Gene expression profiling of cutaneous wound healing.

BACKGROUND: Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events. STUDY DESIGN: This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier--toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days). 17.5K cDNA microarrays were utilized to profile the biopsy material. RESULTS: Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy) were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies) and repair and angiogenesis genes in the later (4 to 8 days) biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated. CONCLUSION: The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominantly M2 macrophages, may help the interpretation of the cellular and molecular events occurring in the microenvironment of serially biopsied tissues.

Sequential gene profiling of basal cell carcinomas treated with imiquimod in a placebo-controlled study defines the requirements for tissue rejection.

BACKGROUND: Imiquimod is a Toll-like receptor-7 agonist capable of inducing complete clearance of basal cell carcinoma (BCC) and other cutaneous malignancies. We hypothesized that the characterization of the early transcriptional events induced by imiquimod may provide insights about immunological events preceding acute tissue and/or tumor rejection. RESULTS: We report a paired analysis of adjacent punch biopsies obtained pre- and post-treatment from 36 patients with BCC subjected to local application of imiquimod (n = 22) or vehicle cream (n = 14) in a blinded, randomized protocol. Four treatments were assessed (q12 applications for 2 or 4 days, or q24 hours for 4 or 8 days). RNA was amplified and hybridized to 17.5 K cDNA arrays. All treatment schedules similarly affected the transcriptional profile of BCC; however, the q12 x 4 days regimen, associated with highest effectiveness, induced the most changes, with 637 genes unequivocally stimulated by imiquimod. A minority of transcripts (98 genes) confirmed previous reports of interferon-alpha involvement. The remaining 539 genes portrayed additional immunological functions predominantly involving the activation of cellular innate and adaptive immune-effector mechanisms. Importantly, these effector signatures recapitulate previous observations of tissue rejection in the context of cancer immunotherapy, acute allograft rejection and autoimmunity. CONCLUSION: This study, based on a powerful and reproducible model of cancer eradication by innate immune mechanisms, provides the first insights in humans into the early transcriptional events associated with immune rejection. This model is likely representative of constant immunological pathways through which innate and adaptive immune responses combine to induce tissue destruction.

Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms.

BACKGROUND: Interferon (IFN)-alpha is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs). This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS) stimulation in vitro. RESULTS: Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-beta, IFN-alpha2b/alpha2, IFN-alphaI, IFN-alpha2, IFN-alphaC, IFN-alphaJ1, IFN-alphaH2, and INF-alpha4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx)-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-gamma. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs. CONCLUSION: Seven IFN-alpha isoforms and IFN-beta participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-alpha in autoimmunity and tumor rejection by including and/or excluding an array of related factors likely to be heterogeneously expressed by distinct sub-populations of individuals in sickness or in response to biological therapy.

Polarized monocyte response to cytokine stimulation.

BACKGROUND: Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate. RESULTS: Circulating CD14+ MPs were exposed to bacterial lipopolysaccharide (LPS) followed by exposure to an array of cytokines, chemokines and soluble factors involved in the immune response. Gene expression was studied by global transcript analysis. Two main classes of cytokines were identified that induced a classical or an alternative pathway of MP activation. Expression of genes affected by NFkappaB activation was most predictive of the two main classes, suggesting that this pathway is a fundamental target of cytokine regulation. As LPS itself induces a classical type of activation, the most dramatic modulation was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components. CONCLUSIONS: This analysis is directly informative of the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative of the way MP maturation may be polarized at the early stages of the immune response.

A genomic- and proteomic-based hypothesis on the eclectic effects of systemic interleukin-2 administration in the context of melanoma-specific immunization.

Among human cancers, melanoma is characterized by an almost unique predisposition to regress in response to immune therapy. Recent clinical studies suggest that the frequency of this favorable event is enhanced by combining T-cell-directed active specific immunization with the systemic administration of interleukin (IL)-2. While waiting for additional clinical experience to confirm this observation, we embraced the working hypothesis that this combination provides superior response rates than either treatment alone. In particular, we have focused our interest on the paradoxical observation that active specific immunization consistently induces circulating CD8+ T cells capable of recognizing in ex vivo assays tumor cells, but cannot induce tumor regression alone. In these settings, it appears that combining the systemic administration of IL-2 is almost an absolute requirement for the induction of clinical responses. Here, we will expand on previous speculations on the postulated mechanism(s) of action of systemic IL-2 administration and, based on original data recently derived through high-throughput transcriptional and post-translational analysis, we will suggest an explanation for the eclectic effects of IL-2 administration in the context of active specific immunization.

Overview of melanoma vaccines and promising approaches.

It is difficult to envision anything better than melanoma vaccines to exemplify the effectiveness of modern biotechnology in developing biologically rational therapeutics. Melanoma vaccines can reproducibly induce cytotoxic T lymphocyte (CTL) responses better than any other anticancer therapy. Anticancer vaccines have been labeled by some as ineffective for the simple reason that they only rarely lead to cancer regression. This oxymoron stems from the naïve expectation that CTLs are all that is needed to reject cancer. Little is known about requirements for CTL localization and effector function within the tumor microenvironment. In the future, more attention should be given to events downstream of immunization (afferent arm of immune response) to identify combination therapies likely to facilitate localization and activation of CTL at the receiving end (efferent arm).

Selection and validation of endogenous reference genes using a high throughput approach.

BACKGROUND: Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines. RESULTS: Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used. CONCLUSIONS: This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.

Mechanism of immune response during immunotherapy.

Tumor immunology embraces an extensive array of biological phenomena that include interactions between neoplastic cells and the innate and adaptive immune response. Among immune cells, T cells have taken the center stage because they can be easily demonstrated to specifically recognize autologous cancer cells. However, their role is limited and other components of the immune response are likely necessary for the completion of cancer rejection. Metastatic melanoma and renal cell carcinoma (RCC) are malignancies strongly predisposed to regress in response to the systemic administration of high-dose interleukin (IL)-2. Several clinical Studies in extensive cohorts of patients have shown that this treatment can induce complete or partial clinical regressions of metastatic disease in 15 to 20% of patients who receive this treatment. Although IL-2 has direct pluri-potent effects on cells with immune and inflammatory function, it remains unexplained which cell subset is implicated in mediating tumor regression. In a quest to characterize the mechanism of action of IL-2 during the course of immunotherapy, we have investigated the early changes in transcriptional profiles of circulating mononuclear cells and microenvironment of melanoma metastases following high dose IL-2 administration (720,000 IU/kg) by serial sampling of blood cells and tumors in the form of fine needle aspirate (FNA). Furthermore, studies are currently ongoing to characterize the proteomic profiling of RCC patients undergoing the same treatment using protein arrays (manuscript in preparation). The predominant activation of genes related to inflammation and activation of mononuclear phagocytes lead us to further characterize this cell subset in the context of stimulation with a panel of soluble factors potentially present in the circulation and tumor microenvironment.

Forecasting the cytokine storm following systemic interleukin (IL)-2 administration.

Extensive clinical experience has shown that systemic interleukin (IL)-2 administration can induce complete or partial regression of renal cell cancer (RCC) metastases in 15 to 20 % of patients. Since IL-2 has no direct anti-cancer effects, it is believed that cancer regression is mediated either by a direct modulation of immune cell effector functions or through the mediation of soluble factors released as a result of IL-2 administration.We previously observed that transcriptional and protein changes induced by systemic IL-2 administration affect predominantly mononuclear phagocytes with little effect, particularly within the tumor microenvironment, on T cell activation, localization and proliferation. It further appeared that mononuclear phagocyte activation could be best explained by the indirect mediation of a secondary release of cytokines by IL-2 responsive cells either in the circulation or in peripheral tissues.To better characterize the cytokine outburst that follows systemic IL-2 administration we followed the serum levels of 68 soluble factors in ten patients with RCC undergoing high dose (720,000 IU/kg intravenously every 8 hours) IL-2 therapy. Serum was collected before therapy, 3 hours after the 1st and 4th dose and assayed on a multiplexed protein array platform. This study demonstrated that 1) the serum concentration of more than half the soluble factors studied changed significantly during therapy; 2) changes became more dramatic with increasing doses; 3) subclasses of soluble factors displayed different kinetics and 4) cytokine patterns varied quantitatively among patients.This study shows that the cytokine storm that follows systemic IL-2 administration is complex and far-reaching inclusive of soluble factors with disparate, partly redundant and partly contrasting effects on immune function. Therefore comparing in parallel large number of soluble factors, it sets a comprehensive foundation for further elucidation of "cytokine storm" in larger patient pools. Based on this analysis, we propose a prospective collection of serum samples in a larger cohort of patients undergoing IL-2 administration with the purpose of discerning patterns predictive of clinical outcome and toxicity.

Quiescent phenotype of tumor-specific CD8+ T cells following immunization.

In a human melanoma model of tumor antigen (TA)-based immunization, we tested the functional status of TA-specific CD8+ cytotoxic T lymphocytes. A "quiescent" phenotype lacking direct ex vivo cytotoxic and proliferative potential was identified that was further characterized by comparing its transcriptional profile to that of TA-specific T cells sensitized in vitro by exposure to the same TA and the T-cell growth factor interleukin 2 (IL-2). Quiescent circulating tumor-specific CD8+ T cells were deficient in expression of genes associated with T-cell activation, proliferation, and effector function. This quiescent status may explain the observed lack of correlation between the presence of circulating immunization-induced lymphocytes and tumor regression. In addition, the activation of TA-specific T cells by in vitro antigen recall and IL-2 suggests that a complete effector phenotype might be reinstated in vivo to fulfill the potential of anticancer vaccine protocols.

Vaccination with T cell-defined antigens.

Tumour immunology encompasses a broad array of biological phenomena including interactions between neoplastic cells and the innate and adaptive immune response. Among immune cells, T cells have taken the centre stage because they can be easily demonstrated to specifically recognise autologous cancer cells. As most tumour-associated antigens are intracellular proteins, T cells appear to be the most suitable tool for cancer-specific attack, as antibodies do not cross the cell membrane and the innate immune response lacks the same level of specificity. Finally, the relative ease in which T cells can be educated through antigen-specific immunisation to recognise cancer cells has elevated them to an even higher stature. In this review, it will be argued that T cells represent a unique anticancer agent, characterised by absolute specificity. Although other therapeutic modalities (antibody-based) have been effectively implemented, a comparison of T cell-based approaches with other modalities goes beyond the purposes of this review and will not be included in the discussion. However, it is obvious that the role of the T cell is limited and other components of the immune response (effector mononuclear phagocytes, natural killer cells, cytokines, chemokines, soluble factors), genetic background and tumour heterogeneity are likely to be necessary for the completion of cancer rejection.

Active-specific immunization against melanoma: is the problem at the receiving end?

The recent progress in tumor immunology is a striking example of the successful application of modern biotechnology to understand the complex phenomenon of immune-mediated cancer rejection. Tumor antigens were identified and successfully utilized in active immunization trials to induce tumor antigen-specific T cells. This achievement has left, however, clinicians and researchers perplexed by the paradoxical observation that immunization-induced T cells can recognize tumor cells in standard assays but cannot induce tumor regression. A closer look at T cell physiology and tumor biology suggests that this observation is not so surprising. Here, we argue that successful immunization is one of several steps required for tumor clearance while more needs to be understood about how T cells localize and are effective within a tumor microenvironment impervious to the execution of their effector function.