MAGE-A3 and MAGE-A4 specific CD4(+) T cells in head and neck cancer patients: detection of naturally acquired responses and identification of new epitopes.

Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlapping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from healthy donors and seven head and neck cancer patients. CD4(+) T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 specific CD4(+) T cells from 7/7 cancer patients analyzed. Moreover, we identified six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3(111-125) and MAGE-A3(161-175) epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were specific for new helper epitopes. These data suggest that naturally acquired CD4(+) T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor.

BAFF and the regulation of B cell survival.

The TNF family member BAFF is a fundamental survival factor for B cells. BAFF binds to three receptors, only one of which, BAFF-R, does not cross-react with the BAFF-related ligand APRIL. The survival function of BAFF on B cells is mediated mainly by BAFF-R and is particularly effective in transitional B cells. BAFF depletion leads to a considerable decrease in mature B cells, without apparent effect on B cell genesis. Consistently, BAFF overexpression results in an expanded B cell compartment and autoimmunity in mice. Elevated amounts of BAFF can be found in the serum of patients suffering from autoimmune diseases. The BAFF system is a promising target for the treatment of autoimmune diseases.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

Pulmonary Sarcoid-like Granulomatosis after Multiple Vaccinations of a Long-term Surviving Patient with Metastatic Melanoma.

Autoimmune side effects are frequent in patients with cancer treated with immune checkpoint-targeting antibodies, but are rare with cancer vaccines. Here, we present a case report on a patient with metastatic melanoma who developed pulmonary sarcoid-like granulomatosis following repetitive vaccinations with peptides and CpG. Despite multiple metastases, including one lesion in the brain, the patient is alive and well more than 13 years after the diagnosis of metastatic disease. The strongly activated tumor-specific CD8(+) T cells showed robust long-term memory and effector functions. It is possible that long-term survival and adverse autoimmune events may become more common for vaccines inducing robust anticancer immune responses as were present in this patient. Cancer Immunol Res; 2(12); 1148-53. ©2014 AACR.

Ultra-fast recovery from right neglect after 'awake surgery' for slow-growing tumor invading the left parietal area

It is now possible to perform resections of slow-growing tumors in awake patients. Using direct electrical stimulation, real-time functional mapping of the brain can be used to prevent the resection of essential areas near the tumor. Simple clinical observations of patients with a resection of slow-growing tumors have demonstrated substantial recovery within a few days of such 'awake surgery'. The aim of this study was to investigate the kinetics of recovery following the resection of slow-growing tumors invading the left parietal area and to focus mainly on its rapidity. Two patients were assessed by standard line bisection tests and compared with eight healthy individuals. Independently of the pure nature of the symptoms, we report that the patients rapidly and substantially recovered from pronounced right neglect. They were tested 48 hours after the surgery and the recovery was significant for both patients after less than 4 hours. Strikingly, for one patient, recovery was ultra fast and substantial in the first practice session within less than 7 minutes: it occurred without verbal feedback and was substantially retained during the following testing session. Its rapidity suggests a process of unmasking redundant networks. With the slow growth of the lesion, the contralesional hemisphere is probably progressively prepared for rapid unmasking of homologue networks. These results have major clinical implications. For patients with an invading left-side tumor, it is now clear that line bisections are required before, during, and after awake surgery to: plan the surgery, control the quality of the resection, and also optimize the rehabilitation of the patient

Development of T-B cell collaboration in neonatal mice.

The neonatal immune response is impaired during the first weeks after birth. To obtain a better understanding of this immaturity, we investigated the development of T cell interactions with B cells in mice. For this purpose, we analyzed the immune response to three T-dependent antigens in vivo: (i) the polyclonal antibody response induced by vaccinia virus; (ii) the production of polyclonal and specific antibodies following immunization with hapten-carrier conjugates; (iii) the mouse mammary tumor virus superantigen (sAg) response involving an increase in sAg-reactive T cells and induction of polyclonal antibody production. After vaccinia virus injection into neonates, the polyclonal antibody response was similar to that observed in adult mice. The antibody response to hapten-carrier conjugates, however, was delayed and reduced. Injection with sAg-expressing B cells from neonatal or adult mice allowed us to determine whether B cells, T cells or both were implicated in the reduced immune response. In these sAg responses, neonatal T cells were stimulated by both neonatal and adult sAg-presenting B cells but only B cells from adult mice differentiated into IgM- and IgG-secreting plasma cells in the neonatal environment in vivo. Injecting neonatal B cells into adult mice did not induce antibody production. These results demonstrate that the environment of the neonatal lymph node is able to support a T and B cell response, and that immaturity of B cells plays a key role in the reduced immune response observed in the neonate.

Notch tumor suppressor function

Cancer development results from deregulated control of stem cell populations and alterations in their surrounding environment. Notch signaling is an important form of direct cell-cell communication involved in cell fate determination, stem cell potential and lineage commitment. The biological function of this pathway is critically context dependent. Here we review the pro-differentiation role and tumor suppressing function of this pathway, as revealed by loss-of-function in keratinocytes and skin, downstream of p53 and in cross-connection with other determinants of stem cell potential and/or tumor formation, such as p63 and Rho/CDC42 effectors. The possibility that Notch signaling elicits a duality of signals, involved in growth/differentiation control and cell survival will be discussed, in the context of novel approaches for cancer therapy

Long circulating half-life and high tumor selectivity of the photosensitizer meta-tetrahydroxyphenylchlorin conjugated to polyethylene glycol in nude mice grafted with a human colon carcinoma.

In a mode of nude mice bearing a human colon carcinoma xenograft, the biodistribution and tumor localization of metatetrahydroxyphenylchlorin (m-THPC) coupled to polyethylene glycol (PEG) were compared with those of the free form of this photosensitizer used in photodynamic therapy (PDT). At different times after i.v. injection of both forms of 125I-labeled photosensitizer, m-THPC-PEG gave on average a 2-fold higher tumor uptake than free m-THPC. In addition, at early times after injection, m-THPC-PEG showed a 2-fold longer blood circulating half-life and a 4-fold lower liver uptake than free m-THPC. The tumor to normal tissue ratios of radioactivity concentrations were always higher for m-THPC-PEG than for free m-THPC at any time point studied from 2 to 96 hr post-injection. Significant coefficients of correlation between direct fluorescence measurements and radioactivity counting were obtained within each organ tested. Fluorescence microscopy studies showed that m-THPC-PEG was preferentially localized near the tumor vessels, whereas m-THPC was more diffusely distributed inside the tumor tissue. To verify whether m-THPC-PEG conjugate remained phototoxic in vivo, PDT experiments were performed 72 hr after injection and showed that m-THPC-PEG was as potent as free m-THPC in the induction of tumor regression provided that the irradiation does for m-THPC-PEG conjugate was adapted to a well-tolerated 2-fold higher level. The overall results demonstrate first the possibility of improving the in vivo tumor localization of a hydrophobic dye used for PDT by coupling it to PEG and second that a photosensitizer conjugated to a macromolecule can remain phototoxic in vivo.

Human CD8(+) T cells engineered with supraphysiological TCRs are functionally inhibited via PD-1 and SHP-1 phosphatase

Purpose/Objective: Protective CD8+ T cell responses rely on TCRdependent recognition of immunogenic peptides presented by MHC I. Cytolytic T lymphocytes directed against self/tumor antigens express TCRs of lower affinity/avidity than pathogen-derived T lymphocytes and elicit less protective immune responses due to mechanisms of central and peripheral tolerance. Anti-tumor T cell reactivity can be improved by increasing the TCR-pMHC affinity within physiological limits, while intriguingly further increase in the supraphysiological range (KD < 1 lM) leads to drastic functional declines. We aim at identifying the molecular mechanisms underlying the loss of T cell responsiveness associated with supraphysiological TCRpMHC affinities in order to improve effectiveness of TCR-engineered T cells used in adoptive cell transfer (ACT) cancer immunotherapy. Materials and methods: Using a panel of human CD8+ T cells engineered with TCRs of incremental affinity for the HLA-A2-resticted tumor cancer testis antigen NY-ESO-1, we performed comparative gene expression microarray and TCR-mediated signaling analysis together with membrane receptors level analysis. Results: As compared to cells expressing TCR affinities generating optimal function (KD from 5to 1 lM), those with supraphysiological affinity (KD from 1 lM to 15 nM) had an overall reduced expression of genes implied in signaling, cell activation and proliferation, and showed impaired proximal and distal TCR signaling capacity. This correlated with a decline in surface expression of CD8b, CD28 and activatory TNFR superfamily members. Importantly, expression of inhibitory receptor PD-1 and SHP-1 phosphatase was upregulated in a TCR affinity-dependent manner. Consequently, PD-L1 and SHP-1 blockade restored the function of T cells with high TCRs affinity. Moreover, SHP-1 inhibition also augmented functional efficacy of T cells with TCRs of optimal affinity. Conclusions: Our findings indicate that TCR affinity-associated regulatory mechanisms control T cells responsiveness at various levels to limit potential auto-reactive cytotoxic effects. They also support the development of ACT therapies combined with blockade of inhibitory molecules such as SHP-1 to enhance effectiveness of T cell immunotherapy.

Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail.

Carcinoembryonic antigen (CEA) is a well-known tumor marker, consisting of a single heavily glycosylated polypeptide chain (mol. wt 200 kD), bound to the cell surface by a phosphatidylinositol-glycan anchor. The hydrophobic domain, encoded by the 3' end of the open reading frame of the CEA gene is not present in the mature protein. This domain is assumed to play an important role in the targeting and attachment of CEA to the cell surface. To verify this hypothesis, a recombinant CEA cDNA lacking the 78 b.p. of the 3' region, encoding the 26 a.a. hydrophobic domain, was prepared in a Rc/CMV expression vector containing a neomycin resistance gene. The construct was transfected by the calcium phosphate technique into CEA-negative human and rat colon carcinoma cell lines. Geneticin-resistant transfectants were screened for the presence of CEA in the supernatant and positive clones were isolated. As determined by ELISA, up to 13 micrograms of recombinant CEA per 10(6) cells was secreted within 72 hr by the human transfected cells and about 1 microgram by the rat cells. For comparison, two human carcinoma cell lines, CO112 and LS174T, selected for high CEA expression, shed about 45 and 128 ng per 10(6) cells within 72 hr, respectively. Western blot analysis showed that the size of the recombinant CEA secreted by the transfected human cells is identical to that of reference CEA purified from human colon carcinomas metastases (about 200 kD). The recombinant CEA synthesized by the transfected rat carcinoma cells has a smaller size (about 144 kD, possibly due to incomplete glycosylation), as has already been observed for CEA produced by rat colon carcinoma cells transfected with full-length CEA cDNA. The 100-fold increase in secretion of rCEA encoded by truncated CEA cDNA transfected in human cells confirms the essential role of this domain in the targeting and anchoring of the glycoprotein. These results suggest a new approach for the in vitro production of large amounts of CEA needed in research laboratories and for immunoassay kits.

Methods for the ex vivo characterization of human CD8+ T subsets based on gene expression and replicative history analysis.

The generation of an antigen-specific T-lymphocyte response is a complex multi-step process. Upon T-cell receptor-mediated recognition of antigen presented by activated dendritic cells, naive T-lymphocytes enter a program of proliferation and differentiation, during the course of which they acquire effector functions and may ultimately become memory T-cells. A major goal of modern immunology is to precisely identify and characterize effector and memory T-cell subpopulations that may be most efficient in disease protection. Sensitive methods are required to address these questions in exceedingly low numbers of antigen-specific lymphocytes recovered from clinical samples, and not manipulated in vitro. We have developed new techniques to dissect immune responses against viral or tumor antigens. These allow the isolation of various subsets of antigen-specific T-cells (with major histocompatibility complex [MHC]-peptide multimers and five-color FACS sorting) and the monitoring of gene expression in individual cells (by five-cell reverse transcription-polymerase chain reaction [RT-PCR]). We can also follow their proliferative life history by flow-fluorescence in situ hybridization (FISH) analysis of average telomere length. Recently, using these tools, we have identified subpopulations of CD8+ T-lymphocytes with distinct proliferative history and partial effector-like properties. Our data suggest that these subsets descend from recently activated T-cells and are committed to become differentiated effector T-lymphocytes.

Notch signaling in the integrated control of keratinocyte growth/differentiation and tumor suppression.

Oncogenesis is closely linked to abnormalities in cell differentiation. Notch signaling provides an important form of intercellular communication involved in cell fate determination, stem cell potential and differentiation. Here we review the role of this pathway in the integrated growth/differentiation control of the keratinocyte cell type, and the maintenance of normal skin homeostasis. In parallel with the pro-differentiation function of Notch1 in keratinocytes, we discuss recent evidence pointing to a tumor suppressor function of this gene in both mouse skin and human cervical carcinogenesis. The possibility that Notch signaling elicits signals with a duality of growth positive and negative function will be discussed.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.

TNF-alpha-dependent loss of IKKbeta-deficient myeloid progenitors triggers a cytokine loop culminating in granulocytosis.

Loss of IκB kinase (IKK) β-dependent NF-κB signaling in hematopoietic cells is associated with increased granulopoiesis. Here we identify a regulatory cytokine loop that causes neutrophilia in Ikkβ-deficient mice. TNF-α-dependent apoptosis of myeloid progenitor cells leads to the release of IL-1β, which promotes Th17 polarization of peripheral CD4(+) T cells. Although the elevation of IL-17 and the consecutive induction of granulocyte colony-stimulating factor compensate for the loss of myeloid progenitor cells, the facilitated induction of Th17 cells renders Ikkβ-deficient animals more susceptible to the development of experimental autoimmune encephalitis. These results unravel so far unanticipated direct and indirect functions for IKKβ in myeloid progenitor survival and maintenance of innate and Th17 immunity and raise concerns about long-term IKKβ inhibition in IL-17-mediated diseases.