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* = Presenting author

P351. The effect of infliximab pre-treated human blood-enriched dendritic cells from patients with active Crohn's disease and healthy controls on subsequent human T-lymphocyte phenotype and cytokine production in vitro

S. Peake1, D. Bernardo2, E. Mann2, J. Landy1, H. Omar2, S. Knight2, A. Hart1, 1St Mark's Hospital, IBD Unit, Harrow, United Kingdom, 2Imperial College, APRG, London, United Kingdom

Background

Dendritic cells (DC) play a key role in discriminating between commensal microorganisms and harmful pathogens. Expression of surface markers and cytokine production by DC at the time of antigen presentation control T-cell differentiation, cytokine profile and homing properties imprinted on stimulated T-cells. This process defines the type of immune response that occurs (regulatory/inflammatory) and its anatomical location. In CD, dysregulation of the immune response to gut microbiota and aberrant immune cell trafficking play a central role in disease pathogenesis. Infliximab (IFX) is a widely used and effective treatment for CD. Its mechanism of action is unclear.

We investigated the effect of IFX pre-treated blood-enriched DC, isolated from patients with active CD and HC, on human T-cell proliferation, phenotype and cytokine production.

Methods

Low density cells (LDC), which are enriched for DC, were obtained following Ficoll and Nycoprep gradient separation of blood obtained from patients with active CD (CDAI >220) and HC. LDC were cultured (0.5×106 cells/ml) with IFX (0, 1, 10, 100 µg/ml) for 24 hr.

T-cells were enriched from allogeneic HC blood by negative immunomagnetic bead separation. 400,000 CFSE-labelled T-cells were cultured for 5 days with 0, 1, 2 and 3% pre-treated LDC.

Following incubation, T-cell proliferation, homing marker expression and cytokine content of T-cells (TNFα, TGFβ, IFNγ, IL-10, -15, -17) was quantified by flow cytometry on stimulated T-cells. Unparied t-test & one-way ANOVA analyses were applied.

Results

Unconditioned LDC from HC and CD patients did not differ in their stimulatory capacity for allogeneic T-cells or in the cytokine profile acquired by T-cells. However, T-cells stimulated by LDC from CD patients decreased β7 IR.

Following culture with IFX, LDC decreased their stimulatory capacity in a dose-dependent, stepwise fashion in both HC and CD.

Culture with IFX did not have any effect on the acquired homing profile of stimulated T-cells, although these T-cells had a trend (not statistically significant) towards lower TNFα & higher IL-17 production.

Conclusion

The marked reduction in the ability of LDC to stimulate T-cells following culture with IFX represents one plausible explanation for the efficacy of anti TNF-alpha therapies in the treatment of CD. This effect was dose-dependent (within our range of test concentrations) suggesting that higher doses of IFX further reduce T-cell stimulation and may provide one explanation of the clinical benefits of dose escalation in refractory CD.