Dear colleagues,
We are going to stimulate human B lymphocytes isolated from PBMCs from blood of healthy subjects (negative selection with MyltenilBiotech magnetic beads). It should be a control experiment just to see if we observe the same effects as we do observe with B cells incubated with HIV and HIV proteins.
We have decided to use the following combinations of reagents and final concentrations that we will have in the medium with B cells:
Combination 1. anti-IgM (10 mkg/ml) and IL-4 (20 ng/ml)
Combination 2. IL-4 (20 ng/ml) and anti-CD40 (1 mkg/ml)
Variant 3. PHA (10 mkg/ml)
We are going to take cell samples at 24, 48 and 72 hours after addition of the activating reagents. After that we will fix the cells and perform a 3D FISH for our genes of interest and at the same time we should check if the cells were activated.
Question 1. What do you think about these combinations and concentrations? For instance, is it OK to use 1 mkg/ml anti-CD40? I have taken it from an article (Lefevre et al, J Immunology, 1999), but I have another article in which they take 20 mkg/ml anti-CD40.
Question 2. Which way is better to check whether our B cells were really activated?
1. I do not think we would be able to use the 3H-thimidine method.
2. We can use FACS or immunostaining with antibodies to an activation marker
3. Is it possible just to count the cells and to check if they have proliferated or not?
Question 3. As for the activation marker, we should know for sure that the marker we select should be present on activated B cells 48-72 h after incubation with antibodies or PHA. Could you tell me, which one would be better?
We have already got an anti-CD71 and anti-CD23. Will it work? A friend of mine advised me to use anti-CD80 or anti-CD86.
I would appreciate your help,
Regards,
T.T.
As there are still people who find this question/answer in the Internet, and write me to know, what we actually did to activate B cells - I will give here a short summary of the discussion and what we have done afterwards.
PHA does not activate human B cells, but it activates very well human T cells (see http://www.ncbi.nlm.nih.gov/pubmed/3263438). LPS actually DOES NOT activate human B cells. However, it can activate murine B cells that express TLR4 unlike human B cells.
For a review see http://www.biomedcentral.com/1471-2172/13/63: "In contrast to mouse B-cells, human B-cells express neither TLR4 nor CD14, the two canonical ligands for Gram– bacteria LPS, and are therefore unresponsive to LPS."
For human B cell activation we have successfully used 10-20 mkg/ml anti-human-IgM (DA4.4) + 20 ng/ml human recombinant IL-4 (Sigma) + 1 mkg/ml anti-human-CD40 (G28.5). We have been also able to activate B cells with two of these components together, for example, IL4 + anti-CD40. It should also be noted for anti-CD40 that not all monoclones work equally well for B cell activation!!! Monoclonal anti-CD40 G28.5 has been shown to work. To access if B cells were really activated we used flow cytometry for B cell surface activation markers (CD23 and CD69), 24 and 48h after activation, and BrDU incorporation studies 72 h after activation.
Here are some useful references - http://www.ncbi.nlm.nih.gov/pubmed/12748040 - a very good work where one can find also activation markers that can be used to assess B cell activation,
http://www.jimmunol.org/content/163/3/1119.long - they activated B cells with different coctails.
Good luck with your work!
Tatyana.
PHA is not good for B cells--you will need to titrate everything first--human B cells die easily. You can check for increases in CD69 and DR expression by flow cytometry. Most human immunologists use SEB or PWM to activate B cells--could also use LPS. Counting is not going to tell you anything--so many B cells will die You might be able to use CFSE staining and look for cell division
What is the question? Is there a difference in HIV? Are you going to isolate them first? Good luck--many will die before you do anything.
Your questions are flawed. Any experiment you set up initially should have a dose response, since the optimal dose depends on the reagent you use, so do the experiment yourself rather than asking someone on the Internet to tell what to do! Many anti-IgM reagents work in the 5-25 microgram/ml range for say 500,000 B cells/well and G28-5 anti-CD40 I know works well usually in the 1-20 microgram/ml range. For activation, we've discussed this quite a bit on this site already, but see my J Immunol paper EA Clark et al in J Immunol 1989 for a range of stimuli and time points and markers. Finally, I'm not sure what your hypothesis is. Clear HIV proteins are not like anti-IgM. So are you simply seeking a good positive control?
For stimulation of B cells you can use SEB (or LPS) as suggested above. You can measure B cell activation after 48 hrs by FACS analyses using CD86, HLA-DR, CD70 or CD38 or by cytokine analyses e.g. IL-6 (eg. CBA assay or ELISA). Hope that helps!
Please give me the reference where LPS stimulates human B cells to be activated. I'm not sure this is correct or is helpful.
I think every stimulus causes its own characteristic effect or activation in B cells, depending on which receptor or intracellular structure you activate with it... It might be important indeed if you use it to compare with HIV infected cells. Because then, you would want to be as close as possible with the B cell signalling to your HIV infection-induced signalling, right?
In our hands, CpG at 5 µM on 100.000 B cells in 200 µl volume induces proliferation (after 5 days) and CD80 upregulation (already after 24 hours). Probably HLA-DR is also up, and CD86 as well. Of course there are also a lot of dead B cells/leftovers :-) but this is normal. Counting does not really make sence (in my humble opinion) because you have a mixture of apoptosis, survival and proliferation...
btw my colleague uses 5 µM, I use 0.5 µM because it also works in my assay set up for my purpose.
Of CD71 and CD23 I do not know whether these are related to activation of B cells.
Good luck,
I heartily thank everybody for comments. I think CFSE staining will be best for us to see if the cells were activated.
For Dr Edward Clark: Thank you for your suggestion. We have already started an experiment and we will adjust the quantity of reagents we use if it does not work.
By the way, has anybody used 1G1 anti-CD40 (Sigma) for B cell activation?
For Dr Dorothy Lewis and Dr Edward Clark:
We are interested in the distance between IgH and c-Myc genes in B cells treated by HIV (method - FISH), as the rearrangement between IgH and c-Myc is the main rearrangement in B cell lymphomas, that are more frequent in HIV patients than in healthy people.
It has been shown that HIV infection causes B cell hyperactivation. It has also been shown that HIV-Tat protein can activate B cells in vitro (Huang L et al. Biochem Biophys Res Commun. (1997)).
That is why we really need a good positive control to know whether we will observe similar patterns in the C-Myc/IgH location in the nucleus upon activation of B cells with a more natural agent.
For Dr Dorothea Dijkstra:
Yes, I think it would be best for us to use a natural activation stimulus that acts similar to the HIV infection. However, as HIV infection is not a natural stimulus, and as it causes hyperactivation and cytokine "misproduction" in B cells, I guess there is no natural stimulus with exactly the same effect.
Hi Tatyana, this is totally clear :-)
Then maybe the best would be to use different B cell activators to compare with the HIV infection effects.
CpG, anti-Ig, CD40L are the most used I guess...
That is exactly what we are going to do. For the moment we have decided to use PHA in one experiment and anti-CD40 with IL-4 in another, as we have already got all of these reagents.
I will also consider using what you have advised (CpG, anti-Ig, perhaps CD40L also if the anti-CD40 we have will not work). If PHA does not work with our cells, we may also try SEB, LPS or PWM that have been mentioned in this discussion.
Thank you for your comments!
T.
You listen to Ed Clark - he is a doyen of human B cell biology and made some of the most important findings on human B cell activation over the past few decades!!
and LPS does not activate human B cells as they do not express TLR4 (unlike mouse B cells). there are some papers saying LPS can be induced on activated B cells but the data is not that convincing (in my opinion)
OK. Thank you for information. :) Does it mean that murine B cells differ greatly from our B cells? That is one of the possible reasons, that not all the vaccines tested in mice provide the same level of protection in human.
Anyway, we did not have any LPS for the moment.
Listen to Stuart Tangye, who has done more to define human memory B cells than anyone I know!
Tatyana, I would just note again that it is hard to get anywhere doing experiments in a piecemeal fashion. It is good that you are asking questions and getting some information about what is done before. But I am still unclear what the rationale is for your experiments and what hypothesis you are testing. Andrea Cerrutti and his coworkers just published a great paper in Nature Immunology showing that neutrophils in human spleen contribute to the activation of presumably red pulp B cells. The neutrophil 'nets' he detects and the significant numbers of neutrophils in human spleens are not present in mouse spleens! The mouse of course is different from human but has been wonderfully useful for human B cell biologists and B cell biology.
there are many significant differences between mouse and human B cells, especially in relation to the function of cytokines in regulating B cell development and differentiation. In terms of development, mouse B cells are absolutely dependent on IL-7 - whereas human B cells development is completely unaffected when signalling through the IL-7R is compromised, as shown by studies of humans with mutations in either the IL7R or the common gamma chain. BAFF deficiency in mice compromises B cell development beyond the transitional stage - but in humans, the requirement for BAFF at this stage of development is less clear. when BAFF in neutralised in humans or monkeys, B cells are reduced by ~2x, but in mice there is a ~10-fold reduction.
For differentiation, human B cells (when co-stimulated with CD40L or BCR engagement) respond to IL-10 and IL-13 (at least in vitro) with heightened proliferation, Ab secretion and also Ig class switch recombination. IL-10 has little effect on mouse B cells (as shown by analysis of IL-10 ko or transgenic mice), while mouse B cells do not express the receptor for IL-13 (unlike human B cells). there are also 2 receptors for IL-4 in humans - one is the IL-4R (comprising IL-4Ra and common gamma chains) while the other is actually the IL-13R (comprising IL-4Ra and IL-13Ra). This latter receptor allows human B cells to respond to IL-4 in the absence of common gamma chain - as IL-13R is absent from mouse B cells, these cells can only respond to IL-4 using the "classic" IL-4R.
IFN-g induces mouse B cells to switch to IgG2a/2c (depending on teh strain) but there is little good evidence that IFNg has a similar effect on human B cells.
most B cells in the marginal zone of mice express unmutated (ie germline) Ig V region genes, while in humans most if not all MZ B cells have undergone somatic hypermutation and belong to the pool of memory B cells (altho there are controversies in this area as well). CD27 is a helpful marker to identify human memory B cells, but this is not the case in mice - CD27 is only transiently expressed on in vivo activated B cells, whereas in humans it comes on on germinal centre B cells and is retained as these cells differentiated to either memory of plasma cells.
there are numerous other differences, but that's enough to get you thinking....
Sorry for such a long period of silence (I've been trying to activate B cells).
For Dr Edward Clark:
As for our aim:
The effects that we observe after treatment of B cells with HIV (24, 48 and 72 h after treatment) are similar to those observed after 5-15 minutes incubation of murine B cells with anti-IgM + anti-CD40 + IL-4 (Osborne et al, PLOS, 2007). As it is known that HIV and its proteins may cause B cell activation, we decided to check if the effects we observe are similar to those observed upon activation of human B cells (24, 48 and 72 h after activation).
As for experiments in a "piecemeal fashion", I did not get it. What do you propose? To check different dosage of anti-CD-40, IL-4 or PHA for B cell activation?
As for PHA, we have tried with 10 mkg/ml, and it appeared to be quite toxic for cells (about 20% entered appoptosis after 24 h of incubation, and only about 5% were in G2 or S phase after 48 h (DNA quantity measured by FACS)). As for anti-CD-40 1G1 (Sigma) + IL-4, it did not induce apoptosis, and there were no cells in S phase 72 h after treatment. We plan to check if there are any activation markers on cell surface of our cells 48 and 72 h after treatment.
For Dr Stuart Tangye:
Thank you very much for such a review on murine and human B cells. That is quite interesting.
My little cotribution on the use of PHA (a mitogen) as a positve control for your work under discussion is that it is best to use at a concentration of 5 micrograms/ml for most immunological assays. This concentration is not usually toxic to the cells up to at least 3 days of culture.
OK. Thank you for your comment. I have seen articles, in which they use 1, 5 or 10 mkg/ml. I have already realized that 10 mkg/ml is too much, and I am going to try 1 and 5 mkg/ml now.
PHA activates lots of cells, including T cells and other cells, not a good choice for human B cell experiments, better to use defined signals, or if you want to use a mitogenic signal that will activate B cells to become antibody forming cells in a TD manner, pokeweed mitogen.
Hi Edward Clark,
I am interested in your talk about the "defined signals" ...to activate B cells into antibody producing cells. What do you mean by "in a TD manner"? And do you know besides pokeweed mitogen, what else we can use to induce B cells to become antibody producing cells?
Thank you!
Let me have a guess "in a TD manner" = "in a T-cell dependent manner" => in the ways similar to interaction between T and B cells. E.g., using anti-CD40 and cytokines.
For B cell stimulation, your 2 first options are relevant. I would not use PHA, large spectrum as others already pointed out. You amy also envisage LPS, this is unexpensive, and well established. Your chioce has to be defined accordingly to what you want to analyse (since it is a control you are looking for, is not it?).
To state activation, if you can use flow cytometry, with any of activation markers such as anti-CD80 or anti-CD86, or CD69; you can also decide to monitor cytokine production by ELISA, or both, this will give you different level of information.
PM
Defined signals as opposed to mitogens: e.g., anti-IgM, anti-CD40 or CD40L, IL-4, IL-6
As Stuart Tangye and I have already pointed out, LPS does not activate human B cells
If you don't' know what 'TD' is and have to guess, you need to take a course in Immunology.
A question for Dr Clark.
When you say that PWM activates B cells in a TD manner do you mean that the signaling is dependent on T cells or that the binding/signaling resembles that of a T cell/B cell interaction?
In my hands when stimulating rhesus and human B cell with PWM in combination with SAC and CpG (also alone) T cells were critically needed to drive antibody production (Sundling et al JEM 2010). Additionally PWM had a similar effect as PHA in B cell/T cell co-cults, due to their effect being dependent on T cell activation. However, my readout was antibody production. I guess the B cells could have gotten activated but not proliferated and differentiated to PCs.
Christopher, I meant that PWM activation of Ig secretion requires T cells. I think PWM induce IgG requires accessory cells as well as T cells and B cells and that PWM is likely to stimulate via more than one receptor, particularly at higher doses (see e.g., A novel role for accessory cells in T cell-dependent B cell differentiation. K Shiba et al. Cell Immunol. 1990. 127:458)
As there are still people who find this question/answer in the Internet, and write me to know, what we actually did to activate B cells - I will give here a short summary of the discussion and what we have done afterwards.
PHA does not activate human B cells, but it activates very well human T cells (see http://www.ncbi.nlm.nih.gov/pubmed/3263438). LPS actually DOES NOT activate human B cells. However, it can activate murine B cells that express TLR4 unlike human B cells.
For a review see http://www.biomedcentral.com/1471-2172/13/63: "In contrast to mouse B-cells, human B-cells express neither TLR4 nor CD14, the two canonical ligands for Gram– bacteria LPS, and are therefore unresponsive to LPS."
For human B cell activation we have successfully used 10-20 mkg/ml anti-human-IgM (DA4.4) + 20 ng/ml human recombinant IL-4 (Sigma) + 1 mkg/ml anti-human-CD40 (G28.5). We have been also able to activate B cells with two of these components together, for example, IL4 + anti-CD40. It should also be noted for anti-CD40 that not all monoclones work equally well for B cell activation!!! Monoclonal anti-CD40 G28.5 has been shown to work. To access if B cells were really activated we used flow cytometry for B cell surface activation markers (CD23 and CD69), 24 and 48h after activation, and BrDU incorporation studies 72 h after activation.
Here are some useful references - http://www.ncbi.nlm.nih.gov/pubmed/12748040 - a very good work where one can find also activation markers that can be used to assess B cell activation,
http://www.jimmunol.org/content/163/3/1119.long - they activated B cells with different coctails.
Good luck with your work!
Tatyana.
The G28.5 was from Biolegend (www.biolegend.com, calatog number 303602), but the last time we ordered it was in 2014, and now when you search for this clone, it seems that this product has been discontinued... Anyway, a monoclone is a monoclone. I think that if another company sells the right clone, it should be OK for your cells, if properly purified.
By the way, there are certainly other monoclonal anti-CD40 that should work, although not all of them will work. It seems that the clone HB14 sold by Biolegend, also activates B cells: http://www.biolegend.com/purified-anti-human-cd40-antibody-2361.html (you can have a look at the references they provide, e.g. http://www.ncbi.nlm.nih.gov/pubmed/20962324?dopt=Abstract).
As for antibodies from Biolegend, we have a positive experience with them in general. Good luck with your activation!
Nice and useful conversation I just stumbled upon - except for some gratuitous rudeness. Thanks all.
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