I have trouble inducing T cell transfer colitis in mice. I have been strugling for many years and tried several troubleshooting without any luck. It doesn't seem mice are responding to transferred T cells. Here are my protocol.
Recipient: BALB/c Rag1 KO mouse 8-10wk
Donor: BALB/c Foxp3-eGFP, WT and gene KO I crossed.
I am trying to match gender between recipient and donor.
T cell sorting: CD4+ GFP- CD25- CD45RBhi CD44low
We don't have sorting facility, so I have to take 1 hour travel to nearby university.
injection: 4 x 10^5 sorted T cell via retro orbital sinus injection in 200ul PBS
The problem is that I cannot see weight loss in WT-transferred recipients in next 5-6 weeks. Threre are sometimes visible loss in one or two mice, but I need to see clear weight loss in all WTmice so that I cen tell whether KO of a gene makes any difference.
I tried several troubleshooting such as,
Changing fluorescent Abs: It turned out I was using depleting clones. I switched to non-depleting ones.
Raising recipients in non-SPF condition: colitis is dependent on gut microbiota so I breed Rag1 KO in non-SPF condition. I also use non-autoclaved water and non-irradiated chow.
T cell viability: since FACS facility is far, I keep cells on ice and count viable cells with trypan blue right before the injection. Also I coat harvesting tube for sorting with FBS as I heard that it helps with cell viability.
It would be a great help if somebody can tell what I am doing wrong. Thanks.
Dear Norman,
Thank you for your opinion. The cells I transfer are naive T cells, actually I try to eliminate Tregs as much as possible because I heard small amount of Tregs could prevent inducing colitis. So I am not sure if viability of naive T cells could be affected by NICD or it may have something to do with recipients not having weight loss. There were several occasions I transferred naive T cells in other experiments and at that time it seemed transferred cells were alive many weeks later.
Wonyong
I do two things differently. First, we use IP injection, they seem to work well in our model. Also, are you ordering your Rag mice, injecting them, and then placing them in non-SPF conditions? When we perform this sequence, we find that it takes ~70 days for those mice to get sick. The model works well if the mice are already in non-SPF conditions prior to transfer of T cells. When we have Rag knockouts born and bred in non-SPF conditions and placed in the model they get sick within 4-5 weeks. Hope this helps.
Douglas
Sorry, I just noticed you raised the recipients in non-SPF conditions. Also, I MACs purify CD4+ T cells then sort CD25loCD45RBhi then do the injections. I change out cages once a weeks. If the mice are not "dirty", colitis development will take longer.
Our adoptive transfer work is in lymphoma, and I will transfer at least between 2-5 million T-cells. Maybe upping your cell number might help?
Dear Douglas,
Thanks for your input. Yes there is concern that my mice might not be "dirty" enough. That is why I started breeding in non-SPF condition. Also I am considering having them co-housing with a hamster or something. I heard that co-housing Rag1 KO with inflammasome-defective mice in SPF condition also helps, but I dont have those strains.
Dear Akshat,
Thanks for your input. It is routine dose for inducing colitis. I heard more than that would actually inhibit colitis but don't know why exactly.