Birgit L. M. G. Senden-Gijsbers

Inflammation restraining effects of prostaglandin E2 on natural killer–dendritic cell (NK-DC) interaction are imprinted during DC maturation

Among prostaglandins (PGs), PGE2 is abundantly expressed in various malignancies and is probably one of many factors promoting tumor growth by inhibiting tumor immune surveillance. In the current study, we report on a novel mechanism by which PGE2 inhibits in vitro natural killer-dendritic cell (NK-DC) crosstalk and thereby innate and adaptive immune responses via its effect on NK-DC crosstalk. The presence of PGE2 during IFN-γ/membrane fraction of Klebsiella pneumoniae DC maturation inhibits the production of chemokines (CCL5, CCL19, and CXCL10) and cytokines (IL-12 and IL-18), which is cAMP-dependent and imprinted during DC maturation. As a consequence, these DCs fail to attract NK cells and show a decreased capacity to trigger NK cell IFN-γ production, which in turn leads to reduced T-helper 1 polarization. In addition, the presence of PGE2 during DC maturation impairs DC-mediated augmentation of NK-cell cytotoxicity. Opposed to their inhibitory effects on peripheral blood-derived NK cells, PGE2 matured DCs induce IL-22 secretion of inflammation constraining NKp44(+) NK cells present in mucosa-associated lymphoid tissue. The inhibition of NK-DC interaction is a novel regulatory property of PGE2 that is of possible relevance in dampening immune responses in vivo.

Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin‐1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti‐MUC1 antibodies. Seventy‐three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation‐dependent and cancer‐associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Klebsiella pneumoniae-triggered DC recruit human NK cells in a CCR5-dependent manner leading to increased CCL19-responsiveness and activation of NK cells.

Besides their role in destruction of altered self‐cells, NK cells have been shown to potentiate T‐cell responses by interacting with DC. To take advantage of NK–DC crosstalk in therapeutic DC‐based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK‐cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5‐dependent manner. An additional mechanism of DC‐induced NK‐cell recruitment is characterized by the induction of CCR7 expression on CD56dimCD16+ NK cells after physical contact with membrane fraction of K. pneumoniae‐matured DC, resulting in an enhanced migratory responsiveness to the lymph node‐associated chemokine CCL19. Bacterial fragment‐matured DC do not only mediate NK‐cell migration but also meet the prerequisites needed for augmentation of NK‐cell cytotoxicity and IFN‐γ production, the latter of which contributes to Th1 polarization.

INCREASED TUMOR-SPECIFIC CD8(+) T CELL INDUCTION BY DENDRITIC CELLS MATURED WITH A CLINICAL GRADE TLR-AGONIST IN COMBINATION WITH IFN-gamma

The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-γ (FI-cocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF-α/PGE-2, i.e. TP-cocktail). In addition to the assessment of CD4+ T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8+ T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Th1 and Th17 polarization, we demonstrate in this study that FMKp/IFN-γ matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.

Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.

The efficacy of donor HSCT is partly reduced as a result of slow post‐transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection‐related mortality in the first years post‐transplantation. Adoptive transfer of in vitro‐generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder‐free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell‐stage arrest occurred. We conclude that neither DLL4‐expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G‐CSF‐mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Combination of radiotherapy with the immunocytokine L19-IL2: Additive effect in a NK cell dependent tumour model.

BACKGROUND AND PURPOSE Recently, we have shown that radiotherapy (RT) combined with the immunocytokine L19-IL2 can induce long-lasting antitumour effects, dependent on ED-B expression and infiltration of cytotoxic T cells. On the other hand, in certain tumours, IL2 treatment can trigger a natural killer cell (NK) immune response. The aim of this study is to investigate the therapeutic effect of our combination therapy in the ED-B positive F9 teratocarcinoma model, lacking MHCI expression and known to be dependent on NK immune responses. MATERIAL AND METHODS In syngeneic F9 tumour bearing 129/FvHsd mice tumour growth delay was evaluated after local tumour irradiation (10Gy) combined with systemic administration of L19-IL2. Immunological responses were investigated using flow cytometry. RESULTS Tumour growth delay of L19-IL2 can be further improved by a single dose of RT administered before immunotherapy, but not during immunotherapy. Furthermore, treatment of L19-IL2 favours a NK response and lacks cytotoxic T cell tumour infiltrating immune cells, which may be explained by the absence of MHCI expression. CONCLUSION An additive effect can be detected when the NK dependent F9 tumour model is treated with radiotherapy and L19-IL2 and therefore this combination could be useful in the absence of tumoural MHCI expression.

Transduction with a fiber-modified adenoviral vector is superior to non-viral nucleofection for expressing tumor-associated Ag mucin-1 in human DC

BACKGROUND DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase the chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. Methodologically, several recombinant DNA delivery techniques have been used. METHODS In this study we compared nucleofection, an optimized form of electroporation, and adenoviral transduction regarding their efficiency to transduce human monocyte-derived (Mo-) DC in vitro. Expression of the tumor-associated Ag mucin-1 (MUC1) after adenoviral transduction (rAd5Fib35-MUC1) was determined using two MAb. RESULTS We showed that the viability of cells and percentage of green fluorescent protein (GFP)-positive cells after transduction with a fiber-modified adenoviral vector (rAd5F35-GFP) was much higher than after nucleofection. Furthermore, phenotype and function of DC were not impaired by infection with adenovirus particles. Cells matured normally; up-regulation of CD40, CD80, CD83, CD86 and HLA-DR was not affected by adenoviral transduction. The capacity to stimulate naive T-cell proliferation was preserved and no change in IL-10 production was observed. Production of IL-12 increased up to 500-fold upon adenoviral transduction, considered to contribute positively to an anti-tumor immune response. Non-transduced mature DC expressed low levels of endogenous MUC1. After transduction with the rAd5F35-MUC1 adenoviral vector, a 100-fold increase in MUC1 expression by DC was observed. DISCUSSION The use of the fiber-modified adenoviral vector presented here may therefore be favorable compared with non-viral gene delivery systems for DC that will be used in cancer immunotherapy.

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector

BACKGROUND DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Fractionated Radiotherapy with 3 x 8 Gy Induces Systemic Anti-Tumour Responses and Abscopal Tumour Inhibition without Modulating the Humoral Anti-Tumour Response

Accumulating evidence indicates that fractionated radiotherapy (RT) can result in distant non-irradiated (abscopal) tumour regression. Although preclinical studies indicate the importance of T cells in this infrequent phenomenon, these studies do not preclude that other immune mechanisms exhibit an addition role in the abscopal effect. We therefore addressed the question whether in addition to T cell mediated responses also humoral anti-tumour responses are modulated after fractionated RT and whether systemic dendritic cell (DC) stimulation can enhance tumour-specific antibody production. We selected the 67NR mammary carcinoma model since this tumour showed spontaneous antibody production in all tumour-bearing mice. Fractionated RT to the primary tumour was associated with a survival benefit and a delayed growth of a non-irradiated (contralateral) secondary tumour. Notably, fractionated RT did not affect anti-tumour antibody titers and the composition of the immunoglobulin (Ig) isotypes. Likewise, we demonstrated that treatment of tumour-bearing Balb/C mice with DC stimulating growth factor Flt3-L did neither modulate the magnitude nor the composition of the humoral immune response. Finally, we evaluated the immune infiltrate and Ig isotype content of the tumour tissue using flow cytometry and found no differences between treatment groups that were indicative for local antibody production. In conclusion, we demonstrate that the 67NR mammary carcinoma in Balb/C mice is associated with a pre-existing antibody response. And, we show that in tumour-bearing Balb/C mice with abscopal tumour regression such pre-existing antibody responses are not altered upon fractionated RT and/or DC stimulation with Flt3-L. Our research indicates that evaluating the humoral immune response in the setting of abscopal tumour regression is not invariably associated with therapeutic effects.

Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy

BACKGROUND AIMS Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required. METHODS Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34(+) hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. RESULTS Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell-derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. CONCLUSIONS Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy.