{'fraction_non_alphanumeric': 0.03306862628896527, 'fraction_numerical': 0.029098020623444354, 'mean_word_length': 4.461165048543689, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A protocol for generating induced T cells by reprogramming B cells in vivo

Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells in vivo. This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research.


Introduction
Current T cell-based anti-tumor therapy, such as chimeric antigen receptor T-Cell Immunotherapy (CAR-T) and tumorassociated antigen-TCR-T (TAA-TCR-T) therapy, uses T cells collected from individual patient' peripheral blood. After in vitro activated amplification and subsequent gene editing, the engineered bulk T cells were transfused back to the patient for expected tumor clearance. The complicated manipulation process of separation, amplification, and gene editing of patients' T cells before transfusion is time-and cost-consuming. Besides, it is challenging to collect sufficient T cells from patients' peripheral blood in some cases, such as infant or T cell-deficient patients. Therefore, it is necessary to explore alternative approaches to generate functional T cells to extend T-cell based therapy.
Several attempts have been reported aiming to generate T cells. Hematopoietic stem cells (HSCs) can naturally differentiate into the entire lymphoid lineages in vivo, including T cells. However, it is unrealistic to derive abundant and functional CD4 þ single positive (SP) and CD8 þ SP T cells simultaneously from HSCs or iPSCs due to the failure of mimicking the thymic microenvironments in vitro. Another concept of generating de novo T cells by direct lineageconversion in vivo takes advantage of the physiological microenvironment of T cell development, which is expected to obtain functional T cells. Pax5-deficient pro-B cells give rise to mature T cells in irradiated Rag2-deficient mice. 1 Combined heterozygous loss of Ebf1 and Pax5 allows for B cell progenitors to generate T cell progenitors in irradiated Rag1-deficient recipients. 2 However, the phenotypic T cells obtained above brought the risk of clonal tumors in vivo. In a recent study, we reported that the enforced expression of transcription factor Hoxb5 converted pro/pre-B cells into fully functional T cells in vivo. 3 Here, we describe a step-by-step protocol of generating functional T cells in vivo from Hoxb5-expressing pro/pre-B cells. The cDNA encoding Hoxb5 was inserted into a retroviral expression cassette with a green fluorescent protein (GFP) reporting sequence. The recombinant vector was transduced into a modified Plat-E cell line containing retroviral packaging elements. Pro/pre-B cells were used as target cells due to their weak epigenetic barriers for reprogramming. 4e6 The pro/pre-B cells sorted from murine bone marrow were transduced with the Hoxb5 retrovirus by two-round spin-infection and subsequently transplanted into sublethally irradiated syngeneic recipient mice via orbital veins. Four weeks after transplantation, GFP þ T cells occurred in the peripheral blood, lymph node, spleen and thymus of individual recipient mice. This protocol provides a simple approach to abundantly generate functional T cells in vivo by reprogramming.

Experimental design
We illustrate a multi-step PROCEDURE for obtaining functional T cells by in vivo reprogramming, in which each part (retrovirus packaging, isolation of pro/pre-B cells, transduction of pro/pre-B cells, cell transplantation, and analysis of induced T cells) should be carefully followed to reach ideal results (Fig. 1).

Retrovirus packaging
The advantages of high expression level and low toxicity of the retrovirus-expression system in mice is the reason why we choose retrovirus for ectopic expression of Hoxb5 in pro/pre-B cells. To make a high titer, Hoxb5-retrovirus is essential for efficient transduction in this experiment. We use a modified plat-E cell line containing the retroviral packaging elements. 7 The accession number of the murine Hoxb5 sequence is NM_008268.2 (Genbank database). The vector used to produce retroviruses is pMYs-IRES-GFP. This vector can stably express ectopic genes in hematopoietic progenitor cells. 8 We inserted the CDS fragment of Hoxb5 into this vector and used the same empty vector as negative control. Induced GFP þ T cells will be detected in the recipients with Hoxb5-retrovirus transduced pro/pre-B cells. We choose calcium phosphate transfection in this protocol due to its easy operation, relative safety for researchers, and low toxicity for cells.

Isolation of pro/pre-B cells
Why do we choose the pro/pre-B cells as the target cell for reprogramming? Firstly, several research groups have reported that B cells can be converted to macrophages, 6 reprogrammed into pluripotent stem cells, 4,9,10 and even de-differentiated into longterm repopulating HSCs. 5 These reports reveal that pro/pre-B cells possess the plasticity for reprogramming. Secondly, the genomic rearrangements of Ig heavy chain variable, diverse and joining regions in individual pro/pre-B cells serve as natural "genetic barcodes" for verifying their origin in reprogrammed cell products. Thirdly, pro/pre-B cells can be abundantly isolated, possess the capacity of homing to bone marrow, and can sustain over several months for B-lymphopoiesis in vivo, which is temporally and spatially beneficial for hematopoietic reprogramming in vivo. One to two million pro/pre-B cells can be collected via sorting from individual mice (4e6 weeks old). To exclude the potential contaminants of other lineages, we added antibodies recognizing Ter119, CD11b, CD3, CD4, CD8 for lineage-depletion (lin À ). Then we sorted the Lin À B220 þ CD19 þ CD93 þ IgM À cells as the pro/pre-B target cells for reprogramming. To reduce the sorting time and enhance cell viability, we enriched the total bone marrow cells using Streptavidin-beads recognizing biotin-conjugated B220 antibody.

Transduction of pro/pre-B cells
Spin infection is preferential for viral transduction of suspended blood cells. Here, we choose two rounds spin infection to increase Plat-E cells to produce viruses. Pro/pre-B cells (Ter119 e Mac1 e CD3 e CD4 e CD8 e B220 þ CD19 þ CD93 þ IgM e ) were sorted from the bone marrow and then cultured in medium (15%FBS, 100 mM GlutaMAX, 10 À4 M b-ME, 10 ng/ml mSCF, 10 ng/ml Flt3L and 10 ng/ml IL7) for 12e16 h before virus transduction. Pro/pre-B cells (1 Â 10 6 per ml) were transduced with the mixed viruses with equal pre-adjusted titers. The pro/pre-B cells transduced with retro-Hoxb5 virus were collected for transfection efficiency analysis and then were transplanted into sublethally irradiated syngeneic recipient mice via orbital veins. Four weeks after transplantation, cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry. the infection rate. A short time of transduction duration is also essential to keep a balance between cell motility and transduction rate. Two hours' spin transduction at 32 C is recommended. Besides, 8 mg/ml polybrene is recommended to guarantee an ideal transduction rate. The density of the pro/pre-B cells is another crucial factor in spin transduction. Three million pro/pre-B cells in 1-ml retrovirus solution is recommended in this experiment.

Transplantation and analysis of the induced T cells
The early phase-reprogramming of the Hoxb5-retrovirus transduced pro/pre-B cells requires the bone marrow niche. Meanwhile, the donors and the recipients should be syngeneic to avoid potential immune rejection. The recipients must be conditioned by irradiation prior to transplantation, to reduce the competitiveness of host cells and evacuate related niches occupied by host cells. We choose a sublethal irradiation dose of 6.5 Gy for 8e10 week old mice (C57BL/6 background). The mature T cells can be detected in the lymph node, spleen, peripheral blood and other immune tissues or organs of the individual recipients four weeks after transplantation with Hoxb5-retrovirus transduced pro/pre-B cells. Here, we choose the thymus to analyze the early development of T cells at double negative stage (DN cells) and monitor the mature induced CD4 þ SP T, CD8 þ SP T (iT) cells in the lymph node, spleen, and peripheral blood of the related recipients. 2j Clean the vial with ethanol, and transfer the cell suspension to a 15 ml tube. 3j Add 9 ml Plat-E medium to the cells suspension dropwise. Mix the suspension by shaking the tube gently. 4j Centrifuge at 250 g for 5 min at room temperature and discard the supernatant. 5j Resuspend the cells with 10 ml Plat-E medium and transfer to a 100-mm dish. Incubate the cells in 37 C, 5% CO 2 until the cell become 80e90% confluent. Aspirate the medium and wash the cells with 2 ml DPBS. 6j Remove DPBS completely, add 1 ml of 0.25% (wt/vol) trypsin and incubate at 37 C for 1 min. 7j Add 4 ml Plat-E medium to neutralize trypsin and suspend the cells by pipetting up and down to single-cell suspension. 8j Transfer the cell suspension to a 50 ml tube. Centrifuge at 250 g for 5 min at room temperature. Count the numbers and adjust the concentration to 9 Â 10 5 cells/ml. Transfer 10 ml suspension to 10 mm dish. Incubate the dish at 37 C 5% CO 2 until the cell become 80e90% confluent. 9j Aspirate the Plat-E medium, and add 7.5 ml fresh Plat-E medium to the dish. Incubate the cells at 37 C 5% CO 2 for 2 h. 10j Prepare a 15 ml tube to set up transfection reagent. The table below provides a typical composition of the transfection reagent for one 10 mm dish of Plat-E cell in step 8.

Preparation of pro/pre-B cells TIMING: 24 h
14j Euthanize the mice by cervical dislocation. ! CAUTION Animal experiments are to be performed in accordance with relevant authorities' guidelines and regulations. 15j Using small scissors and forceps to isolate the femur and tibia bones from C57BL/6 CD45.2 mice and place them in a 60 mm tissue culture dish containing 5 ml ice-cold staining medium. 16j Cut off the end tips of each bone in the dish. 17j Discard the medium from the dish using a 5 ml syringe, and flush out the bone marrow cells with staining medium. 18j Disaggregate bone marrow tissues by repeated aspirations using pipette. 19j Filter the cells through a 70 um strainer into a 50 ml tube. 20j Spin down the cells for 5 min at 500 g at 4 C. Remove the supernatant carefully.
21j Resuspend the cells with 500 ml ACK red blood cell lysis buffer per mouse and incubate them on ice for 3 min. 22j Add 5X volume of staining buffer. Centrifuge for 5 min at 500 g and 4 C.  (Table 1), and incubate on ice for 20 min. 39j Wash with 5 volumes of staining buffer, and centrifuge for 5 min at 500 g and 4 C. 46j Centrifuge the 6-well plate at 805 g, 35 C for 90min. ! CAUTION Warm up the centrifuge to 35 C before usage. 47j Unseal the 6-well plate. Incubate the 6-well plate 37 C, 5% CO 2 for 2 h. ! CAUTION After centrifuge, the 6-well plate must be unsealed and transferred to incubator within 5 min. ! CAUTION Do not shake the 6-well plates. 48j Transfer the cells in 6-well plate into 50 ml tube. Centrifuge 350 g for 5 min. Discard the supernatant. 49j Preheat the pro/pre-B culture medium. Resuspend the cells with pro/pre-B culture medium. Count the number of pro/pre-B cells. Adjust the cell concentration to 5 million/ml in each flask. Incubate the cells at 37 C, 5% CO 2 for 24 h. ! CAUTION The cell concentration should be accurate for cell viability. ? TROUBLESHOOTING. 50j Repeat step 42e48 to infect the pro/pre-B cells again. 54j Anesthetize the Hoxb5-mice using 300 ml avertin by intraperitoneal injection and then collect 1 ml peripheral blood into 1.5 ml anticoagulation tube by ophthalmic vein blood sampling. ! CAUTION Place the tube on ice after sampling. 55j Dissociate the immune organs after sacrifice the recipients. The immune organs include Thymus, spleen and lymph node. Isolate the tibia of wild type mice for preparing single stain control samples for compensation setup. ! CAUTION Place the fresh immune organs in the 2% FBS$DPBS solution and keep on ice. 56j Grind the immune organs in the staining buffer using a syringe plunger. Flush out the bone marrow of the tibia with a 5 ml syringe.
Pass the cell suspension through a 70 mm strainer into the 15 ml centrifuge tubes. 57j Centrifuge the cell suspension at 4 C 500 g for 5 min and then discard the supernatant. ! CAUTION For the cells collected from thymus and lymph node, resuspend the cells using 500 ml staining buffer after discard the supernatant, and then count the cell numbers. 58j Add 1 ml ACK red blood cell lysis buffer to the peripheral blood, spleen cells and bone marrow nucleated cells to lyse the red cells.
For the peripheral blood, the lysis time is 25 min, and for the spleen cells, the lysis time is 3 min. 59j Centrifuge the cells suspension at 4 C 500 g for 5 min and then discard the supernatant. 62j Add 1 ml purified anti-mouse CD16/32 antibody to each sample.
Incubate the samples on ice for 20 min. 63j Add the related antibody mix buffer into the related samples. Incubate on ice in dark for 20 min. The antibodies and their quantity used for staining PB, spleen and lymph node cells were listed in Table 2, and for staining the thymus cells were listed in Table 3. For compensation, CD11b antibodies conjugated each fluorescent dye were added to unstained and individual single stained samples (Table 4).
! CAUTION It's better to make the antibody mix in a volume of 20 ml for each sample.
! CAUTION The staining process should be performed on ice in dark. 64j Add 5X volume staining buffer into the staining tubes and centrifuge at 4 C 500 g for 5 min and then discard the supernatant.
65j Add 800 ml DAPI solution to each sample and resuspend the cells. Place the stained sample on ice. 66j Flow cytometry analysis.
Step 54e66: Flow cytometry analysis of the induced T cells: 5 h.

Anticipated Results
This protocol describes a detailed method for the generation of functional T cells from pro/pre-B cells through reprogramming in vivo. High-purity of pro/pre-B cells and high titer of Hoxb5 virus should be ensured. The purity of sorted pro/pre-B cells  Table 3 Antibodies used for staining thymus cells Table 4 Antibodies used for single staining samples (Ter119 e Mac1 e CD3 e CD4 e CD8 e B220 þ CD19 þ CD93 þ IgM e ) should be close to 100% confirmed by flow cytometry analysis (Fig. 2). We usually collected 10e15 million B220 þ cells per mouse after antibiotin beads enrichment and 3e3.5 million pro/pre-B cells per mouse after sorting. The Titer of Hoxb5 virus or GFP control virus can be adjusted to 0.69 MOI using a NIH/3T3 cell line (Fig. 5). After two-round spin-infection (805 g, 90min, 35 C), the transduction efficiency of Hoxb5 or GFP control virus should reach around 50% in pro/pre-B cells (Fig. 3). The viability of pro/pre-B cells should be 50%e60%.
Then, three million transduced GFP þ pro/pre-cells are transplanted into sublethally irradiated (6.5Gy) recipient mice (C57BL/6, CD45.2) via retro-orbital vein injection. Four weeks after transplantation, abundant mature iT cells would occur in the peripheral blood (PB, 5%e10%), spleen (10%e15%) and lymph node (30%e40%), and GFP þ iDN cells would occur in the thymus of the Hoxb5 virus transduced pro/pre-B recipient mice (Fig. 4). In general, one million Hoxb5-expressing pro/pre-B cells gave rise to approximately ten thousand induced T cell progenitors. Thus, the converting efficiency from B cell to T cell by Hoxb5 is around 1%. Fig. 2. Flow cytometry analysis of the purity of sorted Ter119 e Mac1 e CD3 e CD4 e CD8 e B220 þ CD19 þ CD93 þ IgM e pro/pre-B cells. Bone marrow nucleated cells were first incubated with biotin-conjugated anti-B220 antibody and then enriched by streptavidin microbeads using AutoMACS Pro. The pro/pre-B cells were further sorted from the B220 enriched cells by defined markers. Flow cytometry analysis confirmed the purities of B220 enriched cells (top) and sorted pro/pre-B cells (bottom).   4. Flow cytometry analysis of induced T lymphocytes in irradiated recipients injected with Hoxb5-retrovirus transduced pro/pre-B cells. GFP þ Ter119 e Mac1 e populations obtained from the PB, spleen, and LNs (a) or GFP þ Ter119 e Mac1 e CD19 e iDN populations from the thymus (b) of recipient mice were analyzed. Three million pro/pre-B cells transduced with the retro-Hoxb5 virus were transplanted into sublethally irradiated syngeneic mice. Four weeks after transplantation, the single nucleated blood cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry. Representative plots were shown.