Early Pregnancy Human Decidua Gamma/Delta T Cells Exhibit the Tissue Resident and Specic Functional characteristics

Background: A successful pregnancy is a complicated process that builds upon two aspects of the maternal immune system that needs to be balanced. As one of the dominant groups of cells at the maternal fetal interface, the decidual γδ T cells have attracted great research attention in normal pregnancy or miscarriage. However, the role of γδ T cells in fetal growth still remains poorly studied. Results: In this study, we identied γδ T cells were enriched and resident in decidua during early pregnancy, and early decidual γδ T cells were involved in the secretion of growth factors, including growth differentiation factor 15 (GDF15) and bone morphogenetic protein 1 (BMP1). Decrease of these growth factors could impaire fetal development, resulting in fetal growth restriction. We also observed that the early decidual γδ T cells exhibited stronger cytokine secretion characteristic, but its cytotoxicity against A549 cells was weaker, when compared with the γδ T cells in peripheral blood mononuclear cells (PBMCs). In addition, the functional abilities of early decidual γδ T cells in promoting trophoblast cell proliferation, migration, invasion and tube formation were also signicantly stronger than those in γδ T cells of PBMCs. Conclusions: These ndings highlighted the importance of γδ T cells in fetal growth and maternal immune tolerance during pregnancy, which is different from γδ T cells in PBMCs, and encouraged further research in this eld.

the decidual immune cells are different in composition, phenotype and function, and change with the stage of gestation [12].The immune cells were recruited in human early decidua, including decidua natural killer (dNK) cells (70%), macrophages (20%), and T cells (5-20%).Toward term decidua, the frequency of dNK cells decreased, while the frequency of T cells increased.The dynamic changes of immune cell composition also occurred during pregnancy in mice [13][14][15].So far, NK cells are the relatively well-studied immune cells at the maternal fetal interface [16][17][18][19], while the research of γδ T cells, another dominant cell group at the maternal fetal interface, and its role in pregnancy are far from understood and quite controversial.γδ T cells are an important subset of T lymphocytes that play important roles in innate and adaptive immunity via the secretion of various cytokines [20,21].Different from the traditional CD4 + and CD8 + T cells, γδ T cells express heterodimeric TCRs consist of gamma (γ) and delta (δ) chains [22].γδ T cells represented 5-10% of T lymphocytes in human peripheral blood, while γδ T cells accounted for over 30% of T cells in human early decidua.In addition, decidua γδ T also cells involved in maintenance of pregnancy by recognizing alloantigen without MHC restriction, producing cytokines and connecting the innate and adaptive immune responses as a bridge [23][24][25].However, some authors reported that there was no change in the proportion of γδT cells during pregnancy [26].Other study has observed changes of decidua γδT cell subtypes during pregnancy may associate with recurrent spontaneous abortion [27].These studies indicated that the study of γδT cells at maternal fetal interface was still relatively super cial, and the role of γδT in early pregnancy needed to be further explored [28].Moreover, γδ T cells have been studied for decades as an example of speci c immune cell with regulatory activity during the evolution of mammalian pregnancy [29][30][31].However, whether decidua γδ T cells participate in the early optimization of maternal nourishment of the fetus remains unknown.Here, we focus on whether early decidua γδ T cells are responsible for maintaining the nourishing function in the early fetus.We also examine speci c characteristics of decidual γδ T cells, and their regulatory effects on trophoblasts to evaluate the possible functional roles of decidual γδ T cells in the placentation and maintenance of normal pregnancy.


Results


γδ

enriched and resident in early decidua tissue at the the maternal fetal interface during human early pregnancy

To know chara
teristics of distribution of decidua γδ T at the human maternal fetal interface, immunohistochemical assay was carried out.We observed the decidual γδ T cells were mainly distributed around the glands as clusters, and were scattered in the decidual stromal cells as single cells (Fig. 1A).We also identi ed staining intensity of γδ T in early decidua tissue was signi cantly stronger than in term decidua tissue (Fig. 1B), revealed that γδ T cells were enriched in early decidua probably.So we further pro led the expression of γδ T in early decidua compared with term decidua, PBMCs of pregnant and PBMCs of non-pregnant through ow cytometry.We observed the proportion of γδ T (CD3 + CD45 + ) in early decidua (11.06 ± 1.65%) increased signi cantly compared with term decidua (7.54 ± 0.77%) and PBMCs of pregnant (6.13 ± 1.22%) (Fig. 1C, D).No difference in γδ T cell numbers in PBMCs of pregnant (6.13 ± 1.22%) women was detected compared with Term decidua (7.54 ± 0.77%) and PBMCs of non-pregnant (5.15 ± 1.36%) (Fig. 1C, D).Moreover, we also analyzed the main subsets of γδ T cells in early, term decidua and PBMCs.We identi ed Vδ1 subset accounted for higher percentages of γδ T cells in the early decidua.Conversely, the term decidua and PBMCs were dominated by the Vδ2 subset (Fig. 1E, F, G).These ndings showed γδ T cells were resident cells of decidual tissue showing speci c location and were highly enriched in early decidua.

2. The higher level of resident maker was identi ed in γδ T cells from early deicuda.

In order to further ex lore the resident characteristics of γδ T cells at the maternal fetal interface, human CD49a, CD69 and CD44 expression in γδ T cells were analyzed from decidua of the early pregnancy through ow cytometry compared to those from term decidua and PBMCs.We observed the higher proportion of CD49a in γδ T cells from early decidua (69.26 ± 7.07%) compared with term decidua (52.52 ± 8.53%) and PBMCs (15.04 ± 6.61%).The proportion of CD49a in γδ T cells from term decidua was also higher than from PBMCs (Fig. 2A, B).Moreover, more than 85% of γδ T cells from early decidua (92.15 ± 2.71%) and term decidua (88.13 ± 8.16%) expressed CD69 compared with PBMCs (6.29 ± 3.15%) (Fig. 2A,   B).However, no differences in the percentage of CD44 + γδ T cells were found in the early decidua compared to term decidua and PBMCs (Fig. 2A, B).Our ndings further showed that γδ T cells from decidua have resident characteristics in maternal fetal interface.


RNA-seq analysis of γδ T cell

indicated that signi cant function altered in early decidual

To explore whether γδ T cells
hat enriched in early decidua have speci c functions compared with term decidua and PBMCs, γδ T cells were puri ed and analyzed through mRNA sequencing.There were 1573 upregulated genes and 1391 downregulated genes between the early decidual γδ T cells and γδ T cells of PBMCs (Fig. 3A).There also were 1670 upregulated genes and 1365 downregulated genes between the early and term decidual γδ T cells (Fig. 3D).To further study the function of differentially expressed genes in early decidual γδ T cells, Go analysis was performed.Go analysis revealed that these differentially expressed genes altered in early decidual γδ T cells were mainly enriched in some biological pathways including cytokine activity, growth, growth hormone secretion, cell-cell adhesion mediator activity, growth factor activity (Fig. 3B,3E).Subsequently, we focused on 15 genes related to embryonic development and immune tolerance.Heatmap showing the different expression of selected genes in early decidual γδ T cells compared with that in term decidual γδ T cells and γδ T of PBMCs (Fig. 3C, 3F).These results indicated that signi cant function altered in early decidual γδ T cells compared with term decidual and PBMCs.Therefore, it was meaningful to further study the roles of the γδ T cells during human pregnancy.


Pregnancy-induced alterations in γδ T

ell Phenotype and Function

To know whether γδ T cells phenotype i
different during human pregnancy, we performed comparative analysis of the phenotype of γδ T cells, including NKG2D (a hallmark receptor for γδ T cells), HLA-DR (T cells activation marker), CD38 (immune cell activation maker), CD31 (platelet/endothelial cell adhesion molecule 1).We found that NKG2D, HLA-DR, CD38 and CD31 expressed at signi cantly higher levels in early decidual γδ T cells than γδ T cells from PBMCs (Fig. 4A).In addition, the expression of these makers in early decidual γδ T cells was higher than term decidual γδ T cells, except the expression of HLA-DR (Fig. 4B-4E).Furthermore, we explored the function of γδ T cells in early pregnancy.We rst analyzed the expression of TNF-α, IFN-γ, IL-10, IL-17a and TGF-β1 in γδ T cells via ow cytometry.TNF-α, IFN-γ and IL-17a are pro-in ammatory cytokines that mainly mediate the cellular immune response, while IL-10 is anti-in ammatory cytokines that mediate the humoral immune response and TGF-β1 could inhibit the differentiation of lymphocytes and the production of cytokines.Interestingly, we observed the expressed levels of TNF-α, IFN-γ, IL-10, IL-17a and TGF-β1 in early decidual γδ T cells were signi cantly higher than those in γδ T cells from PBMCs (Fig. 4F).Moreover, the proportion of TNF-α, IFN-γ and IL-17a in early decidual γδ T cells was also higher than those in term decidual γδ T cells (Fig. 4G-4K).

Cytotoxicity is a major function of γδ T cells and also an important indicator of the functional status of γδ T cells.We next explored the cytotoxicity of γδ T cells against A549 cells in early decidua, term decidua and PBMCs when effector cells: target cells were 20:1.We identi ed the cytotoxicity of γδ T cells in early decidua was signi cantly lower than that in term decidua and PBMCs (Fig. 4L, M).Meanwhile, cytotoxicity of γδ T cells in term decidua was also lower than that in PBMCs (Fig. 4L, M).Our ndings indicated that γδ T cells were more activated and secreted more cytokines during early pregnancy, but exhibited lower cytotoxicity.


γδ T cells are involved in secreting growth facto

in early decidua.

To validate the sequencing results, eight genes re
ated to maternal fetal interface were selected to validate using reverse transcription polymerase chain reaction (RT-PCR), including Sprouty2(SPRY2),Kisspeptin-1(KISS1), Insulin Like Growth Factor Binding Protein 5 (IGFBP5), platelet/endothelial cell adhesion molecule 1 (CD31), Insulin Like Growth Factor Binding Protein 2 (IGFBP2), vascular endothelial growth factor C (VEGFC), growth differentiation factor 15 (GDF15), bone morphogenetic protein 1 (BMP1).The qRT-PCR results were similar to those observed in sequencing results (Fig. 5A).Meanwhile, we want to further explore the role of γδ T cells in early embryonic development, which is different from that of immune cells.To identify genes promoting fetal development in early pregnancy, gene expression was measured in sorted γδ T cells from early decidua of pregnancy compared with the sorted γδ T cells from term decidua and PBMCs through immuno uorescence.We identi ed that γδ T cells from early decidua upregulated several growth factors, including IGFBP2, VEGFC, GDF15 and BMP1, compared to these products in γδ T cells from term decidua and PBMCs (Fig. 5B, C, D).In addition, in order to further determine that γδ T cells secrete growth factors to promote embryo growth and development, growth factor genes expression was analyzed in sorted γδ T cells from patients experiencing recurrent spontaneous abortion (RSA).Contrary to the high growth factors expression in early decidua γδ T cells, signi cantly decreased expression of growth factors, including IGFBP2, VEGFC, GDF15 and BMP1, were con rmed in γδ T cells from RSA patients via immuno uorescence and qRT-PCR (Fig. 5E, F).These growth factors are critical for the development of blood vessels, bone, and neurite in the fetus.Therefore, insu cient secretion of growth factors in γδ T cells from RSA patients may be responsible for the con ned fetal development.


Early decidual γδ T cells promote the human trophoblasts

gration and invasion

Extra villous trophoblast cells (EVTs) play a critical rol
in maintaining su cient maternal blood ow to support placental function and EVT invasion is an important process for fetal implantation and placenta formation [1,4,5].In order to explore whether γδ T cells have effects on the invasion and migration of trophoblast, we established γδ T cells and HTR8/SVneo cells, a human trophoblast cell line, co-culture system using a 8 µm Transwell insert.Isolated γδ T cells were placed in the lower chamber, while HTR8/SVneo cells were added the upper chamber with or without matrigel.We identi ed the numbers of migrated and invaded HTR8/SVneo cells increased signi cantly in early decidua compared with those in Control (HTR8/SVneo cells without isolated γδ T cells), term decidua and PBMCs (Fig. 6).Meanwhile, the numbers of migrated HTR8/SVneo cells in term decidua were higher than that in PBMCs (Fig. 6B).No difference in the numbers of migrated and invaded HTR8/SVneo cells between control and PBMCs was found (Fig. 6B, D).Our observations suggested early decidual γδ T cells could signi cantly promote the migration and invasion of trophoblast cells, while that the ability of γδ T cells in PBMCs to affect the migration and invasion of trophoblasts was obviously weaker.


γδ T cells promote the human trophoblasts proliferation and tube

ormation during early pregnancy

Trophoblast cells form villi through division, proliferation and
ifferentiation, and villi are an important part of placental formation [32,33].To evaluate the effect of γδ T cells on the proliferation of trophoblast cells.We established isolated γδ T cells and HTR8/SVneo cells directly co-culture system.Then we analyzed the proliferation ability of HTR8/SVneo cells via ow cytometry.7-AAD + Ki-67 + HTR8/SVneo cells were used to represent the cohort of S/G2-M proliferating cells.After co-culture of γδ T cells isolated from early decidua and trophoblast cells, we found the proportion of proliferating cells (S-G2/M) was signi cantly higher than those in control and the proportion of proliferating cells in term decidua was also higher than that in control (Fig. 7A, B).While the proportion of proliferating cells between PBMCs and control showed no signi cant differences (Fig. 7A, B).Previous studies reported that trophoblast was involved in the process of uterine spiral artery remodeling to form placenta [4,9].To determine whether the effect of γδ T cells on the tube formation of trophoblast cells, tube formation assays were performed.As shown in Figs.7C, decreased tube formation was observed in control and PBMCs compared with early decidua.The quantitative results demonstrated that the total tube length, the total branch length, and the number of nodes in HTR8/SVneo cells from early decidua signi cantly increased compare with those in PBMCs (Fig. 7D).Additionally, the total tube length and the number of nodes of HTR8/SVneo cell in early decidua were also higher than those in control (Fig. 7D).Analysis of the total tube length, the total branch length, and the number of nodes in PBMCs exhibited no differences compared with control (Fig. 7D).Our results showed that γδ T cells from early decidua exhibited the ability to promote trophoblast proliferation and tubule formation compared with γδ T cells in PBMCs.


Discussion

Maintaining a balance in decidual immune microenvironment is essen

al for norm
l pregnancy.Previous studies showed decidual γδ T cells were the key effector immune cells in the maternal-fetal interface in early pregnancy, which were associated with maintaining immune tolerance by producing cytokines [25,34].However, the role of decidual γδ T cells in the maternal fetal interface is not comprehensive, some of them are even a matter of debate.In this study, we found γδ T cells were enriched and resided in early decidua.We also identi ed early decidual γδ T cells were involved in the secretion of growth factors, and the loss of these growth factors impaired fetal development, resulting in fetal growth restriction.In addition, our studies revealed early decidua γδ T cells affected the function of trophoblast, which may have effects on the development of placenta.These ndings highlighted a novel and important role of γδ T cells in decidua tissue during pregnancy, a function independent of its antiviral and anti-infective role.

It has been reported that the percentage of γδ T cells increased during early pregnan y [34,35].Consistent with this, we found the percentage of γδ T cells increased in early decidua compared with term deicua and PBMCs via immunohistochemical assay and ow cytometry.We and others have not found any signi cant uctuation in number of γδ T cells in the blood between pregnant and non-pregnant women.We also identi ed decidua γδ T cells mainly localized and distributed around the decidua glands, which provide growth factors, cytokines and T-cell immunosuppressive factors, as well as nutrients in the intervillous space during early pregnancy [36][37][38].Therefore, we speculate that γδ T cells are a group of tissue resident cells in maternal fetal interface, and the expression of resident maker CD69 and CD49a in the decidua veri es our conjecture.In accordance with previous reports, we con rmed the majority of the human early decidual γδ T cells were Vδ1, while most of term decidua and PBMCs expressed Vδ2 [35,39,40].This result is consistent with the previous work which has described that immune tolerance dominates in the early decidua and gradually revises in the term decidua [41].The main differences in γδ T cell subsets and the inverse pattern of distribution among early decidua, term decidua and PBMCs may re ect their different immunoregulation functions in both compartments.

The percentage of γδ T cells increased in the decidua during the rst trimester of pregnancy ut signi cantly declined after the placenta is formed [25,29].We investigated whether these transient γδ T cells had speci c function during the early pregnancy.NGS and bioinformatic analysis were performed to gure out the differentially expressed mRNA in puri ed γδ T cells.It has been reported that decidua NK cells secreted growth factors to promote the growth and development of early embryos [18].We also focused on whether γδ T cells are involved in the secretion of growth factors to promote fetal growth and development.The data presented here showed that early decidua γδ T cells have speci c functions involved in secretion of growth factors, such as GDF15 and BMP1, and promoting fetal growth in human.

GDF15, a member of the TGF-beta superfamily, has been shown to be implicated in many biological p ocesses such as energy homeostasis and body weight regulation and has key roles in heart development [42,43].BMP1 is an astacin metalloprotease, which has key roles in the development and regeneration of bone and cartilage [44][45][46].Both growth factors are critical for fetal development.

We also found that the expression levels of T cell activated receptor, NKG2D and HLA-DR, on γδ T ce ls in early decidua were higher than that in term decidua and PBMCs.These data demonstrated that the activity of early decidua γδ T cells was higher in the maternal-fetal interface.Moreover, high expression of CD31, an endothelial cell adhesion molecule [47], was also detected in early decidua, which may be related to γδ T cells resident in early decidua.It is an indisputable fact that cytokines in the maternal fetal interface play an important role in the establishment and maintenance of normal pregnancy.Inconsistent with the previous report [25,34,48], we identi ed that both pro-in ammatory and anti-in ammatory cytokines are highly expressed in early decidua, we speculated that the function of secreting cytokines by decidual γδ T cells in early pregnancy might be stronger.Cytotoxicity is an important functional indicator of γδ T cells that arising from an abnormal balance between inhibitory and activating signaling [49].We demonstrated that early decidua γδ T cells exhibited lower cytotoxicity against A549 cells, this might suggest that decidua γδ T cells at the maternal-fetal interface are relatively immunosuppressive during early pregnancy and might also be a sign of enhanced tolerance of early decidua γδ T cells.

Trophoblast cells, as the key cells in placenta, play an important role in the growth and development of fetus [1].Trophoblast proliferation, migration and invasion are the basis of placental formation, which occurred through a series of intercellular signal transduction processes mediated by cytokines and hormones [50,51].At the top of anchored villi, these cytotrophoblasts proliferate and differentiate into extravillous trophoblast (EVT).Invasive EVT plays a positive role in the remodeling of uterine spiral artery.Therefore, insu cient proliferation, migration and invasion of trophoblast can lead to poor placental formation and affect fetal development, which is directly associated with the serious complications of human pregnancy, including preeclampsia and intrauterine growth restriction [52,53].In this study, we investigated whether γδ T cells affected placental trophoblast cells at the maternal-fetal interface during pregnancy.We identi ed early decidua γδ T cells could enhance the proliferation, migration and invasion of trophoblast cell line HTR8 / svneo compared with those in term decidua and PBMCs.Furthermore, Angiogenesis is a key step in the development of placenta and fetus.Insu cient angiogenesis may lead to early abortion [33,54].The data presented here showed that early decidua γδ T cells signi cantly promoted tube formation in HTR8/SVneo cells.These results revealed that early decidual γδ T cells participated in and promoted the function of trophoblast, but this function is weaker in term decidual γδ T cells and γδ T cells of PBMCs.

Although our current results clarify the characteristics of decidual γδ T cells, and decidual γδ T cells present a new role in early fetal development, our research still have some limitations that need to be considered.We found that early decidual γδ T cells are involved in the secretion of growth factors in early pregnancy, but the related mechanism needs to be further studied.In addition, due to the obvious species differences of γδ T cells, we chose clinical samples for indirect veri cation instead of in vivo experiments.

Although HTR8 / svneo cells have been shown to effectively reproduce key aspects of EVTs [55], the validity of using immortalized cell lines to represent the in vivo environment remains questionable [56].It is necessary to isolate and acquire primary trophoblast cells and conduct appropriate animal experiments.The mechanism of how decidua γδ T cells affect the function of trophoblast also require to further explore in order to provide stronger evidence for our current research in the following work.


Conclusions

In summary, we have provided evidence that γδ T cells are enriched and resident in decidua during early

regnancy, an
early decidual γδ T cells were involved in the secretion of growth factors.Moreover, Decrease of these growth factors could impaire fetal development, resulting in fetal growth restriction.

These ndings highlighted a novel and important role of γδ T cells in decidua tissue during pregnancy, a function indep ndent of its antiviral and anti-infective role.


Material And Methods


Study Population and Samples

Healthy pregnant women in early pregnancy, directed to elective

12 weeks, n = 41) and in term
pregnancy, directed to cesarean section (37-40 weeks, n = 30) were involved in the study.10 deciduas from abnormal pregnancies were obtained from patients who experienced recurrent spontaneous abortions.Women with endocrine and metabolic diseases (diabetes, hyperthyroidism, and hypothyroidism), hypertension, or infectious diseases (hepatitis, AIDS, syphilis, or any other bacterial or viral infection) were excluded.Written informed consent was taken from all subjects for the use of blood and tissue samples.The research protocol was approved by the Ethics Committee of West China Second University Hospital of Sichuan University and informed consent was obtained from all participants [Approval number: Medical research 2020 (029)].All samples were collected under sterile conditions.


The Isolation of Decidual Immunocytes and Peripheral Blood Mononuclear Cells

Decidual sample isolation was performed acco

ing to previous report [34].Brie y, fresh decidua samples were washed and min
ed into small pieces.Decidual tissues were released by digesting the tissues with 1 mg/mL collagenase type IV (Sigma, USA) and 150 U/mL of DNase I (Applichem, Germany) in RPMI 1640 medium for 1 hour at 37℃.The suspensions were strained through nylon mesh and then loaded onto a percoll density gradient (60%, 40%, 20%) to purify the lymphocytes.Blood samples were obtained in heparin anti-coagulated vacutainer tubes (BD Biosciences, USA).Peripheral blood mononuclear cells were isolated according to previous report [35].The DICs and PBMCs were re-suspended in RPMI1640 supplemented with 10% FBS, 1% penicillin and streptomycin, and cultured at 37℃, 5% CO2.


Human γδ T cells isolation

γδ T cells were separated from decidua and PBMCs by positively selection using a magnetic isolation

ll-Mediated Cytotoxicity Kit (Invitrogen
USA) was used to quantify dead cells.Brie y, 1*10 6 cells of target cells (A549 cells, a human lung adenocarcinoma cells line) were incubated with 10 mL DiOC for 20minutes at 37℃.Then the effector cells (γδ T) and target cells were mixed at a ratio of 20:1(E:T = 20:1) for 2 hours at 37℃.Dead A549 cells (DIOC + PI + ) were represented as cytotoxic signal in the upper right hand quadrant.


RNA Isolation and qRT-PCR

RNA was extracted using Trizol RNA Isolation Reagents (Invitrogen, USA) according to the manufacturer's instructions.Reverse transcription was conducted with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scienti c, USA) according to the manufacturer's instructions.qRT-PCR was performed using SYBR Green Master Mix (Thermo Fisher Scienti c) on an Applied Biosystems 7500 (Life technologies, USA).GAPDH was used as an internal control.Primers were shown in Table 1.


RNA-Seq

Total RNA was isolated by Trizol RNA Isolation Re

ents (Invitrogen, USA) according to the
anufacturer's protocol.Puri ed γδ T cells were used for the RNA-sEq.A total of 3 µg RNA of each sample was used to prepare libraries.RNA sequencing was performed using HiSeq 4000 (Illumina, San Diego, USA) by Novogene.


Transwell Invasion and Migration Assays

The invasive and migratory ability of HTR8/SVneo cells were evaluated using the Transwell assay as previously described [48].Brie y, Cell invasion was examined in 24-well Transwell chambers (8 µm, Corni g, USA).Each insert was coated with 50 mL Matrigel (Corning, USA) before the assay had been conducted.5*10 4 HTR8/SVneo cells, resuspended in 200 mL RMPI1640, were seeded in the upper chamber.While the same number of isolated γδ T cells, in 500 ml complete medium (with 10% FBS), were added to the bottom well.After 24 h incubation, the cells were xed and stained with crystal violet.The number of stained cells was determined by light microscopy.All experiments were performed in duplicates, and the invasion index was expressed as the proportion (%) of invaded cells compared with the corresponding control (cultured HTR8/SVneo without γδ T cells).The transwell migration assay was the same as for the invasion assay, except without Matrigel.


Proliferati

and Tube Formation A
says

For proliferation Assays, 1*10 5 HTR8/SVneo cells were cultured with RPMI 1640 complete medium (with 10% FBS) in a 24-well plate for adherent culture.Thereafter, 1*105 isolated γδ T cells were directly cocultured with HTR8/SVneo cells for 24 h.Then, the cells were washed twice, collected, xed

permeabilized, and stain
d with 7-AAD and

Assays, HTR8/SVneo c
lls were cultured with RPMI 1640 supplemented with 10% FBS attach to the bottom of the 24-well plate.Afterward, isolated γδ T cells were seeded in the 0.4 µm upper chamber and