Tumor escape and progression under immune pressure.

Although cancers develop and progress in immunocompetent hosts, immunological therapies for cancer have been proposed as alternative or complementary approaches to more standard therapy. It was initially thought that tumors were silent to the immune system, and that breaking immunological tolerance could result in immune-mediated tumor rejection. However, we have learned that cancer patients have preexisting immune responses against their tumor antigens which, nevertheless, fail to protect them, in part because of increased activity of the immune suppressor cells such as myeloid-derived suppressor cells (MDSC). Attempts to develop combinatorial therapies by depleting suppressor cells or blocking suppressor pathways and at the same time actively inducing immune responses in vivo or adoptively transferring tumor-specific T cells have largely failed. Very limited success has been achieved only against melanoma, using adoptive T-cell therapy, or prostate cancer, using a vaccine which improves patient survival but has no apparent inhibitory effect on disease progression. Further progress in the immunotherapy of cancer has been halted because of a poor understanding of the cellular components of the immune responses working together in favor of or against the tumors, as well as our inability to reliably reprogram immune responses towards the most effective phenotypes against cancer. This special issue is focused on understanding the escape mechanisms that malignant cells develop to hijack antitumor immune responses as well as strategies to overcome tumor escape. Four main areas that are covered in this issue include the following. Opposing Functions of the Immune System in Tumor Inhibition and Tumor ProgressionRobert Schreiber proposed the term “cancer immunoediting” in order to broadly describe the dual host-protecting and tumor-sculpting actions of the immune system that not only survey for, and eliminate, nascent malignant cells but also shape neoplastic disease through equilibrium and escape mechanisms. In this issue, M. Aris et al. discuss the dual function of the immune system in controlling and promoting tumor progression in cutaneous melanoma. They propose that tumor evolution is because of a continuous feedback between tumor cells and their environment, and thus different combinatorial therapeutic approaches can be implemented according to the tumor stage. A. Amedei et al. discuss recent knowledge on the contribution of T cells in oncogenesis. They review the different types, “friend or foe,” of T-cell response in gastric cancer. Tumor-Associated Modulation of Immune Checkpoint MoleculesUpon activation, T cells develop negative feedback regulatory mechanisms in order to avoid overstimulation. These include the expression of checkpoint molecules such as PD-1 and CTLA-4. T cells that recognize and respond to tumor antigens produce IFN-γ. A dual function of IFN-γ is the induction of apoptosis in target cells and upregulation of PD-L1 that interacts with PD-1 positive T cells, thereby resulting in the exhaustion of tumor-reactive T cells. Expression of CTLA-4 on activated T cells also results in T-cell anergy upon interaction with costimulatory molecules on DCs. S. Sapozink et al. describe new immunomodulatory approaches currently in the development pipeline, with focus on the novel CEACAM1 immune checkpoint, and compare its potential to the extensively described lymphocyte inhibitory targets, CTLA4 and PD-1. E. Rozali et al. provide an extensive review of the literature on the immunoregulatory role of PD-L2 in cancer-induced immune suppression and discuss the results of recent studies targeting PD-L2 in cancer. L. Cruz-Merino et al. discuss immune escape mechanisms in Hodgkin’s lymphoma (HL) and summarize the clinical, histological, pathological, and biological factors in HL, with special emphasis on the improvement of prognosis and their impact on treatment strategies. L. Farnault et al. introduce various mechanisms involved in the escape of hematological malignancies from NK-cell surveillance. These include NK-cell qualitative and qualitative deficiencies that occur through modulating the inhibitory and activating stimuli. Tumor-Induced Immune SuppressionMalignant cells produce cytokines and chemokines that facilitate the expansion or differentiation of immune suppressor cells such as Tregs, MDSC, and M2 macrophages. G. Zhou and H. Levitsky summarize the findings from some recent preclinical and clinical studies, focusing on how tumor cells advance their survival and expansion by hijacking therapy-induced immune effector mechanisms that would otherwise mediate their destruction. A particularly interesting notion that is touched upon involves tumor-independent treatment-induced homeostatic counter-regulation. M. Jadus et al. cover the escape mechanisms of bronchogenic lung cancer that must be overcome before they can be successfully treated. They also review the history of immunotherapy directed towards lung cancers. N. Hao et al. discuss the role of tumor-associated macrophages including M1 and M2 subsets during tumour progression and metastasis, highlighting the immunosuppressive role of M2 macrophages. V. Levina et al. investigate the role of indoleamine 2,3-dioxygenase (IDO1) in tumor escape and metastasis using 4T1 mammary carcinoma model. They show that IDO1 can not only suppress antitumour immune responses but also promote tumour cell proliferation. Improved Immunotherapeutic Strategies to Overcome Tumor EscapeImmunotherapy combined with blockade of immune suppressor pathways has been developed to overcome tumor-induced immune suppression. Cornelissen et al. discuss the interplay between a dual function of the immune responses against mesothelioma which can either inhibit or stimulate tumor growth and review the challenges associated with immunotherapy. They also discuss possible strategies and opportunities to overcome tumor escape. R. Casalegno-Garduño et al. analyze the expression of the leukemia-associated antigen receptor for hyaluronan acid-mediated motility (RHAMM) in patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Their results suggest that immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the clinical outcome in AML/MDS patients. S.Wallner et al. summarize the current knowledge about the negative regulatory role of Cbl-b in T-cell activation and its potential therapeutic implications for cancer immunotherapy. H. Nagai et al. demonstrate that sorafenib-induced Th1 dominance can prevent the escape of tumor cells from the host immune system in liver cirrhosis (LC) patients with advanced hepatocellular carcinoma (aHCC).Overall, this special issue provides a well-rounded synopsis of representative research efforts addressing the issues related to “tumor escape and progression under immune pressure.

Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase ST6Gal I

Elevation of serum sialic acid and the ST6Gal-1 sialyltransferase is part of the hepatic system inflammatory response, but the contribution of ST6Gal-1 has remained unclear. Hepatic ST6Gal-1 elevation is mediated by P1, 1 of 6 promoters regulating the ST6Gal1 gene. We report that the P1-ablated mouse, Siat1ΔP1, and a globally ST6Gal-1–deficient mouse had significantly increased peritoneal leukocytosis after intraperitoneal challenge with thioglycollate. Exaggerated peritonitis was accompanied by only a modest increase in neutrophil viability, and transferred bone marrow–derived neutrophils from Siat1ΔP1 mice migrated to the peritonea of recipients with normal efficiency after thioglycollate challenge. Siat1ΔP1 mice exhibited 3-fold greater neutrophilia by thioglycollate, greater pools of epinephrine-releasable marginated neutrophils, greater sensitivity to G-CSF, elevated bone marrow CFU-G and proliferative-stage myeloid cells, and a more robust recovery from cyclophosphamide-induced myelosuppression. Bone marrow leukocytes from Siat1ΔP1 are indistinguishable from those of wild-type mice in α2,6-sialylation, as revealed by the Sambucus nigra lectin, and in the expression of total ST6Gal-1 mRNA. Together, our study demonstrated a role for ST6Gal-1, possibly from extramedullary sources (eg, produced in liver) in regulating inflammation, circulating neutrophil homeostasis, and replenishing granulocyte numbers

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients

Establish non-toxic MTX and MMF dose regimens for <i>in vivo</i> selection.

<p>6–8 week old NSG mice (n = 6) were (<b>a</b>), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (<b>b</b>), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (<b>c</b>), white blood cell counts, (<b>d</b>), body weight, (<b>e</b>), Hemoglobin, (<b>f</b>), ALT, and (<b>g</b>), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.</p

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer

Primary human T cells transduced to DHFR^FS/IMPDH2^IY transgenes are resistant to MTX and MPA.

<p>Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt⁺ T cells (99.5% EGFRt⁺; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (<b>a</b>), MPA (<b>b</b>), and a combination of MTX+ MPA (<b>c</b>), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.</p

Primary human T cells transduced to express huEGFRt/DHFR^FS/IMPDH2^IY can be selected <i>in vitro</i> with MTX and MPA.

<p>(<b>a</b>), Representative flow cytometric evaluation of EGFRt transgene expression (grey histograms) on transduced T cells over 10 days of culture in 0.1 µM MTX, 1 µM MPA, or 0.05 µM MTX +0.75 µM MPA. Percentage of positive cells above control staining (open histograms) is indicated in each histogram. (<b>b</b>), Graphical depiction of the percentages of EGFRt⁺ cells shown in (a). Equal numbers of three different gene-modified T cell lines were each plated in a 6-well plate at the indicated drug concentrations. The data represent means ± S.D. There was a significant difference between the cells on D0 vs. D10 at either 0.1 µM MTX, 1 µM MPA, or 0.05 µM MTX +0.75 µM MPA (****, p≤0.0001; *, p≤0.05).</p

Immunomagnetically enriched huEGFRt/DHFR^FS/IMPDH2^IY T cells maintained their cell surface phenotype and effector function upon culture in MTX and MPA.

<p>(<b>a</b>), Cell surface expression of EGFRt, CD4, CD8, CD28, CD45, TCRαβ and CD127 (grey histogram) vs. isotype control antibody (open histogram) on EGFRt-enriched T cells after 14 days culture +/−0.1 µM MTX or 1 µM MPA were analyzed by flow cytometry. Percentage of positive cells is indicated. (<b>b</b>), Cytotoxic activity of the same cells in (<b>a</b>) after 4 hr co-culture with ⁵¹Cr- labeled OKT3-expressing LCL (LCL-OKT3) or negative control LCL targets. Mean percent of ⁵¹Cr release ± S.D. of triplicate wells is depicted. (<b>c</b>), Production of cytokines IL-2, GM-CSF, IFN-γ and TNF-α by the same cells as described in (<b>a</b>). Supernatants were collected after overnight co-culture of the same cells in (a) with LCL-OKT3 or negative control LCL stimulators, and mean (±S.D. of triplicate wells) cytokine levels were quantified by cytometric bead array.</p