Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures.
BACKGROUND
Cytokine-induced killer (CIK) cells are typically differentiated in vitro with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol.


METHODS
Peripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of bona fide regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the in vitro cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells.


RESULTS
CIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells.


CONCLUSIONS
TG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.
Department of Gynecology, Catholic University Med. School, and Department of Blood Transfusion and Cell Therapy, Azienda Ospedaliera S. Camillo-Forlanini, Rome, Italy.
Giuseppina
G
Bonanno
Paola
P
Iudicone
Andrea
A
Mariotti
Annabella
A
Procoli
Annino
A
Pandolfi
Daniela
D
Fioravanti
Maria
M
Corallo
Alessandro
A
Perillo
Giovanni
G
Scambia
Luca
L
Pierelli
Sergio
S
Rutella
D000328:Adult; D000818:Animals; D000961:Antilymphocyte Serum / Q000494:pharmacology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D055612:Cytokine-Induced Killer Cells / Q000166:cytology* / Q000187:drug effects*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007376:Interleukin-2 / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000166:cytology / Q000187:drug effects; D008875:Middle Aged; D009369:Neoplasms / Q000276:immunology; D010641:Phenotype; D011817:Rabbits; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000187:drug effects; D014019:Tissue Donors; D055815:Young Adult
D000961:Antilymphocyte Serum; D007376:Interleukin-2; D007371:Interferon-gamma; C512542:thymoglobulin