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CHAPTER 1
The in-betweeners: a short introduction
Adapted from:
Bispecific antibody platforms for cancer immunotherapy.
Lameris R, de Bruin RC, Schneiders FL, van Bergen en Henegouwen PM, Verheul
HM, de Gruijl TD, van der Vliet HJ.
Crit Rev Oncol Hematol. 2014 Dec;92(3):153-65
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T
raditionally, immune responses were categorized into two types: innate
and adaptive. The traditional opinion that innate immunity is nonspecific
and lacks immunologic memory whereas adaptive immunity is char­
acterized by specific antigen recognition and immunologic memory is no longer
accurate or sufficient. Innate-like lymphocytes, also called ‘in-betweeners’,
including γδ T cells and NKT cells, combine conventional adaptive features with
rapid, innate-like responses, and thereby play an important role in initiating and
orchestrating immune responses [1–3].
Invariant Natural killer T (iNKT) cells
Invariant natural killer T (iNKT) cells represent a distinctive population of
lymphocytes characterized by a (semi-)invariant TCR. Unlike conventional
T-cells, iNKT cells recognize (glyco-)lipids presented by non-polymorphic CD1d
molecules. Upon stimulation, iNKT cells rapidly secrete a wide range of cytokines
and induce activation of effector cells (e.g. NK and CTL) in an IFN-γ dependent
manner. [4–6] iNKT cells were shown to contribute to immune surveillance in
early-stage tumors and chemically induced cancers and appear to play a pivotal
role in controlling different forms of cancer, at least in mice. [7,8] Moreover, iNKT
cells from patients with advanced cancer display quantitative and qualitative
defects and circulating numbers correlate with patient survival. [7–10] Reciprocal
interactions between dendritic cells (DC) and iNKT cells can reverse defects in
the iNKT cell population. Indeed, in vitro results indicate rehabilitation of iNKT
cell function after stimulation with monocyte derived DC (moDC) pulsed with
the agonistic CD1d ligand α-galactosylceramide (α-GalCer) and exogenous IL12. [11,12] It was shown that sustained activation of iNKT cells at the tumor
site could be induced after systemic treatment with α-GalCer loaded on soluble
CD1d fused to an anti-tumor scFv. Potent tumor inhibition of aggressive tumor
grafts expressing the targeted antigen was observed in mice. [13,14] Although
less clear than in mice, clinical studies evaluating injection of α-GalCer-pulsed
moDC with or without adoptive transfer of ex vivo expanded iNKT cells have
reported objective tumor regressions in several patients. [15–18] In patients
with recurrent HNSCC, nasal submucosal administration of αGalCer-pulsed
APCs combined with intra-arterial infusion of activated iNKT cells via tumorfeeding arteries produced an objective response in 5 out of 10 patients. [15]
The number of infiltrating iNKT cells in extirpated tumor tissue correlated with
clinical outcome. [18] The precise role of iNKT cells in human cancer treatment
still has to be determined, however, clinical data underscore their role in tumor
immune-surveillance and indicate beneficial effects with low toxicity in cancer
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The In-betweeners: a short introduction
treatment.
Figure 1. iNKT cells are activated by lipid antigens presented in the context of a CD1d
antigen presenting molecule. Activation of Vγ9Vδ2-T cells is initiated by conformational
changes of CD277 upon intracellular phosphoantigen accumulation (isopentenyl
pyrophosphate (IPP)). Adapted from [19].
Vγ9Vδ2T-cells
γδ T-cells, once regarded an evolutionary redundant T-cell subset en route to
extinction, have recently been demonstrated to hold a unique position in the
immune system. They can directly lyse stressed or infected cells, produce a
diversified set of cytokines and chemokines to regulate both immune and nonimmune cells, and can present antigens for αβ T-cell priming. [2] Vγ9Vδ2-T cells
constitute the predominant γδ-T cell subset in human peripheral blood and
account for 1–5% of peripheral blood mononuclear cells (PBMC) of healthy adults.
Vγ9Vδ2-T cells can be activated and expanded by non-peptidic pyrophosphate
antigens (pAg), of which there are both host and microbe-derived counterparts,
typified by isopentenyl pyrophosphate (IPP) and hydroxymethyl-but-2-enylpyrophosphate (HMBPP) respectively. Furthermore, aminobisphosphonate
compounds (e.g. zoledronic acid and pamidronate) sensitize target cells to
Vγ9Vδ2-T cell killing by promoting the intracellular accumulation of endogenous
IPP by inhibiting mevalonate metabolism. [20] Recently, it was reported that
CD277/ butyrophilin (BTN) 3A1 is required for the presentation of pAg to
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Vγ9Vδ2 T cells. [21,22] In stressed/malignant cells pAg production is frequently
upregulated allowing discrimination from normal tissue. Indeed, Vγ9Vδ2Tcells have been shown to be able to recognize and eliminate malignant cells
from multiple tumors types, including multiple myeloma (MM), NHL, prostate-,
renal cell- and colon cancer. Of interest, quantitative and qualitative defects
in the Vγ9Vδ2 T-cell population have been observed in various malignancies
[20] and negatively impact disease-free survival, e.g. in ovarian carcinoma. [23]
Importantly however, these functional Vγ9Vδ2 T-cell defects are reversible. [20]
In patients with various metastatic cancers treatment with zoledronic acid and
IL-2 promoted the differentiation of peripheral blood Vγ9Vδ2 T-cells toward an
effector/memory-like phenotype with augmented numbers correlating with
arrested disease progression. Observed toxicities were minor and limited to
transient flu-like symptoms. [24,25] Adoptive transfer of Vγ9Vδ2 T-cells following
ex vivo expansion by pAg, aminobisphosphonates or mAbs combined with IL2, in patients with various types of metastatic cancer resulted in some clinical
responses. [20] Of interest, Vγ9Vδ2T-cell mediated lysis of hepatic tumor cell
lines could be significantly enhanced by an anti-EpCAM-anti-CD3 BiTE in vitro.
[26] Although clinical data are still scarce, preliminary findings clearly indicate
that exploiting the natural abilities of Vγ9Vδ2 T-cells in cancer immunotherapy is
feasible and carries low toxicity.
Introduction to the chapters
Both iNKT cells and Vγ9Vδ2-T cells have shown great promise in anti-tumor
immune responses and interest in exploiting these lymphocyte subsets for
the induction of potent and enduring antitumor immune responses has grown
rapidly. Moreover, given the current spectacular results of immune checkpoint
inhibitors and the rising interest in bispecific targeting constructs, specific
activation of iNKT and Vγ9Vδ2-T cells has more potential than ever before.
Multiple studies have shown immunological, biochemical and even clinical
responses in patients treated with specific activating ligands, underscoring
their role in anti-tumor immunity. However, results lack consistency and
many challenges remain to be overcome, since both iNKT and Vγ9Vδ2-T
cells can be influenced greatly by the environment in which they are in.
In this dissertation we have studied iNKT and Vγ9Vδ2-T cells in detail and
investigated cross-talk between these subsets, evaluating whether their
reciprocal interactions can interfere or enhance anti-tumor immune responses.
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The In-betweeners: a short introduction
Chapters 1 and 2 provide an introduction to iNKT and Vγ9Vδ2-T cells and review the
clinical experience that has been obtained in the field of cancer immunotherapy.
In Chapter 3 we provide a detailed description of how immature and mature
moDC can be generated from peripheral blood and are optimally exposed to
α-GalCer to allow activation and biased cytokine production in responding iNKT.
Chapter 4 provides an updated analysis of a study in which circulating iNKT
cell numbers were assessed in a group of HNSCC patients before the start
of curative intent radiotherapy. It was shown that patients with HNSCC that
have a severe deficiency of iNKT cells have a strikingly poor clinical outcome.
We studied the effects of iNKT cell activation on Vγ9Vδ2-T cells in Chapter 5,
and found that co-activation of iNKT cells enhanced the IFN-γ production
as well as the cytolytic potential of phosphoantigen-activated Vγ9Vδ2-T
cells and that this effect was mediated via the production of TNF-α.
In Chapter 6 we explored the capacity of Vγ9Vδ2-T cells to act as antigen presenting
cells and present glycolipid antigen to iNKT cells. Vγ9Vδ2-T APC were indeed found
to be able to present α-GalCer to iNKT cells resulting in their activation. However,
glycolipid antigen presentation was shown not to result from the de novo synthesis
of CD1d by Vγ9Vδ2-T cells, but depends on trogocytosis of CD1d-containing
membrane fragments from pAg-expressing cells with which Vγ9Vδ2-T cells interact.
We further discuss the role of Vγ9Vδ2-T cells in cancer immunotherapy
in Chapter 7, focusing on their putative use as a novel APCplatform for potentiation of iNKT cell based cancer immunotherapy.
In Chapter 8 we investigated the effect of NBP on glycolipid Ag presentation
by moDC and found that treatment of moDC with NBP during DC maturation
resulted in a striking inhibition of iNKT cell activation. This inhibitory effect was
found to be caused by a reduction in moDC apolipoprotein E (apoE, a known
facilitator of trans-membrane transport of exogenously derived glycolipids)
production and could be relieved by supplementing apoE in the culture medium.
Chapter 9 describes the studied effects of intravenous administration of
NBP and statins on circulating Vγ9Vδ2-T cells in advanced cancer patients.
We found that administration of NBP resulted in the rapid activation of
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circulating Vγ9Vδ2-T cells, with circulating numbers dropping immediately
after infusion and recovering within the next week. Pretreatment of
patients with statins did not significantly reduce the observed activation
nor did it prevent the occurrence of an acute phase response (APR).
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