The Immunoregulatory and Regenerative Potential of Activated Human Stem Cell Secretome Mitigates Acute-on-Chronic Liver Failure in a Rat Model.

Acute-on-chronic liver failure (ACLF) is a syndrome marked by sudden liver function decline and multiorgan failure, predominantly acute kidney injury (AKY), in patients with chronic liver disease. Unregulated inflammation is a hallmark of ACLF; however, the key drivers of ACLF are not fully understood. This study explores the therapeutic properties of human mesenchymal stem cell (MSC) secretome, particularly focusing on its enhanced anti-inflammatory and pro-regenerative properties after the in vitro preconditioning of the cells. We evaluated the efficacy of the systemic administration of MSC secretome in preventing liver failure and AKI in a rat ACLF model where chronic liver disease was induced using by the administration of porcine serum, followed by D-galN/LPS administration to induce acute failure. After ACLF induction, animals were treated with saline (ACLF group) or MSC-derived secretome (ACLF-secretome group). The study revealed that MSC-secretome administration strongly reduced liver histological damage in the ACLF group, which was correlated with higher hepatocyte proliferation, increased hepatic and systemic anti-inflammatory molecule levels, and reduced neutrophil and macrophage infiltration. Additionally, renal examination revealed that MSC-secretome treatment mitigated tubular injuries, reduced apoptosis, and downregulated injury markers. These improvements were linked to increased survival rates in the ACLF-secretome group, endorsing MSC secretomes as a promising therapy for multiorgan failure in ACLF.

Serological study of CoronaVac vaccine and booster doses in Chile: immunogenicity and persistence of anti-SARS-CoV-2 spike antibodies.

BACKGROUND: Chile was severely affected by COVID19 outbreaks but was also one of the first countries to start a nationwide program to vaccinate against the disease. Furthermore, Chile became one of the fastest countries to inoculate a high percentage of the target population and implemented homologous and heterologous booster schemes in late 2021 to prevent potential immunological waning. The aim of this study is to compare the immunogenicity and time course of the humoral response elicited by the CoronaVac vaccine in combination with homologous versus heterologous boosters. METHODS: We compared the immunogenicity of two doses of CoronaVac and BNT162b2 vaccines and one homologous or heterologous booster through an ELISA assay directed against the ancestral spike protein of SARS-CoV-2. Sera were collected from individuals during the vaccination schedule and throughout the implementation of homologous and heterologous booster programs in Chile. RESULTS: Our findings demonstrate that a two-dose vaccination scheme with CoronaVac induces lower levels of anti-SARS-CoV-2 spike antibodies than BNT162b2 in a broad age range (median age 42 years; interquartile range (IQR) 27-61). Furthermore, antibody production declines with time in individuals vaccinated with CoronaVac and less noticeably, with BNT162b2. Analysis of booster schemes revealed that individuals vaccinated with two doses of CoronaVac generate immunological memory against the SARS-CoV-2 ancestral strain, which can be re-activated with homologous or heterologous (BNT162b2 and ChAdOx1) boosters. Nevertheless, the magnitude of the antibody response with the heterologous booster regime was considerably higher (induction fold BNT162b2: 11.2x; ChAdoX1; 12.4x; CoronaVac: 6.0x) than the responses induced by the homologous scheme. Both homologous and heterologous boosters induced persistent humoral responses (median 122 days, IQR (108-133)), although heterologous boosters remained superior in activating a humoral response after 100 days. CONCLUSIONS: Two doses of CoronaVac induces antibody titers against the SARS-CoV-2 ancestral strain which are lower in magnitude than those induced by the BNT162b2 vaccine. However, the response induced by CoronaVac can be greatly potentiated with a heterologous booster scheme with BNT162b2 or ChAdOx1 vaccines. Furthermore, the heterologous and homologous booster regimes induce a durable antibody response which does not show signs of decay 3 months after the booster dose.

Phenotypic and functional alterations of peritoneal macrophages in lupus-prone mice.

BACKGROUND: Several studies have demonstrated the contribution of innate immune cells, including macrophages, in promoting systemic lupus erythematosus (SLE). Macrophages, one of the most abundant cell populations in the peritoneal cavity, are considered multifunctional cells with phenotypic plasticity. However, the functional properties of peritoneal macrophages in steady-state and during the progression of SLE remain poorly defined. METHODS AND RESULTS: Using the [NZB × NZW]F1 (BWF1) murine model of SLE, we analyzed the phenotype and function of peritoneal macrophages during the disease's onset. We found a higher frequency of peritoneal macrophages and B1a cells in BWF1-diseased mice than age-matched controls. Additionally, macrophages from diseased animals expressed lower levels of CD206, MHC-II, and Sirpα. RNAseq analysis identified 286 differentially expressed genes in peritoneal macrophages from diseased-BWF1 mice compared to control mice. Functional experiments demonstrate that peritoneal macrophages from diseased-BWF1 mice secrete higher levels of pro-inflammatory cytokines when activated with TLR7 and TLR9 agonists, and they were less efficient in suppressing the activation and proliferation of peritoneal LPS-activated B cells. These data demonstrate that peritoneal macrophages from BWF1-diseased mice present phenotypic and functional alterations shifting to a more pro-inflammatory state. CONCLUSIONS: The increase of macrophages with an altered phenotype and function together with the accumulation of B1a cells in the peritoneal cavity of diseased-BWF1 mice may promote the progression of the disease. Advancing awareness of the role and phenotype of peritoneal macrophages in SLE may contribute to a better understanding of these types of diseases and the development of novel therapies.

The Multifaceted Roles of B Cells in the Thymus: From Immune Tolerance to Autoimmunity.

The thymus is home to a significant number of resident B cells which possess several unique characteristics regarding their origin, phenotype and function. Evidence shows that they originate both from precursors that mature intrathymically and as the entry of recirculating mature B cells. Under steady-state conditions they exhibit hallmark signatures of activated B cells, undergo immunoglobulin class-switch, and express the Aire transcription factor. These features are imprinted within the thymus and enable B cells to act as specialized antigen-presenting cells in the thymic medulla that contribute negative selection of self-reactive T cells. Though, most studies have focused on B cells located in the medulla, a second contingent of B cells is also present in non-epithelial perivascular spaces of the thymus. This latter group of B cells, which includes memory B cells and plasma cells, is not readily detected in the thymus of infants or young mice but gradually accumulates during normal aging. Remarkably, in many autoimmune diseases the thymus suffers severe structural atrophy and infiltration of B cells in the perivascular spaces, which organize into follicles similar to those typically found in secondary lymphoid organs. This review provides an overview of the pathways involved in thymic B cell origin and presents an integrated view of both thymic medullary and perivascular B cells and their respective physiological and pathological roles in central tolerance and autoimmune diseases.

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.

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.

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.

Thymic B Cells Promote Germinal Center-Like Structures and the Expansion of Follicular Helper T Cells in Lupus-Prone Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive T and B cells, autoantibody production, and immune complex deposition in various organs. Previous evidence showed abnormal accumulation of B cells in the thymus of lupus-prone mice, but the role of this population in the progression of the disease remains mostly undefined. Here we analyzed the spatial distribution, function, and properties of this thymic B cell population in the BWF1 murine model of SLE. We found that in diseased animals, thymic B cells proliferate, and cluster in structures that resemble ectopic germinal centers. Moreover, we detected antibody-secreting cells in the thymus of diseased-BWF1 mice that produce anti-dsDNA IgG autoantibodies. We also found that thymic B cells from diseased-BWF1 mice induced the differentiation of thymocytes to follicular helper T cells (TFH). These data suggest that the accumulation of B cells in the thymus of BWF1 mice results in the formation of germinal center-like structures and the expansion of a TFH population, which may, in turn, activate and differentiate B cells into autoreactive plasma cells. Therefore, the thymus emerges as an important niche that supports the maintenance of the pathogenic humoral response in the development of murine SLE.

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.

Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells.

Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-ß1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFß1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the ß7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.

Adoptive transfer of autoimmune splenic dendritic cells to lupus-prone mice triggers a B lymphocyte humoral response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by increased autoantibody production that leads to multiple tissue injuries. Dendritic cells (DCs) are important orchestrators of immune responses and key components in fine-tuning the balance between tolerance and immunity. However, their role in autoimmune disorders such as SLE remains uncertain. We analyzed the contribution of DCs in triggering SLE by adoptively transferring splenic DCs from aged autoimmune [NZB×NZW]F1 (BWF1) mice to young healthy BWF1 mice. We observed that the transfer of DCs from autoimmune mice to pre-autoimmune mice induced high autoantibody titers in the serum of recipient mice. Moreover, autoimmune DCs from aged BWF1 mice were crucial for the expansion and differentiation of plasmablasts and CD5+ B cells or B1-like cells in the peripheral blood, and spleen of recipient BWF1 mice, a phenomenon that is observed in autoimmune BWF1 mice. On the other hand, DCs from aged BWF1 mice participated in the expansion and differentiation of DCs and IFN-γ-producing T cells. These results reveal that DCs from autoimmune BWF1 mice exhibit functional and phenotypic characteristics that allow them to trigger B cell hyperactivation, as well as DC and T cell expansion and differentiation, thereby promoting an exacerbated humoral response in lupus-prone mice.

The histone demethylase inhibitor GSK-J4 limits inflammation through the induction of a tolerogenic phenotype on DCs.

As it has been established that demethylation of lysine 27 of histone H3 by the lysine-specific demethylase JMJD3 increases immune responses and thus elicits inflammation, we hypothesize that inhibition of JMJD3 may attenuate autoimmune disorders. We found that in vivo administration of GSK-J4, a selective inhibitor of JMJD3 and UTX, ameliorates the severity of experimental autoimmune encephalomyelitis (EAE). In vitro experiments revealed that the anti-inflammatory effect of GSK-J4 was exerted through an effect on dendritic cells (DCs), promoting a tolerogenic profile characterized by reduced expression of costimulatory molecules CD80/CD86, an increased expression of tolerogenic molecules CD103 and TGF-ß1, and reduced secretion of proinflammatory cytokines IL-6, IFN-γ, and TNF. Adoptive transfer of GSK-J4-treated DCs into EAE mice reduced the clinical manifestation of the disease and decreased the extent of inflammatory CD4+ T cells infiltrating the central nervous system. Notably, Treg generation, stability, and suppressive activity were all exacerbated by GSK-J4-treated DCs without affecting Th1 and Th17 cell production. Our data show that GSK-J4-mediated modulation of inflammation is achieved by a direct effect on DCs and that systemic treatment with GSK-J4 or adoptive transfer of GSK-J4-treated DCs ex vivo may be promising approaches for the treatment of inflammatory and autoimmune disorders.

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.

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 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.

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.

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-ß. 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-ß, 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-ß, RA, and IL-2. Thus, RA-iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.