Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

doi: 10.1002/adma.202313226

Figure Lengend Snippet: Figure 1. Antigen presenting cell mimetic activating lipid nanoparticles (aLNPs) rapidly activate primary human T cells and transfect them with CAR mRNA in a single step. a) In the body, T cells are activated when they engage with antigen presenting cells (APCs). For complete activation, APCs must provide T cells with a primary and a costimulatory signal. The primary signal occurs when APC peptide-MHC interacts with T cell CD3/TCR. The costimulatory signal occurs when APC CD80/CD86 interacts with T cell CD28. Once activated, the T cell can carry out its effector function in the body. b) Top: To engineer chimeric antigen receptor (CAR) T cells outside of the body with lipid nanoparticles (LNPs), T cells must first be activated. Antibodies against CD3 and CD28, often conjugated to magnetic beads, are used to mimic APC activation before dosing the T cells with mRNA LNPs. Bottom: Activating LNPs (aLNPs) are developed by conjugating CD3 and CD28 antibody fragments to the surface of the LNPs. aLNPs combine the activating properties of the beads and the mRNA-delivering capabilities of traditional LNPs, enabling activation of and CAR mRNA delivery to T cells in a single, rapid step. (a,b) were created with BioRender.com.

Article Snippet: Antibody Cleavage and Disulfide Bond Reduction: Anti-human CD3 antibodies (BioXCell, InVivoMAb anti-human CD3, clone OKT-3, #BE00012) and anti-human CD28 antibodies (BioXCell, InVivoMAb anti-human CD28, clone 9.3, #BE0248) were cleaved with IdeZ protease (New England Biolabs, #P0770S) for 2 h at 37 °C with gentle shaking at 300 rpm.

Techniques: Activation Assay, Magnetic Beads

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

doi: 10.1002/adma.202313226

Figure Lengend Snippet: Figure 2. Formulation and characterization of activating LNPs (aLNPs). a) Molar composition of maleimide-LNPs (mal-LNPs). PEG, polyethylene glycol. DOPE, dioleoylphosphatidylethanolamine. C14-4, an ionizable lipid. b) SN2 synthesis of the ionizable lipid C14-4 from 1,2-epoxytetradecane (top left) and a polyamine core (bottom left). c) The formulation of maleimide-LNPs (mal-LNPs) by microfluidic mixing, the cleavage and reduction of antibody fragments, and the conjugation of antibody fragments onto the mal-LNP surface to generate aLNPs. d) Left, the hydrodynamic diameter (intensity weighted Z-average) distributions of mal-LNPs and 1:1 anti-CD3:anti-CD28 aLNPs. Right, Z-average and polydispersity index (PDI) measurements,

Article Snippet: Antibody Cleavage and Disulfide Bond Reduction: Anti-human CD3 antibodies (BioXCell, InVivoMAb anti-human CD3, clone OKT-3, #BE00012) and anti-human CD28 antibodies (BioXCell, InVivoMAb anti-human CD28, clone 9.3, #BE0248) were cleaved with IdeZ protease (New England Biolabs, #P0770S) for 2 h at 37 °C with gentle shaking at 300 rpm.

Techniques: Formulation, Conjugation Assay

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

doi: 10.1002/adma.202313226

Figure Lengend Snippet: Figure 4. The ratio of CD3 to CD28 antibody fragments on the aLNP surface influences the number and mean fluorescence intensity (MFI) of CAR transfected cells, which effectively kill leukemia cells ex vivo. a) Representative flow cytometry histograms obtained from primary human T cells treated with CD19-directed CAR mRNA aLNPs containing varying ratios of CD3:CD28 antibody fragments on their surfaces. CAR+ cells are defined as those to the right of the dashed line. b) Top: Schematic depicting the various aLNP treatments. Bottom, left: Percentage of single cells that are CAR+ after each aLNP treatment, from the same experiment as the representative histograms. Bottom, right: MFI of single cells after each aLNP treatment, from the same experiment as the representative histograms. n = 1 donor, with n = 2 replicates. For each bar graph, differences between group means were assessed by an ordinary one-way ANOVA with post-hoc t-tests using Tukey’s correction for multiple comparisons. Only comparisons to 1:1 aLNPs are shown. c) Co-culture assay plating setup. d) In vitro transcribed CD19-targeted CAR mRNA. e) Percentage of Nalm6 cancer cells killed when cultured with CAR T cells generated with aLNPs containing 50:1, 10:1, 1:1, 1:10, and 1:50 ratios of CD3:CD28 antibody fragments on their surfaces. n = 1 donor, with n = 3 replicates per donor, from the same experiment as (a) and (b). Differences in treatment mean within each CAR T cell:cancer cell ratio were assessed by a two-way ANOVA with post-hoc t-tests using Tukey’s correction for multiple comparisons. Only significant comparisons to 1:1 aLNPs are shown. f) Percentage of Nalm6 cancer cells killed when cultured with CAR T cells generated with beads + mal-LNPs or 1:10 aLNPs. n = 3 donors, with n = 3 replicates per donor. Assay results for a single donor are shown, with results for additional donors included in Figure S6, Supporting Information. Differences in treatment means within each CAR T cell:cancer cell ratio were assessed by a two-way ANOVA with post-hoc t-tests using Šídák’s correction for multiple comparisons. Not shown = not significant. For (a,b,e,f) data are presented as mean ± SD. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤ 0.0001. (b–d) were created with BioRender.com.

Article Snippet: Antibody Cleavage and Disulfide Bond Reduction: Anti-human CD3 antibodies (BioXCell, InVivoMAb anti-human CD3, clone OKT-3, #BE00012) and anti-human CD28 antibodies (BioXCell, InVivoMAb anti-human CD28, clone 9.3, #BE0248) were cleaved with IdeZ protease (New England Biolabs, #P0770S) for 2 h at 37 °C with gentle shaking at 300 rpm.

Techniques: Transfection, Ex Vivo, Cytometry, Co-culture Assay, In Vitro, Cell Culture, Generated

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Antigen Presenting Cell Mimetic Lipid Nanoparticles for Rapid mRNA CAR T Cell Cancer Immunotherapy.

doi: 10.1002/adma.202313226

Figure Lengend Snippet: Figure 5. Anti-CD19 CAR T cells generated with aLNPs readily proliferate, maintain cytotoxicity following expansion, and have an activated phenotype. a) Flow cytometry histograms of primary human T cells stained with CellTrace Far Red at 2, 4, and 6 days post-treatment with beads + mal-LNPs (B+L) or 1:10 aLNPs (1:10). Each proliferative generation appears as a distinct leftward-shifted peak in the flow cytometry histogram. n = 1 donor, with n = 2 replicates. b) Representative flow cytometry histograms obtained from primary human T cells 4 days after the cells received no treatment (NT) or 1:10 aLNPs. CAR+ cells are defined as those to the right of the dashed line. c) Percentage of Nalm6 cancer cells killed after 48 h when cultured with the 4-day-post-aLNP CAR T cells shown in (b). n = 1 donor, with n = 3 replicates per donor. d) Representative flow cytometry plots showing CCR7 versus CD45RA expression for primary human T cells 3 days following treatment with beads + mal-LNPs (B+L) or 1:10 aLNPs. n = 1 donor, with n = 3 replicates per donor. e) Quantification of Naïve, central memory (CM), effector memory (EM), and TEMRA population sizes for the cells treated in (d). Differences in population sizes between B+L and 1:10 aLNPs were assessed by a two-way ANOVA with post-hoc t-tests using Šídák’s correction for multiple comparisons. f) Percentages of primary human T cells expressing CD25, CD69, and CD44, as assessed via flow cytometry, 24 h after receiving no treatment (NT), LNPs with only CD3 antibody fragments on their surface (𝛼CD3), LNPs with only CD28 antibody fragments on their surface (𝛼CD28), beads + mal-LNPs (B+L), or 1:10 aLNPs (1:10). n = 1 donor, with n = 4 replicates. For each graph, differences between all group means were assessed by an ordinary one-way ANOVA with post-hoc t-tests using Tukey’s correction for multiple comparisons. Only results of comparisons to 1:10 aLNPs are shown. For (b,c,e,f) data are presented as mean ± SD. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001, ns = not significant.

Article Snippet: Antibody Cleavage and Disulfide Bond Reduction: Anti-human CD3 antibodies (BioXCell, InVivoMAb anti-human CD3, clone OKT-3, #BE00012) and anti-human CD28 antibodies (BioXCell, InVivoMAb anti-human CD28, clone 9.3, #BE0248) were cleaved with IdeZ protease (New England Biolabs, #P0770S) for 2 h at 37 °C with gentle shaking at 300 rpm.

Techniques: Generated, Flow Cytometry, Staining, Cytometry, Cell Culture, Expressing

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a Flow cytometry gating strategy used to identify human circulating T follicular helper T cells (cTFH) defined as CD3 + CD8α – CD4 + CXCR5 + ICOS + PD-1 + . Among cTFH cells, cTFH1 cells were defined as CXCR3 + CCR6 – , cTFH2 as CXCR3 – CCR6 – , and cTFH17 as CXCR3 – CCR6 + . b Proportions (%) among CD4 + T cells of cTFH as defined in ( a ) in blood from healthy controls (CT) and inactive (inact.), mild, or active SLE patients ( n = 16/11/12/21) as determined by flow cytometry. Proportions (%) among CD4 + T cells of cTFH2 ( c ), cTFH1 ( d ) and cTFH17 ( e ) as defined in ( a ) in blood from healthy controls and inactive, mild, or active SLE patients ( n = 16/10/12/20) as determined by flow cytometry. f Correlation (and linear regression with 95% confidence intervals) between Log (cTFH2 cell numbers per mL of blood) and SLEDAI (Spearman r = 0.3682, p = 0.02, n = 37). g Left, Histogram plot representing the CD203c expression levels on blood basophils from a healthy control (CT, black), a patient with active SLE (red), and isotype control staining (gray filled). Right, CD203c expression levels on blood basophils from CT and inact, mild or active SLE patients ( n = 43/61/46/98) as determined by flow cytometry. h Left, Representative histogram plot of PD-L1 expression levels on blood basophils as in ( g ). Right, PD-L1 expression levels on blood basophils from CT and inact, mild or active SLE patients ( n = 39/60/45/96) as determined by flow cytometry. ( b – e , g , h ) Data are presented as violin plots with median (solid line) and quartiles (dotted lines). Statistical analyses were Kruskal–Wallis tests followed by Dunn’s multiple comparisons tests and p values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Flow Cytometry, Expressing, Staining

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a – g CD3/CD28-stimulated wild-type (WT) naïve CD4 + T cells cultured for three days without (–, gray) or with basophils from Mcpt8 CT/+ (WT) (blue), Mcpt8 CT/+ Il4 fl/fl (red), Mcpt8 CT/+ Il6 fl/fl (orange), or Mcpt8 CT/+ Pdl1 fl/fl (green) mice. a Proportions (%) of TFH cells among CD4 + T cells ( n = 17/31/12/12/15). Proportions (%) of IL-21- ( b ), IL-6- ( c ), IL-4- ( d ), and IL-13- ( e ) producing cells among TFH cells non-restimulated ( n = 11/16/5/10/8). f ( Top ) Proportions (%) of TFH cells among CD4 + T cells ( n = 10/9/20/9). Bottom RT-qPCR done for the indicated targets on resorted CD4 + T cells. Relative results are presented with the color scale indicated from 0% (blue, the least abundant) to 100% (red, the most abundant). g Ratio of Bcl6 on Bach2 mRNA ( Top ) ( n = 10/5/17/8) and of Batf on Bach2 mRNA ( Bottom ) ( n = 13/5/17/8) as described in ( f ). h Top Proportions (%) of TFH cells among CD4 + T cells cultured without basophils for three days either alone (–, gray) or with coated anti-PD-1 antibody (red) or soluble IL-4 (green) or both (blue) ( n = 10/12/12/8). Bottom. Relative mRNA expression of the indicated targets as in ( f ). i Ratio of Bcl6 on Bach2 mRNA ( Top ) ( n = 10/5/4/6) and of Batf on Bach2 mRNA ( Bottom ) ( n = 13/5/4/6) on samples described in ( h ). b – e Results for PMA/Ionomycin restimulated cells are shown in Supplementary Fig. . a – i Results are from at least three independent experiments and presented as individual values in bars representing the mean ± s.e.m. Statistical analyses were done by Kruskal–Wallis test followed by Dunn’s multiple comparisons tests ( a ) or by one way ANOVA followed by Tukey’s multiple comparisons tests ( b – i ) between the indicated groups. P values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: a Histograms of PD-L1 expression on basophils cultured without (dotted) or with (solid) activated naïve CD4 + T cells as in Fig. (gray filled: isotype control staining). b PD-L1 expression levels on basophils cultured without (–, lighter colors) or with (+, darker colors) activated naïve CD4 + T cells ( n = 14/31/5/12/6/12/5/12). c Left. Intracellular IL-3 staining in WT CD3/CD28-activated naïve CD4 + T cells after 3 days of culture (red line) (gray filled: isotype control staining). Right. Proportions (%) of IL-3 + cells among CD4 + T cells non-restimulated in the same samples as in Fig. ( n = 6/9/16/9). d PD-L1 expression induction on WT basophils after stimulation of splenocytes with 1 ng/mL of IL-4 (red), of IL-3 (blue), both (purple), 100 ng/mL of anti-IgE without (light green) or with (green) IL-3 for 20 h ( n = 8/8/8/8/4/4) (normalized to unstimulated conditions mean value). e Contour plots of IL-6 and IL-4 spontaneous production by basophils of the indicated genotype after co-culture without (–) or with activated CD4 + T cells. Proportions (%) of spontaneous IL-4 + ( f ) and IL-6 + ( g ) basophils of the indicated genotypes co-cultured (+) or not (–) with WT CD3/CD28-activated naïve CD4 + T cells ( n = 11/16/3/5/6/8/4/8). f , g Results for IL-13 and PMA/Ionomycin restimulated cells are shown in Supplementary Fig. . a – g Results are from at least three independent experiments and presented as individual values in bars representing the mean ± s.e.m. b – d , f , g Statistical analyses were by one way ANOVA followed by Tukey’s multiple comparisons tests between the indicated groups. P values are shown above each bracket. NS not significant. A.U. arbitrary units. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Expressing, Cell Culture, Staining, Co-Culture Assay

Journal: Nature Communications

Article Title: PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus

doi: 10.1038/s41467-024-47691-w

Figure Lengend Snippet: Contour plots showing CD3/CD28-activated human CD4 + T cells cultured for three days without ( a ) or with ( b ) purified human basophils at a 5:1 ratio (left) and the condition-induced TFH differentiation of the CD4 + T cells (right). Basophils were defined as FcεRIα + CRTH2 + CCR3 + cells and TFH cells were defined as CD4 + PD-1 + CXCR5 + ICOS + cells. c Proportions (%) of TFH cells among CD3/CD28-activated CD4 + T cells cultured without (0:1) or with the indicated ratio of purified human basophils ( n = 4 per group). d Proportions (%) of TFH cells among CD3/CD28-activated CD4 + T cells cultured without or with purified human basophils at a ratio of 1:5 in the absence (–) (blue) or presence of antibodies blocking IL-4 (αIL-4) (red), IL-6 (αIL-6) (orange) or PD-1 (αPD-1) (green) or the corresponding isotype controls (Iso) (blue) ( n = 4/3/6/6/6/6/5/5/8/8/8/8). e Contour plots showing subsets of TFH cells as defined in Fig. on cells as in ( a , b ). f Proportions (%) of TFH2 cells among TFH cells after culture as described in ( d ) ( n = 5/5/8/8/8/8). TFH2 cells were defined as CD4 + PD-1 + CXCR5 + ICOS + CCR6 – CXCR3 – cells. c , d , f Data are presented as individual values in bars representing the mean values ± s.e.m. c One representative experiment out of two with cells from 4 different donors is shown. d , f Results are from three independent experiments. c , d , f Statistical analyses were done by one-way ANOVA test followed by Tukey’s multiple comparisons tests between the indicated groups. P values are shown above each bracket. NS not significant. Source data are provided in the file.

Article Snippet: For basophil/CD4 + T cell mouse co-culture experiments, F(ab’)2 anti-CD3 (clone145-2C11 at 0,5 µg/mL) and anti-CD28 (clone PV-1 at 0,5 µg/mL) antibodies (BioXcell) were coated overnight at 4 °C in culture 96 well plates (Costar) in sterile-filtered coating buffer (20 mM carbonate buffer (pH 9.6) containing 2 mM MgCl 2 and 0.01% NaN 3 ).

Techniques: Cell Culture, Purification, Blocking Assay