Q-fever is a flu-like illness caused by Coxiella burnetii ( Cb ), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb , consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb .
Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a Langerhans cells in the dermis and CD11c dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.
Lung cancer is the leading cause of cancer-related deaths worldwide. Surgery and chemoradiation are the standard of care in early stages of non-small cell lung cancer (NSCLC), while immunotherapy is the standard of care in late-stage NSCLC.The immune composition of the tumor microenvironment (TME) is recognized as an indicator for responsiveness to immunotherapy, although much remains unknown about its role in responsiveness to surgery or chemoradiation. In this pilot study, we characterized the NSCLC TME using mass cytometry (CyTOF) and bulk RNA sequencing (RNA-Seq) with deconvolution of RNA-Seq being performed by Kassandra, a recently published deconvolution tool. Stratification of patients based on the intratumoral abundance of B cells identified that the B-cell rich patient group had increased expression of CXCL13 and greater abundance of PD1 + CD8 T cells. The presence of B cells and PD1 + CD8 T cells correlated positively with the presence of intratumoral tertiary lymphoid structures (TLS).We then assessed the predictive and prognostic utility of these cell types and TLS
e21026 Background: Comprehensive molecular profiling and the use of biomarkers as companion diagnostics have transformed precision medicine for cancer patients. To identify patient-specific tumor microenvironment and biomarker profiles, we assessed the accuracy of our deconvolution algorithm in identifying cellular compositions from whole exome (WES) and whole transcriptome (RNA-seq) sequencing of solid tumors compared with cell populations identified by Mass Cytometry by Time of Flight (CyTOF) in surgically resected tissue from non-small cell lung cancer (NSCLC) patients. Methods: Resected NSCLC tissue was divided for RNA-seq and WES of whole tissue (n = 9) and for generating tissue single cell suspensions through mechanical dissociation and enzymatic digestion (n = 11). Bulk RNA-seq and CyTOF were performed on all cell suspensions. Cellular phenotypes were identified using clustering algorithms in CyTOF and predicted from bulk RNA-seq using our proprietary computational method. Results: Cellular composition reconstructed from RNA-seq correlated with the composition detected by CyTOF (R2= 0.922, n = 7) from cell suspensions. To recover the cell percentage from bulk RNA-seq, a machine learning framework was trained on the cell compendia comprising 7,117 unique cell type RNA-seq profiles. A two-stage hierarchical learning procedure generated a gradient boosting Light GBM model that included training on artificial RNA-seq mixtures of different cell types. With this method, we found that stromal and malignant cells were depleted during single cell suspension preparation, resulting in statistically significant differences in the tumor cell composition reconstructed from solid tissue and single cell suspensions. Immune cell types namely T cells and macrophages were similarly represented in both the bulk tumor tissue and matched single cell suspensions. Transcriptomics revealed a subgroup of patients whose tumors were B-cell-enriched, which was validated in other NSCLC cohorts and was associated with greater CD4+ and CD8+ T cell infiltration and improved clinical outcomes. Conclusions: Since preparation of single cell suspensions leads to the loss of several cellular components, RNA-seq of tumor bulk tissue better describes the molecular and cellular properties of the tumor microenvironment. The combination of RNA-seq and WES of tumor tissue provides a comprehensive profile of cellular composition, suggesting that this combination is ideal for precision medicine applications.
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