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  • HLA DR resulted highly expressed in


    HLA-DR resulted highly expressed in the surface of immature DC (iDC) as well as in mature DC (48 h-DC) in both groups; this is possibly due to the chronic infection as previously observed in monocytes-derived DC from HIV+[17]. As expected, maturation and activation markers - CD40, CD80, CD83 and CD86 – resulted significantly augmented in mature DC compared to iDC in both groups of R406 (p < 0.05) (Fig. 5A). However, any significant difference in markers expression between A and B groups were observed (p > 0.05), suggesting that, at least from a phenotypic point of view, DC from all the donors are very similar. When looking at surface markers in ex vivo monocytes, as the precursors of in vitro manipulated DC, monocytes from group B donors appeared to be more activated compared to those from group A (HLA-DR: 40% versus 57.6%; CD11c: 49.6% versus 98.2%; CD86: 48.5% versus 94.2%) (Fig. 5B), even if this difference did not achieve significant threshold (p > 0.05) possibly due to the limited size of samples and the well known PBMC inter-individual heterogeneity. Wondering whether B group patients could have all PBMC more activated, peripheral blood lymphocytes were analysed for surface expression of activation molecule CD38, however no differences have been observed (Fig. 5C). To determine the capacity of DC from groups A and B donors to activate in vitro autologous lymphocytes, CD38 surface expression and intracellular IFN-γ were measured in lymphocytes from co-culture assays. CD38 expression was augmented in B group CD3+ T cells compared to A group (21.8% versus 12.3%), however also in this case the difference was not statistically significant (p > 0.05) (Fig. 6A). Intracellular IFN-γ staining revealed that DC from both groups were similarly able to induce IFN-γ production in CD3+ T cells (Fig. 6B). Finally, to evaluate any in vivo effect of studied DC, we compared difference (Δ) in PVL, CD4+ and CD8+ counts after immunotherapy in individuals of groups A and B. Clinical data have been collected before each of the three immunotherapy doses (t1, t2, t3) and after 1, 2 and 6 months from the third dose (t4, t5, t6). PVL did not diminished during treatment. Apparently group A DC induced a better CD4+ and CD8+ (CD3+) increment compared to group B DC (Supplementary Table 3), however this difference was not statistically significant at any of the time points.
    Discussion Liu et coll [16] have demonstrated that DC gene expression profile could be used as a predictor of function and help the design and/or patients selection of DC-vaccine trials in cancer therapy. With a similar purpose the differential expression of a subset of genes involved in host anti-HIV response was analysed in dendritic cells used in the on-going Brazilian clinical trial of anti-HIV immunotherapy. Gene expression analysis revealed a distinct profile in monocyte-to-DC differentiation within HIV+ individuals submitted to immunotherapy. This profile did not apparently correlate with initial clinical data such as CD4+ and CD8+ cells count or plasma viral load (Table 1), nor with well-known genetic factor involved in HIV infection pathogenesis, suggesting the need to investigate novel characterization markers for DC in the contest of immunotherapy. Cells from group B appeared to be chronically activated in term of HIV-response, showing an up-regulation of both restriction and co-factors for HIV-1, but this augmented expression did not significantly vary along differentiation, possibly being an intrinsic characteristic of these patients (genetic background, chronic inflammation state) and not a consequence of dendritic cell preparation protocol. Moreover these cells seemed to be more prone to programmed cell death than group A, as several pro-apoptotic genes (i.e.: BAD, BAX, CASP8) were significantly up-regulated in group B. On the contrary, BAD and CASP8 resulted down-regulated in DC of group A. Giri et coll [7], showed that monocytes from HIV+ individuals are characterized by an anti-apoptotic signature, however, to our knowledge, no data have been reported about monocyte-derived DC. We can hypothesize that monocytes-to DC differentiation protocol may act in different way according to original monocytes expression profile, suggesting that activation state of monocytes could be taken into account as a early predictor of DC characteristics before mature DC viability result, that nowadays represents one of the main quality control data for DC application in patients.