Inducing Humoral Immune Responses Against Regulatory T Cells by Foxp3-Fc(IgG) Fusion Protein

DNA constructs and recombinant proteins as immunogens

DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct pIRES2-EGFP-Foxp3-Fc (IgG) was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by Western blot.⁽¹²⁾ Expression of Foxp3-Fc by pET21a vector harboring the corresponding gene (pET21a-Foxp3-IgG2Fc) in BL21-AI strain of E. coli under the control of IPTG-inducible (and detailed steps for confirmation of recombinant fusion protein by SDS-PAGE followed by Western blot analysis and purification through SDS-PAGE reverse staining) have been described previously.⁽¹²⁾ The protein concentration was determined with BCA assay and Nanodrop spectrophotometry analyzer (Thermo Scientific, Fremont, CA).

Recombinant Foxp3 production for antigen coating

For expression of recombinant protein Foxp3, pET24a-Foxp3 plasmid containing truncated Foxp3 sequence (which lacks a polypeptide segment of nuclear localization signal that leads to inefficient functional properties) was ligated in pET24a expression vector; it was transformed into competent E. coli BL21 (DE3). Transformed cells were grown at 37°C in LB medium containing kanamycin (50 μg/mL) until the exponential phase (OD600 nm = 0.6), followed by induction with 1 mM IPTG (Fermentas, Vilnius, Lithuania). Samples were collected after 3 h and analyzed by SDS-PAGE to follow the protein expression. To purify the recombinant protein, E. coli BL21 (DE3) containing pET24a-Foxp3 plasmid was grown in large scale, and the pellets of bacterial cells expressing protein were harvested and resuspended in lysis buffer (8 M urea, 0.1 M NaH₂PO₄, and 0.01 M Tris [pH 8.0]) containing protease inhibitors. Cell suspension was sonicated and centrifuged for 20 min at 10,000 rpm. After centrifugation, the recombinant protein (∼42 kDa) was purified from supernatant under denaturing conditions via its His-tag using Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Ni-NTA Agarose, Hilden, Qiagen, Germany) according to the manufacturer's instructions.^(13,14) The output fractions were analyzed by SDS-PAGE and the quantity of protein was determined with BCA protein assay and Nanodrop analyzer. The Foxp3 protein solution was dialyzed against 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 72 h to remove urea, filtered (0.22 μm, Sartorius, Goettingen, Germany), and stored at −70°C until use.

Western blot analysis

For Western blot analysis, the separated proteins by SDS-PAGE gel were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was then blocked in Tris-buffered saline (TBS) containing 5% bovine serum albumin (BSA) overnight at 4°C and washed three times with TBS containing 0.05% Tween-20 (TBST). Afterward, the nitrocellulose membrane was incubated for 2 h at room temperature with mouse anti-his tag antibody (Qiagen) diluted 1:10,000 in TBS-T. Then the membrane was washed with TBS-T and incubated with goat anti-mouse immunoglobulin G (heavy and light chain) horseradish peroxidase (HRP) conjugate antibody (diluted 1:5000 in TBS-T) for 2 h at room temperature. After washing three times, the membrane was treated using DAB solution (Sigma, St. Louis, MO) and placed in darkness until the appearance of the protein band.^(15,16)

Experimental groups, immunization procedures, and bleeding

Six- to eight-week-old female C57/BL6 mice were purchased from Pasteur Institute of Iran and kept in standard conditions. The mice were assigned into three groups (n = 10) and immunized with different immunogens, as summarized in Table 1. The mice were immunized intramuscularly (into the quadriceps muscles of leg) with 100 μg of the plasmids (pIRES2-EGFP-Foxp3 or pIRES2-EGFP-Foxp3-Fc(IgG2)) in the final volume of 100 μL (1μg/mL) and subcutaneously (s.c.) in the back of the neck with 30 μg of purified protein immunogen containing Foxp3 or Foxp3-Fc formulated in incomplete Freund's adjuvant (Sigma). Vaccination was performed as heterologous prime/boost immunization using purified plasmid DNA prepared by EndoFree Plasmid Giga Kit (Qiagen, Victoria, Australia) in the first immunization followed by two protein administrations on the second and third immunization dates (Table 1). Control group was injected by pIRES2-EGFP and BSA (Table 1). Two weeks after the last immunization, the mice were bled and serum samples were collected and stored at −70°C until use.

Performing ELISA for specific antibody and subclasses

The enzyme-linked immunosorbent assay (ELISA) was used to determine the presence of anti-Foxp3 antibodies in the sera of immunized mice. Ninety-six well micro-titer plates (Extragene, Taiwan) were coated with 100 μL of 10 μ/mL of recombinant Foxp3 protein (1 μg/well) diluted in PBS, and incubated overnight at 4°C. Each plate was washed five times with PBS–T and blocked with PBS containing 5% BSA (blocking buffer) for 2 h at 37°C. Following blocking and washing, mouse sera were added (diluted in blocking buffer 1:500) and plates were incubated for 1 h at 37°C. The plates were then washed five times and incubated with HRP-conjugated anti-mouse IgG (Sigma) diluted 1:10,000 (as secondary antibody) at 37°C for 1 h. To develop the reaction, plates were washed and incubated with tetramethylbenzidine (TMB) as the substrate for 30 min at room temperature in dark conditions. The reaction was stopped with 100 μL of 2N HCL.

To determine the antibody subtypes, biotinylated antibodies against mouse IgG1, IgG2a, IgG2b, or IgG3 (Sigma, 1:1000 dilution), streptavidin-HRP conjugate (Sigma, 1:3000 dilution), and consequently TMB substrate were used for detection. In both experiments, absorbance was read at an optical density of 450 nm by ELISA reader.

Statistical analysis

Data were summarized using descriptive statistical methods. One-way analysis of variance (ANOVA) was used to compare the mean values. A p value <0.05 or 0.001 represented a significant difference. All data were analyzed and depicted using SPSS Software (v. 20.0) and Graph pad Prism 5.

SDS page analysis of Foxp3

Cells harboring pET24a-Foxp3 plasmid were cultured at 37°C in the presence of IPTG. The whole cell lysates were analyzed by 12% SDS-PAGE. One major band appeared at the ∼42 kDa position in the case of IPTG induction, which was the expected position of Foxp3 (Fig. 1A). Induction of the cells at 37°C for 3 h and IPTG with dosage of 1.0 mmol/L was optimal to achieve the highest level of Foxp3 expression. Afterward, recombinant protein was carefully purified with Ni-NTA affinity chromatography under denaturing conditions (Fig. 1B).

OPEN IN VIEWER

FIG. 1.

(A) SDS-PAGE analysis of recombinant Foxp3 expressed in E. coli BL21. Lane 1, crude transformed bacterial extract; lane 2, crude bacterial extract before induction by IPTG; lane 3, crude bacterial lysate 3 h after induction; lane M, protein MW marker in kDa. (B) Purification of rFoxp3 on Ni-NTA column. Lanes 1–4, elusion samples passed through columns (E1,2,3,4) that show 42 kDa protein (Foxp3). The most concentrated protein is in E2 sample. Recombinant Foxp3 protein bands are indicated by arrows.

Western blot analysis of Foxp3

Western blot analysis was performed to detect the expression of the desired protein. The major band observed in SDS-PAGE (42 kDa) (Fig. 2) was confirmed as Foxp3 protein by Western blot analysis with mouse serum anti-His-tag, which indicates the apparent molecular mass of 42 kDa.

OPEN IN VIEWER

FIG. 2.

Western blot analysis of recombinant Foxp3 protein probed by anti-His (1:10,000). Lane 1, negative control (BL21 transformed with pET24a+); lane 2, pellet of IPTG-induced bacteria; lane M, protein MW marker in kDa.

Humoral immune responses

Total IgG

To determine whether Foxp3-Fc immunization can induce antibody responses, specific antibody production was measured by an optimized ELISA method with sera obtained from three vaccinated groups 2 weeks after the last vaccination (Table 1). All animals immunized with recombinant vaccines (Groups II and III) were able to break humoral tolerance to produce the specific antibody anti-Foxp3. As depicted in Figure 3, the highest antibody titers were observed in the third group of immunization—Foxp3-IgG2Fc vaccinated group (p value = 0.004)—although total IgG titers were also significant in the second group of vaccination compared to control (p value = 0.03). Taken together, this result indicates the superiority of Foxp3-Fc in inducing B cell responses.

OPEN IN VIEWER

FIG. 3.

Comparison of specific total IgG antibody (anti-Foxp3) levels in sera of control and immunized mice indicates significant increase in Foxp3 and Foxp3-Fc groups. mean ± SD,*p ≤ 0.05, **p ≤ 0.001, n = 10.

IgG subclasses

Assessment of subclass antibodies of IgG may elucidate the direction of immunological responses; therefore subclass antibodies were measured following total IgG antibody measurement. As shown in Figure 4, IgG2b subclass, which is one of the T helper 1–dependent subclasses, has the highest serum level in the second and third groups of vaccination compared to the other subclasses in their respective groups. The increase of IgG2b is significant compared to other subclasses in both vaccinated groups II and III (p value <0.0001). Despite the fact that the serum level of IgG3 was also augmented in group III compared to IgG2a subclass, its increase was not significant compared to its level in the control group.

OPEN IN VIEWER

FIG. 4.

Comparison of Foxp3 specific IgG subclass (IgG1, IgG2a, IgG2b, IgG3) levels in sera of vaccinated and control groups. See text for detailed procedure and results. mean±S.D,*p ≤ 0.05, ***p ≤ 0.0001.