Study participants and cell preparation
We obtained up to 300 ml of peripheral blood from 19 HIV positive participants (one on two occasions); three were ultimately excluded from the data presented, because their cells did not show evidence of latency reversal after stimulation with antibodies to CD3 and CD28 (the positive T cell activation control). All participants had a CD4 T cell count of at least 500 cells/µl of peripheral blood and fully suppressed viremia (less than 20 copies/ml) for at least one year. For the eight participants for whom data are available, HIV-infection was diagnosed five to over twenty years before their blood was obtained for this study.
We isolated peripheral blood mononuclear cells (PBMC) by density centrifugation. To ensure optimal antigen presentation to CD4 T cells, we kept professional antigen presenting cells - B cells and monocytes - in the cell population by depleting CD8 T cells from the PBMC rather than isolating the CD4 T cells. Our rationale for removing CD8 T cells was to avoid potential killing of reactivated reservoir cells as well as the potential secretion of suppressive mediators. CD8 T cell depletion from participant PBMC was confirmed by flow cytometry, and residual CD8 T cells were usually < 0.1% (data not shown).
Identification of antigen specific CD4 T cell responses by INF-γ ELISpot assay
Production of IFN-γ by CD4 T cells is a hallmark of the Th1-type CD4 T cell phenotype and is typically associated with an effective host defense against intracellular pathogens (reviewed in [26]). Therefore, we first used an IFNγ ELISpot assay to evaluate the ability of peptide pools to activate and induce an immune response in the CD4 T cells within the CD8-depleted cell populations.
We screened IFN-γ production in response to peptide pools containing 15mer overlapping peptides from: 1) Cytomegalovirus (CMV) matrix protein 65 (pp65); 2) Candida albicans mannoprotein MP65; 3) a mixture of 14 previously identified optimal MHC class-II restricted epitopes from human cytomegalovirus (HHV-5), Epstein-Barr virus (HHV-4), influenza A, and Clostridium tetani toxoid (pool named CEFT); 4) HIV-1 Gag; 5) HIV-1 Pol; 6) HIV-1 Env; and 7) HIV-1 Nef. For each condition, we stimulated 100,000 CD8-depleted PBMC with 1 µg/ml per individual peptide. We used anti-CD3/CD28-coated beads as well as phorbol myristate acetate in combination with ionomycin (PMA/Iono) as a positive control for maximal T cell stimulation. Both positive controls typically saturated the signals on the ELISpot plate and did not allow quantification (Fig. 1A). We used DMSO as a negative control, because the peptide pools and individual peptides were reconstituted in DMSO. Wells treated with DMSO typically showed < 10 SFC/million cells. IFNγ responses of varying magnitudes were measured for these peptide pools in CD8-depleted PBMCs from a subset of participants (Fig. 1A, which shows example ELISpot results for each of the peptide pools; in addition to the example shown, two additional participants tested showed no response to peptides from C.albicans). For three participants, we tested the 123 Gag 15mer peptides singly at a concentration of 12.5 µg/ml per peptide with 100,000 CD8-depleted PBMC per well to identify the specific peptides within the pool that induced IFN-γ production in the CD4 T cells (Fig. 1B and data not shown). The peptides that yielded > 50 SFC/million CD8-depleted PBMC were subsequently pooled to generate a participant-customized ELISpot selected Gag peptide pool used in some of the latency reactivation experiments. We verified for one participant that the observed IFN-γ production was not a result of contamination with CD8 T cells by comparing two ELISpot assays, one with CD8 depleted PBMC and one with isolated CD8 T cells. The ELISpot assay with the isolated CD8 T cells showed IFN-γ production in response to different peptides compared to the assay with CD8 depleted PBMC, suggesting that the epitopes recognized by class I MHC and class II MHC were different (data not shown). Overall, these data indicated that with the possible exception of the peptides derived from Candida MP65, the peptide pools were able to activate a small fraction of CD4 T cells, presumably in an antigen-specific manner.
Virion-release from latently infected cells after antigenic stimulation measured by real-time RT-qPCR
For nine participants, we stimulated five million CD8-depleted PBMC with each peptide pool in quadruplicate for seven days in the presence of the integrase-inhibitor raltegravir [1 µM]. Raltegravir prevented viral spread and ensured that the measured cell-free, virion-associated (cf)-RNA was released directly from reactivated reservoir cells and not a consequence of propagation of virus in the culture. Depending on the yield of isolated cells from each participant, we stimulated the cells with overlapping peptide pools from Gag, CMV, Candida, CEFT, Pol, Env, and Nef at a concentration of 1 µg/ml per peptide. DMSO was used as negative control and platebound antibodies against CD3 and CD28 were used as a maximum-stimulation control.
We first assessed the activity of the peptide pools with respect to immune activation by measuring the early activation marker CD69. After two days of culture, an aliquot of ~ 50,000 cells from each condition was stained for cell-surface CD69 and CD4 and analyzed by flow cytometry. The DMSO controls showed baseline CD69-positive CD4 T cells ranging from 1–12%, whereas the platebound anti-CD3 and anti-CD28-stimulated cells showed the maximum T cell activation for each participant, which ranged from 45–87% CD69-positive CD4 T cells (Fig. 2A). In general, none of the peptide pools activated a substantial fraction of the CD4 T cells.
After seven days of antigen-stimulation in the presence of raltegravir, we measured released virion-associated RNA to assess reversal of latency. The culture supernatants were cleared of debris and cellular contaminants by low-speed centrifugation, and the virions were isolated by ultracentrifugation through 20% sucrose. Cell-free (cf)RNA was isolated and cf-HIV Gag RNA was measured using real-time RT-qPCR (Fig. 2B). The results were normalized to the cf-RNA yields after maximum stimulation with antibodies to CD3 and CD28 for each participant. One participant was evaluated twice, at times approximately one year apart (Fig. 2B, filled circles and open circles). At the first evaluation, this participant's cells showed relatively high fractional levels of latency-reversal in response to several peptide pools including the Gag and CEFT pools, despite a limited up-regulation of CD69 (Fig. 2A, filled circles). At this time, this participant's cells yielded cf-RNA in response to the C. albicans peptide pool, although this pool did not show activity in the IFN-γ ELISpot assay using cells from other participants (Fig. 1A and data not shown). Also at this time, this participant's cells had the highest baseline expression of cfRNA in the DMSO control. At the later time (open circles in Fig. 2B), this participant's cells showed a different pattern of latency reversal: under 5% of the positive control values in all cases except for the Nef peptide pool, which was over 10% of the positive control value. Cells from the other participants shown in Fig. 2 yielded modest latency reversal after exposure to the peptide pools (less than 15% of the positive control values). The HIV Nef peptide pool appeared the most consistent among multiple participants, although the fraction of latency reversal relative to the positive control was less than 15%.
Viral cell-associated mRNA induction after antigenic stimulation measured by droplet digital (dd)PCR
To confirm and extend the results obtained measuring cf-RNA, we evaluated latency reversal in a second group of participants by measuring cell-associated (ca) HIV mRNA using a ddPCR assay that detects multiply spliced Tat/Rev transcripts [27]. We stimulated nine million CD8-depleted PBMC from six participants with each peptide pool, again in the presence of 1 µM raltegravir to prevent viral spread. Depending on the yields of participant cells, CD8-depleted PBMC were stimulated with peptide pools from CMV, CEFT, Gag, or a participant-customized ELISpot-selected Gag peptide pool at a concentration of 1 µg/ml and plated in three-fold limiting dilutions and six replicates. As before, we used DMSO as the negative control and platebound antibodies to CD3 and CD28 as a maximum-stimulation control. Cellular activation was measured by the up-regulation of CD69 after 48 hours of incubation. Consistent with the results above (Fig. 2A), we observed that the peptide pools did not activate a substantial fraction of the CD8-depleted PBMC (Fig. 3A).
After five days of culture in the presence of 1 µM raltegravir, we collected the cells and isolated caRNA to measure the amounts of multiply spliced Tat/Rev mRNA using the ddPCR assay. Overall, the ddPCR mRNA data showed no consistent induction of the expression of multiply spliced Tat/Rev mRNA by the different peptide pools. With some exceptions the peptide pools reversed latency only modestly when compared to the positive control (Fig. 3B). In cells from one participant (indicated by open diamond), the Gag peptide pool induced Tat/Rev mRNA to 75% of the positive control value, but for this participant the DMSO control value was also unusually high (24%). Notably, in cells from another participant (indicated by "x"), the ELISpot-selected Gag-peptide pool induced Tat/Rev mRNA to 50% of the positive control value, while the DMSO control value in this case was low (under 3%). This participant also had partial latency reversal in response to peptide pools of CMV (18% of the positive control) and the CEFT mixture (29% of the positive control).
Antigen Presentation By Autologous Monocyte-derived Dendritic Cells (dc)
We considered that suboptimal antigen presentation might render the above experiments less sensitive to the potential activity of antigens as LRAs. Therefore, we isolated, differentiated, and matured autologous monocyte-derived DC from two additional participants and used these mature DC as antigen-presenting cells. We also used complete proteins in addition to peptide pools as antigens to better simulate natural antigen processing and presentation (Fig. 4 and Table 1). In these co-culture experiments, like those above, we included the integrase inhibitor raltegravir to block the spread of infection during the seven day incubation (Fig. 4B), but for one participant we also omitted the raltegravir and incubated the cells for 18 days to determine how allowing viral propagation would affect the results (Table 1).
Table 1
RNA measurements of latency reversal after presentation of the indicated antigens by dendritic cells for one of the participants shown in Fig. 4; cells were cultured with or without raltegravir. Values in parentheses are the percent of the α-CD3/α-CD28 control.
Assay | DMSO | α-CD3 + α-CD28 | CMV pp65 | HIV-1 p55 Gag | SIV p55 Gag | CMV pp65 peptide pool1 | CEFT2 peptide pool | HIV-1 Gag peptide pool |
cf3-RNA raltegravir4 | n.d.5 (0) | 24,000 (100) | n.d. (0) | 130 (0.5) | n.d. (0) | 107 (0.4) | n.d. (0) | 8300 (35) |
cf-RNA no raltegravir6 | n.d. (0) | 1.2 × 1010 (100) | n.d. (0) | 1 × 1010 (87) | 370 (3 × 10− 6) | 440 (4 × 10− 6) | n.d. (0) | 33,000 (2.7 × 10− 4) |
ca7-RNA raltegravir4 | n.d. (0) | 151 (100) | 3 (2) | n.d. (0) | n.d. (0) | 14 (9) | 30 (20) | 96 (64) |
ca-RNA no raltegravir6 | n.d. (0) | 1.2 × 109 (100) | n.d. (0) | 7.5 × 106 (0.63) | n.d. (0) | n.d. (0) | n.d. (0) | 50 (4 × 10− 6) |
1all peptide pools were used at 1 µg/ml each peptide |
2CEFT: pool of class II restricted peptides from CMV, EBV, influenza virus, and tetanus toxoid. |
3cf: cell-free, virion-associated RNA in copies/ml |
4raltegravir was used at 1 µM to block viral propagation and the cells were stimulated for 7 days |
5n.d.: not detected |
6cells were stimulated for 18 days in the absence of raltegravir |
7ca: cell-associated, Tat/Rev mRNA in copies/µg RNA |
Consistent with our experiments using CD8-depleted PBMC, the use of autologous DCs to present antigen did not cause substantial T cell activation measured by CD69 up-regulation (Fig. 4A), nor did it yield consistent latency reversal by any peptide or protein antigen (Fig. 4B). For both participants, we used cf-RNA for the initial readout (Fig. 4B). The data of one participant indicated that in the presence of raltegravir, the Gag peptide pool reversed latency to almost 40% of the positive control value, but the other peptide pools and complete proteins were inactive.
For the participant in whom the Gag peptide pool appeared active (square symbol in Fig. 4B), we also omitted raltegravir from parallel cultures: in this condition the intact p55 Gag protein yielded substantial cf-RNA (1 × 1010 copies/ml) reflecting marked viral outgrowth, but neither SIV Gag nor any other antigen did (Table 1). Notably, although the Gag peptide pool did not yield amounts of cf-RNA comparable to maximum-stimulation or the p55 Gag protein, it differed from the other conditions, yielding 3 × 104 copies/ml of cf-RNA compared to the low or undetectable copies for DMSO and the other antigens (Table 1). For this participant (square symbol in Fig. 4B), we also used ddPCR of ca Tat/Rev mRNA as the readout (Table 1). The data indicated that in the presence of raltegravir, the CMV, CEFT, and Gag peptide pools each reversed latency substantially. The activity of the Gag peptide pool (64% of maximum-stimulation) measured by the ca-RNA (Tat/Rev mRNA) readout in the presence of raltegravir was consistent with that measured using the cf-RNA (virion-RNA) readout (35% of maximum-stimulation). In contrast, when raltegravir was omitted, only HIV p55 protein yielded detectable induction of Tat/Rev mRNA: stimulation with p55 yielded 7.5 × 106 copies/ml compared to 1.2 × 109 copies/ml after maximum stimulation. Overall, these results suggested that antigen presentation by DCs did not markedly increase the degree or consistency of latency reversal by these peptides and proteins. The results also suggested that prolonged culture can change the conclusions of these latency reversal experiments relative to short-term, "single-cycle" readout, either amplifying or reducing the signals.
Peptides And Peptide Pools
For antigen stimulation of CD8-depleted PBMC we used pools of 15mer peptides overlapping by 11 amino acids. These peptide pools were specific for: HCMV pp65 (138 peptides), PepMix Candida (MP65) (JPT − 92 peptides), PepMix CEFT-MHC-II-pool (JPT − 14 peptides selected from defined HLA class II-restricted T-cell epitopes of Clostridium tetani, Epstein-Barr virus (HHV-4), Human cytomegalovirus (HHV-5), and Influenza A), HIV consensus B Gag (123 peptides), HIV consensus B Env (211 peptides), HIV consensus B Pol (249 peptides), HIV consensus B Nef (49 peptides). The 123 peptides of HIV consensus B Gag peptide-pool were also tested individually using an IFN-γ ELIspot assay (see below).
For experiments using matured monocyte-derived DC, we used proteins of CMV pp65 (Miltenyi Biotec), HIV-1 Gag p55 (Abcam) and SIV Gag p55 (Proteinsciences) as a control.
The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HCMV pp65 Peptide Pool (cat# 11549) [33–35], HIV-1 Consensus B Gag Peptide Pool (cat# 12425), HIV-1 Consensus B Gag Peptide Set (cat# 8117), HIV-1 Consensus B Pol Peptide Pool (cat# 12438), HIV-1 Consensus Subtype B Env Peptide Pool (cat# 12540), HIV-1 Consensus Subtype B Nef Peptide Pool (cat# 12545), and HIV-1 Consensus Subtype B Nef Peptide Set (cat# 5189).
Generation Of Mature Monocyte Derived Dendritic Cells (dc)
CD14 monocytes were isolated from PBMC using magnetically labelled anti-CD14 antibodies (Stemcell Technologies). Monocytes were cultured for five days in Iscove's Modified Dulbecco's Media (Gibco) complemented with 10% fetal bovine serum, penicillin/streptomycin (Gibco), granulocyte-monocyte colony-stimulating factor (GM-CSF, 1000 IU/ml, R&D Systems), and interleukin4 (IL-4, 1000 IU/ml, R&D Systems). On day five, maturation factors [interferon (IFN)-α (1000 IU/ml, R&D Systems), IFN-γ (1000 U/ml, R&D), IL1β (10 ng/ml, R&D Systems), tumor necrosis factor (TNF)-α (25 ng/ml, R&D Systems), and polyinosinic:polycytidylic acid (20 ng/ml, Sigma-Aldrich)] were added to the cultures for 48 hours, as previously described [36]. DC-maturation was confirmed by staining the cells with antibodies against CD11c, CD80, CD83, CD86, CD143, CD169, CD197 (all antibodies were obtained from Biolegend) and analysis by flow cytometry. Mature DC were loaded with the antigen for two hours, washed in IMDM media and subsequently co-cultured with autologous CD4 T cells in complete IMDM in the presence of the integrase inhibitor raltegravir [1 µM] at a ratio of 1:10 (100,000:1 million) for seven days (or in the absence of raltegrvir for 18 days) in 12-well plates. Culture supernatants and cells were collected on day seven (presence of raltegravir) or day 18 (absence of raltegravir) for quantitation of HIV cf-RNA and ca-RNA.
Interferon-gamma ELISpot assay
Freshly isolated CD8 depleted PBMC were seeded in 96-well polyvinylidene difluoride-backed plates (MAIP S45, Millipore) that had been coated with an anti-IFN-γ MAb 1-D1k (0.5 µg/ml, Mabtech) overnight at 4 °C.
HIV-specific CD4 T-cell responses were quantified, as described previously [37]. Overlapping peptides were added to 1 × 105 CD8 depleted PBMC per well at a final concentration of 12.5 µg/ml. The plates were then incubated for a 14–16 hours at 37°C and 5% CO2. IFN-γ positive were detected using a biotinylated secondary anti-IFN-γ MAb 7-B6-1 (0.5 µg/ml, Mabtech), a streptavidin-alkaline phosphatase conjugate (Mabtech) and TMB substrate (Mabtech).
IFN-γ-producing cells were counted and expressed as spot-forming cells (SFC) per 106 PBMC. Negative controls were always < 10 SFC per 106 cells. As positive controls, we incubated PBMC with phytohemagglutinin or anti-CD3/CD28 coated beads. Wells were considered positive if they had at least 20 SFC/106 PBMC.
Real-time Qrt-pcr
Five million CD8 depleted PBMC were stimulated with the respective peptide pools at a concentration of 1 µg/ml and plated on a 6-well cell culture plate. After seven days of culture in the presence of the integrase inhibitor raltegravir [1 µM] supernatant was collected from each well and centrifuged for five minutes at 300 x g to remove cells and debris. Virions were harvested by centrifugation of cell-free culture supernatants through a 20% sucrose cushion at 23,500 x g for one hour at 4 °C. The pelleted virus particles were resuspended in 200 µl PBS and cell-free (cf)-RNA was isolated using the Viral RNA extraction Kit (Roche) following the manufacturer’s instructions. Cell free viral RNA was eluted in 20 µl and real-time RT-qPCR was performed using Superscript III Platinum One-step qRT-PCR Kit (Invitrogen) and 10 µl cf-RNA, in duplicate. The PCR reactions used Cy5-labeled gag probes together with the corresponding forward and reverse primers. Cycling was performed in an 7900HT Sequence Detection System (Applied Biosystems) with the following parameters: 50 °C for 15 minutes for the RT-step and 95 °C for 2 minutes, followed by 50 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds, and a final step of 50 °C for 10 minutes. A standard curve of pNL4-3 plasmid DNA in duplicate, 10-fold serial dilutions was included on each plate, as well as water negative controls. The limit of detection of this assay was 100 copies/ml gag DNA.
The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Raltegravir from Merck & Company, Inc (Cat # 11680).
Droplet Digital Pcr
Study participant samples
We received up to 300 ml blood whole from 19 study participants with the following inclusion criteria: HIV positive, suppressed viremia (< 50 cps/ml blood) for at least one year, and a CD4 T cell count of at least 500 cells/µl blood.
Isolation of PBMC, CD8 T cell depletion, and stimulation with peptide pools
Peripheral blood mononuclear cells (PBMCs) were isolated from 300 ml whole blood donations using Lymphoprep (Stemcell Technologies) density gradient centrifugation. CD8 T cells were depleted by positive selection using the EasySep™ Human CD8 Positive Selection Kit (Stemcell Technologies) and EasySep magnet (Stemcell Technologies). Depletion of CD8 T cells was confirmed by staining an aliquot of 50,000 CD8 depleted PBMC with anti-CD3-FITC (clone: OKT3), anti-CD4-PE (clone: OKT4) and anti-CD8-APC (clone: HIT8a) fluorescent antibodies and analysis with an Accuri C6 flow cytometer and software (BD Biosciences).
Antigen reactivity of CD8 T cell depleted PBMC to the peptide pools was determined where indicated by IFNγ ELISpot assay (see below), and cells were subsequently stimulated with the various antigen peptide pools (see below) at 1 µg/ml each peptide. After 48 hours of antigen stimulation, an aliquot of ~ 50,000 CD8 depleted PBMC of each condition was stained for anti-CD4-APC (clone: OKT4) and anti-CD69-PE (clone: FN50) and analyzed using the Accuri C6 flow cytometer and software (BD Biosciences). All antibodies for flow cytometry were obtained from BioLegend.