Differences in functional phenotype of the HCV-specific CD8 responses during therapy between SVRs and NRs.

<p>(A) Pre- and on-treatment FACS density plots in response to a 5.5 hour incubation with 2 µgml⁻¹ of HCV NS5 peptide pools showing the frequencies of memory CD8⁺ T cells displaying the depicted combinations of CD107a mobilizing and intracellular production of IFNγ, IL-2, and TNF-α for patient P70. Gating was done on viable memory CD14⁻CD19⁻CD3⁺CD4⁻CD8⁺ cells that were not CD27⁺CD45RO⁻. Background activity against CD28/CD49d costimulation alone has been subtracted. (B) Flow cytometric analysis of polyfunctionality within total genotype 1 HCV-specific CD8 memory T-cells is shown at pre-treatment prior to start of therapy. The bar chart shows each of the 15 possible response profiles on the x-axis as the percentage of the total cytokine response on the y-axis. The filled bar represent the interquartile range and the line the median. (C) Summary of functional profile in SVR (red outline) and NR during treatment. The distinct cellular subsets shown in panel B were grouped by number of functions, so each section of the pie charts represent the mean proportion of HCV-specific CD8⁺ T cells grouped by the number of functions expressed independently of any particular function and matching the color code used in panel B. Statistically significant differences at pre-treatment between SVRs an NRs (*P<0.05, by Mann Whitney test) are indicated by the asterix. (D) The frequency of total HCV-specific CD8 memory T-cell responses at pre-treatment (week 0) and during treatment is shown in each individual as a percentage of their total memory CD8 T-cell population. (E) pegIFN-induced breakdown of the immunosuppressive environment is similar in nonresponders versus responders. Relative levels of immune suppressive genes in PBMCs of NRs (black) and SVRs (red), normalized to pretreatment levels, as determined by qPCR. Error bars represent mean ± SEM (Bonferoni's One-way Anova post-test, **P<0.001). (F) Relationship between the proportion of CD107a mobilization on HCV-specific CD8⁺ T cells at 12 weeks after treatment initiation and the 12-week treatment changes of PVL from pre-treatment (n = 10, analyzed using Pearson correlation). Each symbol corresponds to one subject: SVRs are in red, NRs are in black.</p

Lower rate of SVR in HCV chronic patients with impaired MDC TLR functionality.

<p>PBMCs were cultured in the presence of brefeldin A and TLR agonists for 6(A) Representative FACS plots of IL-6⁺ gated lineage⁻CD16⁻CD45⁺CD11c⁺MHC-II^br MDCs positive for TNF-α or IL-12 before treatment are shown following LPS or 3M-002 stimulation as stratified by CP cluster. Numbers on the left side of and above the bracketed lines indicate the geometric MFI and the percentages of cytokine–expressing MDCs in the designated area, respectively. (B) TNF-α and IL-12 expression (mean ± SD) as a log₂ geometric MFI fold-induction above unstimulated control. Statistical comparisons between CP groups and aviremic control group were calculated by the Dunnett one-way ANOVA post test (***P<0.001). (C) MDC cytokine profile-stratified analysis of SVR and NR rates indicates that the difference between CP-N and CP-D patients irrespective of HCV genotypes is significant (n = 34 subjects; *P = 0.0048 by Fisher's exact two-tail test).</p

Characteristics of the twenty HCV chronic patients longitudinally followed during treatment course.

a<p>log₁₀ IU/ml.</p>b<p>By trypan blue count; visits during and following treatment.</p>c<p>Frequencies are total cytokine-producing (IFN-γ, IL-2, TNFα) and degranulating (CD107a⁺) cells out of the CD8⁺ memory subset at pre-treatment. All positive responses to HCV pools were summed to determine the total antigen-specific response within the memory peripheral blood T cell populations. Due to HCV peptide reagent availability, only genotype 1 patients could be stimulated. ND: not determined.</p>d<p>Number of HCV proteins detected by CD8⁺ T cells.</p

MDC TLR functionality is associated with the likelihood of achieving SVR following pegIFN and ribavirin treatment.

<p>(A) Heat maps of week 0 (pre-treatment) FACS measured IL-12 and TNFα (columns) protein expression profiles (CP) for lineage⁻CD16⁻CD45⁺CD11c⁺MHC-II^br MDCs activated with TLR agonists from viremics starting therapy and followed longitudinally (n = 20) as a log₂ fold-change in MFI expression relative to TLR stimulated MDCs from a reference group of aviremic controls that cleared HCV after IFN therapy (n = 12) (yellow, higher than; blue, lower than; white, no change versus protein expression in aviremics). (B-D) Individual HCV plasma viral RNA loads (PVL, B), ALT (C) or AST (D) levels were determined before, during and after antiviral therapy. Red lines represent SVR; black lines NR. Vertical gray lines indicate period of antiviral treatment. (E) Relative levels of IRG mRNAs in PBMCS of NRs and SVRs at 4 and 12 weeks of treatment, normalized to pre-treatment levels, as determined by qPCR. Error bars represent mean ± SEM. (F) Correlation between MDC inhibition (sum of IL-12 and TNF-α TRIF-dependent TLR MFI fold-change, log₂) prior to treatment initiation and the end-of-treatment changes of PVL from pre-treatment (n = 20).</p

Clinical data of the 34 patients clustered according to MDC functionality suffering from chronic HCV and undergoing pegIFN treatment.

<p>In parentheses are %; NA, not applicable.</p>1<p>yr ± SD.</p>2<p>log₁₀ IU x ml⁻¹ ± SD.</p

IFNα therapy leads to reduction in IL-7 plasma concentration.

<p>(A) IL-7 plasma levels were quantified in peripheral blood cells from acutely HCV-infected (light grey symbols), chronically HCV-infected (black symbols) and HIV/HCV co-infected (white symbols) patients at study entry, as compared to healthy donors (HCV-, dark grey symbols). **: p<0.001 for any HCV-infected patients group. (B) Evolution of plasma IL-7 levels over the first 4 months of IFNα therapy in acutely HCV-infected (left panels), chronically HCV-infected (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute IL-7 plasma levels in each individual sample (Wilcoxon matched-pairs signed-ranks test) are shown on top. (C) Soluble CD127 was quantified in plasma from acutely HCV-infected (white symbols, top panel), chronically HCV-infected (black symbols, top panel) and HIV/HCV co-infected (bottom panel) patients at baseline (0) and M2. (D) CD127 expression was measured on circulating CD4⁺ (top panel) and CD8⁺ (bottom panel) and expressed as mean fluorescence intensity (left panels) and percentages of positive cells (right panels) over the 4 first months of IFNα therapy.</p

IFNα therapy leads to naïve T-cell lymphocytopenia.

<p>(A) CD4⁺ (top panel) and CD8⁺ (bottom panel) naïve T-cell counts were quantified in peripheral blood cells from acutely HCV-infected (light grey symbols), chronically HCV-infected (black symbols) and HIV/HCV co-infected (white symbols) patients at study entry, as compared to healthy donors (HCV-, dark grey symbols). (B) Evolution of CD4⁺ (top panels) and CD8⁺ (bottom panels) naïve T-cell counts during the first 4 months of IFNα therapy in acutely HCV-infected (left panels), chronically HCV-infected (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents data from an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute naïve T-cell counts in each individual sample, (Wilcoxon matched-pairs signed-ranks test) are shown on top. The horizontal bars represent median values.</p

Variations in IL-7 plasma levels correlate with evolution of RTE

<p><b>production.</b> Correlations. between variations in IL-7 plasma levels (ΔIL-7) and either variations in (A) total (CD4⁺ + CD8⁺) naïve T-cell counts (Δnaïve T-cell counts), (B) RTE defined as CD31^hi naïve CD4⁺ T-cells (ΔRTE CD4 counts), (C) the sj/βTREC ratio (Δsj/βTREC ratio), (D) the frequency of Ki-67⁺ cells in the RTE CD4⁺ T-cell subset (Δ%Ki-67⁺ in CD4⁺ RTEs) or (E) the number of circulating Ki-67⁺CD4⁺ RTEs (ΔKi-67⁺ RTE counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) HCV-infected patients (left panels) and HIV/HCV co-infected patients (right panels). Correlation coefficients (Spearman's r) and the associated probabilities (p) are shown.</p

IFNα therapy leads to major impairment of thymic function.

<p>(A) The frequency of Ki-67 expressing cells in the CD4⁺ RTE subset (CD31^hi naïve T-cells) was measured in acutely HCV-infected (grey symbols, top panel), chronically HCV-infected (white symbols, top panel) and HIV/HCV co-infected (bottom panel) patients (central panels) and HIV/HCV co-infected (right panels) patients. Each line represents data from an individual patient. Statistical significances of the differences to baseline values (time 0), calculated on the absolute sj/βTREC ratio in each individual sample (Wilcoxon matched-pairs signed-ranks test) are shown on top. The horizontal bars represent median values.</p