CD1d expression level in tumor cells is an important determinant for anti-tumor immunity by natural killer T cells

Invariant natural killer T (iNKT) cells are thought to regulate anti-tumor immunity. Human iNKT (i.e. Valpha24+ NKT) cells have been reported to recognize CD1d on target cells and show cytotoxicity directly on the target cells in vitro. However, the anti-tumor effect of mouse iNKT (i.e. Valpha14+ NKT) cells has been repeatedly reported to be dependent on the activity of natural killer (NK) cells via interferon-gamma, with no evidence of direct cytotoxicity. In the present study, we report that in vitro cytolysis of EL-4 mouse lymphoblastic lymphoma cells by Valpha24+ NKT cells and in vivo eradication of these cells are both dependent on the level of CD1d expression on the tumor cell surface. These observations possibly suggest that direct cytotoxicity of tumor cells by iNKT cells is common to both humans and mice, and that the high expression level of CD1d may be a predictor whether the tumor is a good target of iNKT cells.     

Therapy for minimal residual tumor disease: β‐Galactosylceramide inhibits the growth of recurrent HPV16‐associated neoplasms after surgery and chemotherapy

Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d‐binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor‐inhibitory effects of 12 carbon acyl chain β‐galactosyl ceramide (C12 β‐D ‐Galactosyl Ceramide; β‐GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)‐associated neoplasms transplanted in syngeneic mice. Treatment of tumor‐bearing mice with β‐GalCer(C12) 3–14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatability complex (MHC) Class I‐positive (TC‐1), as well as MHC Class I‐deficient (TC‐1/A9) HPV16‐asssociated tumors. Moreover, administration of β‐GalCer(C12) after surgical removal of TC‐1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. β‐GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL‐12‐producing cellular vaccines. These results suggest that β‐GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.     

Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.     

Increased frequency of circulating invariant natural killer T cells in malignant pleural mesothelioma patients

Invariant natural killer T (iNKT) cells are a distinct subset of human T cells, which expresses an invariant T cell receptor Vα24 Jα18 and recognizes glycolipid antigens in the context of CD1d molecules. iNKT cells exert pivotal regulatory roles in many immune responses, including antitumor immune responses. Alterations in iNKT cell frequency, phenotype, and activation state have been reported in cancer patients. No data are available on the iNKT cells in malignant pleural mesothelioma (MPM), a rare, but very aggressive, malignancy of the pleura with a very poor prognosis. Here, we studied the frequency, phenotype, and cytokine profile of circulating iNKT cells in MPM patients, and correlated results with tumor histological types (epithelioid, sarcomatoid, biphasic) and clinical stages (I-III). We found that the iNKT cell frequency was significantly increased in MPM patients with epithelioid and sarcomatoid types in comparison with healthy volunteers (HV); only three biphasic mesotheliomas were available in this study, thus no conclusions can be drawn for this MPM type. The increased frequency significantly correlates with the clinical stage of tumor with the highest value at the stage III in both epithelioid and sarcomatoid subtypes. According to the histological types, we measured changes in the frequencies of CD4? CD8? (DP) and CD4?CD8? (DN), but not in the cytokine profiles (IFN-γ/IL-4 expression). These results demonstrate that the frequency of iNKT cells is increased in MPM patients and that this increase correlates with MPM type and stage.     

Invariant NKT cells are resistant to circulating CD15+ myeloid‐derived suppressor cells in patients with head and neck cancer

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti‐tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15⁺ granulocytic MDSC (G‐MDSC) and CD14⁺ monocytic MDSC (M‐MDSC) was demonstrated in HNSCC patients. The percentage of G‐MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G‐MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15⁺ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α‐galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15⁺ G‐MDSC. These results indicate that increased G‐MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G‐MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G‐MDSC.     

NY‐ESO‐1 antigen‐reactive T cell receptors exhibit diverse therapeutic capability

The cancer‐testis antigen NY‐ESO‐1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen‐specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross‐reactive to other antigens. We characterized three NY‐ESO‐1 antigen‐reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC‐multimers loaded with NY‐ESO‐1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA‐A2 independent of the bound peptide beside its much stronger recognition of NY‐ESO‐1 bound to HLA‐A2. A third TCR displayed an intermediate but peptide‐specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene‐modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy.     

Antibody‐dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells

Anti‐ganglioside GD2 antibodies mainly work through antibody‐dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high‐risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti‐GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti‐tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti‐GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co‐cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti‐GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK‐NKT cell contact or NK cell‐dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell‐based immunotherapy could be an appropriate candidate for anti‐GD2 antibody therapy for neuroblastoma.     

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy

CD8⁺ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8⁺ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8⁺ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRβ deep sequencing was performed to analyze CD8⁺ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRβ clonotypes were noted in CD8⁺ PD-1⁺ T cells than in CD8⁺ PD-1^- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8⁺ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.     

Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo

We have investigated the role of the host's CD1d-dependent innate antitumor immune response in a murine T-cell lymphoma model in vivo. We found that C57BL/6 wildtype (WT) mice inoculated with RMA/S cells transfected with murine CD1d1 died at the same rate as mice inoculated with vector-transfected cells. In contrast, natural killer T (NKT) cell-deficient CD1d or Jalpha18 knockout mice inoculated with CD1d-transfected RMA/S cells survived significantly longer than mice inoculated with vector-transfected RMA/S cells, implicating the involvement of invariant NKT (iNKT) cells in inhibiting antitumor activity in vivo. In contrast to the mutant mice, which produced more of the proinflammatory cytokines IFN-gamma and GM-CSF, WT mice produced significantly elevated amounts of IL-13. Antitumor activity in the knockout mice was not due to the development of CD1d-specific cytotoxic T lymphocytes or circulating antibodies. However, iNKT cell numbers were elevated in tumor-bearing mice. Thus, iNKT cells may be playing a negative role in the host's antitumor immune response against T-cell lymphomas in a CD1d-dependent manner.     

Chimeric antigen receptor‐engineered cytokine‐induced killer cells overcome treatment resistance of pre‐B‐cell acute lymphoblastic leukemia and enhance survival

Pre‐emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine‐induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft‐versus‐host‐disease. CIK cells are a heterogeneous effector cell population including T cells (CD3⁺ CD56^?), natural killer (NK) cells (CD3^?CD56⁺) and natural killer T (T‐NK) cells (CD3⁺ CD56⁺) that exhibit non‐major histocompatibility complex (MHC)‐restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)‐γ, anti‐CD3 antibody, interleukin‐2 (IL‐2) and interleukin‐15 (IL‐15). To facilitate selective target‐cell recognition and enhance specific cytotoxicity against B‐cell acute lymphoblastic leukemia (B‐ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28‐CD3ζ domain for signaling and a CD19‐specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19‐targeted CIK/63.28.z cells against otherwise CIK‐resistant cancer cell lines and primary B‐ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre‐B‐ALL. Our results demonstrate potent antileukemic activity of CAR‐engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre‐B‐ALL.     

Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line‐derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)‐4 and granulocyte‐macrophage colony‐stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL‐12 and tumor necrosis factor (TNF)‐α production from mature DC, while no effect of SH‐SY5Y cell supernatant was noted. In addition, IL‐6 and IL‐10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α‐Galactosylceramide‐pulsed DC co‐cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)‐γ after stimulation with neuroblastoma cell line supernatant‐cultured DC was reversed by addition of IL‐12. CD40 expression and IL‐12 production in NLF‐sup‐treated DC were increased by addition of exogenous IFN‐γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer‐pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN‐γ production.     

Distinct role of CD8 cells and CD4 cells in antitumor immunity triggered by cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir system

Immune cells can recognize tumor-associated antigens released from dead tumor cells, which elicit immune responses, potentially resulting in tumor regression. Tumor cell death induced by chemotherapy has also been reported to activate immunity. However, various studies have reported drug-induced immunosuppression or suppression of inflammation by apoptotic cells. Thus, this study aimed to investigate whether apoptotic tumor cells trigger antitumor immunity independent of anticancer treatment. Local immune responses were evaluated after direct induction of tumor cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. The inflammatory response was significantly altered at the tumor site after apoptosis induction. The expression of cytokines and molecules that activate and suppress inflammation simultaneously increased. The HSV-tk/GCV-induced tumor cell apoptosis resulted in tumor growth suppression and promoted T lymphocyte infiltration into tumors. Therefore, the role of T cells after inducing tumor cell death was explored. CD8 T cell depletion abrogated the antitumor efficacy of apoptosis induction, indicating that tumor regression was mainly dependent on CD8 T cells. Furthermore, CD4 T cell depletion inhibited tumor growth, suggesting the potential role of CD4 T cells in suppressive tumor immunity. Tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion to elucidate this immunological mechanism. Foxp3 and CTLA4, regulatory T-cell markers, decreased. Furthermore, arginase 1, an immune-suppressive mediator induced by myeloid cells, was significantly downregulated. These findings indicate that tumors accelerate CD8 T cell-dependent antitumor immunity and CD4 T cell-mediated suppressive immunity. These findings could be a therapeutic target for immunotherapy in combination with cytotoxic chemotherapy.     

CD1d is expressed on B-chronic lymphocytic leukemia cells and mediates alpha-galactosylceramide presentation to natural killer T lymphocytes

Generation of immune responses against B cell chronic lymphocytic leukemia (B-CLL) has been the aim of several studies that have demonstrated a poor antigen presenting ability of B-CLL cells and an inconsistent emergence of T cells capable of killing efficiently the leukemic cells. CD1d is a restriction element structurally related to the major histocompatibility complex (MHC) and capable of presenting lipid antigens to CD1d-restricted T cells (also defined as natural killer-T [NKT] cells). The synthetic lipid alpha-galactosylceramide (alpha-GalCer) has been characterized as a potent stimulator of CD1d-restricted T cells. We have investigated the expression of CD1d on B-CLL cells. CD1d was detected by flow cytometric analyses on leukemic cells of all B-CLL cases studied (n = 38) and was expressed at higher density on cells carrying unmutated immunoglobulin variable region (IgV) genes. In addition, CD1d on B-CLL cells mediated the presentation of alpha-GalCer to CD1d-restricted T cells, which in turn induced B-CLL cell death. At variance with another study (Metelitsa et al., Leukemia 2003;17:1068-77), no correlation between expression levels of CD1d and susceptibility to NKT cell lysis was observed. Proliferation and production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) by CD1d-restricted T cells, in the presence of B-CLL cells loaded with alpha-GalCer, were also observed. Our study demonstrates that B-CLL cells express a monomorphic restriction element that is functionally capable of antigen presentation and can be useful to design novel B-CLL immunotherapies.     

Enhanced antitumor activity mediated by human 4‐1BB‐engineered T cells

4‐1BB (CD137) is a costimulatory molecule transiently expressed on the T‐cell surface after TCR engagement, whereas its ligand 4‐1BBL can be found on professional antigen‐presenting cells, but more importantly, also on tumor cells. As the role of the 4‐1BB/4‐1BBL pathway has emerged central to CD8⁺ T‐cell responses and survival, we sought to test its relevance in the context of genetically modified human T cells. To that end, T cells purified from healthy donors and from vaccinated‐melanoma patients were transduced to express high levels of constitutive 4‐1BB. 4‐1BB‐transduced T cells were cocultured with melanoma tumor lines and exhibited enhanced cytokine secretion, upregulation of activation markers as well as increased cytotoxicity in a chick‐chorioallantoic membrane model of human melanoma tumors. In addition, these cells expanded and proliferated at a higher rate, expressed heightened levels of the antiapoptotic molecule Bcl_XL and were also relatively insensitive to immunosuppression mediated by transforming growth factor‐β, compared to control cells. We also show that 4‐1BBL expression on the target cell is essential to 4‐1BB‐mediated functional improvement. Overall, we conclude that the modification of human T cells with 4‐1BB yields enhanced antitumor function which may have important applications in therapies based on the genetic modification of patient lymphocytes.     

CD4+ T cells support polyfunctionality of cytotoxic CD8+ T cells with memory potential in immunological control of tumor

Polyfunctionality/multifunctionality of effector T cells at the single cell level has been shown as an important parameter to predict the quality of T cell response and immunological control of infectious disease and malignancy. However, the fate of polyfunctional CD8⁺ CTLs and the factors that control the polyfunctionality of T cells remain largely unknown. Here we show that the acquisition of polyfunctionality on the initial stimulation is a sensitive immune correlate of CTL survival and memory formation. CD8⁺ T cells with high polyfunctionality, assessed with γ‐interferon and tumor necrosis factor‐α production and surface mobilization of the degranulation marker CD107a, showed enhanced Bcl‐2 expression, low apoptosis, and increased CD127^highKLRG1^low memory precursor phenotype. Consistent with these observations, CD8⁺ T cells were found to acquire high frequency of cells with polyfunctionality when stimulated in conditions known to enhance memory formation, such as the presence of CD4⁺ T cells, interleukin (IL)‐2, or IL‐21. Utilizing T‐cell receptor (TCR) transgenic mouse‐derived CD8⁺ T cells that express a TCR specific for a tumor‐derived neoantigen, we showed that polyfunctional tumor‐specific CTLs generated in the presence of CD4⁺ T cells showed long persistence in vivo and induced enhanced tumor regression when adoptively transferred into mice with progressing tumor. Acquisition of polyfunctionality thus impacts CTL survival and memory formation associated with immunological control of tumor.     

V gamma 9 V delta 2 T cell cytotoxicity against tumor cells is enhanced by monoclonal antibody drugs--rituximab and trastuzumab

V gamma 9 V delta 2 T cells exert potent cytotoxicity toward various tumor cells and adoptive transfer of V gamma 9 V delta 2 T cells is an attractive proposition for cell based immunotherapy. V gamma 9 V delta 2 T cells expanded in the presence of Zoledronate and IL-2 express CD16 (Fc gamma RIII), which raises the possibility that V gamma 9 V delta 2 T cells could be used in conjunction with tumor targeting monoclonal antibody drugs to increase antitumor cytotoxicity by antibody dependent cellular cytotoxicity (ADCC). Cytotoxic activity against CD20-positive B lineage lymphoma or chronic lymphocytic leukemia (CLL) and HER2-positive breast cancer cells was assessed in the presence of rituximab and trastuzumab, respectively. Cytotoxicity of V gamma 9 V delta 2 T cells against CD20-positive targets was higher when used in combination with rituximab. Similarly, V gamma 9 V delta 2 T cells used in combination with trastuzumab resulted in greater cytotoxicity against HER2-positive cells in comparison with either agent alone and this effect was restricted to the CD16(+)V gamma 9 V delta 2 T cell population. Our results show that CD16(+)V gamma 9 V delta 2 T cells recognize monoclonal antibody coated tumor cells via CD16 and exert ADCC similar to that observed with NK cells, even when target cells are relatively resistant to monoclonal antibodies or V gamma 9 V delta 2 T cells alone. Combination therapy involving ex vivo expanded CD16(+)V gamma 9 V delta 2 T cells and monoclonal antibodies may enhance the clinical outcomes for patients treated with monoclonal antibody therapy.