Science topic

T Lymphocytes - Science topic

T Cells are primary agents in immunology and call mediated immunity. Helper, Cytotoxic, Memory, Regulatory, Natural Killer and gamma delta T Cells, their development, mechanism and application
Questions related to T Lymphocytes
  • asked a question related to T Lymphocytes
Question
1 answer
Dear, In my experimental model I will administer a substance intraperitoneally and some time later I need to collect the T lymphocytes from the spleen for phenotypic characterization in order to evaluate the host's adaptive immune response. I need to know: i) on average, how long after administering the substance I should collect the spleen; ii) how to isolate T cells from spleen and, finally, which T lymphocyte activation surface markers I should use (in addition to CD8; CD4 and CD69).
Relevant answer
Answer
Hey!
I am not sure about how long you would need to wait after the injection but in general you can use CD3 dyna beads to isolate T cells. Then stain for CD4 and CD8 to analyze separate populations. For activation you can use CD25 or CD69 antibody.
Hope this helps!!
  • asked a question related to T Lymphocytes
Question
1 answer
We are aware that T cells can specialize into various memory cell types, such as stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and resident memory (Trm) subsets. However, the intracellular signaling regulation involved in the differentiation of these subsets appears to be less understood. Are there any evidence indicating important signaling pathways or key factors that might influence or determine the generation of specific memory cell subsets?
Relevant answer
Answer
Please consider the the hematopoietic defects (in mice & humans) associated with loss of function mutations in Mysm1 and Slfn2 that impair quiescence in the CD8 T cell compartment that do result in activation phenotypes as defined CD62L, CD44, CD69 etc.
For MYSM1 related papers consider:
And for SLFN2 please see:
I hope that helps.
All the best & kind regards,
Michael
  • asked a question related to T Lymphocytes
Question
4 answers
I am currently performing co-culture experiments with murine CD8 T cells isolated from mouse spleens and with skin cancer cells (PDV) in a 24-well plate. I first isolate the CD8 T cells from spleen which are around 70% alive at time of isolation, mix them together with CD3/CD28 T Cell activation beads to activate the CD8 T cells, and plate them 2x10^5 T cells on top of 2x10^5 skin cancer cells that were plated the night before. The cancer cells are treated with mitomycin C prior to plating the T cells in order to halt cancer cell proliferation. Then I let them incubate in a tissue culture incubator with standard parameters (37 degrees Celsius and 5% CO2) for 5 days. In addition to the wells where I co-culture the T cells with the plate cancer cells, I also plate some of the T cells with the activation beads in an empty well to act as a control. I then assess the viability of the T cells by staining with a live dead stain and running flow cytometry. However the results of the flow cytometry show show that most of my T cells are dead (<40% viability). The most problematic issue is that the T cell-only wells come up only 1% viability which render the rest of the experiment null since I would not have a T cell control to compare the T cell viability from the T cell/cancer cell co-culture wells.
The interesting observation is that the T cells co-cultured with cancer cells have greater viability compared to the control T cells cultured by themselves which makes me think that the cancer cells are stimulating the T cells and improving T cell viability that way. I was wondering if there are any adjustments I could make to improve the viability of my T cells especially for the T cells in the T cell only control wells?
Some adjustments I have made already:
1. Increase the speed of the T cell isolation from the spleen to improve baseline viability prior to seeding on the cancer cells
2. Utilize wide bore tips when pipetting T cells to decrease the shear force on the T cells
Thank you for any suggestions and apologies for the length of this question.
Relevant answer
Answer
Dear Matthew,
Your work seems interesting. Which type of cancer are you investigating? The results can be dependant on that too. What are the goals of your study?
Sincerely,
Amar, MSc in MLT, Researcher interested in breast cancer
  • asked a question related to T Lymphocytes
Question
5 answers
Usually I culture mouse T cell with RPMI media and the mouse EC cell lines(C166---adherent cells) always culture with DMEM. I want to establish a in-vitro transendothelial migration model that mouse T cells migrate through C166 monolayer. So I want to know if DMEM medium affect mouse T cells activity and function. Could I use DMEM medium to culture mouse T cells in order to adapt the DMEM culture environment .
Relevant answer
Answer
One thing that I can tell is that you can´t cultivate EC with RPMI. I did it in the co-culture and lost my cells.
  • asked a question related to T Lymphocytes
Question
1 answer
We are planning to check the level of phosphorylation of NF-KB in the CD4+ T cell population after activation with anti-CD3/CD28 and treatment for 15 minutes. However, I constantly see two fixed bands with MW around 65 and 25 KDa which might come from serum proteins but when we removed FBS from the media, they are still there. Since we're looking for p-NF-KB in 65 kDa size, how can I fix this issue?
Relevant answer
Answer
@Mehri,
The 25 KDa might be NFKB isoform due to alternative splicing. If you do not want the 25 KDa isoform, you should look for methods to prevent this alternative splicing.
  • asked a question related to T Lymphocytes
Question
1 answer
I work in a project where we need to knockout a gene at primary T cells targeting an exon that is shared between all variants. However, while the sgRNA design has been optimised ,we still see low KO efficiency. (Note: we are using RNP method and electroporation)
Relevant answer
Answer
The information above is not suffcient for trouble shooting. You may need a positive control to validate your protocol first.
  • asked a question related to T Lymphocytes
Question
1 answer
The untransduced T cells are produced by mock lentiviral transduction of human primary CD4+CD8+ T cells. These cells are subjected to comparable manipulations as CAR-T cells: activation, spinoculation (without lentivirus), and expansion. These T cells are meant to be negative controls in experiments using lentivirus-transduced primary CAR-T cells.
Why not to use Scramble or a vector without a construct? Why UTDs are used?
Relevant answer
Answer
Actually, since viral transduction may affect T cell function, you must always use an empty vector for the transduction of T cells and use it as negative control along with non-transduced T cells, and compare the results.
  • asked a question related to T Lymphocytes
Question
2 answers
I'm currently working with human primary T cells, isolated from whole blood that I get from my university's blood bank, and I'm trying to determine if my experimental treatment can enhance cytotoxicity. I'd like to run some type of in vitro killing assay but am struggling to find a good target cell for the cells that I work with.
In general, I use cells that are not CMV-positive. I've seen lots of literature using T cells from OVA mice and having the target cells pulsed with OVA peptide, but something like this isn't feasible with the cells that I have access to. Does anyone know if it's possible to run a killing assay of this type with run-of-the-mill human T cells? Is there a good target cell line that any human T cell with kill?
Thanks in advance!
Relevant answer
Answer
Check the old publications of Antonio Lanzavecchia and Federica Sallusto. They typically used tetanus antigen to check cytotoxic responses of human primary T cells (many people have somewhere in their live received a tetanus vaccine). You would need to use the B cells or moniocyte-derived dendritic cells from the donors to get the appropriate target cells (having the HLA antigens that match the restriction of the T cells). Again the work of Lanzavecchia provides protocols (usually GM-CSF +/- IL4 and optionally maturation with LPS).
  • asked a question related to T Lymphocytes
Question
2 answers
Does anyone have a protocol for measuring T lymphocyte activation, either by flow cytometry or by measuring cytokine production? I'm working with CAR-T lymphocytes and I want to see if incubating them with patient sera will activate them in a specific manner. Can you suggest different cytokines and activation markers?
Relevant answer
Answer
I briefly wanted to reach out concerning your inquiry.
For a detailed list of flow cytometry agents for the characterization of activation markers on the cell surface please consider the following paper from my old boss.
For quick insights into activation markers for cytotoxic T cells and a basic flow cytometry protocol, please consider:
For a comprehensive cytokine analysis (48plex with absolute quantification) use:
And for a detailed proteomic analysis (using cell culture supernatants or serum (prior to incubation)) please consider the T96 INF, IR and IO panels (albeit with relative quantification of expression) as per:
and more specifically used in the following papers:
I hope that helps.
All the best & good luck with your experiments,
Michael
  • asked a question related to T Lymphocytes
Question
1 answer
Hi- I would like to transiently deplete CD8 T-cells in vivo to allows allografts to establish, but am testing T-cell activation as a therapeutic modality, so need them to come back with appropriate timing. Does anyone have experience with re-population kinetics of T-cells after in vivo administration of mouse anti-CD8 mAb clone 53-6.7?
Relevant answer
Answer
Emily Girard do you find a solution? I'm looking for this also...
  • asked a question related to T Lymphocytes
Question
2 answers
I have attempted various transfection reagents including Lipo3000/Lipo2000/Hieff Trans™ Liposomal Transfection Reagent, both GAPDH positive control and custom siRNA targeting gene of interest at different concentrations (20nM, 40nM, 60nM) in a 24-well plate, FAM-NC, and time points (24h and 48h post-transfection) for numerous trials. However, none of these approaches yielded the expected results. Could it be that non-electroporation methods are not effective for Jurkat T cells? Or does anyone have a more effective and unique experimental protocol? I'm hoping to receive your guidance. Thank you.
Relevant answer
Answer
Yang 明明 您说的或是很重要的原因之一(印象中,siRNA的转染在一定浓度下可能不会触发该通路,当然也可能是我的认知有限),对于免疫或悬浮细胞,正如你说的电转、病毒转染是优选的方案。不过,电转对于经济实力不强的实验室,其成本是较为高昂的。
  • asked a question related to T Lymphocytes
Question
2 answers
I am trying to expand TH17 cells from Naive T cells for the first time. Can you suggest the best expansion kit available in the market to do the same?
Relevant answer
Answer
IL6, IL21, IL23, and TGF-β are the major signaling cytokines involved in Th17 cells differentiation, and retinoic acid receptor-related orphan receptor gamma-T (RORγt) is the master regulator. The differentiation process can be split into 3 stages,
The differentiation stage mediated by TGF-β and IL6,
The self-amplification stage mediated by IL21, and
The stabilization stage mediated by IL23.
If you are interested in mouse, then you may use the kit attached below. The Flow Cellect Mouse Th17 Differentiation Tool Kit Catalog No. FCIM025163 from Millipore.
For human, RnD systems used to provide the Human Th17 Cell Differentiation Kit which has been discontinued. See below.
Nevertheless, you could follow the protocol provided below for human Th17 cell differentiation.
1. You may purify naive CD4+CD45RA+ T cells from PBMCs using the Naive T Cell Isolation Kit (human).
2. You may stimulate the cells by using the T Cell Activation/ Expansion Kit, (human) which is based on MACSi bead particles loaded with the required antibodies.
3. You may culture the cells in the presence of the Th17-polarizing cytokines, like IL-1β (20 ng/mL), IL-6 (30 ng/mL), IL-23 (30 ng/mL), and TGF-β1 (2.25 ng/mL), in addition to anti-IFN-γ (1 µg/mL) and anti-IL-4 (2.5 µg/mL) antibodies. Culture the cells for 7 days at 37 °C in an atmosphere of 5% CO2, without any media exchange.
4. You may collect the cell culture supernatants, filter, and measure the content of IFN-γ, IL-4, IL-17, IL-22, by ELISA method.
For detailed protocol you may want to refer to the link below.
Best.
  • asked a question related to T Lymphocytes
Question
1 answer
In mouse SI-lamina propria T lymphocytes stimulation with PMA (20ng/mL) /Iono(1ug/mL) is not strongly inducing IFN-g after 6hrs. The previous results in our lab had 4-5% and I could have just 2% max, though the protocol and antibodies to stain everything is followed in same way! any suggestions or comments would be greatly appreciated.
Bref A (1ug/mL) after 2hrs of PMA7Iono.
Relevant answer
Answer
Good morning,
I'm having the same problem, did you get any idea on how could we enhace cytokine's production please ?
Thank you
  • asked a question related to T Lymphocytes
Question
5 answers
I want to deplete CD3 T cell from PBMC, how can I do that without magnetic bead separation?
Relevant answer
Answer
Hello,
High levels of anti-CD3 will likely just tolerize the T cells, not deplete them.
I was going to suggest FACS cell sorting but an alternative you could use is the old standby; stain the cells with anti-CD3 antibody of an isotype that fixes complement then expose the cell population to reconstituted rabbit complement which will lyse the vast majority of the CD3 expressing T cells.
However, you should do FACS analysis at least once to see that both your antibody and source of complement are functional and that the CD3+ve cells are truly depleted.
  • asked a question related to T Lymphocytes
Question
3 answers
Hello all,
I'm looking to measure Ag presentation to T cells on a B cell line HLA. I was hoping that I would be able to conjugate my Ag to a protein tag (FLAG, GFP, etc) and get a functional T cell read out as a proxy for presentation of my Ag. But as far as I can tell there isn't a T cell line with a TCR that is specific for anything like that.
Does anyone have any thoughts or know of any cell lines? I'm working with HIV so an Ag specific T cell line isn't really possible.
Thanks!
Relevant answer
Answer
One such line is the T cell hybridoma line B3Z. This line is specific for the chicken ovalbumin peptide SIINFEKL presented by H-2Kb.
Another line is the DO11.10 T cell hybridoma which is specific for the ovalbumin peptide OVA323-339 presented by I-A[b].
  • asked a question related to T Lymphocytes
Question
4 answers
I've been trying to activate and expand naive T cells that have previously been isolated from PBMCs with a pan-T cell isolation kit from Miltenyi and cryopreserved in 90% FBS 10% DMSO. I have decent viability post thaw >85% with ~10% loss in viability from the pre-freeze viability. However, over the 10 days that i'm expanding the T cells, I initially see good activation, but then the T cells start dying off and I end up with abysmal viability by day 10.
Here's the protocol i'm using:
I rapidly thaw the vial of T cells in the water bath and when the vial is almost completely thawed, add 1mL T cell media dropwise. I transfer the full volume to a 15mL tube with more T cell media dropwise, then spin at 300xg for 5 minutes. I activate the T cells in Miltenyi TexMACS medium supplemented with 300 IU/mL IL2 and CD3/CD38 Dynabeads at a 1:1 T cell:bead ratio. I plate the cells at 2 million/mL in a 24 well plate for the initial activation. I expand the T cells for 10 days, checking the viability every 2 days and replenishing media/cytokine if the media starts looking yellow, keeping T cells between 1.5-2 million/mL.
This protocol has worked very well for me from T cells isolated from freshly prepared PBMCs, but hasn't been working well with the cryopreserved T cells. I initially see very good clumps forming that get larger, but towards the second half of the 10 days the clumps don't get very large and the T cells start dying. Over 10 days I had less than a 2-fold expansion, whereas with fresh T cells I could get a 4-5 fold expansion over 10 days.
Does anyone have experience activating and expanding thawed naive T cells or have thoughts on why my T cells may be dying? Whats weird is that I've used the exact same vial of frozen naive T cells to generate CART cells using a 3 day Dynabead activation with IL7 & IL15 followed by viral spinoculation, and the viability of these CART cells is fine after a few days of expansion after spinoculation.
Relevant answer
Answer
Use monoclonal anti-CD3 antibodies and anti-CD28 antibodies to provide a co-stimulatory signal that engages the TCR which can be used for antigen-induced activation.
  • asked a question related to T Lymphocytes
Question
1 answer
Hi! Could someone help me to solve this problem?
I will coat the 6-well plate with anti-CD3 first. Then, add anti-CD28 into T cell culture and remove T cell culture into 6-well plate. However, Jurkat cells always form clumps. Do I need to split the T cells before adding anti-CD28 into individual cells? What should I do? Centrifuge the cell culture?
Besides, I will then test the CD69 and CD25 expressed on activated T cells by flow cytometry. In this test, I will add anti-CD69-FITC and anti-CD25-PE to the cell culture. Do I also need to centrifuge the T cells to split them from clumps?
Thank you in advance!
Relevant answer
Answer
I briefly wanted to reach out concerning your inquiry. Firstly, Jurkat T cells clump a lot (especially when re-stimulated). To that end I would suggest to spin them and gently resuspend them by pipetting up and down to get a single cell suspension.
Follow the extracellular staining protocol outlined here:
Please stain for live/ dead cells (in PBS) and then for your extracellular antibodies in FACS buffer (i.e. PBS + 0.5% BSA + 0.1 % NaAzide (optional for storage).
For a very basic protocol please refer to:
I hope that helps.
All the best & good luck with your experiments,
Michael
  • asked a question related to T Lymphocytes
Question
3 answers
A reviewer wants me to generate CD4Cre CD19Cre cKO mice to complement the individual Cre data in an initial submission. I think that breeding and genotyping here could be a long and painful process. Does anyone know of a Cre driver that is 'pan'-lymphocyte?
Many Thanks in advance
Relevant answer
Answer
Maybe the Vav-iCre strain (originally from the Kioussis lab) may help, pending on your experimental set-up.
For a characterization (of Cre expression) please see:
All the best & good luck with your experiment,
Michael
  • asked a question related to T Lymphocytes
Question
4 answers
Activation of naive T cells generally requires antigen to be presented by dendritic cells in lymph nodes. Activation of naive B cells generally requires opsonized antigen to be displayed by follicular dendritic cells in lymph nodes.
Thus, it seems that dendritic cells and complement (innate immune system) need to recognize a pathogen before an adaptive immune response can be initiated. Is this always the case?
Relevant answer
Answer
Activation of naive T cells generally requires antigens to be presented by dendritic cells in lymph nodes. Unlike the cells of the innate immune system, T cells and B cells can identify specific features of pathogens. Therefore, B or T cells can be raised against pathogens not detected by the innate immune system.
  • asked a question related to T Lymphocytes
Question
1 answer
The isolated T cells will be further activated and expanded in vitro for transduction (with chimeric antigen receptors). The ex vivo culturing will take about 2-3 weeks.
I know that for isolating rare cell populations (I.e. pDCs) it is recommended to pool spleens together to obtain sufficient numbers but not sure if the same can be done for mouse T cells. Would there be any concerns of cross-reactivity despite the cells coming from the same background (C57Bl6 in our case) only different individuals?
Relevant answer
Answer
When isolating mouse T cells, it is generally recommended to use spleens from individual mice rather than pooling them together. This is because T cell populations can vary among individuals, even within the same strain, due to genetic and environmental factors. Pooling spleens from different mice may introduce variability in the T cell populations, including differences in T cell receptor repertoires and activation states.
To obtain sufficient numbers of T cells for downstream applications, you can isolate T cells from multiple individual spleens separately and then combine the isolated T cells together after purification. This approach ensures that the T cell populations remain representative of each individual mouse, minimizing the potential for cross-reactivity or other unwanted effects.
By isolating T cells from individual spleens, you can also control the quality and purity of the T cell population, ensuring consistency in your experiments. Additionally, if any issues arise during the ex vivo culturing process, it allows for easier troubleshooting and identification of potential sources of variability.
In summary, it is generally recommended to isolate T cells from individual mouse spleens rather than pooling them together to maintain consistency and minimize potential issues related to cross-reactivity or variability in T cell populations.
  • asked a question related to T Lymphocytes
Question
3 answers
How many naïve T cells we can get from a C57BL/6J mouse spleen and lymph nodes? or in other word, what percentage of the cells from spleen and lymph nodes are naïve T cells?
Relevant answer
Answer
If you want to have an idea for absolute numbers and distribution, please check our paper on T cell development
We used C57BL/6J littermate controls in our experiments. Moreover, a FACS staining for CD62L and CD44 in the CD4/ CD8 compartment, with CD44 low CD62L high expressing T cells will be naive.
Please check Fig. 3 of the paper for more details.
All the best & kind regards,
Michael
  • asked a question related to T Lymphocytes
Question
4 answers
I am doing multiplex immunofluorescence assay on FFPE section. I used CD3 to identify T cells and observed that some of T cells stained with CD3 showed nuclear staining instead of membranous. I am not sure why the staining pattern is nuclear for CD3? is it possible to see nuclear staining with CD3?
Relevant answer
Answer
This depends on the type of cancer you are investigating and the tissue from which the FFPE sections are made. As mentioned, e.g. T-cell lymphomas show cytoplasmic CD3 staining.
Try to figure out in which tissue/cell type you see the cytoplasmic CD3 staining.
  • asked a question related to T Lymphocytes
Question
2 answers
Why is tetramer only used to test the interaction between T cell and peptide-MHC complex and not to test binding affinity between peptide and MHC?
Relevant answer
Answer
Major histocompatibility complex (MHC) antigens bind peptides of diverse sequences with high affinity. Both MHC class I and MHC class II molecules contain peptide-binding grooves formed by two α-helices and eight β-strands. In the peptide-binding groove, specific amino acids compose pockets that accommodate the corresponding side chains of the anchor residues of the presented peptides. Peptide-binding preferences exist among different alleles of both of MHC I and MHC II molecules, which are mainly dependent on amino acid polymorphisms in the peptide-binding grooves of MHC chains. They do this in order to generate maximal immunological protection by covering the spectrum of peptides that may be seen by a host over the course of its lifetime.
On the other hand, TCR (T cell receptor) has a low avidity and fast off-rates for MHC-peptide complexes. So, MHC-peptide tetrameric complexes (so-called MHC tetramers) have been introduced for the detection of antigen-specific T cells. MHC tetramers have increased avidity for their cognate TCRs and are successfully used to directly visualize antigen-specific T cells ex vivo. MHC tetramer technology is based on the ability of MHC-peptide complexes to recognize the antigen-specific T cells at a single cell level.
Hope this information is helpful!
Best.
  • asked a question related to T Lymphocytes
Question
5 answers
Hello, I isolate PMBCs from blood using Ficoll and plate the cells in a 96-well plate for different treatments for a few hours before T cell activation. At the end of the treatments I centrifuge the plate, discard media and use new media to pipette the cells several times and transfer to an anti CD3 coated 96 well plate for 3 days, new media is also supplemented with anti CD28 ab and IL2.
I can't seem to get the cells into the new plate and rarely get any cells in the plate after the 3 day incubation. I was under the impression that these cells detach easily with pipetting. Any suggestions?
Relevant answer
Answer
Kerry S Vistisen I tried to lower the g and doubled the amount I seeded, and it now looks like I was able to transfer a good amount. Thanks!
  • asked a question related to T Lymphocytes
Question
2 answers
First, we coated 96 well TC-treated plates with 5ug/ml anti-CD3 antibody (17A2, biolegend)
Then, we added Naive CD4+ CD25- CD45RB high T cells (500000) in each well after sorting through flow cytometry. Next, we used anti-CD28 antibody (soluble 2ug/ml, biolegend). Again, different cytokines such as 5ng/ml TGF-β, 20ng/ml IL-6, 10ug/ml anti-IL-4, 10ug/ml anti-IFN-gamma were added in each well. After 5 days, when we detecetd TH17 cells after, we found low ratio of IL17+ cells (1%-2%) and most of cells were dead (>80%). This is the main problem. Thats why we want to know which can cause this problem.
Relevant answer
Answer
Philippe August Robert Thank you sir for your kind response and suggestion
  • asked a question related to T Lymphocytes
Question
1 answer
They don't take up any dyes, and they don't activate when treated with IL-2. I've confirmed it multiple times that these cells are CD4+ T cells by immunophenotyping (FACS). So why won't these cells take up any dyes? Or lenti? Why do they look like opaque black spots under IF?
Argh!
Relevant answer
Answer
Dead cells?
  • asked a question related to T Lymphocytes
Question
2 answers
Hi,
Does anybody know whether effector or memory T cells can de-differentiate into naive T cells, rather than just become exhausted or undergo apoptosis? Thanks
Relevant answer
Answer
Thanks Malcolm. Do you hapoen to know if memory T cells can ever fully revert back to naive T cells?
  • asked a question related to T Lymphocytes
Question
3 answers
Recently I’ve started seeing these clumps in my primary MSC flask (T175). I first noticed these compact clumps in a flask near 80% confluence but I’m also seeing it in less confluent flasks. The clumps vary in size. The medium is neither cloudy nor changing color. I’m using a basic growth media (DMEM, 10% FBS, and 1% antibiotic-antimycotic). When passaging I dissociate the cells using 0.25% trypsi-EDTA for 5 minutes. I’ve been washing the flasks with PBS + 3% antibiotic-antimycotic at media changes, but have not seen any change. Does this look like fungal contamination or could it be cellular contamination (for example, T cells or pluripotent cells clumping) or cellular debris? Whatever it is, it appears to sit a a level above the live, attached cells (with other dead cells).
Thank you!
Relevant answer
Answer
Thanks for the information. Since the cells are from passage 7-8 then I would say the clumps are definitely cell debris. I typically do not use MSCs after passage 6. Past passage 6 the cell morphology looks suspect, their growth slows down and you start to see a lot of floating cell debris compared to earlier passages. I just toss the flasks and break out new vials from LN2.
  • asked a question related to T Lymphocytes
Question
3 answers
I isolated CD3+ Cells from frozen PBMCs and then I seeded them in a 96-well plate in different conditions with some independent variables.
In this experiment, I need to incubate the plate for 7 days and then I do FACS analysis.
The problem is that after 1 week I lost around 70-80% of cells in each condition.
What should I do to lose a low number of cells during the incubation?
Relevant answer
Answer
Firstly, I isolate the CD3+ cells from PBMCs. Then I seed 200.000 cells/well.
In baseline condition only I have IL-2 and cell medium.
In the second and third condition I have , anti-CD8 and IL-2 and my independent variable.
Note: Before seeding I coat the plate (the wells that I need the activation of T cells) with 5 µg/ml anti-CD3 in PBS.
  • asked a question related to T Lymphocytes
Question
2 answers
Is it possible to transduce a human CAR into mouse T cells and have the mouse T cells function when the CAR binds it's antigen? I am thinking that if anything the singling domain of the CAR, as it is human, may be a problem for the mouse T cells.
Relevant answer
Answer
Thank you!
  • asked a question related to T Lymphocytes
Question
1 answer
Hello,
I have been trying to do a lentiviral transduction of mouse primary CD4+ T cells with little luck. We do not see any expression and cells begin to die out.
Current protocol:
Isolate CD4+ T cells, transduce on day 7 with spinoculation (1000G, 1hr)
Thank you!
Relevant answer
Answer
Hi Meisha,
Have you checked whether the cells survive and are sufficiently activated? Just wondering if you have an untransduced control that also starts to die out around the same time (in which case the problem can be with ex vivo culture).
@
  • asked a question related to T Lymphocytes
Question
3 answers
Hello all,
I could really use some advice with T cell intracellular cytokine staining. I've been trying to stimulate CD4+ T cells isolated from mouse spleens and stain intracellularly for cytokines, including IL-2, IFN-gamma, and IL-17A. However, I see very poor cytokine staining in my stimulated T cells, even IL-2 staining. I expected that most of my stimulated T cells would be positive for IL-2 intracellular staining, but only about 3 percent of my T cell population are positive for IL-2-PE when I run my samples on our flow cytometer. I see clear activation of my T cells not treated with Brefeldin A based on increased percentages of T cells positive for CD69 post stimulation, so I doubt that stimulation is the problem. If anyone has tips or tricks on optimizing T cell intracellular staining to share, then that would be most helpful!
Below is my general protocol:
1) Isolate CD4+ T cells from mouse spleen by mashing spleens and using StemCell EasySep CD4+ T cell Isolation Kit on the spleen lysate (I usually get 3-6 million T cells depending on the age and genotype of the mice)
2) Plate CD4+ T cells in 24-well TC-treated plate coated with anti-mouse CD3 antibody (5 ug/mL); soluble anti-mouse CD28 antibody is added (5 ug/mL); usually 400,000 T cells in 1 mL of R10 solution
3) Incubate T cells for 30 minutes at 37 degrees Celsius
4) Add 1 microliter of Brefeldin A (1,000x stock solution) to wells; I've also tried 0.5 microliters
5) Incubate T cells for 6-12 hrs at 37 degrees Celsius; wash after incubation with PBS/BSA
6) Stain cells for cell membrane markers (CD4, CD25, CD69) for 30 minutes
7) Incubate cells in BD Cytofix/Perm for 20 minutes; wash with BD Perm/Wash
8) Stain cells with intracellular cytokines (e.g. IL-2-PE) diluted in BD Perm/Wash for 30 minutes (1:50 antibody dilution)
9) Wash cells with BD Perm/Wash
10) Resuspend cells in 1xPBS 1%BSA
11) Ran cells on BD Accuri Flow Cytometer, collecting 50,000-100,000 events
12) Analyzed flow data and performed compensation using FlowJo software
Relevant answer
Answer
I used to stimulate the cells for 24 hours at 37C with 5% CO2. Add 1 ul 1x Brefeldin in fresh medium incubate for 5 hours at 37C with 5% CO2. The cells were washed three times with staining buffer (Perm wash) and then continued with an intracellular staining procedure.
  • asked a question related to T Lymphocytes
Question
3 answers
I'm new to the cancer immunology field, and trying to develop an antigen dependent T cell : cancer cell killing assay in vitro. I plan to use OT-1/ova. I would isolate T cells from OT-1 transgenic mice. Do I need to use OT-1 homozygous or heterozygous T cells for the T cell killing assay? Thanks in advance!
Relevant answer
Answer
OT-1 mice are a strain of transgenic mice that express the T cell receptor (TCR) specific for the chicken ovalbumin (OVA) peptide presented by the MHC class I molecule H-2Kb. These mice are commonly used as a model to study T cell responses to OVA and to evaluate the effectiveness of cancer immunotherapies targeting OVA-expressing tumors.
For the T cell killing assay you described, you can use either OT-1 homozygous or heterozygous T cells. Homozygous OT-1 mice carry two copies of the transgene encoding the OT-1 TCR, while heterozygous OT-1 mice carry only one copy of the transgene. As a result, homozygous OT-1 T cells will express higher levels of the OT-1 TCR compared to heterozygous OT-1 T cells. However, both homozygous and heterozygous OT-1 T cells will recognize and respond to the OVA peptide presented by H-2Kb.
It is important to note that the choice between using homozygous or heterozygous OT-1 T cells for the T cell killing assay may depend on the specific goals and experimental design of your study. For example, if you are interested in studying the role of TCR expression levels on T cell function, you may want to compare the responses of homozygous and heterozygous OT-1 T cells. In contrast, if you are interested in evaluating the general effectiveness of the T cell killing assay, you may want to use homozygous OT-1 T cells, which are expected to have higher TCR expression levels and potentially stronger responses to the OVA peptide.
Regardless of whether you use homozygous or heterozygous OT-1 T cells, it is important to carefully optimize the conditions of the T cell killing assay, including the ratio of T cells to cancer cells, the duration of the assay, and the presence of other factors that may affect T cell function, such as cytokines or costimulatory molecules.
  • asked a question related to T Lymphocytes
Question
2 answers
T cells
Relevant answer
Answer
Please consider the following suggestions. to improve your cell culture medium by switching to "complete" medium, i.e. RPMI with glutamine, pyruvate, non-essential amino acids, beta-mercaptoethanol and Hyclone FBS as per:
I hope that helps.
All the best & good luck with your experiments,
Michael
  • asked a question related to T Lymphocytes
Question
5 answers
Recently, I am working on a project that stimulate the TCR- transduced Jurkat with peptide, and looking for a activation marker (cytokine or surface marker). What is the activation marker for CD4+ T cells?
Relevant answer
Answer
CD69 and CD25 are among the most used.
Also the production of IL-2 or IFN-gamma could be useful.
Here is a reference you can check: "Shipkova M, Wieland E. Surface markers of lymphocyte activation and markers of cell proliferation. Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49. doi: 10.1016/j.cca.2011.11.006. Epub 2011 Nov 19. PMID: 22120733."
  • asked a question related to T Lymphocytes
Question
3 answers
We want to identify a certain type of lymphoid cells in cancer tissue however for that we need to identify and exclude three different types of cells (T, B and Natural Killers) using immmuno-histochemistry separately applying antibodies (CD56, CD2 and CD 20) on separate )sections/slides by assuming there will not significant difference among four sections at 4µm thickness.
Brief procedure is to make 4µm thickness slides from the same tissue sample obtained from FFPE blocks, first slide will be exposed to CD2 to identify T cells, take photograph using light microscope and export the image to Fiji and identified T cells will be given a color,
Similarly the same procedure will be done on rest of three separate slides to identify B cells, NK and ILC 3 using rorgamma t antibody giving each cell type different colors. At the end we'll have 4 different Figi colored images.
Now the tricky part is how can we merge these four images together to get one single image with all four types of cells exhibiting different colors, so that in a single final merged image we can show all four types of cells WITH DIFFERENT COLORS.
Relevant answer
Heiko Dussmann, Thank you very much, really much help. I'll take the liberty to ask you more if I need more help I hope you are okay with that :)
Have a nice Christmas and season greetings
Tahir
  • asked a question related to T Lymphocytes
Question
3 answers
We just incorporated a Northern Lights Spectral Flow Cytometer, and we want to analyze glucose incorporation in T lymphocytes using the 2-NBGD probe.  The thing is that we cannot find the spectral fingerprint (ribbon plot) from 2-NBDG. Anyone that could help us with this? And of course any information / advice you can give us will be deeply appreciated!
Relevant answer
Answer
Hi Maria,
I have never worked with 2-NBDG, but it seems it is excited by the blue laser (488nm) and detected in the FITC channel on a conventional flow cytometer. I don´t know the laser configuration of your Northern Light flow cytometer, but I am guessing that most likely, you have a blue laser. If that is the case, you can measure 2-NBDG. It will probably be similar to FITC/SparkBlue 550/ AF532, PE(?) (max. emission at 550nm, according to google), so keep that in mind during panel design.
For unmixing, I would recommend to create a single stain control with the probe and using unstained cells (as you would anyways) as a negative control. that will give you the spectral fingerprint for the probe in the unmixing window. and may give you a good idea on how to implement it in future panels.
Make sure the single stain "expresses" the target for the probe and make sure the unstained control is stained exactly the same way as your single stain.
If you want to compare it to other fluorochromes directly, I would just import (or make fresh) other single stains of similar fluorochromes (FITC, SparkBlue550, AF532, maybe even PE). When you are in the unmixing window, you can see the spectra (click "next" after gating your single stains). Additionally, you can calculate the similarity score matrix in that window and export it. It is not as easy as using the spectra viewer from the Cytek Website, but in the end, you´ll get the same results. Perhaps you could ask them to upload the 2-NBDG spectrum to their database, but that would probably be too late for you :)
Hope this helps!
Best
Johannes
  • asked a question related to T Lymphocytes
Question
2 answers
I'm interested in T cell subsets, and ideally, I would be able to capture some cytokine information. Has anyone tried directly injecting a mouse with Brefeldin A, without dissociating, isolating, and culturing T cells? (I always have such poor viability with the culture.)
Relevant answer
Answer
Robert Maile Fantastic, thank you for the reference.
  • asked a question related to T Lymphocytes
Question
4 answers
I plan to check t cells from mouse tumors for IFN-y, TNF-a, IL-2 and granzyme B. Is T cell activation by PMA or anti-CD3/CD28 needed?
Thanks!
Relevant answer
Answer
Johannes Brandi Thank you for sharing the helpful experience and tips! Will definitely give it a try.
Jin
  • asked a question related to T Lymphocytes
Question
1 answer
is there any traditional method to check T cell levels or can we isolate T-cells to check Th17 cell levels using specific markers? I am a little stuck here.
Just to clarify I don't want to use the magnetic separation method. I want to Try the traditional method before I jump to that.
Thank you in advance.
Relevant answer
Answer
In the past, for HLA testing, T cells were separated form PBMC by elution through a nylon mesh column ( we usually used a sirynge). Cells were suspended in medium and the column was incubated at 37%. Adherent cells ( B cells and monocytes) were trapped and T cells were eluted. Believe it or not, if performed correctly, this yielded a nice separation.
Obviously, sorting by flow cytometry or magnetic beads ( positive or negative separation) are more suitable methods nowadays
  • asked a question related to T Lymphocytes
Question
2 answers
Hi,
Is there a way to analyze the VDJ regions that T cell falls into using the scRNA data? R package is preferable.
Relevant answer
Answer
I think you could try this:
doi: 10.1038/s41592-021-01142-2. Epub 2021 May 13.. 2021 Jun;18(6):627-630.Nat Methods
TRUST4: immune repertoire reconstruction from bulk and single-cell RNA-seq data
Li Song 1 2, David Cohen 1, Zhangyi Ouyang 3, Yang Cao 4, Xihao Hu 1 2, X Shirley Liu 5 6 7
Affiliations expand
  • PMID: 33986545
  • PMCID: PMC9328942
  • DOI: 10.1038/s41592-021-01142-2
  • asked a question related to T Lymphocytes
Question
4 answers
I am interested in isolating naïve CD4+ T cells from human PBMCs. I get whole blood (~500 mL blood + 70 mL anticoagulant) from a blood center but I don't receive it till +1 day after extraction. Blood sample is stored and delivered on ice. Once I get the sample, I start performing the Ficoll gradient. However, the amount of PBMCs that I have obtained (~0.25*10^6 cells/mL) is lower than expected (~10^6 cells/mL - this is the amount I used to obtain using fresh whole blood samples). I have been reading about keeping blood samples at room temperature before processing, does anyone have experience with this approach?
On the other hand, does anyone know about the percentage of naïve CD4+ T cells in the PBMCs fraction? Thanks!
Relevant answer
Answer
Hi,
I have tried both and I clearly prefer room temperature. Ficoll should also be heated (37°C) just before use, as well as new culture medium. 24h storage is OK to get PBMCs.
Best regards
  • asked a question related to T Lymphocytes
Question
2 answers
Hello everyone,
I want to do an ELISA assay to detect IFN gamma and IL2 secreted from a primary T cell line.
In order to do so, on day 1 I will thaw these T cells and centrifuge them to get rid of the DMSO and then resuspend them in media consisting of : RPMI, FBS 10%, PenniStrep 1%, 0.5% non essentials amino acids, 1% HEPES, 0.5% L-Glutamine and 0.001% B-mercaptoethanol and 2nm of IL2.
Then on day 2 I will put those cells in 96 well plates with the APC that have been incubating with the antigen prior in the same media.
This incubation will last 48h at 37°C and then I will harvest the supernatant after centrifugation of the plate to perform an ELISA assay.
I read that feeder cells are important when working with primary cells but I don't know if I have to add them when working with such short time, if I don't put any will it prevent the T cells to activate and secrete IL2 and IFN gamma ?
Thanks in advance, I hope I was clear enough.
Best regards,
Sonia
Relevant answer
Answer
It might very well depend on the cell line itself. If I were you, I would do both
  • asked a question related to T Lymphocytes
Question
2 answers
Hi, guys
I have had big problem with Jurkat cell activation.
Protocol is as follow:
1. Seed jurkat at 1X 10^5 cell/ml and incubate for O/N
2. Treat jurkat with PHA(sigma, L1668-5MG) at 1, 10, and 50 μg/mL, and PMA (sigma, P1585-1MG) at 50, 100 ng/mL for 24, and 48 hours
3. Collect cell culture supernatants
4. IL-2 ELISA (abcam, ab270883)
But, IL-2 was not released from jurkat stimulated by PMA/PHA.
Instead of PHA, Jurkat was treated with PMA/ionomycin (invivogen), however, IL-2 secretion was not shown (PMA at 50 ng/ml, Ionomycin at 1 ug/ml).
I doubled the cell density, but it didn’t work out.
What's the matter with my experiments?
Is the cell in bad condition? Or Should I change the ELSIA kit?
Thank you
Relevant answer
Answer
You may be missing stimulation via CD28. We co-stimulate Jurkat T cells with anti-CD3 antibody OKT3 and an anti-CD28 antibody and get a good, ELISA-measured IL-2 response. It also works when we express recombinant murine-derived TCRs in Jurkat cells and stimulate with a murine TCR-specific antibody (H57) or the actual antigen presented by MHC, but again only when adding an anti-CD28 antibody. The anti-CD28 antibdy by itself does not induce IL-2 production.
  • asked a question related to T Lymphocytes
Question
2 answers
I want to detect markers of degranulation of T cells in HIV patients (mainly perforin and granzyme B), but I'm not sure if I need to stimulate the cells or not before test. Both are available in the published literature, what is the difference between them?
Relevant answer
Answer
Dear Qing Xiao
You can check perforin and granzyme on the resting cells as well. We did not stimulate blood in our experiments and found good results. Even in clinical diagnosis of HLH at our centre, we do not stimulate cells for Perforin.
Those who stimulated cells (which you read) might also be checking de-granulations (CD107a) study altogether. And CD107a expression needs stimulations.
Please find attached link of abstract from our centre.
Best wishes.
  • asked a question related to T Lymphocytes
Question
1 answer
how do immune cells(NK cells, CD4 Th1, and CD8 cytotoxic T lymphocyte effector T cells) produce IFN-gamma? Is there a signal pathway?
Relevant answer
Answer
Hello Hongjie Hu
A variety of cytokines, signaling molecules, and transcription factors contribute to IFN-γ production. For instance, multiple lines of evidence have implicated the p38 mitogen-activated protein kinase (MAPK) pathway in this process. It has been suggested that STAT4 is a candidate p38 MAPK target that could regulate IFN-γ production.
You may refer to the article attached below for more information.
IFN-γ production is controlled by cytokines secreted by APCs, most notably interleukin (IL)-12 and IL-18. These cytokines serve as a bridge to link infection with IFN-γ production in the innate immune response. Macrophage recognition of many pathogens induces secretion of IL-12 and chemokines [e.g., macrophage-inflammatory protein-1α (MIP-1α)]. These chemokines attract NK cells to the site of inflammation, and IL-12 promotes IFN-γ synthesis in these cells. In macrophages, NK and T cells, the combination of IL-12 and IL-18 stimulation further increases IFN-γ production.
Negative regulators of IFN-γ production include IL-4, IL-10, transforming growth factor-β, and glucocorticoids.
Best.
  • asked a question related to T Lymphocytes
Question
5 answers
I am identifying cell types on Loupe Cell Browser using various cell surface makers. Are there any specific markers unique to NK cells that differentiate them from T-cells? Right now, I have filtered out the "NK cells" based on the fact that they don't express classic T-cell markers such as CD3E, CD3D, CD3G, CD8A, CD8B, TRAC; but do express genes required for cytotoxic functions such as GNLY, NKG7, etc.
Relevant answer
Answer
The main CD antigens on the surface of T cells are CD2, CD3, CD4/CD8. The surface markers of human NK cells are mainly identified by CD16 and CD56.
At present, CD3-, CD16+, and CD56+ are used as typical markers of NK cells. CD16 molecule is known as a low-affinity IgG Fc receptor. When the IgG class antibody specifically binds to the corresponding epitope on the surface of the target cell, it can bind to the FcR III on the surface of the NK cell through its Fc segment to exert a directional non-specific killing effect on the target cell, namely, antibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells.
  • asked a question related to T Lymphocytes
Question
1 answer
Hello, I am stimulating CD4+ genes with doxorubicin to see effect on FV, but am having real trouble finding a good endogenous control gene for qPCR. I have tried GAPDH, PMM1, 18S and HPRT1. None of these seem very good at all, 18S shows some promise but the others are no good.
I can't seem to find any research on this particular cell stimulation, and based on what I have found I am thinking of trying B- actin or RPLP0 and maybe combine these with 18S...
I am desperate for any kind of advice here.
Best regards, Kaja
Relevant answer
Answer
Control genes are usually selected from β-actin, tubulin, GAPDH, Lamin B, PCNA, Na+/K+-ATPase, Histone H3, etc.
The following articles related to DOX-stimulated CD4+ may be helpful to you.
  • asked a question related to T Lymphocytes
Question
1 answer
Hello everyone, I used several shScremble (from sigma, worldwide use) as a control for knocking
down my target genes in human primary T cells.
To my suprise, two shScremble I use has a negative effect on proliferation than just WT untransduced T cells and empty shRNA vector transduced T cells.
I am confused. Is there any bad things with shScremble? They should be the same with WT cell in theory.
I am wondering that what kind of control should I use? WT cells, empty shRNA vector or shScremble? Which one worth trust most?
Why people usually use shScremble rather than others?
Really hope someone could help to figure out.
Thank you so so so much in advance!
Relevant answer
Answer
This is because there are non-specific effects from shRNA. Using shScremble enable you to see specific effect of target gene knockdown.
  • asked a question related to T Lymphocytes
Question
3 answers
can we genetically modify the memory b cells and t cells for a disease in order to make it work as vaccine.
Relevant answer
Answer
Hi Pranav,
If there are memory cells available for a particular pathogen then it will do its work. To be clear memory B cells are generated during primary responses to T-dependent vaccines. They do not produce antibodies, i.e., do not protect, unless re-exposure to antigen drives their differentiation into antibody-producing plasma cells. As your question suggests the possibility of modifying memory B cells is not easy because the less percentage of that memory B cells would be difficult to isolate and maintain. If you are talking about modifying B cells there are many studies available for cancer immunotherapies. These B cells are used as APCs instead of DCs to avoid toxicity and generate an antigen-specific T cell response.
All the best
  • asked a question related to T Lymphocytes
Question
3 answers
I'm currently testing an Ab panel for T-cell analysis on Attune flow cytometer and I'm facing some strange stuff (see attached picture). I'm using IL4-BV711 Biolegend Ab for intracellular staining and when I apply this Ab I see a huge negative tail in the corresponding channel. I supposed some compensation issues, but it seems that it is not the case, since the tail appears when there is no compensation at all. Has anybody ever faced such kind of problem? What it might be? Too high gain on BV711-channel? I know, that flow data always contain negative values due to baseline correction after each event registration. However, in these data, the percentage of points that are below zero seems to be too high. Interestingly, the problem does not exist on the Fortessa cytometer. I would be grateful for any suggestions.
Relevant answer
Answer
This tail seams to be result of weak washing out of intracellular antibodies. I suppose cells from tail in SSC/IL-4 plot localizes on the right form "0" cloud. check with gates. How have You deterrmined the compensation, with splenocytes or beads?
  • asked a question related to T Lymphocytes
Question
1 answer
Hello,
I am trying to image live primary cells with Annexin V as a detector of PS exposure. I've currently been imaging in RPMI with 5% FBS, but I have realised that the calcium level of this may be too low for realiable Annexin V binding, as RPMI has a low calcium level of 0.42mM. Annexin V binding seems to require 1-3mM.
Other labs have successfully used complete DMEM with 10% FBS, would it be appropriate to use this for imaging even if I usually culture the cells in RMPI 5% FBS? Or should I try adding calcium (via calcium chloride) to my RPMI 5% media?
Thanks for any help.
Relevant answer
Answer
Hi Emily,
DMEM is more nutrient-rich and has higher levels of Calcium(around 1.6-1.8mM). DMEM generally comes in two variants, DMEM low glucose(1g/L) and high glucose(4 g/L). I would recommend you to culture your cells for at least 2 passages in DMEM High Glu and then stain them with Annexin V
Hope this helps!
  • asked a question related to T Lymphocytes
Question
1 answer
Hi everyone!
For human T cells isolated from PBMCs & frozen after isolation, how would you recommend to proceed with them after thawing? Shall I use them immediately (for sorting for instance) or let them recover (overnight or for a few hours) before experiment?
I need naive T cells, and I m not sure I can maintain this phenotype if I keep them too much in culture.
Thanks in advance for your help :)
Relevant answer
Answer
Once thawed, T cells must be rested for a minimum of 8 hours to a maximum of 18 hours to remove any apoptotic cells. A longer duration of resting could separate the dead cells and late apoptotic cells from the lymphocyte population. This would help to increase viability and improve functionality thereby enhancing the specificity and sensitivity of the assay (for instance, flow cytometry assay). The resting process could be helpful to restore the cells as well as have a positive effect on the recovery of the function of lymphocytes.
As you rightly mentioned, resting could also induce phenotypic changes.
Please refer to the research article attached below where in the investigators investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cryopreserved PBMCs.
Hope this will be helpful!
Best.
  • asked a question related to T Lymphocytes
Question
4 answers
My Jurkats Cells looks differents after cytometry and even under the microscope. I just saw this and I am wondering what kind of contamination is this ?!
Thanks
Relevant answer
Answer
Thanks all for your answers ! Definitely Yeast ! I'll decontaminate all the lab and restart with a new culture !
Thx
  • asked a question related to T Lymphocytes
Question
5 answers
Hi all,
I am new to immunology field, need some help to distinguish CD3, CD4, CD8 and CD25. What are differences between them for T cells? How to decide which CDs are specific for T cells, B cells, neutrophils and macrophage.
Relevant answer
Answer
I commonly use CD3 to gate T cells; then CD4 and CD8 to gate T helper cells and T cytotoxic cells, respectively; Then using a CD3+CD4+ gating (Th cells) you could use CD25 and CD127 in combination to distinguish between effector T cells (CD127+CD25low) and regulatory T cells (CD127low CD25high).
You can check one of my articles for that: doi: 10.1038/s41598-019-43622-8
Best
Juanjo
  • asked a question related to T Lymphocytes
Question
3 answers
We want to analyze whether T cells produce ROS under certain pahtophysiologic conditions.
Relevant answer
Answer
Try this for method and alternatives
best of luck
  • asked a question related to T Lymphocytes
Question
1 answer
Hello everyone.
We isolated CD8+PD1+ T cells from the peripheral blood of a metastatic melanoma patient, with 99.5% isolation purity and 98% PD1 expression. We activated cells with Human TransACT "Miltenyi Biotec". After the activation phase had been finished, we left the cells in the expansion phase for 14 days, and then we performed a flow cytometry experiment to identify the phenotypic features of the expanded population. We found out that PD1 expression decreased from 97% to nearly 50%, but we still have CD8+PD1+ purity of 96%.
The part we didn't understand, what was the cause that led CD8+PD1+ T cells to decrease PD1 expression?
Relevant answer
Answer
PD-1 expression on naïve T cells is induced upon TCR activation. This transient expression decreases in absence of TCR signaling but is maintained upon chronic activation with a persisting epitope target. You used Human TransACT "Miltenyi Biotec", it may create a chronic activation on CD8+ Tcells. May be this activation results in lower expression of PD-1. Good luck
  • asked a question related to T Lymphocytes
Question
6 answers
Assessing differential expression of activation-related proteins in naive versus activated T cells is hampered by the fact that apples are compared with melons. Activated T cells vastly increase cell size, cytoskeleton, ER, protein synthesis and metabolism.
A problem occurs when you try to normalize to an internal control like actin, tubulin, Gapdh, etc. Despite equal loading according to Lowry determination all these proteins show increased expression in activated T cells a result understandable in light of the huge changes these cells undergo. What causes the bias? What would be a good loading control in this particular case? Histones, HDAC1? Can someone help with suggestions?
Relevant answer
Answer
Can someone recommend a good HKP for WB analysis
  • asked a question related to T Lymphocytes
Question
1 answer
I noticed that researchers use MOG35-55 to induce a type of T cell-dependent EAE, while for both T cell and B cell-dependent EAE, they choose rhMOG. Some papers report that B cells also contribute to the disease process in rmMOG-induced EAE setting. If these two recombinant proteins both activate B cells, I want to know what's the difference between them.
Relevant answer
Answer
Dear Echo Zhu
Please see the comparison below. There is a single amino acid substitution at position 42 of the MOG protein (position 7 of the peptide respectively) between the rodent (mouse/ rat) and human MOG protein from serine to proline.
rmMOG vs. rhMOG (aa 35-55)
rmMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
rhMOG Met-Glu-Val-Gly-Trp-Tyr-Arg-Pro-Pro-Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys
The difference in B cell dependency of MOG induced EAE seems to majrily be attributed to immunization with full length (human) protein vs. the rodent peptide (in C57BL/6 mice), which may differ in other mouse strains (given their different MHCII) as per:
I hope that helps.
All the best and good luck with your experiments,
Michael
  • asked a question related to T Lymphocytes
Question
1 answer
i need clarify difference between ILC and T lymphocyte Th1,Th2 and Th17
because i found it have the same transcription factor?
like T bet, GATA3 and RORg
Relevant answer
Answer
If your ILCs are largely NK cells, there are a few ways I can think of to approach this issue. T cells express surface CD3, whereas NK cells do not; rather they express cytoplasmic CD3 only. T cells have TCR gene rearrangements, NK cells do not. NK cells express surface CD56 and the majority lack CD4 and CD8, though a normal subset will have CD8 expression. Give the close relationship between ILCs and T cells, I would expect a significant overlap in transcription factors, mRNA expression, et cetera. Please respond with more specific information regarding the ILCs, the tissue source of these cell lines, etc, if you would like to further discuss.
  • asked a question related to T Lymphocytes
Question
1 answer
I have activated human T cells in vitro (with CD3/CD28 for 1-2 days) and then stimulated them shortly with rhTNF (0-30 min). After CD3/CD28 activation, basal p-STAT3 levels incresead compared to the control T cells. However, upon TNF stimulation, preactivated T cells showed decreased p-STAT3 MFI. On the other hand, unactivated T cells had only slightly increased p-STAT3 levels. Any comments?
Relevant answer
Answer
Are your FACS data really convincing enough so that you can make those asumptions?
I would privilege Western Blot instead of intracellular FACS in order to make sure the signal that I look at really is coming from p-STAT3.
Also please consider the fact that activated T cells will endogenously produce TNF and that they produce IL-2 (which induces some STAT3 signaling), while unactivated T cells kept in culture for 2 days start/are dying (which could lead to a poor response to TNF).
  • asked a question related to T Lymphocytes
Question
3 answers
Hi everyone,
Been trying to culture murine T cells isolated from spleen or lymph nodes (iLNs) but been having difficulty in keeping the Pan T cells alive even after only 24hr of culturing (viability drops to ~20%). I process the tissue after harvesting with a plunger and 100um cell strainer in cold cRPMI to ensure single cell suspension, wash with PBS/2% FBS and performed RBC lysis with 3ml ACK lysis for 2 mins, quench with complete RPMI (cRPMI). Then isolate Pan T cells using the Myltenyi Pan T cell isolation kit (cat no:130-095-130 and stimulate with Myltenyi anti CD3 / or anti-CD3/CD28 beads or keep them resting for 48hr iin the 96-well plate at 0.25x10^6/well.
** Everything takes place at 4 C with cRPMI (10% FBS, 2mM Glutamine and 1mM sodium pyruvate, 100 units penicillin and 100μg/mL streptomycin and 1x NEA). For culturing i added to the cRPMI media: 0.05mM 2-Mercaptoethanol (2ME).
I have trouble-shouted the following and have NOT made a difference:
1) processing in cold cRPMI vs PBS or PBS/2%FBS (cRPMI elevated the viability by 20% vs PBS)
2) 2ME concentration: titrated between 50mM to 0.05mM and it seems that 0.05mM is the same with not adding 2ME (which was only around 20%) and anything above that concentration decreased the viability even more.
3) PBS tablets versus premade PBS from Thermo (pH checked)
4) stimulation induced cell death (although you always see some but even the resting cells die )
5) ACK lysis (optimised time required for lysis and quenching and volume) and also compared T-cells that went through RBC lysis (Splenocytes) vs cells not undergone through RBC (LN). Findings: had equally bad viability.
6) compared resuspending the cells with mechanical mixing (racking the tube with cells on a rack to release the pellet and then resuspending with the pippete) vs resuspending the pelleted cells with pippete directly . Findings: still did not make a difference
7) Centrifuge speed 1500rpm vs ~1200rpm (300xg). Did not make a difference.
Thank you all in advance, any help would greatly be appreciated.
Relevant answer
Answer
Hey Charys,
Maybe you can add some IL-2 to the medium? We recommend to add 50U/ml IL-2 in our T cell expansion protocol (see attachment).
Did you compare sorted T cells vs. seeding whole splenocytes? From my experience, the T cells are happier when you expand them in whole splenocytes (after a few days of expansion, you have around 90% T cells anyway).
I usually sorted CD4+ T cells and they didn't like it when the cell density was too low. So maybe you can also try to increase the cell density?
Best,
Alex
  • asked a question related to T Lymphocytes
Question
7 answers
Recently, we have done the macrophage antigen lpresenting experiment by co-culture of macrophages pre-loaded with OVA and CD4+/CD8+ T cells isolated from OT-II/OT-I mouse spleen. Using CFSE to stain the T cells, after 3-5 day co-culture, there is no significant differentiation of the target T cell.
Is there any probelm in the experiment? and is thre any trick in processing the sample?
Thank you very much.
  • asked a question related to T Lymphocytes
Question
2 answers
Hello,
I am looking to use Sytox Green as a substitute for propidium iodide (which I can't use with my microscope) in a live cell imaging experiment with T cells killing Daudi Lymphoma cells.
I know that propidium iodide has to be at high concentrations to enter through perforin pores (100uM).
Should I use the same concentration of Sytox green? Any help would be appreciated.
Relevant answer
Answer
SYTOX Green probe may be substituted for PI. SYTOX Green does not display any side effects on cellular viability, cellular proliferation or cell migration when used in concentration upto 1mM. You may use 150nM SYTOX Green. Please see the paper attached below.
Also, in another article attached below, the investigators explored the interaction of live cells with different fluorescent dyes, over extended periods of time in which cells were continuously cultured in the presence of selected probes: SYTOX Green (SYG, 1–1000 nM), SYTOX Red (SY-R, 1–1000 nM), YO-PRO 1 (1–1000 nM), PO-PRO 1 (1–1000 nM), propidium iodide (PI, 0.1-10 μg/mL), and 7-aminoactinomycin D (7-AAD, 0.1–10 μg/ mL) for up to 5 days. They found that treatment of several different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation.
Best.
  • asked a question related to T Lymphocytes
Question
2 answers
What would be ideal mice strain to study T-cell dependent B-cell activation or KLH immunization?
Relevant answer
Answer
You may use C57BL/6 mice.
Please refer to the two articles attached below.
Best.
  • asked a question related to T Lymphocytes
Question
2 answers
I am studying oxysterol effects on murine T cells and I want to perform a western blot on the gene that is more highly expressed. However, I am unsure which INSIG gene, INSIG1 or INSIG2, is more highly expressed in T cells. Please provide a citation if possible.
Relevant answer
Answer
Dear Natalie,
I hope you are doing well. Please take a look at this link (https://www.proteinatlas.org/ENSG00000186480-INSIG1/immune+cell).
Best regards,
Pooya
  • asked a question related to T Lymphocytes
Question
1 answer
I am investigating the role of the tumor microenvironment in shaping the antitumor immune response. In particular, we see that the T cell immune response is important but inhibited by some tumor-related factors. There is an issue that I was curious about but could not find the answer to: Some T cells remain resident in various tissues. Is this also true for tumor tissue? How long do tumor-infiltrating T cells remain in tumor tissue? Do they leave the tumor tissue and recirculate? How long do they come into contact with tumor antigens in tumor tissue? Does this contact time have an effect on the anti-tumor response?
In my research, I expose T cells to some of the factors they encounter in the tumor microenvironment, but I'm not sure how long I need to do them.
I would appreciate it if you could guide me on this.
Kind regards
Hasan
Relevant answer
Answer
Tumor microenvironement (TME) is very complex environment composed of various cells including tumor-infiltrating CD4/CD8+ T cells (TILs), and regulatory T (Treg) cells. The concern with TME is the mechanism of cancer cell evasion/escaping pathways via hijacking the immune system. Cancer interactions in the tumour microenvironment can stimulate biological components in the environment, such as tumour associated macrophages (TAM), cancer associated fibroblasts (CAF), and mesenchymal cells, which promote drug resistance against cancer. Some cancer biomarkers can also inhibit the production of tumour antigens, resulting in the failure of the targeted medication to attach/penetrate cancer due to cancer's microenvironment's unfavorable and complex mutational landscape, which also creates immunometabolic barriers.
In my point of view if we can block/inactive immune suppressor agents/immune checkpoint–related immune cells such as PD1/PD-L1, CTLA-4 would be a good strategy.
Many studies have reported that the tumor microenvironment plays a critical role in tumor progression and prognosis. Understanding the relationship between the various cellular components of the tumor microenvironment and prognosis is crucial for developing therapeutics aimed at the tumor microenvironment. Perhaps the following articles may be of use in gaining a better understanding:
Ji, X., Lu, Y., Tian, H., Meng, X., Wei, M., & Cho, W. C. (2019). Chemoresistance mechanisms of breast cancer and their countermeasures. Biomedicine & Pharmacotherapy, 114, 108800
Crespo, I., Götz, L., Liechti, R., Coukos, G., Doucey, M. A., & Xenarios, I. (2016). Identifying biological mechanisms for favorable cancer prognosis using non-hypothesis-driven iterative survival analysis. NPJ systems biology and applications, 2(1), 1-11.
Anfray, C., Ummarino, A., Andón, F. T., & Allavena, P. (2020). Current strategies to target tumor-associated-macrophages to improve anti-tumor immune responses. Cells, 9(1), 46.
Davis, R. J., Van Waes, C., & Allen, C. T. (2016). Overcoming barriers to effective immunotherapy: MDSCs, TAMs, and Tregs as mediators of the immunosuppressive microenvironment in head and neck cancer. Oral oncology, 58, 59-70.
  • asked a question related to T Lymphocytes
Question
4 answers
Dear all,
I am just trying to do an overall stimulation of PBMCs. Anti CD28/CD3 to stimulate T cells, but how can I stimulate the innate response? Some TLR agonist should be fine? TLR agonist can alter the natural T cell responses? Can I ask for some papers in which I can check this, please?
Also, if I am checking CD28 marker by flow cytometry, can the addition of anti-CD28 alter the detection of real levels of CD28 by flow cytometry?
Many thanks,
Julen.
Relevant answer
Answer
Hi Giacomo! Thank you so much! Hope everything is fine ;)
  • asked a question related to T Lymphocytes
Question
1 answer
Dear Experts,
We would like to inquire if planned to buy commercialized Anti-HER2/neu (369-377) T Cells to perform a cytotoxic T Lymphocyte (CTL) assay.
Do we need to use APC to activate T cells before CTL assay?
(Co-culture with antigen+APC first?)
We only have limited experience with CAR-NK cells.
In our experience, CAR-NK could use to kill the target cells.
However, we are not sure whether T cell needs to go through activation as we haven't found a proper protocol for the experiment.
Could you help us to solve the myth?
Sincerely,
Relevant answer
Answer
CAR-T cells are synthetic molecules in which the effector function of T lymphocytes combines with the ability of antibodies to identify specific antigens. Thus, CAR T cells do not require antigen presentation by antigen presenting cells (APC) and can recognize intact proteins. Therefore, the creation of genetically engineered T cells redirected to tumor antigens can bypass several mechanisms of immunological tolerance.
Please refer to the article attached for more information.
Best.
  • asked a question related to T Lymphocytes
Question
1 answer
Dear all,
We are starting a project that involves calcium imaging from activated T cells; in the literature people often use ratiometric dyes but I prefer to use OGB or calcium green AM
Does anyone has experience in loading theses cells with these dyes? can anyone reference a previously tested protocol? Many thanks
Relevant answer
Answer
I did use OGB BAPTA II already (octapotassium salt). However, I performed patch-clamp experiments (not sure if that's what you're looking for), so I loaded the cell by adding 50µM OGB in my internal solution. Worked like a charm!
  • asked a question related to T Lymphocytes
Question
1 answer
I have been trying to activate my Jurkat T cells using CD3/CD28 DynaBeads and looking at activation markers such as CD69 and CD62L. However, there have been no signs of activation after multiple experiments.
Do you have any recommendations to improve my results, preferably without adding PMA/ionomycin?
Relevant answer
Answer
Please consider the following suggestions:
Improve your cell culture medium by switching to "complete" medium, i.e. RPMI with glutamine, pyruvate, non-essential amino acids, beta-mercaptoethanol and Hi-clone FBS as per
which may improve your cell culture and activation results as per:
Please also consider the following paper.
As per Figure 5: CD62L is at best mildly upregulated, whereas CD69 does not seem to be upregulated at all following CD3/CD28 agonistic stimulation. They did culture for 24 hours in their experiment and used massspec as their readout, so therefore a time course might be a good experiment to do.
I hope that helps.
All the best & kind regards,
Michael
  • asked a question related to T Lymphocytes
Question
3 answers
I have been trying a protocol for T cell activation and differentiation using murine splenocytes in vitro, but I have been getting a lot of dead cells as indicat