Primed CD4 and CD8 T cell responses

<p>Primed CD4 and CD8 T cell responses</p

HIV-1 does not affect the phenotypic maturation of MDDCs or their T cell stimulatory ability.

<p>(A) Matured MDDCs were unpulsed or pulsed with 1500ng/10⁶ p24 equivalents of AT-2 HIV-1 or infectious HIV-1 for 16–18hrs and washed. The effect of HIV-1 on the mature MDDCs was assessed by staining with isotype control, CD40, CD80, CD83, CD86, HLA ABC, and HLA DR antibodies conjugated to phycoerythrin (PE).B) Mature MDDCs were unpulsed or pulsed with 150ng–1500ng/10⁶ p24 equivalents of AT-2 HIV-1 or infections HIV-1 for 16–18 hrs. The ability stimulate T cells was measured using a HIV-specific CD8⁺ T cell clone recognizing HLA A2.01-restricted SL9 gag p17 and a HIV-specific CD4⁺ T cell clone recognizing HLA DR4-restricted LI15 gag p24. The activation of T cells was measured by IFN- γ Elispot assay. (C) Mature MDDCs from a chronically infected individual were pulsed with 300ng/10⁶ p24 equivalents MV, AT-2 or infectious HIV-1 and cocultured with autologous T cells for 7 days at a DC:T cell ratio of 1:10. At day 7, the different groups of T cells were harvested and restimulated with mature MDDCs infected with vaccinia vector constructs (V ctr, V gag, V pol or V nef) to detect CD8+ T cell expansion or MDDCs pulsed with recombinant proteins (ctr, gp160, p24, p66 or nef) to detect CD4+ T cell expansion. The responding T cells were enumerated by IFN-γ Elispot assay. The data are representative of 4 experiments.</p

Strong and broad HIV-specific CD4 T cell responses exist in acute infection which quickly diminish.

<p>(A and B) Fresh CD8-depleted PBMCs from 12 adult individuals with documented primary HIV infection (PHI) were screened for HIV Gag-specific CD4 responses by IFNγ Elispot using a pool of overlapping peptides spanning all HIV proteins and corresponding to Clade B consensus sequence 2001. Subjects were assessed at baseline (BL) before institution of any antiviral therapy and 1 month, 3 months and 6 months after BL. Data represent total HIV-specific CD4 T cells responses (A) as well as responses to individual gene products (B). Grey circles: subjects treated with HAART; white circles: individuals who remained without therapy. Horizontal black bars: median values. (C) This decline can also be demonstrated by intracellular cytokine staining and affects both IFNγ- and IL-2-secreting CD4+ T cells. Fresh PBMCs from an adult individual with acute HIV infection were stimulated with medium alone, the same HIV Gag as above or a CMV lysate, in the presence of anti-CD28 and anti-CD49 stimulating antibodies and submitted to intracellular cytokine staining after six hour incubation. Similar experiments were performed 12, 24 and 36 weeks after BL. These results are representative of three separate subjects. Numbers in quadrants: percentage of cytokine-producing CD4⁺ T cells.</p

HIV-1 pulsed MDDCs prime HIV-1 specific T cells.

<p>(A) Autologous naïve bulk (CD4⁺ and CD8⁺) T cells were negatively isolated from PBMCs by magnetic beads to remove CD14, CD19, CD56 and CD45RO positive cells. (B) The autologous naïve bulk T cells were co-cultured with the mock (no virus), AT-2 or infectious HIV-pulsed DCs. Cultures were restimulated weekly by the addition of DCs pulsed with mock, AT-2 HIV-1 or Inf HIV-1 for 4 weeks. HIV-specific responses were detected by intracellular cytokine staining for IFN-γ after 4 weeks of culture. (C) HIV-1 specific priming was attempted with bulk T cells from 11 HIV naïve donors to establish the efficiency of in vitro MDDC priming. 8 out of 11 donors were successfully primed. (D) The priming efficiency of MDDCs pulsed with AT-2 HIV-1 vs. infectious HIV-1 of the 8 donors that was successfully primed were compared. (E) Priming cultures were analyzed for the presence of viral replication. Supernatants were collected from the cultures after the 3^rd restimulation on day 21 and virus replication was measured using HIV p24 ELISA kit. Mock, AT2-HIV, and Inf HIV priming cultures were compared.</p

Double-label immunohistochemistry and <i>in situ</i> hybridization of colon.

<p><b>(A).</b> Representative images of SIV ISH (blue) with double-label immunohistochemistry for macrophage marker Ham56 (DAB, brown) in rhesus macaques in groups SIV239E (left), SIV239noE (middle), and SIVΔ<i>vpx</i> (right). SIV+ Ham56+ macrophages were frequent in SIV239E monkeys (left), but absent in the SIVΔ<i>vpx</i> group (right). <b>(B).</b> SIVΔ<i>vpx</i>-infected monkeys had significantly less virus in the colon (mean 10.5 SIV+ cells) compared to monkeys infected with SIV239 (SIV239E mean 105.3 SIV+ cells, SIV239noE mean 33.5 SIV+ cells), but no difference with animals in the SIVΔ<i>nef</i>/SIVΔ3 group. <b>(C).</b> SIVΔ<i>vpx</i> monkeys (mean 0 cells) had significantly fewer SIV+ Ham56+ macrophages compared to SIV239E (mean 66.7 cells), SIV239noE (mean 9.8 cells), and SIVΔ<i>nef</i>/SIVΔ3 groups (mean 3.3 cells) groups. <b>(D).</b> There was a significantly lower percentage of SIV+ cells that were macrophages in SIVΔ<i>vpx</i>-infected rhesus (mean 0%) compared to both the SIV239E (mean 63.3%) and SIV239noE (mean 22.4%) groups and a trend in the SIVΔ<i>nef</i>/SIVΔ3 group (mean 25.6%).</p

Amino acid sequences from chronic infection plasma viral RNA from SIVΔ<i>vpx</i>-infected monkeys.

<p><b>(A).</b> Vpx N-terminal sequences accumulated debilitating mutations in two animals (cases 4 and 5) (*). <b>(B).</b> Novel amino acid sequence changes of Vif were present in case 4 (with highest viral loads) with leucine to proline (L->P) and alanine to threonine (A->T). <b>(C).</b> Analysis of Vpr revealed changes in amino acid sequences detected at the C-terminal region in cases 3 and 4 (I94T, P95L, S99N or G). <b>(D).</b> Alignments of Tat demonstrated multiple non-conservative changes, such as I22T, S32L, L35P.</p

HIV-specific T cells are polyfunctional and recognized a broad repertoire of antigens.

<p>(A) Mapping of epitopes was done using pools of overlapping peptides. The peptides spanned the entire HIV-1 genome and were based on the consensus B sequence (<a href="http://www.hiv.lanl.gov/content/immunology/index.html" target="_blank">http://www.hiv.lanl.gov/content/immunology/index.html</a>). The peptide pool matrices and the HIV-1 primed T cell cultures were added to IFN-γ Elispot assays as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004256#pone.0004256-Kaufmann1" target="_blank">[19]</a>. The assays were developed after overnight culture and evaluated. Reconfirmations of all positive wells were done the following day with the single peptide by flow cytometry to detect intracellular IFNγ production. (B) The reconfirmed HIV-1 specific T cell responses from all donors tested were analyzed to determine which HIV-1 antigens the primed T cells recognized and the percentile they constituted of total primed responses from those screened. (C) The effector functions of the HIV-1 specific polyclonal T cells were examined by stimulation with MDDCs pulsed with AT-2 HIV-1 or infectious HIV-1 and the intracellular production of the effector cytokines/chemokines; IFN-γ, TNF-α, IL-2 and MIP-1β, was measured by flow cytometry. The polyfunctionality of the T cell responses were analyzed as the ability to produce the combination of one, two, three or all four of the cytokines/chemokines tested by the primed CD4⁺ (D) and CD8⁺ (E) T cells in response to HIV.</p

In vitro primed novel HIV epitopes correlates with CD4⁺ T cell responses seen during primary infection.

<p>(A) Summary of responses to the novel epitopes detected in 12 acute and recent HIV-infected subject PBMCs. PBMCs from acute and recent HIV infected individuals were thawed and tested directly ex vivo for reactivity to OLPs 11, 189, 262, 322, 377, and 382 using intracellular staining for IFN-γ, TNF-α, and IL-2 after six hour incubation. Reactivity against the peptide was determined when the percentage of cytokine-secreting cells were at least twice the percentage of cytokine-secreting cells in the unstimulated wells. (B) HIV-infected PBMCs from one donor evaluated at 3 different time points after infection (sample one: 2 weeks after onset of retroviral syndrome: acute infection, sample two: 1 year infected: recent infection, and sample three: 2 years infected: chronic infection) were thawed and cocultured with peptide pools OLP 371-381 or OLP 382-392 for 14 days. The peptide activated PBMCs were then assessed for reactivity to each of the individual peptides in the pools used.</p

Summaryoflentiviral infection of macrophages in spleen, lymph node, anin macaques inoculated intravenously with SIVΔ<i>vpx</i> compared to SIVmac239 (with or without SIV encephalitis) and SIVΔ<i>nef</i> or Δ3 assessed in spleen, lymph node, and colon.

<p>*p<0.05 difference between SIVΔ<i>vpx</i> and both SIVmac239 and SIVΔ<i>nef</i>/Δ3cases.</p

Correlation of primed T cell responses with in vivo data

<p>Correlation of primed T cell responses with in vivo data</p