T cells for adoptive transfer were genetically modified to induce expression of FOXP3, and/or GFP for in vivo tracking, as appropriate. The choice of gene transfer vector was determined by the need of achieving strong and permanent transgene expression in antigen- activated T cells as target cells. Since activated T cells undergo very fast cell cycle, we expected simple plasmid transfection methods originating episomes to lead to a loss of transgene expression upon target cell proliferation, and we therefore deemed such methods as not valid for our purposes. Thus we chose commercially available, replication-incompetent retroviral vectors, derived from murine oncoretroviruses. Such vectors are devoid of the env, gag and pol genes encoding the viral envelope and capsid proteins, and the retro-transcriptase, but preserve the necessary sequences for viral RNA encapsidation and for retro-transcribed DNA integration into the host cell genome, and transgene expression. Such kept elements include the retroviral 5’ and 3’ long terminal repeats (LTRs), the packaging signal for RNA encapsidation and the tRNA binding site. These retrovectors are issued as DNA plasmids provided with a multiple cloning site (MCS) containing a variety of restriction sites, which allows the user to insert the complementary DNA sequence (cDNA) encoding the product of interest. The plasmids are also provided with: a ColE1 origin of replication for replication in prokaryote competent cells; an ampicillin resistance gene (encoding a β-lactamase) for the selection of transformed bacteria, for vector DNA amplification; and a puromycine resistance gene that allows to select transfected cells for the preparation of packaging cell lines in eukaryotes.
To produce virion particles for transfer to the target cells, packaging cell lines expressing the env, gag and pol structural genes were generated. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chances of producing replication-competent viruses due to recombination events during cell proliferation. The resulting retroviruses
can infect target cells and induce permanent expression of the gene of interest, but they cannot replicate within these cells due to the absence of viral structural genes.
The retroviruses employed are RNA viruses that require target cells to be under active proliferation in order to transduce the cells, that is, to integrate into the host cell genome leading to permanent transgene expression. Oncoretroviruses take advantage of the absence of nuclear envelope during target cell mitosis (metaphase and anaphase) to integrate the retro-transcribed viral DNA into the host DNA. This mechanism can be used to selectively transduce proliferating cells within a cell population. In our experiments, we used this retrovirus property to selectively transduce antigen-specific CD4+ T cells. Following i.p. sensitization of the donor animals, splenocytes comprising antigen-presenting cells and antigen-specific CD4+ T cells are stimulated with antigen in vitro, leading to proliferation of the antigen-specific CD4+ T cells. By coupling antigen stimulation in vitro with retrovirus infection, only the antigen-specific CD4+ T cells are transduced with high specificity.
4.1. Expression Vectors
A Murine Stem Cell Virus (pMSCV) retroviral expression vector (Clontech Inc., Saint-Germain-en-Laye, France) was used to induce GFP expression on target CD4+ T cells. GFP, a 238-aminoacid protein first extracted from jellyfish Aequorea victoria, exhibits bright green fluorescence when exposed to ultraviolet light. More efficient and spectrally shifted mutant isoforms have been designed or discovered, such as the AcGFP1 variant used in this work. The cDNA encoding GFP was amplified from a pAcGFP1 vector (Clontech Inc.) using sticky end primers (Table 2, primers 01 and 02) and subcloned into the MCS of pMSCV between XhoI and EcoRI restriction sites. For all experiments, primers were designed and checked using TibMol tools (available at www.tib-molbiol.com). Polymerase chain reactions (PCR)s were performed in a conventional thermocycler (Primus 96, Peqlab Biotechnologie GmbH, Erlangen, Germany) using Expand High Fidelity PLUS PCR System (Roche Diagnostics, Mannheim, Germany) (see appendix). Restriction sites were checked and designed using Saccharomyces Genome Database (SCG) and New England BioLabs tools (available at http://www.yeastgenome.org and http://www.neb.com/nebecomm/default.asp respectively). Ligation reactions were performed using T4 DNA Ligase (Invitrogen, Life Technologies, Madrid, Spain) according to manufacturer’s instructions. PCR products and plasmids were purified with QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) according to manufacturer’s instructions. Reactions with restriction enzymes and alkaline phosphatase were performed as appropriate (enzymes purchased from Roche Diagnostics and used according to manufacturer’s instructions).
Table 2. Primers
Primer Name Sequence (5´ – 3’) Sticky end
01 AcGFP/F cgcctcgagGTCGCCACCATGGTGAGC XhoI
02 AcGFP/R ggggaattcCGGCCGCTCACTTGTACAG EcoRI
03 Dir ZsGreen gggctcgagGCGGCTTCGAGGATAAA XhoI
04 Rev ZsGreen gggctcgagATTATCATCGTGTTTTTCAAAGG XhoI
05 Upper AcGFP gggctcgagATGGTGAGCAAGG XhoI
06 Lower Puromic gggctcgagTCAGGCACCGGGCTTG XhoI
07 Forward CheqGFP CTGATCTGGGGCCTCGGTG –
08 Rev CheqGFP TATCGGGCAGCAGCACAGG –
09 Foxp3 F ggggcggccgcATGCCCAACCCTAGGCC NotI
10 Foxp3 R gggatcgatTCAAGGGCAGGGATTGGA ClaI
Transgenic, antigen-specific Treg cells with forced FOXP3 expression were generated according Hori et al. (411) with minor modifications. Antigen-specific effector CD4+T cells were transduced using a bicistronic construct containing Foxp3 cDNA plus gfp as a reporter, expressed under the same promoter by means of an internal ribosome entry site (IRES). Foxp3 cDNA was kindly provided by Dr. Shimon Sakaguchi (Kyoto University, Japan). We used commercial bicistronic vector pRetroX-IRES-ZsGreen1 (Clontech) to obtain the IRES sequence. In the final construct, Foxp3 was subcloned upstream the IRES, and its expression could be monitored from the GFP signal. ZsGreen fluorescent protein, a GFP variant present in the commercially available pRetroX-IRES-ZsGreen1, was not of interest for our purposes because we had previously optimized immunohistochemical GFP detection in tissues for the AcGFP1 variant (see section 8.6). To replace ZsGreen by AcGFP1 cDNA we performed inverse amplification of the pRetroX-IRES-ZsGreen vector using primers 03 and 04 in Table 2 (with sticky ends for XhoI). We called the resulting vector, provided with a MCS upstream the IRES and the XhoI restriction site in frame downstream, pRetroX-IRES-Y. The fragment encoding GFP and puromycin resistance, from the pMSCV-GFP vector, was amplified using primers with XhoI sticky ends (Table 2, primers 05 and 06), and
subcloned into the Y site. Correct insert orientation was checked by PCR (Table 2, primers 07 and 08). Then, Foxp3 mRNA was amplified using primers with sticky ends for NotI and ClaI restriction enzimes (Table 2, primers 09 and 10), at the 3’ and 5’ ends respectively. These restriction sites were then used to insert the Foxp3 mRNA into the MCS, giving as a result the pRetroFOXP3-IRES-GFP vector.
4.2. Virion particle production
The pMSCV-GFP and pRetroFOXP3-IRES-GFP vectors were separately replicated in competent DH5αTM Escherichia coli (Invitrogen) that became ampicilin resistant due to the expression of the ampicilin resistance gene present at the vectors. Transformed bacteria were selected with ampicillin in Petri dishes (Corning, New York, USA) using Luria Bertani agar (LBA) medium (see appendix), and then cultured in liquid Luria Bertani (LB) medium (see appendix) at 37ºC with gentle agitation. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions. A HEK293T "Phoenix" (φNX) ecotropic packaging cell line (Orbigen, San Diego, USA) was transfected with pMSCV-GFP using Lipofectamine (LipofectamineTM 2000, Invitrogen) according to manufacturer’s instructions. To generate a stable packaging cell line, transfected Phoenix cells carrying plasmid integration events were selected by puromycin resistance using 2 µg/mL puromycin (Sigma-Aldrich) (see appendix for a detailed protocol). Surviving puromycin resistant cells were checked in a FacsCalibur cytometer (BD Biosciences, Madrid, Spain) for GFP expression, and then cultured and expanded. Cellular stocks were frozen in culture media with 10% DMSO (Sigma-Aldrich), and stored in liquid nitrogen for later use.
Retrovirus production was performed according to Phoenix cell provider’s instructions:
transfected Phoenix cells were cultured and expanded using 60 mL of D-MEM culture medium with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine (PSG) (all from Gibco, Invitrogen) into 15 cm culture plates (Corning). Once cells reached 95% confluence, culture medium was changed by production medium, D-MEM plus 10% calf serum (Gibco, Invitrogen), and the cells were maintained for 24 hours. Cell culture supernatant was collected and filtered through 0.45 µm filter (Stericup, Millipore) and maintained at 4ºC to maximize virion viability.
Virion particles were concentrated by centrifuging the supernatants in Centricon© 100.000 NMWL membrane filters (Millipore) at 3500 g. Virion stocks were stored at -80ºC until use.
Viral titers in the frozen stocks were estimated using a murine NIH3T3 cell line (ATCC-LGC Standards partnership, USA). For this purpose, flow cytometry assays were performed in NIH3T3 cells after transduction with serial dilutions of the pMSCV-GFP and pRetroFOXP3-IRES-GFP
stocks. Additionally, a western blot assay was performed on pRetroFOXP3-IRES-GFP transduced NIH3T3 cells to check for Foxp3 expression. Antibodies against FOXP3, GFP, and α-tubulin were used, followed by a horseradish peroxidase-conjugated goat antibody (see appendix for a detailed protocol).
4.3. Lymphocyte cell culture
For CD4+ T cell transduction and adoptive transfer experiments, donor mice were i.p. sensitized to OVA or HDM as described (see section 3 on sensitization and challenge). One week after sensitization, the mice were euthanized and the spleens were removed in aseptic conditions and digested with collagenase IV (Sigma-Aldrich) during 60 minutes in a CO2 incubator (37ºC). Tissue was disrupted using a 5 mL pipet, and the resulting cell suspension was passed through a 70 μm cell strainer (Falcon, BD Biosciences) and collected into 50-mL centrifuge tubes. Red blood cells were lysed using Red Blood Cell Lysing Buffer (Sigma-Aldrich) according to manufacturer’s instructions. Splenocytes were cultured at 5x106 cells/mL in 6-well plates (Corning). To favor Th2 differentiation, cultures were supplemented with 10 ng/mL recombinant IL4 (PeproTech, Rocky Hill, USA), 100 U/mL IL2 (BD Bioscience), 250 ng/mL anti-IL12 (eBioscience, Hatfield, UK), 2%
non essential aminoacids (Invitrogen) and 100 μM β-mercaptoetanol (Merck, Madrid, Spain). To stimulate antigen-specific CD4+ T cells, 40 μg/mL OVA (for CD4+ effector T cell experiments) or 8 ng/mL HDM extract derived Der p 1 (for CD4+Foxp3+ T cell experiments) were added as appropriate.
4.4. T cell retroviral infection
After 24 hours of stimulation with antigen in vitro, the cells were infected with pSCMV-GFP or pRetroFOXP3-IRES-GFP using a spin infection procedure. Viral supernatant was added to the splenocyte cultures with 6 µg/mL polybrene (Sigma-Aldrich), at a multiplicity of infection (MOI, ratio of infectious viral particles to cells) of 1. The cultures were then centrifuged at 455g for 1 hour at 37ºC. After centrifugation, the cells were returned to the cell culture incubator and maintained in stimulation medium for 7 days, and then sorted by FACS to separate the GFP-expressing cells for in vivo transfer. Cell sorting was done with BD Bioscience FacsAria equipment.
Analytic flow cytometry was performed to check FOXP3, cell surface markers (CD4, CD25) and/or GFP expression in transduced cells, using a BD Bioscience FacsScan cytometer. For this purpose, cells were resuspended in flow cytometry buffer (see appendix). PE-Conjugated Rat Mouse CD4, allophycocyanin(APC)-conjugated Rat Mouse CD4 and PE-Conjugated Rat Anti-Mouse CD25 antibodies (all from BD Bioscience) were used for the staining of cell surface antigens
at 0.8 μg/106 cells. For FOXP3 staining, APC-anti-mouse/rat FOXP3 Staining Kit (eBioscience) was employed at 0.5μg/106 cells.