MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2
2021; Springer Nature; Volume: 23; Issue: 1 Linguagem: Inglês
10.15252/embr.202052234
ISSN1469-3178
AutoresGisela Gordino, Sara Costa‐Pereira, Patrícia Corredeira, Patrícia Alves, Luís Costa, Anita Quintal Gomes, Bruno Silva‐Santos, Julie C. Ribot,
Tópico(s)Quinazolinone synthesis and applications
ResumoReport24 November 2021Open Access Source DataTransparent process MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2 Gisela Gordino Gisela Gordino Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Sara Costa-Pereira Sara Costa-Pereira Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Patrícia Corredeira Patrícia Corredeira Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Patrícia Alves Patrícia Alves orcid.org/0000-0002-0650-2445 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Luís Costa Luís Costa orcid.org/0000-0002-4782-7318 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Medical Oncology Division, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal Search for more papers by this author Anita Q Gomes Anita Q Gomes Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Escola Superior de Tecnologia da Saúde de Lisboa, Lisbon, Portugal Search for more papers by this author Bruno Silva-Santos Corresponding Author Bruno Silva-Santos [email protected] orcid.org/0000-0003-4141-9302 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Julie C Ribot Corresponding Author Julie C Ribot [email protected] orcid.org/0000-0002-7852-343X Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Gisela Gordino Gisela Gordino Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Sara Costa-Pereira Sara Costa-Pereira Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Patrícia Corredeira Patrícia Corredeira Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Patrícia Alves Patrícia Alves orcid.org/0000-0002-0650-2445 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Luís Costa Luís Costa orcid.org/0000-0002-4782-7318 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Medical Oncology Division, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal Search for more papers by this author Anita Q Gomes Anita Q Gomes Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Escola Superior de Tecnologia da Saúde de Lisboa, Lisbon, Portugal Search for more papers by this author Bruno Silva-Santos Corresponding Author Bruno Silva-Santos [email protected] orcid.org/0000-0003-4141-9302 Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Julie C Ribot Corresponding Author Julie C Ribot [email protected] orcid.org/0000-0002-7852-343X Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal Search for more papers by this author Author Information Gisela Gordino1, Sara Costa-Pereira1, Patrícia Corredeira1, Patrícia Alves1, Luís Costa1,2, Anita Q Gomes1,3, Bruno Silva-Santos *,1 and Julie C Ribot *,1 1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal 2Medical Oncology Division, Hospital de Santa Maria, Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal 3Escola Superior de Tecnologia da Saúde de Lisboa, Lisbon, Portugal *Corresponding author. Tel: +351 217999466; E-mail: [email protected] *Corresponding author. Tel: +351 217999411; E-mail: [email protected] EMBO Reports (2022)23:e52234https://doi.org/10.15252/embr.202052234 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies. Synopsis The miR-181a targets Map3k2 and Notch2 and thereby regulates human γδ T cell differentiation. This limits the expression of NKG2D and TNF-α, both involved in γδ T cell-mediated cancer surveillance. High levels of miR-181a associate with low expression of NKG2D in circulating γδ T cells from prostate cancer patients. miR-181a expression negatively correlates with activated type 1 effector profiles in human γδ T cell differentiated cultures. miR-181a overexpression limits the induction of NKG2D and TNF-α expression in differentiating γδ T cells. Map3k2 and Notch2 are direct miR-181a targets. Introduction Despite the recent advances in therapeutic strategies against cancer, early tumor recurrence and novel metastasis formation indicate resistance to current treatments. This urges the development of alternative treatments for advanced stages of the disease. γδ T cells possess multiple anti-tumor characteristics, making them promising candidates to be used in cellular and combination therapies (Gentles et al, 2015; Silva-Santos et al, 2015, 2019). They provide IFN-γ-associated type 1 responses against cancer and express critical determinants of tumor cell recognition, including their signature γδ TCR but also a variety of NK cell receptors (NKRs), among which the natural killer group 2 member D (NKG2D) is of utmost importance (Lança et al, 2010; Correia et al, 2013; Silva-Santos et al, 2015; Wu et al, 2019). This notwithstanding, the γδ T cell-based clinical trials completed to date have shown objective responses of only 10–33% (Gomes et al, 2010; Lo Presti et al, 2017). This modest outcome could be explained by their deficient expansion and/or dysregulated effector functions in vivo (Argentati et al, 2003; Bryant et al, 2009; Gaafar et al, 2009; Kuroda et al, 2012). Parallel investigations in both mice and humans have suggested that the tumor microenvironment can subvert the anti-tumor type 1 effector γδ T cell phenotype, either inactivating it (Marten et al, 2006; Gonnermann et al, 2015) or diverting into immunosuppressive phenotypes (Peng et al, 2007; Hao et al, 2011; Ma et al, 2012; Ye et al, 2013a, 2013b) or promoting the expansion of distinct pro-tumor type 17 effector γδ T cells (Rei et al, 2014; Wu et al, 2014; Silva-Santos et al, 2019). Collectively, these limitations stress the need to restore or enhance γδ T cell type 1 inflammatory and cytotoxic properties in future immunotherapeutic approaches. Based on this background, we have previously demonstrated that the anti-tumor type 1 effector properties of γδ T cells are selectively acquired upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7 (Ribot et al, 2014). The effects of IL-2/IL-15 depended on MAPK/ERK signaling and induced de novo expression of the type 1 transcription factors T-bet and eomesodermin. We followed those studies by exploring an additional layer of regulation of γδ T cell differentiation, namely, post-transcriptional mechanisms mediated by microRNAs (miRNAs or miRs), which are still poorly characterized in γδ T cells (Fiala et al, 2020), especially in the human setting (Zhu et al, 2017). MiRNAs are naturally occurring and evolutionarily conserved endogenous small non-coding RNAs (18–25 nucleotides) that typically downregulate post-transcriptional gene expression by binding to the 3ʹ untranslated region (UTR) of their target mRNAs and promoting their degradation or inhibiting their translation (Ambros, 2004). Each precursor miRNA consists of two mature RNA sequences—the 5p and 3p strands—whose designation is attributed according to the directionality of the miRNA strand (Kozomara & Griffiths-Jones, 2014). Although it has long been proposed that the 5p strand is the one being loaded into the RNA-induced silencing complex (RISC), recent evidence has disproven the idea that the 3p strand is mainly degraded during miR biogenesis (Kozomara et al, 2019). In fact, both the 5p and 3p strands can be loaded onto the Argonaute (AGO) family of proteins in an ATP-dependent manner (Yoda et al, 2010) and can show differential expression levels according to the pathophysiological context under study, namely, in cancer (Mitra et al, 2015, 2020). Importantly, miRNAs exert their regulatory functions in a highly combinatorial way: One miRNA can regulate several mRNAs in parallel, and different miRNAs can target one mRNA simultaneously, thus repressing its expression more efficiently (Pons-Espinal et al, 2017). Since this post-transcriptional process controls the expression of most mammalian genes, it is particularly relevant to analyze its role in human γδ T cell differentiation. To date, only one study has highlighted a role for miR-125b-5p and miR-99a-5p in human γδ T cell activation and cytotoxicity (Zhu et al, 2017), while the involvement of other miRs and their potential impact on γδ T cell responses to cancer remains unclear. Here, we identify miR-181a as a novel molecular regulator of human γδ T cell functional differentiation. We demonstrate that both its -5p and -2-3-p strands control γδ T cell type 1 effector differentiation and responsiveness by targeting Map3k2 and Notch2 mRNAs, and suggest a potential implication of this process in metastatic cancer patients. Results and Discussion miR-181a is upregulated in peripheral γδ T cells from metastatic cancer patients We investigated potential associations between miRNA expression patterns and γδ T cell dysfunction in metastatic cancer patients compared to healthy controls. Reduced number and impaired pro-inflammatory cytokine production have been previously reported in circulating γδ T cells from patients with melanoma (Argentati et al, 2003), glioblastoma (Bryant et al, 2009), breast (Gaafar et al, 2009), and gastric (Kuroda et al, 2011) carcinomas. Here, we analyzed patients of the cancer types with highest incidence in women (breast) and men (prostate), both in the metastatic (stage IV) setting. We found lower numbers of γδ T cells in the peripheral blood of both cohorts, and a decrease in their expression of NKG2D (significant in the prostate cohort), when compared to the respective (female or male) healthy controls (Fig 1A and B). We also observed a tendency for reduced IFN-γ production in the peripheral γδ T cells from the breast cancer patient cohorts, but this did not reach statistical significance (Fig 1C). Whereas the expression of miR-125b-5p and miR-99a-5p, which have previously been reported to modulate γδ T cell activation and cytotoxicity (Zhu et al, 2017), was very low and comparable to healthy controls (Fig 1D), we found—among other candidates under study—miR-181a-5p and miR-181a-2-3p to be upregulated in metastatic cancer patients, especially in the prostate cancer cohort (Fig 1E), thus providing an interesting association with γδ T cell dysfunction (Fig 1A and B). Of note, the effects observed were independent of patient age and treatment history. Figure 1. Peripheral γδ T cells from metastatic cancer patients display impaired effector functions and increased expression of miR-181a γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients. RT–PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples. RT–PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients. RT–PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples. Data information: (A–E) Error bars represent the mean ± SEM. CTRL F = Control Females (n = 25–27 independent biological samples); BC=Breast Cancer (n = 79–83 independent biological samples); CTRL M = Control Males (n = 19–20 independent biological samples); PC = Prostate Cancer (n = 29–36 independent biological samples). Statistical analysis was performed using the unpaired Student's t-test with Welch's correction. ns, not significant. *P < 0.05, **P < 0.01, and ****P < 0.0001. All experiments were performed with two technical replicates. Source data are available online for this figure. Source Data for Figure 1 [embr202052234-sup-0002-SDataFig1.xlsx] Download figure Download PowerPoint miR-181a is known for its pleiotropic functions on αβ T cell differentiation, including controlling the Th1 response of human CD4+ T cells (Sang et al, 2015); however, the role of miR-181a has never been addressed in human γδ T cells. miR-181a is downregulated upon type 1 effector γδ T cell differentiation Given each individual's history of infections, circulating γδ T cells in healthy donors are mostly functionally mature cells (Gibbons et al, 2009; deBarros et al, 2011). By contrast, we have previously shown that human γδ thymocytes isolated from pediatric biopsies of thymus are functionally immature and can be induced to acquire a type 1/cytotoxic profile upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7 (Ribot et al, 2014). Based on our data described above, we hypothesized that miR-181a might be downregulated during type 1 effector differentiation of human γδ thymocytes. Consistent with our hypothesis, we observed that the expression of both miR-181a strands was significantly lower in in vitro differentiated (IL-2 cultured) γδ thymocytes compared to immature (IL-7 cultured) controls (Fig 2A). In fact, all miR-181a species, including pre-miRNAs and mature 5p and 3p strands, were downregulated upon IL-2 treatment (Fig EV1A and B). Moreover, this downregulation of miR-181a (-5p and 2-3p) expression was also found when comparing freshly isolated (immature) γδ thymocytes versus (mature) peripheral γδ T cells ex vivo (Fig 2B). Figure 2. miR-181a is downregulated upon type 1 effector γδ T cell differentiation A. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n = 12–15 independent biological samples). B. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n = 6–14 independent biological samples). C, D. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4–6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n = 4–10 independent biological samples). E, F. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel), and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n = 5 independent biological samples). Data information: (A–D) Error bars represent the mean ± SEM. (A, C, D) Statistical analysis was performed using the paired Student's t-test. (B) Statistical analysis was performed using the Mann–Whitney U test. (E, F) The Pearson's correlation coefficient (r) was used to measure the strength of association between two variables. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All experiments were performed with two technical replicates. Source data are available online for this figure. Source Data for Figure 2 [embr202052234-sup-0003-SDataFig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. miR-181a-5p and 3p strands are downregulated upon IL-2 stimulation RT–PCR analysis of miR-181a-5p, miR-181a-1-3p and miR-181a-2-3p copy numbers in γδ thymocytes cultured with IL-7 versus IL-2 (n = 6 independent biological samples). RT–PCR analysis of pre-miR-181a-1-3p and pre-miR-181a-2-3p expression in γδ thymocytes cultured with IL-7 versus IL-2 (n = 6 independent biological samples). Data information: Error bars represent the mean ± SEM. Statistical analysis was performed using the paired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. All experiments were performed with two technical replicates. Source data are available online for this figure. Download figure Download PowerPoint Given that human γδ T cells comprise two major subsets, Vδ1+ cells, that are more abundant in the thymus and non-lymphoid tissues, and Vδ2+ cells, which represent 60–95% of γδ T cells in the peripheral blood and in lymph nodes (Fichtner et al, 2020), we assessed whether the composition of these populations could influence the levels of miR-181a expression in total γδ T cell samples. Although we found a higher level of miR-181a expression in the Vδ1+ γδ T cell subset (Fig EV2A), the Vδ1/Vδ2 ratio did not correlate with miR-181a expression levels (Fig EV2B). We next anticipated that, besides IL-2, other cytokines might regulate miR-181a expression and thus cultured γδ thymocytes with a set of pro- and anti-inflammatory mediators. IL-7 was used as control condition, allowing cell survival without promoting their functional differentiation (Ribot et al, 2014). Like IL-2, IL-15, which is also known to promote γδ T cell functional differentiation (Ribot et al, 2014), substantially downregulated the expression of both miR-181a strands (Fig 2C). By contrast, TGF-β upregulated miR-181a expression, while other cytokines such as IL-4, IL-12, IFN-γ, and TNF-α showed no impact (Fig 2C). Collectively, these data revealed that miR-181a expression is downregulated by drivers of type 1 effector γδ T cell differentiation, while being induced by immunosuppressive cytokines, namely, TGF-β, which is typically enriched in the cancer setting (Batlle & Massagué, 2019). This may have pathophysiological relevance, as TGF-β significantly enhanced miR-181a expression in γδ T cells isolated from the peripheral blood of healthy donors (Fig 2D), thus mimicking the increased miR-181a levels displayed by γδ T cells from patients with prostate cancer (Fig 1E). Click here to expand this figure. Figure EV2. miR-181a expression in Vδ1+ versus Vδ2+ γδ T cells RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) in freshly isolated Vδ1+ versus Vδ2+ sorted γδ PBLs (n = 4–8 independent biological samples). Correlation between Vδ1/Vδ2 ratio versus miR-181a(-5p and -2-3p) expression in freshly isolated γδ PBLs (n = 39–46 independent biological samples). Data information: (A) Error bars represent the mean ± SEM. Statistical analysis was performed using the unpaired Student's t-test. (B) The Pearson's correlation coefficient (r) was used to measure the strength of association between two variables. ns, not significant. *P < 0.05. All experiments were performed with two technical replicates. Source data are available online for this figure. Download figure Download PowerPoint To further document the negative association of miR-181a with type 1 effector γδ T cells, we next analyzed the expression of molecular hallmarks in various γδ T cell samples at different stages of differentiation. Namely, we used the same samples (i.e., freshly isolated versus in vitro IL-7- or IL-2-cultured γδ thymocytes) to measure the expression of miR-181a-5p and -2-3p, as well as the surface protein levels of the type 1 cytotoxic mediator NKG2D, and the intracellular expression of the type 1 cytokines, IFN-γ and TNF-α. We observed a striking inverse correlation between the expression of both miR-181a strands and the percentages of γδ thymocytes positive for IFN-γ, TNF-α, and NKG2D (Fig 2E), fully consistent with our hypothesis that miR-181a negatively regulates type 1 effector γδ T cell differentiation. Interestingly, this inverse correlation was not sustained in ex vivo peripheral blood γδ T cells (Fig 2F), suggesting a role for miR-181a during the early differentiation process, after which cytokine production becomes constitutive in mature cells. On the other hand, NKG2D showed a more dynamic profile in peripheral γδ T cells, which maintained the inverse correlation between NKG2D and miR-181a levels (Fig 2F) as observed in γδ thymocytes (Fig 2E). Consistently, the upregulation of miR-181a expression observed in TGF-β cultured peripheral γδ T cells (Fig 2D) associated with a lower percentage of NKG2D+ cells (Fig EV3). While this may suggest a segregation of the molecular mechanisms that regulate NKG2D and the pro-inflammatory cytokines in fully mature peripheral γδ T cells, our results collectively pointed toward a role for miR-181a during effector γδ T cell differentiation, which we set out to test using a gain-of-function approach. Click here to expand this figure. Figure EV3. TGF-β signals reduce NKG2D expression FACS analysis of the expression of NKGD2 in γδ T cells isolated from PBLs cultured with the indicated cytokines for 4–6 days (n = 5–6 independent biological samples, paired Student's t-test). Data represent the mean ± SEM. ns, not significant. **P < 0.01. All experiments were performed with two technical replicates. Source data are available online for this figure. Download figure Download PowerPoint miR-181a overexpression impairs γδ T cell differentiation To formally test whether miR-181a is able to regulate effector γδ T cell differentiation, we used retroviral transduction to overexpress miR-181a. We designed a construct in order to overexpress the native stem-loop of miR-181a (containing both -5p and -2-3p strands) in γδ thymocyte cultures. Cells were freshly isolated, activated, transduced, and maintained in the presence of IL-7 and IL-2 to promote type 1 cytotoxic differentiation (Fig 3A). The expression of the GFP reporter allowed us to identify the subpopulation that integrated the retroviral construct (Fig 3B). As a technical control of this set of experiments, we verified that sorted GFP+ cells transduced with miR-181a vector displayed a significant increase in the expression of miR-181a (both the -5p and -2-3p strands) when compared to cells transduced with the empty virus (Fig 3C). On the other hand, as an internal control, we consistently analyzed the untransduced GFP− population, where miR-181a expression remained at baseline (Fig 3C), and thus, no changes in any of the below-mentioned readouts were expected. Figure 3. miR-181a overexpression impairs γδ T cell functional differentiation Retroviral transduction workflow for γδ thymocytes. Gating strategy for identification of the GFP+ versus GFP− cells. RT–PCR analysis of the expression of miR-181a(-5p and -2-3p) (normalized to the values obtained with the empty virus) in transduced (GFP+) and untransduced (GFP−) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n = 4 independent biological samples). FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP−) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n = 5 independent biological samples). RT–PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n = 5 independent biological samples). FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP−) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n = 7–11 independent biological samples). Data information: (C–F) Error bars represent the mean ± SEM. Statistical analysis was performed using the paired Student's t-test. ns, not significant. *P < 0.05 and **P < 0.01. All experiments were performed with two technical replicates. Source data are available online for this figure. Source Data for Figure 3 [embr202052234-sup-0004-SDataFig3.xlsx] Download figure Download PowerPoint miR-181a overexpression seemingly impaired γδ T cell survival, given the increase in Annexin V+ cells (Fig 3D) and the reduction in expression of the anti-apoptotic genes Bcl2 and Bcl-xL (Fig 3E) among the transduced (but not the untransduced) γδ T cells. On the other hand, miR-181a overexpression increased the percentage of cells with a central memory profile (CD27+CD45RA−), while reducing the percentage of cells displaying a typical naïve (CD27+CD45RA+) and effector phenotype (CD27−CD45RA+; Fig 3F). Since previous studies reported that memory T cells arise from the presence of high concentration levels of IL-2 (Berard & Tough, 2002; Yurova et al, 2017), our results support a functional crosstalk between miR-181a and IL-2-dependent signaling. From a functional standpoint, we demonstrated that miR-181a overexpression led to significant reductions in the percentages of TNF-α+ and NKG2D+ γδ T cells (Fig 3F). Interestingly, while the percentage of IFN-γ+ γδ T cells was not affected, we observed a decrease in the percentage of TNF-α+ IFN-γ+ subset, suggesting that, in the type 1 effector differentiation pathway, miR-181a could also regulate a step where IFN-γ production would be induced from the TNF-α+ population, as previously proposed for Vδ2+ γδ T cells and NK cells (Skeen & Ziegler, 1995; Li et al, 2008). Of note, we observed that miR-181a overexpression equally affected the percentage of TNF-α+ cells within Vδ1+ and Vδ2+ population (Fig EV4). Click here to expand this figure. Figure EV4. miR-181a overexpression impact on TNF-α cytokine production in different γδ T cell subpopulations Gating strategy for the identification of the Vδ1 versus Vδ2 subpopulations in miR-181a versus empty transduced (GFP+) γδ thymocytes (left panel) and TNF-α expression gated on either Vδ1+, Vδ2+, or Vδ1−Vδ2− populations, in (GFP+) miR-181a versus empty transduced γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (right panel, n = 11 independent biological samples). Data Information: Error bars represent the mean ± SEM. Statistical analysis was performed using the paired Student's t-test. **P < 0.01. All experiments were performed with two technical replicates. Source data are available online for this figure. Download figure Download PowerPoint We further overexpressed miR-181a in mature γδ T cells isolated from healthy donor peripheral blood (Fig EV5A and B) and found a significant albeit minor decrease in NKG2D levels, while type 1 differentiation program remained intact (Fig EV5C). This is consistent with our previous data pointing at an inverse correlation in peripheral γδ T cells between the expression of miR-181a and NKG2D, but not type 1 cytokine expression (Fig 2F). Of note, by comparing GFPLow and GFPHigh subsets, either in γδ thymocyte (Fig EV5D) or PBL (Fig EV5E) cultures, we did not observe any dose effect of the levels of miR-181a-bearing vector transduction. Overall, the differences between PBL and thymocyte cultures suggest that the maturation status of γδ T cells conditions their sensitivity to miR-181a action, which is maximized at early stages of functional γδ T cell differentiation. Click here to expand this figure. Figure EV5. Absence of dose effect of miR-181a-bearing vector transduction in γδ T PBLs and thymocytes A. Retroviral transduction workflow for γδ PBLs. B. Gating strategy for the identification of the GFP+ versus GFP− cells. C. FACS analysis of the expression of indicated surface a
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