CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.

Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy.

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell-like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell-like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell-like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma-bearing mice. These findings provide a rationale for clinical exploitation of ex vivo-generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice.

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

siRNA silencing of PD-1 ligands on dendritic cell vaccines boosts the expansion of minor histocompatibility antigen-specific CD8(+) T cells in NOD/SCID/IL2Rg(null) mice.

Allogeneic stem cell transplantation (allo-SCT) can be a curative therapy for patients suffering from hematological malignancies. The therapeutic efficacy is based on donor-derived CD8(+) T cells that recognize minor histocompatibility antigens (MiHAs) expressed by patient's tumor cells. However, these responses are not always sufficient, and persistence and recurrence of the malignant disease are often observed. Therefore, application of additive therapy targeting hematopoietic-restricted MiHAs is essential. Adoptive transfer of MiHA-specific CD8(+) T cells in combination with dendritic cell (DC) vaccination could be a promising strategy. Though effects of DC vaccination in anti-cancer therapy have been demonstrated, improvement in DC vaccination therapy is needed, as clinical responses are limited. In this study, we investigated the potency of program death ligand (PD-L) 1 and 2 silenced DC vaccines for ex vivo priming and in vivo boosting of MiHA-specific CD8(+) T cell responses. Co-culturing CD8(+) T cells with MiHA-loaded DCs resulted in priming and expansion of functional MiHA-specific CD8(+) T cells from the naive repertoire, which was augmented upon silencing of PD-L1 and PD-L2. Furthermore, DC vaccination supported and expanded adoptively transferred antigen-specific CD8(+) T cells in vivo. Importantly, the use of PD-L silenced DCs improved boosting and further expansion of ex vivo primed MiHA-specific CD8(+) T cells in immunodeficient mice. In conclusion, adoptive transfer of ex vivo primed MiHA-specific CD8(+) T cells in combination with PD-L silenced DC vaccination, targeting MiHAs restricted to the hematopoietic system, is an interesting approach to boost GVT immunity in allo-SCT patients and thereby prevent relapse.

Coinhibitory molecules in hematologic malignancies: targets for therapeutic intervention.

The adaptive immune system can be a potent defense mechanism against cancer; however, it is often hampered by immune suppressive mechanisms in the tumor microenvironment. Coinhibitory molecules expressed by tumor cells, immune cells, and stromal cells in the tumor milieu can dominantly attenuate T-cell responses against cancer cells. Today, a variety of coinhibitory molecules, including cytotoxic T lymphocyte-associated antigen-4, programmed death-1, B and T lymphocyte attenuator, LAG3, T-cell immunoglobulin and mucin domain 3, and CD200 receptor, have been implicated in immune escape of cancer cells. Sustained signaling via these coinhibitory molecules results in functional exhaustion of T cells, during which the ability to proliferate, secrete cytokines, and mediate lysis of tumor cells is sequentially lost. In this review, we discuss the influence of coinhibitory pathways in suppressing autologous and allogeneic T cell-mediated immunity against hematologic malignancies. In addition, promising preclinical and clinical data of immunotherapeutic approaches interfering with negative cosignaling, either as monotherapy or in conjunction with vaccination strategies, are reviewed. Numerous studies indicate that coinhibitory signaling hampers the clinical benefit of current immunotherapies. Therefore, manipulation of coinhibitory networks is an attractive adjuvant immunotherapeutic intervention for hematologic cancers after standard treatment with chemotherapy and hematopoietic stem cell transplantation.

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Homing characteristics of donor T cells after experimental allogeneic bone marrow transplantation and posttransplantation therapy for multiple myeloma.

Relapse and graft-versus-host disease remain major problems associated with allogeneic bone marrow (BM) transplantation (allo-BMT) and posttransplantation therapy in patients with multiple myeloma (MM) and other hematologic malignancies. A possible strategy for selectively enhancing the graft-versus-myeloma response and possibly reducing graft-versus-host disease is to increase the migration of alloreactive T cells toward the MM-containing BM. In the present study, we characterized the BM-homing behavior of donor-derived effector T cells in a novel allo-BMT model for the treatment of MM. We observed that posttransplantation immunotherapy consisting of donor lymphocyte infusion (DLI) and vaccination with minor histocompatibility antigen-loaded dendritic cells (DCs) was associated with prolonged survival compared with allo-BMT with no further treatment. Moreover, CD8(+) effector T cells expressing inflammatory homing receptors, including high levels of CD44, LFA-1, and inflammatory chemokine receptors, were recruited to MM-bearing BM. This was paralleled by strongly increased expression of IFN-γ and IFN-γ-inducible chemokines, including CXCL9, CXCL10, and CXCL16, especially in mice treated with DLI plus minor histocompatibility antigen-loaded DC vaccination. Remarkably, expression of the homeostatic chemokine CXCL12 was reduced. Furthermore, IFN-γ and TNF-α induced BM endothelial cells to express high levels of the inflammatory chemokines and reduced or unaltered levels of CXCL12. Finally, presentation of CXCL9 by multiple BM endothelial cell-expressed heparan sulfate proteoglycans triggered transendothelial migration of effector T cells. Taken together, our data demonstrate that both post-transplantation DLI plus miHA-loaded DC vaccination and MM growth result in an increased expression of inflammatory homing receptors on donor T cells, decreased levels of the homeostatic BM-homing chemokine CXCL12, and strong induction of inflammatory chemokines in the BM. Thus, along with increasing the population of alloreactive T cells, post-transplantation immunotherapy also might contribute to a more effective graft-versus-tumor response by switching homeostatic T cell migration to inflammation-driven migration.

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation.

Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8(+) T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.

siRNA silencing of PD-L1 and PD-L2 on dendritic cells augments expansion and function of minor histocompatibility antigen-specific CD8+ T cells.

Tumor relapse after human leukocyte antigen-matched allogeneic stem cell transplantation (SCT) remains a serious problem, despite the long-term presence of minor histocompatibility antigen (MiHA)-specific memory T cells. Dendritic cell (DC)-based vaccination boosting MiHA-specific T-cell immunity is an appealing strategy to prevent or counteract tumor recurrence, but improvement is necessary to increase the clinical benefit. Here, we investigated whether knockdown of programmed death ligand 1 (PD-L1) and PD-L2 on monocyte-derived DCs results in improved T-cell activation. Electroporation of single siRNA sequences into immature DCs resulted in efficient, specific, and long-lasting knockdown of PD-L1 and PD-L2 expression. PD-L knockdown DCs strongly augmented interferon-γ and interleukin-2 production by stimulated T cells in an allogeneic mixed lymphocyte reaction, whereas no effect was observed on T-cell proliferation. Moreover, we demonstrated that PD-L gene silencing, especially combined PD-L1 and PD-L2 knockdown, resulted in improved proliferation and cytokine production of keyhole limpet hemocyanin-specific CD4(+) T cells. Most importantly, PD-L knockdown DCs showed superior potential to expand MiHA-specific CD8(+) effector and memory T cells from leukemia patients early after donor lymphocyte infusion and later during relapse. These data demonstrate that PD-L siRNA electroporated DCs are highly effective in enhancing T-cell proliferation and cytokine production, and are therefore attractive cells for improving the efficacy of DC vaccines in cancer patients.

Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells.

CD8(+) T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. We found that P2X5 is significantly expressed in CD34(+) leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. Here, we demonstrate that LRH-1-specific CD8(+) T-cell responses are frequently induced in myeloid leukemia patients following donor lymphocyte infusions. Patients with high percentages of circulating LRH-1-specific CD8(+) T cells had no or only mild graft-versus-host disease. Functional analysis showed that LRH-1-specific cytotoxic T lymphocytes (CTLs) isolated from 2 different patients efficiently target LRH-1-positive leukemic CD34(+) progenitor cells from both CML and AML patients, whereas mature CML cells are only marginally lysed due to down-regulation of P2X5. Furthermore, we observed that relative resistance to LRH-1 CTL-mediated cell death due to elevated levels of antiapoptotic XIAP could be overcome by IFN-gamma prestimulation and increased CTL-target ratios. These findings provide a rationale for use of LRH-1 as immunotherapeutic target antigen to treat residual or persisting myeloid malignancies after allogeneic SCT.

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis.

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.

Intratumoral rhIL-12 administration in head and neck squamous cell carcinoma patients induces B cell activation.

The objectives of this study were to investigate the effects of intratumorally (i.t.) administered recombinant human interleukin-12 (rhIL-12) on the distribution and function of B cells in the primary tumors, the locoregional lymph nodes and peripheral blood of head and neck squamous cell carcinoma (HNSCC) patients. The initial characterization of the patients participating in the phase Ib and phase II studies has previously been reported. After rhIL-12 treatment, fewer secondary follicles with a broader outer region of the mantle zones and an increase in interfollicular B-blasts were seen in the enlarged lymph nodes compared with control HNSCC patients. The size of the germinal center (GC) was diminished, partly due to a decrease in the number of CD57+ GC cells that have been associated with immune suppression. These changes did not correlate with signs of apoptosis or CXCR5 expression by B cells. Strikingly, in 3 out of 4 IL-12 treated patients, increased IFN-gamma mRNA expression by B cells was detected. In addition, a highly significant IgG subclass switch was seen in the plasma with more IgG1, less IgG2 and more IgG4, indicating a switch to T helper 1 phenotype. Finally, peritumoral B cell infiltration was a positive prognostic sign for overall survival in the 30 HNSCC patients investigated, irrespective of IL-12 treatment. In conclusion, these data indicate that after i.t. IL-12 treatment in HNSCC, significant activation of the B cell and the B cell compartment occurred and that the presence of tumor infiltrating B cells correlated with overall survival of HNSCC patients.

Expression of P2X5 in lymphoid malignancies results in LRH-1-specific cytotoxic T-cell-mediated lysis.

Minor histocompatibility antigens (MiHA) selectively expressed by haematopoietic cells are attractive targets for specific immunotherapy after allogeneic stem cell transplantation (SCT). Previously, we described LRH-1 as a haematopoietic-lineage restricted MiHA that is capable of eliciting an allogeneic cytotoxic T-lymphocyte (CTL) response after SCT and donor lymphocyte infusion. Importantly, the gene encoding LRH-1, P2X5, is not expressed in prominent graft-versus-host-disease target tissues such as skin, liver and gut. Here, we investigate whether LRH-1-specific immunotherapy may be exploited for the treatment of lymphoid malignancies. We examined P2X5 mRNA expression in a large panel of patient samples and cell lines from different types of lymphoid malignancies by real-time quantitative reverse transcription polymerase chain reaction. P2X5 mRNA was highly expressed in malignant cells from all stages of lymphoid development. Furthermore, all LRH-1-positive lymphoid tumour cell lines were susceptible to LRH-1 CTL-mediated lysis in flow cytometry-based cytotoxicity assays. However, interferon-gamma production was low or absent after stimulation with some cell lines, possibly due to differences in activation thresholds for CTL effector functions. Importantly, primary cells from patients with lymphoid malignancies were effectively lysed by LRH-1-specific CTL. These findings indicate that MiHA LRH-1 is a potential therapeutic target for cellular immunotherapy of lymphoid malignancies.

Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines, toll-like receptors and rheumatoid synovial fluid.

BACKGROUND: The T cell attracting chemokine CCL18 is produced by antigen presenting cells and a role for CCL18 has been suggested in the pathogenesis of a variety of diseases. Rheumatoid arthritis (RA) is one of these conditions, in which abundant CCL18 production is present. Although Th2 cytokines and IL-10 are known to have an effect on CCL18 production, there are several gaps in our knowledge regarding the exact regulation of CCL18 secretion, both in general and in RA. In this study we provide new insights in the regulation of CCL18 secretion by monocytes and dendritic cells. RESULTS: In contrast to a large panel of pro-inflammatory stimuli (IL-1beta, TNF-alpha, IL-10, IL-13, IL-15, IL-17, IL-18, IFN-gamma), T cell mimicking molecules (RANKL, CD40L) or TLR driven maturation, the anti-inflammatory IL-10 strongly stimulated DC to secrete CCL18. On freshly isolated monocytes, CCL18 secretion was induced by IL-4 and IL-13, in strong synergy with IL-10. This synergistic effect could already be observed after only 24 hours, indicating that not only macrophages and dendritic cells, but also monocytes secrete CCL18 under these stimulatory conditions. A high CCL18 expression was detected in RA synovial tissue and incubation of monocytes with synovial fluid from RA patients clearly enhanced the effects of IL-4, IL-13 and IL-10. Surprisingly, the effect of synovial fluid was not driven by IL-10 of IL-13, suggesting the presence of another CCL18 inducing factor in synovial fluid. CONCLUSION: In summary, IL-10 synergistically induces CCL18 secretion in combination with IL-4 of IL-13 on monocytes and monocyte derived cells. The effects of IL-14, IL-13 and IL-10 are strongly enhanced by synovial fluid. This synergy may contribute to the high CCL18 expression in RA.

Novel monoclonal antibodies detect elevated levels of the chemokine CCL18/DC-CK1 in serum and body fluids in pathological conditions.

CC chemokine ligand 18/dendritic cell-chemokine 1 (CCL18/DC-CK1) is a CC chemokine, preferentially expressed by DC, which acts as a chemoattractant for naive T cells and mantle zone B cells. Applying a newly developed CCL18/DC-CK1 sandwich enzyme-linked immunosorbent assay, we demonstrate that DC secrete high amounts of CCL18/DC-CK1 and that this expression can be increased by interleukin-10. High levels of CCL18/DC-CK1 were also detected in human serum (average of 88 ng/ml). Moreover, elevated CCL18/DC-CK1 levels were detected in synovial fluid from rheumatoid arthritis patients and in drain fluid (average of 254 ng/ml and 122 ng/ml, respectively). Immunoprecipitation experiment using anti-CCL18/DC-CK1 monoclonal antibodies revealed a protein of 6-7 kDa in serum and drain fluid that was indistinguishable from recombinant CCL18/DC-CK1 on Western blot and in re-aggregation assays. The concentration of CCL18/DC-CK1 found in human serum is in the same order of magnitude as was previously reported to completely inhibit CCL11/eotaxin-induced CC chemokine receptor 3 (CCR3) activation and consequent migration of eosinophils. CCL18/DC-CK1 may therefore function as an agonist (for naive T and B cells) and as an antagonist for CCR3-expressing leukocytes such as eosinophils.

In situ tumor ablation creates an antigen source for the generation of antitumor immunity.

Tumor-destructing techniques, like radiofrequency ablation (RFA), allow eradication of large tumors. Potentially, in situ tumor destruction also can provide the immune system with an antigen source for the induction of antitumor immunity. Antigen-presenting cells could take up antigens in the periphery after which they induce specific immune responses. Recent data show that especially antigen-presenting dendritic cells are crucial for the induction of potent immune responses. However, virtually nothing is known regarding the induction of immune responses after in situ tumor destruction in mice or humans. We used the well-defined murine B16-OVA melanoma cell line to develop a novel tumor model to explore: (a). the immunologic consequences of in situ tumor destruction; and (b). the efficacy of a combination approach of tumor destruction and immunostimulation. Applying this model system we demonstrate that following RFA, a weak but detectable immune response develops, directed against OVA, but also against a broader range of B16 antigens. Adoptive transfer experiments further indicate that antitumor reactivity can be transferred to naïve mice by splenocytes. To augment the response observed, we administered a blocking monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 at the time of tumor destruction. Interestingly, this strongly enhanced antitumor immunity, resulting in long-lasting tumor protection. These results illustrate that in situ tumor destruction can provide a useful antigen source for the induction of antitumor immunity, provided that additional immunostimulatory signals are coadministered.

Interleukin-12 has no effect on vascular density, perfusion, hypoxia and proliferation of an implanted human squamous cell carcinoma xenograft tumour despite up-regulation of ICAM-1.

BACKGROUND: Interleukin-12 is an anti-angiogenic and antitumor agent in many transplanted murine tumour models. In a previous clinical study in head and neck squamous cell carcinoma patients treated with rhIL-12 the tumour turned pale, after an initial reddening. The aim of this study was to investigate the effects of rmIL-12 on the vasculature, blood perfusion, hypoxia and proliferation of tumour cells in an implanted human head and neck squamous cell carcinoma xenograft tumour, with a relatively large diameter, in Balb/c nu/nu mice over time. MATERIALS AND METHODS: Established human squamous cell carcinoma xenograft tumours were intratumorally injected for 3 days with either 200 ng rmIL-12 or PBA. Mice were sacrificed at 4 different time points (between 8 hours and 8 days after the last injection), after administration of Pimonidazole, BrdUrd and Hoechst 33342. The tumour sections were quantitatively analysed with a semi-automatic method based on a computerised digital image analysis system, after immunohistochemical staining. RESULTS: Despite a faster and higher up-regulation of anti-mouse ICAM-1 in the IL-12-treated tumours, no significant differences in vascular density, perfusion fraction, hypoxic fraction and BrdUrd labelling index were detected between IL-12-treated tumour and control tumours. CONCLUSION: We suggest that the main reason why the observation made in humans could not be confirmed in this mice study is the combination of a lack of an intact immune system in the Balb/c nu/nu mice and a relatively large tumour with probably a lot of mature vessels.

Efficient nontoxic delivery of PD-L1 and PD-L2 siRNA into dendritic cell vaccines using the cationic lipid SAINT-18.

Dendritic cell (DC)-based vaccination is an appealing strategy to boost graft-versus-tumor immunity after allogeneic stem cell transplantation (allo-SCT), and thereby prevent or counteract tumor recurrence. By exploiting minor histocompatibility antigens (MiHA) presented on hematopoietic cells, donor CD8 T-cell immunity can be selectively targeted to patient's hematological tumor cells without the risk of inducing graft-versus-host disease. Previously, we demonstrated that silencing RNA (siRNA) of programmed death-ligand 1 (PD-L1) and PD-L2 on DCs markedly augments the expansion and function of MiHA-specific CD8 T cells. However, previously applied methods based on electroporation or lipid nanoparticles were either incompatible with target antigen mRNA delivery or required complex manufacturing compliant to Good Manufacturing Practice. Here, we investigated whether transfection using lipoplexes composed of PD-L1 and PD-L2 siRNAs plus SAINT-18:DOPE (ie, SAINT-RED) is an effective and feasible clinical-grade method in DC vaccine manufacturing. We observed that a single siRNA/SAINT-RED transfection resulted in efficient and long-term knockdown of the PD-1 ligands without affecting DC maturation or viability. Furthermore, we demonstrated that SAINT-RED can be heat sterilized without loss of function, facilitating its use in aseptic DC vaccine production. Finally, we showed that the established transfection method can be combined with target antigen mRNA or peptide loading to efficiently stimulate MiHA-specific T-cell expansion and cytokine production. Together, these findings indicate that the developed PD-L siRNA/SAINT-RED transfection protocol in combination with MiHA mRNA or peptide loading can be applied in the generation of clinical-grade DC vaccines to boost antitumor immunity after allo-SCT.

Combined IL-15 and IL-12 drives the generation of CD34+-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer.

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34+ hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.