Establishment of T-cell clones

Summary

This unit provides a method for establishing specific T cell clones. Note that the key to the success of this method is the selection and preparation of conditioned media. Author(s): J.E. Colligan et al, Translator(s): Xuitao Cao et al. This experiment is from the "Comprehensive Immunology Laboratory Guide".

Operation method

Establishment of the T cell clone

Move

Basic Program 1 Generate and maintain homozygous responsive T h and CTL clones

Although homoreactive T cells can be generated from unstimulated splenocytes or lymph node cells, the yield of reactive cells is higher if the cells are first stimulated with the same antigen in the initial mixed lymphocyte (M L C ). See the following description.

Material

Spleen T-cells from homozygous reactive mice

V H B S S (optional)

V D M E M -20 Complete Culture Media

Irradiated (2000 rad, unit 2.1 1 ) homozygous mouse spleen T cells

Growth factors: MLC or Con A-stimulated cell supernatant (see Supplementary protocols 1 or 2), recombinant cytokines

9 6-well flat-bottomed culture plates (Costar)

2 4-well cell culture plates (Linbro, Costar)

1 . In vitro sensitization of homoreactive T cells with alloantigens for 10 to M d (see Option 2, Step 1) is best performed with HBSS or DMEM solutions (i.e. isotonic with mouse serum).

2 . Prepare the MLC for re-stimulation (see Additional protocol 2, step 3) and incubate for 36 to 48 h.

3. Add irradiated (2000 rad) isotype splenic T cells to a 96-well flat-bottomed plate at a concentration of ^IO7 cells/m l with 100 ul per well in DMEM-20 medium.

4 . Collect T cells from the MLC system used for restimulation (step 2), resuspend in DMEM-20 and count (Appendix 3A).

5. Dilute the resuspended T cells with supernatant activated by MLC or Con A (as cytokine) to 1-10 cells/m l, 50ul per well in a 96-well plate from step 3, and incubate at 37°C for 4 days.

6 . Add 50ul of MLC-activated supernatant and 50 M D M E M -I O medium to each well. Incubate for 7 ~IOd until visible cell colony formation can be observed in some wells under an inverted microscope.

7a. Detection of cytotoxic activity by the 51Cr microrelease assay: Cytotoxic activity is detected by the 51Cr microrelease assay (Engers and Fitch; module 2.10), which distinguishes between homozygous reactive C T L and T h cells. If the cytotoxic activity test is negative, the presence of T h cells is further detected by the proliferation assay shown in Module 2.1 1.

7b. Detection of IL-2 or IL-4 production: Detection of IL-2 or IL-4 production by re-stimulation with antigen (Module 5.1) or screening with surface expression of C D 4 or C D 8 (Modules 4.1 and 4.2).

8 . Characterize the homozygous reactive T-cell clone as it should be (specific cytotoxic activity and specific cytokine production).

9 . Maintain the clones as follows: Transfer IOOiUl cell suspension (containing 2.5 X ^IO4 to I X ^lO5 cells) from the original 96-well plate to 0.9 ml of a 24-well cell culture plate containing 6 X ^lO6 irradiated allogeneic splenocytes and 0.5 ml of MLC-activated supernatant, and replenish with DMEM-20 medium to a final volume of I.5 ml per well. Cells were passaged in this manner every other week.

Basic Scheme 2 Induction of T h Cloning with Soluble Protein Antigen

Do not conclude that T C R transgenic mice are monoclonal because they will contain cells with endogenous T C R gene rearrangements.

MATERIALS

m a n , 1982).
9. Once a sufficient number of cells have been obtained, identify the newly generated clone by examining the production of IL-2, IL-4, and IFN-Y (Unit 5.1). Once a sufficient number of cells have been obtained, the newly generated clones are identified by examining IL-2, IL-4 and IFN-Y production (Tab 5.1).

Conditions of Growth Factor Origin Preparation of Culture Media

An important factor that must be considered before selecting a conditioned medium is that each medium contains a different cytokine profile.

Auxiliary Scheme 1 Preparation of Knife Bean Protein A-Activated Supernatant (C c m A Supernatant)
Auxiliary Program 2 Preparation of Mixed Lymphocyte Culture Supernatant (M L C Supernatant)

Adjuvant Scheme 3 Preparation of P M A Activated E L -4 Lymphoma Cell Supernatant (E L -4 Supernatant)

Auxiliary Program 4 Cloning by Micromanipulation

The use of repetitive micromanipulation methods to grow clones is more recommended than the limited dilution method, and it is the only method that ensures the ability to form clones.

Additional materials (see Basic Scheme 2 for additional materials)

Microcapillary tubes (e.g., microhematocrit tubes, BectonDickinson)

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