Fusion of ubiquitin to HIV gag impairs human monocyte-derived dendritic cell maturation and reduces ability to induce gag T cell responses

The efficient induction of CD8 T cell immunity is dependent on the processing and presentation of antigen on MHC class I molecules by professional antigen presenting cells (APC). To develop an improved T cell vaccine for HIV we investigated whether fusing the ubiquitin gene to the N terminus of the HIV gag gene enhanced targeting to the proteasome resulting in better CD8 T cell responses. Human monocyte derived dendritic cells (moDC), transduced with adenovirus vectors carrying either ubiquitinated or non-ubiquitinated gag transgene constructs, were co-cultured with autologous naïve T cells and T cell responses were measured after several weekly cycles of stimulation. Despite targeting of the ubiquitin gag transgene protein to the proteasome, ubiquitination did not increase CD8 T cell immune responses and in some cases diminished responses to gag peptides. There were no marked differences in cytokines produced from ubiquitinated and non-ubiquitinated gag stimulated cultures or in the expression of inhibitory molecules on expanded T cells. However, the ability of moDC transduced with ubiquitinated gag gene to upregulate co-stimulatory molecules was reduced, whilst no difference in moDC maturation was observed with a control ubiquitinated and non-ubiquitinated MART gene. Furthermore moDC transduced with ubiquitinated gag produced more IL-10 than transduction with unmodified gag. Thus failure of gag ubiquitination to enhance CD8 responses may be caused by suppression of moDC maturation. These results indicate that when designing a successful vaccine strategy to target a particular cell population, attention must also be given to the effect of the vaccine on APCs

IFN gamma production by T cells treated with gag constructs.

<p>MoDCs were transduced with either the non-ubiquitinated (0x, hatched bar) or ubiquitinated (1x, grey bar) HIV-1 CN54 gag constructs and co-cultured for 7 days with autologous T cells for cells from A) HIV infected patients or C) for healthy control donors. T cells were expanded in culture for 4 weeks, harvested and restimulated with peptide pools and IFNγ production determined by ELISPOT. In addition, mice were injected with either construct and one week post injection, spleens were harvested, stimulated with peptide pools and IFNγ production was determined by ELISPOT (B). IFNγ production by T cells from healthy human controls was also determined by intracellular cytokine staining of CD8 or CD4 T cells (D and E). Graph A) represents one donor of 3± SD, B) represents 3 mice±SD and is representative of one experiment out of three, C) represents 2 individual donors out of 6, D) represents 4 donors±SD and E) is a representative CD8+ intracellular flow cytometry plot.</p

Expression of constructs and T cell responses.

<p>A) Human moDCs were transduced with either the non-ubiquitinated (0x) or ubiquitinated (1x) HIV-1 ZM96 gag constructs for 24 h or untransduced (control). Cells were then cultured for a further 18 h in the absence (hatched bar) or presence (grey bar) of the proteasome inhibitor, MG132. Cells were then harvested and stained intracellularly with anti-gag-PE antibody and analysed by flow cytometry. Results shown are mean±SD for 3 donors. B) MoDCs were transduced with either the non-ubiquitinated (0x, hatched bar) or ubiquitinated (1x, grey bar) HIV-1 ZM96 gag constructs and co-cultured with autologous naïve T cells. After 4 weeks of T cell expansion in culture, T cells were harvested and restimulated with single individual peptides and IFNγ production was determined by ELISPOT. Overall fusion with ubiquitin lowered the response although responses to 7 peptides were increased. For the 3 donors not shown only 2/49, 5/49 and 1/49 peptides gave a better response with Ub-gag. Graph represents 1 of 4 donors.</p

Expression of maturation markers on transduced moDCs.

<p>MoDCs were transduced with either the A) non-ubiquitinated (0x, hatched bar and dotted line) or ubiquitinated (1x, grey bar and grey line) HIV-1 CN54 gag constructs or B) non-ubiquitinated (0x, hatched bar and dotted line) or ubiquitinated (1x, grey bar and grey line) MART-1 constructs or untransduced (control, open bar). Following an overnight incubation, DCs were then matured with LPS and IFNγ for a further 24 hrs and then harvested and stained with the indicated antibodies and analysed by flow cytometry. A) represents 9 donors, and the histogram represents one donor, B) represents 4 donors and the histogram represents one donor and box plots represent the median with upper and lower quartiles, and whiskers represent the min and max values.</p

Phenotype of CD8 and CD4 T cells stimulated with either non-ubiquitinated or ubiquitinated constructs.

<p>MoDCs were transduced with either the non-ubiquitinated (0x, hatched bar) or ubiquitinated (1x, grey bar) HIV-1 CN54 gag constructs and co-cultured with autologous naïve T cells. After each subsequent week in culture, cells were removed and stained with antibodies to A) CD8 or B) CD4 and the indicated surface markers. Cells were analysed by flow cytometry, and the box plots represent the median with upper and lower quartiles, and whiskers represent the min and max values of 9 donors.</p

Expression of constructs and T cell expansion.

<p>A, B and D) Human moDCs were transduced with either the non-ubiquitinated (0x) or ubiquitinated (1x) HIV-1 CN54 gag constructs for 24 h or untransduced (control) and then cultured for a further 18 h in the presence (grey bar) or absence (hatched bar) of the proteasome inhibitor, MG132 (A and B). Cells were then harvested and stained intracellularly with anti-gag-PE antibody and analysed for the frequency (A) and density (B) of gag by flow cytometry. Results shown are the mean±SD for 3 donors and representative flow cytometry plots are shown in D. C) MoDCs were transduced with either the non-ubiquitinated (0x, closed circle) or ubiquitinated (1x, closed square) HIV-1 CN54 gag constructs and co-cultured with autologous naïve T cells. On a weekly basis, cells were harvested and counted by Trypan blue exclusion and co-cultured with freshly transduced moDCs. Graph shows the mean±SD of 4 donors.</p

Detection of immune modulators in supernatants of cultures, or by ICS, treated with either construct.

<p>MoDCs were transduced with either the non-ubiquitinated (0x, hatched bar) or ubiquitinated (1x, grey bar) HIV-1 CN54 gag constructs and co-cultured with autologous naïve T cells. After each week, supernatants were removed and the presence of the indicated cytokines was determined by CBA and analysed by flow cytometry (A). After 4 weeks in culture, the cells were harvested and restimulated with peptide pools and the expression of TNF-α and IL-10 were determined by ICS. The box plots (A) represent the median with upper and lower quartiles, and whiskers represent the min and max values of 5 donors. Graph B show the results of 4 donors±SD.</p

Phenotype of CD8 cells and their expression of IFN gamma from HLA-A*0201 individuals.

<p>MoDCs from HLA-A*0201 individuals were transduced with either the non-ubiquitinated (MART 0x, hatched bar) or ubiquitinated (MART 1x, grey bar) MART construct, as well as the dominant peptide ELAGIGILTV (peptide, open bar) as a positive control. DCs were then co-cultured with autologous naïve T cells and after each subsequent week in culture, T cells were harvested and stained with antibodies to the indicated surface markers (A). B) After 3 weeks of T cell expansion in culture, T cells were harvested and restimulated with the dominant peptide, ELAGIGILTV, and IFNγ production was determined by intracellular cytokine staining and ELISPOT. Cells were analysed by flow cytometry, and A) box plots represent the median with upper and lower quartiles, and whiskers represent the min and max values of 5 donors and B) shows individual donors.</p

Expression of PDL1 and production of IL-10 by transduced moDCs.

<p>MoDCs were transduced with either the non-ubiquitinated (0x, hatched bar or closed circle) or ubiquitinated (1x, grey bar or closed square) HIV-1 CN54 gag constructs or untransduced (control, open bar). Following an overnight incubation, DCs were then matured with LPS and IFNγ for a further 24 hrs. Cells and supernatants were harvested and A) cells were stained with anti-PDL1 and analysed by flow cytometry and B) the production of IL-10 was determined by ELISA. Graph A) represents the mean±SD of 5 donors and graph B) represents 6 individual donors.</p