TO STUDY THE ROLE OF CD4+ SAMHD1low MEMORY CELLS IN HIV-1 INFECTION

La mise en évidence du rôle de la molécule SAMHD1 dans l’infection par le VIH-1 en tant que facteur de restriction a ouvert de nouvelles perspectives dans la compréhension de la pathogénicité du virus.En effet, il a été clairement démontré que dans les cellules myéloïdes comme les monocytes/macrophages et les cellules dendritiques ainsi que les lymphocytes T CD4+ quiescents, SAMHD1 jouait un rôle important dans la protection de ces cellules de l’infection. En revanche, le rôle de cette molécule dans l’infection des lymphocytes activés, qui sont souvent la cible préférentielle du virus, n’est pas élucidé.Nos résultats ont révélé l'existence d'une sous-population de lymphocytes T CD4+ mémoires exprimant de faibles niveaux de SAMHD1 (CD4+ CD45RO+ SAMHD1low), tandis que la grande majorité des lymphocytes expriment cette molécule à des niveaux plus élevés (94±0.7%). Nous montrons également que ces cellules sont hautement différenciées, qu’elles expriment en larges proportions le marqueur de cycle cellulaire Ki67 et qu’elles sont enrichies en cellules « T helper 17 » (Th17) dans le sang périphérique.De plus, la fréquence de la population CD4+ CD45RO+ SAMHD1low, est diminuée de manière significative chez les patients infectés par le VIH-1 par rapport aux sujets sains. De manière intéressante, nous montrons que dans les ganglions, les cellules T follicular helper (Tfh) expriment faiblement SAMHD1 et sont plus susceptibles à l’infection par le VIH-1 in vitro.L’ensemble de ces résultats suggère que les cellules SAMHD1 low représentent une cible préférentielle pour le virus et pourraient contribuer au réservoir viral.Les objectifs de ce projet sont:1. Déterminer si les cellules SAMHD1low contiennent plus de virus par comparaison aux cellules mémoires SAMHD1high et comparer les séquences virales isolées des cellules mémoires SAMHD1low et SAMHD1high.2. Caractérisation des cellules SAMHD1low au niveau moléculaire par une analyse transcriptomique qui permettra la mise en évidence de marqueurs membranaires.We have previously reported the presence of memory CD4+ T cells that display low levels of SAMHD1 (SAMHD1low ) enriched in Th17 and Tfh cells. Here we investigated gene expression profile and the size and composition of HIV DNA population in SAMHD1 low cells.A total of 36 individuals on c-ART (median: 7y) with median CD4+ counts and nadir of 549 cells/ul and 210 cells/ul respectively, including 6 elite controllers (EC, CD4+: 900 cells/ul) and 8 healthy donors were studied. Blood memory CD4+ CD45RO+ SAMHD1low, CD45RO+ SAMHD1high and naive CD45RO- SAMHD1high cells were sorted. Cell associated HIV-1 DNA levels were quantified (HIV DNA Cell, Biocentric) and ultra-deep-sequencing (UDS, 454/Roche) of partial env (C2/V3) HIV-1 DNA was performed. Gene expression profile on sorted cells was deternined with RNA-Sequencing (Illumina RNASeq technology). Levels of HIV-1 DNA were significantly higher in memory SAMHD1low cells compared to SAMHD1high cells (4.5 [3.1-6.2] vs 3.8 [2.9-5.7] log/10 6 cells, respectively, p=0.02) among c-ART individuals, while naïve CD45RO- SAMHD1high showed lower levels (3.1 [1.6-4.4]). EC exhibited low HIV-1 DNA level in both SAMHD1low and SAMHD1high (1.6 and 2.3 log/10 6 cells respectively p>0.05). Naïve CD45RO - SAMHD1 high cells from EC showed lower DNA compared to naïve cells from c-ART pts (1.6 and 3.1 log/10 6 cells, respectively, p=0.01). Phylogenetic analyses revealed well-segregated HIV-DNA populations between subsets with significant compartmentalization between SAMHD1low and SAMHD1high cells in all but 2 participants (p<0.001) and limited viral exchange. Moreover SAMHD1low cells exhibited a distinct gene profile as compared to SAMHD1high allowing thus further characterisation of these cells.This pilot study revealed distinct HIV DNA populations in size and composition associated with unique genes profile in memory SAMHD1low cells. We show that memory SAMHD1low cells exhibit distinct genes profile which segregates them from the SAMHD1 high counterpart, and contain the highest level of HIV-1 DNA. We reveal distinct/well-segregated HIV-1 DNA populations in both subsets, suggesting minimal viral exchange

Caractérisation et rôle des lymphocytes T CD4+ mémoires SAMHD1low au cours de l'infection par le VIH-1

We have previously reported the presence of memory CD4+ T cells that display low levels of SAMHD1 (SAMHD1low ) enriched in Th17 and Tfh cells. Here we investigated gene expression profile and the size and composition of HIV DNA population in SAMHD1 low cells.A total of 36 individuals on c-ART (median: 7y) with median CD4+ counts and nadir of 549 cells/ul and 210 cells/ul respectively, including 6 elite controllers (EC, CD4+: 900 cells/ul) and 8 healthy donors were studied. Blood memory CD4+ CD45RO+ SAMHD1low, CD45RO+ SAMHD1high and naive CD45RO- SAMHD1high cells were sorted. Cell associated HIV-1 DNA levels were quantified (HIV DNA Cell, Biocentric) and ultra-deep-sequencing (UDS, 454/Roche) of partial env (C2/V3) HIV-1 DNA was performed. Gene expression profile on sorted cells was deternined with RNA-Sequencing (Illumina RNASeq technology). Levels of HIV-1 DNA were significantly higher in memory SAMHD1low cells compared to SAMHD1high cells (4.5 [3.1-6.2] vs 3.8 [2.9-5.7] log/10 6 cells, respectively, p=0.02) among c-ART individuals, while naïve CD45RO- SAMHD1high showed lower levels (3.1 [1.6-4.4]). EC exhibited low HIV-1 DNA level in both SAMHD1low and SAMHD1high (1.6 and 2.3 log/10 6 cells respectively p>0.05). Naïve CD45RO - SAMHD1 high cells from EC showed lower DNA compared to naïve cells from c-ART pts (1.6 and 3.1 log/10 6 cells, respectively, p=0.01). Phylogenetic analyses revealed well-segregated HIV-DNA populations between subsets with significant compartmentalization between SAMHD1low and SAMHD1high cells in all but 2 participants (p<0.001) and limited viral exchange. Moreover SAMHD1low cells exhibited a distinct gene profile as compared to SAMHD1high allowing thus further characterisation of these cells.This pilot study revealed distinct HIV DNA populations in size and composition associated with unique genes profile in memory SAMHD1low cells. We show that memory SAMHD1low cells exhibit distinct genes profile which segregates them from the SAMHD1 high counterpart, and contain the highest level of HIV-1 DNA. We reveal distinct/well-segregated HIV-1 DNA populations in both subsets, suggesting minimal viral exchange.La mise en évidence du rôle de la molécule SAMHD1 dans l’infection par le VIH-1 en tant que facteur de restriction a ouvert de nouvelles perspectives dans la compréhension de la pathogénicité du virus.En effet, il a été clairement démontré que dans les cellules myéloïdes comme les monocytes/macrophages et les cellules dendritiques ainsi que les lymphocytes T CD4+ quiescents, SAMHD1 jouait un rôle important dans la protection de ces cellules de l’infection. En revanche, le rôle de cette molécule dans l’infection des lymphocytes activés, qui sont souvent la cible préférentielle du virus, n’est pas élucidé.Nos résultats ont révélé l'existence d'une sous-population de lymphocytes T CD4+ mémoires exprimant de faibles niveaux de SAMHD1 (CD4+ CD45RO+ SAMHD1low), tandis que la grande majorité des lymphocytes expriment cette molécule à des niveaux plus élevés (94±0.7%). Nous montrons également que ces cellules sont hautement différenciées, qu’elles expriment en larges proportions le marqueur de cycle cellulaire Ki67 et qu’elles sont enrichies en cellules « T helper 17 » (Th17) dans le sang périphérique.De plus, la fréquence de la population CD4+ CD45RO+ SAMHD1low, est diminuée de manière significative chez les patients infectés par le VIH-1 par rapport aux sujets sains. De manière intéressante, nous montrons que dans les ganglions, les cellules T follicular helper (Tfh) expriment faiblement SAMHD1 et sont plus susceptibles à l’infection par le VIH-1 in vitro.L’ensemble de ces résultats suggère que les cellules SAMHD1 low représentent une cible préférentielle pour le virus et pourraient contribuer au réservoir viral.Les objectifs de ce projet sont:1. Déterminer si les cellules SAMHD1low contiennent plus de virus par comparaison aux cellules mémoires SAMHD1high et comparer les séquences virales isolées des cellules mémoires SAMHD1low et SAMHD1high.2. Caractérisation des cellules SAMHD1low au niveau moléculaire par une analyse transcriptomique qui permettra la mise en évidence de marqueurs membranaires

Negative modulation of suppressive HIV-specific regulatory T cells by IL-2 adjuvanted therapeutic vaccine.

The potential benefit in using IL-2 in immunotherapy for cancer and autoimmunity has been linked to the modulation of immune responses, which partly relies on a direct effect on Tregs populations. Here, we revisited the role of IL-2 in HIV infection and investigated whether its use as an adjuvant with therapeutic vaccination, impacts on HIV-specific responses. Antiretroviral therapy treated-patients were randomized to receive 4 boosts of vaccination (ALVACHIV/Lipo-6T, weeks 0/4/8/12) followed by 3 cycles of IL-2 (weeks 16/24/32) before treatment interruption (TI) at week40. IL-2 administration increased significantly HIV-specific CD4+CD25+CD134+ T-cell responses, which inversely correlated with viral load after TI (r = -0.7, p <0.007) in the vaccine/IL-2 group. IL-2 increased global CD25+CD127lowFoxP3+Tregs (p <0.05) while it decreased HIV- but not CMV- specific CD39+FoxP3+CD25+CD134+Tregs (p <0.05). HIV-specific Tregs were inversely correlated with IFN-γ producing specific-effectors (p = 0.03) and positively correlated with viral load (r = 0.7, p = 0.01), revealing their undesired presence during chronic infection. Global Tregs, but not HIV-specific Tregs, inversely correlated with a decrease in exhausted PD1+CD95+ T-cells (p = 0.001). Altogether, our results underline the negative impact of HIV-specific Tregs on HIV-specific effectors and reveal the beneficial use of IL-2 as an adjuvant as its administration increases global Tregs that impact on T-cell exhaustion and decreases HIV-specific CD39+Tregs by shifting the balance towards effectors

IL6/IL6R genetic diversity and plasma IL6 levels in bipolar disorder: An Indo-French study

Reports of association of genetic variants of IL6 and its receptor (IL6R) with psychiatric disorders are inconsistent, and there are few population-based studies thus far in bipolar disorder (BD). We genotyped the IL6 rs1800795 and IL6R rs2228145 polymorphisms in two independent sets of patients exposed to different environmental stimuli such as climatic conditions or specific infectious burden – a French sample and a south Indian Tamil sample of BD with quantitation of circulating plasma IL-6 levels in the latter sub-sample.In both populations, allele and genotype frequencies did not differ significantly between cases and controls for either polymorphism. Upon stratifying based on age at onset, we found no associations with the IL6 rs1800795 variant. However, the IL6R rs2228145 C allele and CC genotype were associated with early onset of disease in the French sample when compared to late onset BD. A similar trend was observed in the Indian population where we also found that plasma IL-6 levels were significantly higher in BD and also in patients who were in residual phase or remission both as compared to controls.Our findings are in favour of a possible trans-ethnic implication of the IL6R genetic diversity in BD and reinforce the notion that IL-6 is an important marker of the operating inflammatory processes in the disease

Subcutaneous injections of IL-2 expand peripheral CD4⁺CD25⁺CD127^lowFoxp3⁺ naïve and total memory Tregs.

<p>(A) Gating strategy for naïve CD4⁺ CD45RO^- CD25⁺ CD127^low FoxP3⁺, total memory CD4⁺ CD45RO⁺ CD25⁺ CD127^low FoxP3⁺ and memory CD4⁺ CD45RO⁺ CD25⁺ CD127^low FoxP3⁺ CD39⁺. (B) Proportions of total CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs, (C) naïve CD45RO^-CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs, (D) total memory CD45RO⁺CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs and (E) memory CD39⁺Tregs, were measured in eighteen patients at wk0, wk16 and wk36 after vaccination and IL-2 injections. Each patient can be distinguished by a colored symbol in the figures. (F) Pie chart showing comparison for compartments of Tregs subsets expansion at wk0, wk16 and wk36. Prism 5.0, version 5.0d, (GraphPad Software, Inc.) was used for statistical analyses. P values were considered significant when < 0.05.</p

CD25+CD134+ CD39+FoxP3+ cells have bona fide Tregs phenotype.

<p>(A) Gating strategy used for detecting CMV-specific CD4+CD25+CD134+ CD39+FoxP3+ Tregs, and IFN-γ producing CD25+CD134+CD39-FoxP3- and CD25+CD134+ CD39-FoxP3+. PBMCs from CMV+ individuals were stimulated with CMV lysate for 44hrs. Flow cytometry was used to detect CMV-specific cells as described in the methods. (B) Mean fluorescence intensity (MFI) of Helios, CD15s, CTLA-4, ICOS, PD-1 and T-bet molecules were measured by flow cytometry in 8 CMV+ individuals to detect CMV-specific subsets (CD134+CD25+Foxp3+CD39+, CD134+CD25+Foxp3+CD39- and CD134+CD25+Foxp3-CD39-). P values were considered significant when < 0.05.</p

Correlations between Í4⁺CD25⁺CD134⁺CD39⁺FoxP3⁺ Tregs and p24 IFNγ-ELISpot at wk36.

<p>IFN-γ producing cells (SFC) were measured by ELISpot as described in the methods. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p

HIV-specific CD4⁺ T-cell responses were amplified by IL-2 treatment and inversely correlate with VL after treatment interruption.

<p>(A) Example of gating strategy used for detecting antigen-specific CD4⁺CD25⁺CD134⁺ cells after in vitro stimulation with p24 or none (OX40 assay), at wk0, wk16 and wk36. (B and D) Representation of HIV- and CMV- specific CD4⁺CD25⁺CD134⁺ percentages in patients at wk0, wk16 and wk36 using the gating strategy shown in (A). (C and E) Correlations between Í4⁺CD25⁺CD134⁺ and viral load at resuming ART, at wk36 for p24 and CMV, respectively. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p