Phase I trial evaluating safety and efficacy of intratumorally administered inflammatory allogeneic dendritic cells (ilixadencel) in advanced gastrointestinal stromal tumors.

BACKGROUND: The majority of patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Ilixadencel (allogeneic inflammatory dendritic cells) is a cell-based immune primer injected intratumorally that previously has been clinically investigated in metastatic renal cell carcinoma and hepatocellular carcinoma. METHODS: The trial was a single arm phase I trial assessing safety and efficacy of ilixadencel in subjects with progressing advanced/metastatic GIST despite ongoing treatment with second or later lines of tyrosine kinase inhibitors (TKI). Three patients were progressing while on sunitinib (second line), one on regorafenib (third line), and two on pazopanib (fourth line). TKI treatment was maintained throughout, while two intratumoral injections of ilixadencel (10 × 106 viable and HLA-DR expressing cells per dose) were administered. RESULTS: No severe adverse events were found to be related to ilixadencel administration. Four patients showed continued tumor progression at 3 months per RECIST 1.1 and Choi criteria. One patient (on third line regorafenib) had stable disease for 9 months and another patient (on second line sunitinib) had stable disease at end of study (12 months) as per RECIST 1.1. These two patients developed a partial response as per Choi criteria with a duration of 3 and 6 months, respectively. The median progression-free survival (PFS) was 4.0 months. CONCLUSION: Ilixadencel treatment presented an acceptable safety profile among advanced GIST patients who developed resistance to TKI. Encouraging radiological tumor responses were detected in 33% of treated patients, supporting further investigation. Clinical trial registration www.clinicaltrials.gov ; NCT: 02432846; registration date: February 22, 2016.

Ilixadencel - an Allogeneic Cell-Based Anticancer Immune Primer for Intratumoral Administration.

Intratumoral administration of an immune primer is a therapeutic vaccine strategy aimed to trigger dendritic cell (DC)-mediated cross-presentation of cell-associated tumor antigens to cytotoxic CD8+ T cells without the need for tumor antigen characterization. The prevailing view is that these cross-presenting DCs have to be directly activated by pathogen-associated molecular patterns (PAMPS), including Toll-like receptor ligands or live microbial agents like oncolytic viruses. Emerging data are however challenging this view, indicating that the cross-presenting machinery in DCs is suboptimally activated by direct PAMP recognition, and that endogenous inflammatory factors are the main drivers of DC-mediated cross-presentation within the tumor. Here we present preclinical mode of action data, CMC and regulatory data, as well as initial clinical data on ilixadencel. This cell-based drug product is an off-the-shelf immune primer, consisting of pro-inflammatory allogeneic DCs secreting high amounts of pro-inflammatory chemokines and cytokines at the time of intratumoral administration. The mechanism of action of ilixadencel is to induce recruitment and activation of endogenous immune cells, including NK cells that subsequently promotes cross-presentation of cell-associated tumor antigens by co-recruited DCs.

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists.

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.

Melan-A specific CD8+ T lymphocytes after hyperthermic isolated limb perfusion: a pilot study in patients with in-transit metastases of malignant melanoma.

PURPOSE: Isolated limb perfusion (ILP) with hyperthermia is an effective treatment for in-transit metastases of malignant melanoma in the extremities. Preclinical studies have shown that hyperthermia may induce an immunogenic death of tumour cells. We therefore decided to study whether ILP may induce tumour-specific immune responses in the clinical setting. METHOD: The number of Melan-A/Mart-1 specific CD8+ T cells, as well as other phenotypically different immune cells, was recorded in peripheral blood in 12 HLA-A2+ patients with in-transit metastases undergoing hyperthermic ILP with melphalan. RESULTS: All patients underwent ILP without any complication and with an overall response rate of 83%. No substantial changes in the number of circulating T-cells, B-cells, NK-cells or monocytes were observed during follow-up. Four out of 12 patients showed an elevation of Melan-A+ CD8+ T-cells 4 weeks after ILP. CONCLUSION: We here report our preliminary observations that a small increase in tumour-specific T-cells could be seen in a subpopulation of patients after ILP. However, much more work is necessary to fully delineate the systemic immune response to hyperthermic ILP.

Proinflammatory allogeneic dendritic cells enhance the therapeutic efficacy of systemic anti-4-1BB treatment.

As an immune adjuvant, proinflammatory allogeneic dendritic cells (AlloDCs) have demonstrated promising immune-priming effects in several preclinical and clinical studies. The effector cells, including NK cells and T cells are widely acknowledged as pivotal factors in the effectiveness of cancer immunotherapy due to their ability to selectively identify and eradicate malignant cells. 4-1BB, as a costimulatory receptor, plays a significant role in the stimulation of effector cell activation. This study evaluated the anti-tumor effects when combining intratumoral administration of the immune-adjuvant AlloDCs with systemic α4-1BB treatment directly acting on effector cells. In both the CT-26 murine colon carcinoma model and B16 murine melanoma model, AlloDCs demonstrated a significant enhancement in the therapeutic efficacy of α4-1BB antibody. This enhancement was observed through the delayed growth of tumors and prolonged survival. Analysis of the tumor microenvironment (TME) in the combined-treatment group revealed an immune-inflamed TME characterized by increased infiltration of activated endogenous DCs and IFNγ+ CD8+ T cells, showing reduced signs of exhaustion. Furthermore, there was an augmented presence of tissue-resident memory (TRM) CD8+ T cells (CD103+CD49a+CD69+). The combination treatment also led to increased infiltration of CD39+CD103+ tumor-specific CD8+ T cells and neoantigen-specific T cells into the tumor. Additionally, the combined treatment resulted in a less immunosuppressive TME, indicated by decreased infiltration of myeloid-derived suppressor cells and Tregs. These findings suggest that the combination of intratumoral AlloDCs administration with systemic agonistic α4-1BB treatment can generate a synergistic anti-tumor response, thereby warranting further investigation through clinical studies.

Intratumoral administration of pro-inflammatory allogeneic dendritic cells improved the anti-tumor response of systemic anti-CTLA-4 treatment via unleashing a T cell-dependent response.

Immune checkpoint inhibitors (ICIs) have revolutionized the oncology field. However, a significant number of patients do not respond, at least partly due to the lack of preexisting anti-tumor T-cell immunity. Therefore, it is emergent to add an immune-priming step to improve efficacy. Here, we report a combined approach consisting of intratumoral administration of pro-inflammatory allogeneic dendritic cells (AlloDCs) and systemic treatment with αCTLA-4 that can drastically improve the anti-tumor efficacy compared to αCTLA-4 monotherapy. When evaluated in mice with large established CT-26 tumors, monotherapy with αCTLA-4 neither delayed tumor progression nor improved mice survival. However, combination treatment of AlloDCs and αCTLA-4 drastically improved the effectiveness, with 70% of mice being cured. This effect was T cell-dependent, and all survived mice rejected a subsequent tumor re-challenge. Further investigation revealed an immune-inflamed tumor microenvironment (TME) in the combination treatment group characterized by enhanced infiltration of activated antigen-presenting endogenous DCs and CD8+ T cells with a tissue-resident memory (TRM) phenotype (CD49a+CD103+). This correlated with elevated levels of tumor-specific CD39+CD103+CD8+ T cells in the tumor and "tumor-matching" NKG2D+CD39+CX3CR1+CD8+ T cells in peripheral blood. Moreover, splenocytes from mice in the combination treatment group secreted significantly higher IFN-γ upon stimulation with the peptide from the endogenous CT-26 retroviral gp70 (model neoantigen), confirming the induction of a tumor-specific CD8+ T-cell response. Taken together, these data indicate a strong anti-tumor synergy between AlloDCs and αCTLA-4 that warrant further clinical investigation with the corresponding human AlloDC product (ilixadencel) for patients receiving αCTLA-4 therapy.

Ilixadencel, a Cell-based Immune Primer, plus Sunitinib Versus Sunitinib Alone in Metastatic Renal Cell Carcinoma: A Randomized Phase 2 Study.

Background: The prognosis of patients with synchronous metastatic renal cell carcinoma (mRCC) is poor. Whereas single-agent tyrosine kinase inhibition (TKI) is clearly insufficient, the effects can be enhanced by combinations with immune checkpoint inhibitors. Innovative treatment options combining TKI and other immune-stimulating agents could prove beneficial. Objective: To evaluate the clinical effects on metastatic disease when two doses of allogeneic monocyte-derived dendritic cells (ilixadencel) are administrated intratumorally followed by nephrectomy and treatment with sunitinib compared with nephrectomy and sunitinib monotherapy, in patients with synchronous mRCC. Design setting and participants: A randomized (2:1) phase 2 multicenter trial enrolled 88 patients with newly diagnosed mRCC to treatment with the combination ilixadencel/sunitinib (ILIXA/SUN; 58 patients) or sunitinib alone (SUN; 30 patients). Outcome measurements and statistical analysis: The primary endpoints were 18-mo survival rate and overall survival (OS). A secondary endpoint was objective response rate (ORR) assessed up to 18 mo after enrollment. Statistic evaluations included Kaplan-Meier estimates, log-rank tests, Cox regression, and stratified Cochran-Mantel-Haenszel tests. Results and limitations: The median OS was 35.6 mo in the ILIXA/SUN arm versus 25.3 mo in the SUN arm (hazard ratio 0.73, 95% confidence interval 0.42-1.27; p = 0.25), while the 18-mo OS rates were 63% and 66% in the ILIXA/SUN and SUN arms, respectively. The confirmed ORR in the ILIXA/SUN arm were 42.2% (19/45), including three patients with complete response, versus 24.0% (six/25) in the SUN arm (p = 0.13) without complete responses. The study was not adequately powered to detect modest differences in survival. Conclusions: The study failed to meet its primary endpoints. However, ilixadencel in combination with sunitinib was associated with a numerically higher, nonsignificant, confirmed response rate, including complete responses, compared with sunitinib monotherapy. Patient summary: We studied the effects of intratumoral vaccination with ilixadencel followed by sunitinib versus sunitinib only in a randomized phase 2 study. The combination treatment showed numerically higher numbers of confirmed responses, suggesting an immunologic effect.

Tumour-loaded α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia produce a superior NK-, NKT- and CD8+ T cell-attracting chemokine profile.

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1ß/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1ß/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.

The peptide AF-16 decreases high interstitial fluid pressure in solid tumors.

BACKGROUND: The high interstitial fluid pressure (IFP) in solid tumors restricts the access to nutrients, oxygen and drugs. MATERIAL AND METHODS: We investigated the ability of the peptide AF-16, involved in water and ion transfer through cell membranes, to lower the IFP in two different solid rat mammary tumors, one chemically induced, slowly growing, and the other transplantable, and rapidly progressing having high cellularity. AF-16 was administered either in the tumor capsule, intranasally or intravenously. The IFP was measured by a miniature fiber optic device. RESULTS: AF-16 significantly lowered the IFP in both the slowly and the rapidly progressing tumors, whether administrated locally or systemically. The AF-16 induced IFP reduction was maximal after 90 min, lasted at least 3 h, and returned to pretreatment levels in less than 24 h. Topical AF-16 transiently reduced the IFP in the DMBA tumors from 17.7 ± 4.2 mmHg to 8.6 ± 2.1 mmHg. CONCLUSION: We conclude that AF-16 transiently and reversibly lowered the high IFP in solid tumors during a few hours, which might translate into improved therapeutic efficacy.

Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Phase 1 Trial With the Cell-Based Immune Primer Ilixadencel, Alone, and Combined With Sorafenib, in Advanced Hepatocellular Carcinoma.

Several lines of evidence support immunotherapy in hepatocellular carcinoma (HCC). We have shown that intratumoral injections of the immune primer ilixadencel (pro-inflammatory allogeneic dendritic cells) are safe in renal-cell carcinoma. Here, we assessed ilixadencel as a single agent and combined with sorafenib in advanced HCC. Of 17 HCC patients enrolled, 12 patients received ilixadencel at the dose of 10 × 106 cells (six as monotherapy and six in combination with sorafenib), and five received ilixadencel at the dose of 20 × 106 cells as monotherapy. The primary objective was to evaluate tolerability. All patients had at least one adverse event, with 30% of such events considered as treatment-related, with one single treatment-related grade three event. The most common toxicity was grade 1 and 2 fever and chills. Eleven of 15 evaluable patients (73%) showed increased frequency of tumor-specific CD8+ T cells in peripheral blood. Overall one patient had a partial response (with ilixadencel as monotherapy), and five had stable disease as overall best response per mRECIST. The median time to progression was 5.5 months, and overall survival ranged from 1.6 to 21.4 months. Our study confirms the safety of ilixadencel as single agent or in combination with sorafenib and indicates tumor-specific immunological responses in advanced HCC. Clinical Trial Registration: www.ClinicalTrials.gov, identifier: NCT01974661.

Pro-inflammatory allogeneic DCs promote activation of bystander immune cells and thereby license antigen-specific T-cell responses.

Accumulating evidence support an important role for endogenous bystander dendritic cells (DCs) in the efficiency of autologous patient-derived DC-vaccines, as bystander DCs take up material from vaccine-DCs, migrate to draining lymph node and initiate antitumor T-cell responses. We examined the possibility of using allogeneic DCs as vaccine-DCs to activate bystander immune cells and promote antigen-specific T-cell responses. We demonstrate that human DCs matured with polyI:C, R848 and IFN-γ (denoted COMBIG) in combination with an infection-enhanced adenovirus vector (denoted Ad5M) exhibit a pro-inflammatory state. COMBIG/Ad5M-matured allogeneic DCs (alloDCs) efficiently activated T-cells and NK-cells in allogeneic co-culture experiments. The secretion of immunostimulatory factors during the co-culture promoted the maturation of bystander-DCs, which efficiently cross-presented a model-antigen to activate antigen-specific CD8+ T-cells in vitro. We propose that alloDCs, in combination with Ad5M as loading vehicle, may be a cost-effective and logistically simplified DC vaccination strategy to induce anti-tumor immune responses in cancer patients.

Cancer vaccine based on a combination of an infection-enhanced adenoviral vector and pro-inflammatory allogeneic DCs leads to sustained antigen-specific immune responses in three melanoma models.

Autologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens (TAAs) are frequently used as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidence indicate that ex vivo engineered DCs are poor in migration and in fact do not directly present TAA epitopes to naïve T cells in vivo. Instead, it is proposed that bystander host DCs take up material from vaccine-DCs, migrate and subsequently initiate antitumor T-cell responses. We used mouse models to examine the possibility of using pro-inflammatory allogeneic DCs (alloDCs) to activate host DCs and enable them to promote antigen-specific T-cell immunity. We found that alloDCs were able to initiate host DC activation and migration to draining lymph node leading to T-cell activation. The pro-inflammatory milieu created by alloDCs also led to recruitment of NK cells and neutrophils at the site of injection. Vaccination with alloDCs combined with Ad5M(gp100), an infection-enhanced adenovirus encoding the human melanoma-associated antigen gp100 resulted in generation of CD8+ T cells with a T-cell receptor (TCR) specific for the gp10025-33 epitope (gp100-TCR+). Ad5M(gp100)-alloDC vaccination in combination with transfer of gp100-specific pmel-1 T cells resulted in prolonged survival of B16-F10 melanoma-bearing mice and altered the composition of the tumor microenvironment (TME). We hereby propose that alloDCs together with TAA- or neoepitope-encoding Ad5M can become an "off-the-shelf" cancer vaccine, which can reverse the TME-induced immunosuppression and induce host cellular anti-tumor immune responses in patients without the need of a time-consuming preparation step of autologous DCs.

Intratumorally injected pro-inflammatory allogeneic dendritic cells as immune enhancers: a first-in-human study in unfavourable risk patients with metastatic renal cell carcinoma.

BACKGROUND: Accumulating pre-clinical data indicate that the efficient induction of antigen-specific cytotoxic CD8+ T cells characterizing viral infections is caused by cross-priming where initially infected DCs produce an unique set of inflammatory factors that recruit and activate non-infected bystander DCs. Our DC-based immunotherapy concept is guided by such bystander view and accordingly, we have developed a cellular adjuvant consisting of pre-activated allogeneic DCs producing high levels of DC-recruiting and DC-activating factors. This concept doesn't require MHC-compatibility between injected cells and the patient and therefore introduces the possibility of using pre-produced and freeze-stored DCs from healthy blood donors as an off- the-shelf immune enhancer. The use of MHC-incompatible allogeneic DCs will further induce a local rejection process at the injection site that is expected to further enhance recruitment and maturation of endogenous bystander DCs. METHODS: Twelve intermediate and poor risk patients with newly diagnosed metastatic renal cell carcinoma (mRCC) where included in a phase I/II study. Pro-inflammatory allogeneic DCs were produced from a leukapheresis product collected from one healthy blood donor and subsequently deep-frozen. A dose of 5-20 × 106 DCs (INTUVAX) was injected into the renal tumor twice with 2 weeks interval before planned nephrectomy and subsequent standard of care. RESULTS: No INTUVAX-related severe adverse events were observed. A massive infiltration of CD8+ T cells was found in 5 out of 12 removed kidney tumors. No objective tumor response was observed and 6 out of 11 evaluable patients have subsequently received additional treatment with standard tyrosine kinase inhibitors (TKI). Three of these 6 patients experienced an objective tumor response including one sunitinib-treated patient who responded with a complete and durable regression of 4 brain metastases. Median overall survival (mOS) is still not reached (currently 42.5 months) but has already passed historical mOS in patients with unfavourable risk mRCC on standard TKI therapy. CONCLUSIONS: Our findings indicate that intratumoral administration of proinflammatory allogeneic DCs induces an anti-tumor immune response that may prolong survival in unfavourable risk mRCC-patients given subsequent standard of care. A randomized, multi-center, phase II mRCC trial (MERECA) with INTUVAX in conjuction with sunitinib has been initiated. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01525017.

Characterization of Innate Immunity in an Extended Whole Blood Model of Human Islet Allotransplantation.

The instant blood-mediated inflammatory reaction (IBMIR) has been studied in whole blood models of human allo-islet transplantation for short periods (<6 h). Beyond this time frame the innate response to intraportally transplanted islets is less well described. A novel whole blood model was applied to study blood-islet-graft interactions up to 48 h. Heparinized polyvinyl chloride tubing was sealed into small bags containing venous blood together with allogeneic human islets and exocrine tissue, respectively. The bags were attached to a rotating wheel (37°C). Concentrated glucose and sodium hydrogen carbonate were added every 12 h to maintain physiological limits for sustained immune cell functions. Plasma was collected at repeated time points for analyses of coagulation/complement activation and chemokine/cytokine production. Immune cell infiltration was analyzed using immunohistochemistry. Coagulation and platelet activation markers, thrombin-antithrombin complex (TAT) and soluble CD40 ligand (sCD40L) showed early high concentrations (at 6-12 h). sC5b-9 steadily increased over 48 h. At 6 h neutrophils and monocytes surrounded the clotted cellular grafts with a following massive infiltration of neutrophils. High and increasing concentrations of CXCR1/2 ligands [IL-8 and growth-regulated oncogene α/ß/γ (Gro-α/ß/γ)] and IL-6 were produced in response to human islets and exocrine tissue. The CCR2 ligand monocyte chemoattractant protein 1 (MCP-1) exhibited increasing concentrations in response to exocrine tissue. The CXCR3 ligand interferon-inducible T cell α chemoattractant (I-TAC) was produced in response to both human islets and exocrine tissue from 6 h. Monokine induced by γ interferon (Mig) and interferon γ-induced protein 10 (IP-10) showed a later response, preferentially to exocrine tissue and with larger variations among preparations. An extended blood model of clinical islet transplantation allowed characterization of early immune activation in response to human islets and exocrine tissue. Increased production of chemokines targeting CXCR1/2, CCR2, and CXCR3 was observed, accompanied by massive intraislet neutrophil infiltration over 48 h. The model proved to be useful in exploring early blood-mediated reactions to cellular transplants and has relevance for evaluation of pharmacological interventions to prevent graft loss.

Early onset of rejection in concordant hamster xeno hearts display signs of necrosis, but not apoptosis, correlating to the phosphocreatine concentration.

BACKGROUND: The importance of apoptosis contra necrosis for ischemia/reperfusion (RP) and acute rejection in concordant rodent xenotransplantation is largely unknown. We explored this question by comparing rodent allo and concordant xenotransplants with different morphological methods to detect apoptosis and biochemical data on the levels of high-energy phosphates obtained with in vitro 31Phosphorous Magnetic Resonance Spectroscopy (31P MRS). More specifically, we applied a hitherto unused method in transplantation research, apoptosis specific biotin labeled oligonucleotides designed with a 10 base pair stem region and a 20 nucleotides large loop that form a hairpin like shape. The results obtained with this method were compared to results obtained with the more widely used in situ 3'-end labeling of DNA (TUNEL) assay and extraction and gel electrophoresis of labeled DNA (DNA laddering). METHODS: Cervical heart transplantations were performed between inbred Lewis (L) (RT1l) to L, L to DA (RT1a) rats, hamster (H) to H and H to L (X) (n=5 for all groups except for X, n=9). All hearts were subjected to 30 min of cold ischemia (+4 degrees C) and 6 h of RP before explantation. In vitro 31P MRS was used to determine the phosphocreatine (PCr), beta-adenosine triphosphate (beta-ATP) concentrations and the PCr/beta-ATP ratio of the transplants. We correlated the biochemical data to haematoxylin and eosin (H & E) stained tissue slides scored for rejection, infiltration of antibodies and complement depositions, DNA extraction and gel electrophoresis of labeled DNA (DNA laddering), in situ 3'-end labeling of DNA (TUNEL) and the apoptosis specific hairpin probe assays scoring. RESULTS: The rejection score of the xeno grafts differed significantly compared to their syngeneic hamster to hamster controls (2.40 +/- 0.25 vs. 1.20 +/- 0.20; P=0.005) and they had a significantly higher TUNEL score, 228 +/- 15 vs. 2.44 +/- 0.32 (P=0.009), that correlated to changes in PCr concentration (P<0.001) and to the PCr/beta-ATP ratio (P=0.01). The uptake was mainly (90-95%) located to 1-2 microm large extra cellular 'granule'. A picture resembling early necrosis was seen on the H & E stainings and reflected in the Billingham rejection score above. CONCLUSIONS: After 6 h of RP the onset of acute rejection in the concordant hamster xeno hearts displayed features of early, possibly mitochondrial, necrosis, but not apoptosis, which correlated to changes in the PCr concentration and the PCr/beta-ATP ratio. The mechanism for the early rejection observed is unclear and might be caused by other factors in the sera apart from cellular components, antibodies and complement factors. Identification of the underlying mechanisms could enable us to design rational therapies that prevent activation of the recipient's innate immune response.

Viral triggering of anti-NMDA receptor encephalitis in a child - an important cause for disease relapse.

Herpes simplex encephalitis (HSE) in children is a potentially devastating condition which is occasionally complicated by a clinical relapse. An autoimmune component has long been suspected in these relapses and recent findings suggest that antibodies against N-methyl-D-aspartate receptors (NMDARs) may be part of this mechanism. We here report an 11 months old girl with acute HSE and with negative NMDAR antibody serology at presentation who after an initial response to antiviral treatment deteriorated with seizures, abnormal movements, focal neurologic deficits and psychiatric symptoms. We show that this relapse occurred as production of NMDAR antibodies developed and that clinical improvement followed immunotherapy with a concomitant decrease in NMDAR antibody titers in CSF. She also developed a characteristic 15-20 Hz activity over both hemispheres which has been previously described as an electroencephalographic presentation of anti-NMDAR encephalitis. We conclude that relapse or persisting symptoms in HSE in children may represent an immune-mediated mechanism rather than a viral reactivation and that NMDAR antibodies should be analyzed as this may be of importance for the choice of therapy.

Allogeneic lymphocyte-licensed DCs expand T cells with improved antitumor activity and resistance to oxidative stress and immunosuppressive factors.

Adoptive T-cell therapy of cancer is a treatment strategy where T cells are isolated, activated, in some cases engineered, and expanded ex vivo before being reinfused to the patient. The most commonly used T-cell expansion methods are either anti-CD3/CD28 antibody beads or the "rapid expansion protocol" (REP), which utilizes OKT-3, interleukin (IL)-2, and irradiated allogeneic feeder cells. However, REP-expanded or bead-expanded T cells are sensitive to the harsh tumor microenvironment and often short-lived after reinfusion. Here, we demonstrate that when irradiated and preactivated allosensitized allogeneic lymphocytes (ASALs) are used as helper cells to license OKT3-armed allogeneic mature dendritic cells (DCs), together they expand target T cells of high quality. The ASAL/DC combination yields an enriched Th1-polarizing cytokine environment (interferon (IFN)-γ, IL-12, IL-2) and optimal costimulatory signals for T-cell stimulation. When genetically engineered antitumor T cells were expanded by this coculture system, they showed better survival and cytotoxic efficacy under oxidative stress and immunosuppressive environment, as well as superior proliferative response during tumor cell killing compared to the REP protocol. Our result suggests a robust ex vivo method to expand T cells with improved quality for adoptive cancer immunotherapy.

Rapid increase of interleukin-10 plasma levels after combined auxiliary liver-kidney transplantation in presensitized patients.

BACKGROUND: After transplantation, donor dendritic cells (DCs) in the grafted organ are activated by an ischemia/reperfusion-induced inflammatory process that induces their migration to the recipient's secondary lymphoid tissues. The subsequent interaction between migrated and mature donor DCs, recipient T cells, and natural killer (NK) cells is proposed to be crucial in directing host immune reactions toward allograft rejection. A liver transplant is less prone to induce rejection compared with most other solid organ transplants, and simultaneous transplantation of liver and kidney is known to improve the clinical outcome of kidney transplantation. METHODS AND RESULTS: Here we show that liver as well as combined auxiliary liver-kidney transplantation in patients induces a rapid increase in plasma interleukin-10 (IL-10) to levels that are significantly higher than those seen after standard kidney transplantation. Addition of IL-10 during in vitro maturation of human monocyte-derived DCs with ischemia/reperfusion-associated factors was found to affect phenotypic DC maturation significantly. Addition of IL-10 inhibited DC production of the NK cell- and T cell-recruiting chemokines CXCL9, CXCL10 and CXCL11. CONCLUSION: Our findings indicate that liver transplantation induces a substantial systemic release of IL-10, which may inhibit T cell- and NK cell-mediated rejection processes toward the transplanted liver and concurrently transplanted kidney.

Postischemic inflammatory response in an auxiliary liver graft predicts renal graft outcome in sensitized patients.

BACKGROUND: The liver is considered a tolerogenic organ that favors the induction of peripheral tolerance and protects other organs from the same donor from rejection. This has been exploited in combined auxiliary liver-kidney transplantation, where a renal graft is transplanted against a positive crossmatch under the protection of a liver transplanted from the same donor. METHODS: To elucidate mechanisms behind the liver protective effect, we studied early transcriptional changes of inflammatory mediators in the grafts during combined auxiliary liver-kidney transplantation using microarrays and real-time polymerase chain reaction. The results were correlated to clinical data. RESULTS: Liver and kidney grafts both exhibited an upregulation of the leukocyte-recruiting chemokines CCL2, CCL3, and CCL4. Notably, liver grafts strongly upregulated CCL20, a dendritic cell, and T-cell recruiting chemokine. By comparing the gene expression in liver grafts with the clinical outcome, we found that 14 of 45 investigated inflammatory genes were expressed significantly higher in patients without early rejection when compared with those with early rejections. This included the above-mentioned chemokines and the T-cell-recruiting CX3CL1, NFKB1, and the tolerance-inducing gene indoleamine 2,3-dioxygenase. CONCLUSIONS: In this study, the protective role of the liver was associated with a proinflammatory reaction within this organ after ischemia-reperfusion. In particular, we found an increased expression of leukocyte-recruiting chemokines in patients without rejection, indicating a protective role of host inflammatory cells infiltrating the auxiliary liver graft in presensitized patients. Second, gene expression profiling of transplant biopsies shortly after reperfusion predicted the risk of early rejection in these patients.