Immune responses to disease are complex and the only way to understand such a response is to consider the behavior of multiple different cell types across both the innate and adaptive branches of the immune system. We developed Barcode Enabled Antigen Mapping (BEAM) technology to enable easy enrichment of antigen-specific cells. This technology is fully integrated with the 10x Genomics Chromium Single Cell Immune Profiling Solution to allow multimodal evaluation of immune cells including full-length, paired sequences of T and B cell receptor genes while simultaneously screening their specificity for a set of genes. Our antigen-specific TCR workflow (BEAM-T) identifies antigen-specific CD8+T cells. We demonstrate the assay’s performance with spike-in experiments using various percentages of HLA-A*02:01-restricted peptide-stimulated T cells (targeted mixture being 40% T cells with specificity for cytomegalovirus peptide, 10% T cells with specificity for flu peptide and 0–5% anti-SARS-CoV-2 T cells) into a background of human PBMCs. Cells were labeled with a panel of five viral pathogen peptide-loaded multimers, plus a HLA-A*02:01 negative control peptide to assess antigen specificity and were then sorted for CD8+PE+ T cells. We demonstrated that the major VDJ-T clonotypes identified for the SARS-CoV2 cells were retained, albeit with roughly proportionally fewer cells, as the percentage of spiked cells decreased. We further feature the robustness of the protocol with various sample types and models including H2Kb-restricted OT-1 transgenic mouse splenocytes, as well as healthy human donor PBMCs and dissociated tumor cells to demonstrate the utility of an integrated reagent-to-data analysis offering across a range of samples.

Multi-analyte single cell technologies have potential to greatly accelerate antibody discovery by identifying antigen-specific B cells. Barcode Enabled Antigen Mapping (BEAM) provides a high-throughput, multimodal analysis of antigen-bound B cells (BEAM-Ab) using 10x Genomics Chromium Single Cell Immune Profiling v2 Solution and novel computational approaches to discover B-cell receptor (BCR) sequences for further functional characterization. To demonstrate the antigen sensitivity of BEAM-Ab, we spiked in 5% Hen Egg Lysozyme (HEL) and 5% gp120 transgenic B cells in 90% wild type non-transgenic splenocytes. The cells were screened using a panel of barcoded antigens and then CD19+PE+ (antigen+) B cells were isolated using flow cytometry. Single cell gene expression, BCR, and antigen barcode analysis indicated clonotype specificity to the respective antigens in the sorted cells. Furthermore, we demonstrated BEAM-Ab specificity by analyzing a convalescent COVID patient sample against an antigen panel containing five different COVID antigens and a negative control. Analysis of this COVID sample suggests the clonal expansion of B cells recognizing the ectodomain of the wild type SARS-CoV2 spike protein, receptor binding domain of spike protein, and the ectodomain of the D614G mutant spike protein. We did not observe any clonal expansion to Omicron-specific antigen or the non-specific negative control in our dataset. Our data demonstrates that BEAM-Ab is an effective antibody discovery tool. BEAM-Ab unlocks the ability to rapidly screen large numbers of samples with an accurate single cell resolution and antigen specificity, with the entire workflow generating the candidate sequences just one week after sample processing.

Continued advances in single cell gene detection sensitivity and the ability to analyze data quickly with biological context are critical for discoveries in therapeutic research. The second version of the Chromium Single Cell Immune Profiling Solution by 10x Genomics enables highly sensitive detection of gene expression, full-length paired T-cell alpha- and beta-chain and immunoglobulin sequences, T-cell antigen specificity, and cell surface protein expression from the same single cells, allowing a comprehensive view of the immune response at the cellular level. The new workflow provides a 45% increase in the number of genes detected per cell and up to a 25% increase in the cells detected with paired full-length V(D)J receptor sequences in melanoma tumor derived cells. Using a new version of Cell Ranger (v5.0), clonotypes were grouped from >30,000 cells, increasing the power to detect small clonotype expansions with fewer total cells. In addition, the new software enabled comparison of pre- and post-influenza vaccination B-cell receptor sequences from a single donor, identifying post-vaccination specific clonotypes. These technological and informatics advancements enhance a researcher’s ability to perform a broad characterization of immune cell populations at unprecedented throughput and resolution.

Profiling the complex interactions of immune infiltrate with tumor cells in a tumor microenvironment is critical for advancing our understanding of tumor biology for developing personalized cancer therapies. Using a droplet-based single cell RNA sequencing (scRNA-seq) platform, we profiled the transcriptome and immune repertoire of gastric, kidney, and lung cancer cells that are primarily immune cells from three different donors. scRNA-seq analysis also identified similar fractions of CD45+ immune cells, CD4+ and CD8+ T cells, CD19+ B cells, and myeloid cells compared to flow. Targeted scRNA-seq was also used to identify paired, full length B-cell (BCR) and T-cell (TCR) receptors. In the gastric adenocarcinoma tumor cells, gene expression analysis identified a large population of B cells, but with no clonal expansion. T cells expressing CD8A constituted about 10% of cells with the top clonotype being 1% of all clones. The clear cell RCC sample had a modest fraction of infiltrating T cells with the top clonotype representing 7.4% of all clones, and no B cell infiltrate. The NSCLC cells were mainly T and B cells but limited clonal expansion was observed; the top TCR clonotype was present on 2.2% of all T cells, no expansion was seen in any of the B cell clonotypes. These findings highlight the value of profiling of tumor immune cells holistically and not relying on the presence of B or T cells alone to understand the immune dynamics of the tumor microenvironment. The presence of tumor-infiltrating lymphocytes is associated with favorable clinical outcomes in some cancers, but understanding their cellular subtype and clonality by high resolution profiling is key in the development of immune-based cancer treatments

The flow of a suspension through a bifurcating channel is studied experimentally and by computational methods. The geometry considered is an ‘asymmetric T’, as flow in the entering branch divides to either continue straight or to make a right angle turn. All branches are of the same square cross-section of side length $D$ , with inlet and outlet section lengths $L$ yielding $L/D=58$ in the experiments. The suspensions are composed of neutrally buoyant spherical particles in a Newtonian liquid, with mean particle diameters of $d=250~\unicode[STIX]{x03BC}\text{m}$ and $480~\unicode[STIX]{x03BC}\text{m}$ resulting in $d/D\approx 0.1$ to $d/D\approx 0.2$ for $D=2.4~\text{mm}$ . The flow rate ratio $\unicode[STIX]{x1D6FD}=Q_{\Vert }/Q_{0}$ , defined for the bulk, fluid and particles, is used to characterize the flow behaviour; here $Q_{\Vert }$ and $Q_{0}$ are volumetric flow rates in the straight outlet branch and inlet branch, respectively. The channel Reynolds number $Re=(\unicode[STIX]{x1D70C}DU)/\unicode[STIX]{x1D702}$ was varied over $00.5$ for $\unicode[STIX]{x1D719}_{0}<0.16$ while $\unicode[STIX]{x1D719}_{0}=0.16$ crosses from $\unicode[STIX]{x1D6FD}\approx 0.5$ to $\unicode[STIX]{x1D6FD}>0.5$ at $Re\approx 100$ . For $\unicode[STIX]{x1D719}_{0}\geqslant 0.2$ , $\unicode[STIX]{x1D6FD}<0.5$ at all $Re$ studied. A complex dependence of the mean solid fraction in the downstream branches upon inlet fraction $\unicode[STIX]{x1D719}_{0}$ and $Re$ is observed: for $\unicode[STIX]{x1D719}_{0}<0.1$ , the solid fraction in the straight downstream branch initially decreases with $Re$ , before increasing to surpass the inlet fraction at large $Re$ ( $Re\approx 500$ for $\unicode[STIX]{x1D719}_{0}=0.05$ ). At $\unicode[STIX]{x1D719}_{0}>0.1$ , the solid fraction in the straight branch satisfies $\unicode[STIX]{x1D719}_{\Vert }/\unicode[STIX]{x1D719}_{0}>1$ , and this ratio grows with $Re$ . Discrete-particle simulations employing immersed boundary and lattice-Boltzmann techniques are used to analyse these phenomena, allowing rationalization of aspects of this complex behaviour as being due to particle migration in the inlet branch.

The motion of a rigid spherical particle in a sheared polymeric fluid is studied via experiments and numerical simulations. We study particle mobility in highly elastic fluids, where the deformation due to the sphere's movement and the shear flow both result in significant stretching of the polymer. The shear flow is imposed in a plane perpendicular to the sphere's movement, resulting in regions of high polymer tension in the wake of the sphere that can extend well into the shear flow and gradient directions. We observe that these viscoelastic wake structures, resembling wings, are linked to an increase in the form drag, providing a mechanism for a dramatic decrease in the particle mobility.

Viscoelastic particulate suspensions play a key role in many energy applications. Our goal is to develop a simulation-based tool for engineering such suspensions. This study is concerned with fully resolved simulations, wherein all flow scales associated with the particle motion are resolved. The present effort is based on Immersed Boundary (IB) methods, in which the domain grids do not conform to the particle geometry. The particles are defined on a separate Lagrangian mesh that is free to move over an underlying Eulerian grid. An immersed boundary forcing technique for moving bodies within an unstructured-mesh, non-Newtonian viscoelastic flow solver is thus developed and described. This method is implemented in a massively parallel, finite-volume-based incompressible fluid solver. A number of flows, simulated using this method are presented to assess the accuracy and correctness of the algorithm.

•Simulations of the bulk stress of a dilute suspension of spheres in Boger fluids.•Observed shear-thickening in all the viscometric functions.•Resolved a discrepancy in the theory for the second normal stress difference.•Investigated the effect of hydrodynamic interactions on the suspension rheology.

This project idea is motivated from the image based lighting technique presented in the class, where a single spherical mirror is used as a light probe for image based lighting. The project used an array of spherical mirrors arranged in a fixed pattern to capture the light field of the surrounding. The main steps in the project involved construction of a spherical mirror array of a fixed pattern, taking pictures using a high resolution camera and recreating the light field by processing the data obtained using axial cone modeling.

Two-phase flows in which solid particles are sedimenting in a visco-elastic fluid are common in drilling muds of oil and gas wells. Typically, in the numerical simulation of such flows, one employs Lagrangian formulation for the dispersed phase and Eulerian formulation of the carrier phase. Depending on the relative resolution of the grid resolution (Δ) and the characteristic length scale of the particle (dp), three different regimes [1] can be identified: (i) subgrid, when dp Δ. This study is concerned with fully resolved simulations (FRS) of particles in Newtonian and viscoelastic fluids. In FRS, all scales associated with the particle motion are resolved and the drag forces are directly evaluated, unlike in the other approaches where drag and lift correlations are used to estimate forces on the particles. A variety of methods have been proposed and used for implementing FRS in laminar and turbulent flows of Newtonian fluids. The present effort is based on the fictitious domain approach, based on the algorithm proposed by Patankar [2]. In this approach, the Navier Stoked equations, which include both Newtonian and non-Newtonian stresses, are solved over the entire domain including the region occupied by the particles. It is assumed that this region is filled with a fluid with density equal to the particle density. The particle is defined on a separate mesh that is free to move over the underlying grid. Both the fluid and the particle are assumed incompressible and the motion of the material inside the particle is constrained to be a rigid body motion. This is accomplished by adding a rigidity constraint body force in the momentum equation. The method requires transfer of information between the underlying fluid grid and the particle mesh; both interpolation and integration operators are suitably defined to preserve the second-order accuracy of the entire algorithm. The method is currently being implemented in a massively parallel, unstructured finite-volume-based incompressible fluid solver developed at Stanford University's Center for Turbulence Research [3,4]. In a previous effort [5] we have added viscoelastic effect by including the FENE-P constitutive model, which includes a tensorial partial differential equation representing the transport and dynamics of the components of the viscoelastic stresses. The combination of immersed boundary forcing formulations within a non-Newtonian Navier-Stokes solver has not been presented before and this represents one of the major challenges tackled herein. The algorithm is only briefly described here. In the first step, we solve the Navier-Stokes equation without any rigidity constraint force; we solve the momentum equations (without considerations of mass conservation) and size transport equations for the (symmetric) tensor of the nonNewtonian stresses. Then the solution in terms of the velocity field inside the particle domain is projected onto a rigid body motion. This is accomplished by computing the translation and rotation components of the rigid body motion using the statement of conservation of linear and angular momentum. From the rigid body motion, we can then evaluate the rigidity constraint force (the immersed boundary forcing), which is applied as a correction to the velocity field together with the usual constraint that enforces mass-conservation, e.g. the solenoidal condition for the velocity field. Finally using the body motion velocity we update the particle position. The algorithm has been implemented and preliminary simulations of flows around single particles have been considered for validating the numerical procedure. The flow around a fixed sphere (Fig. 1) is solved at different Reynolds using uniform grids with 128 elements. Comparisons of the drag coefficient are reported in Fig. 2. The same methodology has been already successfully applied to simulations of multiple moving particles. In Fig. 3 an example of the flow of a sphere sedimenting in a well is reported. Both particle-particle and particle-wells-wall interactions are treated considering a “soft” collision approach in which a short-range repulsive force is implemented. In addition, the various components of the algorithm have been designed to enable efficient parallelization. Some details of the implementation of the data transfer between particle-mesh and the fluid-grid will be also described. Current work is focused on the extension and validation of the immersed boundary forcing to non-Newtonian fluids. This will be the main focus of the presentation at the Workshop. Figure 1. Pressure field and streamlines for the flow around a fixed sphere. Figure 2. Comparisons of the coefficient of drag at different Reynolds numbers Figure 3. Sphere sedimenting in a well under gravity.

Physics of ultrasound-assisted augmentation of saturated nucleate boiling through the interaction of multiphase fluid flow is revealed in the present work. Different regimes of influence of ultrasound, ranging from augmentation to deterioration and even no effect, as reported in literature in a contradictory fashion, have been observed. However unlike the previous studies, here it has been clearly demonstrated that this apparent anomaly lies in the different natures of interactions between the influencing parameters like heat flux, ultrasonic frequency, and pressure amplitude. The present results clearly bring out an interactive effect of these operating parameters with surface parameter like surface roughness. A mechanistic model unifying all these parameters has been presented to explain quantitatively the physics of the interaction. The model-based predictions match experimental results quite well suggesting the validity of the hypothesis on liquid–vapor-surface interaction through the process of nucleation and its site density, on which the model is built, and thus revealing the underlying physics.

This paper envisages a mechanism of heat conduction behind the thermal conductivity enhancement observed in graphene nanofluids. Graphene nanofluids have been prepared, characterized, and their thermal conductivity was measured using the transient hot wire method. The enhancements in thermal conductivity are substantial even at lower concentrations and are not predicted by the classical Maxwell model. The enhancement also shows strong temperature dependence which is unlike its carbon predecessors, carbon nanotube (CNT) and graphene oxide nanofluids. It is also seen that the magnitude of enhancement is in-between CNT and metallic/metal oxide nanofluids. This could be an indication that the mechanism of heat conduction is a combination of percolation in CNT and Brownian motion and micro convection effects in metallic/metal oxide nanofluids, leading to a strong proposition of a hybrid model.