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CHAPTER 7
Vγ9Vδ2-T cells as antigen presenting cells for iNKT cell
based cancer immunotherapy
CD1d-restricted invariant natural killer T cells (iNKT) constitute an important
immunoregulatory T-cell subset involved in the induction of antitumor immune
responses. Here, we provide a view on the recent observation that Vγ9Vδ2-T cells,
through trogocytosis of CD1d-containing membrane fragments, have the capacity to
act as antigen presenting cells for iNKT.
Inge M. Werter, Famke L. Schneiders, Emmanuel Scotet, Henk M.W. Verheul, Tanja
D. de Gruijl, Hans J. van der Vliet.
OncoImmunology. 2014 Dec; 3(9): e955343.
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CHAPTER 7
I
nvariant natural killer T cells (iNKT) represent an important immunoregulatory
T-cell subset. In humans, iNKT cells are mostly CD4C, or CD4¡CD8¡ double
negative (DN). CD4C iNKT produce both T helper type 1 (Th1) cytokines, such
as granulocyte macrophage colony stimulating factor (GM-CSF), interferon g
(IFNγ) and tumor necrosis factor a (TNFα) and T helper type 2 (Th2) cytokines
(e.g., the interleukins IL-4 and IL13), whereas DN iNKT primarily produce Th1
cytokines. Depending on their predominant cytokine profile, iNKT can be
important in autoimmunity, transplantation, anti-viral, anti-microbial, and
anti-tumor immune responses. (1) Through development of long-lasting Th1biased pro-inflammatory immune responses, iNKT were shown to play a
role in various tumor-metastasis models (e.g., lymphoma, melanoma, colon
carcinoma) (reviewed in ref 1 and 2). Furthermore, deficient iNKT numbers
predict poor clinical outcome in various tumor types, including colon carcinoma,
neuroblastoma, and head and neck squamous cell carcinoma. (3-5)
iNKT can be activated by the synthetic glycolipid ligand a-galactosylceramide
(α-GalCer) presented by CD1d-expressing antigen presenting cells (APC) and
various strategies to exploit the antitumor characteristics of iNKT have been
employed in immunotherapy clinical trials for advanced cancer. In a Phase I
trial, intravenous (i.v.) administration of α-GalCer alone was tested and found
to consistently induce the rapid depletion of circulating iNKT, with signs of
immunological activation in a subset of patients with reasonable pretreatment
iNKT numbers. No clinical responses were observed (reviewed in ref. 1). A second
approach studied i.v. administration of immature and mature α-GalCer-pulsed
monocyte derived dendritic cells (moDC) as APC. This approach appeared to
be more potent as compared to i.v. α-GalCer alone, resulting in a transient
immune activation (i.e., inducing changes in iNKT-, NK and T-cell numbers
and stimulating higher levels of IFNγ) in patients treated with α-GalCer-pulsed
immature moDC. Immune activation was further increased and prolonged in
patients treated with α-GalCer-pulsed mature moDC with a >100 fold expansion
of iNKT. In some of these patients these immune features correlated with signs
of antitumor activity, including decreases in the presence of tumor markers
and tumor necrosis (reviewed in ref. 1). Other related approaches studied i.v.
or intranasal administration of α-GalCer-pulsed APC fractions, generated from
peripheral blood mononuclear cells (PBMC) using GM-CSF and IL-2, either alone
or in combination with intra-arterially administered autologous iNKT. These
approaches also efficaciously increased iNKT and NK cell responses, as well as
antitumor activity, in several patients. Of note, major side effects have not been
observed in the trials performed to date (reviewed in ref. 1). As certain known
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Vγ9Vδ2-T cells as antigen presenting cells for iNKT cell based cancer immunotherapy
caveats restrict the utility of moDC (e.g., relatively long in vitro culture period
and functional heterogeneity) or the probably even more heterogeneous and
poorly defined APC fractions generated from GM-CSF and IL-2 their therapeutic
predictability appears to be limited. Therefore, efforts to develop more potent
platforms for iNKT-based cancer immunotherapy are required. (1,6)
Vγ9Vδ2-T cells, the predominant γδ-T-cell subset of PBMC, have been
reported to exhibit characteristics of professional APCs, including antigen
processing and presentation, as well as the provision of costimulatory signals.
(7) Vγ9Vδ2-T are directly activated by natural (isopentenyl pyrophosphate (IPP))
and synthetic (bromohydrin pyrophosphate (BrHPP)), phosphoantigens (pAgs)
presented in the context of CD277/BTN3A1. (8) Activated Vg9Vd2T cells secrete
a variety of chemokines and cytokines, thereby contributing to anti-inflammatory
processes and cytolysis. (9) We thus reasoned that Vγ9Vδ2-T could be more
suitable for clinical applications than moDCs, as they are comparatively more
frequent within the peripheral blood (1–5% of PBMCs in healthy adults), mature
more rapidly into professional APCs (<24 hours), home to the lymph node more
efficiently and exhibit a more uniform and consistent proinflammatory status.
(6,7) To address this, we set out to study the glycolipid Ag presentation capacity
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of Vγ9Vδ2-T cells activated using different modes, including pAg. In contrast to
prior reports for MHC Class I and MHC Class II mediated Ag presentation,
α-GalCer-pulsed Vγ9Vδ2-T cells were not able to directly activate iNKT. (10)
Phenotypical analyses of α-GalCer activated Vγ9Vδ2-T cells confirmed the
upregulation of costimulatory markers, but showed no expression of CD1d
molecules, which is required for the presentation of glycolipid Ag to iNKT.
Importantly, when Vγ9Vδ2-T cells were activated using pAg-expressing cells that
co-expressed relatively high levels of CD1d, we found that the so activated
Vγ9Vδ2-T cells subsequently expressed CD1d and had obtained the capacity to
present α-GalCer to iNKT. Additional experiments demonstrated that this
acquisition of CD1d resulted from trogocytosis of CD1d-containing membrane
fragments from pAg-expressing cells by Vγ9Vδ2-T. (10)
Although we found that α-GalCer loaded moDC clearly outperformed
Vγ9Vδ2-T cells functioning as APCs on a per cell basis, Vγ9Vδ2-T cells have the
advantage of a quicker maturation into APCs as well as that of quantitative
superiority, being more numerous as compared to moDC precursors.
Furthermore, culture supernatants of iNKT activated via Vγ9Vδ2-T cells as APCs
were more biased toward a Th1 type cytokine profile –with a higher IFNγ/IL-4
ratio– than those iNKT cells stimulated by moDCs. However, expression of
CD1d on Vγ9Vδ2-T with APC functional capacity requires trogocytosis of CD1dcontaining membrane fragments from pAg-expressing donor cells, thereby
causing inherent limitations in level, membrane distribution and duration of
CD1d expression on Vγ9Vδ2-T APCs, thus clearly limiting their practical and
widespread use. In vitro transfection of Vγ9Vδ2-T with CD1d could overcome
this hurdle and thereby pave the way for future clinical assessment of the
potential superiority of CD1d+ Vγ9Vδ2-T cells over moDCs with respect to iNKTbased immunotherapeutic approaches (Fig. 1A). Finally, Vγ9Vδ2-T cells are well
established antitumor effector cells with the capacity to secrete proinflammatory
cytokines and cytolytic molecules upon recognition of pAg expressing tumor
cells. (6,7,9,10) It is therefore conceivable that Vγ9Vδ2-T combine their lytic
“kiss of death” to kill pAg-expressing tumor cells with tumor-cell trogocytosis,
thus acting as cytotoxic effectors and APCs in the tumor microenvironment and
propagating the presentation of tumor-associated Ags to the immune system
(Fig. 1B).
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Vγ9Vδ2-T cells as antigen presenting cells for iNKT cell based cancer immunotherapy
Acknowledgements
This work is supported by grant nr 90700309 from The Netherlands Organization
for Health Research and Development (ZonMw) and grant VU 2010– 4728 from
the Dutch Cancer Society (KWF).
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