I would like to separate the in vivo IFN-ß effects observed in the EAE model attending to different subsets. For that purpose, I would need to specifically deplete the intracellular pathway for the IFN-ß response in T cells. Is there any way to make T cells that do not response to IFN-ß?
Thank you for your help.
Carol
Using CD4cre+/- IFNARfl/fl mice looks to me one of the best way to selectively delete type I IFN signal in CD4 cells.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054447/
CD8 depletions
Hybridoma supernatants were clarified by centrifugation, concentrated with Amicon filters (Millipore), and protein G purified (Pierce). The Ab preparations were quantified by spectrophotometry. IFN-α/βR-/- mice were injected i.p. with 250 μg of SFR3 or 2.43 in PBS (250-μl total volume) 3 days and 1 day before infection. At day 6 after infection, 85-95% of CD8+ T cells had been depleted.
Peptide immunizations
IFN-α/βR-/- mice were immunized s.c. at the base of the tail with 50 μg of each DENV peptide plus 100 μg of helper peptide (I-Ab-restricted hepatitis B virus core 128-140) emulsified in IFA (Difco). Mock-immunized mice received helper peptide and DMSO emulsified in IFA. Mice were infected 12-13 days after immunization.
In vivo cytotoxicity assay
IFN-α/βR-/- mice (recipients) were infected with 1010 GE S221. Splenocytes and lymph node cells (targets) were harvested from donor B6.SJL-congenic mice (CD45.1) 7 days later. RBC were lysed, and the target cells were pulsed with 0.5 μg/ml of the irrelevant vaccinia virus (VACV) peptide, C51-59, NS2A8-15, NS4B99-107, NS5237-245, or a pool of C51-59, NS2A8-15, NS4B99-107, and NS5237-245 for 1 h at 37°C. The cells were then washed and labeled with CFSE (Invitrogen) in PBS/0.1% BSA for 10 min at 37°C. DENV peptide-pulsed cells were labeled with 1 μM CFSE (CFSEhigh) and the irrelevant peptide-pulsed cells with 100 nM CFSE (CFSElow). After washing, the two cell populations were mixed at a 1:1 ratio and 5 × 106cells from each population were injected i.v. into naive or infected recipient mice. After 4 h, the mice were sacrificed and splenocytes were stained with CD45.1-allophycocyanin and analyzed by flow cytometry, gating on CD45.1 cells. The percentage killing was calculated as follows: 100 - ((percentage DENV peptide-pulsed in infected mice/percentage irrelevant peptide-pulsed in infected mice)/(percentage DENV peptide-pulsed in naive mice/percentage irrelevant peptide-pulsed in naive mice)) × 100.
Quantitation of DENV burden in mice
Mice were euthanized by isoflurane inhalation and blood was collected via cardiac puncture. Serum was separated from whole blood by centrifugation in serum separator tubes (Starsted). Small tissue pieces were immediately placed into RNAlater (Qiagen) and subsequently homogenized for 3 min at 4°C in 1 ml of tissue lysis buffer (Qiagen Buffer RLT) using a Mini-Beadbeater-8 (BioSpec Products). Immediately after homogenization, all tissues were centrifuged (5 min, 4°C, 16,000 × g) to pellet debris and RNA was isolated using the RNeasy Mini Kit (Qiagen). Serum RNA was isolated using a QIAamp Viral RNA Mini Kit (Qiagen). After elution, viral RNA was snap frozen in liquid N2 and stored at -80°C. Quantitative RT-PCR was performed according to a published protocol (27), except a MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) with an iScript One-Step RT-PCR Kit for Probes (Bio-Rad) were used. The DENV standard curve was generated with serial dilutions of a known concentration of DENV genomic RNA which was in vitro transcribed (MAXIscript Kit; Ambion) from a plasmid containing the cDNA template of S221 3′ untranslated region. After transcription, DNA was digested with DNase I and RNA was purified using a RNeasy Mini Kit and quantified by spectrophotometry. To control for RNA quality and quantity when measuring DENV in tissues, the level of 18S rRNA was measured using an 18S rRNA Control Kit (Eurogentec) in parallel real-time RT-PCR. A relative 18S standard curve was made from total splenic RNA.
Statistical analyses
Data were analyzed with Prism software version 5.0 (GraphPad Software). Statistical significance for CD8+ T cell numbers and viral load were determined using the unpaired t test with Welch's correction; for the ELISPOT, the unpaired t test was used.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2674070/
Using CD4cre+/- IFNARfl/fl mice looks to me one of the best way to selectively delete type I IFN signal in CD4 cells.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054447/
Barbara Capuccini's comment above along with CD8cre+/- matings.
But if you want the T cells themselves deficient in IFNb, rather than their ability to respond that is more iffy, since there are many type I IFN genes you would need to target to totally eliminate ability of T cells to produce type I IFN. Selective depletion of IFNaR in the target cells often is used for this.
I would also say, Like Barbara Capuccini, that CD4-Cre with IFNAR fl mice would give you the most elegant model. Keep in mind that CD4 cre deletes the gene of interest in both CD4 and CD8 due to the DP phase in T cell development. If you want CD8 specific deletion you can look for a CD8a-Cre. However, as elegant as these models are... They might be difficult to access.
An alternative is to run your experiment in a transfer system and compare transferred WT and IFNAR deficient cells.
Hope this helps.
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