Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment.
OBJECTIVE
Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T(reg)). To study the role of T(reg) in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10(+) T(reg )type 1 (Tr1) was established.


METHODS
An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4(+)CD25(- )T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) +/- 1 nM rapamycin. Autologous iDC and CD4(+)CD25(-) T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays +/- neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-beta and IFN-gamma in ELISA.


RESULTS
In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+) cells with suppressor activity (mean suppression +/- SD = 58 +/- 12%). These suppressor cells produced IL-10 (mean +/- SD = 535 +/- 12 pg/ml) and TGF-beta (mean +/- SD = 512 +/- 38 pg/ml), but no IL-4 or IFN-gamma. Suppressor function of co-cultures correlated with the percent of expanding IL-10(+) Tr1 cells (r (2 )=( )0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent.


CONCLUSIONS
The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4(+)CD25(high)FoxP3(+) nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.
Bergmann
Christoph
C
Research Pavilion at the Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213-1863, USA.
Strauss
Laura
L
Zeidler
Reinhard
R
Lang
Stephan
S
Whiteside
Theresa L
TL
D002294:Carcinoma, Squamous Cell / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism / Q000494:pharmacology; D003713:Dendritic Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006258:Head and Neck Neoplasms / Q000276:immunology; D006801:Humans; D016753:Interleukin-10; D008213:Lymphocyte Activation; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology*
D016207:Cytokines; D016753:Interleukin-10; D020123:Sirolimus