Adenosine increases PD‐L1 expression in mesenchymal stromal cells derived from cervical cancer through its interaction with A2AR/A2BR and the production of TGF‐β1

Mesenchymal stromal cells (MSCs) together with malignant cells present in the tumor microenvironment (TME), participate in the suppression of the antitumor immune response through the production of immunosuppressive factors, such as transforming growth factor beta 1 (TGF‐β1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF‐β1 by interacting with A2AR/A2BR. In the present study, we provide evidence that Ado induces the production of TGF‐β1 in MSCs derived from CeCa tumors (CeCa‐MSCs) by interacting with both receptors and that TGF‐β1 acts in an autocrine manner to induce the expression of programmed death ligand 1 (PD‐L1) in CeCa‐MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T lymphocytes. The addition of the antagonists ZM241385 and MRS1754, specific for A2AR and A2BR, respectively, or SB‐505124, a selective TGF‐β1 receptor inhibitor, in CeCa‐MSC cultures significantly inhibited the expression of PD‐L1. Compared with CeCa‐MSCs, MSCs derived from normal cervical tissue (NCx‐MSCs), used as a control and induced with Ado to express PD‐L1, showed a lower response to TGF‐β1 to increase PD‐L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF‐β1, and the induction of PD‐L1 in CeCa‐MSCs to suppress the antitumor response of CD8+ T lymphocytes. The findings of this study suggest that this pathway may have clinical importance as a therapeutic target.


| INTRODUCTION
Cervical cancer (CeCa) is the fourth most common type of cancer in women and represents a major public health problem worldwide.In 2020, approximately 600,000 new cases and 340,000 deaths were reported, more than 80% of which occurred in developing countries. 1,2Persistent high-risk human papillomavirus (HR-HPV) infection is an important risk factor for the development of low-grade squamous intraepithelial lesions (LSILs), which can progress to highgrade squamous intraepithelial lesions (HSILs) and eventually to CeCa. 3 The immune response against HPV antigens can eliminate most precursor infections and lesions; however, some women exposed to HR-HPV will develop cancer, suggesting that other risk factors may be involved. 4Recently, it was proposed that the intrinsic factors of tumor cells and immunosuppressive cells present in the tumor microenvironment (TME), such as mesenchymal stromal cells (MSCs), may participate in the suppression of the antitumor immune response. 5,62][13][14][15][16] MSCs have been reported to exert important functions to promote and sustain tumor growth: (a) they favor the proliferation of tumor cells 17,18 ; (b) they propitiate conditions to increase the number of tumor stem cells 19 ; (c) they promote angiogenesis and epithelialmesenchymal transition (EMT) of tumor cells to promote metastasis 20 ; (d) they suppress the antitumor immune response through the production of Th2 (IL-4, IL-5, IL-10) and Th3 (TGF-β) type cytokines 6,21 ; and (e) they favor the recruitment of regulatory T lymphocytes (Tregs) and the production of immunosuppressive factors such as PGE2, Indoleamine 2,3-dioxygenase (IDO), and nitric oxide (NO). 22,23 recent years, it has been shown that MSCs use the adenosinergic pathway to exert immunosuppressive effects on cytotoxic T lymphocytes (CTLs). 24,25Through this pathway, ATP/ ADP nucleotides found in high concentrations in the TME (>100 μM) 26 are converted to adenosine monophosphate (AMP) by the hydrolytic activity of the ectoenzyme CD39 (ectonucleoside triphosphate diphosphohydrolase − 1, ENTPD1; EC 3.6.1.5)and later to adenosine (Ado) through 5′-ectonucleotidase (CD73, EC 3.1.3.5). 27,28Most of the signaling activities of Ado are mediated by specific receptors (ARs) arranged in the membrane of target cells, where they are coupled to G proteins.These receptors are divided into four subtypes: A 1 R, A 2A R, A 2B R, and A 3 R. 29 It has been reported that Ado produced by MSCs, through the CD73-Ado pathway, suppresses the proliferation, maturation, and cytotoxic function of T lymphocytes by activating A 2A R. 24,30 The expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells, tumor-associated macrophages, dendritic cells (DCs), T regs and MSCs present in the TME can attenuate the ability of the immune system to successfully eliminate tumor cells because when the PD-1 receptor binds to its ligands, PD-L1 and/or PD-L2, Tcell receptor (TCR) signaling is negatively regulated, resulting in the inhibition of effector T lymphocytes. 31,32Cytokines IL-1a, IL-10, IL-27, and IL-32g produced in the TME can upregulate the expression of PD-L1 in both monocytes and tumor cells in culture. 33,346][37] It has been reported that the expression of PD-1 and PD-L1 in cervical T cells and DCs, respectively, is associated with HR-HPV positivity and with

Significance statement
This study provides the first evidence that Ado, when interacting with A 2A R or A 2B R in CeCa-MSCs, induces the production of TGF-β1 and PD-L1 expression through autocrine action, resulting in an increase in the immunosuppressive capacity of CeCa-MSCs on activated CD8+ T lymphocytes.
the degree of development of CeCa. 38In addition, TGF-β plays a crucial role in the development of CeCa by generating a local immunosuppressive microenvironment in the cervix in HR-HPV infection and inhibiting the proliferation and activation of T lymphocytes with antitumor activity. 39r research group has previously reported that CeCa-MSCs produce higher amounts of Ado than do NCx-MSCs because they present a higher content of CD73 in the cell membrane. 24Likewise, we reported that Ado induced the production of TGF-β in CeCa cells when interacting with A 2A R/A 2B R. 40 Therefore, in this study, we analyzed the participation of Ado in the induction of PD-L1 in CeCa-MSCs and the ability of these cells to inhibit the proliferation and activation of CD8+ T lymphocytes.We found that Ado, through the interaction with A 2A R/A 2B R and the production of TGF-β, induced the expression of PD-L1 in CeCa-MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T cells.

| MSCs
NCx-MSCs were obtained from tissue samples from three people who underwent a noncancer hysterectomy.CeCa-MSCs were obtained from biopsies of three patients with stage IIIB CeCa whose histopathological diagnosis was confirmed by the Department of Pathology.The local ethics committee approved these procedures.

| Expression of PD-L1 molecules in MSCs
Negative Control Compensation Particles Set (BD Biosciences).

| Statistical analysis
For the analysis of the expression of PD-L1 molecules by flow cytometry, the Kruskal-Wallis test was used.To analyze the differences between the treatments, one-way ANOVA and Fisher's exact test were used.p < .05 was the threshold for significant differences.

| Ado induces the expression of PD-L1 in
CeCa-MSCs through the interaction with A 2A R/A 2B R and the autocrine action of TGF-β1 Our research group previously reported that CeCa-MSCs express higher amounts of CD73 in the cell membrane than do NCx-MSCs, and through the generation of Ado, they strongly inhibit the effector functions of CD8+ CTLs. 246][37] Taking that information into account, in this study, we analyzed the effect of Ado on the production of TGF-β1 and in the induction of PD-L1 in CeCa-MSCs.
NCx-MSCs cultured under the same conditions were used as a control.The basal expression of PD-L1 in CeCa-MSCs was higher than that in NCx-MSCs, with mean fluorescence intensity (MFI) values of 15226 and 9247, respectively (Figure 1A).In contrast, the Mv1Lu cell line derived from N. vison, 42 which was used as a negative control, showed very low labeling with the anti-PD-L1 antibody (Supporting Information: Figure 1).Ado induced the expression of PD-L1 in both MSC types in a dose-dependent manner.When using 1 mM Ado, the expression of PD-L1 in the membrane of NCx-MSCs and CeCa-MSCs doubled and tripled, respectively, in relation to the baseline expression of this protein (Figure 1B).The increase in the expression of PD-L1 in both MSC types was dependent on the culture time in the presence of Ado, with the highest expression of this molecule occurring at 72 h (Figure 1C).Therefore, the culture of For determination of whether TGF-β1 induces the expression of PD-L1, MSCs were cultured for 24 h in the presence of different concentrations of rhTGF-β1 (0, 5, 10, 15, and 20 ng/mL).The higher induction of PD-L1 on NCx-MSCs and CeCa-MSCs was observed using 20 ng/mL of rhTGF-β1 (Figure 3A).The effect of 20 ng/mL of rhTGF-b in the induction of PD-L1 on MSCs was also determined at 48 and 72 h of culture.After 48 h of culture, the expression of PD-L1 in CeCa-MSCs decreased gradually.In contrast, NCx-MSCs marginally increased PD-L1 expression after 72 h of culture (Figure 3B).For determination of whether TGF-β1 acts in an autocrine manner to induce the expression of PD-L1, MSCs were cultured in the presence or absence of 1 mM Ado and in the presence or absence of 25 μM SB-505124, a selective inhibitor of the TGF-β1R.The expression of TGF-β1R was previously analyzed in MSCs, using an anti-TGFβ R type II (anti-TGF-β RII), and CeCa-MSCs showed higher expression of TGF-β1R than did NCx-MSCs, with MFI values of 5127 and 420.

Interestingly both types of MSCs cultured in the presence of 1 mM
Ado for 72 h showed strongly increased expression of TGF-β1R (Supporting Information: Figure 3).The addition of SB-505124 to the cell cultures decreased the expression of PD-L1 induced by Ado by more than 80% in CeCa-MSCs and by approximately 15% in NCx-MSCs (Figure 3C).Moreover, blocking A 2A R and A 2B R in MSCs cultured in the presence of 1 mM Ado reduced the expression of PD-L1 by 95% and 60% in CeCa-MSCs, respectively, and by 50% and 20% in NCx-MSCs, respectively (Figure 3D).In summary, these results suggest that the interaction of Ado with A 2A R and A 2B R in CeCa-MSCs plays an important role in the induction of PD-L1 expression through the production of TGF-β1.

| Ado-stimulated CeCa-MSCs inhibit the proliferation of CD8+ T lymphocytes through the expression of PD-L1
For analysis of the immunosuppressive effect of MSCs through the expression of PD-L1, NCx-MSCs, and CeCa-MSCs were cultured

| DISCUSSION
Elucidation of the mechanisms that promote PD-L1 expression in the TME is of interest because the PD-1:PD-L1 pathway has been recognized as the dominant immune checkpoint (ICP) in cancer. 43,44though this pathway has been targeted with some success in cancer therapy, current drug development strategies aim to overcome the failure of many drugs designed to block the PD-1 pathway and address relapses that can occur in cancer patients after initial tumor regression. 45,46The PD-L1 molecule can be expressed in various types of cells resident in the TME, including tumor cells, immune cells, endothelial cells, and even MSCs.Therefore, the expression of this ligand in tumors has been proposed as a predictive biomarker for the therapeutic effects of anti-PD-1 and anti-PD-L1 drugs. 47Tumor cells have been proposed to express PD-L1 through innate or adaptive resistance to immunity. 48Innate resistance refers to the constitutive expression of PD-L1 in tumor cells, resulting from the amplification of the PDL1 gene or the aberrant activation of oncogenic signaling pathways [49][50][51][52][53] ; adaptive resistance to immunity refers to the expression of PD-L1 in tumors or immune cells in response to inflammatory factors secreted in the TME during antitumor immune responses, where it is known that IFN-γ is one of the main cytokines responsible for inducing the adaptive expression of PD-L1. 54However, several additional TME-resident cytokines that can upregulate PD-L1 expression have been identified, including IL-1a, IL-10, IL-27, and IL-32g. 33,34,55,56Similarly, there is evidence that TGF-β regulates the expression of PD-L1 in different tumor models [35][36][37] and that this cytokine promotes the enrichment of PD-L1 in exosomes derived from breast cancer tumor cells. 57 the particular case of CeCa, the expression of PD-L1 is increased in LSIL and HSIL as well as in HR-HPV-positive CeCa, with PD-L1 considered a marker of poor prognosis. 38,58,59Likewise, the expression of PD-1 and PD-L1 in T lymphocytes and CDs, respectively, has been associated with HR-HPV positivity and with the degree of development of squamous intraepithelial lesions (SILs).
In fact, in CeCa tumors, PD-1 has been found to be expressed in a large number of infiltrating CD8 T lymphocytes. 60In a recent study, CeCa patients with tumors with high levels of PD-L1 or PD-L2 mRNA and high IFN-γ signaling activity had better overall survival than did patients with high PD-L1 expression and low IFN-γ activity, 61 suggesting that other factors present in the TME may play individual or synergistic roles in the regulation of PD-L1/PD-L2. 62Ado is produced at sites of metabolic stress associated with hypoxia, ischemia, trauma or inflammation and even in the TME through the activity of the adenosinergic pathway.In previous studies, we reported that CeCa-MSCs produce higher amounts of Ado than do NCx-MSCs because they present a higher CD73 content in the cell membrane. 24We also reported that Ado induces the production of TGF-β1 in CeCa cells when interacting with A 2A R/ A 2B R. 40 In the present study, evidence was provided, for the first time, that Ado induces the production of TGF-β1 in CeCa-MSCs by interacting with A 2A R and A 2B R and that this cytokine acts in an autocrine manner to increase the expression of PD-L1 molecules in the membrane and favor the immunosuppressive activity of CeCa-MSCs on CD8+ T lymphocytes (Figure 5).Interestingly, this phenomenon was also observed in NCx-MSCs, although with less The development of CeCa has been strongly associated with the production of TGF-β1, whose expression is directly correlated with the degree of progression of CeCa and suppression of the antitumor immune response. 3937]63 TGF-β1 is an immunoregulatory factor that is involved in tumor progression and immunosuppression, and the sources of this factor in the TME include several types of stromal cells, in addition to cancer cells. 64,65We recently reported that CeCa-MSCs are capable of inducing the expression and secretion of TGF-β in CeCa tumor cells. 66and that this factor is responsible for maintaining the expression of CD73 in CeCa cells. 40Based on our results, we suggest that Ado, as a participant in this feedback loop, may also participate in inducing and maintaining the expression of PD-L1 and increase the immunosuppressive capacity of the cells present in the CeCa TME, as was observed by culturing CeCa-MSCs in the presence of Ado and coculturing them with activated CD8+ T lymphocytes.In recent studies, through the design of bispecific recognition proteins, joint TGF-β and PD-L1 blockade has been proposed for the treatment of cancer. 67In this sense, the use of bintrafusp alfa (BA), a dual blocking protein against TGF-β and PD-L1, has been shown to decrease the size of tumors in a model of colorectal cancer. 68Furthermore, encouraging results have been obtained in phase I clinical trials with patients with advanced CeCa when using this drug. 69Therefore, blocking the activity of the adenosinergic pathway to inhibit the production of Ado, as well as its signaling to induce the production of TGF-β and the subsequent expression of PD-L1 in the TME, could contribute to improving the design of combinatorial therapies against CeCa and other types of cancer.

| CONCLUSION
In the present study, the results indicate, for the first time, that Ado induces the production of TGF-β1 in CeCa-MSCs by interacting with A 2A R and A 2B R and that this factor acts in an autocrine manner to induce the expression of PD-L1 in CeCa-MSCs and increase their immunosuppressive capacity on activated CD8+ T lymphocytes.

2. 3 | 24 2. 4 |
Obtaining activated CD8+ T lymphocytes (ATLs) CD8+ T lymphocytes were obtained from normal donor peripheral blood mononuclear cells (PBMCs) by negative selection (EasySep Enrichment Cocktail, Stem Cell Technologies).CD8+ T lymphocytes were resuspended in supplemented ISCOVE'S medium (SIM), composed of ISCOVE'S-modified medium (Sigma-Aldrich), 10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 20 μM 2mercaptoethanol and a mixture of nonessential amino acids (Gibco BRL), in addition to antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).Subsequently, the CD8+ T lymphocytes were cultured for 72 h in the presence of beads coated with anti-CD2/CD3/CD28 antibodies (Miltenyi BioteGmbH) at a ratio of 2:1 bead:T-lymphocyte and 100 U/mL recombinant interleukin-2 human (hrIL-2) in a total volume of 200 μL, as previously reported.Inhibition of ATLs by MSCs For analysis of the immunosuppressive effect of MSCs, NCx-MSCs and CeCa-MSCs were cultured with 1 mM Ado (NCx-MSCs/Ado and CeCa-MSCs/Ado) or without Ado (NCx-MSCs and CeCa-MSCs) for 72 h.The cells were subsequently fixed 4% paraformaldehyde (Sigma-Aldrich), washed two times with SIM and co-cultivated with activated CD8+ T lymphocytes (ATLs) in different ratios ATLs: MSCs (5:1, 10:1, and 20:1) for 96 h.For determination of whether the Ado signaling-induced expression of PD-L1 participates in the inhibition of the proliferation of ATLs, ATLs were cocultured with MSCs or MSCs/Ado for 96 h in a ratio 5:1 in the presence or abscence of the neutralizing anti-PD-L1 antibody (Novus) according to the Novus protocol.The proliferation of ATLs was determined using the CellTiter 96 ® Aqueous One Solution reagent (Promega) following the manufacturer's instructions.The bioreduction of tetrazolium [3-(4,5-dimethylthiazol-2-yl)−5-(3 carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium, inner salt; MTS] to formazan was analyzed at a wavelength of 490 nm in an ELISA plate reader on GloMax equipment (Promega).The proliferation index (PI) of the ATLs was determined every 24 h and was calculated based on the optical density (OD) of the bioreduction of MTS to formazan by ATLs in culture.The OD obtained at the beginning of the culture was normalized to 1.

MSCs for 72 h in the presence of 1 mM
Ado was used in subsequent experiments.NCx-MSCs and CeCa-MSCs cultivated for 72 h in the presence of 1 mM Ado increased the production of TGF-β1.In fact, after 24 h of culture, there was a significant increase in TGF-β1 content in the supernatants of the cultures of the NCx-MSCs (black bars) and the CeCa-MSCs (gray bars) with respect to that in the cells cultured in the absence of Ado, which were used as controls (Ctl) (Figure2A).To analyze whether A 2A R or A 2B R participate in the Ado signalinginduced production of TGF-β1, MSCs were cultured for 72 h in the presence or absence of 10 µM ZM241385 and MRS1754, antagonists specific for A 2A R and A 2B R, respectively, and in the presence or absence of 1 mM Ado.The effect of Ado on the expression of A 2A R and A 2B R in the MSCs was previously analyzed.CeCa-MSCs showed higher basal expression of A 2A R and A 2B R than did NCx-MSCs.However, when the MSCs were cultured in the presence of 1 mM Ado for 72 h, the expression of A 2A R was essentially not modified in NCx-MSCs, but increased more than 65% in CeCa-MSCs; moreover, the expression of A 2B R increased more than 75% on NCx-MSCs and decreased in approximately 30% on CeCa-MSCs (Supporting Information: Figure2).In contrast, blocking A 2A R or A 2B R significantly reduced TGF-β1 production in CeCa-MSCs, whereas only the blocking of A 2B R significantly reduced the production of TGF-β1 in NCx-MSCs (Figure2B).These results indicate that Ado signaling through A 2A R and A 2B R is important to induce TGF-β1 production in CeCa-MSCs, whereas in NCx-MSC is through A 2B R.

with 1 mM
Ado (NCx-MSCs/Ado and CeCa-MSCs/Ado) or without Ado (NCx-MSCs and CeCa-MSCs) for 72 h.The cells were subsequently fixed with paraformaldehyde, washed, and cocultured with activated CD8+ T lymphocytes (ATLs) in different ratios with MSCs (5:1, 10:1, and 20:1) for 96 h.The proliferation exhibited by the independently grown ATLs was considered a positive control.The PI of the ATLs determined at the beginning of the culture was normalized to 1.After 96 h of culture, the PI for the ATLs was 2.2-2.6 (Figure4A-C, black lines).The addition of NCx-MSCs at only a ratio of 5:1 (ATLs:NCx-MSCs) inhibited the proliferative capacity of the ATLs, with a PI of 1.6.In contrast, the addition of CeCa-MSCs inhibited the proliferative capacity of the ATLs at all ratios, with PIs of 1, 1.6, and 1.5 at ratios of 5:1, 10:1, and 20:1, respectively (Figure4A, red lines).Interestingly, the higher inhibition of the proliferation of ATLs was observed with the addition of NCx-MSCs/Ado or CeCa-MSCs/Ado to the cultures.At ratios of 5:1, 10:1, and 20:1 of ATLs: NCx-MSCs/Ado, PIs of 1.2, 1.7, and 1.6, respectively, were obtained; while the PIs of ATLs cultured with CeCa-MSCs/Ado were 0.6, 0.9, and 1,25, respectively (Figure 4A, blue lines).For determination of whether the Ado signaling-induced expression of PD-L1 participates in the inhibition of the proliferation of ATLs, ATLs were cocultured with MSCs or MSCs/Ado for 96 h in a ratio 5:1 in the presence or abscence of the neutralizing anti-PD-L1 antibody.The addition of F I G U R E 1 Effect of Ado on the expression of PD-L1 in MSCs.(A) The baseline expression of PD-L1 in MSCs obtained from normal cervical tissue (NCx-MSCs) and CeCa tumors (CeCa-MSCs) is shown.The Mv1Lu cell line was used as a negative control for PD-L1 expression.The MSC types, 1 × 10 5 of each, were cultured for 72 h in the presence or absence of 0.01, 0.1, and 1 mM Ado (B) or 1 mM Ado for 24, 48, and 72 h (C).The expression of PD-L1 was analyzed by flow cytometry.The baseline expression of PD-L1 in each MSC type was normalized to 1. Representative data from three independent experiments ±SEM.*p < .05,**p < .01,# p < .05,one-way ANOVA.anti-PD-L1 to ATL cultures with NCx-MSCs and CeCa-MSCs reversed the inhibition of ATL proliferation with PIs of 1.4 and 1, respectively (Figure 4A,B red line), to PIs of 1.55 and 1.5, respectively (Figure 4A,B, purple line).Interestingly, the addition of anti-PD-L1 to ATL cultures with NCx-MSCs/Ado and CeCa-MSCs/Ado significantly reversed the inhibition of ATL proliferation with PIs of 1.2 and 0.65, respectively (Figure 4A,B, blue line), to PIs of 2.4 and 2.25, respectively (Figure 4A,B, green line).Importantly, the NCx-MSCs, CeCa-MSCs, NCx-MSCs/Ado, and CeCa-MSCs/Ado used in the cultures with ATLs did not present proliferative activity when they were cultured independently for 96 h (data not shown).The expression of PD-1 in the ATLs was verified by flow cytometry (Supporting Information: Figure 4).These results suggest that Ado strongly increases the immunosuppressive capacity of CeCa-MSCs through the induction of PD-L1 expression promoted by TGF-β1 production.

F I G U R E 2
Ado induces the production of TGF-β1 in MSCs by interacting with A 2A R and A 2B R. (A) NCx-MSCs and CeCa-MSCs, 1 × 10 5 each, were cultured for 24, 48, and 72 h in the presence or absence (Ctl) of 1 mM Ado. (B) The MSCs were previously incubated for 30 min in the presence of 10 µM ZM241385 and MRS1754, antagonists specific for the A2a and A2b receptors, respectively, and subsequently cultured for 72 h in the presence or absence of 1 mM Ado.Cells cultured in the absence of Ado and AR antagonists were used as controls.The TGF-β1 content in the supernatants was determined by ELISA.Representative data from three independent experiments ±SEM.*p < .01,**p < .001,***p < .0001,# p < .05,one-way ANOVA.
intensity because unlike CeCa-MSCs, NCx-MSCs stimulated with Ado produced lower amounts of TGF-β1 and showed a lower response to the induction of PD-L1 expression when they were cultured in the presence of rhTGF-β1.The production of TGF-β1 in CeCa-MSCs stimulated with Ado and cultured in the presence of specific antagonists for A 2A R or A 2B R was inhibited by more than 95%, even though the contents of this cytokine were lower than those detected in the culture supernatants of CeCa-MSCs used as controls.TGF-β1 production in the CeCa-MSCs cultured only in the presence of the A 2A R or A 2B R antagonist also decreased significantly (data not shown).The reduced TGF-β1 production caused by the addition of the A 2A R or A 2B R antagonist or by the addition of a TGF-β1 receptor blocker (SB-505124) to CeCa-MSC cultures was key to reversing the effect of Ado on the induction F I G U R E 3 TGF-β1 induces the expression of PD-L1 in CeCa-MSCs.NCx-MSCs and CeCa-MSCs, 1 × 10 5 each, that were cultured for 24 h in the presence of different concentrations (0, 5, 10, 15, and 20 ng/mL) of recombinant human TGF-β1 (rhTGF-β1) (A) or in the presence of 20 ng/ mL of rhTGF-β for 24, 48, and 72 h (B).The MSCs were cultured for 72 h in the presence or absence of 1 mM Ado and in the presence or absence of 25 μM SB-505124, a selective TGF-β1receptor inhibitor (C).The MSCs were previously incubated for 30 min in the presence of 10 µM ZM241385 and MRS1754 and subsequently cultured for 72 h in the presence or absence of 1 mM Ado (D).The expression of PD-L1 was analyzed by flow cytometry.Representative data from three independent experiments ±SEM.*p < .01,**p < .001,***p < .0001,one-way ANOVA.F I G U R E 4 Ado-stimulated MSCs inhibit the proliferation of CD8+ T cells through the expression of PD-L1.(A) The proliferation indices (PIs) of activated CD8+ T lymphocytes (ATLs) cultured for 24, 48, 72, and 96 h are shown in the absence (black lines) or in the presence of MSCs at different ratios (ATLs:MSCs, 5:1, 10:1, and 20:1) cultured either in the absence (NCx-MSCs or CeCa-MSCs) (red lines) or in the presence of 1 mM Ado (NCx-MSCs/ Ado or CeCa-MSCs/Ado) (blue lines).In some cultures, the neutralizing anti-PD-L1 antibody was added to inhibit the effect of PD-L1 on the proliferation of ATLs in coculture with NCx-MSCs (B) or CeCa-MSC (C) previously cultivated in the absence (purple lines) or presence (green lines) of 1 mM Ado.The PI for the ATLs determined at the beginning of the cultures was normalized to 1. Representative data from three independent experiments ±SEM.*p < .05,**p < .01,***p < .001two-way Fisher's exact test.ofPD-L1 expression in these cells.This finding suggests that TGF-β1, present in the CeCa TME, may be involved in adaptive resistance to immunity that promotes the expression of PD-L1 in CeCa-MSCs and probably in other cells present in the TME.Additionally, compared with CeCa-MSCs, NCx-MSCs showed a lower response to TGF-β1 with regard to the increase in the expression of PD-L1.However, stimulation with Ado increased the expression of PD-L1 in these cells.This interesting phenomenon may be associated with factors other than TGF-β1 that can regulate the expression of PD-L1 on NCx-MSCs, since the addition of SB-505124 to NCx-MSC cultures in the presence of Ado did not reverse the effect of this nucleoside to induce the expression of PD-L1, and treatment with Ado also strongly increased the expression of TGF-β1R on these cells.