Fig 2: FasL AM treatment of MPBCs decreases B cells, myeloid cells and activated T cells populations, reducing the antigen presentation potential and T cell activation potential.MPBCs graft characterization following 100 ng/ml FasL AM treatment: (a) Percentage of B-lymphocytes (CD19+), (b) Myeloid cells (CD33+) and (c) Activated T cells (CD25+) per total CD45+ population. The percentage of HLA-DRhigh expressing on B-lymphocytes (d) and on myeloid cells (e) as marker for antigen presentation potential per total CD19+ or CD33+ population. N = 7, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; Paired T-test. f Concentration of IFN-γ secreted to the medium, measured 24 h post T cell specific and normalized per seeded CD3+ cell number in culture. N = 8, *P ≤ 0.05; Paired T-test.

Fig 3: Description and characterization of the pMorpheus-V5 viral reporter vector(A) Representation of the HIV reporter viral vector pMorpheus-V5 (pLAI2-V5-NGFR HSA-mCherry-IRES-Nef).(B) Gating strategy to identify cells latently or productively infected by pMorpheus-V5 using flow cytometry or CyTOF is depicted.(C) Latently (V5+, NGFR+) or productively (HSAPOS, mCherryPOS, V5POS, NGFRPOS, p24POS) infected, IL-2 + α-CD3/CD28-stimulated CD4+ T cells were identified using flow cytometry after infection with pMorpheus-V5. The data show one representative example of four independent experiments using cells from different healthy donors. The percentage of CD4+ T cells in each quadrant is indicated.(D) Nef down-regulates CD4 levels during pMorpheus-V5 infection. CD4+ T cells from two different donors were stimulated with IL-2 + α-CD3/CD28 and infected with pMorpheus-V5. CyTOF was performed 5 days post-infection (dpi). Histogram plots show the CD4 expression for uninfected cells (shaded light gray), p24POS cells (orange), and p24NEG cells (light blue). The data were analyzed using FlowJo.(E) Cells were stimulated with IL-2 + α-CD3/CD28 prior to infection with p-Morpheus-V5. Histogram analysis of CD4 expression levels of NGFR (magenta), V5 (blue), mCherry (red), or HSA (green) positive cells. The data were analyzed using FlowJo. Data from two independent donors.

Fig 4: Quantification of proviruses and multiply spliced HIV transcripts in FACS-sorted CD4+ T cells(A) Representation of the experimental design to analyze provirus integration or multiply spliced transcripts in productive or latently infected cells from three different healthy donors. CD4+ T cells were activated with IL-2 and α-CD3/CD28 antibody-coated beads for 3 days prior to infection with pMorpheus-V5. CD4+ T cells were infected with pMorpheus-V5 in the presence and absence of raltegravir. Latently (orange), productively (green), and the reporter negative cell populations were sorted using FACS 5 dpi with pMorpheus-V5. RNA and DNA were extracted from the same samples.(B) Gag p24 concentration of culture supernatants was measured using ELISA at 5 dpi. Bar graph shows the production of p24 in pMorpheus-V5 or mock-infected cells in the presence and absence of raltegravir (RAL). Each donor is identified by a specific symbol. The average of three donors is shown (±SD) (∗∗∗∗p < 0.0001, unpaired t test).(C) HIV integration was measured using a two-step quantitative Alu-PCR assay using total DNA from each sorted cell population (latent infection, yellow; productive infection, green; reporter expression-negative cells, gray). The bar graph depicts relative amounts of integrated proviruses. J-Lat 6.3 cells were used as a positive control. The mean ± SD of three independent experiments/donors are shown. Sorted cells from the same donor (N = 3) are identified by the same symbol.(D) Multiply spliced (MS) HIV transcripts were quantified using semi-nested real-time qPCR. RNA was extracted from the same samples as in (C). RNA extracted from stimulated J-Lat 6.3 cells was used as a positive control. The bar graph depicts the mean ± SD from three independent experiments/donors. Sorted cells from the same healthy donor (N = 3) are identified by the same symbol.

Fig 5: Identification of latently and productively infected CD4+ T cells(A) Experimental design to analyze CD4+ T cells infected with pMorpheus-V5. CD4+ T cells from four healthy donors were stimulated with IL-2 or a combination of IL-2 with α-CD3/CD28 antibody-coated beads for 3 days prior to infection with pMorpheus-V5. The frequency of latently and productively infected cells was determined 5 dpi using flow cytometry.(B) Frequency of latently (V5POS, NGFRPOS; yellow) and productively (mCherryPOS, V5POS, NGFRPOS, p24POS; green) infected CD4+ T cells stimulated with IL-2 alone or IL-2/α-CD3/CD28. Cells were analyzed using flow cytometry 5 dpi. Each of the four healthy donors is identified by symbol. Error bars represent the average ± SD, and statistical significance was established using unpaired t test.(C) Relative proportion of pMorpheus-V5 latently (yellow) or productively (green) infected CD4+ T cells stimulated with IL-2 is shown. The data represent the average of the four healthy donors. Average LTR-silent 58% ± 4% versus average LTR-active 42% ± 4% (p = 0.0014, unpaired t test).(D) Relative proportion of pMorpheus-V5 latently (yellow) or productively (green) infected CD4+ T cells activated with α-CD3/CD28. The data represent the average of the four individual healthy donors. Average LTR-silent 52% ± 8% versus average LTR-active 48% ± 8% (p = 0.61, t test).