Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

The overall goal of this procedure is to assess both effector and memory T-cell responses using short and long-term cultured interferon gamma L spot assays respectively. The assessment of the central memory response is accomplished by culturing bovine peripheral blood mononuclear cells under antigenic stimulation for 13 days. The long-term culture allows effector T-cell responses to occur and decline.

Interleukin two is added to the cultures for maintenance of memory T-cells. On day 13, long-term cells are transferred to L spot plates coated with IFN gamma capture antibody. The response of long-term cells to individual mycobacterial antigens is evaluated either in the presence or absence of autologous antigen presenting cells.

APCs APCs are isolated from fresh p BMCs by adherence on day 13. Additionally, the response of freshly isolated pbmc under ex vivo conditions is evaluated as a correlate of effector responses. In several studies, the bovine long-term cultured sbot response to mycobacterial antigens after vaccination has been shown to positively correlate with vaccine efficacy.

Also, long-term T-cell cultures contain mainly central memory T cells in both humans and cattle In order to perform the Long-term cultured L spot assay. 60 mls of blood is collected from m bovis infected or vaccinated animals by jugular, venipuncture and P BMCs are isolated by density. Gradient centrifugation.

Cell concentration should be adjusted to 4 million cells per ml. In complete RPMI Media begin by plating 500 microliters of microbacterial antigens, in this case TB 10.4 and antigen 85 A and the purified protein derivative of em bovis culture called PPDB. Then add 500 microliters of pbmc isolated from each animal with four replicates per animal.

Note that all wells must be stimulated. Controls such as no stimulation or mitogens are not needed in This step, incubate the plate at 39 Celsius the normal temperature of cattle for 13 days. On days three and seven, discard 500 microliters from each well pipetting carefully to not disturb the cell layer at the bottom of the wells.

Replenish the volume with complete PMI containing IL Two. Place the plate in the incubator On day 10, discard 750 microliters of the supernatant from each well replenish with complete RPMI without any IL two on day 12, pipette and discard one ml of supernatant from each well replenished with complete RPMI without IL two. Also prepare the L spot plate to be coated with IFM Gamma capture antibody first pipette 15 Microliters of 35%alcohol into each well using a multi-channel pipetter this step increases the spot definition.

Wash well six times with 300 microliters of PBS. Add 100 microliters of capture antibody at eight micrograms per ml in PBS. Pipetting should be done carefully during all procedures, not allowing pipette tips to touch or damage the membrane at the bottom of the well place plate inside a plastic bag and incubate at four Celsius overnight.

On day 13, collect 60 mls of blood by jugular vena puncture from the same animals sampled in the first day. Isolate the pbmc and adjust the concentration to 2 million cells per ml. Label the L spot plate properly For each set of samples.

Long-term cells plus APCs long-term cells without APCs and ex vivo cells. Discard capture antibody from the Ellis spot plate wash wells with 300 microliters of PBS containing tween. Repeat Six times, discard, wash fluid and Pipette.

50 microliters of freshly isolated p BMCs into the wells allotted for long-term cells. Plus APCs. Block other wells of the L spot Plate with 200 microliters per well of complete RPMI To evaluate the Phenotype of cells by flow cytometry in parallel with the L spot assay plate.

50 microliters of freshly isolated cells in a properly labeled round bottom 96 well plate. This is an ancillary assay to be performed in conjunction with the yellow spot assay to determine the phenotype of responding cells. Incubate plates for 90 minutes at 39 Celsius in a 5%CO2 incubator, allowing antigen presenting cells to adhere.

Also incubate 200 mls of complete RPMI to warm it up during this 90 minute incubation step. Harvest the long-term cultured cells from the 24 well plate. Combining the quad duplicates from each animal into a single 15 mil conical tube.

Centrifuge the Tubes for five minutes at 400 Gs.Discard the supernatant by tube inversion, dislodge the cell pellet and add five mls of PBS gently resuspend the pellet if necessary. Wash cells twice and then adjust the cell concentration to 200, 000 cells per ml. After the 90 minute incubation, take the LPO plate and the complete RPMI from the incubator.

Place the LPO plate on a plate shaker for 30 seconds. Then remove non-adherence cells by plate inversion pipette 150 microliters per well of warm complete RPMI. Shake the plate and discard wash fluid.

Repeat three times, discard as much liquid as possible, and add a hundred microliters per well of each antigen into the appropriate wells containing adherent APCs. Then pipette 100 microliters per well of long-term cultured cell suspension. 200, 000 cells per ML into the wells labeled as long-term cells plus APCs.

Be sure that the long-term cells added in this step are from the same animal as the APCs already in the well. Do not mix APCs and long-term cultured cells from different animals. Also, pipette 100 microliters per well of long-term cultured cell suspension at a concentration of 200, 000 cells per ml into wells without APCs for assessing long-term responses.

In the absence of antigen presentation, add 100 microliters per well of short-term cells, plus freshly isolated and adjusted to 2 million cells per ML for ex vivo response assessment. If performing flow cytometry analysis, wash the round bottom 96, well plate and add the long-term freshly isolated cells and antigens to the wells following the same procedures. Employed for IFN Gamma LPO plate Incubate Plates overnight at 39 Celsius in a 5%CO2 incubator.

Make sure that the plates lay flat and do Not stack plates on day 14. Discard fluids from wells and wash plates six times with 300 microliters per well of PBS containing tween placing on the plate shaker each time for 10 seconds before and in between washes. Then wash one time with the same volume of distilled water and an additional six times with PBS containing tween.

Finally, wash cells twice with 300 microliters per well of PBS. Remove wash fluid by tapping the plate on paper Towels or absorbent pads. Add 100 microliters per well of detection antibody at five micrograms per ml diluted in PBS with 1%bovine serum albumin.

Incubate the plates for 120 minutes At 39 Celsius. During this incubation step, prepare the alkaline phosphatase solution from the VTA stain A BC AP kit standard following the manufacturer's instructions 30 minutes before use or 90 minutes after the addition of detection antibody to the wells. Dilute the reagent in PBS containing tween after 120 minute incubation, discard detection antibody by plate inversion wash plates six times with 300 microliters per well of PBS containing tween.

Remove wash fluid by tapping the plate on paper towels. Then add 100 microliters per well of alkaline phosphatase solution. Incubate the plates for 45 minutes at room temperature.

Discard the fluid and wash each plate six times with PBS Containing tween. Prepare the substrate Solution from the vector blue AP substrate three kit following the manufacturer's instructions. Dilute the reagents in 0.1 molar tris HCL buffer at pH 8.2.

Pipette 50 microliters of substrate D.Each well incubate at room temperature until the blue color starts to develop approximately 30 minutes. Discard fluid and wash plates with copious amounts of distilled water. Remove the bottom plate to expose the membrane wash back of the wells and allow the plate to air dry.

Measure the response using an immuno spot image analyzer or keep plates in the dark at room temperature until the response is measured. Because of the high stability of the reaction substrate quality is preserved for several years. If evaluating the phenotype and cytokine response by flow cytometry, perform standard staining procedures of the cells plated on the round bottom 96, well plate representative TCM in the presence or absence of APCs, manned ex vivo IFN gamma L spot responses from infected animals and non-infected animals are shown.

Specific responses to m bovis are assessed by PBDB or ESAT six, CFP 10 antigenic stimulation. The spots on the bottom of the plates are the colored product of the yellow spot assay. Each spot typically represents an individual cytokine producing cell, providing a quantitative measure of T-cell producing IFN gamma with cells from infected animals.

Long-term IFN gamma L spot responses presented as spot forming cells or SFCs are clearly visualized little to no IFN gamma production in response to antigen stimulation is seen with cells from non-infected cattle and with non stimulated cells from either infected or non-infected cattle. Robust T-cell response should occur in response to poke weed mitogen as shown TCM and ex vivo responses to PPDB and ESAT six. CCFP 10 were detected from all 3M bovis infected animals and minimal to no TCM and ex vivo responses were detected from the control animal.

A presence of APCs was required for optimal TCM responses by infected animals as demonstrated by the greatly reduced responses by long-term cells cultured. In the absence of autologous APCs, spot forming cells from em, bovis infected and non-infected animals were presented. The number of SFCs from each animal was calculated by an average number of SFCs at 10 to the sixth cells in duplicate samples in response to PPDB minus the respective response to media alone responses differed based on the infection status, presence, or absence of APCs and culture length, IE the long-term culture versus ex vivo culture.