T Cell Biology - Science topic

A vaccine is a shot of a disease into someone's body, to summon antibodies to fight the illness. If the exact and specific cause of aging could be identified then t cells may mass produce themselves for anti aging. First, aging, as a disease, must have an identified parsimonious cause.

I am looking into selecting T cells with microbeads for isolation and subsequent adoptive transfer in immunodeficient NRG mice. I am hoping to do positive selection with microbeads. After column selection, I am wondering how the T cells will act in vivo after transfer. Do antibody/microbead-bound T cells act differently or are eliminated in immunodeficient NRG mice?

I am looking into selecting T cells with microbeads for isolation and subsequent adoptive transfer in mice. Before labeling with microbeads, I usually block Fc receptors to ensure a more specific binding of antibodies. If I block Fc receptors, do magnetic selection, and then do adoptive transfer, will the fact that the Fc receptors are block affect anything in vivo (e.g. proper T cell function in vivo)?

It's more by curiosity, but I didn't find a convincing mechanism for explaining the opposite effects of IL10 (inhibiting [1]) versus IL6/IL23/IL21 (potentiating) Th17 differentiation on T cells. All these cytokines signal (mainly) through STAT3 which, once phosphorylated, is known to activate Th17 associated transcription factors, so one should expect that IL10 also increases Th17 differentiation, but it's not the case...

Several hypotheses could be:

- that the receptors for these cytokines differentially activate parallel signaling pathways on T cells (such as p38 Map kinase, other STATs). However, several papers claim that the anti-inflammatory effect of IL10 is through STAT3, but they only quote effects on macrophages, not on T cells ... [2]

- that the receptors phosphorylate different loci in STAT3 (and indeed there are different ones [3]), but I didn't find literature on their role on Th17 differentiation

- that IL10R/signaling is not inhibited by SOCS3 while IL6/23/21 is. One could say then that IL6/23/21 induce SOCS3 that suppresses IL6/23/21 signaling but not IL10 signaling, and that IL10 could further increase the amount of SOCS3 in Th17 for instance. But then, would low IL10 doses promote Th17 differentiation ?

Would you have some papers to suggest on this question ?

Best,

References :

[1] Qu et al. 2012 (Experimental Hematology), Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion

http://dx.doi.org/10.1016/j.exphem.2012.05.006)

[2] Murray 2006 (Curr Opin Pharmacol) Understanding and exploiting the endogenous interleukin-10/STAT3-mediated anti-inflammatory response - see references 31-33

[3] Ng et al. 1997 (JBC) STAT3 Is a Serine Kinase Target in T Lymphocytes

It seems to be an apparent consensus that the activation and function of AhR influences the landscape of TC populations, but is there a group that is more severely affected?

Hello!

I isolated some T-cells with some very interesting TCRs from primary cultures. I sent them to Genewiz to get chromium single cell TCR sequencing done, however the sample viability was super low. I sent them 8 more vials so that they could do a dead cell removal and then isolate the live population and perform single cell sequencing on the remaining cells. The sequencing results show that whatever is left over after DCR is most likely another contaminant cell type, not a TCR. I now only have one vial left, so whatever I choose to do next is very critical and essentially has to work the first try.

At this point I dont care about chain pairing, I can piece that back together afterwards by trial and error, I just want to get some data from these cells. I was wondering if anyone knows if I can thaw my cells directly into RNA later and then do either normal NGS or another single cell sequencing method to get any info on the TCR sequences? Should I just amplify the TCR regions on thawing with some kind of primer pool and then send that for NGS? In general, what's the most robust process for getting out TCR information from low viability samples?

Some other notes:

1. I didnt personally do the T cell isolation but my thinking is they were pretty much exhausted at the time of freezing which is why we have viability issues on thawing

2. They were frozen in 10% glycerol + 10% FBS in a Mr Frosty at -80C and then maintained in LN2 and shipped on dry ice.

3. Observed viability is ~30% on thawing however this could just be the contaminant cell population....

Interested in MHC I

What is signal width (i.e. ssc-w) used for?

I use height (FSC-H) plotted against FSC-A (area) to gate singlets, then analyzing PBMCs or T cells but I see there is the option of w for FSC and SSC. Is it useful?

Hello everyone. I am wondering which surface markers can be used for MDSC cell identification in humans. In literature, they mentioned S1009, CD14. Does anyone have any experience? Thank you!

I intend to study the in vitro T cell response just to dengue virion for which I need to stimulate PBMCs with dengue virus (DENV). I need to inactivate it. I have purified dengue virus in NTE buffer through 30% glycerol cushion. 

Authors have reported the use of 0.025% gluteraldehyde (Kurane JClinInvest 1989), UV for 2 hrs (Correa 2015)  and heat treatment at 55C for 1 hr (Fink 2007), 56C 30min (Mosquera 2005). I have certain reservations regarding the use of chemical reagents for DENV inactivation since I have to analyse PBMCs on flow cytometer and in my experience use formalin for inactivation and further stimulation with PBMCs with such an inactivated virus gives horrible FSC vs SSC scatter plots. 

So please advise me regarding the use of heat inactivated DENV for stimulation or do you recommended any other method?

I am interested in culturing murine dendritic epidermal T cells (DETCs) after isolating cell "soup" from skin scalp. I have an overall idea, but maybe someone has a protocol and would be so kind to share it with me?

I think, many investigators observed T cell markers on the cells of myeloid lineage (neutrophils, monocytes, dendritic cells, eosinophils, macrophages). Evidently, many of these observations can not be fully explained by artifacts due to co-aggregation with T cells. In my laboratory, we really observed TCR beta and gamma chains on 1-3% of neutrophils and CD4 on CD11c+ (dendritic) cells. In available literature, evidence for expression of rearranged chains of TCR and immunoglobulin molecules in myeloid leukemia cells can be found. My interest to these findings was stressed by recently published works by Kerstin Puellmann and Wolfgang Kaminski with co-authors, showing functionality of recombinatorial immune receptors on neutrophils and macrophages (original publication has appeared in PNAS, 2006, 103(39):14441-6.). However, I see continuation of this history still in one laboratory. My personal doubts are evoked by own practice and the reason that we do not see any support for this idea in responses to allogeneic MHC molecules. Although neutrophils can accumulate in the spleen and blood of animals, immunized with MHC class I disparate tumor cells, they cannot do this without CD8 T cells and do not contain elevated percent of TCR bearing cells. So, I am inclined to think, that T cell markers on myeloid cells represent reminders of developmental history of individual cells, deciding to change lineage too late. I would very appreciate further discussion on this issue.

NKG2D is a receptor expressed on NK and T cells. I would be grateful if anybody could suggest a suitable cell line that can express NKG2D on its surface for in vitro studies.

I am currently optimising a plate-bound adhesion assay to study the adhesiveness of polyclonal human T-cells to differing concentrations of ICAM-1. 

I start with coating the plate with ICAM-1-Fc. I usually do this overnight, and then, after 3 washes with PBS (at room temp) + 40mM HEPES + 2mM Mg2+, I block the plate with PBS-3%BSA.

I then seed CFSE labeled cells onto the plate. The assay/binding buffer I use contains RPMI + 0.1% BSA + 2mM Mg2+ + 40mM HEPES.

After 20 minutes of incubation at 37C, I gently wash off the cells (x3), and then read the fluorescence on a plate reader.

What I have consistently noticed is that in the blank or 0 ug ml-1 ICAM-1-Fc condition, adhesion is the greatest. I have no idea what these cells are adhering to, but I am wondering if it is the BSA, perhaps? 

So, folks who commonly do plate-bound adhesion assays, do you or do you not block the plate after seeding your ligand? 

The plates I am using are opaque-walled, 96 well plates, polystyrene bottomed. 

Thanks, folks!

A.

First, I got the cell line from ATCC and thawed it and seeded in DMEM medium with 10% horse serum and the cells gradually died. I then bought another vial and I split them in two different media, one is DMEM with 10% horse serum and the other one is DMEM with 10% FBS. Neither of them worked. I then requested one vial from another lab and thawed it and grew it in DMEM with 10% FBS and the cells grew very well. I froze some in 5% DMSO. A couple of days ago, I thawed one vial and the cells didn't grow, and I noticed it started to die like the one bought from ATCC. I was wondering what happened with the cells. I can't figure it out.

Hi

I'm designing a flow cytometry panel including markers for Innate Lymphoid Cells, to use on human samples. As far as I can tell, they are:

ILC1:   CRTH2−   CD117−         NKp44−

ILC2:   CRTH2+   CD117+or−    NKp44−

ILC3:   CRTH2−   CD117+         NKp44+or−

Question: can I gate for ILC1, ILC2, ILC3 using

(Follow up - if not, what is NKp44 for?)

Thanks!

I want to stain intracellular cytokines and exhaustion markers (e.g. PD-1/CTLA-4) in mouse LN/spleen samples. I'm wondering whether ex vivo Ag restimulation or anti-CD3/28 stimulation affect expression levels of exhaustion markers ?

I found a lymph node-like granule under the thymus. Is it a lymph node of MLN?

I am looking for the activation of Jurkat T-cell and if you activate it then how can you measure their activation  I could not find anything related to these following: CD69 IL2 The other question is how to inhibit activation (turning it on and turning it off?

Anything in relation to cytokines with Jurkat T cell.

If you could kindly direct me in any direction please, that would be amazing of you please.

Thank you very much.

Hello,

Could someone point me to an article or tell me the surface copy numbers of most common T cell markers (TCR, CD3, CD4, CD8, CD28, ...)?

Many thanks,

Vincent

I am establishing a Tcell co-cultured 3D human cancer spheroid model. 1 hour after addition of HLA-matched Tcells to spheroids, I see that the Tcells infiltrate the spheroids. I used this model to check if immunotherapy using anti-PD1 inhibitor is a viable option to treat this cancer. On performing cyrotoxicity assay, I found that Tcells alone was killing the cancer cells and the addition of anti-PD1 checkpoint inhibitor actually suppressed the cytotoxicity. I wonder if the Tcells were compatible with the cancer cells even though they were HLA matched. what additional test should be done to confirm that. What could be the other possible reasons for the observed cytotoxicity?

Hi, guys. I want to use MACS-sorted mouse CD4+ T cells to test the effect of an drug and some directors asked me to culture T cells without IL-2/CD3/28 to exclude the effect of these stimulation. However, I cultured it and find that cells under 16h culture broken into pieces when I am using flow cytometry for analysis. May I know how long (how many hours) can I keep MACS-sorted CD4 T cells without IL-2? I found if the culture time is too long they will die, such as 24h. But how long is enough without killing them? May I know your experience in T cells culture?

I am planning to adoptively transfer 1*10^6 gamma delta T cells, I enrich CD3 T cells with T cell enrichment kit (Stem Cell) and then sort CD3+TCRgd+ cells with FACS. However, the number of gamma delta T cells isolated from spleen is very small (about 2*10^5 at most from a mouse). To obtain sufficient number of cells, I want to expand the cells in vitro, and then transfer them.

Does anyone know any good method to expand gamma delta T cells? Is it possible to expand them with plate-bound anti-CD3 antibody, soluble CD28 and IL-2 as everyone else does for T cell expansion?

I am new to immunology and T cell biology. I know that in vivo T cells have different subsets based on CD44 and CD62 expression, e.g. memory T cells express CD44. How about in vitro cultured T cells? Mice spleen CD3 T cells are isolated by Pan T cell isolation kit (negative selection), and activated for 48 hour via plate-bound CD3/CD28 antibodies (2ug/ml and 1ug/ml, respectively). Then the cells are transferred to uncoated plate and maintained in culture (RPMI medium, with 10% FBS, 30ng/ml IL-2, P/S, 2-BME, non-essential AA). How will the cells differentiate by CD44 and CD62 expression? Thanks a lot.

Typically IL-12 plus anti-IL-4 are needed in Th1 induction from naive T cells and IL-4 plus anti-IFN-gamma for Th2, but what about IL-2? Is it also necessary for T cell differentiation?

Does it work fine? Any recommendations?

Hello

Which T cell line is better for studying Autophagy pathways?

Thanks in advance for your help

Dear all,

I'm interested to perform in vitro of naive OT1 cells to get effector cells (CD44high CD62Llow) before I use them for adoptive transfer into mice. I tried stimulating 2million cells/ml with 1ug/ml N4 peptide for three days but ended up with mainly memory cells (CD44high CD62Lhigh). Does anyone know of any protocol I can use? I can't have IL-2 in the culture though as I'm testing some other substances.

Any advice will be much appreciated, thanks!

Hi For the CD8+ CD28- T cell expansion ( terminally differentiated memory T cells), in addition to the anti-cd3, what cytokines or other substances should i add?How about IL-2, IL-15, T+I or IL-7 ?

Thanks in advance for your advice

Hi all,

I am new to T cell differentiation and have of course been having some difficulty.  

My main problem is that when I differentiate for Th2 Conditions, I see FoXP3 expression . I tried a few different medias; I used regular RPMI with heat inactivated FBS (and other additives), Aim 5 and Ex-Vivo 15.  Regarding cell number, Ex-Vivo 15 seems superior.  I used human cells in round bottom 96 well plates for 5 days at 100,00 cells starting per well.

I used CD4+ cells (CD3/CD28 bead stimulated) and PBMC (soluble CD3/CD28 stimulated).  I used the following cocktails:

1) CD3/CD28, anti IFN-y (1ug/ml) , anti IL-4 (1ug/ml) and TGF-b at 20ng/ml (lower concentration prevent cell death??). I used this for Treg differentiations.

2) CD3/CD28, anti-IFNy (1ug/ml) and IL-4 at 20ng/ml. I used this for Th2 differentiation.

I'm sure my approach requires much optimisation, but I thought I should post for some suggestions from researchers experienced in T cell polarisation. Do you use different media? Do you use additional cytokines/blocking antibodies? Do you use different concentrations? Etc..

Thanks in advance for your help and suggestions

Hi, 

I can't find anywhere what is the T helper profile induced by pOVA(323-339) in DO11.10 splenocytes. I was wondering what is the "starting point" of the cytokine profile using this model to study antigen-specific responses.

Thanks,

Martin

The following is the protocol I used:

Dynabeads Human T expander CD3/CD28 (Invitrogen) at a 1:1 ratio (T cell:bead) in 25 mL X Vivo15 containing 10% FCS, then cultured in the presence of 50 U/mL rhIL-2  and 0.5 ng/ml rhIL-15  keep cell density between 3×105 and 2×106 viable cells/mL, with cytokine

supplementation (final concentration of 50 U/mL rhIL-2 and 0.5 ng/mL rhIL-15) every

Monday, Wednesday and Friday of culture.

The cells expand rapidly during the first week,but after 10 days,they grow very slowly, after 14days,they expand to approximately 20-fold. I have read all kinds of protocols, they can expand the T cells to 100 fold easily within 2weeks, I don’t know where the problem is? Anyone who has the experience about expanding T cells efficiently ? thanks.

Hi,

We do transduction on t cells to express a specific target domain. The plasmid expressing the domain has a selecting marker. After selecting the tranduced cells using a specific isolation kit, these cells undergo a REP with irradaited PBMS. I am just missing why and how these feeder cells work to increase the transduced cells?

Thanks

I want to track the MHC-TCR signaling in resting memory T cells. Which protein in the downstream signaling I should track to differentiate it from the cognate Ag specific MHC-TCR signaling? 

I am keen to use some spare stock of vedolizumab, and stain peripheral blood lymphocytes using vedo as the primary antibody and conjugating it to a secondary antibody (?anti-human). I am hoping that this will identify alpha4beta7 positive cells, but am concerned re the antibody attachment causing internalisation of the intern.

Does anyone have any experience of this?

Can anyone share some insights about

1. activation of T cells using feeder cells, like a protocol. My goal is to use activated t cells against tumor cells (in my project myeloma cells)

2. is it better to use feeder cells than CD3/CD28 cocktail to activate T cells?

3. I am using CAM releasing assay to measure T cell cytotoxicity after activation. Is there any other assay to measure T cell function after activation?

Thanks

I'm trying to make a Treg supression assay, to evaluate de ability of autologous Treg to inhibit the proliferation of Teff after treatment of patients with our product. To differentiate autologous monocytes into APCs would take me one week and I cant wait that long to do the assay.

So, Can I use human CD14+ freshly purified monocytes (without differentiating them) as APC in a Treg supression assay? Can someone help me with this trouble or give me a new idea? In addition, should they be irradiated?

Thanks

I am attempting to stimulate mouse T-cell proliferation in vitro using plate-bound CD3 (10ug/ml, clone 145-2C11) and soluble CD28 (2ug/ml, clone 37.51) antibodies. I can induce some activation as assessed by forward and side scatter parameters, but not to levels I would expect. It turns out that mice of the 129 background (for which my model is on) have an amino acid substitution within CD3e that compromises binding of the 145-2C11 clone. This leads to suboptimal activation. See reference https://www.ncbi.nlm.nih.gov/pubmed/20638133 for the data on this.

Does anyone know of another mouse-specific CD3e antibody that can be used to induce in vitro proliferation of T-cells or perhaps a way around this? Or, does anyone know if any of the human CD3e antibodies (OKT3, UCH1, etc) can induce proliferation of mouse cells? Thanks for any insights you can provide.

I would like some help figuring out if my working concentration of PHA was too much, so that my cells died. I used 2% PHA in an overnight culture with some T cells that have 48hrs before gotten anti-CD3, IL-2, IL-12, anti-IL4 and anti-CD28.

Thanks

I recently did IHC staining of intracranial GBM tumors with anti-CD3 (a marker for T-cells, as I understand) and anti-CD4 (T-helper cells). The tumors are grown in Nu/Nu mice from M.D. Anderson's in-house colony at the Department of Experimental Radiation Oncology.

It was my understanding that nude mice should not have T-cells at all. I am therefore surprised to see that there are, a few, scattered, CD3-positive and CD4-positive cells scattered throughout the tumor. As far as I can tell this is not an artefact...the positive control (immunocompetant BALBc mice) show strong staining of CD3 in hemopoetic compartments. So here is my question: is it typical to see CD3-positive cells in tumors of Nude mice?

As part of an effort to determine whether a certain proliferative response of virtual/innate memory T cells (CD44hi) I tried to knock-out the TCRa of these primary T cells and to culture them with my stimulation of question or with what I thought to be a TCR-independent stimulation, PMA/Ionomycin. I wanted to then, after 96 hours of culture, check for the ratios of TCRb-positive vs. TCRb-negative T cells in both cultures, assuming the PMA/Ionomycin stimulation will, on one hand, keep those primary T cells alive and well during those 96 hours, but on the other hand will get them to proliferate in a completely TCR-independent fashion. This does not seem to work, however, and more than 90% of the cells in that culture die (should I try it without ionomycin maybe?).

My questions are, therefore:

1) What would be a proliferation-inducing stimulus which I can use for T cells and which is practically/completely TCR-independent? As these are innate/virtual memory CD8+ T cells, maybe I should use IL-15/IL-7/both?

2) Do you have any suggestion as to how I shall examine the TCR-specificity of a reaction of innate/virtual memory CD8+ T cells without manipulating MHC presentation (KO of b2m from the immunogenic cells gave ambivalent results, which is the reason I wanted to check the other side of that coin, the TCR-dependency)?

I would really appreciate any effort to help!

Hi,

As my knowledge, TCRb is crucial for the beta-selection in DN (CD25/44) stage on T-cell development. But, my question is how the surface expression of TCRb is going.

As the TCRb expression is key check point for the T-cell survival during DN3-4 stage, latter development such as DP stage T-cells would have TCRb surface expression. But when I stain thymocyte, I found majority of DP cells are TCRb negative. So I am confusing about the TCR expression during thymic development of T-cells. Please somebody answer for my naive question. 

Can anyone help me to know whether CD4 count drops <500 in any other infections or illnesses apart from HIV positive?

I would like to do a co-transfer of B and T cells in mice (colitis model). I have done it once using a 1:1 ratio, injection i.p., and it resulted in no B cells in the intestine when analyzed by flow cytometry.

Unfortunately, there is not much data available in the literature about co-transferring these two cell populations and I wonder if anyone has a good protocol? Route of administration? Cell ratio? Timing of injection (co-injection, separate on different days), etc.

Thank you very much.

Dear college,

Recently we performed ex vivo analysis of IFN-g T cell response in immunized mice. As we did not expect high frequency of T cells to our target antigen and as we wanted to see CD4+ and CD8+ T cell responses we enriched T cell populations with appropriate (CD4 or CD8)T cell separation columns and performed IFN+ Elispots. The purity of separation was 85% and we did not mind that because we reasoned that non-T cells can serve as antigen-presenting cells in our in vitro stimulation. My question is can those non-T cells seriously affect T cell response in a way that that compasion of of the results in immunized and non immunized will be not correct. Will be very greatful for your suggestions.

I am trying to infect mouse primary CD8 T cells using lentivirus. Here is what I did. I activated CD8 T cells using plate bound CD3 (1 ug/ml) and CD28 (5 ug/ml) for 24 hours. Then I tested MOI ( from 1 to 20) in 48 well plate with 10000 cells/well. Cells were cultured for 24 hours with or without spin ( 2500 rpm 90 min 37 C). I checked GFP expression at 72 hours after infection. GFP positive cells are not a separate peak, they look like a tail of the negative cells. The positive cells, I think, are less than 12% . Any one can offer any suggestion in how to improve the efficient of lentivirus infection? I appreciate discussion as well.

Hello,

I am planning to perform a cytotoxic assay with porcine splenocytes as effector cells. I have tried to find the proper concentration of porcine IL-2 that I should use for NKs stimulation but I think there is not a consensus for that. Does anyone have experience with this type of experiments? In this case, what concentration of porcine IL-2 you normally use?

Thanks in advance.

Iker

Is 4 hours too long?

Population is majority CD8 T cells (but not 100% pure CD8 T cells)

I am studying the effect of PD-1 inhibition in GBM cell lines that have mismatch repair mutations

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.

I'm interested in identifying T-cell populations in mouse tumor sections. I can do IHC or IF and I have paraffin or frozen sections.

Hi everyone,

We are performing some intracellular staining for flow with Ag-stimulated (5 days) human PBMCs. We use cytofix/cytoperm for intracellular staining of IFN-gamma (BV711) in CD4 T cells.  We then observe a very high nonspecific binding.  Of note, we don’t see this binding with the same clone conjugated to other fluorochromes. Any idea of how to manage this issue? Thanks a lot!

We are isolating PBMC from equine blood and I have had a number of recent isolations that resulted in low T cell percentages (CD4 = 20% of lymphocytes). 

We use fresh blood collected into ACD vacutainers, allowed to settle for 30 min at room temperature or 4*C if not processed within an hour. The LRPR layer is separated, spun, washed, then layered over Ficoll Paque Plus. If the aqueous layer is pink after the Ficoll spin (consistent with hemolysis of the few RBC carried over), then the T cell component will be markedly reduced. This is confirmed by flow cytometry with CD4, CD8, CD14, and CD21 (B cell) markers. Our wash solution is DAB with 1% FBS. The reagents have not changed between the first 10 times I ran the protocol with no problem and the last 4 times I ran it with apparent lysis. The flow antibodies have not changed either. I have gotten the same lysis results on 5 different horses and they appear to be completely proportional among horses tested on the same day. Some horses with lysis have had previously normal samples. All horses were healthy. 

Hi All,

So I need to create a mouse line where I can use the DTR system to ablate CD4+ T cells. I was able to find a CD4-cre line, an lck-cre line, and a line of mice with the DTR gene downstream of a stop codon flanked by 2 loxP sites under the control of a ubiquitous promoter. My understanding is if I were to cross the CD4-cre line with the floxed DTR line, this would produce a line with the DTR gene under the  control of the CD4 promoter. I could then add DT to ablate CD4-DTR cells. However, this approach would not just ablate CD4 T cells but other CD4 expressing cells (monocytes, macrophages, DCs). If I want to just ablate CD4+ T cells, while leaving non T cells intact, how do I go about this? is there a line of CD4 promoter driven floxed DTR mice that I was just unable to find while searching online? 

Thanks so much!

I currently isolate human T-cells from various tissues (blood, intestine, liver), and have a cell surface staining protocol which involve blocking FcR blocker. As I do not analyse B-cells, or other cells which have FcR on them, is there any point in my blocking them before I stain with antibody? 

I do not purify the cell suspension for T-cells beforehand, so there are monocytes/B-cells present, so I wonder whether this just means I block the FcR on them, meaning any antibody I use is able to be used directly not he antigen of interest on the T-cells etc, rather than being used up by non-specific FcR binding on B-cells etc? Is this the reason?

Also, I use a commercial FcR blocking reagent - is the just human AB serum usually? And what is it in the human sera that blocks the FcR? Is it just antibodies?

Thank you.

Immunecheckpoint inhibitors such as Pembrolizumab, nivolumab (anti-PD-L1 agents) and Ipilimumab (anti-CTLA4 antibody) can cause myocarditis as the side effect. The same T cell clone population is expected to exist both in the tumor and the heart tissue which responds and exhibits the proliferation upon the exposure to the similar epitope. However, why other organs are spared and the heart tissue tend to harbor such epitope as to cause myocarditis? 

Hi everyone.

I am using Invitrogen CTS CD3/CD28 Dynabeads to simultaneously activate and isolate T cells from cryopreserved PBMCs. I thawed PBMCs in AIM-V medium, viability was 80% by trypan blue staining. During incubation with Dynabeads (RT, 1hr, with rocking) a big clump formed which did not dissociate upon pipetting. I then proceeded to removal of unbound cells and cultured the bound (presumably CD3+CD28+ T cells) cells in IL-2 supplemented medium. The big clump remained when I checked my cells the next day. Recovery of live cells was pretty poor and I suspect most of the cells either died/got trapped within the big clump.

Is this normal for cyropreserved PBMCs?

I will study calcium flux in T cells using fluorescence, so I`ll need cells to stick to a plastic 96-well plate, without any spontaneous triggering of calcium currents.

Which substratum is best for T cell adhesion in this case?

.

From what I've read there are 3 hypotheses to characterize Th17 cells:

1) Incubate PBMC with PMA and ionomicine in a tissue incubator, then surface staining for CD3 and CD8 and intracellular staining with IL-17. (Yang et al. 2009)

2) Expression of chemokine receptors - CD4+ CCR4+ CXCR3- CCR6+ (Clark et al. 2011)

3) Quantify cytokines, namely IL-17, in peripheral plasma/serum and relate that with alterations in Th17 population, through ELISA or Multiplex-bead analysis.

If I harvest a spleen from a WT C57BL/6 mouse and use CD4+ T cell isolation kit from MACS Miltenyi kit, what is the ideal number of T cells I should get?

Does there is any drugs that can specific kill T cells without harm NK cells?

Dear all,

I am now looking for the sequence of Ras-binding domain (RBD) to construct vector using for detection of Ras activation in mouse CD8 T cells. Would you please inform me some references about it? 

thank you very much.

P/S: I found some commercial Ras activation kits, however they are quite costly.

It is well known that cells that are Propidium Iodide (PI) -ve and annexin V (AV) -ve are alive cells; PI-ve AV +ve are early apoptotic cells; PI+ve AV+ve late apoptotic cells and PI+ve AV-ve are damaged cells (necrotic cells). So, does this apply for the staining with LIVE/DEAD and AV?

Attached is an example of staining with LIVE/DEAD and AV.

I would like to address whether my genetically engineered human AD293 cells are more or less attacked by T-cells. Is there a human T-cell line that can be used for a cytolysis assay?

I’m struggling with a cellular assay using human PD-L1 protein to inhibit human T cell activation through the inhibition of IFN gamma release. 

I stimulated human total T cells with anti-CD3/28 (100ng/ml) followed by treating cells with human PD-L1 (10ug/ml ) for 72h, then measured the IFN gamma by ELISA.   The signal of IFN gamma from stimulation samples is decent, but PD-L1 did not show the significant reduction of the cytokine production. What factors could cause the problem?

I’m developing a cellular assay to screen and identify small molecule inhibitors of PD-L1.  Are there any alternative cellular assays available for the purpose?

Hi there! When culturing T hybridoma cells I am often faced with the challenge that the colony starts downregulating the T cell receptor or even stops expressing it at all after a while. Can anyone recommend a medium supplement or another technique to prevent this phenomenon? Thanks in advance! 

We are working with primary human T cells and are searching for a good assay to look at their lytic function. The chromium release assay is an option, but we're looking to avoid radioactivity where possible. 

Hi. I have to use tat-Cre on islated T cells. Does anybody know whetehr decraesing the temperature of incubation with a given concentration of tat-Cre in vitro from 37 to RT decreases the toxicity?

In my experiments, I have observed that stimulation of thawed PBMCs from young healthy donors with PMA/ionomycin for 6 hours leads to an increase in naive (CD27+ CD45RA+) T cell subset and decrease in the other 3 T cell subsets (CM,EM,TEMRA).

However, from my knowledge, I understand that T cell stimulation should activate naive T cells and probably drive their differentiation into the memory subsets.

Hence, I would like to check is there everyone else who also made similar observations to mine? Or is there a mistake in my understanding of T cell stimulation, as I am pretty new to the field of immunology...

Will replacing CD45RA with CD45RO make a huge difference to the results, as I have heard that CD45RA expression is increased with PMA/ionomycin stimulation. 

Thank you in advance for any advice given!! 

I want to positively select NKG2D+ NKT CELLS

I need to titrate mouse CD107a antibody to be used for my future CD8+ T cell degranulation assays. After checking the literature, it is recommended to stimulate the cells with some antigen and add the CD107a antibody to the culture at the beginning of the stimulation. So, the strategy I will follow is :

1. Stimulate the CD107a stained splenocytes using anti-CD3/CD28 (plate bound) for total 5 hrs.

2. Add 1uM Monensin to the cultured cells for the last four hrs of incubation.

3. Harvest cells, wash and surface stain with anti-CD3 and anti-CD8 antibodies.

4. Fix and permeabilize the cells and acquire by Flow cytometer.

I was wondering if this is the right protocol to follow?

Also, is it fine to stain the splenocytes with anti-CD107a in complete RPMI medium as you need to stain the cells with this antibody in the beginning of the culture stimulation?

Is it necessary to fix and permeable the cells for checking the expression of CD107a antibody?

Please suggest.

Hi,

I am ex vivo activating CD8 T cells for an adoptive cell therapy model. I would like to freeze down batches of activated cells to use at later timepoints and in other assays. How well do the activated cells respond after freeze/thawing and how long should they be cultured after thawing before I look for cytokine production?

Thanks,

I am debating whether I should use BrdU or CFSE to measure T-cell proliferation in vivo after stimulation with superantigen SEB.  BrdU is appealing because you don't have to stain the cells ex vivo but I am concerned that BrdU is not sensitive enough.  Any strong preferences?

I need to find two cell line that expresses CD4 alone or in combination with CD28.

Many thanks to all

Francesco

I'm new to T cell culture and would like to know how people generally culture antigen specific T cells. My plan is to isolate PBMCs from donor derived peripheral blood. Add specific peptides to activate the PBMC. After culturing for like 10 days (I'm not sure how long I should be culturing the T cells. This is a number that I saw most of the times in literature), do a flow cytometry to isolate the antigen specifc T cells I want. That is the plan and I would like to know as much as possible about the details in T cell culturing. If you could provide a protocol, that would be AWESOME. Thank you very much in advance.

We are currently using the FASCIA method for in vitro stimulation of whole blood (ref: Marits P. et al. Clin Immunol PubMed ID 24909732). We use PHA, PWM and ConA for stimulation but I don't really understand where the differences in responses come from with these mitogens. Some times the same donor has a perfect response to PHA but only a moderate response to ConA. Does anyone happen to know why?

We'd like to have a stimulant that would be TCR specific but the CD3-CD28-antibody stimulation does not work in this setting since the incubation time is very long and the cells downregulate CD3 making it impossible to gate on them in flow cytometry analysis. Any recommendations for CD3-CD28 replacement that would still be TCR-specific and give robust responses?

All input is greatly appreciated!

I am trying to detect stimulated Tregs and their IL-10 expression in cultured PBMCs (not isolated from the cells mixture) by flow cytometry. For other T cells phenotyping and evaluating of cytokine production, I usually stimulate PBMCs with a-CD3/a-CD28 for 5 days, then re-stimulate with PMA/I+ Brefeldin A in the last 5h, and then stain for surface markers and intracellular cytokines. Is this applicable to Tregs as well? and are a-CD3/a-CD28+ PMA/I good Tregs stimulants (knowing that PMA may alter CD4, CD25?, and foxp3? expression)? 

I am setting up an experiment where I use magnetic beads to positively select for CD4+ T cells from a C57BL/6 mouse (with a tumour, either untreated or with treatment) spleen, followed by expanding the population. I then want to do a direct coculture with a cancer cell line, where I will conduct an ELISA on the supernatant to assess cytokine production (IL-12 etc.). My question is, by expanding the CD4+ T cells ex vivo (using antiCD3/antiCD28 and IL-2), would I be selectively expanding a specific subset (Th1 over Tregs for example), thereby skewing my results? If so, how might I go about avoiding that?

If CD8 T cells have been activated in vivo, would in vitro stimulation with an agonistic anti-CD3 antibody alone (no agonistic anti-CD28 stimulation) cause degranulation (stainable CD107a protein) during a 4-6 hour co-incubation?