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Invariant natural killer T cells and immunotherapy of cancer
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Invariant natural killer T cells and immunotherapy of cancer



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  • 1. Clinical Immunology (2008) 129, 182–194 a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / y c l i m SHORT ANALYTICAL REVIEW Invariant natural killer T cells and immunotherapy of cancer Johan W. Molling 1 , María Moreno, Hans J.J. van der Vliet, Alfons J.M. van den Eertwegh, Rik J. Scheper, B. Mary E. von Blomberg, Hetty J. Bontkes ⁎ Cancer Center Amsterdam, VUMC Institute for Cancer and Immunology (V-ICI), Division of Immune Therapy, Department of Medical Oncology, The Netherlands Cancer Center Amsterdam, VUMC Institute for Cancer and Immunology (V-ICI), Division of Immune Therapy, Department of Pathology, The Netherlands Received 21 November 2007; accepted with revision 29 July 2008 Available online 9 September 2008 KEYWORDS Abstract Invariant CD1d restricted natural killer T (iNKT) cells are regulatory cells that express iNKT cell; a canonical TCR-Vα-chain (Vα24.Jα18 in humans and Vα14.Jα18 in mice) which recognizes Cancer; glycolipid antigens presented by the monomorphic CD1d molecule. They can secrete a wide Prognosis; variety of both pro-inflammatory and anti-inflammatory cytokines very swiftly upon their Immune surveillance; activation. Evidence for the significance of iNKT cells in human cancer has been ambiguous. Still, Immunotherapy the (pre-)clinical findings reviewed here, provide evidence for a distinct contribution of iNKTcells to natural anti-tumor immune responses in humans. Furthermore, clinical phase I studies that are discussed here have revealed that the infusion of cancer patients with ligand-loaded dendritic cells or cultured iNKT cells is well tolerated. We thus underscore the potential of iNKT cell based immunotherapy in conjunction with established modalities such as surgery and radiotherapy, as adjuvant therapy against carcinomas. © 2008 Elsevier Inc. All rights reserved. Introduction The complex relationship between the immune system and human cancer has been thoroughly investigated for decades. An effective anti-tumor immune response appears to be ⁎ Corresponding author. Vrije Universiteit Medical Center, Depart- important to eradicate malignant cells from the body. This is ment of Hematology, De Boelelaan 1117, 1081 HV Amsterdam, The illustrated by the observations in immunocompromised Netherlands. Fax: +31204442601. patients, who have a higher incidence of tumors (reviewed E-mail address: hj.bontkes@vumc.nl (H.J. Bontkes). 1 Current affiliation of Johan W. Molling: Radboud University in [1]). The microenvironment of progressing tumors that Nijmegen, NCMLS (Nijmegen Centre for Molecular Life Sciences), have evaded eradication by effector cells shares some Department of Tumor Immunology, NCMLS/278 TIL, 6500HB Nijmegen, features with sites of chronic inflammation. It is characte- The Netherlands. rized by the presence of angiogenic and tumor growth 1521-6616/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2008.07.025
  • 2. Invariant natural killer T cells and immunotherapy of cancer 183 factors which promote tumor growth and the induction of Studies combining intravenous (i.v.) free αGalCer treat- immunosuppressive cells (reviewed in [2–4]). An immune ment with protein vaccination demonstrated that αGalCer response mediated by effector cells without the develop- acts as an adjuvant for the induction of antigen specific CD4+ ment of a state of chronic inflammation is vital for a and CD8+ T cell mediated immunity [26,27]. Furthermore, successful anti-tumor immune response. Here we provide i.v. injection of dying hematologic tumor cells together with evidence that a relatively recently discovered T cell subset αGalCer induced long lasting protective immunity, depend- with immune controlling capacity holds promise in this ing on conventional CD4+ and CD8+ T cells. In this model respect. αGalCer-activated iNKT cells enhanced the maturation of Invariant CD1d restricted natural killer T (iNKT) cells can DC, that subsequently were more efficient in (cross-) express natural killer (NK) receptors and express a canonical priming CD4+ and CD8+ T cells [28]. T cell receptor (TCR)-Vα-chain (Vα24.Jα18 in humans, These intriguing findings from pre-clinical studies preferentially paired with Vβ11; Vα14.Jα18 in mice, paired prompted several groups to study iNKT cell numbers and with Vβ2, Vβ7 or Vβ8.2) recognizing glycolipid antigens function in cancer patients and to perform clinical phase I presented by the monomorphic CD1d molecule [5,6]. The studies in these patients to modulate the iNKT cell popula- glycolipid α-galactosylceramide (αGalCer) was originally tion through administration of αGalCer, αGalCer-pulsed DC isolated from the marine sponge Agelas mauritianus. or iNKT cell-enriched autologous peripheral blood mono- Synthetically produced αGalCer, also known as KRN7000 nuclear cells (PBMC). This review will translate these studies has been used in most pre-clinical and clinical studies to possible future strategies of iNKT cell mediated immu- performed so-far and it has been shown to induce prolifera- notherapy. In the first part we will provide evidence for the tion of, and cytokine production by, iNKT cells [5,7]. iNKT relevance of iNKT cells in human cancer. In the second part cells are activated by microbial pathogen-derived glycolipids we will discuss several clinical phase I studies on iNKT cell presented by CD1d on dendritic cells (DC) [8–11]. Activation activation and, in particular, we will illustrate the ther- by endogenous antigens such as the (currently disputed apeutic potential of autologous adoptive transfer of purified [12,13]) lysosomal glycosphingolipid isoglobotrihexosylcera- iNKT cell lines in patients that are severely deficient in iNKT mide (iGb3), is amplified by DC activation through bacterial- cells. derived toll-like receptor (TLR) ligands such as LPS [11,14]. These findings underscore the role iNKT cells play in the Part I: Relevance of invariant Natural Killer immune response against microorganisms. On the other hand iNKT cells can protect against autoimmunity most probably T lymphocytes in human cancer due to secretion of anti-inflammatory cytokines (reviewed in [15]). The response towards microorganisms may very well Selective decrease of iNKT cell numbers in be linked to this protection since immune responses against peripheral blood of carcinoma patients infections may be accompanied by anti-self responses causing autoimmunity (reviewed in [16]). As listed in Table 1, Kawano et al. were the first to report a Human and mouse iNKT cells can either be CD4+ or numeric defect in circulating iNKT cells in cancer [29]. These CD4−CD8− [double negative (DN)] and in humans a small findings were confirmed by some [30–32] and contradicted proportion can express CD8. Direct ex vivo analyses by others [33–36]. suggested that CD4+ iNKT cells produce both Th1 cytokines Most of these studies however, investigated small (e.g. GM-CSF, IFN-γ and TNF-α) and Th2 cytokines (e.g. IL-4 cohorts and the effects of age and gender, as reported by and IL-13), whereas the DN and CD8+ iNKT cell subsets Delarosa et al. [37] and Sandberg et al. [38], were not primarily produce Th1 cytokines [17–20]. While the always taken into account. We therefore studied circulat- occurrence of different iNKT cell subsets may be the ing iNKT cell levels in a cohort of 120 patients with various most likely explanation for their dichotomous regulatory epithelial cancers [melanoma, breast-, colorectal-, renal nature, conclusive evidence for this distinction is lacking to cell-cancer and head and neck squamous cell carcinoma date. (HNSCC)] and 69 healthy controls using multivariate Apart from protection against microorganisms and auto- analysis and confirmed that after correction for the strong immunity, an important physiological role for iNKT cells was influence of both age and gender, cancer patients had a shown in the immuno-surveillance of cancers. Mice deficient selective numeric iNKT cell deficiency within the circulat- in iNKT cells (Jα18−/− mice) were more susceptible to ing T cell pool (average 47% reduction compared to healthy chemically [methylcholanthrene (MCA)] induced sarcomas, controls; p = 0.013, linear regression analysis) [39]. Crough while protection could be restored by adoptive transfer of et al. reported similar findings and observed that while iNKT cells isolated from wild-type animals. Protection chemotherapy did not affect iNKT cell numbers, radio- depended on CD1d, IFN-γ production by iNKT cells, and NK therapy induced a decline of circulating iNKT cells in and CD8 Tcell function [21]. The capacity of in vivo activated melanoma patients [40]. We observed a mild and transient iNKT cells to enhance protection against experimental effect of radiotherapy on iNKT circulating cell levels in tumors has been studied extensively. Depending on the patients with HNSCC (Molling JW, unpublished results). model, resident iNKT cells can augment innate as well as iNKT cell numbers were not influenced by tumor type or adaptive anti-tumor immune responses. Several groups disease stage [31,33,39], nor were iNKT cells restored after demonstrated that systemic injection of αGalCer or αGal- tumor de-bulking by surgery or radiotherapy [39]. Taken Cer-loaded DC activates iNKT cells, leading to the inhibition together these findings indicate that circulating iNKT cell of metastasis formation predominantly via the downstream levels are at most only mildly affected by chemotherapy, activation of NK cells [22–25]. radiotherapy or by the outgrowing tumor.
  • 3. 184 J.W. Molling et al. Table 1 Immunologic studies on iNKT cells in cancer patients n Peripheral Related to iNKT cells Relation to Peripheral Proliferative Cytotoxic blood disease in tumor clinical blood iNKT response to against iNKT levels stage infiltrate outcome IFN-γ αGalCer tumorXI production Melanoma 13 ↓ ? ? ? ? ↔ yes (Kawano et al.) [29] Prostate cancer 6 ↓ ? ? ? ↓VIII ↓X ? (Tahir et al.) [30] Lung cancer 60 ↓ no yes ? ↔IX ? ? (Motohashi et al.) [31] Lung cancer 55 ↔ no ? ? ? ↓ ? (Konishi et al.) [33] Myeloma 23 ↔ n/a yes yesV ↓ ↔ yes (Dhodapkar et al.) [34] Glioma 9 ↔ n/a ? ? ↔ ↔ yes (Dhodapkar et al.) [35] Neuroblastoma 8 (blood) ↔ n/a yes yesVI ? ? ? (Metelitsa et al.) [36] 98 (tumor) Various carcinomas 109 ↔I; ↓II n/aI; ?II n/aI; ?II n/aI; ?II n/aI; ?II n/aI; ?II n/aI; ?II (Crough et al.) [40] Various carcinomas 120 ↓ noIII ? ? ↔ ? ? (Molling et al.) [39] Various carcinomas 21 ↓ n/a ? ? ? ↓X ? (Yanagisawa et al.) [32] Head and neck squamous 47 ↓III ? ? yesVII ? ? ? cell carcinoma (Molling et al.) [60] Colorectal cancer 103 ? n/a yesIV yesVI ? ? ? (Tachibana et al.) [77] Overview of the results from various studies on the number and function of circulating invariant natural killer T (iNKT) cells in cancer patients, their capacity to infiltrate tumors, or their relation to prognosis. I: Colorectal, renal cell and other cancers; II: Breast cancer and melanoma; III: Not restored upon tumor de-bulking (18 weeks); IV: Vα24+ T cells, assumed to be invariant natural killer T (iNKT) cells; V: IFN-γ secretion by iNKT in peripheral blood or tumor bed; VI: Size of tumor infiltrating iNKT cell pool; VII: Size of peripheral blood iNKT cell pool; VIII: After in vitro expansion using α-Galactosylceramide (αGalCer) loaded autologous peripheral blood mononuclear cells (PBMC) as antigen presenting cells (APC); IX: Detection of messenger RNA, not protein; X: Autologous αGalCer-loaded PBMC used as APC, not monocyte derived dendritic cells; XI: Upon in vitro expansion; ↓, ↔: reduced, unaffected compared to healthy controls respectively; ?: unknown (analysis was not performed); n/a: not applicable (no reduction in iNKT cells). Some controversy exists with regard to the capacity of the broad variety of cancers [43–46] and that they can suppress residual iNKT cells of cancer patients to respond to αGalCer anti-tumor responses (reviewed in [47]). An additional factor in vitro (Table 1). It is noteworthy, in this respect, that the of influence might be the limited presence and poor function decline in ex vivo IFN-γ secretion [39] and expansion [41] of of DC in the circulation of cancer patients [48–50]. The use of iNKT cells in response to αGalCer were also reported to be autologous PBMC as APC might thus induce in vitro iNKT cell age related. Unfortunately, information regarding subjects' anergy. In line with this, Tahir et al. demonstrated that IFN-γ age is not always provided. secretion by iNKT cells from prostate cancer patients was Substantial in vitro proliferation of iNKT cells from cancer restored when recombinant IL-12 was added to iNKTcell/APC patients was achieved in studies using αGalCer-pulsed co-cultures [30]. Actually, in all reported studies, iNKT cells monocytes or immature monocyte derived DC (moDC) as from cancer patients had retained their capacity to either antigen presenting cells (APC) [29,35]. In contrast, iNKT cell produce IFN-γ ex vivo upon αGalCer stimulation, or after expansion using αGalCer-pulsed autologous PBMC did not repeated or highly active stimulation by professional lead to adequate expansion of iNKT cells from carcinoma antigen presenting cells [30,31,34,35,39]. Furthermore, patients [30,32,33]. The latter might be explained by the expanded iNKT cells of cancer patients can display in presence of other, suppressive, T cells in peripheral blood of vitro cytotoxicity against various CD1d expressing tumor these patients, as proposed by Yanagisawa et al. [32]. targets (Table 1) [29,32,34,35]. This is in line with our Naturally occurring regulatory T cells (nTreg) have been observations that in vitro cultured iNKT cells of healthy demonstrated to be capable of directly inhibiting iNKT cell donors contain granules consisting of cytotoxic effector proliferation, cytokine secretion and cytotoxic activity via molecules such as perforin and granzyme B [51–53]. cognate interactions [42]. Furthermore, it has been well However, cultured iNKT cells do not kill very efficiently established that circulating nTreg numbers are enhanced in a and often require loading of CD1d+ tumor cells with
  • 4. Invariant natural killer T cells and immunotherapy of cancer 185 αGalCer [54–56]. Furthermore, the ability to directly kill peripheral blood were also reduced in this cohort as tumor cells has not been established to be a physiological compared to historical age and gender matched controls. function of circulating iNKT cells [57]. However, no evidence was obtained for a relation of In summary, although some discrepancies have been peripheral blood T or NK cell levels and clinical outcome. noted regarding iNKT cell number and function in different This was in line with previous reports on HNSCC [61–63]. cancers, evidence is accumulating that circulating iNKT cells Reduced functionality, rather than reduced numbers of of carcinoma patients are substantially reduced compared to circulating or tumor infiltrating T and/or NK cells was related healthy controls. Notwithstanding, residual iNKT cells in to a poor prognosis in HNSCC in additional studies [64–68]. these patients still possess the capacity to proliferate, to Interestingly, Reichert et al. demonstrated a strong correla- secrete IFN-γ and to gain some cytotoxic activity when tion between a loss of function in peripheral T cells and a loss properly stimulated in vitro. This suggests that the residual of function in tumor infiltrating lymphocytes (TILs) in HNSCC iNKT cells of carcinoma patients might still be capable of patients [67]. A similar observation was made in myeloma, taking part in patho-physiological anti-tumor responses. where a marked defect in IFN-γ secretion by iNKT cells in the Therapies aimed at their increase and activation in cancer circulation as well as in the tumor bed of progressive, but not patients could thus facilitate more potent anti-tumor of non-progressive myeloma patients was found [34]. immunity. These two aspects will be discussed further in As mentioned above, we found evidence that peripheral the remaining sections of this review. blood iNKT cells of HNSCC patients can still secrete IFN-γ. Taken together these findings [34,39,60,67] suggest that the level of circulating iNKT cells is indicative of their relative Peripheral blood iNKT cells in relation to clinical contribution to local anti-tumor immune responses. It would outcome of cancer thus be relevant to study the correlation between peripheral and intra-tumor iNKT cell number (and function) in future The reduction in circulating iNKT cells of carcinoma patients cohorts, and to relate these parameters to (local) T and NK suggests their contribution to anti-tumor immune responses. cell function. Furthermore, prospective studies are war- However, it has not been demonstrated in the studies re- ranted to provide more insight in how findings of such studies viewed above, whether a reduction of circulating iNKT cells would relate to patient survival. can be regarded as a risk factor for carcinoma development. This also remains to be elucidated for an elevated level of circulating nTregs, which may inhibit iNKT cell number and Tumor infiltrating iNKT cells in relation to clinical function [42]. We therefore studied the relation between outcome of cancer peripheral blood iNKT cell or nTreg frequencies and the natural course of pre-invasive cervical intraepithelial neopla- To make a significant contribution to local anti-tumor sia (CIN), in a prospective nonintervention cohort study of 82 responses, iNKT cells would most likely have to be located women with abnormal cervix cytology [58]. Persistent infec- in the tumor draining lymph nodes and/or in the tumor tion with human papillomavirus type 16 (HPV) is a major risk microenvironment. Both in humans and in germ free mice factor for the development of high grade CIN (i.e. CIN 3) iNKT cells were found to acquire an effector (memory) which, if left untreated, may lead to invasive cervical phenotype before birth, allowing for their distribution to carcinoma [59]. Circulating nTreg numbers were increased in sites of inflammation [69,70]. As such, they resemble tissue- individuals with persistent HPV16 infection, compared to infiltrating Th1 cells and CD8+ cytotoxic T lymphocytes (CTL) women who had cleared the infection, and were possibly and indeed have a corresponding chemokine receptor associated with the development of a CIN 3 lesion. The expression pattern [18,71,72]. In mouse models, the initial number of circulating iNKT cells was not related to these pre- influx of iNKT cells was required for the formation of malignant events of cervical carcinoma, or to the level of granulomatous lesions caused by Mycobacterium tuberculo- circulating nTregs. However, this does not rule out that iNKT sis [73] or Cryptococcus neoformans [74]. In the latter study, cells could be involved in immune responses directed at more the iNKT cells were attracted by the CCR2 ligand monocyte advanced carcinomas. chemoattractant protein (MCP)-1 (i.e. CCL2). Metelitsa et al. While the carcinoma patient cohort introduced earlier in demonstrated that human neuroblastomas expressing CCL2, this review [39] as a whole was significantly deficient in iNKT were well infiltrated with iNKT cells. Patients with iNKT cell- cell numbers, some individuals had iNKT cell levels resem- enriched tumors had a significantly prolonged long-term bling those observed in age-matched healthy controls survival, compared to patients with less iNKT cells at the whereas others had very low to undetectable numbers of tumor site [36]. Interestingly, they recently reported that iNKT cells in their circulation. We therefore tested the amplified MYCN oncogene, which is a hallmark of aggressive hypothesis that a severe iNKT cell deficiency was related to a neuroblastoma [75], repressed CCL2 expression leading to poor clinical outcome after radiation therapy in a prospec- impaired iNKT cell infiltration. This was strikingly evident in tive study of 47 patients with HNSCC [60]. Indeed patients patients with bone marrow metastases of neuroblastoma, with a severe iNKT cell deficiency prior to radiation therapy suggesting an important contribution of iNKT cells to the had a poor 3 year disease specific survival, compared to local immune attack against these metastases [76]. patients with above average iNKT cell levels (43 vs. 92% In addition, Tachibana et al. demonstrated that colorectal 3 year survival rate; p = 0.0027, Log Rank test). This effect carcinomas were well infiltrated with activated CD69+ TCR- was independent of clinical T stage and age (hazard ratio Vα24+ T cells compared to the patients' control tissue and [p value] = 17 [0.022], 5.3 [0.024] and 1.1 [0.030] respectively, established by Cox regression analysis that high TCR-Vα24+ T Cox regression analysis). Pre-therapy T or NK cell levels in cell infiltration was predictive of prolonged (disease free)
  • 5. 186 J.W. Molling et al. survival, independently of other prognostic variables like carcinoma patients [92,93]. Given that iNKT cells are clinical T stage [77]. Although the authors did provide positioned early in the immune cascade, that they express indirect evidence that the TCR-Vα24+ T cells were indeed CCR5 and CXCR3 [71], can readily release IFN-γ in carcinoma iNKT cells, they did not clarify whether these cells infiltrated patients [31,39] and apparently infiltrate colorectal carci- the tumor more readily than other T cells. This is relevant, nomas [77], some challenging clues arise regarding the since a high number of T cells infiltrating the tumor is by nature of the before mentioned “early arriving” T cells itself related to favorable prognosis in colorectal cancer (Fig. 1Aiii). In addition, several groups have demonstrated (reviewed in [78]). the capacity of iNKT cells to stimulate DC activation and maturation [26–28,94,95]. At the tumor site, iNKTcells could thus facilitate proper DC maturation, resulting in improved Proposed mechanisms behind the role of iNKT cells migration of more mature DC towards the tumor draining in anti-tumor responses lymph nodes (Fig. 1B). This would result in more effective priming of additional T effector cells with tumor associated A few putative mechanisms via which iNKT cells could take antigens [96–99]. part in anti-tumor responses will be discussed below. Heat shock proteins (Hsp), which may be released by tumor cells [79,80], have been proposed to enhance CD1d Therapies aimed at the activation of iNKT cells in expression on epithelial cells [81–83] as well as DC [84]. In cancer patients vivo studies suggest that apoptotic tumor cells releasing Hsp may induce an effective anti-tumor response [85]. Locally Attempts have been made to target iNKT cells in vivo in produced Hsp at sites of (metastasized) malignant lesions, by clinical phase I studies, based on the use of αGalCer as a damaged epithelial or tumor cells, might therefore enhance stimulatory ligand (summarized in Table 2). We established in CD1d mediated antigen presentation within the tumor 24 advanced cancer patients with solid tumors that 3 weekly microenvironment. For example, GD3 ganglioside is highly i.v. injections of soluble αGalCer did not reach dose limiting expressed on human tumors of neuroectodermal origin toxicity over a wide dose range (50–4800 μg/kg) [100]. (which include neuroblastoma and melanoma) but not on Although no clinical responses were recorded, a transient normal tissues. Wu et al. described that this glycolipid can be increase in serum levels of immunostimulatory cytokines (IL- effectively cross-presented by CD1d+ DC to mouse iNKT cells 12, IFN-γ, TNF-α and GM-CSF) was observed after the first in vivo resulting in their activation [86]. αGalCer injection in patients with relatively high iNKT cell iNKTcell activation leads to the subsequent augmentation levels. This was preceded by a rapid loss of detectable iNKT of innate, e.g. NK cell mediated, anti-tumor effector cells from the circulation within 24 h after αGalCer mechanisms. In addition, iNKT cells can enhance antigen administration and iNKT cell levels remained low for up to specific B cell responses, leading to elevated IgG levels 21 days after the first administration. In line with the decline [87,88]. These combined effects may lead to opsonization of in iNKT cell levels, no increase in serum cytokines was cancer cells with immunoglobulins, leading to antibody- observed after a second and third injection with αGalCer (7 dependent cellular cytotoxicity (ADCC) mediated by NK cells and 14 days after the first injection respectively). This might (reviewed in [2]). iNKT cells might thus be able to promote a be explained by the relatively high dose of the glycolipid tumoricidal environment within the vicinity of the tumor injected and the short interval between repeated injections. (Fig. 1Ai). In a clinical phase I/II trial in chronic Hepatitis C virus Angiogenesis and lymphangiogenesis are of vital impor- infected patients we observed a re-appearance of circulating tance for a malignant lesion to develop into a substantial iNKT cells within 2 weeks after i.v. αGalCer given at a lower tumor mass [89]. iNKT cell activation via systemic injection dose (0.1–10 μg/kg). Furthermore, we observed a cytokine of αGalCer significantly inhibited angiogenesis of intrader- response even to a second and third αGalCer injection given mal tumors in mice, which depended on IFN-γ release by at 4 and 8 weeks after the first injection respectively [101]. both iNKT cells and subsequently activated NK cells [90] The lack of detectable iNKT cells quickly after i.v. injection (Fig. 1Aii). Interestingly, Dhodapkar et al. showed that the of the glycolipid might reflect their antigen-specific activa- neo-vasculature of gliomas expressed CD1d, identifying tion by αGalCer, leading to TCR internalization, especially in them as targets for this anti-angiogenic response [35]. It the case of the administration of high doses of αGalCer. This would therefore be interesting to determine neo-vasculature has been demonstrated previously in mice [102,103] and CD1d expression in a broader panel of human tumors, in the additional studies revealed that in vivo treatment with a light of local iNKT and NK cell activation. high dose of soluble αGalCer can lead to long-term iNKT cell It has been proposed that once human colorectal cancers anergy, even after a single injection of the glycolipid become clinically detectable, and thus have escaped early [104,105]. innate immune surveillance, the adaptive arm plays a The relatively low in vivo responsiveness of circulating predominant role in preventing disease progression. Color- iNKT cells upon i.v. injection of free αGalCer parallels the ectal cancer tumors are initially infiltrated with CCR5+ reduced in vitro responsiveness of iNKT cells when stimu- CXCR3+ T cells [91]. IFN-γ release by these “early arriving” T lated with autologous αGalCer-loaded PBMC [30,32,33]. Both cells was proposed in this study to trigger events leading to phenomena may be explained by the impaired function of the influx of effector T cells. The presence of many Th1 APC in cancer patients. Analogous to the in vitro data which oriented memory/activated T cells in both the tumor center showed restored iNKT proliferation upon stimulation with and in the tumor's surrounding tissue invasive margin was αGalCer-loaded, in vitro generated, fully functional mature related to prolonged (disease specific) survival of colorectal DC [54], the injection of αGalCer-pulsed DC gave rise to more
  • 6. Invariant natural killer T cells and immunotherapy of cancer 187 Figure 1 Proposed role of iNKT cells in enhancing immune function in tumor microenvironment. (A) iNKT cells recognize endogenous ligands presented by CD1d on tumor cells, damaged epithelial cells or APC and subsequently release IFN-γ locally. As a result chemokine release is enhanced, facilitating considerable influx of anti-tumor effector cells. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) arrive at the tumor site. i) iNKTcells in the (lymphatic) circulation recognize, and are activated by, CD1d on migrating tumor cells. IFN-γ, released by the activated iNKT cells enhances antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells and antibodies specific for cell surface tumor associated antigens (Ig/Fc-R). ii) iNKT cells that are activated by CD1d on endothelial cells secrete IFN-γ to activate NK cells that subsequently release higher levels of IFN-γ, resulting in the inhibition of neo-angiogenesis initiated by the developing tumor. iii) NK cells and CTL cross the endothelial barrier to directly kill the tumor cells. (B) Chemokine (CHEM) release from the tumor microenvironment results in the influx of iNKTcells which subsequently co-localize with residing immature dendritic cells (immDC) that might be kept in an immature state by tumor derived soluble factors. Binding of the iNKT TCR to the APC CD1d, presenting endogenous glycolipids triggers IFN-γ release by the iNKTand IL-12 release by the immDC, creating a positive feedback loop that results in elevated iNKT derived IFN-γ and DC maturation. As a result, mature DC (matDC) up-regulate CCR7 and migrate towards the tumor draining lymph node (TDLN), where they can prime CD4+ T helper cells and CD8+ cytotoxic T lymphocytes and facilitate B cell activation.
  • 7. 188 J.W. Molling et al. Table 2 Clinical phase I studies regarding activation of peripheral blood iNKT cells in cancer patients Therapeutic n Clinical responses Serum Effects on Effects on other approach (when observed) cytokines circulating circulating cells after therapy iNKT cells after therapy after therapy Solid tumors i.v. αGalCer 24 Stable disease Elevated IL-12, Decline Transient reduction (Giaccone et al.) (in 7 pt; IFN-γ (in 1/10 pt (in all pt) in NK cell [100] 83–216 days) tested) TNF-α number + cytotoxicity and GM-CSF (in all pt tested) (in 5/21 pt tested) Metastatic i.v. immature 4 ? ? Decline, ? malignancy αGalCer-pulsed followed by (Okai et al.) moDC mild increase [106] (in all pt) Metastatic i.v. immature 12 Decreased serum Elevated IL-12 Decline, T + NK activation carcinoma αGalCer-pulsed tumor markers (in all pt) and followed by and increased (Nieda et al.) moDC (in 2 pt; IFN-γ (in 6/9 mild increase NK cytotoxicity [107] 4–12 months), patients tested) (in all pt) (in 5/11 pt tested) tumor necrosis (in 1 pt), decreased serum liver enzymes (in 2 pt with hepatic metastases) Myeloma and i.v mature 3 and Decreased serum Elevated N 100 fold Expansion of carcinoma αGalCer-pulsed 2 tumor markers IL-12p40 increase at CMV specific (Chang et al.) moDC (in 3 pt; MIP-1β and peak (in all pt) CD8+ T cells [108] 9–10 months) IP-10 (in all pt (in 3/3 pt and stable disease tested) tested) (in 1 pt; 8 months) Advanced lung i.v. autologous 12 Stable disease ? N 20 fold ? canceri PBMC enriched (in 3/9 pt tested; increase at (Ishikawa et al.) for moDC 23–26 weeks) peak (in 3/12 pt) [109] pulsed with and increase in αGalCer iNKT IFN-γ mRNA (tested in 1 pt) Non small cell i.v. autologous 6 Stable disease ? Mild increase Mild increase lung cancerII PBMC enriched (in 4/6 pt; up to (in 3/6 pt) and in NK cells (Motohashi et al.) for iNKT cells 12 moths) elevated IFN-γ (in 1/4 pt tested) [110] in response to αGalCer in vitro in 5/5 pt tested) Head and neck Intra mucosal 9 Stable disease Elevated IFN-γ Mild increase Increase in NK cancerIII (nasal) injection (in 5/9 pt; (in 8/9 patients (in 4/9 pt) and cells (in 1/9 pt (Uchida et al.) of autologous PBMC 8 weeks); tested) elevated IFN-γ tested; case with [111] enriched for moDC partial response in response to partial response) pulsed with (in 1/9 pt; αGalCer in vitro αGalCer 8 weeks) (in 8/9 pt tested) Overview of the clinical phase I studies conducted regarding activation of peripheral blood invariant natural killer T (iNKT) cells in cancer patients. Patients were treated with αGalCer, monocyte derived dendritic cells (moDC) or ex vivo activated iNKT cells. No adverse events were recorded in any of these studies. I: The method of DC preparation does not exclude the presence of ex vivo activated iNKTcells at time of injection; II: Preparations contain large amounts of CD3+ Va24− T cells and NK cells; III: The method of DC preparation does not exclude the presence of ex vivo activated (iNK)T cells at time of injection?: unknown (parameter not tested); pt: patients. potent in vivo expansion of endogenous iNKT cells. Three respectively with metastatic carcinomas with autologous independent groups have now shown that this approach αGalCer-pulsed immature moDC and found that this was well was well tolerated in humans. In two consecutive studies, tolerated and resulted in a mild increase in circulating iNKT the group of Nicol treated 4 [106] and 12 [107] patients cell numbers. In the latter study, transient but potent pro-
  • 8. Invariant natural killer T cells and immunotherapy of cancer 189 inflammatory effects were observed in peripheral blood towards a type 1 cytokine profile by stimulation of isolated samples after DC treatment, consisting of elevated levels of TCR-Vα24+ T cells with mature αGalCer-pulsed moDC in the IL-12 and IFN-γ, reduced levels of IL-4, activation of T and NK presence of IL-15 [54]. These type 1 polarized iNKT cultures cells and an increase in NK cell number and cytotoxicity. secreted large amounts of the pro-inflammatory cytokines These effects were reproduced upon a second injection of IFN-γ, TNF-α and GM-CSF. Although some patient derived immature moDC pulsed with αGalCer. Next, Chang et al. were iNKT cell cultures showed an initial delay in proliferation, as able to induce dramatic expansion of circulating iNKT cells in reported previously [30,32,33], this could be overcome by 5 out of 5 advanced cancer patients who received i.v. repeated stimulation with αGalCer-pulsed moDC, resulting injections with high purity, properly matured, αGalCer- in functionally competent iNKTcells. This suggests that, even pulsed autologous moDC (N100 fold expansion at peak level in patients with a putative state of iNKT cell anergy, in vitro in all cases) [108]. Strikingly, despite having undetectable expansion of autologous iNKT cells using mature DC may peripheral blood iNKT cell counts at the time of study allow for subsequent adoptive transfer of these cells. enrollment, the iNKT cell level remained above baseline Another advantage of autologous adoptive transfer of ex after DC treatment for more than 85 days in all patients and vivo expanded iNKT cells would be that their in vitro for up to 6 months in two patients with longer follow up. expansion and activation does not depend on autologous Ishikawa et al reported activation and transient expansion of DC, since CD1d is monomorphic. This allows for the use of DC resident iNKT cells in 3 out of 12 advanced lung cancer cell lines as more standardized in vitro APC leading to more patients by injecting αGalCer-pulsed APC [109]. However, standardized iNKT cells for adoptive transfer. We have given the culturing protocol of their APC preparations (low previously demonstrated that human iNKT cells can be purity of APC in autologous PBMC cultured with GM-CSF, IL-2 expanded using αGalCer-pulsed DC derived from the CD34+ and αGalCer for 7 to 14 days) this could also have resulted human acute myeloid leukemia derived cell line MUTZ-3 (M3- from a direct iNKT cell infusion. In a follow up study the DC) [112]. In successive experiments we stimulated iNKT authors indeed demonstrated that this method provided cells with M3-DC over-expressing both CD1d and IL-12 preparations that were enriched for iNKTcells. In 6 non-small (M312CD1d-DC). The obtained iNKT cells had an increased cell lung cancer patients they re-confirmed that infusion was activation phenotype and were capable of producing high well tolerated [110]. Furthermore, a transient increase in levels of IFN-γ. Addition of these iNKTcells to autologous CD8 circulating iNKT cells and direct ex vivo IFN-γ production in and tumor associated antigen (TAA) peptide-loaded moDC response to αGalCer in an ELISPOTassay were observed. More leads to an enhanced TAA specific CTL response [113]. In recently, this group demonstrated that the administration of similar experiments we found enhanced NK cell functionality these αGalCer-pulsed APC into the nasal sub mucosa was also upon adding expanded iNKT cells to co-cultures of NK cells well tolerated [111]. and moDC [114]. The effect of the iNKT cells depended in It can be concluded from these clinical phase I studies that both cases on the presence of αGalCer on the moDC. Thus, injection of preparations containing αGalCer-pulsed DC and/ M312CD1d-DC provide us with an attractive “off the shelf” or iNKT cells is feasible, since it can be performed safely in DC source for the large scale expansion of functional iNKT advanced cancer patients and results in distinct activation of cells from cancer patients. iNKT and downstream effector cells. Retained capacity of long-term mouse iNKT cell Part II: Towards autologous adoptive transfer of cultures to enhance the in vivo immune response highly purified and well defined against experimental tumor metastases pro-inflammatory iNKT cells Although the strategy of treating carcinoma patients with autologous adoptive transfer of ex vivo expanded iNKT cells Ex vivo establishment of iNKT cell lines from healthy is very appealing, long-term in vitro culture would be controls and carcinoma patients necessary in order to obtain sufficient numbers to repopulate patients with iNKT cells up to healthy control levels. This Although the phase I clinical trials performed thus far led to would especially be the case in those individuals who would promising results there is ample room for improvement. probably benefit most from this therapy, namely those with a When mature DC are used as “in vivo iNKT cell activators“, a severe deficiency in circulating iNKT cell number and/or a high iNKT cell expansion can be achieved, but the ability to poor in vitro proliferative response towards αGalCer. control the functional aspects of the expanded iNKT cells in However, the effect of repetitive in vitro stimulation with vivo (e.g. cytokine profile or the capacity to home towards DC pulsed with the strong agonist αGalCer on in vivo iNKTcell tumor sites) is limited. For instance, in the clinical phase I functionality had not previously been investigated. We study of Chang et al., iNKT cells that were isolated after the therefore developed a method to generate long-term high administration of αGalCer-pulsed mature moDC lacked the purity oligoclonal mouse iNKT cell cultures [115]. Although capacity to secrete IFN-γ in an αGalCer ELISPOT assay [108]. other studies previously demonstrated that it is possible to In addition, the use of highly purified and well defined iNKT generate from mice either short lived cultures containing cells for adoptive transfer enables to ascribe any immuno- bona fide iNKT cells [57,116,117], or long lived clone derived logical or clinical effects observed to the injected iNKT cells. iNKT cell hybridomas [118–120], our study was the first to As already mentioned, we have developed a method to make available large scale, highly pure oligoclonal mouse expand peripheral blood iNKTcells of healthy controls as well iNKT cell lines representative of in vivo iNKT cells. The iNKT as advanced cancer patients in vitro, and to polarize them cell cultures retained their most important functional
  • 9. 190 J.W. Molling et al. aspects since they could bind to αGalCer-loaded mouse certain HLA type or expressing a particular tumor antigen is CD1d:IgG1 dimers and readily released substantial amounts necessary. The risk of selection of TAA and/or HLA negative of IFN-γ, GM-CSF, IL-4, IL-5, IL-6, IL-10 and IL-13 upon tumor cells is expected to be low, compared to TAA peptide antigen specific TCR triggering. In a subsequent study we based immunotherapy. Furthermore, safety issues always demonstrated that these expanded iNKT cell lines could need to be addressed with the application of TCR gene induce a partial NK cell-dependent protection against B16. transfer because of the use of retroviral vectors [126]. F10 lung metastases, upon their adoptive transfer into wild- iNKT cells can be expanded in vitro using the “off the type mice shortly after tumor injection [121]. Crowe et al. shelf” MUTZ-3 cell line derived DC pulsed with αGalCer. Using investigated the effect of transfer of freshly isolated iNKT this ex vivo approach, high purity iNKT cells can be obtained cells on B16.F10 lung metastases in iNKT cell deficient mice. and their potential to enhance anti-tumor responses can be In their study, the activation of NK cells and thus the strengthened by defining the desired culture conditions (e.g. inhibition of metastasis formation required additional treat- the use of IL-12 over expressing MUTZ-3 DC). More impor- ment of mice with αGalCer. We demonstrated that additional tantly, their capacity to secrete high amounts of type 1 αGalCer injection was not required when iNKT cells had been cytokines (e.g. IFN-γ and GM-CSF), to enhance the function- pre-activated in vitro with αGalCer-pulsed DC [122]. This is ality of other immune cells (e.g. DC, antigen specific CTL or in line with the findings by Shin et al., who observed NK cells), or their expression of “tumor homing receptors” inhibition of experimental B16.F10 liver metastases upon (e.g. CXCR3 and CCR5 for carcinomas or CCR2 for neuro- adoptive transfer of in vitro IL-12 pre-activated iNKT cells blastomas) can be checked prior to adoptive transfer. into iNKT cell deficient mice without additional αGalCer There are many questions remaining that could lead to a treatment [123]. In contrast to these results we found, as more efficient therapeutic outcome when answered. For described in the previous paragraph, that additional trigger- instance: How do peripheral and intra-tumor iNKT cell ing of expanded human iNKT cells by αGalCer was necessary numbers and function relate? What is the impact of systemic to achieve enhanced CTL and NK cell responses in vitro. or local (tumor site) enhancement of iNKT cell numbers on Whether this discrepancy is due to a difference in species (tumor infiltrating) DC, T and NK cell function, also with (mouse vs. human) or experimental setting (in vitro vs. in regard to disease outcome? Does additional treatment with vivo) remains to be established. αGalCer enhance these responses and should it then be Interestingly, Shin et al. also demonstrated that iNKT cell administered systemically (e.g. loaded on DC) or locally? Can transfer mediated protection was superior to the injection of responses be enhanced when the negative influence of nTreg high dose IFN-γ or a combination of high dose IFN-γ, IL-2 and on anti-tumor responses is abrogated (e.g. by blocking CTLA4 IL-4. This protection is possibly due to a selective recruit- function [127–130], by TLR2 triggering [131,132]) or by nTreg ment of iNKT cells to the tumor site followed by their local depletion using e.g. denileukin diftitox [133])? release of chemokines and cytokines, allowing for the Nonetheless, we already observed that the amount of pre- recruitment and trans-activation of downstream effector therapy circulating iNKT cells has a dramatic impact on cells. Although direct evidence for this is still lacking, we did disease specific survival upon curative radiotherapy of observe that long-term iNKTcell lines can express the “tumor HNSCC patients. Immuno-adjuvant treatment of iNKT cell homing receptors” CXCR3 and CCR5 (Molling JW, unpublished deficient HNSCC patients, by radiotherapy accompanied by results) and that they secrete a broad variety of cytokines in re-constitution of their peripheral blood iNKT cell compart- vitro (as described above [115]). These observations, in ment using autologous adoptive transfer of iNKT cells, could combination with the finding that NK cells were selectively provide a solid base from which questions as those above recruited to the lungs after i.v. iNKT injection, are in favor of could be further addressed. the sequential influx of iNKT cells and NK cells into the lungs of B16.F10 tumor challenged mice. These pre-clinical studies Acknowledgments illustrate the potential of adoptive transfer of autologous ex vivo expanded and activated iNKT cells as an immunother- Funded by Dutch Cancer Society: Grant # VU2002-2607 apeutic strategy for the treatment of cancer patients. (AJMvdE, BMEvB and RJS); Netherlands Organization for Scientific Research: NWO-TALENT grant and Grant # 920-03- 142 (HJJvdV). Concluding remarks References Based on the (pre-)clinical data reviewed here, re-constitu- tion of the circulating iNKT cell pool as an immunotherapeutic [1] I. 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