Human Glomerular Endothelial Cells Treated With Shiga Toxin Type 2 Activate γδ T Lymphocytes

The hemolytic uremic syndrome associated with diarrhea, a consequence of Shiga toxin (Stx)-producing Escherichia coli infection, is a common cause of pediatric acute renal failure in Argentina. Stx type 2a (Stx2a) causes direct damage to renal cells and induces local inflammatory responses that involve secretion of inflammatory mediators and the recruitment of innate immune cells. γδ T cells constitute a subset of T lymphocytes, which act as early sensors of cellular stress and infection. They can exert cytotoxicity against infected and transformed cells, and produce cytokines and chemokines. In this study, we investigated the activation of human peripheral γδ T cells in response to the incubation with Stx2a-stimulated human glomerular endothelial cells (HGEC) or their conditioned medium, by analyzing in γδ T lymphocytes, the expression of CD69, CD107a, and perforin, and the production of TNF-α and IFN-γ. In addition, we evaluated by confocal microscopy the contact between γδ T cells and HGEC. This analysis showed an augmentation in cellular interactions in the presence of Stx2a-stimulated HGEC compared to untreated HGEC. Furthermore, we observed an increase in cytokine production and CD107a expression, together with a decrease in intracellular perforin when γδ T cells were incubated with Stx2a-treated HGEC or their conditioned medium. Interestingly, the blocking of TNF-α by Etanercept reversed the changes in the parameters measured in γδ T cells incubated with Stx2a-treated HGEC supernatants. Altogether, our results suggest that soluble factors released by Stx2a-stimulated HGEC modulate the activation of γδ T cells, being TNF-α a key player during this process.


Cell-surface CD69 expression
Recovered γδ T cells (0.6x10 5 ) from previously described assays were incubated with saturating concentrations of mouse monoclonal antibodies anti-human CD69 conjugated to PE.Cy5 in PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (staining buffer) (30 minutes at 4°C). Then, cells were washed with 300 µl of staining buffer, then washed with PBS, and fixed in 1% paraformaldehyde. Afterward samples were analyzed by flow cytometry (FACSCalibur, BD Bioscience, CA, San Diego).
Cytokine production assay IFN-γ and TNF- production by  T cells (0.6x10 5 ), were quantified by ELISA in the supernatants recovered from the previously described assays, following conventional protocols provided by the manufacturer (Supplementary material, Table 2). For IL-6, IL-8 and TNF- production by HGEC, supernatants were collected after 24 hours of incubation with or without Stx2a (0.01 or 1 ng/ml) in growth-arrested conditions media, according to the provider recommendations (Supplementary material, Table 2).
CD107a expression γδ T cells were cultured with conditioned media obtained from monolayers of HGEC prestimulated or not with Stx2a (0.01 ng/ml, 24 h) as mentioned before, in the presence of saturating concentrations of mouse monoclonal antibodies anti-human CD107a conjugated to PE and monensin (2 µM) for 5 h at 37°C as previously described (Alter G et al., 2004). After the incubation time, cells were recovered, washed with 300 µl of staining buffer, then washed with PBS, and fixed in 1% paraformaldehyde. Afterward samples were analyzed by flow cytometry.

Intracellular cell staining of perforin and flow cytometry
After 4 h of incubation with the conditioned medium obtained from monolayers of HGEC pre-stimulated or not with Stx2a (0.01 ng/ml, 24 h), γδ T cells (0.6x10 5 ) were recovered and fixed in 2% paraformaldehyde for 20 minutes at 4°C. Then, cells were washed with PBS and permeabilized with PBS containing 0.5% BSA and 0.05% saponin (permeabilization buffer), for 20 minutes at 4°C. After incubation, cells were centrifuge and immunostained with saturating concentrations of mouse monoclonal antibodies antihuman perforin in permeabilization buffer, for 30 minutes at 4°C. Then, cells were washed with 300 µl of permeabilization buffer, and after that washed in PBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry. For the intracellular staining of TNF-α, γδ T cells (0.6x10 5 ) cultured with monolayers of HGEC (final concentration: 1x10 6 /ml), were treated with Brefeldin A (1µg/ml). After 5 hours of incubation, cells were recovered with PBS/5 mM EDTA, centrifuge, fixed in 1% paraformaldehyde (15 minutes at 4°C), and permeabilized with permeabilization buffer (20 minutes at 4°C). Afterward, cells were centrifuged, incubated with saturating concentrations of mouse monoclonal antibodies anti-human TNF-α conjugated to PE, in permeabilization buffer (30 minutes at 4°C). Then, cells were washed with 300 µl of permeabilization buffer, and then washed in PBS, fixed with 1% paraformaldehyde, and analyzed by flow cytometry. A gate based on size was done in the analysis to evaluate the expression of TNF-α in the γδ T cells.

Intracellular perforin expression by confocal microscopy
HGEC were seeded on fibronectin-coated glass coverslips (12 mm), overnight at 37°C. The next day, cells were stimulated or not with Stx2a (0.01 ng/ml) for 24 h. After that, the coverslips were washed with PBS, and γδ T cells (0.6x10 5 ) were incorporated and incubated at 37°C in 5% CO2 for 5 h. After incubation, the coverslips were carefully washed with PBS to discard non-adherent cells, and then adherent cells were fixed in 2% paraformaldehyde and stained for perforin. Briefly, after fixation, the samples were permeabilized with permeabilization buffer for 20 minutes. Afterward, samples were incubated with saturating concentrations of mouse monoclonal anti-human perforin or the corresponding isotype control, in permeabilization buffer for 45 minutes at 4°C. After that, cells were washed with permeabilization buffer and incubated with a DyLight549goat anti-mouse IgG antibody (9 µg/ml) for 30 minutes at 4°C. Finally, samples were washed with permeabilization buffer and fixed in 1% paraformaldehyde, and the coverslips were mounted onto glass slides using Fluoromount-G solution. Immunofluorescence images were acquired with a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) using a Plapon 60X/1.42 NA oil immersion objective. Images were analyzed using FIJI software (National Institutes of Health, Bethesda, MD).
Neutral red cytotoxicity assay: HGEC monolayers were treated or not during 24 h with Stx2a (0.01 and 1 ng/ml), in growth-arrested conditions. After treatment, freshly diluted neutral red (10 µg/ml) was added to the cells and incubated for an additional 1 h at 37°C in 5% CO2. Cells were then washed and fixed with 1% CaCl2 + 1% formaldehyde, and lysed with 1% acetic acid in 50% ethanol to solubilize the neutral red. Absorbance was measured in an automated plate spectrophotometer at 540 nm. Results were expressed as a percentage of cell viability, where 100% represents cells incubated under identical conditions but without toxin.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism v6.00 for Windows, GraphPad Software (La Jolla, CA, USA). Statistical significance was defined as p<0.05, by using nonparametric tests, with Dunn posttest for multiple competitions when necessary. Supplementary material, Table 1 A) Blood donors to purified  T cells:

Inclusion criteria
1) The donor of blood must be between 18 and 65 years old. Minors between 16 and 18 years old must have the written and signed authorization of their parents or legal representatives, expressing their consent to the donation process.
2) Hg ≥ 12.5 g/dl 3) Hematocrit ≥ 38%. 4) Beats per minute between 50 and 100. 5) Systolic blood pressure, between 90 and 180 mmHg. Diastolic pressure between 60 and 100 mmHg. People who have no other health considerations and who are taking medications to control their blood pressure can donate blood if their blood pressure is within acceptable limits. 6) Body weight equal to or greater than 50 kg.

Exclusion criteria
1) Having had viral hepatitis after age 10, other than Hepatitis A.
2) Have or have had clinical or laboratory evidence of infections by Tripanosoma cruzi, HIV, HTLV, HCV, and/or HBV.
3) Injecting drug users not prescribed by doctors. 4) Persons who suffer from Hemophilia or are hemodialysis or periodically receive transfusions of blood, its components or derivatives. 5) Have had repeatedly suffered from syphilis or gonorrhea. Those potential donors who report having suffered a single episode with complete and adequate treatment may be included in a readmission protocol with a medical interview and a negative screening test. 6) Are at risk for Creutzfeldt-Jakob disease, or its variant. Have a family history of the disease. 7) Have received pituitary hormone of human origin between 1958 and 1966. 8) Has received a brain tissue or membrane transplant. 9) Have been reside for more than one year (adding all the periods of stay) in the United Kingdom during the period from 1980 to 1996, or in countries that have had foci of infection by CJ V. 10) Not having suffered, or have been at risk of contracting infections liable to be transmitted by transfusion (ITT). Information related to travel or stay in areas with a high prevalence of endemic ITTs (leishmania, borrelia, dengue, Variant of the agent of Creutzfeldt-Jakob disease, West Nile virus, among others) should be collected. 11) Pregnancy contraindicates donation. Women will be excluded for 6 weeks after a normal delivery, 12 months after a caesarean section or an abortion followed by an evacuation curettage. It is recommended that nursing mothers do not donate blood. 12) People who have undergone endoscopies will be excluded for a period of 6 months. Regarding laparoscopies and surgeries, a medical evaluation is necessary before accepting as a donor. When it comes to uncomplicated surgeries, it should be postponed for six months after the intervention. The deferral should be extended to 12 months if the person received transfusions. 14) At the time of evaluation, no show signs or symptoms of fever.