Abstracts (complete list) - Wissenschaft Online

Manuel N. D. M. Guerreiro, Anne Marie Asemissen, Gianna Schulz, Il-Kang Na, Jochen
Hühn, Sandra Bauer, Eckhard Thiel, Ulrich Keilholz, Carmen Scheibenbogen
IL-2 induces IL-10 producing regulatory CD3+ T cells in vitro
and in vivo
Purpose:
IL-10-producing regulatory T cells have recently gained much interest as important
players in immune regulation. In this study we have analyzed in melanoma patients the
influence of IL-2 treatment and of in-vitro IL-2 exposure on IL-10-producing regulatory
T cells.
Materials and methods:
PBMC from 6 melanoma patients who had been treated with IL-2 and from healthy
subjects were analyzed ex vivo or after 72h incubation with IL-2 (50 U/ml) for IL-10
producing capacity of T cells by intracellular cytokine staining. Further phenotypic and
functional characterization was performed by flow cytometry.
Results:
Less than 1% of unprimed PB T cells produced IL-10 in response to PMA/Ionomycin
(n=9 healthy subjects). IL-10-producing T cells could be primed by short-term
incubation with IL-2. After 3 days median 6.29% (range 0.79 – 23.04) of CD4+ and
10.64% (range 0.64 – 34.99) of CD8+ T cells produced IL-10 in response to PMA/Iono
(n=9 healthy subjects). CFSE staining revealed that the capacity to produce IL-10 is
due to priming by IL-2 and not due to expansion of this subset. In PBMCs cultured with
IL-2 for longer periods further upregulation of IL-10 producing capacity was observed
on day 7. We next studied IL-10-producing capacity in PBMC from 6 melanoma patients
who had been treated with IL-2. In accordance with the in vitro studies less than 0.2%
of unstimulated CD3+T cells produced IL-10 before, and after IL-2 therapy. Upon PMA/
Ionomycin stimulation both CD3+CD4+ and CD8+T cells showed enhanced IL-10 but
not IFN-g-producing capacity in samples obtained 5 days after IL-2 treatment as
compared to pretreatment samples in 4 of 6 patients. Phenotypic characterization of IL-
10-producing T cells showed lack of FOXP3 expression, failure to produce IL-4 and IFNg
and lack of CD25. A major subset of IL-10+ T cells expressed the mucosal chemokine
receptors CCR6 and/or CCR9, and approximately 30% the inflammatory chemokine
receptor CXCR3. Interestingly, CD8+ IL-10+ T cells were not detectable in bone marrow
suggesting that the IL-10+ T cells have a restricted migratory capacity. PBMC primed in
the presence of IL-2 and stimulated by anti-CD2/-CD3/-CD28 were able to substantially
inhibit the proliferation of stimulated PBMCs as assed by CFSE proliferation assay.
Further we could demonstrate the in vitro generation of IL-10-producing influenzapeptide-specific
T cells by IL-2 with a mean of 1.35 % influenza-specific CD8+IL-10+ T
cells (range 0.14 – 3.33 %, n= 3) after 7 days. We also could generate MART-1 and
Tyrosinase-peptide specific CD3+CD8+ IL-10 producing T cells in 4 of 6 melanoma
patients (0.03-0.13%). The study of the single nucleotide polymorphism –1082 (G/A)
located in the proximal promoter of the IL-10 gene from 8 healthy subjects did not
correlate with the frequency of the IL-10+ T cells, suggesting a predominantely
adaptive response. The effect of IL-7, IL-15, IL-21, also belonging to the gamma chain
cytokines family, on IL-10-producing and secreting capacity were compared.
Interestingly, IL-15 had similar IL-10 priming capability as IL-2, while IL-21 did not
enhance IL-10 production.
Conclusion
IL-2 primes IL-10-producing T cells in vivo and in vitro. This finding has important
implications for the use of IL-2 as vaccine adjuvant in cancer immunotherapy as well as
for adoptive T cell therapy. Importantly, the gamma chain cytokine IL-21 does not
enhance IL-10 production.