medfusion是什么牌子Regulatory T cells expressing CD19-targeted chimeric antigen receptor restore homeostasis in Systemic Lupus Erythematosus

This study complies all the required ethical requirements. Healthy donors’ Peripheral Blood Mononuclear Cells and CD34+ cord-blood hematopoietic stem cells were employed after the collection of a written informed consent approved by the San Raffaele Ethical Committee. The in vivo studies involving mice were approved by the Italian Ministry of Health.

Acute lymphocytic leukemia cell line (ALL-CM) was grown in X-VIVO 15 (Lonza) supplemented with 10% FBS (fetal bovine serum, Euroclone), 1% penicillin/streptomycin (Lonza) and 1% glutamine (Lonza). HEK293T and CD40L⁺ 3T3 cells were grown in IMDM (Lonza) supplemented with 10% FBS (fetal bovine serum, Euroclone), 1% penicillin/streptomycin (Lonza) and 1% glutamine (Lonza). Cells were counted every 2–4 days by Trypan blue dye exclusion according to the cell line and plated at a concentration of 0.5-1 × 10⁶ cells/ml.

Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors, after written informed consent, according to the San Raffaele Scientific Institutional Ethical Committee guidelines. PBMCs were isolated by Ficoll-Hypaque gradient separation (Lymphoprep; Fresenius) and were freshly used for the following protocols of specific T cell expansion. After the activation, cells were incubated at 37 °C, 5% CO2, in a humidified cell culture incubator.

CD4⁺CD25⁺ regulatory T cells were isolated by magnetic cell separation (Miltenyi), according to manufacturer’s instructions. Sorted CD4⁺CD25⁺ were activated with cell-sized anti-CD3/anti-CD28 magnetic beads (Dynabeads) in a 3:1 bead: T cell ratio and cultured in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin/streptomycin (Lonza) and glutamine (Lonza) with Ramapacyn (100 nM). At day 2 after activation, IL-2 (500 UI, Proleukin, Novartis) was added to cell culture and replenished every 2-3 days. After 14 days of cultures, beads were magnetically removed according to the manufacturer’s instructions. Starting from day 14, cells were employed for phenotypical evaluation and functional assays.

Naïve Tregs were initially isolated from PBMCs by magnetic cell separation of CD4⁺CD25⁺ cells. A subsequent selection of CD127^–CD45RA⁺ elements was done employing a Miltenyi Tyto cell sorter. Naïve Tregs were subsequently activated with cell-sized anti-CD3/anti-CD28 magnetic beads (Dynabeads) in a 1:1 bead: T cell ratio and cultured in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin/streptomycin (Lonza) and glutamine (Lonza) without Ramapacyn. At day 2 after activation, IL-2 (1000 UI, Proleukin, Novartis) was added to cell culture and replenished every 2–3 days. After 9 days, cells were restimulated with anti-CD3/anti-CD28 magnetic beads in a 1:1 bead: T cell ratio. At day + 14 after stimulation, beads were magnetically removed according to the manufacturer’s instructions. Starting from day +14, cells were employed for phenotypical evaluation and functional assays.

Conventional T Lymphocytes were enriched from isolated PBMCs and stimulated with anti-CD3/CD28 magnetic beads in a 3:1 bead: T cell ratio for 6 days. After 6 days of stimulation, beads were magnetically removed, and cells were maintained in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin/streptomycin (Lonza) and glutamine (Lonza). T cells were cultured in the presence of IL-7 and IL-15 (5 ng/ml; PeproTech) to preserve early differentiated stem cell memory and central memory phenotype, as previously reported (Cieri et al., 2013). Medium was replaced every 3–4 days and cells were counted by Trypan blue dye exclusion. Starting from day +14, cells were employed for phenotypical evaluation and functional assays.

CD4⁺CD25^– T cells were collected from freshly isolated PBMCs after magnetic cell separation (Miltenyi) according to manufacturer’s instructions and were stimulated with cell-sized anti-CD3/anti-CD28 magnetic beads in a 3:1 bead: T cell ratio. Cells were cultured with either IL-7/IL-15 (5 ng/ml, Peprotech) or IL-2 (500 UI, Proleukin, Novartis) and rapamycin (100 nM) in X-VIVO 15 supplemented with 10% human serum (Lonza), 1% penicillin/streptomycin (Lonza) and glutamine (Lonza). Beads were magnetically removed at day +14. Starting from day +14, cells were employed for phenotypical evaluation and functional assays. Medium was replaced every 3–4 days and cells were counted by Trypan blue dye exclusion.

CAR19.28z construct was generated by cloning the antigen-specific single chain fragment variable (scFv) into bi-directional lentiviral vector encoding for CAR backbone containing a NGFR-derived mutated-short spacer (NMS) (Casucci et al. 2018), a CD28 transmembrane and co-stimulatory domain and a CD3zeta endodomain under the control of a hPGK promoter. Green Fluorescent Protein (GFP) was cloned in anti-sense under the control of a mCMV promoter. Fox19CAR LV construct was generated by cloning in a unidirectional lentiviral vector both the human FoxP3 gene and an anti-CD19 second-generation CAR under the control of a hPGK promoter. Anti-CD19 CAR was composed by a CD19-specific scFv, a linker peptide derived from the mutated constant portion of IgG4 (Hudechek et al.), the transmembrane and intracellular portion of the CD28 and the CD3z chain. The two transgenes were linked by a thosea-asigna virus 2 A (T2A) peptide. As control, we generated a unidirectional lentiviral vector encoding for the FoxP3 gene under the control of a hPGK promoter.

All employed lentiviral vector backbones were kindly provided by L. Naldini’s group.

Third generation replication-defective and self-inactivating lentiviral vectors (LV) were produced by transient transfection of HEK-293T cells. Briefly, a solution containing the packaging plasmids pMDLg/pRRE (containing HIV-1 gag/pol genes), the pILVV01-Rev plasmid (encoding Rev protein), the envelope plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), the pAdvantage (that enhances transient protein expression) and the plasmids carrying the transgene of interest was transfected in sub-confluent 293 T cells using the calcium chloride precipitation method. Supernatants containing lentiviral particles were collected 48 h later and filtered using a 0.22 μm filter, ultracentrifuged at 8000 g for 18 h at 4 °C (Beckman Optima XL-100K Ultracentrifuge), aliquoted and cryopreserved.

At day +2 after stimulation, activated T cells (Treg, Tconv or CD4⁺CD25^– lymphocytes) from healthy donors were collected and re-suspended at the concentration of 2,5 × 10⁶ cells/ml in X-VIVO 15. LV vectors were added accordingly to viral titration to achieve an Multiplicity of Infection (MOI) of 10. T cells were kept 24 h at 37 °C and then fresh medium was added in each condition. Transduction efficiency was measured by flow cytometry at day +10/ +14. Recombinant CD19 reagent (Miltenyi) was used for CAR surface staining according to manufacturer’s instructions.

Autologous T lymphocytes (target cells) and engineered T cells (effector cells) were co-cultured at decreasing Effector-to-Target ratio in the presence of anti-CD3/anti-CD28 magnetic beads at 1:10 bead: T cell ratio. To detect proliferating cells, target cells were stained with VioBlue proliferation dye (ThermoFisher), while engineered cells were stained with FarRed proliferation dye (ThermoFisher), according to manufacturer’s instruction. After 6 days, cells were analyzed by flow cytometry. Target and effector cells were discriminated according to the specific proliferation dye. Suppression index was calculated as follows: (1-(percentage of proliferating cells in the effector group)/(percentage of proliferating target cells alone)) x 100.

CD19⁺ B cells (target cells) were negatively sorted from cryopreserved autologous PBMCs using B cell isolation kit II (Miltenyi), according to manufacturer’s instruction. To detect proliferating cells, isolated B lymphocytes were labeled with VioBlue proliferation dye (ThermoFisher), according to manufacturer’s instruction, whereas engineered T cells (effector cells) were stained with FarRed proliferation dye (ThermoFisher), according to manufacturer’s instruction. Labeled target cells and effector cells were co-cultured either in a 1:1 Effector-to-Target ratio in the presence of irradiated CD40L⁺ 3T3 cells (a fibroblast-derived murine cell line) or at different Effector-to-Target ratios and stimulated with anti-CD40 (0.5 ug/ml), anti-IgM/IgG (20 ug/ml) stimulating antibodies. Cells were analyzed by flow cytometry 3 days later. Suppression Index was calculated as follows: (1-(percentage of proliferating cells in the effector group)/(percentage of proliferating target cells alone)) x 100.

For antigen-specific killing assay with tumor cell lines, CD19⁺ ALL-CM cells were co-cultured for 3 days with either engineered or untransduced T cells at 1:5 Effector-to-Target ratio. Cells were analyzed by flow cytometry. The number of residual ALL-CM cells and effector lymphocytes were counted according to the number of elements acquired with flow cytometry and to the total well volume. The anti-tumor activity was expressed as Elimination index as follows: 1—(number of viable target cells in presence of redirected T cells/number of viable target cells in presence of untransduced T cells).

The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) with the number 1127. At day 0, 1 day-old NSG mouse (NSG, JAX 005557, RRID IMSR_JAX: 005557, Charles-River Italia) pups were sub-lethally irradiated and infused intra-liver with 0.8-1 × 10⁵ cord-blood derived human CD34⁺ hematopoietic stem cells. Mice were clinically monitored and weighed every week. Starting from week 5 after cord-blood infusion, the frequency of human cells in the peripheral blood was monitored every week. After the establishment of a full human immune system, chronic inflammation was induced in mice by intra-peritoneal injection of pristane (300 μL, Sigma-Aldrich). A control group of humanized mice did not receive pristane to monitor the dynamics of the immune cells without a chronic inflammatory stimulus. Three weeks after pristane injection, mice were treated with 3.5 × 10⁶ of anti-CD19 CAR Tregs or UT Tregs or PBS. Human chimerism and human cord-blood derived lymphoid compartment on peripheral blood were assessed weekly by flow cytometry. Mouse weight was monitored every week and, in the presence of weight loss >20%, animals were euthanized. Spleen, bone marrow and kidneys were harvested for flow cytometry and pathology evaluations.

At day 0, 8 weeks-old SGM-3 mice (SGM-3, JAX 013062, RRID IMSR_JAX: 013062, Charles-River Italia) were sub-lethally irradiated and intra-venously infused with 0.8-1 × 10⁵ cord-blood derived human CD34⁺ hematopoietic stem cells. Mice were clinically monitored and weighed every week. Starting from week 5 after cord-blood infusion, the frequency of human cells in the peripheral blood was monitored every week. After the establishment of a full human immune system, chronic inflammation was induced in mice by intra-peritoneal injection of pristane (500 μL, Sigma-Aldrich). Mice were subsequently randomized and divided either in the early or late treatment group. Early treatment group received 3 weeks after pristane injection 3.5 × 10⁶ of anti-CD19 CAR-Tregs or anti-CD19 CAR-Tconvs or PBS. After 5 weeks (8 weeks after pristane), CAR-Treg treated mice received a second infusion of 3.5 × 10⁶ of anti-CD19 CAR-Tregs. Late treatment group received 3.5 × 10⁶ of anti-CD19 CAR-Tregs or anti-CD19 CAR-Tconvs 8 weeks after pristane injection.

SGM-3 human chimerism and human cord-blood derived lymphoid compartment on peripheral blood were assessed weekly by flow cytometry. Mouse weight was monitored every week and, in the presence of weight loss >20%, animals were euthanized by cervical dislocation. Spleen, liver, bone marrow ear pinnae and kidneys were harvested for flow cytometry and pathology evaluations.

For both strains, at euthanasia single-cell suspension from kidney was obtained by using multi-tissue dissociation kit II (Miltenyi) according to manufacturer’s instructions with GentleMACS apparatus. Single-cell suspension from spleen was obtained using 70 nm cell strainer. Cells from bone marrow were collected by flushing femurs with PBS. After red-blood cell lysis, cells were suspended in PBS supplemented with 5% FBS. Tregs phenotype and lymphoid immune cells were analyzed by flow cytometry.

All the employed animals were kept in the same animal house under germ-free conditions.

The presence of human anti-double-strand DNA (dsDNA) IgG auto-antibodies in mouse serum was assessed at euthanasia by immunofluorescence assay according to manufacturer’s instruction (QUANTA Lite dsDNA, Werfen Group). A dilution of 1:10 was employed as a threshold for the positivity.

At euthanasia, mice were euthanized by cervical dislocation. Collected organs fixed in buffered 4% formalin and embedded in paraffin. Haematoxylin and eosin stained 3 μm paraffin sections were examined for histopathological analysis. The evaluation of the specimens was performed in double-blind by a pathologist specifically trained in mouse pathology. We assessed: the normal anatomy, the presence of lesions or fibrosis, the presence and the characteristics of immune cells. For each organ, we evaluated the presence of:

Microscopic lesions were classified on a scale of 0–5 as minimal (1), mild (2), moderate (3), marked (4), or severe (5); minimal referred to the least extent discernible and severe the greatest extent possible.

Immunohistochemical analysis was performed on selected sections of spleen using rabbit anti-human CD3 (2GV6; Ventana) and mouse anti-human CD20 (L26; Ventana) in automated Ventana Discovery ULTRA system.

For Treg phenotype, cells were labeled with titrated fluorescent monoclonal antibodies specific for CD3 (eFluor506, Invitrogen, OKT3, 69-0037-42, 1:75), CD4 (PE-Vio615, Miltenyi, REA623, 130-113-226, 1:150), FoxP3 (PE-Cy5, eBioscience, PCH101, 15-4776-42, 1:1500), CD45RA (BUV496, BD Bioscience, 5H9, 741182, 1:150), CD62L (BUV737, BD Bioscience, SK11, 749210, 1:300), TIGIT (BV786, BD Bioscience, 741182, 747838, 1:100), LAP (Per-CP-eFluor710, eBioscience, FNLAP, 46-9829-42, 1:100), Helios (PE, Biolegend, 22F6, 137216, 1:75), CD25 (PE-Vio770, Miltenyi, REA945, 130-116-205, 1:75), CD27 (APC, Biolegend, M-T271, 356410, 1:150), CD137 (Alexa Fluor 700, Biolegend, 4B4-1, 309816, 1:150), CD127 (APC-Vio770, Miltenyi, REA614, 130-113-416, 1:75), GARP (BV421, BD Bioscience, 7B11, 563956, 1:50), CTLA-4 (BV605, Biolegend, BNI3, 369610, 1:200), ICOS (BV650, BD Bioscience, DX29, 563832, 1:1500), GITR (BV711, Biolegend, 108-17, 371212, 1:400). For 19CAR engineered cells, transduction efficiency was assessed with GFP or NGFR spacer (CD271, BD Bioscience, clone C40-1457), conjugated in BB515 (564580, 1:200), PE (557196, 1:100) or PE-Cy7 (562122, 1:200). Fox19CAR engineered cells were identified using biotinylated recombinant human CD19 reagent (Miltenyi, 130-129-550, 1:67). Biotin was detected using VioBright515-conjugated anti-biotin secondary antibody (Miltenyi, Bio3-18E7, 130-113-298, 1:50). For intra-nuclear staining, lymphocytes were stained with surface antibodies, washed, fixed and permeabilized with FoxP3 staining buffer set (Miltenyi, 130-093-142), according to manufacturer’s instructions.

For in vivo experiments, whole blood was lysed with ACK (Ammonium-Chloride-Potassium) buffer for 10 min at room temperature to remove red blood cells. The reaction was then stopped with PBS supplemented with 5% FBS. Subsequently, samples were stained with recombinant CD19 reagent. After washing with PBS supplemented with 5% FBS, fluorochrome-conjugated monoclonal antibodies specific for mouse CD45 (PerCP, Biolegend, 30-F11, 103130, 1:300), human CD45 (PE-Cy7, Invitrogen, HI30, 25-0459-42, 1:300), CD3 (eFluor506, Invitrogen, OKT3, 69-0037-42, 1:150), CD14 (APC-Cy7, Biolegend, 63D3, 367108, 1:300), CD19 (APC, Biolegend, 4G7, 392504, 1:300), CD56 (PE, Biolegend, 5.1H11, 362508, 1:300) were added to samples. Human T cells were counted on peripheral blood using Flow Count fluorescent beads (Beckman Coulter), according to manufacturer’s instruction.

For Treg analysis in harvested organs, single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies specific for CD3 (eFluor506, Invitrogen, OKT3, 69-0037-42, 1:75), CD4 (PE-Vio615, Miltenyi, REA623, 130-113-226, 1:150), FoxP3 (PE-Cy5, eBioscience, PCH101, 15-4776-42, 1:1500), CD45RA (BUV496, BD Bioscience, 5H9, 741182, 1:150), CD62L (BUV737, BD Bioscience, SK11, 749210, 1:300), TIGIT (BV786, BD Bioscience, 741182, 747838, 1:100), LAP (Per-CP-eFluor710, eBioscience, FNLAP, 46-9829-42, 1:100), Helios (PE, Biolegend, 22F6, 137216, 1:75), CD25 (PE-Vio770, Miltenyi, REA945, 130-116-205, 1:75), CD27 (APC, Biolegend, M-T271, 356410, 1:150), CD137 (Alexa Fluor 700, Biolegend, 4B4-1, 309816, 1:150), CD127 (APC-Vio770, Miltenyi, REA614, 130-113-416, 1:75), GARP (BV421, BD Bioscience, 7B11, 563956, 1:50), CTLA-4 (BV605, Biolegend, BNI3, 369610, 1:200), ICOS (BV650, BD Bioscience, DX29, 563832, 1:1500), GITR (BV711, Biolegend, 108-17, 371212, 1:400). Recombinant CD19 reagent (Miltenyi, 130-129-550) was used to detect CAR⁺ T cell, as previously described. Prior to the staining, mouse FC blocking reagent was employed according to the manufacturer’s instructions (Miltenyi) to avoid specific binding and to reduce noise.

For immune cells infiltrating the harvested organs at sacrifice, single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies specific for human CD45 (PE-eFluor610, eBioscience, 2D1, 61-9459-42, 1:150) CD3 (eFluor506, Invitrogen, OKT3, 69-0037-42, 1:150), CD4 (BV785, Biolegend, RM4-5, 100552, 1:75), CD19 (FITC, Biolegend, 4G7, 392508, 1:150), CD20 (PE-Cy7, Biolegend, 2H7, 302312, 1:150), CD138 (PE-Cy5, BeckmanCoulter, B-A38, A54191, 1:300), CD27 (APC-Cy7, Biolegend, M-T271, 356424, 1:300), CD14 (PerCP, Biolegend, M5E2, 301824, 1:150), HLA-DR (BUV805, BD Bioscience, G46-6, 748338, 1:150), PD-1 (BV650, Biolegend, EH12.2H7, 329950, 1:75), CD163 (BV605, BD Bioscience, GHI/61, 745091, 1:300), CD56 (PE, Biolegend, 5.1H11, 362508, 1:300), CD206 (APC, Biolegend, 15-2, 321110, 1:800).

For the in vivo studies, human leukocytes were defined as human CD45+ cells. T cells were defined as huCD45+CD3+ cells. B cell subsets were defined as huCD45⁺CD19⁺ lymphocytes. B cell sub-populations were defined as: pre-B cells CD19⁺CD20^–CD27^– cells, naïve B cells CD19⁺CD20⁺CD27^– cells, memory B cells CD19⁺CD20⁺CD27⁺ cells, plasmablasts CD19⁺CD20^–CD27⁺ cells, plasma cells CD138⁺ cells.

For each experiment, dead cells were excluded by DAPI positive staining. The amount of each antibody has been titrated for each lot as suggested on the data sheet, before the use.

Data were acquired using a BD FACS Canto II or a Symphony A5 (BD Biosciences) and analyzed with FlowJo version 10 software (TreeStar).

Flow cytometry data were analyzed using cytoChain algorithm for unbiased high-dimensional analysis³⁶. Briefly, acquisition stability was evaluated using Flow_iQC algorithm and all the channels’ fluorescence intensities transformed by the arcSin function. Successively, the optimized flowSet was analyzed by FlowSOM-based clustering algorithm: the resulting 50 clusters were collapsed into 20 meta-clusters by Consensus Cluster Plus. Marker expressions in each cluster were then organized in a heat map. The frequency of cluster composition in each group was then plotted and analyzed for statistically significant differences using Prism 10 (GraphPad Software).

Engineered cells were stimulated in an antigen-specific manner, according to the specific experiment. Culture supernatants were collected after 72 h of stimulation and analyzed with a bead-based assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instruction by flow cytometry.

Mouse blood samples were collected at the baseline and 3 or 7 days after the cell injection according to the specific experiment. The samples were centrifugated and stored at −20 °C. At the moment of the cytokine analysis, mouse sera were thawed and analyzed with a bead-based assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions by flow cytometry.

Statistical analyses were performed with Prism 10 (GraphPad Software). Student t-test was used when comparing two independent groups. Two-way ANOVA was used when comparing three or more independent groups. For non-parametric variables, Mann–Whitney or Kruskall–Wallis tests were employed. P-value adjusted tests were employed to identify significant differences between groups. For categorical variables, chi-square test was used. For all comparisons, two-sided p-values were used, and p-value < 0.05 was considered statistically significant.

Sample size for in vivo studies was calculated with the G-power software to obtain a significant difference in circulating B cells, considering a power of 0.8 and type I error of 0.05. Mice were randomized according to the humanization level and the sex of the animals. In the in vivo experiment involving SGM-3 mice, the randoMice software (Github) was employed to improve this approach, always considering the humanization level and the sex of the animals.

The detailed statistical analysis is reported in the description of each figured.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.