We eluted peptides from class I molecules of HLA-A2.1+ breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8+ T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8+ T cells from another HLA-A2.1+ healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1–P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.

Purpose. Pars plana vitrectomy (PPV) has been reported to reduce macular thickness and improve visual acuity in patients with diabetic macular edema (ME). The hypothesis for the study was that after PPV, clearance is accelerated and VEGF concentrations are reduced. To test this hypothesis, hVEGF(165) injections were performed in rabbit eyes, with and without PPV, and vitreous VEGF levels were measured as a function of time. Methods. The PPV group rabbits had a bilateral 25-gauge PPV, and in the no-PPV group, rabbits had intact vitreous. Intravitreal injections of hVEGF(165) were performed, and the animals were euthanatized at time points up to 7 days. The vitreous was isolated and an enzyme-linked immunosorbent assay was used to measure the VEGF levels. Pharmacokinetic parameters were determined in a noncompartmental analysis approach. Results. Mean vitreous VEGF levels decreased more rapidly in eyes subjected to PPV than in no-PPV eyes. The vitreous VEGF half-life (t([)(1/2)(])) in PPV eyes was 10 times shorter than that in normal eyes. In addition, mean clearance and mean area under the curve (AUC) increased and decreased, respectively, in eyes that underwent PPV. Conclusions. VEGF clearance is increased after PPV. Reducing VEGF concentrations in the vitreous post-PPV may partially explain the improvement in macular thickness in some patients with ME. Unexpectedly, the half-life of VEGF in the vitreous, even in no-PPV eyes, was <3 hours, whereas compounds of similar molecular weight typically have longer vitreous half-lives. The back of the eye may be uniquely adapted with rapid-clearance mechanisms to regulate vitreous VEGF levels. Further study is suggested.

Protein kinase C-θ (PKCθ) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCθ in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCθ in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCθ in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCθ−/− mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCθ−/− thymocytes in bone marrow chimeras revealed a more dramatic defect, with a &gt;80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCθ−/− mice. Inefficient positive selection of n3.L2 PKCθ−/− CD4 single-positive cells resulted from “weaker” signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCθ−/− mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCθ−/− mice. These findings now place PKCθ as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.

Introduction: Recent studies indicated that specific intestinal microbiota may modulate efficacy of anti-PD-1 and anti-CTLA-4 immunotherapy in preclinical tumor models (Sivan et al, Science. 2015;350:1084-9; Vétizou et al, Science. 2015;350:1079-84). However, little is known regarding the association of the oral microbiome with checkpoint blockade immunotherapy. In CheckMate 141 (NCT02105636), a randomized global phase 3 study comparing nivolumab with investigator’s choice (IC) therapy in patients with platinum-refractory squamous cell carcinoma of the head and neck (SCCHN), nivolumab improved median overall survival compared with IC (7.5 vs 5.1 months; P=0.01) (Ferris et al, NEJM. 2016;375:1856-67). This analysis assessed if oral microbiome profiling would yield prognostic biomarkers of response to anti-PD-1 immunotherapy in patients with SCCHN treated in CheckMate 141. Methods: Saliva samples were obtained at screening (nivolumab n=85, IC n=31) and week 7 of treatment (nivolumab n=77, IC n=28), and profiled using high-throughput 16S ribosomal RNA sequencing. Bacterial abundance was estimated using a combined differential abundance modeling approach, normalized using cumulative sum scaling to correct for sequencing depth, and analyzed using a linear model for association with response (complete response/partial response vs stable disease vs progressive disease), tumor PD-L1 expression, HPV16 status, treatment history, and patient demographics. Results: Among 221 saliva samples analyzed, bacteria from 13 phyla and 542 species were detected. At baseline, no significant associations were detected in richness of bacterial diversity with best overall response, tumor PD-L1 expression, or HPV16 status. No associations in microbial alpha and beta diversity were detected with treatment modality (nivolumab vs IC) or treatment duration (baseline vs week 7). Patients with prior radiation therapy (n=97) had lower abundance of bacteria from the families Prevotellaceae and Flavobacteriaceae than patients without prior radiation therapy (n=17). The abundance of bacteria from the families Desulfobulbaceae and Flavobacteriaceae was higher in European (n=56) than North American (n=46) patients. Conclusion: Differences in certain bacterial species were observed in patients with prior radiation therapy and between different geographical locations. However, no significant associations were detected between oral bacterial diversity and clinical response, tumor PD-L1 expression, HPV16 status, or treatment modality. This exploratory analysis is the first to evaluate the oral microbiome as a biomarker in a randomized, phase 3 clinical trial for immunotherapy in SCCHN and may serve as a basis for future assessments and experimental design. Correlation of salivary and intestinal microbiota with that from tumor specimens is warranted. Citation Format: Robert L. Ferris, George Blumenschein, Kevin Harrington, Jérôme Fayette, Joël Guigay, A. Dimitrios Colevas, Lisa Licitra, Everett Vokes, Maura Gillison, Caroline Even, Cheryl Ho, Makoto Tahara, Robert Haddad, Mark Lynch, Manish Monga, Somnath Bandyopadhyay, Omar Jabado, Henry Kao. Evaluation of oral microbiome profiling as a response biomarker in squamous cell carcinoma of the head and neck: Analyses from CheckMate 141 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT022. doi:10.1158/1538-7445.AM2017-CT022

The CD4 coreceptor works together with the T cell receptor (TCR) to deliver signals to the developing thymocyte, yet its specific contribution to positive selection and CD4 lineage commitment remains unclear. To resolve this, we used N3.L2 TCR transgenic, RAG-, and CD4-deficient mice, which are severely impaired in positive selection, and asked whether altered peptide ligands can replace CD4 function in vivo. Remarkably, in the presence of antagonist ligands that normally deleted CD4+ T cells in wild-type mice, we induced positive selection of functional CD4 lineage T cells in mice deficient in CD4. We show that the kinetic threshold for positive and negative selection was lowered in the absence of CD4, with no evident skewing toward the CD8 lineage with weaker ligands. These results suggest that CD4 is dispensable as long as the affinity threshold for positive selection is sustained, and strongly argue that CD4 does not deliver a unique instructional signal for lineage commitment.