Skip to main content
NCBI home page
Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 1994 Jul;38(4):253–258. doi: 10.1007/BF01533516

Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment

Wilhelmina M C Mulder 1,✉, Hans Koenen 1, Adrie J C van de Muysenberg 1, Elisabeth Bloemena 1, John Wagsfaff 2, Rik J Scheper 1
PMCID: PMC11038093  PMID: 8168120

Abstract

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.

Key words: Tumour-infiltrating lymphocytes, Protease contamination, CD molecules

References

  • 1.Applegate KG, Balch CM, Pellis NR. In vitro migration of lymphocytes through collagen matrix: arrested locomotion in tumor-infiltrating lymphocytes. Cancer Res. 1990;50:7153. [PubMed] [Google Scholar]
  • 2.Baars JW, Fonk JCM, Scheper RJ, von Blomberg-van der Flier BME, Bril H, van de Valk P, Pinedo HM, Wagstaff J. Treatment with tumour-infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: a pilot study. Biotherapy. 1992;4:289. doi: 10.1007/BF02172659. [DOI] [PubMed] [Google Scholar]
  • 3.Berd D, Maguire HC, Jr, McCue P, Mastrangelo MJ. Treatment of metastatic melanoma with an autologous tumor-cell vaccine: clinical and immunologic results in 64 patients. J Clin Oncol. 1990;8:1858. doi: 10.1200/JCO.1990.8.11.1858. [DOI] [PubMed] [Google Scholar]
  • 4.De Jong R, Stokkers P, Lamme E, Kool JM, Borst F, Brouwer M, Miedema F, Van Lier RAW. Regulation of T-cell differentiation by CD2 and CD28 accessory molecules. Immunology. 1989;67:333. [PMC free article] [PubMed] [Google Scholar]
  • 5.Feeney RE, Stevens FC, Osuga DT. The specificities of chicken ovomucoid and ovoinhibitor. J Biol Chem. 1963;238:1415. [PubMed] [Google Scholar]
  • 6.Hanna MG, Jr, Peters LC. Immunotherapy of established micrometastases withBacillus Calmette Guérin tumor cell vaccine. Cancer Res. 1978;38:204. [PubMed] [Google Scholar]
  • 7.Hoover CH, Jr, Surdyke GG, Dangel RB, Peters LC, Hanna MG., Jr Prospectively randomized trial of adjuvant active-specific immunotherapy for human colorectal cancer. Cancer. 1985;55:1236. doi: 10.1002/1097-0142(19850315)55:6<1236::aid-cncr2820550616>3.0.co;2-#. [DOI] [PubMed] [Google Scholar]
  • 8.Knazek RA, Wu YW, Aebersold PM, Rosenberg SA. Culture of human tumor infiltrating lymphocytes in hollow fiber bioreactors. J Immunol Methods. 1990;127:29. doi: 10.1016/0022-1759(90)90337-u. [DOI] [PubMed] [Google Scholar]
  • 9.Leerling MF, Vaessen LMB, Reubsaet CHK, Weimar W, Ettekoven H, Marsman FR, Kreeftenberg JF. Quality control of antihuman CD3 and CD4 monoclonal antibodies. Dev Biol Stand. 1990;71:191. [PubMed] [Google Scholar]
  • 10.Lineweaver H, Murray W. Identification of the trypsin inhibitor of egg white with ovomucoid. J Biol Chem. 1947;171:565. [PubMed] [Google Scholar]
  • 11.Meuer CS, Hussey RE, Fabbi M, Fos D, Acuto O, Fitzgerald KA, Hodgdon JD, Protentis JP, Schlossman SF, Reinherz EL. An alternative pathway of T-cell activation: a functional role for the 50 kD T11 sheep erythrocyte receptor protein. Cell. 1984;36:897. doi: 10.1016/0092-8674(84)90039-4. [DOI] [PubMed] [Google Scholar]
  • 12.Miescher S, Stoeck M, Qiao L, Barras C, Barrelet L, Von Fliedner V. Proliferative and cytolytic potentials of purified human tumor-infiltrating T lymphocytes. Impaired response to mitogendriven stimulation despite T-cell receptor expression. Int J Cancer. 1988;42:659. doi: 10.1002/ijc.2910420504. [DOI] [PubMed] [Google Scholar]
  • 13.Newman I, Wilkinson PC. Methods for phenotyping polarized and locomotor human lymphocytes. J Immunol Methods. 1992;147:43. doi: 10.1016/s0022-1759(12)80027-5. [DOI] [PubMed] [Google Scholar]
  • 14.Peters LC, Brandhorst JS, Hanna MG., Jr Preparation of immunotherapeutic antologous tumor cell vaccines from solid tumors. Cancer Res. 1979;39:1353. [PubMed] [Google Scholar]
  • 15.Pichler WJ, Walker C, Wolff-Vorbeck G, Koponon M, Tax WJM, de Weck AL. Characterization of T8 epitopes by enzymatic digestion, cross-blocking and involvement in cell-mediated lympholysis. Cell Immunol. 1985;96:398. doi: 10.1016/0008-8749(85)90370-3. [DOI] [PubMed] [Google Scholar]
  • 16.Riddell SR, Greenberg PD. The use of antiCD3 and antiCD28 monoclonal antibodies to clone and expand human anti-gen-specific T-ceels. J Immunol Methods. 1990;128:189. doi: 10.1016/0022-1759(90)90210-m. [DOI] [PubMed] [Google Scholar]
  • 17.Rodrigues M, Nussenzweig RS, Romero P, Zavala F. The in vivo cytotoxic activity of CD8+ T-cell clones correlates with their levels of expression of adhesion molecules. J Exp Med. 1992;175:895. doi: 10.1084/jem.175.4.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Rosenberg SA, Spiess P, Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes. Science. 1986;233:1318. doi: 10.1126/science.3489291. [DOI] [PubMed] [Google Scholar]
  • 19.Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular adhesion molecule (ICAM-1) distinet from LFA-1. J Immunol. 1986;137:1270. [PubMed] [Google Scholar]
  • 20.Sánchez-Madrid F, De Landázuri MO, Morago G, Cebrián M, Acevedo A, Bernabeu C. VLA-3: a novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization. Eur J Immunol. 1986;16:1343. doi: 10.1002/eji.1830161106. [DOI] [PubMed] [Google Scholar]
  • 21.Stoeck M, Miescher S, Qiao L, Capasso P, Barras C, von Fliedner V. Stimulation of FACS-analyzed CD4+ and CD8+ human tumour-infiltrating lymphocytes with ionomycin + phorbol-12, 13-dibutyrate does not overcome their proliferative deficit. Clin Exp Immunol. 1990;79:105. doi: 10.1111/j.1365-2249.1990.tb05135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Sutherland DR, Abdullah KM, Cyopick P, Mellors A. Cleavage of the cell-surfaceO-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease fromPasteurella haemolytica . J Immunol. 1992;148:1458. [PubMed] [Google Scholar]
  • 23.Van Lier RAW, Brouwer M, Aarden LA. Signals involved in T-cell activation. T-cell proliferation induced through the synergistic action of antiCD28 and antiCD2 monoclonal antibodies. Eur J Immunol. 1988;18:167. doi: 10.1002/eji.1830180125. [DOI] [PubMed] [Google Scholar]
  • 24.Van Noesel C, Miedema F, Brouwer M, de Rie MA, Aarden LA, Van Lier RAW. Regulatory properties of LFA-1 α and β chains in human T-lymphocyte activation. Nature. 1988;333:850. doi: 10.1038/333850a0. [DOI] [PubMed] [Google Scholar]
  • 25.Whiteside TL, Miescher S, Hurlimann J, Moretta S, von Fliedner V. Separation, phenotyping and limiting dilution analysis of T-lymphocytes infiltrating human solid tumors. Int J Cancer. 1986;37:803. doi: 10.1002/ijc.2910370602. [DOI] [PubMed] [Google Scholar]
  • 26.Vanelli JR. The preparation of effector cells for use in the adoptive cellular immunotherapy of human cancer. J Immunol Methods. 1991;139:1. doi: 10.1016/0022-1759(91)90345-g. [DOI] [PubMed] [Google Scholar]

Articles from Cancer Immunology, Immunotherapy : CII are provided here courtesy of Springer

RESOURCES