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. Author manuscript; available in PMC: 2017 Nov 3.
Published in final edited form as: Bio Protoc. 2014 Jul 5;4(13):e1171. doi: 10.21769/BioProtoc.1171

Murine In vitro Memory T Cell Differentiation

Myoungjoo V Kim 1,2,3,4,5,*, Weiming Ouyang 1,2,3,4,5, Will Liao 1,2,3,4,5, Michael Q Zhang 1,2,3,4,5, Ming O Li 1,2,3,4,5
PMCID: PMC5669376  NIHMSID: NIHMS913652  PMID: 29104880

Abstract

Upon pathogen encounter, naïve CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian’s group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.

Materials and Reagents

  1. Recombinant mouse IL-15 (rmIL15) (BioLegend, catalog number: 566302)

  2. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-119)

  3. Fetal bovine serum (Atlanta Biologicals, catalog number: S11055H)

  4. Penicillin/streptomycin (Gemini Bio-Products, catalog number: F52M00E)

  5. L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081)

  6. 100x 1 M Hepes (Life Technologies, Gibco®, catalog number: 15630-080)

  7. 100x MEM Non-essential amino acids (Life Technologies, Gibco®, catalog number: 11140-050)

  8. 100x sodium pyruvate (100 mM) (Life Technologies, Gibco®, catalog number: 11360-070)

  9. 100x 2-mercaptoethanol (Life Technologies, Gibco®, catalog number: 21985-023)

  10. OVA257-264 synthetic peptide (Sigma-Aldrich, catalog number: S7951)

  11. Ficoll-Paque™ Premium 1.084 (GE Healthcare, catalog number: 17-5446-02)

  12. Antibodies:

    1. Anti-CD44 PerCpCy5.5 (clone: IM7) (eBioscience, catalog number: 45-0441)

    2. Anti-CD62L APC (clone: MEL-14) (eBioscience, catalog number: 17-0621)

  13. RBC lysis buffer (eBioscience, catalog number: 00-4333-57)

  14. Bovine serum albumin (Thermo Fisher Scientific, catalog number: BP1605-100)

  15. NaN3 (Sigma-Aldrich, catalog number: S8032)

  16. T cell media (see Recipes)

  17. Staining buffer (in PBS) (see Recipes)

Equipment

  1. Centrifuge (Thermo Fischer Scientific, Sorvall™ Legend RT)

  2. 70 μm cell strainer (BD Biosciences, Falcon®, catalog number: 352350)

  3. 15 ml and 50ml Falcon tubes

  4. 24 well plates (BD Biosciences, Falcon®, catalog number: 353226)

  5. T75 culture flask (Corning, catalog number: 430641)

  6. 37 °C 5% CO2 Cell Culture incubator

Procedure

A. CD44hiCD62Llo Memory T cell differentiation proceeds under sterile tissue culture conditions

  • 1

    Euthanize a OT-1 CD8 TCR transgenic mouse and take spleen, and (optional) lymph nodes.

  • 2

    Splenocytes are RBC lysed followed by washing with PBS twice.

B. OT-1 TCR stimulation with cognate peptide antigen

  • 3

    Resuspend cells in 1 ml of T cell media and add OVA257-264 synthetic peptide to 1 μM.

  • 4

    Incubate in the 5% CO2 at 37 °C for 1 h.

  • 5

    Spin down cells at 1,500 rpm for 3 min at 4 °C and wash once with T cell media.

  • 6

    Resuspend cells in 12 ml of T cell media and plate 1ml/well of a 24 well plate.

  • 7

    Incubate in the 5% CO2 at 37 °C for 2 days.

  • 8

    Harvest the cells by pipetting up and down, and pellet cells.

  • 9

    Resuspend cells in 5 ml of T cell media, and load on to 2.5 ml of Ficoll.

  • 10

    Spin down at 400 x g for 15 min at 4 °C.

  • 11

    Transfer live cells on the interphase to a new 15 ml tube and fill up the tube with T cell media.

  • 12

    Spin down cells at 1,500 rpm for 3 min at 4 °C.

C. Memory T cell culture in the presence of IL-15

  • 13

    Resuspend cells in 24 ml of T cell media containing rmIL15 (20 ng/ml). Culture cells in T75 flask for four days.

  • 14

    Harvest and pellet cells for Ficoll gradient (repeat steps 9–12).

  • 15

    Resuspend cells in 40 ml of T cell media containing rmIL15 (20 ng/ml). Culture in T75 flask for two days.

  • 16

    Staining cells with anti-CD44 and CD62L antibodies in staining buffer for 15 min on ice.

  • 17

    Wash with staining buffer twice, then proceeds flow cytometry analysis.

Recipes

  1. T cell media

    RPMI-1640

    10% fetal bovine serum

    1% penicillin/streptomycin

    1% L-Glutamine

    1x 1 M Hepes

    1x MEM non-essential amino acids

    1x sodium pyruvate 100 mM

    1x 2-mercaptoethanol

  2. Staining buffer (in PBS)

    1% BSA

    0.02% NaN3

Figure 1.

Figure 1

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CD44 and CD62L expression of differentiated memory T cells

Acknowledgments

The protocol was adapted from a previously described study (Manjunath et al., 2001). This work was supported by the Starr Cancer Consortium (13-A123 to M.O.L. and M.Q.Z.), the Rita Allen Foundation (M.O.L.), the NBRPC (2012CB316503 to M.Q.Z), and the NIH (HG001696 to M.Q.Z.).

References

  • 1.Kim MV, Ouyang W, Liao W, Zhang MQ, Li MO. The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection. Immunity. 2013;39(2):286–297. doi: 10.1016/j.immuni.2013.07.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Manjunath N, Shankar P, Wan J, Weninger W, Crowley M, Hieshima K, Springer T, Fan X, Shen H, Lieberman J. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J Clin Invest. 2001;108(6):871–878. doi: 10.1172/JCI13296. [DOI] [PMC free article] [PubMed] [Google Scholar]

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