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.

The Unfolded Protein Response Sensor IRE1 Regulates Activation of In Vitro Differentiated Type 1 Conventional DCs with Viral Stimuli.

Type 1 conventional dendritic cells (cDC1s) are leukocytes competent to coordinate antiviral immunity, and thus, the intracellular mechanisms controlling cDC1 function are a matter of intense research. The unfolded protein response (UPR) sensor IRE1 and its associated transcription factor XBP1s control relevant functional aspects in cDC1s including antigen cross-presentation and survival. However, most studies connecting IRE1 and cDC1 function are undertaken in vivo. Thus, the aim of this work is to elucidate whether IRE1 RNase activity can also be modeled in cDC1s differentiated in vitro and reveal the functional consequences of such activation in cells stimulated with viral components. Our data show that cultures of optimally differentiated cDC1s recapitulate several features of IRE1 activation noticed in in vivo counterparts and identify the viral analog Poly(I:C) as a potent UPR inducer in the lineage. In vitro differentiated cDC1s display constitutive IRE1 RNase activity and hyperactivate IRE1 RNase upon genetic deletion of XBP1s, which regulates production of the proinflammatory cytokines IL-12p40, TNF-α and IL-6, Ifna and Ifnb upon Poly(I:C) stimulation. Our results show that a strict regulation of the IRE1/XBP1s axis regulates cDC1 activation to viral agonists, expanding the scope of this UPR branch in potential DC-based therapies.

Normal tissue homeostasis and impairment of selective inflammatory responses in dendritic cells deficient for ATF6α.

The initiation of adaptive immunity relies on the performance of dendritic cells (DCs), which are specialized leukocytes with professional antigen presenting capabilities. As such, the molecular mechanisms safeguarding DC homeostasis are matter of intense research. Sensors of the unfolded protein response (UPR) of the endoplasmic reticulum, a three-pronged signaling pathway that maintains the fidelity of the cellular proteome, have emerged as regulators of DC biology. The archetypical example is the IRE1/XBP1s axis, which supports DC development and survival of the conventional type 1 DC (cDC1) subtype. However, the role of additional UPR sensors in DC biology, such as the ATF6α branch, has not been clearly elucidated. Even though Xbp1 is transcriptionally induced by ATF6α under ER stress, it is unclear if cDCs also co-opt the ATF6α branch in tissues. Here, we examine the role of ATF6α in cDC homeostasis in vivo and upon innate stimulation in vitro. In steady state, animals lacking ATF6α in CD11c+ cells (Itgax Cre x Atf6 fl/fl mice) display normal cDC frequencies in spleen, intestine, liver, and lung. Also, ATF6α deficient cDCs express normal levels of Xbp1 mRNA and additional UPR components. However, a reduction of lung monocytes is observed in Itgax Cre x Atf6 fl/fl conditional deficient animals suggesting that ATF6α may play a role in the biology of monocyte subsets. Notably, in settings of DC activation, ATF6α contributes to the production of IL-12 and IL-6 to inflammatory stimuli. Thus, although ATF6α may be dispensable for tissue cDC homeostasis in steady state, the transcription factor plays a role in the acquisition of selective immunogenic features by activated DCs.

Human cDC1s display constitutive activation of the UPR sensor IRE1.

The intracellular mechanisms safeguarding DC function are of biomedical interest in several immune-related diseases. Type 1 conventional DCs (cDC1s) are prominent targets of immunotherapy typified by constitutive activation of the unfolded protein response (UPR) sensor IRE1. Through its RNase domain, IRE1 regulates key processes in cDC1s including survival, ER architecture and function. However, most evidence linking IRE1 RNase with cDC1 biology emerges from mouse studies and it is currently unknown whether human cDC1s also activate the enzyme to preserve cellular homeostasis. In this work, we report that human cDC1s constitutively activate IRE1 RNase in steady state, which is evidenced by marked expression of IRE1, XBP1s, and target genes, and low levels of mRNA substrates of the IRE1 RNase domain. On a functional level, pharmacological inhibition of the IRE1 RNase domain curtailed IL-12 and TNF production by cDC1s upon stimulation with TLR agonists. Altogether, this work demonstrates that activation of the IRE1/XBP1s axis is a conserved feature of cDC1s across species and suggests that the UPR sensor may also play a relevant role in the biology of the human lineage.

The Unfolded Protein Response in Immune Cells as an Emerging Regulator of Neuroinflammation.

Immune surveillance is an essential process that safeguards the homeostasis of a healthy brain. Among the increasing diversity of immune cells present in the central nervous system (CNS), microglia have emerged as a prominent leukocyte subset with key roles in the support of brain function and in the control of neuroinflammation. In fact, impaired microglial function is associated with the development of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Interestingly, these pathologies are also typified by protein aggregation and proteostasis dysfunction at the level of the endoplasmic reticulum (ER). These processes trigger activation of the unfolded protein response (UPR), which is a conserved signaling network that maintains the fidelity of the cellular proteome. Remarkably, beyond its role in protein folding, the UPR has also emerged as a key regulator of the development and function of immune cells. However, despite this evidence, the contribution of the UPR to immune cell homeostasis, immune surveillance, and neuro-inflammatory processes remains largely unexplored. In this review, we discuss the potential contribution of the UPR in brain-associated immune cells in the context of neurodegenerative diseases.

The demethylase inhibitor GSK-J4 limits inflammatory colitis by promoting de novo synthesis of retinoic acid in dendritic cells.

Dendritic cells (DCs) promote T-cell mediated tolerance to self-antigens and induce inflammation to innocuous-antigens. This dual potential makes DCs fundamental players in inflammatory disorders. Evidence from inflammatory colitis mouse models and inflammatory bowel diseases (IBD) patients indicated that gut inflammation in IBD is driven mainly by T-helper-1 (Th1) and Th17 cells, suggesting an essential role for DCs in the development of IBD. Here we show that GSK-J4, a selective inhibitor of the histone demethylase JMJD3/UTX, attenuated inflammatory colitis by reducing the inflammatory potential and increasing the tolerogenic features of DCs. Mechanistic analyses revealed that GSK-J4 increased activating epigenetic signals while reducing repressive marks in the promoter of retinaldehyde dehydrogenase isoforms 1 and 3 in DCs, enhancing the production of retinoic acid. This, in turn, has an impact on regulatory T cells (Treg) increasing their lineage stability and gut tropism as well as potentiating their suppressive activity. Our results open new avenues for the treatment of IBD patients.

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.

IRE1α Activation in Bone Marrow-Derived Dendritic Cells Modulates Innate Recognition of Melanoma Cells and Favors CD8+ T Cell Priming.

The IRE1α/XBP1s signaling pathway is an arm of the unfolded protein response (UPR) that safeguards the fidelity of the cellular proteome during endoplasmic reticulum (ER) stress, and that has also emerged as a key regulator of dendritic cell (DC) homeostasis. However, in the context of DC activation, the regulation of the IRE1α/XBP1s axis is not fully understood. In this work, we report that cell lysates generated from melanoma cell lines markedly induce XBP1s and certain members of the UPR such as the chaperone BiP in bone marrow derived DCs (BMDCs). Activation of IRE1α endonuclease upon innate recognition of melanoma cell lysates was required for amplification of proinflammatory cytokine production and was necessary for efficient cross-presentation of melanoma-associated antigens without modulating the MHC-II antigen presentation machinery. Altogether, this work provides evidence indicating that ex-vivo activation of the IRE1α/XBP1 pathway in BMDCs enhances CD8+ T cell specific responses against tumor antigens.

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.

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.

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.

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.