Shreffler:Milk Program

Overview

Milk allergy is the most common cause of food allergy in infants and young children, affecting 2.5% of infants in industrialized countries. Previous studies suggest that the majority will tolerate heat-denatured products without harmful effects. Our preliminary findings that ingestion of extensively baked-milk products may enhance tolerance induction. This study is designed to determine whether frequent dose escalation of milk protein in allergic patients would increase the tolerance of their immune system.

Through the baked milk 2 study, we plan to study the immunologic mechanism associated with oral tolerance induction. We propose to investigate the effect of introducing baked-milk products by comparing the rate of tolerating less heated milk in children subjected to more frequent dose-escalation (every 6 months) and in children subjected to less frequent maintenance increases (every 12 months).There will be a comparison of the tolerance of an individual patient longitudinally over a period of time. Also, the changes in T regs would be evaluated in five groups, who are given different doses of baked milk proteins.

CFSE is used to fluorescently label live cells in order to track them. 5,6-CFDA/SE is membrane permeable, but trapped in the cell when converted by intracellular esterases. CFSE is equally partitioned to daughter cells during division and can be used to measure cell proliferation.

T regulatory Assay

Materials

• 15 mL polypropylene tubes
• PBS
• 5,6-CFDA/SE Invitrogen C1157 dissolved in DMSO at 5 mM and stored at -20°C.

Procedure

Basophil Assay and Isolation of Mononuclear Cells (done simultaneously)

1. Label 4 (50mL) tubes as PBS: Blood, AIM-V, PBMC, Ficoll and 2(15mL) tubes as Basophil blood and RPMI
2. Bring 15/10mL of Ficoll(sterile) to room temperature and put 5mL RPMI in water bath.
3. Collect pre-made stimulants from the freezer (Rm 40 in clear boxes with rubber bands). LABEL each one
4. Collect 6 epindorph tubes. Label 1 of these tubes "basophil cocktail." Make a cocktail as follows: Combine 180 uL RPMI + 3.6 uL IL-3. MIX WELL.
5. Label the 5 remaining tubes "Basophil medium." Add 30 uL of the cocktail into these tubes.
6. Add 270 uL of the warmed RPMI to the five basophil tubes(made above), fMLP, milk extract, and anti-IgE.
7. Add 135 uL of RPMI to PMA and CaI and then combine them.
8. Prepare a 10-fold dilution of milk stimulants:

• Transfer 270 uL of basophil medium from the aliquotes prepared in step 2 to each of the milk 2-5 epindorph tubes.
• Transfer 30 uL from tube "milk 1" to "milk 2". VORTEX.
• Take 30 uL from "milk 2" and add to "milk 3" etc. Vortex after each step.

9. Label 5 mL polypropylene tubes A-K. Remember to include subject number on tube A as BM055-06. list of stimulants

10. Transfer 250 uL of warm RPMI to tubes A & B.

11. Transfer 250 uL of each stimulant to the appropriate polypropolene tube (C-K) and keep them in incubator until ready to start.

12. Get the blood from the clinic, open the two green top tubes under the hood.

13. (keep sterile) Set aside 3mL of blood into pre-labeled tube for basophil assay.

14. Transfer remaining blood in pre-labeled 50mL tube and dilute 1:1 with PBS

15. Overlay with 30 mL of diluted blood on the 15mL ficoll.

16. Centrifuge at 500 g for 30 minutes at 23°C with acceleration slow and brake off. now back to basophil protocol

17. Take out the labelled tubes from incubator

18. Transfer 250 uL of participant blood to each tube A-K.

19. Incubate tubes for 30 minutes at 37°C (in incubator).

20. Prepare the cocktail. Label a 5 mL polypropylene tube "staining cocktail" Add 700 uL of staining buffer.

   a. 35 uL of HLA-DR-PE-Cy7 mAb
   b. 70 uL of CD63- FITC
   c. 70 uL of CD203c-PE
   d. 70 uL of CD123 PE-cy5
   e. 70 uL of CD41a
   f.  70 uL of CD3
   g. 70 uL of CD14
   h. 70 uL of CD19-APC

21. Remove tubes from incubator and add 50 uL cold PBS w/20 mM EDTA to each tube to stop degranulation

22. Stain cells by adding 110 uL of the prepared Ab Cocktail to tubes B-K. Do NOT add to Tube A.

23. Put it in the refrigerator(4°C) for 30 minutes.

• Return to isolation of PBMC's. Remember to keep conditions STERILE!

24. Remove ficoll tubes from centrifuge. Using a sterile transfer pipette , collect PBMCs (cloudy gray layer) into a new 50 mL conical tube.

25. Under the hood, add sterile PBS to double the volume and invert the tube to mix. Centrifuge at 500 g for 20 minutes at room temperature (max acceleration and deceleration).

26. Aspirate and discard the supernatant. Resuspend the pellet by tapping the tube until no clumps are visible, then adding 1 mL of PBS.

27. Set aside a 10 uL aliquot of cells for counting: 10 uL of cells into 90 uL of PBS in a STERILE eppendorf tube.

28. Add PBS to cells to make a total volume of 20 mL.

29. Centrifuge at 300 g for 15 minutes at room temperature (maximum acceleration and deceleration).

30. While the cells are in the centrifuge, you must determine the volume to use for resuspending the PBMCs after this wash. This in turn requires knowledge of the total number of cells in the sample:

a. Combine the 100 uL aliquot of cells in PBS set aside in Step 28 with 100 uL of 0.2% Trypan solution (if using the automated counter) or 0.4% Trypan soplution (if maual counting).

b. Mix well with a pipette.

c. Place 20 uL of the stained cells onto a disposable slide.

e. To take the cell count, in the computer program, write dilution as a whole number (20x). [1st diluted 10 μL of cells in 100 μL total volume PBS + Cells, then added an additional 100 μL of Trypan solution

           = 10uL/200uL = 1/20 = 20 fold dilution).

f. Choose cell type from drop down menu ("Human")

g. Pick "display"

h. Focus image so the cells appear yellowish

i. Hit "Count."

31. After centriguation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clumps are visible. Suspend PBMCs at 10 x 10^6 cells/mL in PBS. To calculate this: Take number counted in step 31i divided by 10 x 10^6 cells/mL. This gives you the the total volume of PBS you must add to the cells.

32. Divide Cells as follows: a. 10 x 10^6 cells for measurement of Th1/Th2 gene transcription in allergen-activated cells by RT-PCR (after 48 hr culture). b. 10 x 10^6 cells for measurement of frequency of PBMC-derived, milk-specific CD4+, CD25+, FoxP3+ T cells (after 7 day culture). c. If indicated, remaining cells will undergo CD25 Depletion Experiments.

• Note, if you collect less than 2 mL of PBMC's in step 32, just divide the volume equally to the 48 hour and 7 day test tubes in step 33.

Now prepare a T-Regulatory Cell Assay for 48/ 7 DAY CULTURE PREPARED ABOVE.

33. In a 15 mL conical tube, make a PBS: CFSE solution: a. 1 mL PBS b. 2 uL of 5 uM CFSE

34. Add the PBS: CFSE solution just prepared to the 7 DAY CULTURE PBMC's.

35. Place in a 37°C water bath for 10 minutes.

Proliferation Assay 36. STERILE CONDITIONS. Label two 24 well plates/their lids with specimen ID and date. Label each well with the appropriate condition, ordered by priority (for cases where there are insufficient cells to test all stimulants). a. Allergens- Caseins (C+): 50 ug/mL purified casein proteins (alpha, beta, kappa) in AIM-V medium b. Negative Control- AIM-V medium (A+): Medium alone c. Positive Control- Beads (B+): 1 ug/mL anti-CD3, anti- CD28 beads in AIM-V d. Egg white (E+): 20 ug/mL egg white in AIM-V

37. Label one plate for 48 hour culture and a second plate for a 7 day culture.

38. Add 10 mL of AIM-V to both 7 and 48 hour culture tubes and spin at 300 g for 10 minutes at room temperature.

39. STERILE CONDITIONS: Aspirate the cells.

40. Under the hood, resuspend cells in 2.5 mL of AIM-V medium to obtain a concentration of 4 x 10^6 cells/mL (For plating, each well should contain at 2-2.5 x 10^6 cells and a total volume of 1 mL).

41. Prepare solutions for each stimulant condition in sterile, 5 mL polypropyene tubes. Label each tube with a notation for 48 hour culture and A+, B+, C+, E+. Also, prepare tubes for the 7 day culture samples and also label A+, B+....

42. 48 Hours Antigen Stimulation Preparation a. Place 500 uL of AIM-V in each of the test tubes. b. Add 500 uL of the 48 hr-labelled cells in AIM-V to each tube and mix by pipetting up and down. c. Then add to the respective tubes:

A+: Medium Alone. Do not add any other substances.

B+: Add 5 uL of CD3, CD28, being sure vortex first to resuspend the beads in their container.

C+: Add 2.5 uL of EACH casein (alpha, beta, kappa).

• • Be sure to mix by pippetting up and down.**

43. 7 Day Antigen Stimulation Preparation: Unlike 48 hour culture, this contains CFSE labeled cells and IL-2.

a. Create a cocktail. Place 2 mL of AIM-V + 4 uL of IL-2 in an appropriately labelled test tube. Vortex gently. b. Add 500 uL of the cocktail to the labelled A+, B+, etc. test tubes. c. To each tube, add 500 uL of the 7 day cells set aside in step 33. Mix. d. Then add to the respective tubes:

A+: AIM-V medium + IL-2 alone. Do not add any other substances.

B+: Add 5 uL of CD3, CD28 expander beeds. Be sure to vortext the beeds int their container. Then suspend the beeds in the AIM-V/IL-2 solution by pipetting up and down.

C+: Add 2.5 uL of each casein (alpha, beta, kappa). Pipette up and down.

E+: Add 10 uL of egg white. Pipette up and down.

46. Plate the stimulants for both the 48 hr and 7 day samples into the wells labeled in step 37. Then place the tissue culture plate in the incubator.

CFSE labelling of 7day culture cells

1. Suspend PBMCs at 10x10⁶ cells/mL in PBS alone.
2. Ensure that cells are uniformly suspended when CFSE is added.
3. If CFSE negative control is needed, remove cells now.
4. Make '2X' concentration (10 μM) in PBS
   • For example: add 5 mL PBS + 10 μL 5 mM CFSE
5. Combine 1:1 PBMC cells + CFSE (e.g. 5 mL PBMCs + 5 mL CFSE) in 15mL tube and mix completely.
6. Place in 37°C H₂O bath x 10 min.
7. Wash in 10 mL complete medium.
8. Resuspend in medium at desired density

Discussion

discuss this protocol

References

1. Lyons AB and Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994 May 2;171(1):131-7. DOI:10.1016/0022-1759(94)90236-4 |
2. Fuss IJ, Kanof ME, Smith PD, and Zola H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol. 2009 Apr;Chapter 7:7.1.1-7.1.8. DOI:10.1002/0471142735.im0701s85 |
3. Shreffler WG, Wanich N, Moloney M, Nowak-Wegrzyn A, and Sampson HA. Association of allergen-specific regulatory T cells with the onset of clinical tolerance to milk protein. J Allergy Clin Immunol. 2009 Jan;123(1):43-52.e7. DOI:10.1016/j.jaci.2008.09.051 |
4. Sicherer SH and Sampson HA. 9. Food allergy. J Allergy Clin Immunol. 2006 Feb;117(2 Suppl Mini-Primer):S470-5. DOI:10.1016/j.jaci.2005.05.048 |
5. Sainte-Laudy J, Boumediene A, Touraine F, Orsel I, Brianchon C, Bonnaud F, and Cogné M. Use of both CD63 up regulation and IgE down regulation for the flow cytometric analysis of allergen induced basophil activation. Definition of an activation index. Inflamm Res. 2007 Jul;56(7):291-6. DOI:10.1007/s00011-007-7014-5 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare