Fig 2: MTX-101 activates CD8 regulatory T cells (Tregs) in celiac and healthy peripheral blood mononuclear cells (PBMCs) and induces lysis of gliadin-specific target cells in celiac T-cell co-culture assay. (A) Percentage of CD8 Tregs detected in healthy (n = 2) and celiac (n = 3) PBMCs. (B) MTX-101 dose-dependent CD8 Tregs binding across the same donors as in panel (A) in total PBMCs. p-Values were determined with an unpaired t-test. ns: p > 0.05, *p < 0.05, **p < 0.01. (C) MTX-101 dose-dependent activation of CD8 Tregs in celiac PBMCs (n = 3 donors) following 48 hours of incubation. (D, E) Celiac KIR+ CD8 Tregs were expanded in IL-7+IL-15+ gliadin peptides. Sorted CD8 Tregs were then cultured with autologous gliadin peptide-expanded CD4 T cells and peptide-pulsed antigen-presenting cells (APCs) +/− 100 nM MTX-101 for 7 days. (D) Percentage of CD8 Treg and Granzyme B and CD107a expression in CD8 following MTX-101 treatment. (E) CD4 readouts in co-cultures including number of total CD4 T cells and IFN-γ-producing CD4 T cells following 72-hour incubation with MTX-101. (F) Meso Scale Discovery (MSD) quantification of secreted IFN-γ, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant of co-cultures stimulated with gliadin peptides in the presence of MTX-101 shown relative to untreated controls. (D–F) Results are representative of two independent experiments (n = 3 donors). p-Values were determined with an unpaired t-test. ns: p > 0.05, *p < 0.05, **p < 0.01. (G, H) Activation of CD8 T cells (G) or NK (H) cells as determined percentage CD25 positive is shown for two healthy donor PBMCs following 48-hour incubation in the shown concentrations of anti-KIR single-arm antibody, anti-CD8 single-arm antibody, or MTX-101 in either untreated (resting) or anti-CD3-treated (0.1 µg/mL, activated) PBMCs.

Fig 3: MTX-101 restores CD8 regulatory T-cell (Treg) functions and reduces antigen-induced proinflammatory responses in Crohn’s donor peripheral blood mononuclear cells (PBMCs) and organoids. (A) Healthy or Crohn’s donors (n = 6 each) CD8 Tregs were stained for Granzyme B and Helios. CD4 T cells were stained for co-expression of CD161+CXCR3+CD39+ in PBMCs from eight healthy (n = 8) or Crohn’s donors (n = 11). (B, C) Crohn’s PBMCs were incubated for 7 days with a mixture of IL-7+IL-15, bacterial flagellin, and OmpC peptide in the presence or absence of MTX-101 at 100 nM. (B) The percentage increase in concentration of Granzyme B in the supernatant of antigen-responsive or non-responsive Crohn’s donors (t = 5 days). (C) Carboxyfluorescein succinimidyl ester (CFSE) dilution of antigen-responsive CD4 T cells (t = day 7) antigen-expanded CD4 T cells as determined by diluted CFSE is also shown on day 7 for both responders and non-responders relative to untreated controls. Secreted IFN-γ and TNF-α (t = day 7) in MTX-101-treated donors relative to untreated. (D) Helios expression on CD4 T cells in PBMCs from Crohn’s donors stimulated with anti-CD3 (1 μg/mL) overnight in the presence and absence of MTX-101 (100 nM). (A–D) p-Values were determined with an unpaired t-test. ns: p > 0.05, *p < 0.05. (E) Change in antigen-induced epithelial cell death in both celiac and Crohn’s donor-derived organoids +/− 100 nM MTX-101. Results represent celiac (n = 3) and Crohn’s donors (n = 2). (F) Healthy or celiac PBMCs (n = 3 each) were restimulated with CEFT, Influenza HA, SARS-CoV-2, or AVV5/6/8 peptide (0, 0.1, or 1 µg/mL), in the presence and absence of MTX-101. IFN-γ detected in PBMC culture supernatant on day 5. (G) IFN-γ concentrations in supernatants following 5-day bacterial stimuli of tetanus toxoid (5 µg/mL) or SEB (100 ng/mL). Each point represents the mean of two technical replicates. Values untreated and with MTX-101 were compared using unpaired t-tests and two-way ANOVA followed by Šídák’s multiple-comparisons test.

Fig 4: KIR CD8 regulatory T cells (Tregs) express the surface markers NKG2C, Helios, and KLRG1 and, when enriched from celiac donor peripheral blood mononuclear cells (PBMCs), specifically eliminate a gliadin T-cell receptor (TCR)-transduced SKW line in a dose-dependent manner. (A, B) Healthy donor PBMCs were plated overnight and then stained for KIRs, NKG2C, Helios, and KLRG1 (A) and the cytolytic marker, Granzyme B (B). p-Values were determined with a paired t-test. (B) The percentage positive of each of these markers is shown for the KIR+ CD8 Treg and KIR− CD8 non-CD8 Treg populations from seven healthy donors. (C) KIR+ CD8 Tregs were isolated from celiac PBMCs following expansion with IL-7, IL-15, and gliadin peptides. Sorted CD8 Tregs were then cultured with gliadin-expanded CD4 T cells and autologous antigen-presenting cells (APCs) with or without (unstimulated) additional gliadin peptide stimulation for 3 days and evaluated for CD8 Treg number. Results are representative of two independent experiments across three different celiac donors. p-Values were determined with an unpaired t-test. (D) CD8 Tregs were enriched from celiac donor PBMCs following expansion in IL-7 and IL-15 for 7 days and sorting for CD5+NKp46-CD4-CCR7-CD28- T cells. CD8 Tregs were quantified, and increasing ranges of CD8 Tregs were cultured with 20,000 GFP+ gliadin-responsive TCR-transduced SKW target cells. Percent change in GFP+ objects (as determined by Incucyte software) from baseline (t = 8 hours) is shown for target cells in the absence of CD8 Tregs and with increasing CD8 Treg number. Results are representative of three independent experiments. (E) CD8 Treg-enriched cells were cultured with either green fluorescent protein (GFP)-expressing parental SKW or gliadin TCR-transduced SKW target cells for 48 hours and the percent change in GFP signal relative to baseline (t = 8 hours, n = 2 independent experiments). (A–E) ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D, E) p-Values were determined through a one-way ANOVA of calculated areas under the curves followed by Šídák’s multiple-comparisons test.

Fig 5: MTX-101 specifically binds to target surface receptors on CD8 regulatory T cells (Tregs). (A) Table shows the binding affinities of MTX-101 for human KIR2DL1/2/3 and CD8α based on association and dissociation rate constants measured for each binding interaction, and KD binding affinity calculated from the measured rate constants. (B) Dose–response curves of MTX-101 binding to stably expressed KIR2DL1 and CD8α SKW cell lines. EC50 values were calculated for each cell line using Prism GraphPad software with non-linear regression [agonist vs. response curve (three parameters)]. Binding curves are representative of binding to KIR2DL1 expressing (n = 14 independent experiments) and CD8a expressing (n = 10) cell lines. (C) Simultaneous co-binding of antigens to MTX-101 as determined by Bio-Layer Interferometry. MTX-101 was captured with either KIR2DL or CD8α, and then additional antigen binding was detected with CD8α or KIR2DL, respectively. (D) Dose-dependent MTX-101 binding to immune cell populations within total human peripheral blood mononuclear cells (PBMCs) is shown as detected with an anti-human Fc secondary antibody. Data are representative of seven independent experiments with different donors. (E) Quantification of CD8 and KIR binding sites per CD8 Treg target cell as determined by receptor mean fluorescence intensity (MFI) and quantitative counting beads is shown in the figure. p-Values were determined through a one-way ANOVA of calculated areas under the curves followed by Šídák’s multiple-comparisons test. ns: p > 0.05. (F) Detection of unbound KIR sites with a saturating dose of fluorescently labeled single-arm KIR-Fc antibody on CD8 Tregs in total PBMCs following incubation with increasing doses of MTX-101. IC50 values were calculated for KIR-Fc using GraphPad Prism software and non-linear fit log (inhibitor) vs. response with variable slope and four parameters. The graph is representative of binding data across three independent healthy donors.