Both CT^lo and cAMP-matured DCs are protective in EAE.

<p>(A) CT^lo (0.1 μg/ml) or cAMP (100 μM) treated BM-DCs loaded with 40 μg/ml MOG_35-55 peptide were injected <i>i</i>.<i>v</i>. at days -7, -5, -3 (2×10⁶/mouse) before MOG-specific EAE induction at d0. Control mice received PBS injections. Average disease scores were monitored for a total of 10 mice per group from two independent experiments. Error bars represent mean ± SEM. (B), (C) and (D) Statistical analyses of different other clinical parameters derived from A. Error bars represent mean ± SD of pooled results from n = 10 mice. (E) CT^lo (0.1 μg/ml), cAMP (100 μM) treated BM-DCs or untreated BM-DCs were injected <i>i</i>.<i>v</i>. at days -7, -5, -3 (2×10⁶/mouse) before MOG-specific EAE induction at d0. Control mice received PBS injections. Average disease scores were monitored for a total of 6 mice per group of two independent experiments. Error bars represent mean ± SEM. (F), (G) and (H) Statistical analyses of different other clinical parameters derived from E. Error bars represent mean ± SD of pooled results from n = 6 mice. (I) Spleen cells of EAE mice treated like in A were analyzed 8 or 16 days after EAE induction for their Foxp3⁺ Treg relative frequencies or Ki67⁺ proliferating Foxp3⁺ Treg frequencies. (J) Like in I, but the spleen cells were re-stimulated with 10 μg/ml MOG_35-55 peptide and then the relative frequencies of Treg expansion were determined after 5 days. (K) Like in I, but splenocytes were re-stimulated with graded concentrations of MOG_35-55 peptide and after 3 days their supernatants analyzed by ELISA for IL-17A and IFN-γ production. (I-K) Error bars represent mean ± SD of pooled results from n = 6 mice. One Way ANOVA, Dunnett post-test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant.</p

Cathepsin L expression in CT/cAMP-DCs does not influence iTreg conversion.

<p>(A) BM-DCs were differentially matured with the indicated stimuli for 10 h (CT^lo 0.1 μg/ml; cAMP 100 μM; LPS 0.1 μg/ml). Total cell lysates were analyzed for mature Ctsl protein content by Western Blot. One representative experiment is shown. (B) Densitometric analysis of Western blot data of 3 mice per group from 3 independent experiments performed like the one shown in A, normalized to β-actin and relative to untreated control. (C) BM-DCs were stimulated with CT^lo or cAMP for 4 h. CTLA-2α and -2β mRNA expression was determined by RT-sqPCR, representative experiment, (D) Densitometric analysis of mRNA data of 3 mice per group from 3 independent experiments performed like the one shown in C, normalized to β-actin and relative to WT untreated control. (E) OT-II T cells were co-cultured with WT or <i>Ctsl</i>^<i>-/-</i> BM-DCs-treated with CT^lo or cAMP for 4 h in the presence of 100 ng/ml OVA_327-339 peptide and 2 ng/ml TGF-β. After 5 days cells were analyzed by flow cytometry for Foxp3 and CD25 expression in a CD4⁺Vβ5⁺ gate. Data represent the change in the frequency of iTreg conversion using <i>Ctsl</i>^<i>-/-</i> DCs normalized to WT DCs of n = 5 experiments. Error bars represent mean ± SD. (B) One Way ANOVA, Dunnett post-test. (E) Two-tailed Student’s t test. ns: not significant.</p

CT^lo- or cAMP-DCs enhance TGF-β dependent Foxp3⁺ iTreg conversion via CTLA-2 <i>in vitro</i>.

<p>OT-II T cells were co-cultured with C57BL/6 BM-DCs matured with the indicated stimuli (CT^lo 0.1 μg/ml; CT^hi 1 μg/ml; cAMP 100 μM; LPS 0.1 μg/ml) in the presence of (100 ng/ml) OVA_327-339 peptide and TGF-β (2 ng/ml) + IL-6 (20 ng/ml) for Th17 polarizing conditions or TGF-β (2 ng/ml) for iTreg conversion conditions. (A) After 5 days cells were re-stimulated for 6 h with 30 ng/ml PMA + 1 μg/ml ionomycin in the presence of 5 μg/ml brefeldin A + 2μM monensin and IL-17A and IFN-γ producing OT-II cells were analyzed by flow cytometry in a CD4⁺Vβ5⁺ gate. (B) Statistics of A with respect to LPS-DCs used as negative control for n = 5 experiments. (C) After 5 days cells were analyzed by flow cytometry for Foxp3 and CD25 expression in a CD4⁺Vβ5⁺ gate. (D) Statistics of C with respect to LPS-DCs used as negative control for n = 5 experiments. (E) OT-II naïve T cells were co-cultured with BM-DCs first transfected with 5 μM siRNA against CTLA-2, scrambled siRNA or mock-electroporated (pooled controls) and then, treated with different stimuli. Graphs represent changes in the frequency of iTreg (n = 7), Th17 (n = 6) and Th1 (n = 3) normalized to control. Numbers inside each dot plot represent the cell frequency in each quadrant. Error bars represent mean ± SD. One Way ANOVA, Dunnett post-test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. (F) C57BL/6 naïve CD4⁺CD25^- T cells were cultured in the presence of TGF-β plus recombinant CTLA-2α. After 3 days cells were analyzed by flow cytometry for Foxp3 and CD25 expression in a CD4⁺ gate (n = 6). Error bars represent mean ± SD. Two-tailed paired Student’s t test. *p<0.05.</p

Model on the differential effects of CT and cAMP on DC-directed induction of Th17 or Foxp3⁺ Treg cells.

<p>Immature DCs treated with high (CT^hi) or low (CT^lo) doses of cholera toxin or its known secondary mediator cAMP induced partial DC maturation measured as upregulation of MHC II- and costimulatory molecules such as CD86. CT^lo-DCs and cAMP-DCs produced no pro-inflammatory cytokines (semi-mature DCs) but instead the tolerogenic molecule CTLA-2. Under Treg polarizing conditions <i>in vitro</i>, using naive TCR-transgenic CD4⁺ T cells, CTLA-2 produced by CT^lo-DCs and cAMP-DCs supports the TGF-β mediated conversion into Foxp3⁺ Tregs. In contrast, CT^hi-DCs also released CTLA-2 but additionally the Th17-polarizing cytokines IL-1β, IL-6 and IL-23 (mature DCs). Consequently CT^hi-DCs mainly generated Th17 cells <i>in vitro</i>. <i>In vivo</i>, a polyclonal repertoire of T cells triggered by CT^lo-DCs and cAMP-DCs results in both Th17 and Tregs activation pointing to competing effects at early or late time points in the EAE model.</p

CT and cAMP induces both CTLA-2α and -2β during DC maturation.

<p>BM-DCs were differentially matured with the indicated stimuli for 16h unless otherwise specified. (A) Principal component analysis comparing untreated BM-DCs, DCs matured for 6h with the Th2-inducing stimuli TNF, the <i>Trypanosoma brucei</i> antigens Mitat or mVSG, and the Th1-inducing LPS from our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178114#pone.0178114.ref017" target="_blank">17</a>] with CT^hi (1 μg/ml) matured Th17-inducing DCs investigated in this study. (B) Heat map displaying the top 25 genes regulated in DCs after CT^hi or TNF stimulation compared with untreated DCs. Data show single time point values of a single microarray. (C) Densitometric analysis of CTLA-2α and -2β expression levels determined by RT-sqPCR normalized to β-actin and relative to untreated control for n = 4 experiments; CT^lo (0.1 μg/ml). (D) CT^hi stimulation of BM-DCs ± the cAMP inhibitor KH7 before RT-sqPCR normalized to β-actin of n = 3 independent and pooled experiments. (E) DC maturation analysis by flow cytometry of CD86 and MHC-II on CD11c positive cells. (F) Statistical evaluation of D with respect to untreated control for n = 5 experiments. (G) IL-1β, IL-6 and IL-23 secretion by differentially stimulated DCs measured by ELISA n = 3 experiments. Statistical analysis was performed with respect to untreated control. Error bars represent mean ± SD. One Way ANOVA, Dunnett post-test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. For D, student's t test, n = 3, *p<0.05.</p

Long-term persistence of RAM.

<p>Of the 16 patients who presented with RAM at baseline, we analyzed plasma specimens collected at later time points (“follow-up 1”, “follow-up 2”) for the presence of RAM. We detected three different scenarios, including persistent RAM, disappearing RAM, and newly emerging RAM (some samples appear in more than one scenario). Time to follow-up sample is indicated in months. In some cases, no PCR product from plasma samples could be generated. The investigation for long-term stability of RAM is insofar incomplete as plasma specimens collected at later time points were not available (“n.a.”) for all patients, referred to as “n.d.” (not determined).</p

Phylogenetic analysis of RT and PR sequences from the Tanzanian cohort.

<p>A neighbour-joining phylogenetic tree <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Saitou1" target="_blank">[30]</a> was constructed from the 88 patient derived HIV-1 sequences from the Tanzania cohort and the two partner-derived sequences. Reference sequences were obtained from the Los Alamos HIV sequence database. The analyzed 1302 bp region includes the complete Protease and Reverse Transcriptase coding region. The tree was constructed using Mega software version 4, and the evolutionary distances were calculated using the Kimura 2-parameter method. The bootstrap consensus tree was inferred from 50000 replicates and values greater than 70% are indicated on the branch lengths. The scale at the bottom left indicates the calculated genetic distances between the branches of the phylogenetic tree. Circles represent the 88 samples from our cohort. Black-dotted circles are without RAM, open-circled sequences are with RAM, open triangles are sequences with RAM from HIV-infected partners of two study subjects, which were not included in the determination of HIVDR as these patients received ART. Sequences without symbols are subtype reference sequences derived from Los Alamos database. The subtype is indicated at the end of each sequence name. Relative subtype frequency: A1: 34%, A1D: 7%, C: 26%, CRF10_CD: 4%, D: 28%, B: 1%. Sequences isolated from two couples (couple I, couple II) with NVP resistances.</p

HIVDR in the Mwanza cohort.

<p>RAM according to the Stanford HIV Drug Resistance Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Stanford1" target="_blank">[17]</a> in 16/88 baseline samples. Mutations associated with a score of 60 were attributed as high-level resistance-associated mutations (RAM) and mutations with a score of 10–35 were attributed as low-level RAM. Mutations listed for WHO HIVDR surveillance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Bennett3" target="_blank">[18]</a> are indicated in bold. Superscripted numbers identify the reasons for excluding the respected mutations from the HIVDR list: 1) nonpolymorphic, but at highly polymorphic position; 2) polymorphic position in subtypes B, F, CRF01_AE; 3) polymorphic in multiple subtypes. ABC: Abacavir; ATV: Atazanavir; AZT: Zidovudine; ddI: Didanosine; DLV: Delaviridine; D4T: Stavudine; EFV: Efavirenz; FTC: Emtricitabine; LPV: Lopinavir; NFV: Nelfinavir; NVP: Nevirapine; SQV: Saquinavir; 3TC:Lamivudine <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023091#pone.0023091-Joint1" target="_blank">[29]</a>. <u>Underlined</u> drugs are part of the local first line regimens, double line underlined drugs are part of local second line regimens. Data derived from 88 sequenced samples. Therapeutic drug monitoring (TDM) was performed from plasma samples collected at baseline for NNRTIs (Efavirenz and Nevirapine) and PIs (Nelfinavir, Saquinavir, Atazanavir and Lopinavir).</p

Demographic patient characteristics of the study population.

<p>The study population consisted of 120 ART-naive HIV-1 infected adults. PBMC- or plasma samples from 88 patients yielded amplicons for the bulk sequencing reaction. Data are expressed as means ± S.D. and range in parentheses. Patients with CD4 counts <200/ml at sample date initiated ART if CD4 counts remained below 350/ml four weeks later.</p

NVP resistance and PMTCT.

<p>Patients with NVP resistance mutations were analyzed regarding PMTCT (with NVP monotherapy) as a possible cause for the emergence of the mutation. PMTCT as a cause for the mutation's appearance is discussed as “possible” if the mother received PMTCT; it is discussed as “unclear” if the PMTCT status and the dates of birth of the children are unknown; for plausibility reasons PMTCT was excluded (“no”) as a trigger of the NVP mutation if the patient is either male or if a female patient presented with unknown PMTCT history combined with the HIV infection being diagnosed only since the date of birth of the youngest child.</p