Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy

Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate the fate of CAR-T cells after infusion remain poorly understood. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8+ CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.Published versio

Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering

Distribution of MscL Subunits under Different Growth Conditions at Various Stages of Growth.

<p>The red curves show the fitting of the gamma distribution to the histograms. The OD₆₀₀ and the Fano factor b_Fano for a given sample are listed. (A) MLG910-Δ<i>rpoS</i> strain grown in M9+glucose to OD 0.46, 0.81, and 1.18, respectively. (B) MLG910 strain grown in M9+glucose to OD 0.3, 0.67, and 1.23, respectively. (C) MLG910 strain grown in M9+glucose+0.5 M NaCl to OD 0.26, 0.71, and 1.2, respectively.</p

Mean channel counts per cell determined by fluorescence microscopy (FM) for various media versus OD₆₀₀.

<p>(A) M9+glycerol. Fusion strains with (MLG910, light blue squares) and without RpoS (MLG-Δ<i>rpoS</i>, yellow squares). (B) M9+glucose. Fusion strains with (MLG910, green squares) and without RpoS (MLG-Δ<i>rpoS</i>, red squares). (C) LB-Miller. Fusion strains with RpoS (MLG910, black squares). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone-0033077-g003" target="_blank">Figures 3A, 3B, and 3C</a>, the corresponding mean number of channels determined by Western blots (WB) for the MLG910 strain (open squares) and the MG1655 strain (open triangles) are shown for reference. (D) Comparison of fluorescence microscopy results from MLG910 grown in three different media. (E) Comparison of fluorescence microscopy results from MLG910 grown in M9+glucose supplemented with four different NaCl concentrations: 0 mM (green squares), 100 mM (dark blue squares), 250 mM (gray-blue squares), and 500 mM (dark gray squares). The error bar of each fluorescence microscopy results measurement is dominated by systematic uncertainties in the absolute calibration related to single-molecule fluorescence calibration. The standard error of the mean of the uncalibrated fluorescence counts per cell is typically less than 5% of the total error bar.</p

Representative Western blots showing expression of MscL, MscL-sfGFP, and RpoS.

<p>Arrows indicate the protein of interest. Other bands are the result of non-specific binding. The strains of interest were cultured to exponential (exp) and stationary (stat) phase in LB-Miller (LB), M9+glucose (U), and M9+glycerol (Y). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone-0033077-g001" target="_blank">Figures 1A, 1B, and 1C</a> lanes 1 through 5 are a concentration series of a known number of purified channels diluted into lysate from the MJF612 strain (References). The numbers under lanes 6 through 12 represent the average number of channels from three independent Western blots for the respective conditions. The total error of each measurement includes contributions from the standard deviation of 3 repetitions and the systematic uncertainties in the absolute calibration related to chemiluminescence linearity, initial cell culture density, and lysis efficiency. (A) Western blot performed with MscL antibodies. Lysate from the MJF612 strain (612) was used as a negative control (lane 6). Lanes 7 through 12 show the MscL levels in the MG1655 strain (WT). (B) Western blot performed with GFP antibodies. Lysate from the MJF612 strain (612) was used as a negative control (lane 6). Lanes 7 through 12 show the MscL-sfGFP levels in the MLG910 strain (MLG). (C) Western blot performed with GFP antibodies. Lysate from MJF612 strain (612) was used as a negative control (lane 12). Lanes 6 to 11 show the MscL-sfGFP levels in the MLG910-Δ<i>rpoS</i> strain (ΔR). (D) Western blot performed with RpoS antibodies. Lysate from the MLG910-Δ<i>rpoS</i> strain (ΔR) was used as a negative control (lane 13). Lanes 1 to 12 show the RpoS levels in the MLG910 (MLG) and MG1655 (WT) strains. The numbers under the lanes are the relative amount of RpoS, as compared to the lysate from M9+glucose (lane 11), determined by the average of three independent repetitions. The errors are the standard deviation.</p

Summary of reports on the number of MscL channels per <i>E. coli</i> cell.

<p>Summary of reports on the number of MscL channels per <i>E. coli</i> cell.</p

The dependence of critical tension needed to open one channel on the total number of channels.

<p>Estimates of E_open-E_closed show considerable variation due to experimental uncertainties and the effect of lipid composition <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Perozo1" target="_blank">[15]</a>. For illustration, we chose representative values found in the literature that reflect the range of values: 10.0 k_BT (red solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Ursell3" target="_blank">[62]</a>, 18.6 k_BT (green solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Sukharev2" target="_blank">[63]</a>, and 51.0 k_BT (blue solid line) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033077#pone.0033077-Chiang1" target="_blank">[64]</a>. Dashed lines show τ_1/2 the tension, where P_open = 0.5. The area change ΔA was taken to be 10 nm² .</p