Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Augmentation of antitumor immune responses after adoptive transfer of bone marrow derived from donors immunized with tumor lysate-pulsed dendritic cells.

We demonstrated previously that tumor lysate-pulsed dendritic cells (TP-DC) could mediate a specific and long-lasting antitumor immune response against a weakly immunogenic breast tumor during early lymphoid reconstitution. The purpose of this study was to examine the potential therapeutic efficacy of bone marrow transplants from TP-DC-vaccinated donors. In 2 aggressive metastatic models, bone marrow transplantation with donor bone marrow cells from TP-DC-immunized mice mediated a tumor-specific immune response in the recipient, and this caused regressions of preexisting tumor metastases. After vaccination with TP-DC, donors harbored increased numbers of both activated CD4+ and CD8+ T-cell populations in the bone marrow. Adoptive transfer of T cells purified from the bone marrow of TP-DC-vaccinated mice led to a reduction in preestablished lung metastases, whereas depletion of T cells from bone marrow abolished this effect. By using T cells derived from the bone marrow of TP-DC-vaccinated major histocompatibility complex class I and class II knockout mice, the effector cells required for the observed antitumor effect were determined to be major histocompatibility complex class I-restricted CD8+ T cells. Additionally, the tumor burden in TP-DC-immunized transplant recipients could be reduced further by repetitive TP-DC immunizations after bone marrow transplantation. Collectively, these results demonstrate an important therapeutic role of bone marrow from TP-DC-immunized donors and raise the potential for this approach in patients with advanced cancer.

Myocardial viability as a determinant of the ejection fraction response to carvedilol in patients with heart failure (CHRISTMAS trial): randomised controlled trial.

BACKGROUND: The improvement in left-ventricular ejection fraction (LVEF) in response to beta blockers is heterogeneous in patients with heart failure due to ischaemic heart disease, possibly indicating variations in the myocardial substrate underlying left-ventricular dysfunction. We investigated whether improvement in LVEF was associated with the volume of hibernating myocardium (viable myocardium with contractile failure). METHODS: We did a double-blind, randomised trial to compare placebo and carvedilol for 6 months in individuals with stable, chronic heart failure due to ischaemic left-ventricular systolic dysfunction. We enrolled 489 patients, of whom 387 were randomised. Patients were designated hibernators or non-hibernators according to the volume of hibernating myocardium. The primary endpoint was change in LVEF, measured by radionuclide ventriculography, in hibernators versus non-hibernators, on carvedilol compared with placebo. Analysis was by intention to treat. RESULTS: 82 patients dropped out of the study because of adverse events, withdrawal of consent, or failure to complete the investigation. Thus, 305 (79%) were analysed. LVEF was unchanged with placebo (mean change -0.4 [SE 0.9] and -0.4 [0.8] for non-hibernators and hibernators, respectively) but increased with carvedilol (2.5 [0.9] and 3.2 [0.8], respectively; p<0.0001 compared with baseline). Mean placebo-subtracted change in LVEF was 3.2% (95% CI 1.8-4.7; p=0.0001) overall, and 2.9% (0.7-5.1; p=0.011) and 3.6% (1.7-5.4; p=0.0002) in non-hibernators and hibernators, respectively. Effect of hibernator status on response of LVEF to carvedilol was not significant (0.7 [-2.2 to 3.5]; p=0.644). However, patients with more myocardium affected by hibernation or by hibernation and ischaemia had a greater increase in LVEF on carvedilol (p=0.0002 and p=0.009, respectively). INTERPRETATION: Some of the effect of carvedilol on LVEF might be mediated by improved function of hibernating or ischaemic myocardium, or both. Medical treatment might be an important adjunct or alternative to revascularisation for patients with hibernating myocardium.

Tumor lysate-pulsed dendritic cells can elicit an effective antitumor immune response during early lymphoid recovery.

Dendritic cells (DC) can serve to immunize the newborn immune system to foreign antigen. In a lymphopenic environment, naive T cells undergoing homeostasis-driven proliferation can acquire increased sensitivity to antigen stimulation. Here, we evaluated the capacity of DC to effectively prime the host immune system to elicit antitumor effects in the setting of early lymphoid reconstitution after bone marrow transplantation (BMT). Indeed, bone marrow-derived, cytokine-driven DC pulsed with whole tumor lysates (TP-DC) could, early on, prime a specific and long-lasting antitumor immune response, which mediated the rejection of a lethal challenge of a weakly immunogenic breast tumor. In the therapeutic setting, TP-DC could also inhibit the growth of preexisting breast tumor metastases by repetitive immunizations initiated early after BMT. Spleen T cells obtained from mice immunized with TP-DC early after BMT showed a substantial increase in tumor-specific IFN-gamma production. Our findings demonstrate that it is possible to promote effective antitumor immunity in a defined lymphopenic environment through DC-based immunization.

Immune evasion by muscle-specific gene expression in dystrophic muscle.

Muscle tissue from Duchenne muscular dystrophy patients and the Dmd(mdx/mdx) (hereafter referred to as mdx) mouse is characterized by an abundance of necrotic myofibers and infiltrating macrophages. Both features may provide additional stimulus to the immune response directed against novel antigens, such as those delivered by gene therapy vectors. It has previously been shown that the immune evasion achieved by adeno-associated virus in healthy muscle fails in one model of muscular dystrophy. Here, we examined the immune response to adenoviral vectors and their transgenes in normal and mdx mice. We found that mdx mouse muscles contain 20 times more macrophages and 7 times more dendritic cells than healthy muscles. This higher professional antigen-presenting cell content results in a stronger immune response to antigens that can be directly presented by those cells, including viral antigens and constitutively expressed transgene products. However, we did not detect a significant immune response to beta-galactosidase expressed specifically in muscle, even at high expression levels. This result suggests that cross-presentation is not more effective in mdx mouse muscle, and that targeted vectors and tissue-specific promoters may be useful tools for evasion of the immune response in dystrophic muscle.

Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression.

Dendritic cells (DCs) have been shown to be a promising adjuvant for inducing immunity to cancer. We evaluated tumor lysate-pulsed DC in a Phase I trial of pediatric patients with solid tumors. Children with relapsed solid malignancies who had failed standard therapies were eligible. The vaccine used immature DC (CD14-, CD80+, CD86+, CD83-, and HLA-DR+) generated from peripheral blood monocytes in the presence of granulocyte/monocyte colony-stimulating factor and interleukin-4. These DC were then pulsed separately with tumor cell lysates and the immunogenic protein keyhole limpet hemocyanin (KLH) for 24 h and then combined. A total of 1 x 10(6) to 1 x 10(7) DC are administered intradermally every 2 weeks for a total of three vaccinations. Fifteen patients (ages 3-17 years) were enrolled with 10 patients completing all vaccinations. Leukapheresis yields averaged 2.8 x 10(8) peripheral blood mononuclear cells (PBMC)/kg, and DC yields averaged 10.9% of starting PBMC. Patients with neuroblastoma, sarcoma, and renal malignancies were treated without obvious toxicity. Delayed-type hypersensitivity (DTH) response was detected in 7 of 10 patients for KLH and 3 of 6 patients for tumor lysates. Priming of T cells to KLH was seen in 6 of 10 patients and to tumor in 3 of 7 patients as demonstrated by specific IFN-gamma-secreting T cells in unstimulated PBMCs. Significant regression of multiple metastatic sites was seen in 1 patient. Five patients showed stable disease, including 3 who had minimal disease at time of vaccine therapy and remain free of tumor with 16-30 months follow-up. Our results demonstrate that it is feasible to generate large numbers of functional DC from pediatric patients even in those highly pretreated and with a large tumor burden. The DC can be administered in an outpatient setting without any observable toxicity. Most importantly, we have demonstrated the ability of the tumor lysate/KLH-pulsed DC to generate specific T-cell responses and to elicit regression of metastatic disease.

The dynamics of the T-cell antitumor response: chemokine-secreting dendritic cells can prime tumor-reactive T cells extranodally.

Direct administration of dendritic cells (DCs) genetically modified to express secondary lymphoid tissue chemokine (SLC) into growing B16 melanoma could result in a substantial, sustained influx of T cells within the mass with only a transient increase in T-cell numbers in the draining lymph node (DLN). DCs were retained at the tumor site with only a very small percentage trafficking to the DLN. The T cells infiltrating the tumor mass expressed the activation marker CD25 within 24 h and developed IFN-gamma-secreting function within 7 days as tumor growth was inhibited. Similar results were obtained in lymphotoxin alpha-/- mice, which lacked peripheral lymph nodes. Our data demonstrate that effective T-cell priming can occur extranodally and result in measurable antitumor effects in vivo.

Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization.

There is considerable controversy as to whether necrotic (lysate) or apoptotic tumor cells serve as the superior source of multiple tumor-associated antigens (TAAs) to pulse dendritic cells (DCs) for immunotherapeutic applications. Here, we show that standard procedures to induce apoptosis by UVB irradiation unequivocally result in a mixed population of viable, apoptotic, and necrotic tumor cells, necessitating additional purification. We used highly enriched apoptotic versus lysate of B16 melanoma cells to examine whether or not there are important distinctions between these two sources of TAAs for loading of DCs. Our results demonstrate that although some differences exist between the two forms of TAAs in expression of heat shock proteins, as well as production of interleukin-12 by pulsed DCs, their respective capacities to mature DCs phenotypically, as well as to elicit both effective immune priming and antitumor therapeutic efficacy in vivo when presented by DCs, are equivalent.

Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis.

Dendritic cells (DCs) are specialized antigen-presenting cells that migrate from the periphery to lymphoid tissues, where they activate and regulate T cells. Genetic modification of DCs to express immunoregulatory molecules would provide a new immunotherapeutic strategy for autoimmune and other diseases. We have engineered bone marrow-derived DCs that express IL-4 and tested the ability of these cells to control murine collagen-induced arthritis (CIA), a model for rheumatoid arthritis in which Th1 cells play a critical role. IL-4-transduced DCs inhibited Th1 responses to collagen type II in vitro. A single injection of IL-4-transduced DCs reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses, despite only transient persistence of injected DCs in the spleen. In contrast, control DCs and IL-4-transduced T cells or fibroblastic cells failed to alter the course of the disease. The functional effects correlated well with the differential efficiency of DC migration from various sites of injection to lymphoid organs, especially the spleen. The ability of splenic T cells to produce IL-4 in response to anti-CD3 was enhanced after the administration of IL-4-transduced DCS: These results support the feasibility of using genetically modified DCs for the treatment of autoimmune disease.

Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein.

We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.

T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that is selective in its recruitment of naive T cells and dendritic cells (DCs). In the lymph node, SLC is believed to play an important role in the initiation of an immune response by colocalizing naive T cells with DC-presenting antigen. Here, we used SLC as a treatment for tumors established from the poorly immunogenic B16 melanoma. Intratumoral injections of SLC inhibited tumor growth in a CD8+, T cell-dependent manner. SLC elicited a substantial infiltration of DCs and T cells into the tumor, coincident with the antitumor response. We next used SLC gene-modified DCs as a treatment of established tumors. Intratumoral injections of SLC-expressing DCs resulted in tumor growth inhibition that was significantly better than either control DCs or SLC alone. Distal site immunization of tumor-bearing mice with SLC gene-modified DCs pulsed with tumor lysate elicited an antitumor response whereas control DCs did not. We also found that s.c. injection of lysate-pulsed DCs expressing SLC promoted the migration of T cells to the immunization site. This report demonstrates that SLC can both induce antitumor responses and enhance the antitumor immunity elicited by DCs.

Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents.

Dendritic cells (DCs) can efficiently acquire foreign antigen(s) from apoptotic cells and induce MHC class I-restricted, antigen-specific CTLs. An accumulation of DCs within solid tumor masses in situ has been associated indirectly with a more favorable prognosis. Therefore, DCs may offer an efficient means for triggering immune responses within tumors, particularly in those masses containing significant apoptosis. We examined whether delivery of DCs could, alone, impact on the progressive growth of a tumor with a relatively high apoptotic index. We detected significant early apoptosis within the mass of a s.c. growing murine MT-901 breast carcinoma. DCs could efficiently engulf MT-901 tumor apoptotic cells in vitro. Intratumoral injections of syngeneic but not allogeneic DCs resulted in significant inhibition of MT-901 tumor growth. Histological examination of the tumor revealed intense mononuclear cell infiltration during and after DC injections. Tumor growth inhibition was relatively radiosensitive and dependent on host-derived CD8+ T cells. The baseline level of tumor apoptosis could be increased substantially by tumor necrosis factor alpha administration, leading to a greater DC-mediated antitumor effect. The antitumor effect could also be enhanced by first pulsing DCs with the foreign helper protein, keyhole limpet hemocyanin, prior to intratumoral delivery and combining it with the systemic administration of interleukin 2. Splenocytes from treated animals showed heightened levels of specific CTL activity and production of cytokines. The level of in situ tumor apoptosis appears to play a critical role in DC-mediated antitumor effects. The potential implication of these findings in DC-based tumor therapy strategies is discussed.

Treatment of solid tumours in children with tumour-lysate-pulsed dendritic cells.

Dendritic cells are potent stimulators of antigen-specific immune responses, including antitumour responses. We explored the use of tumour-lysate-pulsed dendritic cells in children with relapsed solid tumours. Dendritic cell treatment in children was feasible and apparently not toxic. The treatment was able to produce significant tumour regression in a child with metastatic fibrosarcoma.

Gene-modified dendritic cells for use in tumor vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells capable of priming activation of naive T cells. Because of their immunostimulatory capacity, immunization with DCs presenting tumor antigens has been proposed as a treatment regimen for cancer. The results from translational research studies and early clinical trials point to the need for improvement of DC-based tumor vaccines before they become a more broadly applicable treatment modality. In this regard, studies suggest that genetic modification of DCs to express tumor antigens and/or immunomodulatory proteins may improve their capacity to promote an antitumor response. Because the DC phenotype is relatively unstable, nonperturbing methods of gene transfer must be employed that do not compromise viability or immunostimulatory capacity. DCs expressing transgenes encoding tumor antigens have been shown to be more potent primers of antitumor immunity both in vitro and in animal models of disease; in some measures of immune priming, gene-modified DCs exceeded their soluble antigen-pulsed counterparts. Cytokine gene modification of DCs has improved their capacity to prime tumor antigen-specific T cell responses and promote antitumor immunity in vivo. Here, we review the current status of gene-modified DCs in both human and murine studies. Although successful results have been obtained to date in experimental systems, we discuss potential problems that have already arisen and may yet be encountered before gene-modified DCs are more widely applicable for use in human clinical trials.

Potentiation of immunologic responsiveness to dendritic cell-based tumor vaccines by recombinant interleukin-2.

PURPOSE: Dendritic cells (DC) can elicit potent immune responses to tumors through their capacity to efficiently process and present tumor-associated antigens. In a variety of animal tumor models, vaccines based on tumor lysate-pulsed DC (TP-DC) have been shown to effectively immunize against lethal tumor challenges as well as to treat established growing tumors at skin and organ sites. The antitumor effects elicited by TP-DC-based vaccines in vivo have been shown to be mediated by tumor-specific proliferative, cytotoxic, and cytokine-secreting host-derived T cells. Because of the critical involvement of T cells in the antitumor immune response, we have been investigating whether the systemic administration of recombinant interleukin (IL)-2 can enhance the therapeutic efficacy of TP-DC-based tumor vaccines. MATERIALS AND METHODS: Immunization with TP-DC plus IL-2 administration was evaluated to determine if this combination could enhance protective immunity toward a weakly immunogenic sarcoma (MCA-207) and a poorly immunogenic subline (D5) of the B16 melanoma and mediate therapeutic rejection of established tumors in C57BL/6 (B6) mouse models. RESULTS: We have demonstrated in our murine models that the addition of IL-2 at relatively nontoxic doses can markedly augment the antitumor activity of TP-DC-based tumor vaccine therapies against both a weakly immunogenic sarcoma and a poorly immunogenic melanoma. Animals treated with the combination exhibited significantly greater protection from tumor-cell challenge, significantly greater regression of established tumors, and significantly longer mean survival time than with either TP-DC or IL-2 therapy alone. The mechanism operative in vivo appears to involve the enhancement of immune T-cell function. CONCLUSION: These preclinical studies demonstrate the potential of this novel treatment strategy and support the rationale for planned phase I/II clinical trials of TP-DC-based vaccines plus IL-2 in patients with advanced melanoma and colorectal cancer.

Prevention of epidural fibrosis with ADCON-L in presence of a durotomy during lumbar disc surgery: experiences with a pre-clinical model.

A potential complication of lumbo-sacral surgery is the inadvertent tear of the dura mater, which sometimes eludes intra-operative detection. ADCON-L, a bioabsorbable gel used in lumbosacral laminectomies or laminotomies, is a physical barrier to post-operative epidural fibrosis. Three experimental lumbar laminectomy studies were designed to assess in vivo the effects of ADCON-L when applied in presence of dural punctures in a rat model. In the first study, the durotomy was repaired with fibrin sealant, in the second experiment the dural defect was microsurgically sutured, while in a third protocol the durotomy was left unrepaired. In each study, dural healing was assessed respectively at 4, 8, or 12 weeks post-operatively. Blinded anatomical dissection and histopathology were used to compare results between treatments (sham operated control vs. ADCON-L). In the fibrin sealant experiment, an additional treatment group (fibrin sealant used together with ADCON-L) was included. The results of these studies consistently demonstrate that ADCON-L is an effective anti-fibrotic agent, and does not interfere with the normal dural healing processes following a meningeal puncture. The application of the gel may therefore be safe in presence of dural incisions, even when they are not identified during surgery, as demonstrated in these in vivo studies.