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Another Step Forward for Pluripotent Stem Cell-Derived Treatment for HIV

Review of “Expression of Chimeric Receptor CD4zeta by Natural Killer Cells Derived from Human Pluripotent Stem Cells Improves In Vitro Activity but Does Not Enhance Suppression of HIV Infection In Vivo” from Stem Cells by Stuart P. Atkinson

Highly active antiretroviral therapy (HAART) has been highly successful in decreasing morbidity and mortality of HIV/AIDS, although other synergistic strategies are required to completely remove any cellular reservoirs of latent virus. One interesting strategy is the use of engineered immune cells which are designed to specifically target and destroy HIV. Previous research from the laboratory of Dan S. Kaufman at the Stem Cell Institute, University of Minnesota, USA has demonstrated that large numbers of Natural Killer (NK) immune cells with potent anti-HIV activity [1] can be derived from hESCs and iPSCs [2, 3], although they lack antigen specific recognition receptors [4, 5]. They now show that engineering hESC- and iPSC-derived NK cells with a specific HIV CD4ζ chimeric receptor enhances the suppression of HIV replication in vitro and study these cells in a mouse xenograft model [6].

The group first transduced a CD4ζ construct (CD4 extracellular and transmembrane domains fused with cytoplasmic domain of TCR CD3ζ chain linked to GFP – See Figure) into hESCs and hiPSCs using the Sleeping Beauty (SB) transposon system and positive clones selected for by antibiotic resistance, giving clones with strong CD4ζ-GFP expression (37). Positive clones were then differentiated using defined serum-free conditions outlined in Ng, et al [7], which overall gave rise to a CD45+CD56+CD117−CD94+ population of cytotoxic NK cells [8] expressing the CD4 construct and other surface markers in a similar manner to peripheral blood NK cells. Importantly, stimulation of the crosslinking of CD4ζ chimeric receptors led to tyrosine phosphorylation as would be expected for a fully functional chimeric receptor.

The researchers next assessed the function of the NK cells to inhibit HIV replication using the CEM-GFP T-cell line which when infected with HIV-1 expresses GFP. Excitingly, the CD4ζ modified hESC- and iPSC-NK cells inhibited HIV replication by up to 90%, a significantly higher level than unmodified hESC- and iPSC-NK cells. Furthermore, HIV infection of CD4 cells, measured by intracellular staining for the viral gag protein (p24), was also significantly blocked when using CD4ζ-modified hESC- and iPSC-NK cells compared to unmodified hESC- and iPSC-NK cells. NK cell function, as measured by expression of CD107a, was also significantly higher in CD4ζ-modified hESC- and iPSC-NK cells compared to unmodified hESC- and iPSC-NK cells

In vivo analysis used the human PBL-NSG mouse model of HIV-1 infection [9]. One day after infection of mice with HIV, CD4ζ-hESC-, hESC-, or PB-NK cells were injected and blood assessed at 6, 9, and 12 days post-NK cell treatment. CD4ζ-hESC-NKs cells were observed in peripheral blood early at day 6 at which point CD4 T-cell counts rose compared in NK treated mice compared to control, which gained significance by day 12, demonstrating suppression of HIV activity.  However, no differences between the use of CD4ζ-modificed and unmodified cells were observed for HIV infection levels and CD4 T-cell levels, although the data does indicate that both NK cells populations can inhibit HIV replication and prevent CD4+ T-cell depletion in vivo. Evaluation of viral RNA in mouse plasma and proviral DNA in different tissues at day 12 of NK treatment found significantly lower RNA and proviral DNA in the peripheral blood of mice receiving NK cells. Proviral DNA from peritoneal washes and spleens also demonstrated lower proviral DNA levels in NK-treated mice demonstrating a significant suppression of HIV replication in several tissues/organs in the hPBL NSG mouse model.  Finally, iPSC-NK cells were assessed using the same model, which found that CD4ζ-iPSC-, and iPSC-NK cells were able to maintain CD4+ T-cell number and significantly suppress viral load in the peripheral blood compared to controls.

Overall, the engineered hESC- and iPSC-derived natural killer cells demonstrated greater efficacy in suppression HIV replication that unmodified cells in vitro and showed utility in in vivo analyse, although did not improve on the non-engineered NK cells effect. This suggests that further engineering efforts may be effective to radically inhibit HIV replication and therefore provide a cure to this disease. This may require further investigation into the innate immune responses during viral infection, and the application of this knowledge using hESC/iPSC-based technologies. This could include optimising homing and tracking through enforced expression of specific receptors [10, 11], which could also potentially help to clear viral reservoirs, which cause major problems related to relapse after therapy.

Discussion Questions

  • Will long term treatment with NK cells abolish HIV infection?
  • What other strategies can we adapt to target HIV?
  • Is this strategy a viable option for many HIV sufferers?

References ▾

  1. Ni Z, Knorr DA, Clouser CL, et al. Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms. J Virol 2011;85:43-50.
  2. Knorr DA, Ni Z, Hermanson D, et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl Med 2013;2:274-283.
  3. Knorr DA and Kaufman DS Pluripotent stem cell-derived natural killer cells for cancer therapy. Transl Res 2010;156:147-154.
  4. Caligiuri MA Human natural killer cells. Blood 2008;112:461-469.
  5. Iannello A, Debbeche O, Samarani S, et al. Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS. J Leukoc Biol 2008;84:1-26.
  6. Ni Z, Knorr DA, Bendzick L, et al. Expression of Chimeric Receptor CD4zeta by Natural Killer Cells Derived from Human Pluripotent Stem Cells Improves In Vitro Activity but Does Not Enhance Suppression of HIV Infection In Vivo. Stem Cells 2014;32:1021-1031.
  7. Ng ES, Davis R, Stanley EG, et al. A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protoc 2008;3:768-776.
  8. Woll PS, Grzywacz B, Tian X, et al. Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood 2009;113:6094-6101.
  9. Mosier DE, Gulizia RJ, Baird SM, et al. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 1988;335:256-259.
  10. Beziat V, Traherne JA, Liu LL, et al. Influence of KIR gene copy number on natural killer cell education. Blood 2013;121:4703-4707.
  11. Beziat V, Liu LL, Malmberg JA, et al. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood 2013;121:2678-2688.