Elsevier

Cytotherapy

Volume 22, Issue 5, May 2020, Pages 276-290
Cytotherapy

FULL-LENGTH ARTICLE
Basic Research
Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

https://doi.org/10.1016/j.jcyt.2020.01.011Get rights and content

Highlights

  • Human iNKT cells robustly expand for cytotherapy via an IL-2/IL-7-based protocol.

  • iNKT cells expanded through this protocol retain regulatory capacity.

  • Post-expansion, CD2/CD3/CD28 stimulation enhances iNKT cell Th2 cytokine secretion.

  • CD2/CD3/CD28 stimulation also enhances expanded iNKT cell anti-tumor cytotoxicity.

Abstract

Background aims

Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells.

Methods

We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vβ11+ iNKT cells.

Results

Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells.

Discussion

These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

Introduction

Natural killer T (NKT) cells are innate regulatory lymphocytes that also participate in tumor immune editing [1], [2], [3], [4], [5], [6], [7], [8], [9]. Unlike human adaptive regulatory T cells, invariant NKT (iNKT) cells have a conserved Vα24-Jα18-Vβ11 TCR [10], are not restricted by classic major histocompatibility complex (MHC) antigens and can be activated by glycolipids such as α-galactosyl ceramide (α-GalCer) presented by CD1d [11]. iNKT cells can regulate both self- and allogeneic tolerance [10]; their lack of canonical MHC restriction allows allo-regulation across histocompatibility barriers, as we first reported in murine transplants [12], [13], [14].

Preventing graft-versus-host disease (GVHD) while maintaining graft-versus-tumor activity remains a “holy grail” of allogeneic hematopoietic cell transplantation. iNKT cells regulate GVHD while maintaining graft-versus-tumor [12], [13], [14], [15], [16]. We defined that iNKT cell-derived T-helper type 2 (Th2) cytokines facilitate this process indirectly by maintaining myeloid populations that expand naturally occurring Foxp3+ Treg [13,14]. This finding has since been confirmed by others [17], [18], [19], [20]. Large-scale clinical studies have also demonstrated strong associations of graft iNKT cell content [21,22] and post–hematopoietic cell transplantation donor iNKT cell reconstitution [23,24] with reduced GVHD [21], relapse [24] and mortality [21,23,24], supporting potential roles for therapeutically expanded human iNKT cells in immunotherapy [4,7,9,[25], [26], [27], [28], [29]]. Notably, early CD4+ iNKT cell expansion, CD161 expression and interleukin (IL)-4 and interferon (IFN)-γ secretion capacity were identified as positive predictive biomarkers [22], [23], [24].

Two significant challenges in iNKT cell immunotherapeutics include (i) the paucity of circulating iNKT cells and (ii) poor understanding of key functions in therapeutically expanded (as opposed to freshly isolated) human iNKT cells. One of the most salient translational aspects of our current approach is the lack of upfront sorting of iNKT cells (allowing peripheral blood mononuclear cell [PBMC]-derived APCs to present α-GalCer to expand iNKT cells through day 7), allowing a robust log-fold expansion and low failure rate of expansions compared with other existing protocols [30], [31], [32], [33], [34], [35], [36]. Murine iNKT cells expand using α-GalCer with IL-2 and IL-15 (two cytokines sharing a receptor β-chain, CD122) [37,38]; IL-15 also expands NK cells [39]. Although CD122 is well documented on both CD4neg and CD4+ iNKT cell subsets, IL-7 receptor-α (CD127) is mainly expressed on the CD4+ subset and drives human iNKT cell differentiation [40,41]. We chose rhIL-7-driven over conventional rhIL-15-based expansions with specific intent to optimize a CD4+ iNKT cell expansion. The rationale include (i) human CD4+ iNKT cells are thymically produced [40], and many therapeutic applications are envisioned in settings of thymic dysfunction (e.g., post-transplant); (ii) human CD4+ iNKT cells display greater propensity for Th2 cytokine secretion [42], [43], [44], which we and subsequently others have shown regulates systemic inflammation in murine models [10,13,16] and supports therapeutic application [45], and (iii) the CD4+ subset is dominant in most cell therapy sources [40,46].

We report highly reproducible and robust expansion of human peripheral blood iNKT cells, which we have functionally characterized post-expansion. Further, we delineate a single mechanism to simultaneously enhance the expression of Th2 cytokines alongside other cytokines (a phenotype associated with iNKT cell alloregulatory potential) and to enhance killing capacity of expanded iNKT cells as compared to unstimulated cells following therapeutic expansion.

Section snippets

iNKT cell expansion

iNKT cell expansion media was RPMI 1640 (Cellgro, Manassas, VA, USA) with 10 mmol/L HEPES (HyClone, Logan, UT, USA), 0.02 mg/mL gentamicin (Life Technologies, Grand Island, NY, USA), and 10% human AB serum (CellGro, Manassas, VA, USA). Peripheral blood apheresis units were obtained from de-identified blood donors at St. Jude Blood Donor Center, under St. Jude Institutional Review Board–exempted protocols. PBMCs were isolated by Ficoll-Paque Plus density-gradient (GE Healthcare, Piscataway, NJ,

iNKT cells from random healthy blood donors can be robustly and reproducibly expanded using IL-2 and IL-7 in tandem

The expansion of iNKT cells was quantified at day 0, 7, 14 and 21 to determine whether iNKT cells could be consistently expanded to clinically therapeutic levels. On the basis of a literature search (Table 1), initial experiments assessed whether greater yields of iNKT cells could be achieved using an IL-2/IL-7 or an IL-2/IL-15 based protocol (N = 4). Total iNKT cell yields at day 7 did not differ between IL-7 and IL-15 (Supplementary Figure 1A), and both methods appear to preferentially expand

Discussion

To address existing obstacles to iNKT cell immunotherapy, we have rigorously optimized expansion of human iNKT cells, phenotyped expanded cells and delineated a specific post-expansion stimulation mechanism to simultaneously enhance multiple therapeutic iNKT cell functions. We have demonstrated through these approaches that the expanded iNKT cells have the capacity to robustly secrete cytokines upon TCR stimulation, as well as to kill hematolymphoid targets. Additionally, at least one subset of

Funding

This work was funded by grants 5P30CA021765-36 (ABP), R12/94-000 (Assisi Foundation) (ABP, KA), the V Scholar Award of the V Foundation for Cancer Research (ABP), the Hyundai Scholar Award (ABP), the American Lebanese Syrian Associated Charities (ABP, KA) and the Batchelor Foundation for Pediatric Research (ABP, KA, AAJH). This research was conducted in collaboration with and using the Biostatistics and Bioinformatics Shared Resource of the Sylvester Comprehensive Cancer Center, University of

Declaration of Competing Interest

The authors have no commercial, proprietary or financial interest in the products or companies described in this article.

Author Contributions

KA and AAJH performed experiments, analyzed results, created the figures and wrote the manuscript. GN, KV, PT, KN, SP and DG performed experiments and analyzed results. XS analyzed results. ABP designed and supervised the research, analyzed results and wrote the manuscript. All authors have approved the final article.

Acknowledgments

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award no. P30CA240139. We thank Jim Houston of the Department of Bone Marrow Transplantation and Cellular Therapy (BMTCT) and the St. Jude Shared FACS Facility for FACS sorting and instrument support; Dr. Mark Exley for early discussions on reagents; and Drs. Helen Heslop, Nelson Chao, Randy Brutkiewicz and John Koreth for their critiques.

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