HLA binding results. The IC₅₀ value (μM) and affinity interpretation for each of the epitopes is shown. Based on comparisons with known peptides IC₅₀ scores lower than 25 μM indicate high binding, while an IC₅₀ above 400 μM is indicative of a weak or nonbinding interaction.

Expansion of T_Reg (CD4/CD25^Hi cells) following coincubation with Tregitope. The expansion of CD4⁺CD25^Hi T cells among PBMC restimulated with dust mite lysate following primary incubation of PBMC with either dust mite lysate alone or dust mite lysate and hTregitope 289, or dust mite lysate and hTregitope 167. FoxP3 was not assessed. Numbers in boxes are the percentage of cells in that gate.

Depletion of CD4⁺CD25^Hi Treg population reduces suppression of Teff response. Tregitope suppression is dependent on CD4⁺CD25^Hi T cells. A. PBMC from allergic individuals were stained with anti-CD4 and anti-CD25 antibodies and analyzed by flow cytometry. The CD4⁺CD25^Hi subset (gate 100) was sorted and discarded. B. CD4⁺CD25^Hi depleted and nondepleted PBMC were costimulated with HDM lysate with or without hTregitope 289. CD4⁺CD25^Hi-depleted PBMC were less able to suppress IFN-γ than were nondepleted PBMC. The levels of IFN-γ were reduced from 33.5 pg/mL to 11.8 pg/mL in the whole PBMC and from 16.5 pg/mL to 12.4 pg/mL in the CD4⁺CD25^Hi-depleted subset.

In vivo studies with mTregitopes. IL-4 and antibody responses to HDM lysate and dust mite antigen are shown. Purified HDM antigen DerP2 is a component of HDM lysate. HLA DR4 transgenic mice were immunized 3 times with (1) HDM lysate alone, (2) HDM lysate plus Tregitope 289, or (3) sham control. In an additional arm, mice were first pre-sensitized to HDM lysate by 3 weekly injections followed by 3 additional weekly injections of an equal mixture of HDM lysate plus Tregitope 289. HDM lystate immunization (■) provokes a robust response by as assessed by IL-4 ELISpot (A) and anti HDM Antigen antibody titers (B). These responses are both significantly reduced (▩) when HDM lysate is coadministered with the murine homologue of Tregitope 289. Responses to HDM lysate in sham-immunized animals are negative, as expected. Antibody response (B) and IL-4 ELISpot (A) are correlated. Panel A shows 38% suppression of ELISpot response in HDM-naive mice (P < .001) and 84% in the case of HDM presensitized mice (P < .001). Panel B shows 61% suppression of HDM antigen antibody response in HDM-naive mice (P = .138); ELISA was not performed for HDM-presensitized mice. Error bars indicate SD.

EpiMatrix analysis of human Tregitopes. EpiMatrix Z scores for eight common HLA alleles are shown, for each of the overlapping 9 mer frames for hTregitope 289 (A) and 167 (B). The EpiMatrix Z score indicates the potential of a 9-mer frame to bind to a given HLA allele; the strength of the score is indicated by the shading. The top 5% of scores are shaded medium and the top 1% of scores are shaded darkest. All scores in the Top 5% (Z-Score ≥ 1.64) are considered “Hits.” Scores in the top 10% (shown but not highlighted) are considered elevated; other scores are masked for simplicity. Frames containing four or more alleles scoring above 1.64 are referred to as EpiBars and are highlighted. These frames have an increased likelihood of binding to HLA. Flanking amino acids, added to stabilize the cluster during in vitro testing, are underlined.

Activation of natural Tregs in the presence of Tregitope. Human PBMC were stimulated directly in vitro for 4 days in the presence of tetanus toxin peptide (TT830-844), Tregitope, or no stimulus. A. Cells were stained extracellularly with anti-CD4 and anti-CD25 and intracellularly with FoxP3 and analyzed by flow cytometry. Incubation with Tregitope increased the percentage of CD4⁺CD25⁺FoxP3⁺ T cells (54%) compared to TT830-844 (13%) or no stimulus (20%). Numbers in boxes are the percentage of cells in that gate. B. From top to bottom, FoxP3 expression is shown in PBMC from the same subject following incubation with Tetanus toxin epitope (13%); hTregitope 289 (54%) and no stimulus (20%).

Response to immunogenic peptides in the presence of Tregitope 167. Responses to antigen restimulation following initial stimulation with a pool of immunogenic peptides derived from C3d protein (■); C3d peptides plus Tregitope 134 (); and C3d peptides plus Tregitope 167 (). Responses are shown as fold increase over background, which was no stimulus (control) in the secondary incubation. The respective baseline (background) values in pg/ml are indicated within the x-axis labels. There was no difference in levels of IL-4, TNFα, or TGFβ1 (data not shown).

Birch pollen epitope-specific immune responses in the presence of Tregitopes. Coculture of PBMC with Bet v 1 allergen with either hTregitope 289 or hTregitope 167 leads to a Th2 to Th1/TReg shift. PBMC from three birch tree-pollen-allergic subjects were costimulated with Bet v 1_141-155 peptide with or without hTregitope. A. Ten to 14 day Tregitope costimulation led Bet v 1_141-155 tetramer positive CD4⁺ cells to decrease Th2-associated surface markers and to increase regulatory-associated surface markers GITR and CTLA4 (average of donors I, II, and III). B. Coincubation also led to a decrease of Bet v 1_141-155 tetramer–positive CD4⁺ cells secreting IL-5 (left axis) and a modest increase in Bet v 1_141-155 tetramer–positive CD4⁺ cells secreting IL-10 (right axis). C. PBMC from Donor I were tested under prolonged culture for 30 days; under these conditions, coculture with Tregitope 167 led to a reduction in the number of Bet v 1_141-155–specific cells secreting IL-5 and to an increase in Bet v 1_141-155–specific cells that were Th0 (neither IL-5– nor IFN-γ–secreting, 42%) or Th1 (IFN-γ–secreting, 44%). Numbers in boxes are the percentage of cells in the respective quadrant.

Hypothesized tolerizing mechanism of IgG. We have discovered conserved T-cell epitopes in IgG that engage natural regulatory T cells. We hypothesize that antibody-derived Treg epitopes (dark blue epitope) activate regulatory T cells, which leads to suppression of effector T cells that recognize effector epitopes (red epitope), like those of IgG hypervariable regions to which central tolerance does not exist. Whether this suppression is mediated by regulatory cytokines alone or by contact-dependent signaling, or both, has yet to be determined.