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Ex vivo purification and characterization of human invariant Natural Killer T cells.

ABSTRACT: Natural Killer T (NKT) cells have gained widespread attention among immunologists because of their distinct ability to regulate anti-tumor responses and to influence the outcome of infections or autoimmunity. Type I (also called invariant) NKT cells (iNKT) are best characterized mainly because of the availability of lipid antigen-loaded CD1d-tetramer detection reagents. Human iNKT cells present important phenotypic differences relative to their murine counterpart, restricting the extrapolation of findings from experimental murine models to human health and disease states. Particularly, the ontogeny and early life phenotype of iNKT cells largely differ between human and mice, indicating divergent functional properties between species. The high therapeutic potential offered by manipulation of iNKT cells in disease warrants a better understanding of human iNKT cell biology. Here, we discuss characteristics of human iNKT cells and present an efficient and rapid method for their ex vivo purification and characterization.

SUBMITTER: Sharma AA

PROVIDER: S-EPMC4619040 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Ex vivo purification and characterization of human invariant Natural Killer T cells.

Sharma Ashish Arunkumar AA Chew Leon L Ladd Mihoko M Jen Roger R Lavoie Pascal M PM

Journal of immunological methods 20110811 1-2

Natural Killer T (NKT) cells have gained widespread attention among immunologists because of their distinct ability to regulate anti-tumor responses and to influence the outcome of infections or autoimmunity. Type I (also called invariant) NKT cells (iNKT) are best characterized mainly because of the availability of lipid antigen-loaded CD1d-tetramer detection reagents. Human iNKT cells present important phenotypic differences relative to their murine counterpart, restricting the extrapolation o ...[more]

PMID: 21854781

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Project description:NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

Project description:Introduction:Ex vivo-expanded natural killer (NK) cells are a potential candidate for cancer immunotherapy based on high cytotoxicity against malignant tumor cells. However, a limited understanding of the migration of activated NK cells toward solid tumors is a critical dilemma in the development of effective and adoptive NK cell-based immunotherapy. Methods:Ex vivo-expanded NK cells from healthy donors were stained with near-infrared fluorophores at different concentrations. NK cell proliferation and cytotoxicity were assessed using a WST-8 assay, while the expression levels of surface molecules were analyzed by flow cytometry. To investigate the biodistribution of NK cells in both normal and tumor-bearing NSG mice, NK cells labeled with ESNF13 were subjected to NIR fluorescence imaging using the Mini-FLARE imaging system. Finally, mice were sacrificed and histopathological tests were performed in resected organs. Results:The signal intensity of ESNF-stained NK cells was long-lasting at 72 h using concentrations as low as 0.04 µM. At a low dose range, ESNF13 did not affect NK cell purity, expression levels of surface receptors, or cytotoxic functions against MDA-MB-231 cancer cells. Ex vivo-expanded NK cells labeled with ESNF13 had a 4-h biodistribution in non-tumor-bearing NSG mice that mainly localized to the lungs immediately after injection and then fully migrated to the kidney after 4 h. In an MDA-MB-231 tumor-bearing NSG mice with extensive metastasis in both lungs, the fluorescence signal was dominant in both lungs and steady at 1, 2, and 4 h post-injection. In a early phase of tumor progression, administered NK cell migrated to the lungs and tumor sites within 30 min post-injection, the signal dominated the tumor site after 1 h, and remained steady at 4 h. Conclusion:Optical imaging with NIR fluorophore ESNF13 is a highly sensitive, applicable, and inexpensive method for the real-time tracking of ex vivo-expanded NK cells both in vitro and in vivo. Administered NK cells had different patterns of NK cell distribution and accumulation to the tumor site according to tumor progression in triple-negative breast cancer xenograft models.

Dataset Information

Ex vivo purification and characterization of human invariant Natural Killer T cells.

Publications

Ex vivo purification and characterization of human invariant Natural Killer T cells.

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