Nowadays other Brazilian HIV patients were
Nowadays other 20 Brazilian HIV+ patients were enrolled in the second-phase of Lu et al. clinical trial , and biologic samples are becoming available for novel investigation.
All this considered and taken into account that the intrinsic ability of each HIV+ individual to counteract HIV-1 results in a different rate of immune Dig-11-utp activation [7,8] and consequently in a different capacity of HIV+ to be responsive toward exogenous stimulation (i.e.: immunotherapy), we decided to study differential expression of genes involved in host anti-HIV response in available cells from 6 HIV+ patients included in the phase-I/II clinical trial. To determine whether this expression profile is different among vaccinated individuals and if an alteration of this profile could eventually be prejudicial to immunotherapy, HIV restriction genes expression was evaluated in different steps of monocytes-to-DC preparation according to immunotherapy protocol  and correlated with DC characteristics and functions, HIV restriction factors genetics, and with clinical trial results.
Material and methods
Results Gene expression of monocytes-derived dendritic cells from 6 HIV+ patients submitted to immunotherapy was examined comparing, in each individual, 5 differentiation\'s steps (iDC, 4 h-DC, 14 h-DC, 24 h-DC and 48 h-DC) versus monocytes. Clustering analysis obtained for the 6 patients on all DC time-points showed the segregation of DC in two independent clusters according to the donor rather than to differentiation step or genes (group A: P1, P5 and P6; group B: P2, P3 and P4). DC from group A presented a general down-regulation of anti-HIV response genes, while an up-regulation was observed in DC from group B (Fig. 1), in an apparently uniform way along differentiation. This finding was intriguing because HIV+ individuals selected for the clinical trial were clinically homogeneous (seropositive for at least 5 years, absence of anti-retroviral treatment, CD4+ >500,000, PVL>3log; see Table 1), moreover the 6 studied patients are all males, with a similar age and race. First we investigated the possible cause of this difference looking at the available clinical data. Correlation analysis between PVL, CD4+ and CD8+ T lymphocytes counts and gene expression did not evidence any association with expression profile in DC from A and B groups (data not shown). However, taking in account the limited size of studied individuals and the absence of clinical significant reduction of PVL (<1 log), we can observe a higher, even not statistically significant (p > 0.05), mean levels of T CD4+ lymphocytes during the treatment in group A compared to B (Table 2, and Supplementary File 1). Polymorphisms in HIV restriction factor genes (APOBEC3G, CCL4, CCL3, CCL5, CCR5, CUL5, CXCL12, CXCR6, HLA-C, IFNG, PARD3B, PROX1, TRIM5, ZNRD1) were previously evaluated in the context of immunotherapy  suggesting that, at least PARD3B, appeared to be associated to a “good” response in the phase-I clinical trial. So we considered whether HIV restriction factors could affect the immunotherapy out-come influencing DC biology in HIV+ individuals. For this purpose, frequency of selected polymorphisms in APOBEC3G, CCL4, CCL3, CCL5, CCR5, CUL5, CXCL12, CXCR6, HLA-C, IFNG, PARD3B, PROX1, TRIM5 and ZNRD1 genes was analysed, however their distribution did not varied in patients between group A and group B (Supplementary File 2). Based on clustering, we decided to analyse differential gene expression separately in the two groups of donors, A and B. Within the 84 genes of the RT2 Profiler PCR Array there were at least three groups of modulated genes: genes with a p-value <0.05 and an absolute log2FC > 2 (significantly and highly modulated), genes with an absolute log2FC > 2 but not significantly different (p > 0.05) and genes with a p-value <0.05 but scarcely modulated (log2FC < 2) (Fig. 2). We decided to considered gene expression significantly different only when supported by a p-value <0.05 and an absolute log2FC > 2. In Supplementary Table 3 complete gene expression data are reported. Table 3 reports selected genes at all the differentiation\'s steps according to above-mentioned criteria. At some time points gene modulation did not reach a statistical significant p-value, however we have included these values (indicated with an asterisk) to emphasize that the direction of gene modulation did not vary along differentiation, neither after virus stimulation (4 h) nor after cytokines maturation cocktail (14 h, 24 h, 48 h).