Function of aTreg

Although the differentiation source and pathway is difference, the function of aTreg is very similar to nTreg. The major function of Treg is to suppress the immune

responses and this suppression must be triggered by TCR activation. Interestingly, once activated, the suppression seems to be antigen-nonspecific. The mechanism of

the suppression is cell- contact dependent. This contact educates the CD4⁺ T helper cells to become induced suppressor cells. It might mediate directly to the T effector cells by GITR and CTLA-4 [49]. On the other way, the contact suppression can also mediate indirectly. Ligation between CD80/CD86 on the DCs and CTLA-4 on Treg could modulate DC function by inducing the expression of IDO (indoleamin 2, 3-dioxygenase) which degrades tryptophan. The reduced Try concentration results in reduction of T cell activity.

Chapter 2 Strategy

Combine with the possible immune suppression function, the indispensable character, and the high conserve homology, Hp Hsp60 might be the unidentified factor which helps all the H. pylori strain survive in the host. In this study, we want to know the effect of Hp hsp60 on T cells, especially on the T cell differentiation toward Treg.

At first, we examined the effect of Hp hsp60 on the immune cell proliferation.

Thus, we used PBMC and CD3⁺ T cells as our targets. PBMC are composed of many kinds of immune cells from both innate and adaptive immunity. We used PBMC as our model to mimic the environment of gastric infection area. CD3⁺ T cells involve in many areas of immunity including B cell activation, T cell differentiation, and the survive of innate immunity. Thus, we also used CD3⁺ T cells as our investigate target.

If Hp hsp60 has an inhibitory effect on the cell proliferation, we want to demostrate whether the mechanism of this inhibition is cell arrest or death. In our experiment, we confirmed it by Annexin V/PI double staining assay to detect the death signal pathway.

Next, we will see whether the percentage of CD4⁺CD25⁺ T cells would be increase in CD4⁺ T cells after the treatment of Hp hsp60 by CD4/CD25 double staining. Because the surface staining is not powerful to identify the differentiation of Treg, thus, the foxp3 mRNA expression level would be detected by combination of reverse

transcription and real-time PCR.

Chapter 3 Material and Method

3.1 Material

3.1.1 Reagent

The following reagents and chemicals were obtained as indicated: RPMI 1640, Fetal Bovine Serum (FBS), BSA, and Tryzol from Invitrogen (Gaithersburg, MD, USA). Penicillin/ streptomycin/ amphotericin (PSA) from Biological industries (Beithaemek, Israel). Restriction enzymes from Promega (WI,USA). Kanamycin, pfu polymerase, DNA agarose, Tryptone and Tris bas from MDBio Inc. (Rockville, MD, USA). Ethidium bromide (EtBr), Isopropyl-beta- D-thiogalactopyranoside (IPTG), NaCl, yeast extract, agar, Tris-HCl, Triton X-100, 40% 29:1 acrylamide:

Bis-acrylamide, TEMED and imidazole from Amresco (Solon, OH, USA).

Ficoll-Paque^TM Plus from GE healthcare (Uppsala, Sweeden). Recombinant human interleukin-2 (rhuman IL-2) and rhTGF-β from Peprotech (Rocky Hill, NJ). Primers from Protech (headquartered in Taipei, Taiwan). Sephadex G-25 Medium from Amersham Bioscciences (Uppsala, Sweeden). Nitrocellulose (NC) paper from

PALL(Ann Arbor, MI, USA). Developer and fixer from Kodak (Rochester, NY, USA).

Isopropanol from E-ECHO (Miaoli,Taiwan). Propidium iodide (PI), RNase A,

Sodium deoxycholate, APS (ammonium persulfate), and SDS (sodium dodecyl sulfate) from SIGMA-ALDRICH (Steinheim, Germany). EDTA and chloroform from TEDIA

(Fairfield, OH, USA). NaOH, H3PO4, KH2PO4, Na2HPO4, Tween 20, KHCO3, NaN3, and KAc from SHOWA (Saitama, Japan). KCl from Scharlau (Barcelona, Spain).

Na2HPO4 from J. T. Baker (Phillipsburg, NJ, USA).

3.1.2 Antibody

The following antibodies were obtained as indicated: Goat anti-mouse IgG MicroBead from Miltenyi Biotec (Bergisch Gladbach, Germany). Mouse anti-human CD3 (UCHT1), mouse anti-human CD3-FITC (HIT3a), mouse anti-human CD4- FITC (RPA-T4), and mouse anti-human CD25-PE (BC96) from Biolegend (Sandiego, CA, USA). HRP- conjugated rabbit anti 6X His antibody from Novus (Littleton, CO, USA). HRP-conjugated goat anti rabbit IgG from MP Biomedicals (Aurora, OH, USA). Mouse anti-human CD3 (OKT3) was kindly provided from Dr. Steve R.

Roffler (ACADEMIA SINICA, Institute of BioMedical Sciences).

3.1.3 Kit

The following kits were obtained as indicated: superscript III RT kit from Invitrogen. Human TGFβ1 ELISA kit, and MTS ( 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium ) assay kit from Promega. RealQ-PCR master mix kit from Ampliqon (Copenhagen, Denmark).

Coomasie Plus^TM Protein Assay Reagent kit and Enhanced chemiluminescence (ECL) system from Pierce (Rockford, IL, USA). Annexin V-FITC apoptosis detection kit from Invitrogen.

3.1.4 Instrument

HisTrap^TM HP column from GE healthcare. NeucleoBond ion-exchange resin from Macherey-Nagel (Düren, Germany). MACS® separation LS column from Miltenyi Biotec (Bergisch Gladbach, Germany). UV photography system from EZlab.

Sunrise remote control (TECAN). ABI prism 7000 from ABI (USA). FASCan from BD (Bedford, MA, USA).

3.1.5 Others

Escherichia coli (BL21 and DH5α) from Yeastern Biotech Co. H. pylori genome

from Department of Internal Medicine, College of Medicine, National Taiwan University. Human cDNA (complementary DNA) library were kindly provided from Dr. Chich-Sheng Lin (NCTU, Laboratory of Biomedical Engineering, Biological Science & Technology Lab). Nitrocellulose paper (NC paper) and 0.45μm syringe filter from PALL. X ray film from Midsci. Human white blood cells were obtained from Hsinchu Blood Center.

3.2 Method

3.2.1 pET-Hp Hsp60 plasmid construct

For rHp Hsp60 expression, pET-HpHsp60 was constructed (see Appendix). The vector is derived from pET-30a which carries an N-terminal His-tag and multiple cloning sites under the control of T7 promoter. The full-length of Hp Hsp60 was amplified from H. pylori genomic DNA by pfu DNA polymerase. H. pylori genome was isolated clinical strain (HC28) which obtained from gastric cancer patient. The full-length H. pylori was amplified with forward primer 5’- ATC GAA TTC ATG GCA AAA GAA ATC AAA TTT TCA - 3’ and backward primer 5’- GAT CTC GAG TTA CAT CAT GCC GCC CAT G -3’ by PCR (polymerase chain reaction). PCR consisted of a 94 denaturation step followed by 35 cycles of 45℃ s at 95 , ℃ 45 s at 50 and 2℃ min at 72 . After these cycles, incubate the PCR mixture ℃ at 72 10℃ min for complete elongation. The PCR product was checked with 0.8 % DNA agarose gel by electrophoresis (Figure 1A) and the DNA length is about 1641 bps. The full gene and pET-30a vector were digested with EcoR I and Xho I at 37 ℃ for 3 hr and checked with gel electrophoresis (Figure 1B). The digested fragment length of vector and insert should be 5422 and 1641 bps, respectively. After ligation and transformation, we picked several single colonies from the plate and checked the

insert by screening with RE digestion (Figure 1C). After check, the plasmids were sequenced with primers. The DNA sequence inserted into vector has been submitted to NCBI nucleotide data base (DQ674277).

3.2.2 RE digestion

Restriction enzyme digestion was used for cloning and checking. We used 0.5 μl

RE (EcoRI and XhoI) (10 U/μl) to digest 1 μg DNA in 20 μl volume at 37 for 3℃ hr.

The reaction mixture consists of DNA, 10 X RE buffer H, 100 X BSA, RE, and DDW.

The digested DNA fragments were checked with 0.8 % DNA agarose gel by

electrophoresis. The gels were analyzed with UV after staining with EtBr for 10 min.

3.2.3 Transformation

Remove the appropriate number of competent cells tubes from the -80 freezer. ℃ DH5α was used for cloning and DNA amplification. BL21 was used for protein expression. After the cells were thawed, add 1 ng DNA into the cells, mix by gently swirling the tip. Incubate the competent cell on ice for 30 min. Heat shock the cell at 42 ℃ for 90 s. Place the cells on ice for 2 min and add 250 μl LB (10 g tryptone, 10 g NaCl, 5 g Yeast extract per liter) and incubate at 37 ℃ with shaking 225 rpm for 1 hr.

Spread 100 μl mixture onto each LB agar plate (10 g tryptone, 10 g NaCl, 5 g Yeast

extract, 20 g agar per liter) containing kanamycin (30 mg/ml) and incubate at 37 ℃ for 12~16 hr.

3.2.4 Midi-preparation

After transforming the competent cell with plasmid, we picked single colony

from the LB agar plate and incubate with 100 ml LB containing kanamycin (30 μg/ml) at 37 Ԩ for 16 hr at 225 rpm shaking incubator. The broth was harvested by

centrifuging at 8000 rpm for 15 min. The supernatant was discarded and the pellet was resuspended in 8 ml buffer S1 (50 mM Tris-HCl, 10 mM EDTA, 100 μg/ml RNase A, pH 8.0). 8 ml buffer S2 (200 mM NaOH, 1 % SDS) was added for lysis, gently mixed by inverting 6~8 times, and incubate at room temperature for 3 min. 8 ml ice-cold buffer S3 (2.8 M KAc, pH 5.1) was added for neutralizing, gently mixed by inverting 6~8 times, and incubate on ice for 2 min. Centrifuge the cell lysates at 12000 rpm for 30 min at 4Ԩ. Wash NeucleoBond ion-exchange resin with 5 ml buffer N2 (100 mM Tris, 15 % ethanol, 900 mM KCl, 0.15 % Triton X-100, adjusted to pH 6.3 with H3PO4) while the lysate centrifuging. After centrifugation, apply the

supernatant to the pre-rinsed resin, followed by washing with 12 ml buffer N3 (100 mM Tris, 15 % ethanol, 1.15 M KCl, adjusted to pH 6.3 with H3PO4) twice. Elute the column with 5 ml buffer N5 (100 mM Tris, 15 % ethanol, 1 M KCl, adjusted to pH

8.5 with H3PO4) and separate the eluted mixture into six fractions equally (~800μl).

Precipitate DNA by adding 0.7 volume of isopropanol (about 700 μl) and kept on ice for 10 min. Centrifuge at 13000 rpm for 30 min at 4Ԩ. The DNA pellet was washed by 1 ml ice-cold 70% ethanol. Discard the ethanol, air-dried, and dissolve with 30 μl DDW. Measure the absorbance at 260 and 280 nm to check the DNA quantity and quality.

3.2.5 rHp hsp60 Protein expression

E. coli [BL21 (DE3)] freshly transformedwith pET-Hp hsp60 were grown onLB plates containing kanamycin (30 mg/ml) at 37°C.After 16 h incubation, 5 colonies grown on the LBplates were picked and inoculated into 100 ml LB medium

containing kanamycin (30 mg/ml) at 37 °C for 16 hr. Refresh the culture broth in 900 ml LB medium with shaking at 37 until the value of OD600 reaches 0.6 (abou℃ t 40 min). Add IPTG from a 800 mM stock to a final concentration of 1 mM and continue the incubation for 4 hr. Harvest the cells by centrifugation at 5000 rpm for 15 min at 4

. Discard the supernatant and resuspend the pellet with 30

℃ ml binding buffer (20

mM Na2HPO4, 0.5 M NaCl, 40 mM imidazole, pH 7.4). Total cell lysates were sonicated with short burst of 1 sec followed by intervals 1 sec and the sonication processing was maintained for 15 min. Centrifuge the solution at 12000 rpm for 30

min at 4 . ℃ Harvest the supernatant. In this experiment, we purified our proteins with HisTrap^TM HP column. All the solutions used in this experiment were needed to be filtered with 0.45 μm syringe filter. To prepare the column, wash the column with 5 column volumes of DDW and equilibrate the column with 5 column volume of binding buffer at the flow rate about 1ml/min. Apply the pretreated sample and wash with wash buffer (20 mM Na2HPO4, 0.5 M NaCl, 60 mM imidazole, pH 7.4) about 60 volume. Elute with elution buffer (20 mM Na2HPO4, 0.5 M NaCl, 200 mM imidazole, pH 7.4) for 10 volumes. Detect which fractions contain proteins with coomasie

reagent. Collect the positive fractions and use G25 column to remove the unnecessary salt from the solution and replace the buffer with PBS (Phosphate Buffered Saline, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). To prepare the G25 column, we need to swell the 7 g Sephadex G-25 Medium with filtered PBS at room temperature for overnight. Fill the column with PBS. Resuspent the medium and pour the medium into the column. We poured the protein into the G25 column and eluted with PBS. Detect which fractions contain proteins with

coomasie reagent and collect the fractions. Poll the fractions together and filtered with 0.22 μm syringe filter. Check the protein concentration of each part with coomassie reagent (Figure 2) and dilute the product to the final concentration to 1 mg/ml. The recombinant protein was checked by SDS-PAGE (sodium dodecyl sulfate

polyacrylamide gel electrophoresis) and Western blotting with anti-His conjugate HRP (Figure 3).

3.2.6 SDS-PAGE and Western blotting

The purified proteins were mixed with 3X protein sample dye and denature at 95

℃ for 10 min. In our experiment, we used the 10 % acrylamide gel to analysis the protein size. Each lane was loaded with 10 μl mixture and the electrophoresis was processed with 130 Volt about 80min. the gel were stained with staining buffer for 1 hr and followed by destaining with destain buffer I for 30 min and destain buffer II overnight. For Western blotting, after electrophoresis, the gel was transferred with nitrocellulose paper (NC paper) with 200 mA for 2 hr at room temperature. After transfer, the NC paper was blocked by blocking buffer (5 % skim milk, 0.05 % tween 20 in PBS) at room temperature for 1 hr. Because the recombinant proteins contain histidine tag, we used HRP- conjugated rabbit anti 6X His antibody (1000X dilution) to recognize the recombinant protein. Wash the NC paper with PBST (0.05 % tween 20 in PBS) at room temperature on shake for 5min three times for all washing steps.

The membrane was developed with ECL system in dark at room temperature for 5 min and exposed to X ray film for 10 s. Develop the film with developer until band was present and fixed the film with fixer for 1 min. Wash the film with water.

3.2.7 PBMC isolation

Peripheral blood mononuclear cells (PBMCs) were separated from human white blood cell solution by using Ficoll-Paque^TM Plus. Dilute human white blood cells with equal volume of PBS. Add Ficoll-Paque Plus (6 ml) into the 15ml centrifuge tube and carefully load the diluted blood sample (8 ml) on Ficoll-Paque Plus. Centrifuge the tubes at 400 g for 40min at 18 . Remove the ℃ plasma layer and collect the PBMC layer. Wash the cells with 2 volume of PBS for centrifuging at 1500 rpm for 15 min.

Discard the supernatant and lyse the red blood cells by ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in DDW) at room temperature for 10 min and followed by centrifuging at 1500 rpm for 10 min. Discard the supernatant and wash the cell with 10 ml PBS. Centrifuge for another 10 min. Discard the supernatant and count the cell number. The purified PBMC were checked by flow cytometry as shown in Figure 4A.

3.2.8 CD3⁺ T cell isolation

CD3⁺ T cells were purified by magnetic bead cell sorting with mouse anti-human CD3 and Goat anti-mouse IgG MicroBead. Magnetic staining buffer (0.5 % BSA, 2 mM EDTA in PBS, pH 7.2) was the only buffer used in this experiment. Count 10⁸

PBMC and suspend in 800 μl staining buffer. Stain the cells with 200 μl mouse anti human CD3 antibody to capture the CD3⁺ T cells at 4 for 30℃ min. Wash the cells with 5 ml buffer twice. Resuspend the cells with 800 μl buffer and stain the cells with 200 μl Goat anti-mouse IgG MicroBead at 4 for 5℃ min. Wash the cells twice and resuspend in 1 ml buffer. Put the column on the separator and wash the LS column with 3 ml buffer. Load the sample into the column and wash the column with 3 ml buffer for three times. Remove the LS column from the separator and add another 5 ml buffer. Immediately flush out the cells with plunger. Centrifuge the cells at 1500 rpm for 15 min and count the cell number. The purified CD3⁺ T cells were checked by staining with anti-human CD3 conjugated FITC and analyzed by flow cytometry.

Compare with the unstrain cells, the purity of our purified CD3⁺ T cell is over 99 % (Figure 4B).

3.2.9 Cell culture

Jurkat is an acute T cell leukemia cell line cultured with RPMI 1640 containing 10 % heat-inactivated FBS, 1 % PSA. The PBMC and CD3⁺ T cells were cultured with RPMI 1640 containing 10 % heat-inactivated FBS, 1 % PSA, 100 U/ml rhuman IL-2. All the cells were incubated at 37 , 5% CO℃ 2 condition.

3.2.10 MTS assay

To evaluate the growth rate of Hp Hsp60-treated cells, MTS assay was used in this experiment. PBMC (2*10⁵/well), CD3⁺ T cells (2*10⁵/well) and Jurkat

(2*10⁴/well) were seeded in anti-CD3 mAb-precoated (1 μg/ml, 30 μl per well at 37 for 2

℃ hr.) 96 well and incubated with various doses of Hp Hsp60 (1 μg/ml, 5 μg/ml, and 10 μg/ml). After incubated for 48 hr (Jurkat) and 4 day (PBMC), 40 μl MTS was added into wells and incubated for another 4 hr in 37 . The OD ℃ absorbance was measured at 490 nm with sunrise remote control.

3.2.11 Annexin V-FITC/PI apoptosis assay

To evaluate the survive rate of Hp Hsp60-treated cells, Annexin V-FITC/PI apoptosis detection kit was used in this experiment. PBMC (2*10⁵/well) and CD3⁺ T cells (2*10⁵/well) were seeded in anti-CD3 mAb-precoated 96 well plate and incubated with various doses of Hp Hsp60 (1 μg/ml, 5 μg/ml, and 10 μg/ml) for 4 day. After 4 day, cells were washed with 1 ml PBS. Each sample was stained with 1μl Annexin V-FITC, 25 ng PI, and suspended in 20 μl staining buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4) for 15 min at room temperature. Add 800 μl PBS into the mixture and analyze the sample on FACScan in FL1 and FL3 channels with dot plots with quadrant line.

3.2.12 TGF-β cytokine secretion detection

10⁶/ml PBMC and CD3⁺ T cells were seeding in one well of 24-well plate.

PBMC was treated with different doses (100 pg/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 μg/ml, and 10 μg/ml) of Hp hsp60 for 24 hr. Harvest the supernatant and stock at -80

℃ refrigerator until the assay beginning. Before assay, we acidified 100 μl sample with 5 μl 1N HCl for 15 min at room temperature. Next, 5 μl 1N NaOH was added to neutralize the sample. The 96 well ELISA plate was coated with 1000 X coating mAb and incubate at 4℃ for overnight. Remove the coated plate from refrigerator and allow it to room temperature. Flick out the contents of the wells and slap the plate upside down on a paper towel until the well dry. Add 270 μl 1 X block buffer to each well at 37℃ for 35 min. Wash the plate with PBST five times and flick out the contents of the wells and slap the plate upside down on a paper towel until the well dry. Add 100 μl standard or sample to each well at room temperature for 2 hr. Repeat wash procedure. Add 100 μl 1000 X anti-TGF-β pAb at room temperature for2 hr.

Repeat wash procedure. Add 100 μl 100 X TGF-β-HRP conjugate at room

temperature for 2 hr. Repeat wash procedure. Add 100 μl 1000 X anti-TGF-β pAb at room temperature for 2 hr. Add 100 μl TMB for each well at room temperature for 15 min in the dark. Next, add 100 μl 1 N HCl to stop the reaction and measure the

absorbance at OD 490nm.

3.2.13 Cell surface marker staining

PBMC (10⁶ cells/well) and CD3⁺ T cells (10⁶ cells/well) were treated with 10 μg/ml Hp Hsp60 and 2 ng/ml TGF-β for 6 days. Centrifuge the cells at 1500 rpm for 10 min. Count the cell number and take 2*10⁵ cells for this experiment. Resuspend the cells with 500 μl staining buffer (1% BSA, 0.05 % NaN3 in PBS). Stain the cells with antibody mixture (2 μl mouse anti-human CD4-FITC and 1 μl mouse anti-human CD25-PE in 500 μl staining buffer (1 % BSA in PBS)) on ice in the dark for 30 min.

For compensation, we also stained cells with 2 μl anti-human CD4-FITC and 1 μl mouse anti-human CD25-PE respectively as control. After washing the cells with 500 μl staining buffer, centrifuge the cells at 1500 rpm for 5 min. Repeat again. Analyze the cells on FACScan in FL1 and FL3 channels with dot plots with quadrant line.

3.2.14 RNA isolation

PBMC and CD3⁺ T cells were seeded with the same condition of the surface marker staining assay. Count the cell number. Take 10⁶ cells and freeze the cell pellet in -80 freezer. In this experiment, we used Trizol reagent for RNA isolation. The ℃ cell pellets were homogennizated by adding 1ml Trizol. Shake violently and incubate

at room temperature for 5 min. Add 0.2 ml chloroform and shake vigorously by hand.

Incubate at room temperature for 3 min and 12000 rpm for 15 min at 4 . Transfer ℃ the aqueous phase to a fresh tube, precipitate the RNA from the aqueous phase by mixing with 0.5 ml isopropanol. Incubate the sample at room temperature for 10 min.

Centrifuge at 1200 rpm for 10 min and discard the supernatant. Wash the pellet with 1ml 70 % ethanol by vortex and centrifuge at 7500 rpm for 5 min. Discard the supernatant and air-dry for 15 min. Dissolve the pellet with 10 μl DEPC water and incubate at 60 for 10℃ min. Check the RNA quality and quantity by measuring the OD 260 and 280.

3.2.15 Reverse transcription

After quantitating the RNA level, 1 μg RNA from each sample was used for

reverse transcription by using the superscript III RT kit. By following the manufacturer's recommendation, we mix the RNA with 1 μl dNTP, 1 μl random haxamer and DEPC water up to 10 μl. Incubate the sample at 65 for 5℃ min. Then, each sample is added with 2 μl 10X RT buffer, 4 μl 25mM MgCl2, 2 μl 0.1M DTT, 1 μl RT (200 U/μl), and 1μl RNaseOUT (40 U/μl). Incubate the mixture at 25 for 10 ℃ min and followed by 50 , 50 min. To terminate the reaction, incubate the mixture at ℃

reverse transcription by using the superscript III RT kit. By following the manufacturer's recommendation, we mix the RNA with 1 μl dNTP, 1 μl random haxamer and DEPC water up to 10 μl. Incubate the sample at 65 for 5℃ min. Then, each sample is added with 2 μl 10X RT buffer, 4 μl 25mM MgCl2, 2 μl 0.1M DTT, 1 μl RT (200 U/μl), and 1μl RNaseOUT (40 U/μl). Incubate the mixture at 25 for 10 ℃ min and followed by 50 , 50 min. To terminate the reaction, incubate the mixture at ℃