Journal: bioRxiv

Article Title: Human Alveolar and Monocyte-derived Human Macrophage Responses to Mycobacterium tuberculosis

doi: 10.1101/2024.02.20.581265

Figure Lengend Snippet: (A) AMs and MDMs stimulated with LPS for 6 hours with IFNA1, IFNA8, IFNB, IL1B and IL6 measured by RT-PCR. Pairwise contrast comparisons between AM and MDMs unstimulated and LPS stimulated were completed using linear mixed effects modeling paired by donor. (**FDR<0.01, *FDR<0.05) (B) Schematic of study design. MDMs from healthy individuals treated with recombinant IFNA8, IFNB, IFNE and IFNG (C) Secreted cytokines: After 24 hours of IFN treatment, cells were infected with H37Rv and TNF, IL6, and IL1B were measured from supernatants harvested overnight. Representative data from two independent experiments are shown. An interaction model of IFN stimulation and Mtb infection was completed using linear mixed effects modeling paired by donor (interaction term **p<0.01, *p<0.05). (D) Cells were treated with IFNs for 6 hours and RNA expression was assessed by RNAseq. Venn diagram depicts IFN-specific and IFN-overlapping DEGs (FDR<0.05). (E) Hypergeometric mean enrichment pathway analysis of IFN DEGs as in (D) against MSigSB Hallmark gene sets. Gene sets specific to a single IFN’s DEGs are shown (FDR < 0.05 for one IFN, FDR > 0.05 for all other IFNs). Color indicates proportion enriched (k/K), and the vertical dashed line indicates FDR = 0.05.

Article Snippet: MDMs were treated with recombinant interferons (IFNs): IFNA8 (10ng/mL) (SinoBiological), IFNG (10ng/mL), IFNB (10ng/mL) (Peprotech), and IFNE (100ng/mL) (R&D Systems) for 6 hours before RNA extraction and sequencing.

Techniques: Reverse Transcription Polymerase Chain Reaction, Recombinant, Infection, RNA Expression

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: A. Diagram illustrating the effect of RhoA(Q63L) mutation on GTPase regulation. B. Schematic of lentiviruses construct encoding control (top) and RhoA(Q63L) mutant (bottom), all driven by the MND promoter. T2A and P2A encode two self-cleavage peptides. RQR8 is used for detection and purification of transduced cells. C. Representative flow cytometry data of purified RQR8+ T cells using PE -conjugated anti-CD34 (QBend10) antibody. D. Representative Western blots of whole cell lysate and RhoA-GTP pulldown samples with RBD beads with antibodies against RhoA and β-actin. Rho activating treatments were included to assess basal protein expression and activation levels in T cells. E. Representative immunofluorescence images of T cells stained with RhoA (red) and RhoA-GTP (green) antibodies, and counter stained with DAPI (blue). Scale bar: 50μm. F. Proliferation rate and viability of human T cells expressing control or RhoA(Q63L) over 7 days. G. Representative images of infiltrated T cell morphology in 3D collagen gels. Quantification of cell morphology parameters demonstrated reduced circularity and increased cell size in RhoA(Q63L) T cells compared to the control. Each dot represents an individual cell pooled from three independent experiments. Data are presented as mean ± s.e.m.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Mutagenesis, Construct, Control, Purification, Flow Cytometry, Western Blot, Expressing, Activation Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: A. Fluorescence-lifetime images of control and RhoA(Q63L) T cells with Flipper-TR to assess cortical membrane tension. Cells are color-coded by fluorescence lifetime (red, shorter lifetimes; blue, longer lifetimes). B. Quantification of average fluorescence lifetimes across Z-stacks from Flipper-TR–stained T cells shows significantly longer lifetimes in RhoA(Q63L) cells, consistent with increased cortical contractility. C. Physics-based mathematical modeling predicts that increased cortical contractility results in increased T cell migration. D. Multiphoton time-lapse images of human control and RhoA(Q63L) T cells migrating in 3D collagen matrices. Representative single-cell migration tracks are highlighted in blue. Images were acquired every 1 min for 1 h. Colors: green, T cells; gray, collagen fibers. E. Motility coefficient for human control and RhoA(Q63L) T cells migrating in 3D collagen matrices. Each dot represents an individual tracked T cell. F. Average area sampled per frame by T cells migrating in 3D collagen matrices. n = 4 biological replicates. G. Multiphoton time-lapse images of murine control and RhoA(Q63L) and CD8⁺ T cells migrating within live KPCT pancreatic tumor slices. Colors: red, PDA carcinoma cells; green, T cells; gray, fibrillar collagen. H. Motility coefficients of murine control and RhoA(Q63L) CD8⁺ T cells migrating in tumor slices, showing a significant increase in motility in RhoA(Q63L) T cells. I. Representative multiphoton microscopy images showing the cumulative area sampled by infiltrating T cells in tumor slices over a 2-h period. Regions of high T-cell presence are outlined in green. J. Quantification of the percentage of area sampled per field of view per T cell. Data are presented as mean ± s.e.m. Statistical comparisons were performed using unpaired two-tailed t-tests with Welch’s correction.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Fluorescence, Control, Membrane, Staining, Migration, Single Cell, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: ( A ) Representative multiphoton time-lapse images of murine RhoA(Q63L) and control CD8⁺ T cells migrating in 3D collagen matrices. Cell trajectories are overlaid and color-coded by total distance traveled (blue = short; red =long). Images were acquired every 1 min for 1 h. Colors: green, T cells; gray, collagen fibers. ( B ) Motility coefficient of murine RhoA(Q63L) and control CD8⁺ T cells migrating in 3D collagen matrices. Each dot represents an individual tracked T cell. ( C ) Total distance traveled by murine RhoA(Q63L) and control CD8⁺ T cells in 3D collagen matrices. Each dot represents an individual tracked T cell. ( D ) Average circularity of murine RhoA(Q63L) and control CD8⁺ T cells in 3D collagen matrices during the imaged period. A value of 1.0 indicates a perfect circle, and the value approaches 0 for elongated cells. ( E ) Cell size of murine RhoA(Q63L) and control CD8⁺ T cells in 3D collagen matrices measure using particle analyzer in FIJI. Data are presented as mean ± s.e.m.; statistical significance was determined using unpaired two-tailed t-tests with Welch’s correction.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Control, Two Tailed Test

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: A. Fluorescence lifetime imaging of control and RhoA(Q63L) T cells inside 3D collagen matrices. Left: unstimulated; right: stimulated with anti-CD3/CD28 antibodies; Color bar indicates percentage of NAD(P)H α1from FLIM analysis (red: low percentage; blue: high percentage). B. Quantification of NAD(P)H α1 percentage in unstimulated and CD3/CD28-stimulated control and RhoA(Q63L) T cells. RhoA(Q63L) T cells showed significantly elevated baseline α1 levels, indicating increased activation. C-D. Multiparameter flow cytometry analysis of T cell subpopulations. RhoA(Q63L) modified T cells display a higher frequency of CD8⁺ T cells (C) and effector subsets (D) compared to control T cells. E. Heatmap of T cell percentage across differentiation stages: naïve (Tn), central memory (Tcm), effector memory (Tem), and terminally differentiated effector (E) cells, showing as heatmap. RhoA(Q63L) modified T cells exhibited a more differentiated phenotype, with the majority residing in the Tem3 (∼60%) and E (∼15%) subsets. F. Expression of exhaustion markers PD-1 and LAG-3 on T cells. RhoA(Q63L) modified T cells exhibit reduced PD-1 and LAG-3 expression compared to control T cells, suggesting a less exhausted phenotype.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Fluorescence, Imaging, Control, Activation Assay, Flow Cytometry, Modification, Expressing

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: ( A ) Schematic representation of vector constructs encoding mesothelin-targeted CAR (mesoCAR) T cells with or without the RhoA(Q63L) mutation. ( B )Representative flow cytometry data of purified mesoCAR RQR8+ T cells using PE -conjugated anti-CD34 (QBend10) antibody. ( C ) Western blots of whole cell lysate and RhoA-GTP pulldown samples with RBD beads with antibodies of RhoA and β-actin.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Plasmid Preparation, Construct, Mutagenesis, Flow Cytometry, Purification, Western Blot

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: A. Migration of RhoA(Q63L) modified and control mesoCAR T cells in 3D collagen matrices. Migration trajectories are overlaid on time-lapse images and color-coded by total distance traveled (blue, short; red, long). RhoA(Q63L) modified CAR T cells display increased motility and more persistent movement. B. Quantification of mesoCAR T-cell motility coefficients in 3D collagen matrices. RhoA(Q63L) modified CAR T cells exhibit significantly higher motility coefficients than control CAR T cells. C. Quantification of 2D cytotoxicity. mesoCAR and mesoCAR RhoA(Q63L) modified T cells. Both CAR T-cell groups show similar increases in target-cell killing with increasing effector ratios. D. Multiphoton images of mesoCAR and mesoCAR RhoA(Q63L) modified T cells migrating and interacting with AsPC-1 cells in 3D collagen matrices. Colors: green, T cells; red, AsPC-1 cells; gray, collagen fibers. E. Quantification of T-cell–AsPC-1 interaction events per T cell for mesoCAR, and mesoCAR RhoA(Q63L) modified T-cell groups in 3D collagen matrices. F. Quantification of T-cell–AsPC-1 interactions events per carcinoma cell for mesoCAR and mesoCAR RhoA(Q63L) modified T-cell groups in 3D collagen matrices. G. Quantification of mesoCAR and mesoCAR RhoA(Q63L) modified T-cell motility in collagen matrices containing AsPC-1 cells. H. Quantification of mesoCAR and mesoCAR RhoA(Q63L) modified T-cell motility in collagen regions lacking AsPC-1 cells. I. Multiphoton microscopy images showing the cumulative area explored by mesoCAR, and mesoCAR RhoA(Q63L) modified T cells in AsPC-1–embedded 3D collagen matrices. Overlaid tracks and projected cumulative areas over 90 min are shown (red, AsPC-1 cells; yellow, T-cell area coverage). Data are presented as mean ± s.e.m. Statistical comparisons were performed using unpaired two-tailed t-tests with Welch’s correction.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Migration, Modification, Control, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: ( A ) The mesoCAR RhoA(Q63L) cells showed increased displacement, ( B ) mean speed, and ( C ) less confined movement.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques:

Journal: bioRxiv

Article Title: Engineering “physically optimized” T cells for increased sampling of complex tumor microenvironments

doi: 10.64898/2026.01.28.702394

Figure Lengend Snippet: MesoCAR and mesoCAR RhoA(Q63L) T cells were co-cultured with AsPC-1 at varying effector-to-target ratios. The percentage of viable AsPC-1 cells is shown relative to no-T-cell controls.

Article Snippet: To examine total RhoA and RhoA-GTP, 1:50 rabbit anti-total RhoA (Novus Bio,Centennial, CO) and 1:100 mouse anti-RhoA-GTP (NewEast Biosciences, King of Prussia, PA) diluted in the blocking buffer were incubated with the T cells at 4°C overnight.

Techniques: Cell Culture