut homing receptors CCR9 and alpha4beta7. The number of Foxp3(+) T cells generated with TGF-beta and RA was at least 3 times higher than in the cultures with TGF-beta alone and 15 times higher than in controls without exogenous cytokines. Also, supplementation of the cultures with RA induced the expression of the intestinal homing receptors CCR9 and alpha4beta7. Our results showed that coculture of naïve T cells with antigen-presenting cells in the presence of TGF-beta and RA represents a powerful approach to generate Treg with specific homing receptors.

T helper type 17 cells contribute to anti-tumour immunity and promote the recruitment of T helper type 1 cells to the tumour

T helper type 17 (Th17) lymphocytes are found in high frequency in tumour‐burdened animals and cancer patients. These lymphocytes, characterized by the production of interleukin‐17 and other pro‐inflammatory cytokines, have a well‐defined role in the development of inflammatory and autoimmune pathologies; however, their function in tumour immunity is less clear. We explored possible opposing anti‐tumour and tumour‐promoting functions of Th17 cells by evaluating tumour growth and the ability to promote tumour infiltration of myeloid‐derived suppressor cells (MDSC), regulatory T cells and CD4+ interferon‐γ+ cells in a retinoic acid‐like orphan receptor γt (RORγt) ‐deficient mouse model. A reduced percentage of Th17 cells in the tumour microenvironment in RORγt‐deficient mice led to enhanced tumour growth, that could be reverted by adoptive transfer of Th17 cells. Differences in tumour growth were not associated with changes in the accumulation or suppressive function of MDSC and regulatory T cells but were related to a decrease in the proportion of CD4+ T cells in the tumour. Our results suggest that Th17 cells do not affect the recruitment of immunosuppressive populations but favour the recruitment of effector Th1 cells to the tumour, thereby promoting anti‐tumour responses.

Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells.

Dendritic cells (DCs) generated in vitro from bone marrow precursors using granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) secrete interleukin‐2 (IL‐2) upon activation, an event probably associated to the initiation of adaptive immune responses. Additionally, they produce IL‐12, a cytokine related to T‐cell polarization. To analyse the effect of IL‐4 on DC differentiation and function, we assessed the capacity of murine bone marrow dendritic cells (BMDCs) differentiated with GM‐CSF in the presence or absence of IL‐4 to produce IL‐2 and IL‐12 upon lipopolysaccharide (LPS) activation. We found that although IL‐4 enhanced DC IL‐12p70 production, it strongly impaired IL‐2 secretion by BMDCs. This inhibition, which depends on the presence of IL‐4 during LPS activation, is DC specific, as IL‐4 did not affect IL‐2 secretion by T cells. Interestingly, inhibition of DC IL‐2 production did not prevent DC priming of T lymphocytes. These results illustrate a new putative role for IL‐4 on the regulation of the immune response and should help clarify the controversial reports on the effect of IL‐4 on DCs.

Differential Regulation of Notch Ligands in Dendritic Cells upon Interaction with T Helper Cells

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta‐like and Jagged, in antigen‐presenting cells. However, whether stimuli other than pathogen‐derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta‐like 4 and Jagged 2 expression in bone marrow–derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta‐like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL‐4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2‐promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL‐4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL‐4. These data suggest that Notch ligands are differentially regulated in DCs: Delta‐like 4 is regulated by T helper cells and by pathogen‐derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen‐derived Th2‐promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.

Vitamin A Impairs the Reprogramming of Tregs into IL-17-Producing Cells during Intestinal Inflammation

Maintaining the identity of Foxp3+ regulatory T cells (Tregs) is critical for controlling immune responses in the gut, where an imbalance between Tregs and T effector cells has been linked to inflammatory bowel disease. Accumulating evidence suggests that Tregs can convert into Th17 cells and acquire an inflammatory phenotype. In this study, we used an adoptive transfer model of Ag-specific T cells to study the contribution of different factors to the reprogramming of in vitro-generated Treg cells (iTreg) into IL-17-producing cells in a mouse model of gut inflammation in vivo. Our results show that intestinal inflammation induces the reprogramming of iTreg cells into IL-17-producing cells and that vitamin A restrains reprogramming in the gut. We also demonstrate that the presence of IL-2 during the in vitro generation of iTreg cells confers resistance to Th17 conversion but that IL-2 and retinoic acid (RA) cooperate to maintain Foxp3 expression following stimulation under Th17-polarizing conditions. Additionally, although IL-2 and RA differentially regulate the expression of different Treg cell suppressive markers, Treg cells generated under different polarizing conditions present similar suppressive capacity.

Dendritic and stromal cells from the spleen of lupic mice present phenotypic and functional abnormalities

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increase in the percentage of autoreactive B and T lymphocytes. Since dendritic cells (DCs) are essential for B cell and T cell function, we hypothesized that changes in DC biology may play a critical role in the pathogenesis of the disease. We analyzed the phenotype and distribution of two main DC subsets, conventional (cDC) and plasmacytoid (pDC), in lupus prone (NZW × NZB)F1 (BWF1) mice and age-matched NZW × BALB/c control mice. Our results show that both subsets of lupic DCs displayed an abnormal phenotype, characterized by an over-expression of the co-stimulatory molecules CD80, CD86, PD-L1 and PD-L2 compared with control mice. Accordingly, spleen CD4(+) T cells from lupic mice exhibit an activated phenotype characterized by a higher expression of PD-1, CD25, CD69 and increased secretion of IFN-γ and IL-10. Interestingly, lupic mice also present an increase in the percentage of cDC in peripheral blood and an increase in the percentage of pDCs in spleen and mesenteric lymph nodes (MLNs) compared with control and pre-lupic mice. Homing experiments demonstrate that lupic and pre-lupic DCs migrate preferentially to the spleen compared to DCs from control mice. This preferential recruitment and retention of DCs in the spleen is related to an altered expression of different chemokine and chemokine receptors on both, DCs and stromal cells from lupic mice. Our results suggest that this altered phenotype and migratory behavior shown by DCs from lupic mice may account for the abnormal T cell and B cell responses in lupus.