Profiling of Tumor-Infiltrating Immune Cells and Their Impact on Survival in Glioblastoma Patients Undergoing Immunotherapy with Dendritic Cells.

Glioblastomas (GBM) are the most common primary malignant brain tumors, comprising 2% of all cancers in adults. Their location and cellular and molecular heterogeneity, along with their highly infiltrative nature, make their treatment challenging. Recently, our research group reported promising results from a prospective phase II clinical trial involving allogeneic vaccination with dendritic cells (DCs). To date, six out of the thirty-seven reported cases remain alive without tumor recurrence. In this study, we focused on the characterization of infiltrating immune cells observed at the time of surgical resection. An analytical model employing a neural network-based predictive algorithm was used to ascertain the potential prognostic implications of immunological variables on patients' overall survival. Counterintuitively, immune phenotyping of tumor-associated macrophages (TAMs) has revealed the extracellular marker PD-L1 to be a positive predictor of overall survival. In contrast, the elevated expression of CD86 within this cellular subset emerged as a negative prognostic indicator. Fundamentally, the neural network algorithm outlined here allows a prediction of the responsiveness of patients undergoing dendritic cell vaccination in terms of overall survival based on clinical parameters and the profile of infiltrated TAMs observed at the time of tumor excision.

Adjuvant Vaccination with Allogenic Dendritic Cells Significantly Prolongs Overall Survival in High-Grade Gliomas: Results of a Phase II Trial.

Immunotherapy for cancer treatment has gained increased attention in recent years. Recently, our group reported the case of a patient with glioblastoma who underwent vaccination based on dendritic cells and experienced a strong Th1 immune response together with near-complete tumor remission. Here we report the results of a phase I/II prospective, non-controlled clinical trial with 37 patients harboring glioblastoma or grade 4 astrocytomas. At the time of first recurrence after surgery, patients began receiving monthly intradermal injections of allogenic DC-autologous tumor cell hybridomas. Overall survival, quality of life, and immunological profiles were assessed prospectively. Compared with patients in the Genomic Data Commons data bank, overall survival for vaccinated patients with glioblastoma was 27.6 ± 2.4 months (vs. 16.3 ± 0.7, log-rank p < 0.001, hazard ratio 0.53, 95%CI 0.36-0.78, p < 0.01), and it was 59.5 ± 15.9 for vaccinated astrocytoma grade 4 patients (vs. 19.8 ± 2.5, log-rank p < 0.05, hazard ratio 0.18, 95%CI 0.05-0.62, p < 0.01). Furthermore, seven vaccinated patients (two IDH-1-mutated and five wild type) remain alive at the time of this report (overall survival 47.9 months, SD 21.1, range: 25.4-78.6 months since diagnosis; and 34.2 months since recurrence, range: 17.8 to 40.7, SD 21.3). We believe that the data reported here can foster the improvement of treatment protocols for high-grade gliomas based on cellular immunotherapy.

Near-Complete Remission of Glioblastoma in a Patient Treated with an Allogenic Dendritic Cell-Based Vaccine: The Role of Tumor-Specific CD4+T-Cell Cytokine Secretion Pattern in Predicting Response and Recurrence.

Immunotherapy has brought hope to the fight against glioblastoma, but its efficacy remains unclear. We present the case of CST, a 25-year-old female patient with a large right-hemisphere glioblastoma treated with a dendritic-tumor cell fusion vaccine. CST showed a near-complete tumor response, with a marked improvement in her functional status and simultaneous increases in tumor-specific CD8+ and CD4+ T cells. Two months before recurrence, the frequency of tumor-specific T cells decreased, while that of IL-17 and CD4+ T cells increased. CST passed away 15 months after enrollment. In this illustrative case, the tumor-specific CD4+ T-cell numbers and phenotype behaved as treatment efficacy biomarkers, highlighting the key role of the latter in glioblastoma immunotherapy.

Frequency determination of breast tumor-reactive CD4 and CD8 T cells in humans: unveiling the antitumor immune response.

As cancer immunotherapy gains importance, the determination of a patient's ability to react to his/her tumor is unquestionably relevant. Though the presence of T cells that recognize specific tumor antigens is well established, the total frequency of tumor-reactive T cells in humans is difficult to assess, especially due to the lack of broad analysis techniques. Here, we describe a strategy that allows this determination, in both CD4 and CD8 compartments, using T cell proliferation induced by tumor cell-lysate pulsed dendritic cells as the readout. All 12 healthy donor tested had circulating CD4 and CD8 tumor cell-reactive T cells. The detection of these T cells, not only in the naïve but also in the memory compartment, can be seen as an evidence of tumor immunosurveillance in humans. As expected, breast cancer patients had higher frequencies of blood tumor-reactive T cells, but with differences among breast cancer subtypes. Interestingly, the frequency of blood tumor-reactive T cells in patients did not correlate to the frequency of infiltrating tumor-reactive T cells, highlighting the danger of implying a local tumor response from blood obtained data. In conclusion, these data add T cell evidence to immunosurveillance in humans, confirm that immune parameters in blood may be misleading and describe a tool to follow the tumor-specific immune response in patients and, thus, to design better immunotherapeutic approaches.

Elucidating the role of leptin in systemic inflammation: a study targeting physiological leptin levels in rats and their macrophages.

To elucidate the role of leptin in acute systemic inflammation, we investigated how its infusion at low, physiologically relevant doses affects the responses to bacterial lipopolysaccharide (LPS) in rats subjected to 24 h of food deprivation. Leptin was infused subcutaneously (0-20 µg·kg-1·h-1) or intracerebroventricularly (0-1 µg·kg-1·h-1). Using hypothermia and hypotension as biomarkers of systemic inflammation, we identified the phase extending from 90 to 240 min post-LPS as the most susceptible to modulation by leptin. In this phase, leptin suppressed the rise in plasma TNF-α and accelerated the recoveries from hypothermia and hypotension. Suppression of TNF-α was not accompanied by changes in other cytokines or prostaglandins. Leptin suppressed TNF-α when infused peripherally but not when infused into the brain. Importantly, the leptin dose that suppressed TNF-α corresponded to the lowest dose that limited food consumption; this dose elevated plasma leptin within the physiological range (to 5.9 ng/ml). We then conducted in vitro experiments to investigate whether an action of leptin on macrophages could parallel our in vivo observations. The results revealed that, when sensitized by food deprivation, LPS-stimulated peritoneal macrophages can be inhibited by leptin at concentrations that are lower than those reported to promote cytokine release. It is concluded that physiological levels of leptin do not exert a proinflammatory effect but rather an anti-inflammatory effect involving selective suppression of TNF-α via an action outside the brain. The mechanism of this effect might involve a previously unrecognized, suppressive action of leptin on macrophage subpopulations sensitized by food deprivation, but future studies are warranted.

Respiratory gas exchange as a new aid to monitor acidosis in endotoxemic rats: relationship to metabolic fuel substrates and thermometabolic responses.

This study introduces the respiratory exchange ratio (RER; the ratio of whole-body CO2 production to O2 consumption) as an aid to monitor metabolic acidosis during the early phase of endotoxic shock in unanesthetized, freely moving rats. Two serotypes of lipopolysaccharide (lipopolysaccharide [LPS] O55:B5 and O127:B8) were tested at shock-inducing doses (0.5-2 mg/kg). Phasic rises in RER were observed consistently across LPS serotypes and doses. The RER rise often exceeded the ceiling of the quotient for oxidative metabolism, and was mirrored by depletion of arterial bicarbonate and decreases in pH It occurred independently of ventilatory adjustments. These data indicate that the rise in RER results from a nonmetabolic CO2 load produced via an acid-induced equilibrium shift in the bicarbonate buffer. Having validated this new experimental aid, we asked whether acidosis was interconnected with the metabolic and thermal responses that accompany endotoxic shock in unanesthetized rats. Contrary to this hypothesis, however, acidosis persisted regardless of whether the ambient temperature favored or prevented downregulation of mitochondrial oxidation and regulated hypothermia. We then asked whether the substrate that fuels aerobic metabolism could be a relevant factor in LPS-induced acidosis. Food deprivation was employed to divert metabolism away from glucose oxidation and toward fatty acid oxidation. Interestingly, this intervention attenuated the RER response to LPS by 58%, without suppressing other key aspects of systemic inflammation. We conclude that acid production in unanesthetized rats with endotoxic shock results from a phasic activation of glycolysis, which occurs independently of physiological changes in mitochondrial oxidation and body temperature.

Hypometabolism and hypothermia in the rat model of endotoxic shock: independence of circulatory hypoxia.

We tested the hypothesis that development of hypothermia instead of fever in endotoxic shock is consequential to hypoxia. Endotoxic shock was induced by bacterial lipopolysaccharide (LPS, 500 µg kg(-1) i.v.) in rats at an ambient temperature of 22 °C. A ß3-adrenergic agonist known to activate metabolic heat production, CL316,243, was employed to evaluate whether thermogenic capacity could be impaired by the fall in oxygen delivery (DO2) during endotoxic shock. This possibility was rejected as CL316,243 (0.15 mg kg(-1) i.v.) evoked similar rises in oxygen consumption (V̇O2) in the presence and absence of endotoxic shock. Next, to investigate whether a less severe form of circulatory hypoxia could be triggering hypothermia, the circulating volume of LPS-injected rats was expanded using 6% hetastarch with the intention of improving tissue perfusion and alleviating hypoxia. This intervention attenuated not only the fall in arterial pressure induced by LPS, but also the associated falls in V̇O2 and body temperature. These effects, however, occurred independently of hypoxia, as they were not accompanied by any detectable changes in NAD(+)/NADH ratios. Further experimentation revealed that even the earliest drops in cardiac output and DO2 during endotoxic shock did not precede the reduction in V̇O2 that brings about hypothermia. In fact, DO2 and V̇O2 fell in such a synchrony that the DO2/V̇O2 ratio remained unaffected. Only when hypothermia was prevented by exposure to a warm environment (30 °C) did an imbalance in the DO2/V̇O2 ratio become evident, and such an imbalance was associated with reductions in the renal and hypothalamic NAD(+)/NADH ratios. In conclusion, hypometabolism and hypothermia in endotoxic shock are not consequential to hypoxia but serve as a pre-emptive strategy to avoid hypoxia in this model.

Dendritic cell membrane CD83 enhances immune responses by boosting intracellular calcium release in T lymphocytes.

CD83 is a marker of mDCs directly related to their lymphostimulatory ability. Some data suggest that it has a central role in the immune system regulation, but how this function is performed remains to be determined. This work aimed to analyze the influence of CD83, present in mDCs, in the modulation of calcium signaling in T lymphocytes. Mo were differentiated into iDCs and activated with TNF-α. iDCs were treated, 4 h before activation, with siRNACD83, to reduce CD83 expression. Purified allogeneic T lymphocytes were labeled with the calcium indicator Fluo-4-AM, and calcium mobilization in the presence of mDCs was analyzed. CD83 knockdown mDCs induced lower calcium signal amplitude in T lymphocytes (29.0±10.0) compared with siRNAscr-treated mDCs (45.5±5.3). In another set of experiments, surface mDC CD83 was blocked with a specific mAb, and again, decreased calcium signaling in T lymphocytes was detected by flow cytometry and microscopy (fluorescence and confocal). In the presence of antibody, the percentage of responding T cells was reduced from 58.14% to 34.29%. As expected, anti-CD83 antibodies also reduced the proliferation of T lymphocytes (as assessed by CFSE dilution). Finally, in the absence of extracellular calcium, CD83 antibodies abrogated T cell signaling induced by allogeneic mDCs, suggesting that the presence of CD83 in mDC membranes enhances T lymphocyte proliferation by boosting calcium release from intracellular stores in these cells.