Purpose A phase I study was conducted to determine safety, clinical efficacy, and anti-tumor immune responses in patients with advanced non-small cell lung carcinoma (NSCLC) following intratumoral (IT) administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen-specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222). Experimental Design Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 106, 5 × 106, 1 × 107, or 3 × 107 dendritic cells/injection) by CT- or bronchoscopic-guided IT injections (days 0 and 7). Immune responses were assessed by tumor antigen-specific peripheral blood lymphocyte induction of IFN-γ in ELISPOT assays. Tumor biopsies were evaluated for CD8+ T cells by immunohistochemistry (IHC) and for PD-L1 expression by IHC and real-time PCR (RT-PCR). Results Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor associated antigens (TAA). Tumor CD8+ T cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8+ T cells per mm2). Patients with increased CD8+ T cells following vaccination showed significantly increased PD-L1 mRNA expression. Conclusions Intratumoral vaccination with Ad-CCL21-DC resulted in 1) induction of systemic tumor antigen-specific immune responses, 2) enhanced tumor CD8+ T cell infiltration, and 3) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination.

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5' flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in Drosophila and other animals. Approximately 300 bases of proximal 5' flanking DNA control expression in Drosophila CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of acj6 mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5' flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional trans factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.

The purpose of this study was to determine the dynamic contributions of different immune cell subsets to primary and abscopal tumor regression after hypofractionated radiation therapy (hRT) and the impact of anti-PD-1 therapy. A bilateral syngeneic FSA1 fibrosarcoma model was used in immunocompetent C3H mice, with delayed inoculation to mimic primary and microscopic disease. The effect of tumor burden on intratumoral and splenic immune cell content was delineated as a prelude to hRT on macroscopic T1 tumors with 3 fractions of 8 Gy while microscopic T2 tumors were left untreated. This was performed with and without systemic anti-PD-1. Immune profiles within T1 and T2 tumors and in spleen changed drastically with tumor burden in untreated mice with infiltrating CD4+ content declining, while the proportion of CD4+ Tregs rose. Myeloid cell representation escalated in larger tumors, resulting in major decreases in the lymphoid:myeloid ratios. In general, activation of Tregs and myeloid-derived suppressor cells allow immunogenic tumors to grow, although their relative contributions change with time. The evidence suggests that primary T1 tumors self-regulate their immune content depending on their size and this can influence the lymphoid compartment of T2 tumors, especially with respect to Tregs. Tumor burden is a major confounding factor in immune analysis that has to be taken into consideration in experimental models and in the clinic. hRT caused complete local regression of primary tumors, which was accompanied by heavy infiltration of CD8+ T cells activated to express IFN-γ and PD-1; while certain myeloid populations diminished. In spite of this active infiltrate, primary hRT failed to generate the systemic conditions required to cause abscopal regression of unirradiated microscopic tumors unless PD-1 blockade, which on its own was ineffective, was added to the RT regimen. The combination further increased local and systemically activated CD8+ T cells, but few other changes. This study emphasizes the subtle interplay between the immune system and tumors as they grow and how difficult it is for local RT, which can generate a local immune response that may help with primary tumor regression, to overcome the systemic barriers that are generated so as to effect immune regression of even small abscopal lesions.

BACKGROUND: Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. KRAS mutations are known to activate the RAF-MEK-ERK pathway. Fos-related antigen-1 (FRA1) is a MEK/ERK-dependent transcription factor and member of the AP-1 transcription factor superfamily. Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in immune-mediated tumor evasion. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation and FRA1 may be an important transcriptional mediator. METHODS: In order to assess the role of KRAS mutation independent of other somatic mutations and to begin to understand potential immune suppressive pathways operative in pulmonary premalignancy, premalignant human bronchial epithelial cell lines (HBEC) were used instead of human lung cancer cell lines. Four immortalized HBEC (2, 3, 7, and 11 cell lines) with KRAS v12-mutation (HBEC-KRAS) compared to control (HBEC-vector) were used to assess mRNA and protein expression levels of PD-L1 by RT-qPCR, flow cytometry, and western blot. Cell-lines were treated with MEK (ERK kinase) inhibitor (PD0325901) for 24 hours (h) up to 1μM. FRA1 was silenced by transfection with siRNA at 100nM. RESULTS: In comparing HBEC-KRAS to HBEC-vector (wild-type) cells, PD-L1 mRNA (1.3∼3.4 fold) and surface protein expressions (1.2∼2.3 fold by flow cytometry) were increased in all 4 cell lines and confirmed by western blot analyses. PD-L1 mRNA and protein levels were highest in HBEC3-KRAS. MEK inhibition resulted in decreased PD-L1 expression by 10 fold (HBEC3-vector) and 11 fold (HBEC3-KRAS) in mRNA levels and 3 fold (HBEC3-vector and HBEC3-KRAS) in surface protein levels. In comparing HBEC3-KRAS to HBEC3-vector, FRA1 increased mRNA (2.7 fold) and protein level expression (2.8 fold). In comparing HBEC3-KRAS to HBEC3-vector cells, FRA1 silencing (FRA1 siRNA) resulted in reduced PD-L1 (53% at 48 h; 73% at 72 h) and FRA1 (94% at 48 h; 99% at 72 h) by western blot. In comparing HBEC3-KRAS to HBEC3-vector cells, ERK phosphorylation increased 7.9 fold and FRA1 silencing led to inhibition of ERK phosphorylation by 2.8 fold at 72 h by western blot. CONCLUSIONS: Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates KRAS mutation driven up regulation of PD-L1 and at least in part through FRA1 dependence. Our data suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway through FRA1 and MEK/ERK-dependent transcriptional regulation. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential new combination immune therapies. Citation Format: Mi-Heon Lee, Jane Yanagawa, Tonya C Walser, Jonathan W. Goldman, Edward B. Garon, Gang Zeng, Sherven Sharma, Boning Gao, John Minna, Steven M. Dubinett, Jay M Lee. FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2324.

Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4+CD25+ regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4+ BDC2.5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c+ or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c+ dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4+ T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.