Nuanced role for dendritic cell intrinsic IRE1 RNase in the regulation of antitumor adaptive immunity.

In cancer, activation of the IRE1/XBP1s axis of the unfolded protein response (UPR) promotes immunosuppression and tumor growth, by acting in cancer cells and tumor infiltrating immune cells. However, the role of IRE1/XBP1s in dendritic cells (DCs) in tumors, particularly in conventional type 1 DCs (cDC1s) which are cellular targets in immunotherapy, has not been fully elucidated. Here, we studied the role of IRE1/XBP1s in subcutaneous B16/B78 melanoma and MC38 tumors by generating loss-of-function models of IRE1 and/or XBP1s in DCs or in cDC1s. Data show that concomitant deletion of the RNase domain of IRE1 and XBP1s in DCs and cDC1s does not influence the kinetics of B16/B78 and MC38 tumor growth or the effector profile of tumor infiltrating T cells. A modest effect is observed in mice bearing single deletion of XBP1s in DCs, which showed slight acceleration of melanoma tumor growth and dysfunctional T cell responses, however, this effect was not recapitulated in animals lacking XBP1 only in cDC1s. Thus, evidence presented here argues against a general pro-tumorigenic role of the IRE1/XBP1s pathway in tumor associated DC subsets.

Ecto-5'-Nucleotidase (CD73) Regulates the Survival of CD8+ T Cells.

Ecto-5'-nucleotidase (CD73) is an enzyme present on the surface of tumor cells whose primary described function is the production of extracellular adenosine. Due to the immunosuppressive properties of adenosine, CD73 is being investigated as a target for new antitumor therapies. We and others have described that CD73 is present at the surface of different CD8+ T cell subsets. Nonetheless, there is limited information as to whether CD73 affects CD8+ T cell proliferation and survival. In this study, we assessed the impact of CD73 deficiency on CD8+ T cells by analyzing their proliferation and survival in antigenic and homeostatic conditions. Results obtained from adoptive transfer experiments demonstrate a paradoxical role of CD73. On one side, it favors the expression of interleukin-7 receptor α chain on CD8+ T cells and their homeostatic survival; on the other side, it reduces the survival of activated CD8+ T cells under antigenic stimulation. Also, upon in vitro antigenic stimulation, CD73 decreases the expression of interleukin-2 receptor α chain and the anti-apoptotic molecule Bcl-2, findings that may explain the reduced CD8+ T cell survival observed in this condition. These results indicate that CD73 has a dual effect on CD8+ T cells depending on whether they are subject to an antigenic or homeostatic stimulus, and thus, special attention should be given to these aspects when considering CD73 blockade in the design of novel antitumor therapies.

CD73 Ectonucleotidase Restrains CD8+ T Cell Metabolic Fitness and Anti-tumoral Activity.

CD39 and CD73 are ectoenzymes that dephosphorylate ATP into its metabolites; ADP, AMP, and adenosine, and thus are considered instrumental in the development of immunosuppressive microenvironments. We have previously shown that within the CD8+ T cell population, naïve and memory cells express the CD73 ectonucleotidase, while terminally differentiated effector cells are devoid of this enzyme. This evidence suggests that adenosine might exert an autocrine effect on CD8+ T cells during T cell differentiation. To study the possible role of CD73 and adenosine during this process, we compared the expression of the adenosinergic signaling components, the phenotype, and the functional properties between CD73-deficient and WT CD8+ T cells. Upon activation, we observed an upregulation of CD73 expression in CD8+ T cells along with an upregulation of the adenosine A2A receptor. Interestingly, when we differentiated CD8+ T cells to Tc1 cells in vitro, we observed that these cells produce adenosine and that CD73-deficient cells present a higher cytotoxic potential evidenced by an increase in IFN-γ, TNF-α, and granzyme B production. Moreover, CD73-deficient cells presented a increased glucose uptake and higher mitochondrial respiration, indicating that this ectonucleotidase restrict the mitochondrial capacity in CD8+ T cells. In agreement, when adoptively transferred, antigen-specific CD73-deficient CD8+ T cells were more effective in reducing the tumor burden in B16.OVA melanoma-bearing mice and presented lower levels of exhaustion markers than wild type cells. All these data suggest an autocrine effect of CD73-mediated adenosine production, limiting differentiation and cytotoxic T cells' metabolic fitness.

P2X7 Receptor at the Crossroads of T Cell Fate.

The P2X7 receptor is a ligand-gated, cation-selective channel whose main physiological ligand is ATP. P2X7 receptor activation may also be triggered by ARTC2.2-dependent ADP ribosylation in the presence of extracellular NAD. Upon activation, this receptor induces several responses, including the influx of calcium and sodium ions, phosphatidylserine externalization, the formation of a non-selective membrane pore, and ultimately cell death. P2X7 receptor activation depends on the availability of extracellular nucleotides, whose concentrations are regulated by the action of extracellular nucleotidases such as CD39 and CD38. The P2X7 receptor has been extensively studied in the context of the immune response, and it has been reported to be involved in inflammasome activation, cytokine production, and the migration of different innate immune cells in response to ATP. In adaptive immune responses, the P2X7 receptor has been linked to T cell activation, differentiation, and apoptosis induction. In this review, we will discuss the evidence of the role of the P2X7 receptor on T cell differentiation and in the control of T cell responses in inflammatory conditions.

In Vitro-Generated Tc17 Cells Present a Memory Phenotype and Serve As a Reservoir of Tc1 Cells In Vivo.

Memory CD8+ T cells are ideal candidates for cancer immunotherapy because they can mediate long-term protection against tumors. However, the therapeutic potential of different in vitro-generated CD8+ T cell effector subsets to persist and become memory cells has not been fully characterized. Type 1 CD8+ T (Tc1) cells produce interferon-γ and are endowed with high cytotoxic capacity, whereas IL-17-producing CD8+ T (Tc17) cells are less cytotoxic but display enhanced self-renewal capacity. We sought to evaluate the functional properties of in vitro-generated Tc17 cells and elucidate their potential to become long lasting memory cells. Our results show that in vitro-generated Tc17 cells display a greater in vivo persistence and expansion in response to secondary antigen stimulation compared to Tc1 cells. When transferred into recipient mice, Tc17 cells persist in secondary lymphoid organs, present a recirculation behavior consistent with central memory T cells, and can shift to a Tc1 phenotype. Accordingly, Tc17 cells are endowed with a higher mitochondrial spare respiratory capacity than Tc1 cells and express higher levels of memory-related molecules than Tc1 cells. Together, these results demonstrate that in vitro-generated Tc17 cells acquire a central memory program and provide a lasting reservoir of Tc1 cells in vivo, thus supporting the use of Tc17 lymphocytes in the design of novel and more effective therapies.

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.

Purinergic Signaling as a Regulator of Th17 Cell Plasticity.

T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.

Towards autotrophic tissue engineering: Photosynthetic gene therapy for regeneration.

The use of artificial tissues in regenerative medicine is limited due to hypoxia. As a strategy to overcome this drawback, we have shown that photosynthetic biomaterials can produce and provide oxygen independently of blood perfusion by generating chimeric animal-plant tissues during dermal regeneration. In this work, we demonstrate the safety and efficacy of photosynthetic biomaterials in vivo after engraftment in a fully immunocompetent mouse skin defect model. Further, we show that it is also possible to genetically engineer such photosynthetic scaffolds to deliver other key molecules in addition to oxygen. As a proof-of-concept, biomaterials were loaded with gene modified microalgae expressing the angiogenic recombinant protein VEGF. Survival of the algae, growth factor delivery and regenerative potential were evaluated in vitro and in vivo. This work proposes the use of photosynthetic gene therapy in regenerative medicine and provides scientific evidence for the use of engineered microalgae as an alternative to deliver recombinant molecules for gene therapy.

Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance.

The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.

CD73-mediated adenosine production promotes stem cell-like properties in mouse Tc17 cells.

The CD73 ectonucleotidase catalyses the hydrolysis of AMP to adenosine, an immunosuppressive molecule. Recent evidence has demonstrated that this ectonucleotidase is up-regulated in T helper type 17 cells when generated in the presence of transforming growth factor-ß (TGF-ß), and hence CD73 expression is related to the acquisition of immunosuppressive potential by these cells. TGF-ß is also able to induce CD73 expression in CD8(+) T cells but the function of this ectonucleotidase in CD8(+) T cells is still unknown. Here, we show that Tc17 cells present high levels of the CD73 ectonucleotidase and produce adenosine; however, they do not suppress the proliferation of CD4(+) T cells. Interestingly, we report that adenosine signalling through A2A receptor favours interleukin-17 production and the expression of stem cell-associated transcription factors such as tcf-7 and lef-1 but restrains the acquisition of Tc1-related effector molecules such as interferon-γ and Granzyme B by Tc17 cells. Within the tumour microenvironment, CD73 is highly expressed in CD62L(+) CD127(+) CD8(+) T cells (memory T cells) and is down-regulated in GZMB(+) KLRG1(+) CD8(+) T cells (terminally differentiated T cells), demonstrating that CD73 is expressed in memory/naive cells and is down-regulated during differentiation. These data reveal a novel function of CD73 ectonucleotidase in arresting CD8(+) T-cell differentiation and support the idea that CD73-driven adenosine production by Tc17 cells may promote stem cell-like properties in Tc17 cells.

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.

[Levels of inflammatory cytokines and plasma cortisol in respiratory syncytial virus bronchiolitis]. / Aumento de interleuquinas proinflamatorias y de cortisol plasmático en bronquiolitis por virus respiratorio sincicial: relación con la gravedad de la infección.

BACKGROUND: An increased inflammatory innate response may play a role in pathogenesis of respiratory syncytial virus (RSV) infection. AIM: To quantify pro-inflammatory cytokines (IL-6-IL-8, ÍL-2-P and TNF-a) in nasopharyngeal aspirate (NPA) and plasma, and plasma cortisol in previously healthy infants with RSV bronchiolitis. PATIENTS AND METHODS: We studied 49 infants aged less than one year of age with RSV bronchiolitis and 25 healthy controls. Severity was defined using a previously described modified score. We quantified interleukins in NPA and plasma by flow cytometry and plasma cortisol by radioimmunoanalysis. RESULTS: Among patients with RSV bronchiolitis, 25 were classified as severe and 24 as moderate or mild. Significantly higher levels of IL-6 and IL-8 in NPA and plasma and IL-lfi in NPA were found in children classified as severe, when compared to those with moderate or mild disease and controls. There was a positive correlation between IL-6 and cortisol in plasma (r = 0,55; p < 0,0001) and both were correlated with the severity of the disease. CONCLUSIONS: RSV bronchiolitis severity was associated with higher levéis of inflammatory interleukins and plasma cortisol.

Trichostatin A promotes the generation and suppressive functions of regulatory T cells.

Regulatory T cells are a specific subset of lymphocytes that suppress immune responses and play a crucial role in the maintenance of self-tolerance. They can be generated in the thymus as well as in the periphery through differentiation of naïve CD4(+) T cells. The forkhead box P3 transcription factor (Foxp3) is a crucial molecule regulating the generation and function of Tregs. Here we show that the foxp3 gene promoter becomes hyperacetylated in in vitro differentiated Tregs compared to naïve CD4(+) T cells. We also show that the histone deacetylase inhibitor TSA stimulated the in vitro differentiation of naïve CD4(+) T cells into Tregs and that this induction was accompanied by a global increase in histone H3 acetylation. Importantly, we also demonstrated that Tregs generated in the presence of TSA have phenotypical and functional differences from the Tregs generated in the absence of TSA. Thus, TSA-generated Tregs showed increased suppressive activities, which could potentially be explained by a mechanism involving the ectonucleotidases CD39 and CD73. Our data show that TSA could potentially be used to enhance the differentiation and suppressive function of CD4(+)Foxp3(+) Treg cells.

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.

Aumento de interleuquinas proinflamatorias y de cortisol plasmático en bronquiolitis por virus respiratorio sincicial: relación con la gravedad de la infección / Levels of inflammatory cytokines and plasma cortisol in respiratory syncytial virus bronchiolitis

Background: An increased inflammatory innate response may play a role in pathogenesis of respiratory syncytial virus (RSV) infection. Aim: To quantify pro-inflammatory cytokines (IL-6-IL-8, ÍL-2-P and TNF-a) in nasopharyngeal aspirate (NPA) and plasma, and plasma cortisol in previously healthy infants with RSV bronchiolitis. Patients and Methods: We studied 49 infants aged less than one year of age with RSV bronchiolitis and 25 healthy controls. Severity was defined using a previously described modified score. We quantified interleukins in NPA and plasma by flow cytometry and plasma cortisol by radioimmunoanalysis. Results: Among patients with RSV bronchiolitis, 25 were classified as severe and 24 as moderate or mild. Significantly higher levels ofIL-6 and IL-8 in NPA and plasma and IL-lfi in NPA were found in children classified as severe, when compared to those with moderate or mild disease and controls. There was a positive correlation between IL-6 and cortisol in plasma (r = 0,55; p < 0,0001) and both were correlated with the severity of the disease. Conclusions: RSV bronchiolitis severity was associated with higher levéis of inflammatory interleukins and plasma cortisol.

Epithelial control of the human pDC response to extracellular bacteria.

Plasmacytoid pre-dendritic cells (pDCs) are specialized in responding to nucleic acids, and link innate with adaptive immunity. Although the response of pDCs to viruses is well established, whether pDCs can respond to extracellular bacteria remains controversial. Here, we demonstrate that extracellular bacteria such as Neisseria meningitidis, Haemophilus influenzae, and Staphylococcus aureus activate pDCs to produce IFN-α, TNF-α, IL-6, and to upregulate CD86 expression. We observed that pDCs were present within tonsillar crypts and oro-nasopharyngeal epithelium, where they may contact extracellular bacteria, in situ. Tonsil epithelium-conditioned supernatants inhibited IFN-α, TNF-α, and IL-6 triggered by the direct contact of N. meningitidis or S. aureus with pDCs. However, pDC priming of naive T cells was not affected, suggesting that tonsil epithelium micro-environment limits local inflammation while preserving adaptive immunity in response to extracellular bacteria. Our results reveal an important and novel function of pDCs in the initiation of the mucosal innate and adaptive immunity to extracellular bacteria.

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.

Cyclosporin A-treated dendritic cells may affect the outcome of organ transplantation by decreasing CD4+CD25+ regulatory T cell proliferation.

One of the mechanisms for generation of tolerance involves immature dendritic cells (DCs) and a subpopulation of regulatory CD4+ CD25+ T lymphocytes (T REG). The purpose of this work was to analyze how Cyclosporine A (CsA), a widely used immunosuppressive drug, may affect T REG proliferation. Purified and activated murine DCs obtained from bone marrow precursors differentiated with rGMCSF were co-cultured with purified CFSE-labeled T REG from OTII mice, and their phenotype and proliferation analyzed by flow cytometry. Our data indicate that DCs differentiated in the presence of CsA show an altered phenotype, with a lower expression of MHC-II and a lower activating capacity. Additionally, these CsA-treated DCs show decreased production of IL-2 and IL-12 and increased IL-10 secretion when stimulated with LPS, indicating an effect on the polarization of the immune response. Interestingly, CsA-treated DCs show an anti-tolerogenic effect since they reduce the proliferation of T REG cells from 72 to 47%. Further inhibition to a 24% of T REG proliferation was obtained as a direct effect of CsA on T REG. In conclusion, the anti-tolerogenic effect of CsA should be considered in the planning of immunosuppression in the context of clinical transplantation.

Cyclosporin A-treated Dendritic Cells may affect the outcome of organ transplantation by decreasing CD4+CD25+ regulatory T cell proliferation

One of the mechanisms for generation of tolerance involves immature dendritic cells (DCs) and a subpopulation of regulatory CD4+ CD25+ T lymphocytes (T REG). The purpose of this work was to analyze how Cyclosporine A (CsA), a widely used immunosuppressive drug, may affect T REG proliferation. Purified and activated murine DCs obtained from bone marrow precursors differentiated with rGMCSF were co-cultured with purified CFSE-labeled T REG from OTII mice, and their phenotype and proliferation analyzed by flow cytometry. Our data indicate that DCs differentiated in the presence of CsA show an altered phenotype, with a lower expression of MHC-II and a lower activating capacity. Additionally, these CsA-treated DCs show decreased production of IL-2 and IL-12 and increased IL-10 secretion when stimulated with LPS, indicating an effect on the polarization of the immune response. Interestingly, CsA-treated DCs show an anti-tolerogenic effect since they reduce the proliferation of T REG cells from 72 to 47 percent. Further inhibition to a 24 percent of T REG proliferation was obtained as a direct effect of CsA on T REG. In conclusion, the anti-tolerogenic effect of CsA should be considered in the planning of immunosuppression in the context of clinical transplantation.

Vitamin C is an essential antioxidant that enhances survival of oxidatively stressed human vascular endothelial cells in the presence of a vast molar excess of glutathione.

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.