FULL-LENGTH ARTICLEBasic ResearchExpansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity
Graphical abstract
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.
References (78)
Anti-tumor potential of type-I NKT cells against CD1d-positive and CD1d-negative tumors in humans
Clinical immunology (Orlando, Fla.)
(2011)- et al.
Natural killer T (NKT) cells and their role in antitumor immunity
Critical reviews in oncology/hematology
(2002) - et al.
Invariant natural killer T cells and immunotherapy of cancer
Clinical immunology (Orlando, Fla.)
(2008) - et al.
Host natural killer T cells induce an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft-versus-host disease
Blood
(2009) - et al.
TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors
Blood
(2009) - et al.
CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells
Blood
(2014) - et al.
Low doses of natural killer T cells provide protection from acute graft-versus-host disease via an IL-4-dependent mechanism
Blood
(2011) - et al.
Modulation of acute graft-versus-host disease and chimerism after adoptive transfer of in vitro-expanded invariant Valpha14 natural killer T cells
Immunology letters
(2006) - et al.
Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation
Blood
(2012) - et al.
Early posttransplantation donor-derived invariant natural killer T-cell recovery predicts the occurrence of acute graft-versus-host disease and overall survival
Blood
(2012)
Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy
Clinical immunology (Orlando, Fla.)
Synergistic effect of KRN7000 with interleukin-15, -7, and -2 on the expansion of human V alpha 24+V beta 11+ T cells in vitro
Hum Immunol
Potent expansion of human natural killer T cells using alpha-galactosylceramide (KRN7000)-loaded monocyte-derived dendritic cells, cultured in the presence of IL-7 and IL-15
J Immunol Methods
In vitro expanded human invariant natural killer T-cells promote functional activity of natural killer cells
Clinical immunology (Orlando, Fla.)
The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation
Blood
Rapid ex vivo expansion of highly enriched human invariant natural killer T cells via single antigenic stimulation for cell therapy to prevent graft-versus-host disease
Cytotherapy
Expansion of human Valpha24+ NKT cells by repeated stimulation with KRN7000
J Immunol Methods
Distinct homeostatic requirements of CD4+ and CD4- subsets of Valpha24-invariant natural killer T cells in humans
Blood
Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity
Blood
Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality
Blood
An integrated genotyping approach for HLA and other complex genetic systems
Hum Immunol
Human natural killer T cells acquire a memory-activated phenotype before birth
Blood
IL-7 promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to support T-cell survival
Blood
Distinct subsets of human Valpha24-invariant NKT cells: cytokine responses and chemokine receptor expression
Trends in immunology
pro-apoptotic, antiviral and antitumor functions
Curr Opin Immunol
A critical role for natural killer T cells in immunosurveillance of methylcholanthrene-induced sarcomas
J Exp Med
Combined natural killer T-cell based immunotherapy eradicates established tumors in mice
Cancer research
Janus-like role of regulatory iNKT cells in autoimmune disease and tumour immunity
Nat Rev Immunol
Sequential activation of NKT cells and NK cells provides effective innate immunotherapy of cancer
J Exp Med
Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2
J Exp Med
Valpha24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages
J Clin Invest
The Biology of NKT Cells
Annual Review of Immunology
Recognition of CD1d-restricted antigens by natural killer T cells
Nat Rev Immunol
Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation
J Immunol
Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation
J Immunol
Protective conditioning for acute graft-versus-host disease
The New England journal of medicine
Adoptive therapy by transfusing expanded donor murine natural killer T cells can suppress acute graft-versus-host disease in allogeneic bone marrow transplantation
Transfusion
Pre-transplant donor CD4- invariant NKT cell expansion capacity predicts the occurrence of acute graft-versus-host disease
Leukemia
Invariant NKT cell reconstitution in pediatric leukemia patients given HLA-haploidentical stem cell transplantation defines distinct CD4+ and CD4- subset dynamics and correlates with remission state
J Immunol
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