ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs

(DR2 × Ob1)F1 transgenic mice homozygous for DRA*0101/DRB1*1501 (HLA-DR2) and MBP_84–102-specific human T-cell receptor (TCR) clone (Ob1) were generated in C57Bl/6 mice [20] and maintained and bred at Charles River Laboratories (Wilmington, MA, USA); the F1 progeny of these animals were used in all experiments. Before each experiment, expression of human MHC and TCR were confirmed in peripheral blood by phenotyping with fluorescein isothiocyanate (FITC)-labelled mouse anti-human HLA-DR, HLA-DP, HLA-DQ (BD Pharmingen, San Jose, CA, USA) and IOTest anti-TCR VB2-PE clone MPB2D5 (Beckman Coulter, Indianapolis, IN, USA). Only double-positive mice with a frequency of human MHC and TCR expression > 15% in peripheral leukocytes were used in the studies.

On arrival, mice were group-housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited vivarium with a 12:12 light–dark cycle and food and water provided ad libitum. The mice were allowed to acclimatize for at least 48 h before any experiments were started. All procedures were approved by the EMD Serono Institutional Animal Care and Use Committees (IACUC) or by the Massachusetts General Hospital IACUC in the case of magnetic resonance imaging (MRI) studies.

Mice reaching a predefined clinical endpoint for humane withdrawal from the study (Table 1) and mice that were not required for tissue sampling were euthanized by slow replacement of the air in their cage by an air mixture containing CO₂. Death by CO₂ asphyxiation was confirmed by cervical displacement. All animal experiments complied with the National Institutes of Health guide for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and to optimize their well-being.

All treatments with ATX-MS-1467 were performed from a reconstituted sterile, freeze-dried formulation of a mixture (1:1:1:1) of four synthetic peptides (MBP epitope 30–44: PRHRDTGILDSIGRF; MBP epitope 83–99: ENPVVHFFKNIVTPRTP; MBP epitope 130–144: RASDYKSAHKGFKGV; MBP epitope 140–154: GFKGVDAQGTLSKIF). After reconstitution of the lyophilized formulation to the original volume with sterile water, the solution was further diluted with PBS to the required doses. The concentration of the ATX-MS-1467 mixture was adjusted for a 100-µL injection volume, administered subcutaneously in all cases. The doses corresponded to the sum of each peptide in the mixture; therefore, a dose of 100 µg contained 25 µg of each peptide. The treatment regimen with ATX-MS-1467 varied; please refer to specific sections for the dose regimen used in each experiment.

In some experiments, HLA-binding peptide (HLAbp) was used as a control for ATX-MS-1467. Although HLAbp can bind directly to MHC on the surface of antigen-presenting cells without requiring antigen processing, the peptide sequence of HLAbp has no similarity to the epitopes involved in the adaptive immune responses to MS or EAE. The peptide sequence used (NPILLWQPIPVHTVPLSEDQ) was provided by Professor David Wraith (University of Bristol, UK).

Clinical score and body weight were monitored and recorded daily from 7 dpi by observers blind to treatment. Neurological disability and humane endpoints were monitored daily using a subjective 0–5-point clinical score scale (Table 1) [21]. Whenever possible, a single investigator scored the animals in each study, and in any single day the same investigator scored all groups of animals. However, because clinical scoring sometimes progressed for several weeks, including weekends, up to four different investigators potentially provided input into a single study. Each investigator’s scoring was routinely calibrated, to ensure the results were comparable across different investigators.

EAE was induced on 0 dpi (n = 35). On 9 or 10 dpi, MRI was obtained, and those animals with radiological evidence of leakage in the cerebellum/brainstem seen on T1-weighted gadolinium-enhanced (Gd⁺) MRI with a 9.4-Tesla magnet scanner (Bruker Biospin, Billerica, MA, USA) were randomized into two groups for treatment with either ATX-MS-1467 (100 µg/mouse, n = 7) or vehicle (n = 7). Treatment was given every other day, starting at 10 dpi (mice scanned on 9 dpi) or 11 dpi (mice scanned at 10 dpi). T1-weighted Gd⁺ MRI was repeated after three doses (15 or 16 dpi). The T1-weighted gradient echo sequence with a short echo time (TE) and a large flip angle [repetition time (TR)/TE/flip angle = 100 ms/2.7 ms/50 degrees] was used to assess Gd leakage.

Pathological analysis was done on samples of spinal cords from animals treated at 7 or 14 dpi with either ATX-MS-1467 or vehicle. Samples were obtained at the termination date of these experiments (30 post-immunization). A 1.6 g/mL 2,2,2-tribromoethanol (T48402, Sigma, St. Louis, MO, USA) stock solution was prepared in 2-methyl-2-butanol (A1685, Sigma, St. Louis, MO, USA). A working solution at 20 mg/mL was obtained by subsequent dilution with PBS. At predefined time points, mice were overdosed with anesthetic (400 mg/kg intraperitoneal 2,2,2-tribromoethanol) and perfused through the left cardiac ventricle with saline followed by 10% neutral buffered formalin. The vertebrae were partially removed to obtain the spinal cord, and the tissues were post-fixed with 10% neutral buffered formalin for 48 h and then stored in PBS before dissecting out the spinal cords. Spinal cords were cut into five segments, spanning the cervical (one piece), thoracic (two pieces) and lumbar (two pieces) regions, which were processed for paraffin embedding. Histological analyses were done on 10 cross-sections of the spinal cord for each animal. These cross-sections were 7 µm thick and at least 280 µm apart.

Hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining were performed, as well as immunohistochemistry staining against CD3 (Abcam, Cambridge, MA, USA) and CD45R (BD Pharmingen, San Jose, CA, USA) surface antigens using Leica BOND-III autostainer (Leica, Wetzlar, Germany). High-quality images obtained with a Nanozoomer 2.0 HT digital slide scanner (Hamamatsu, Hamamatsu City, Shizuoka, Japan) were semi-quantitatively scored for each stain by an experimenter who was blinded to the treatment groups.

The H&E scoring was performed as set out in Table 2. CD3 and CD45R scoring were similar to the H&E scoring, except that only immunostained cells were scored. For the LFB scoring, each cross section of the spinal cord was analyzed as four independent regions (ventral, dorsal, right lateral and left lateral). In all cases, the scores for each of the ten sections was averaged and entered as a single data point for each mouse in the experiment.

Mice were treated with either ATX-MS-1467 (100 µg/mouse, three times per week) or vehicle for 7 days. At 0 dpi, mice were also immunized as if for induction of EAE, but these mice were euthanized before they displayed signs of disease. Mice received the last dose of ATX-MS-1467 or vehicle at 7 dpi, 1 h before sacrifice for spleen removal and cell culture. On the day of sacrifice, mice were anaesthetized with 400 mg/kg intraperitoneal 2,2,2-tribromoethanol, and their spleens were harvested and processed in a gentle MACS Octo dissociator (Myltenyi Biotec, Birgisch Gladback, Germany). The splenocytes underwent several cycles of washing and centrifugation after lysis of red blood cells with red blood cell lysis buffer (Roche, Basel, Switzerland) and were counted manually in a Neubauer chamber using trypan blue solution to exclude dead cells. The concentration of the cell suspension was adjusted to 3 × 10⁶ cells/mL in RPMI medium (Gibco, Waltham, MA, USA) supplemented with fetal calf serum (10%), penicillin–streptomycin (1:100; Gibco, Waltham, MA, USA), HEPES (final concentration 10 mM; Gibco, Waltham, MA, USA), MEM non-essential amino acid solution (1:100; Sigma, St. Louis, MO, USA), l-glutamine (final concentration 2 mM; Gibco, Waltham, MA, USA), sodium pyruvate (final concentration 1 mM; A&E Scientific PAA, San Jose, CA, USA) and β-mercaptoethanol (final concentration 2.5 µM; Sigma, St, Louis, MO, USA). For cell culture, 100 µL of the cell suspension was prepared as described, together with 100 µL of each appropriate stimulant (ATX-MS-1467 0.03–30 µg/mL final concentrations) diluted in medium and incubated at 37 °C for 48 or 72 h, corresponding to the optimum time points for evaluation of cytokine or proliferative responses, respectively. Cytokines were quantified in supernatants from 48-h splenocyte cultures with commercial enzyme-linked immunosorbent assay (ELISA) kits (mouse IL-2 Duoset ELISA, mouse IL-10 Quantikine ELISA, mouse IL-17 Duoset ELISA and mouse IFNγ Duoset ELISA, all purchased from R&D Systems, Minneapolis, MN, USA). Splenocyte proliferation was assessed by replacing 100 μL of the medium from wells that had been stimulated for 72 h with 100 μL of ³H thymidine solution and further incubating for 12 h. Incorporation of ³H thymidine was quantified in a scintillator. The results were expressed as counts per minute (cpm).

The experiments that were conducted to assess antigen-specific cytokine release in vivo were performed in mice that had not been immunized with adjuvant in order to avoid background inflammation caused by this agent. Serum samples were collected from untreated mice or mice that had received repeated treatment with ATX-MS-1467 (100 µg/mouse, three times per week) or HLAbp (100 µg/mouse three times per week) receiving a total of 10 injections, followed or not by a wash-out period lasting from 2–42 days for assessment of the concentrations of IL-2, IL-10, IL-17 and IFNγ. Samples were collected 2 h after the last injection, which was experimentally predetermined as the time point at which the concentrations of most cytokines had reached a maximum. To ascertain the in vivo cytokine secretion of (DR2 × Ob1)F1 mice to an acute challenge with MBP, serum was collected 2 h after acute treatment with MBP at doses ranging 30–1000 µg/mouse. To ascertain the effect of chronic dosing with ATX-MS-1467 on in vivo pro-inflammatory cytokine secretion, mice received repetitive treatment (from one up to ten doses) with ATX-MS-1467 (100 µg/mouse, three times per week) with serum collected 2 h after the last dose. To ascertain the effect of chronic treatment with ATX-MS-1467 on the ratio of IL-10-to-pro-inflammatory cytokines after an acute challenge with MBP, mice received ten doses of ATX-MS-1467 (100 µg/mouse, three times per week) or ten doses of HLAbp (100 µg/mouse, three times per week). At 2, 7, 21 or 42 days after the last injection with ATX-MS-1467 or HLAbp, mice received a single injection of MBP (300 µg/mouse) and serum was collected 2 h later. Acute cytokine responses in serum were quantified using a customized MCYTOMAG-70 K | MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay (Millipore, Billerica, MA, USA).

Flow cytometric analysis was used to assess the effect of treatment with ATX-MS-1467 (100 µg/mouse, three times per week) or vehicle for 2 weeks on cell surface and intracellular markers associated with regulatory cell function. Two different staining panels were used (Table 3).

The first panel focused on cell surface markers and was applied in freshly isolated splenocytes. The second panel included intracellular markers, IL-10 and the transcription factor forkhead box P3 (Foxp3). To enable better identification of the IL-10-secreting cells, the second panel was applied in splenocytes incubated for 3 h with phorbol-myristate acetate (PMA, 5 ng/mL; Sigma, St. Louis, MO, USA), ionomycin (500 ng/mL; Sigma, St. Louis, MO, USA) and the protein transport-inhibitor monensin (4 µL/6 mL of cell culture; GolgiStop, BD Biosciences, San Jose, CA, USA) for 3 h. The splenocytes were isolated as previously described. B220 and CD8 staining were used as a negative control for the T lymphocyte subpopulations of interest. Samples were measured on a LSRFortessa X20 flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed in FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA).

In experiments with multiple groups, clinical score data were analyzed via daily comparison of group averages using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Clinical score data were also integrated as area under the curve, and the results analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparison test. MRI, EAE imaging and clinical score data were analyzed daily by Mann–Whitney test. The mean semi-quantitative histological scores of spinal cord sections were input as a single data point that was used to calculate group averages, which were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni test. Data on concentration-dependent cytokines and proliferation response ex vivo were analyzed with two-way ANOVA followed by the Bonferroni test. In vivo cytokine-release data were analyzed with one-way ANOVA followed by Dunnett’s multiple comparison test.

We treated (DR2 × Ob1)F1 mice with different doses of ATX-MS-1467, starting either 7 or 12 dpi and observed the clinical signs of disease. Compared with vehicle, ATX-MS-1467 dose-dependently reduced clinical disability from the empirically determined disease onset (7 dpi, Fig. 1a, c) and peak disease (12 dpi, Fig. 1b, d) time points, as assessed by the mean daily score and cumulative scores on the subjective 0–5-point clinical score scale during the treatment period. As can be observed in Fig. 1a, d, if treatment starts early in the course of disease, it is possible to completely prevent the appearance of clinical signs. Even if the treatment is initiated when mice exhibit full-blown disease (tail–hand–hind limb paralysis, matching an average score of 3), partial, albeit significant, recovery from the natural course of EAE was seen in this mouse strain (Fig. 1b, d). The optimum dose (100 µg) significantly improved the clinical score in mice showing advanced signs of neurological disability, and this dose was selected for subsequent experiments.

We performed two independent experiments, with treatment started at either 7 or 14 dpi. This allowed us to generate a large tissue bank to assess the effects of treatment with ATX-MS-1467: spinal cord samples were obtained from 20–28 mice from each treatment group, with each spinal cord being stained and analyzed at ten different levels for each staining. In this experiment, we used histological evaluation with subjective scoring (Table 2) to confirm the observed efficacy. The average score for the vehicle-treated groups, which varied from 2 to 3, depending on the marker being analyzed, could wrongly suggest that the level of tissue damage is not compatible with overt disease pathology. However, due to the unpredictable location where the lesions occur in this model, the average score is the result of both highly affected spinal cord sections and other sections that might have remained intact. Indeed, it was a common feature of all vehicle-treated mice that one or multiple areas with intense cellular infiltration and demyelination were observed at various, albeit not all, sections examined. In agreement with the attenuation in clinical score, treatment with ATX-MS-1467 from the empirically determined disease onset (7 dpi) or peak of disease (14 dpi) time points reduced spinal cord inflammation, demyelination and cellular infiltration, unsurprisingly with the greatest effect for all outcomes reported when treatment was initiated at disease onset (p < 0.001 for all measures; Fig. 2). The reduction in inflammation and demyelination seen in the CNS was also accompanied by an improvement in the clinical score, for which the greatest improvement was observed following early (7 dpi) treatment (Fig. 2a). Treatment with ATX-MS-1467 from the peak of disease (14 dpi) partially reduced the histological markers indicative of inflammation (H&E score; Fig. 2b), demyelination (LFB score; Fig. 2c) and T-cell infiltration (CD3 score; Fig. 2d; p < 0.05) and histology scores indicative of B-cell infiltration (p < 0.01; Fig. 2e).

MRI was applied as a translational approach to investigate the efficacy of ATX-MS-1467 in mice. Gd administered intravenously during the acute phase of disease allows the detection of leakage, which can be seen as bright areas of the spinal cord in T1-weighted scans, indicating disruption of the BBB. Because BBB disruption tends to be more active during the beginning of the acute phase of disease, and tends to naturally subside at later phases, we opted for a shorter window of treatment, in order to better appreciate the drug effect. In a longitudinal MRI experiment (Fig. 3a), therapeutic dosing with ATX-MS-1467 (100 µg subcutaneous) every other day (three doses) from 10 dpi significantly (p < 0.05 on 13 and 15 dpi and p < 0.01 on 14 and 16 dpi) reversed disruption to the BBB, as measured by Gd leakage, compared with vehicle-treated animals (Fig. 3c). In group analyses at follow-up, Gd-leakage volume was significantly reduced in ATX-MS-1467-treated mice (Fig. 3e) compared with baseline but not in vehicle-treated mice [Fig. 3e; ATX-MS-1467-treated mice: 77.4% reduction (Gd-leakage volume 1.1 ± 0.3 mm³) from baseline, p < 0.01; vehicle-treated mice: 39.7% reduction (Gd-leakage volume 4.45 ± 0.3 mm³) from baseline, p = 0.23]. Figure 3b shows representative T1-weighed MRI scans of the spinal cords of the mice in this study.

ATX-MS-1467 binds to surface-expressed MHC-II on antigen-presenting cells. Following binding, ATX-MS-1467 is hypothesized to drive either the differentiation of MBP-specific regulatory cells from naïve cells or to induce the conversion of effector into type 1 regulatory cells (Tr1). In both cases, inhibition of the release of the pro-inflammatory cytokines IL-17, and IFNγ from Th1 and Th17 T-cell subsets, which are known to promote the inflammatory processes in MS, along with an increase in the release of IL-10, which is indicative of a tolerogenic state, would be expected. In vivo and in vitro strategies were employed to demonstrate whether these changes occurred in mice treated with ATX-MS-1467. For the in vitro analysis, splenocytes were recovered from animals treated four times between 0 to 7 dpi with ATX-MS-1467 (on days 0, 2, 4 and 7); the last administration took place 1 h prior to take down. The spleens were processed, as described in the methods section, and the cytokine concentrations in the supernatant were assayed (Fig. 4a–d). The cultures were stimulated in the presence of a range of concentrations of each antigen, to allow the assessment of the response over a broad range of concentrations.

It is important to differentiate the purpose of ATX-MS-1467 as a treatment (Fig. 4a–e filled squares), when it was administered in vivo with a vehicle-administered group as a control (Fig. 4a–e filled circles), from its use here as an antigen-specific stimulus for cellular proliferation and cytokine release (x axis, various concentrations). The in vitro antigen recall assay showed that the in vivo treatment with ATX-MS-1467 caused a dose-dependent inhibition in pro-inflammatory cytokine (IL-17 and IFNγ) and IL-2 secretion and splenocyte proliferation. IL-10 secretion was significantly increased only after stimulation with concentrations of ATX-MS-1467 of 3 µg/mL (p < 0.05) and above (p < 0.001) in the ex vivo antigen recall assay (Fig. 4).

The effect of ATX-MS-1467 persists when the drug is cleared from the circulation. To extend the results for ex vivo cytokine secretion, we wanted to assess whether a favorable shift in cytokine balance also occurred in vivo. To achieve this, we performed three types of experiments.

The first type (Fig. 5) aimed to define the optimal dose of MBP needed to elicit robust serum cytokine changes. In the naïve wild-type mouse strain, MBP injection would not normally induce cytokine release at levels that can be detected in serum. However (DR2 × Ob1)F1 mice were genetically engineered to constitutionally express both MHC and TCR recognizing one MBP epitope; therefore, circulating effector and regulatory cells that respond to MBP are already present in (DR2 × Ob1)F1 mice, even when they have not been immunized against this protein. Untreated (DR2 × Ob1)F1 mice responded to an acute challenge with MBP with a dose-dependent increase in pro-inflammatory cytokines IL-17 (Fig. 5c) and IFNγ (Fig. 5d) and also IL-2 (Fig. 5a) compared with vehicle-treated mice. Although the concentration of IL-10 was also increased in response to low doses of MBP, this effect plateaued at MBP concentrations of 100–300 µg and then declined as the concentration of MBP increased (Fig. 5b).

The second type of experiment on in vivo cytokine release (Fig. 6) assessed how cytokine release shifted after repeat treatments. The insert summarizes how the injections were performed in each group (Fig. 6a). A single dose of ATX-MS-1467 elicited an increase in the concentrations of all cytokines in serum (Fig. 6b–e), and the concentrations returned to baseline values within a few hours after reaching their peak at 2 h after the injection (results not shown). Similar to explanation given above for MBP-induced cytokine release, naïve mice with the transgenic mouse construct also respond to ATX-MS-1467 with quantifiable cytokine levels in serum without the need of concomitant exposure with adjuvant. Further doses of ATX-MS-1467 did change the serum IL-2 (Fig. 6b), IL-17 (Fig. 6d) or IFNγ (Fig. 6e) cytokine concentrations in comparison with vehicle for up to ten consecutive doses of ATX-MS-1467. However, up to five consecutive doses of ATX-MS-1467 were still capable of inducing statistically significant drug-induced IL-10 release in serum (Fig. 6d). The use of ratios of signature cytokines for the predicted response is an accurate index of how an organism responds to antigens or disease: how the ratios change over time or how they compare between different groups under controlled conditions shows how a system is responding to the challenge [22]. If treatment with ATX-MS-1467 does indeed induce tolerance, the persistence of the tolerogenic effect, as deduced from changes in the ratios of IL-10-to-pro-inflammatory cytokines, could be assessed by the changes in these ratios. From the second dose to the fifth dose, the concentration of IL-10 relative to IL-17 and IFNγ, respectively, was higher than in vehicle-treated animals. However, the concentration of IL-10 relative to IL-2 was lower for vehicle-treated mice after one dose, and was not different from vehicle-treated animals from the second dose onwards (Fig. 6).

Finally, a third type of experiment was done to assess how mice reacted to the native antigen (MBP) after they received a course of 10 injections with 300 µg of ATX-MS-1467, which was previously determined as the highest dose to elicit the maximum response (Fig. 7). Because all previous evidence indicates that ATX-MS-1467 treatment is associated with induction of Tregs, we hypothesized that the effect of the treatment is long-lasting; therefore, the effects should persist for at least as long as this pool of regulatory cells remained expanded, counteracting the natural emergence of Th1 or Th17 effector cells. To test this hypothesis, we challenged a group of mice with MBP following a wash-out period of 2 days (equivalent to the 3×/weekly regimen treatment with ATX-MS-1467), a group with wash-out periods of either 7, 21 or 42 days from the last ATX-MS-1467 treatment, and also mice in which HLAbp was used as a control for ATX-MS-1467. To help interpret this study (as depicted in Fig. 7), we compared the cytokine levels (or ratios) between the third bar, i.e., mice treated with control peptide (HLA-bp) then challenged with MBP, which could also be regarded as “untolerized”, against those levels in the four last columns representing mice treated chronically with ATX-MS-1467 prior to MBP challenge (performed from 2, 7, 21 or 43 days after the last treatment). When naïve mice were challenged with MBP (Fig. 7a–d; second column in each plot) there was always a large induction of serum cytokines. The same was true for the mice that received ten doses of HLA-bp (Fig. 7a–d; third column in each plot). However, for all cytokines tested, the serum cytokine concentrations were always lower for the groups of mice that received a course of ATX-MS-1467 than those mice that received HLA-bp treatment prior to the challenge, regardless of the duration of the wash-out period. Furthermore, the effector cytokines tended towards a slight increase after longer wash-out periods, whereas the opposite occurred for IL-10. This suggests a down-regulation of the pool of IL-10-secreting cells and a re-emergence of the pool of effector cytokine-secreting cells with time. From this observation, one question arises: at which point does the balance of regulatory/effector cytokines return to that seen in the absence of tolerization (equivalent to the HLB-bp group, third column in each plot)? This can be assessed by replotting the data as cytokine ratios (Fig. 7f–h). The results vary depending on the analyzed cytokine ratio. For the IL-10-to-IL-2 ratio, tolerization with ATX-MS-1467 can still induce higher ratios at wash-out periods of up to 7 days, but the ratios return to untolerized levels (HLA-bp treated) after a wash-out of 21 days (Fig. 7f). For the IL-10-to-IL-17 ratio, the return to baseline values occurs very quickly (Fig. 7g); the ratio has normalized after a wash-out of 7 days. However, the effects on the IL-10-to-IFNγ ratio are more persistent; this ratio did not return to baseline values until a wash-out period of 42 days. This result indicates that, at least with regard to this cytokine pair ratio, the effect of ATX-MS-1467 is long-lasting. On the other hand, the effect on the IL-10-to-IL-17 ratio was less persistent.

The mechanism of tolerance induction can be investigated by quantification of the intracellular and/or extracellular markers unique to each of these Treg subsets (i.e., state-specific markers and subsets). We relied on two different approaches to define the induced regulatory T cells (iTreg) subset: one was the co-expression of LAG3 and CD49 in CD4⁺ lymphocytes; the other was the presence or absence of FOXP3 in IL-10 secreting CD4 lymphocytes. In the second approach, we assumed that the IL-10⁺FOXP3⁺ CD4 lymphocytes are natural T regulatory cells (nTregs), and the remaining IL-10⁺FOXP3⁻ are, by exclusion, iTregs. Staining was performed in splenocytes of mice that had been treated in vivo with ATX-MS-1467 or HLA-bp for 2 weeks. Figure 8a, b shows the representative gating leading to assessment of the frequency of LAG3⁺CD49b⁺CD4⁺ lymphocytes from an HLA-bp-treated mouse and an ATX-MS-1467-treated mouse, respectively. Figure 8c shows mean data for both groups. The results indicate that chronic treatment with ATX-MS-1467 expanded the iTreg compartment in the spleen compared with mice treated with HLAbp. The spleens of mice treated with ATX-MS-1467 had a higher proportion of LAG3⁺C49b⁺ cells compared with HLAbp-treated mice (p < 0.05). Figure 8d–g shows the gating and the means for the upper-left and upper-right quadrants, denoting IL-10⁺FOXP3⁻ and IL-10⁺FOXP3⁺ lymphocytes, respectively, for each group. In conclusion, in vivo ATX-MS-1467 treatment increased the pool of IL-10⁺Foxp3⁻ (iTregs), but it had no effect on IL-10⁺Foxp3⁺ (nTregs).