Background: Blockade of the PD-1/PD-L1 pathways using monoclonal antibodies (mAb) to PD-1 has been reported to activate HIV expression from latently infected CD4 T cells ex vivo. To further evaluate this approach, we tested the ability of the human anti-PD-L1 mAb BMS-936559 to activate virion production from mononuclear cells obtained from patients on suppressive ART.
Methods: PBMC, total (t) CD4, and resting (r) CD4 cells were purified by negative selection of large-volume blood draws from HIV-infected donors. Freshly isolated PBMCs were cryopreserved and immunophenotyped for PD-1/PD-L1 expression. The remaining cells were incubated (1 million cells/well in triplicate) for 1 week with 20, 5, or 1.25µg/ml anti-PD-L1 mAb, with 20µg/ml isotype control [Zymogen DT-1D12g-4P (hIgG4)], or with anti-CD3/CD28-coated microbeads plus either anti-PD-L1 mAb or isotype control. On Day 8, cells were assessed for viability and supernatants tested for HIV RNA using the Roche Taqman v.2.0. A virologic response was defined as a >3-fold increase in HIV RNA over isotype control. Donors whose cells responded initially to anti-PD-L1 mAb were redrawn and tested again.
Results: PBMC, tCD4, and rCD4 cells purified from ten long-term (mean 8 years) suppressed donors. Cell viability was not reduced by treatment with anti-PD-L1 mAb. 9 of 10 donors responded to anti-CD3/CD28 in all cell types (mean fold-increases of 742, 1353, and 272 for PBMC, tCD4 and rCD4, respectively). Anti-PD-L1 mAb did not enhance responses to anti-CD3/CD28. PBMC from 2 of 10 donors (donor 3: 61-fold; donor 4: 7-fold) showed an initial response to anti-PD-L1 mAB that was not reproduced upon repeat blood draw. tCD4 cells from 2 of 10 donors (donor 2: 583-fold; donor 6: 84-fold) initially responded to anti-PD-L1. However, cells from donor 2 did not respond after a repeat draw. Cells from donor 6 responded on the first repeat draw but not the second. rCD4 from 0 of 10 donors responded to anti-PD-L1. PD-1/PD-L1 expression on CD4 and CD8T cells was evident on the day of cell isolation in all donors, but expression levels did not differ between responders, non-responders, and a healthy control.
Conclusions: Despite detectable PD-1/PD-L1 expression, increased HIV production from PBMC, total CD4 T cells or resting CD4 T cells after treatment with anti-PD-L1 antibody was infrequent and not reproduced longitudinally. Alternate strategies will be needed to activate proviral expression from latently infected CD4 T cells.