HIVBr27 immunization elicits polyfunctional CD4⁺ and CD8⁺ T cells.

<p>Two weeks after the last immunization with HIVBr27 or empty pVAX1, pooled spleen cells from 6 BALB/c mice were collected, labeled with CFSE (1.25 µM) and cultured for 4 days in the presence of pooled HIV-1 peptides (5 µM) or medium only. On day 4, cells were pulsed for 12 hours with pooled peptides in the presence of Brefeldin A and costimulatory antibody (anti-CD28). A) Multiparameter flow cytometry strategy used to determine the frequency of IFN-γ, IL-2 or TNF-α producing CFSE^low CD4⁺ and CD8⁺ T cells. B) Frequency of IFN-γ, IL-2 or TNF-α producing CFSE^low CD4⁺ (left) and CD8⁺ (right) T-cells. C) Boolean combinations of IFN-γ, IL-2 and TNF-α producing CFSE^low CD4⁺ and CD8⁺ T cells from HIVBr27 immunized mice. Background responses detected in negative control tubes (cells stimulated with medium and cells from pVAX1 immunized mice stimulated with pooled peptides) were subtracted from those detected in stimulated samples. Data are representative of three independent experiments.</p

HIVBr27 immunization provides cross-clade immunity.

<p>Two weeks after the last immunization with HIVBr27 or empty pVAX1, pooled spleen cells from 6 BALB/c mice were cultured in the presence of HIV-1 M-group consensus peptides (5 µM) (white bars) or their variants (colored bars), from diverse HIV-1 subtypes. Frequency of IFN-γ secreting cells was assessed by ELISPOT assay (A) and proliferative CD4⁺ (B) and CD8⁺ (C) T-cell responses were assessed by CFSE dilution assay. Dotted lines represent ELISPOT or proliferation cutoff, which were calculated as median +3 SD of unspecific responses obtained with splenocytes from pVAX1 immunized mice stimulated with HIV-1 peptides. Data are representative of two independent experiments.</p

Immunization with HIVBr27 elicits T-cell responses in BALB/c mice.

<p>Two weeks after the last immunization with HIVBr27 or empty pVAX1, pooled spleen cells from 6 BALB/c mice were cultured in the presence of individual or pooled HIV-1 peptides (5 µM). Frequency of IFN-γ secreting cells (A) and proliferating CD4⁺ and CD8⁺ T-cells (B) against pooled HIV-1 peptides. C) Frequency of IFN-γ secreting cells against individual HIV-1 peptides. Dotted line represents ELISPOT cutoff values. Data are shown as mean of three independent experiments for ELISPOT assays. Data from proliferation assay are representative of three independent experiments.</p

HLA-DR binding assay for M-group consensus peptides.

<p>Peptide binding assays were performed by incubating purified HLA-DR molecules (5–500 nM) with different concentrations of unlabeled peptide inhibitors and 1–10 nM ¹²⁵I-radiolabeled probe peptides for 48 h. Significant affinity threshold <1000 nM are shown in gray. A dash represents 50% inhibitory concentration (IC50) >30000 nM.</p

M-group consensus peptides are recognized by PBMC from HIV-1-infected patients.

<p>IFN-γ ELISPOT assay was performed to evaluate T-cell responses against the 27 M-group consensus peptides by stimulating PBMC from patients infected with different variants of HIV-1, which are represented by colored symbols. A) IFN-γ secretion against Pol peptides. B) IFN-γ secretion against Gag peptides. C) IFN-γ secretion against Vif, Rev, Vpr, Vpu and Nef peptides. B subtype-infected patients (n = 14), BC recombinant-infected patient (n = 1), BF recombinant-infected patients (n = 9), F subtype-infected patient (n = 1). Dotted lines represent IFN-γ ELISPOT cutoff, which is ≥50 spots/10⁶ cells. Only positive responses are shown in the graphs.</p

Chronological age and p16 expression in CD4⁺ and CD8⁺ T cells.

<p>Correlation between chronological age and per-cell level of p16 expression in each subject group is shown. (A-E) Correlation between chronological age in HC, HS, C, IP, NC and MFI p16 CD4⁺, respectively; (F-J). Correlation between chronological age in HC, HS, C, IP, NC and MFI p16 CD8⁺, respectively. HC: Healthy controls; HS: HAART suppressed; C: controllers; IP: Immunological progressors; NC: non-controllers. p and Spearman rho (r) values are shown for each plot.</p

HIV drives higher per-cell p16 protein levels in CD4⁺ and CD8⁺ T lymphocytes among HIV-1-infected subjects.

<p>PBMC from healthy controls (HC), HAART suppressed (HS), Controllers (C), Immunological progressors (IP) and Non-controllers (NC) were stained for surface markers as well as the intracellular p16 marker. The median intensity of fluorescence (MFI) showing per-cell level expression of p16 in CD4⁺ and CD8 T⁺ cells is also shown (A and B, respectively). (C) Correlation between p16 MFI in CD4⁺ and CD8⁺ T cells in the whole cohort (Spearman rho, r = 0.74/p < 0.0001). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Clinical data.

<p>Clinical data.</p

p16 expression in memory T cell subsets.

<p>(A and B) p16 MFI in CD4⁺ and CD8⁺ memory subsets, respectively, for all groups tested. HC: Healthy controls; HS: HAART suppressed; C: controllers; IP: Immunological progressors; NC: non-controllers. T_CM: central memory T cells; T_SCM: stem cell memory T cells, T_EM: effector memory T cells. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.</p

p16 expression is positively correlated with T cell activation.

<p>(A-B) Correlation between CD4⁺ T cell activation and p16 levels on CD4⁺ and CD8⁺, respectively; (C-D) Correlation between CD8⁺ T cell activation and p16 levels on CD4⁺ and CD8⁺, respectively. R and p values are shown for each plot.</p