Blood was collected from 4 unrelated healthy subjects (aged 32.5±7.05-year-old) from a blood vessel in the arm, inside of the elbow or wrist, at the laboratory of the Nephrology Department, University of Thessaly. In order to exclude any pre-sensitization event, all subjects were males without a history of blood transfusion. Written informed consent was obtained from each individual enrolled; the present study was approved by the Ethics Committee of the Faculty of Medicine, University of Thessaly (Larissa, Greece) (approval no. 558/10-2-2017).

PBMCs were isolated from whole blood samples by Ficoll-Hypaque density gradient centrifugation at 600 x g for 25 min at 18-20˚C using Histopaque-1077 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The interface was collected and washed with RPMI-1640 medium (Sigma Aldrich; Merck KGaA). To count the isolated PBMCs, a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and an optical microscope at x40 (x4 objective) were used. Cell viability was assessed using the trypan blue exclusion assay (Sigma-Aldrich; Merck KGaA) and for each PBMC sample cells were counted in the four fields of the Neubauer chamber. All cell cultures were performed using RPMI-1640 medium, supplemented with 2 mM L-glutamine, 10 mM HEPES, 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA) and 1% antibiotic-antimycotic solution containing penicillin, streptomycin and amphotericin B (Sigma-Aldrich; Merck KGaA). Cultures were incubated at 37˚C in an atmosphere of 95% relative humidity and 5% CO₂.

The concentrations of 1-MT (100 µM), TRP (0.25 mM) and CH223191 (3 µM) were selected according to previous studies (8,29,30). In particular, the concentration of 100 µM for 1-MT was selected according to the results that were repeatedly obtained from several of our previous studies, which revealed efficacy in inhibiting IDO and reduced L-tryptophan consumption in mixed lymphocyte reactions (MLRs) without toxicity (9-12,29). In addition, an LDH release assay for the selected concentrations of the aforementioned substances was performed in resting PBMCs seeded in 96-well plates (1x10⁵ cells/well) and cultured at 37˚C for a 7-day period. The LDH release assay was performed using the Cytotox Non-Radioactive Cytotoxic Assay kit (cat no. G1780; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols. LDH release was calculated by the following equation: LDH release (%) = (LDH in the supernatant / total LDH) x 100. All experiments were performed in triplicate and the results were presented as the mean of the three measurements.

Of note, our previous study originally planned to apply the sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) assay to analyze cell proliferation. This assay comprises a colorimetric technique that assesses actual cytosolic NADH content, a key compound in the mitochondria, in which the tricarboxylic acid cycle takes place (31). The results revealed that IDO inhibition by 1-MT significantly decreased mitochondrial function in PBMCs stimulated with lymphocyte-specific stimulus tetanus toxoid (TT), which was unexpected. In addition, compared with untreated cells, TT stimulation notably increased the optical density (OD) value from the XTT assay, indicating enhanced mitochondrial function. On the contrary, in cells treated with TT and 1-MT, the XTT assay presented a significantly lower OD value compared with TT-treated cells. This decrease suggested that IDO enhances mitochondrial function in stimulated PBMCs, and its inhibition by 1-MT may markedly abrogate this effect. Additionally, as 1-MT did not alter the proliferation of TT-stimulated lymphocytes, the use of tetrazolium dyes for assessing cell proliferation may not be reliable (32).

MLRs are ex vivo cellular immunoassays that occur between two genetically distinct allogeneic lymphocyte populations of the same species. In a one-way MLR, only one lymphocyte population can respond or proliferate. In a two-way MLR, the two populations can proliferate. MLRs are performed to assess the response of T cells to external stimuli. The assay comprises the purification of responder lymphocytes from peripheral blood followed by co-culturing with stimulator cells. Stimulator cell populations that also contain T cells (two-way MLR) can replicate in the presence of the responder cells. Therefore, for a one-way MLR, stimulator cells are prevented from replicating by mitomycin C, a DNA crosslinker to restricT cell replication. The maximal measurable cellular proliferation occurs ~5-7 days of the MLR (33).

In the present study, two-way MLRs were performed in 96-well plates for 7 days in the presence or absence of 100 µΜ 1-MT, 0.25 mM TRP or 3 µM CH223191. A total of 6 different pairs of PBMC samples of the 4 different healthy aforementioned individuals. For each pair, lymphocytes from a subject were mixed with lymphocytes from a different subject. In particular, PBMCs from subject #1 were co-cultured with PBMCs from subject #2, 3 or 4; thus three different MLRs could be obtained. PBMCs from subject #2 were cultured with PBMCs from subject #3 or 4, which provided two more MLRs. Additionally, PBMCs from subject #3 were cultured with PBMCs from subject #4 yielding only one MLR that differed from the others. The number of PBMCs from each member of the MLR pairs was 5x10⁴, which comprised 1x10⁵ PBMCs in each well. Cultures of 1x10⁵ resting PMBCs per well were used as controls. At the end of the 7-day period, cell proliferation was assessed by chemiluminescence with Cell Proliferation ELISA (Roche Diagnostics, Indianapolis, IN, USA) using bromodeoxyuridine (BrdU) labeling overnight at 37˚C and immunoenzymatic detection according to the manufacturer's protocols. For determining the results of the BrdU Cell Proliferation Assay, the fluorescence intensity was measured and analyzed using an EnSpire^® Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA). The proliferation index was calculated by the following equation: Proliferation index (%) = (OD derived from each MLR / the mean OD derived from the control resting PBMCs cultures of the two subjects that constituted the specific MLR) x 100. All aforementioned MLRs were performed in triplicate and the results were representative of the mean of three measurements.

In order to evaluate humoral alloimmunity, the following method was developed. One-way MLRs were performed in 24-well plates. Mitomycin-C-treated PBMCs (0.5x10⁶ cells) from one subject were used as stimulator cells. For the mitomycin-C treatment, PBMCs were incubated for 30 min at 37˚C with 50 µg/ml mitomycin C (Sigma Aldrich; Merck Merck KGaA) and then washed three times with complete RPMI-1640 medium supplemented with 2 mM L-glutamine and 10% FBS. As responder cells, 0.5x10⁶ PBMCs from another individual were used. A total of 12 different pairs of PBMC samples of the 4 healthy aforementioned subjects using one-way MLR cultures. For each pair, lymphocytes from a responder subject were mixed with inactivated lymphocytes from a stimulator subject and cultured as follows: Pair 1, responder subject #1 + stimulator subject #2; pair 2, stimulator subject #1 + responder subject #2; pair 3, responder subject #1 + stimulator subject #3; pair 4, stimulator subject #1 + responder subject #3; pair 5, responder subject #1 + stimulator subject #4; pair 6, stimulator subject #1 + responder subject #4; pair 7, responder subject #2 + stimulator subject #3, pair 8, stimulator subject #2 + responder subject #3; pair 9, responder subject #2 + stimulator subject #4; pair 10, stimulator subject #2 + responder subject #4; pair 11, responder subject #3 + stimulator subject #4 and pair 12, stimulator subject #3 + responder subject #4. MLRs lasted for 7 days in culture at 37˚C in the presence or absence of 100 µΜ 1-MT, 0.25 mM TRP or 3 µM CH223191. Of note, a 7-day period was reported as adequate for the production of IgM and IgG alloantibodies in human mixed lymphocyte cultures (34). Following this period, the supernatants from each one-way MLR were harvested and expected to contain antibodies produced against the stimulator PBMCs.

In parallel to the aforementioned one-way MLRs, resting untreated PBMCs were cultured in 6-well plates. At the end of the 7-day one-way MLRs, resting PBMCs similar to those used as stimulator cells in MLRs but untreated, were counted using a Neubauer chamber (Paul Marienfeld GmbH & Co. KG) and an optical microscope at x40 (x4 objective). Cell viability was assessed using the trypan blue exclusion assay and for each PBMC sample, cells were counted in the four different fields of the Neubauer chamber. Then, cells were placed in 96-well plates at a number of 0.5x10⁵ in a volume of 50 µl of RPMI-1640 medium, supplemented with L-glutamine, 10 mM HEPES, 10% fetal bovine serum and 1% antibiotic-antimycotic solution containing penicillin, streptomycin and amphotericin B. Cultures were incubated at 37˚C in an atmosphere of 95% relative humidity and 5% CO₂. For assessing antibody-mediated complement-dependent cytotoxicity (CDC) a modified protocol, which was initially developed for the assessment of antigen specific antibodies in serum samples was conducted (35). In brief, 50 µl of each supernatant collected from each one-way MLR, undiluted or diluted 1:2 with complete RPMI-1640, was added into 96-well plates that were pre-seeded with the resting target PBMCs. The plates were incubated on ice for 30 min. Subsequently, 11 µl of rabbit complement (Low-Tox-H rabbit complement, Cedarlane Corporation, Burlington, Canada) was added to each well at a final concentration of 10%. The 96-well plates were incubated for another 2 h at 37˚C. As a control, 50 µl of complete RMPI-1640 was added instead of the one-way MLR supernatant, along with 11 µl of rabbit complement.

As cell-mediated cytotoxicity occurs in MLRs (36) and the used compounds (1-MT, TRP and CH223191) affected the intensity of MLRs leading to the release of various quantities of LDH in the supernatants, antibody-mediated CDC induced by these supernatants in cells was not investigated directly. Instead, the present study analyzed cell survival. As the exact time-points of cell death for each of the differenT cell types involved in MLRs have not been clearly defined yet, Annexin V staining for the detection of apoptosis was not selected in the present study; however, it is considered to be the gold standard for the analysis of cell survival/death. Therefore, cell survival was assessed colorimetrically by measuring the reduction of XTT, a yellow tetrazolium salt, to orange formazan via the metabolic targeting of cells. Target cells were incubated with XTT reagent for 1 h at 37˚C. For this purpose, the TACS XTT assay kit (Trevigen, Inc., Gaithersburg, MD, USA) was used according to manufacturer's protocols. Cell survival was calculated by the following equation: Cell survival (%) = (XTT assay OD of the control / XTT assay OD of the evaluated condition) x 100. A total of twelve experiments were performed, each in triplicates and the results were representative of the mean of the three measurements.

Statistical analyses were performed using SPSS Statistics, Version 20 (IBM Corp., Armonk, NY, USA). The normality of the evaluated variables was assessed and confirmed by the one-sample Kolmogorov-Smirnov test. For the comparison of means, a paired t-test or one-way repeated measures analysis of variance followed by Bonferroni's correction test were used. The results were expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

In some cases, to avoid the violation of the prerequisite for normal distribution of the compared variables when applying parametric statistical tests, we compared the OD values as derived from the aforementioned assays prior to normalization to the control; however, the results were expressed and depicted following the normalization of values to the control group.

At the concentrations of 100 µM for 1-MT, 0.25 mM for TRP or 3 µM for CH223191, none of the evaluated compounds induced a statistically significant change in the LDH release of resting PBMCs. The LDH release assay revealed a value of 12.83±1.01% for the control, 11.50±0.75% for 1-MT (P>0.05), 10.83±1.64% for TRP (P>0.05) and 13.50±1.56% for CH223191 (P>0.05; Fig. 1).

The IDO inhibitor 1-MT increased cellular alloimmunity, as assessed by cell proliferation in two-way MLRs. The proliferation index in 1-MT-treated MLRs was significantly higher than that of untreated MLRs (359.37±32.93 vs. 238.99±20.55% respectively, P<0.001; Fig. 2).

On the contrary, compared with MLRs of untreated cells, treatment with the GCN2 kinase activator TRP significantly decreased the proliferation index to 132.89±21.65% (P<0.001; Fig. 2). Similarly, the AhR inhibitor CH223191 significantly reduced the proliferation index to 111.07±7.36% (P<0.001; Fig. 2).

The antibody-mediated CDC assay revealed that one-way MLR-antibodies specific for the stimulator PBMCs were produced by the responder PBMCs. The cell survival of untreated PBMCs, similar to stimulator cells, decreased to 62.25±6.76% compared with the control when supernatants from the respective MLRs were undiluted (P<0.001), and to 77.71±8.62% when diluted 1:2 (P<0.001). Interestingly, the diluted supernatants exhibited less antibody-mediated CDC against the untreated PBMCs, similar to stimulator cells, target PBMCs (P<0.001; Fig. 3).

The antibody-mediated CDC assay revealed that the treatment of one-way MLRs with 1-MT increased the production of specific for the stimulator PBMCs antibodies. When undiluted supernatants from untreated MLRs were used, the survival of target PBMCs was 62.25±6.76%, which was significantly decreased to 35.39±7.75% following treatment with 1-MT (P<0.001; Fig. 4A). Similar results were obtained from the 1:2 diluted supernatants compared with the control; however, decreased antibody-mediated CDC was observed, (cell survival of diluted supernatant, 77.71±8.62% versus diluted supernatant with 1-MT, 47.48±10.72%, P<0.001; Fig. 4B).

In one-way MLRs, the GCN-2 kinase activator TRP did not markedly affect the production of specific antibodies against the stimulator PBMCs. The antibody-mediated CDC assay revealed that when undiluted supernatants from untreated MLRs were used, the survival of untreated, target PMBCs, which are similar to stimulatory cells, was 62.25±6.76%, while in the case of undiluted supernatants from TRP-treated MLRs cell survival was 69.63±12.01% (P>0.05; Fig. 4A). In the case of 1:2 diluted supernatants, cell survival was 77.71±8.62 and 83.04±10.58% in the untreated as TRP-treated groups, respectively (P>0.05; Fig. 4B).

Similarly, in one-way MLRs, the AhR inhibitor CH223191 did not notably alter the production of specific antibodies against the stimulatory PBMCs. The antibody-mediated CDC assay demonstrated that the cell survival of untreated target PMBCs, which are similar to stimulatory cells, was 62.25±6.76%, while that of undiluted supernatants from CH223191-treated MLRs, cell survival was 65.87±6.17% (P>0.05; Fig. 4A). In the case of 1:2 diluted supernatants, cell survival was 77.71±8.62 and 84.13±5.21%, in the untreated and CH223191-treated groups, respectively (P>0.05; Fig. 4B).