Decreased HIV-specific T-regulatory responses are associated with effective DC-vaccine induced immunity

Abstract

The role of regulatory T cells (Tregs) in vaccination has been poorly investigated. We have reported that vaccination with ex vivo-generated dendritic-cells (DC) loaded with HIV-lipopeptides (LIPO-5-DC vaccine) in HIV-infected patients was well tolerated and highly immunogenic. These responses and their relation to viral replication following analytical treatment interruption (ATI) were variable. Here, we investigated whether the presence of HIV-specific Tregs might explain these differences. Co-expression of CD25, CD134, CD39 and FoxP3 was used to delineate both antigen-specific Tregs and effectors T cells (Teffs). Median LIPO-5 specific-CD25+CD134+ polyfunctional T cells increased from 0.1% (IQR 0-0.3) before vaccination (week -4) to 2.1% (IQR 1.1-3.9) at week 16 following 4 immunizations (p=0.001) and were inversely correlated with maximum viral load following ATI (r=-0.77, p=0.001). Vaccinees who displayed lower levels of HIV-specific CD4+CD134+CD25+CD39+FoxP3+ Tregs responded better to the LIPO-5-DC vaccine. After vaccination, the frequency of HIV-specific Tregs decreased (from 69.3 at week -4 to 31.7% at week 16) and inversely correlated with HIV-specific IFN-γ-producing cells (r=-0.64, p=0.002). We show that therapeutic immunization skewed the HIV-specific response from regulatory to effector phenotype which impacts on the magnitude of viral replication following ATI.

Fig 1. HIV-specific responses are significantly upregulated after the vaccination with LIPO-5-DC vaccine.

Each patient is designated with its individual color/symbol code (refer to S1 Table). Symbols are attributed based on maximum viral load rebound after treatment interruption as indicated in the figure. (A) Representative plots for a single patient prior (wk -4) and after (wk 16) the vaccination. Cells were stimulated with gag p24 pool, LIPO-5 vaccine or CMV lysate as a control, stained and analyzed by flow cytometry 44 hours later. Plots show viable CD4⁺ T cells. (B) Graphs show the percentages of antigen-specific cells (CD134⁺CD25⁺) among CD4⁺ T cells after the stimulation with the indicated antigens (n = 14). (C) IFN-γ, TNF-α and IL-2 production among LIPO-5- specific cells (CD134⁺CD25⁺) (n = 14). (D) Graph shows the correlation between the relative increase in LIPO-5-specific response (response after the vaccination-response before vaccination) and maximal viral load rebound after HAART interruption (n = 14). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.

Fig 2. Overall strength of the HIV-specific responses is inversely correlated with maximum viral load after ATI.

(A) Graph showing the responses for all individual patients to indicated antigens. (B) Correlation between the relative increase in the strength of the response (sum of the strengths of the response post vaccination–sum of the strengths of the response prior to vaccination) and maximal viral load rebound after HAART interruption (n = 9). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.

Fig 3. Antigen-specific Tregs originate from CD25^hi cells.

(A) Plots showing the sorting strategy for CD4⁺CD25^hi, CD4⁺CD25^int and CD4⁺CD25^lo populations as well as CD4^neg. (B) Pre-sorted CD25 high, intermediate or low (left side) fraction were mixed with CD4^neg cells and stimulated for 44h with CMV lysate. Gating strategy is given for each fraction. Antigen-specific Tregs (CD39⁺FoxP3⁺IFN-γ^-) originate from CD25^hi fraction while Teffs (CD39^-FoxP3^-IFN-γ⁺) originate from CD25^lo fraction. The figure is representative of 3 individual experiments.

Fig 4. Tregs can suppress HIV-specific responses in vitro.

(A) Percentages of gag p24-specific Tregs (CD134⁺CD25⁺CD39⁺FoxP3⁺) or IFN-γ-producing cells after stimulation in whole or Tregs-depleted fractions. (B) Representative plots of suppression assays in which depleted Tregs from (A) (lower panel) or CD25^lo fraction (upper panel) were cocultured in 1:2 ratio with CFSE-labeled PBMCs in overnight culture in the presence of 2μg/mL of gag p24 peptide pool, 1μg/mL of αCD28 and αCD49d and 10μg/mL of Brefeldin A. (C) Graphs showing percent of both CD4⁺ and CD8⁺ IFN- γ-secretion suppression (n = 9). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 5. HIV-specific Tregs/Teffs ratio is affected by vaccination.

(A) Gag p24-, LIPO-5- and CMV-Tregs responses prior and after vaccination (n = 14). (B) Pie chart showing LIPO-5-specific Tregs among LIPO-5-specific (CD134⁺CD25⁺) cells prior and after vaccination (n = 14). (C) Representative profiles after in vitro stimulation with LIPO-5 showing good and poor responder to vaccination (based on a viral load rebound). (D) Correlation between IFN-γ-producing and Tregs cells among LIPO-5-specific cells after in vitro stimulation. Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.

Fig 6. Patients with lower HIV-specific Tregs respond better to vaccination.

(A) Correlation between the proportions of LIPO-5-specific Tregs among LIPO-5-specific CD4⁺ T cells at the baseline (wk -4) and immune score at wk 16. (B) Correlation between the proportions of LIPO-5-specific Tregs among LIPO-5-specific CD4⁺ T cells at the wk 16 and immune score at wk 16. Spearman coefficient for each correlation is indicated (r) as well as p value.