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Persistent Rel/nuclear factor-κB (NF-κB) activity is a hallmark of many human cancers, and the Rel proteins are implicated in leukemia/lymphomagenesis but the mechanism is not fully understood. Microarray analysis to identify transformation-impacting genes regulated by NF-κB's oncogenic v-Rel and c-Rel proteins uncovered that Rel protein expression leads to transcriptional repression of key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for phosphoinositide 3-kinase (BCAP) and immunoglobulin λ light chain (Igλ), and is accompanied by a block in BCR-mediated activation of extracellular signal-regulated kinase, Akt, and c-Jun-NH2-kinase in response to anti-IgM. The BLNK and BCAP proteins were also down-regulated in lymphoid cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA-binding studies identified functional NF-κB–binding sites, and chromatin immunoprecipitation (ChIP) data showed binding of Rel to the endogenous blnk and bcap promoters in vivo. Importantly, restoration of either BLNK or BCAP expression strongly inhibited transformation of primary chicken lymphocytes by the potent NF-κB oncoprotein v-Rel. These findings are interesting because blnk and other BCR components and signaling molecules are down-regulated in primary mediastinal large B-cell lymphomas and Hodgkin's lymphomas, which depend on c-Rel for survival, and are consistent with the tumor suppressor function of BLNK. Overall, our results indicate that down-regulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for Rel's transforming activity. [Cancer Res 2008;68(3):808–14]

BackgroundAchieving robust responses with adoptive cell therapy for the treatment of the highly lethal pancreatic ductal adenocarcinoma (PDA) has been elusive. We previously showed that T cells engineered to express a mesothelin-specific T cell receptor (TCRMsln) accumulate in autochthonous PDA, mediate therapeutic antitumor activity, but fail to eradicate tumors in part due to acquisition of a dysfunctional exhausted T cell state.MethodsHere, we investigated the role of immune checkpoints in mediating TCR engineered T cell dysfunction in a genetically engineered PDA mouse model. The fate of engineered T cells that were either deficient in PD-1, or transferred concurrent with antibodies blocking PD-L1 and/or additional immune checkpoints, were tracked to evaluate persistence, functionality, and antitumor activity at day 8 and day 28 post infusion. We performed RNAseq on engineered T cells isolated from tumors and compared differentially expressed genes to prototypical endogenous exhausted T cells.ResultsPD-L1 pathway blockade and/or simultaneous blockade of multiple coinhibitory receptors during adoptive cell therapy was insufficient to prevent engineered T cell dysfunction in autochthonous PDA yet resulted in subclinical activity in the lung, without enhancing anti-tumor immunity. Gene expression analysis revealed that ex vivo TCR engineered T cells markedly differed from in vivo primed endogenous effector T cells which can respond to immune checkpoint inhibitors. Early after transfer, intratumoral TCR engineered T cells acquired a similar molecular program to prototypical exhausted T cells that arise during chronic viral infection, but the molecular programs later diverged. Intratumoral engineered T cells exhibited decreased effector and cell cycle genes and were refractory to TCR signaling.ConclusionsAbrogation of PD-1 signaling is not sufficient to overcome TCR engineered T cell dysfunction in PDA. Our study suggests that contributions by both the differentiation pathways induced during the ex vivo T cell engineering process and intratumoral suppressive mechanisms render engineered T cells dysfunctional and resistant to rescue by blockade of immune checkpoints.

Glia are the most abundant cell type in the mammalian brain. They regulate neuronal development and function, CNS immune surveillance, and stem cell biology, yet we know surprisingly little about glia in any organism. Here we identify over 40 new Drosophila glial genes. We use glial cells missing (gcm) mutants and misexpression to verify they are Gcm regulated in vivo. Many genes show unique spatiotemporal responsiveness to Gcm in the CNS, and thus glial subtype diversification requires spatially or temporally restricted Gcm cofactors. These genes provide insights into glial biology: we show unc-5 (a repulsive netrin receptor) orients glial migrations and the draper gene mediates glial engulfment of apoptotic neurons and larval locomotion. Many identified Drosophila glial genes have homologs expressed in mammalian glia, revealing conserved molecular features of glial cells. 80% of these Drosophila glial genes have mammalian homologs; these are now excellent candidates for regulating human glial development, function, or disease.

T-cell tolerance to self antigens is required to prevent autoimmunity. Since most tumor antigens are self antigens a critical challenge is to develop strategies that break T-cell tolerance to such antigens without causing autoimmune injury. A hallmark of tolerant T-cells is the failure to respond to antigen, but the precise regulatory mechanisms remain largely unknown. Previously we showed that strategies which “force” tolerant T-cells to proliferate abrogate tolerance. To understand the mechanisms by which repetitive cell division rescues tolerant T-cells, we studied the effect of homeostasis-driven proliferation (HP) on tolerant T cells. Following adoptive transfer into lymphopenic hosts, tolerant T cells became functional, even in a tolerogenic environment. However, T-cell rescue was only transient and tolerance was re-imposed after the host became lymphoreplete, even if no self antigen was present. The finding that rescued T-cells reacquired tolerance in non-tolerogenic hosts challenges the prevailing paradigm that tolerance maintenance requires continuous exposure of T-cells to self antigen. Instead, mRNA and microRNA profiling revealed that tolerant T-cells harbor a tolerance-specific molecular signature; while the programming can be temporarily overridden, it is eventually reasserted, dictated by an “epigenetic memory”. These studies on the regulation of T-cell tolerance have revealed novel targets for cancer immunotherapy and the treatment of autoimmune disease.