Cancer Immunology - Science topic

Hi,

Check the references below:

HCMV UL83/pp65 (NLVPMVATV)

Reference: UNIPROT https://www.uniprot.org/uniprotkb/P04439/entry

CMV pp65 peptide NLVPMVATV (HLA-A*0201) is a single peptide for stimulation of T cells. The peptide from human cytomegalovirus (CMV) phosphoprotein (pp65) is synthesized as it is presented by the MHC class I HLA-A*0201 allele and can be used for targeted in vitro generation and expansion of antigen-specific CD8+ T cells.

Reference:

"Although the anti-pp65 antibodies and the pp65 antigens are detected in immune-depressed patients with active viral infection [16], the antibody response to the pp65 antigen in normal infected individuals is not always detectable by immunoblotting [17]. It has been reported that the pp65 antigen is targeted by a cell-mediated immune response and that its vaccination can induce a pp65-specific CTL response"

Reference:

or https://academic.oup.com/cei/article/143/1/167/6440089

Hope it helps,

Best regards

Tomasz

No, there are essentially 3 major types of immune response to infection: an antiviral response which illicits interferon, a pro inflammatory response that makes il1, il6, tnfalpha etc wich is normally directed against bacterial and fungal infections ( and can lead to cytokine storm) and then the allergic response directed against allergens like ova that generates il4, il10, il13.

In mouse models, balb/c mice are typically used to study allergic responses as thy are inherently more allergic and c57bl/6 are used to study proinflammatory responses and diseases because they are inherently more prone to this type of immune reaction.

As far as I understand, your data points/conditions are nested within individuals (donors) creating non-independence. In other words, the same individuals provide data for each condition. If that is true, then a paired test such as repeated measures ANOVA would be most appropriate. One way to examine this is by looking at whether the observations across conditions are correlated. For paired observations, you will typically find a positive correlation of the DV scores across conditions reflecting the non-independence of the data points.

Sorry it is outside my aera of expertise

Hi,

Very important question. Moreover, the question is why (.... from my current knowledge please correct if I'm wrong) from more less 100 mAbs approved by FDA only 2 are bispecific ..... I think another trend is competitive for the "multispecificity" of mAbs. Its cocktails of mAbs (cocktails therapy). See some examples below:

1 https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-ebola-virus

2 https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-monoclonal-antibodies-treatment-covid-19-0

3 https://www.fda.gov/media/106517/download

4 https://www.fda.gov/drugs/drug-safety-and-availability/fda-authorizes-bamlanivimab-and-etesevimab-monoclonal-antibody-therapy-post-exposure-prophylaxis

I think that cocktails therapy will be a more dynamic trend than "multispecificity" (because of higher and higher availability of different monospecific monoclonals)... and many other reasons,

Best regards

Tomasz

Interested

IsoFlow in Beckman coulter or sheath fluid in BD machine are the isotonic buffers solutions which may be replaced by Autoclaved and filter sterilized PBS.

You should be extra cautious while using in-house sheath buffer as any particle or growth may block your tubes in flowcytometer.

We have used Autoclaved and filter sterilized PBS in BD instrument and it never caused any issue.

Once you use any in-house buffer, the parent company will consider it void of warranty. So, never let the company people know that your are not using their isoflow.

Good luck,

Go to the web page of your journal of interest and register as a reviewer

First thing i would like to comment on it that you may change the clone of CD20, check CD19 as well if they are even few?

Secondary, The absence B cells might be the drug effect (Like: Rituximab or any alternative/traditional medicine).

I see that Alexandr and Maria have botj given a recipe for freeze medium above and i would like to add that post-thaw viability is increased if the freeze medium also has FBS.

Our recipe for freeze media is

- RMPI (supplemented with 1% antibiotic/antimycotic solution)

- 20% FBS (10% is fine though)

- 10% DMSO.

Anyway, i have seen each laboratory has it's own recipe so as long as it makes you happy, it's fine.

Lauren Pecorino

Molecular Biology of Cancer: Mechanisms, Targets, and Therapeutics

3rd Edición

ISBN-13: 978-0199577170, ISBN-10: 019957717X

Hi

LGR5 is the best biomarker for detection of cancer stem cells in colorectal patients .

u can browse our my profile and see our paper about that

Regards

Short answer, yes there is. The first two is almost always global, with the last one... with the potential of being more specific.

For coated antibodies, it is almost always CD3/CD28, and the coating is for crosslinking. Otherwise, it doesn't work.

As for protein... do you mean an mitogen? Like ConA? or a cytokine like IFNg? Either way, the activation pathways are different and for these, there is no need to coat the plate. They will work in suspension.

Cell stimulation using cell lines...Unless you are looking at stimulation using unmatched HLAs...Most of this is using cell lines preloaded with a specific antigen or antigenic peptide.

If you can give more information about your goal... we can give you an idea of what would be the best way to go about it.

I am so sorry for such a delay, I had not reviewed Researgate in many weeks.

Could you solve your problems ?. We have performed similar experiments and we have not had that problem, so I am not sure what it could be. One question, does your cell therapy promote tumor eradication?

I have seen that some researchers use treatments with IL-7 / IL-15 to favor memory phenotypes that last over time favoring the antitumor response.

It could also be that your B16 cells have lost the expression of OVA, do they have a probe to follow the expression of OVA ?.

I stay tuned in case you need help.

Hi Dominique,

As far as I can tell, it should work to simply add your Ki67 as a separate staining step following permeabilization. Otherwise, your protocol looks fine to me.

Good luck,

Max

Hello

CASTp, Autodock vina, Swissmodel, Qsite, Chimera, RasMol or JMOL, MOE, BIOVIA Discovery Studio.

Check in these links. It might help you.

Good Luck.

Hi Sarah Ratkovich-Gonzalez , I remember that you shoul perform the surface staining first and then stain with AV (and 7AAD for example). That is because for AV staining you need to you a "special" binding buffer.

This article may be useful:

Dear Sangeet Bhaumik

microRNAs have to provide potential signature model for various cancers and other diseases. While miRNA in terms of sequencing is difficult , though being done provides huge number of miRNA in a specific cancer as you have exemplified some in your discussion. Few important learning points:

1- Specific sets of miRNAs usually express or otherwise together and they are termed miRNA families.

2-Some miRNA have the potential to act as pan cancer biomarkers but still no specificity is provided.

3-Some biomarkers are like positive or negative acute phase reactant and may rise or fall with non-cancerous disease.

4-There is some but as i experienced little correlation between blood and tissue based miRNAs

5- The science of miRNAs is still emerging and a lot more has to be learnt i guess before they are available, if available for clinical use.

6-Also need to have complete data about pre, pri and miRNA and he cleaving proteins like DROSHA, DICER and factors incorporated in RISC complex.

The whole picture is yet to appear and but hope is there that someday they may be appearing as both for diagnostic use and therapeutic targets like Riversin (spelling ?) for treating hepatitis C.

So potential is there but more research is needed to quantify and deal associated aspects of miRNA

Sorry, that I could not help you straight as i interpret the knowledge about this subject is still evolving.

Kind regards

For more information,

IC50 for a compound/inhibitor for a cell line depends on several factors, including the cell line in question, solvent for the inhibitor, culture conditions, and proposed endpoint for the treatment. Therefore, it needs to be determined empirically under the desired conditions for the proposed endpoint (when known). If a study has used much higher concentration of the inhibitor for a similar endpoint, it could be due to due to other variables (cell line and culture conditions etc). It is always better to find out an optimum IC50 before performing an experiment.

Dear Chandrasekharan Giridhar

Aceria pongamiae a species of mite causes gall formation on the leaves of Millettia pinnata (= Pongamia pinnata).

Please have a look at these links and PDF attachment.

Good luck!

We use NOD/SCID mice in our commercial A375 xenografts (http://altogenlabs.com/xenograft-models/melanoma-xenograft/a375-xenograft-model/) and they tend to work well, with stable growth curves as well. I would make sure your starting injection is done properly (we use matrigel with 1 million cells) and then monitor for any kinds of symptoms that wouldn't correspond with normal tumor growth.

Samrat Das Thank you for our response Mr. Das. More specifically I wanted to know about Caner and Immunology, and which areas of therapeutics would be more beneficial for the future.

Hi Zoabi, I am wondering about how to extract myeloma-associated macrophages directly from the bone marrow, and I saw that you had posted this similar question a while ago. Did you find an answer to this? Best, Kristian

Following

There are multiple mechanisms that the tumor uses in order to escape immunosurveillance. One of the more important ones is the acidity of the extracellular mathrix. This is mainly due to proton extrusion. Low pH decreases the activity of macrophages and T cell lymphocytes and impedes the immunologic atack on cancer.

Hello,

I worked on invariant natural killer T cells, and I found that these cells become anergic upon activation with a specific antigen and they down regulate their specific receptor. you may have the same situation with CD8 cells (anergy + down regulating their receptors). And this is not the case when isolating healthy NKT cells.

Hi Anne and Christoph,

Thank you so much your comments and references to literature, I found it very helpful!!

Kind Regards,

Martyna

You have NOT changed the K prime (Capacity Factor) at all. You reduced the flow to half of what it was (from 1.0 ml/min to 0.5 ml/min). Your T zero time has now doubled from what it was before. Your previous T0 was ~ 2.9 minutes with a peak showing NO RETENTION at 3 minutes. Now your flow rate is 0.5 ml/min and the new T0 time is ~ 5.8 minutes with your peak continuing to show NO RETENTION at all (as we would predict) at ~ 6 minutes.

Previously, you stated that 30% ACN resulted in no retention. Yet you continue to try mobile phases with even more ACN on a RP column. Again, you would want to try mobile phase mixtures with less ACN (or MeOH mixes or just buffer) to try and retain the sample first.

I understand that you are brand new to chromatography, and apologize for sounding harsh, but you still have not taken the time to read the linked articles I offered, follow my suggestions or demonstrate any basic understanding of the fundamentals of HPLC at this point. Note: Adjusting the flow rate does not change the peak's capacity factor (it is still close to zero). Reducing the flow rate simply slows down the entire chromatography process. Your peak took twice as long to come off the column (because the column volume does not change) as before because you reduced the flow rate by a factor of two. Please change the method and most importantly of all, get some local help in learning the basic fundamentals of HPLC before you start to run samples. You will greatly benefit from some basic training at this point so you can begin to understand what you are doing. Randomly making changes is not the best way to learn. Experimenting without understanding the basic concepts about what you are doing is both unsafe and unscientific. Please get help before continuing.

To boost ADCC or direct lysis with NK you can use IL-2 and/or IL-15. But if you want only a positive control of the NK lysis you have to use a good target for NK cells such as Daudi cell line in the presence of IL-2.

If you want to produce cytokines (IFNg or TNFa) by NK, you can use IL-12 + IL-18

Hello,

I recommend an antibody database available at labome.com. For antibodies to RET for IHC you can use the following link: https://www.labome.com/review/gene/human/RET-antibody.html. Epitomics has RET antibody (Epitomics, 3454-1) used in immunohistochemistry. Also, Abcam offers RET antibody (Abcam, ab1840) was used in immunohistochemistry - paraffin section on human samples (Ding et al, Exp Mol Pathol. 2013). In addition, R and D Systems provides RET antibody (R&D systems, MAB718) used in immunocytochemistry on human samples (Fattahi et al., Nature 2016). You can check the references and the description of these and other RET antibodies following the links.

Hello,

I recommend an antibody database available at labome.com. For antibodies to GATA2 for WB you can use the following link: https://www.labome.com/gene/human/GATA-2-antibody.html. In particular, Santa Cruz Biotechnology has mouse monoclonal (CG2-96) for WB. Also, Proteintech Group offers GATA-2 ab (catalog: 11103-1-AP Proteintech Group) for WB. Invitrogen provides GATA-2 ab (catalog: PA1-100). Also, there are GATA-2 antibodies from GeneTex (catalog: GTX113441), Abnova (catalog: H00002624-D01P),  US Biological (catalog: G2021-40), Novus Biologicals (catalog: NBP1-82581), LifeSpan Biosciences (catalog: LS-C31147) and OriGene (catalog: TA312431) suitable for WB. You can check the references and the description of these and other GATA-2 antibodies following the links.

Dear Jens,

if I have understood correctly, your objective is to find lymphocytes with speficic TCR that are expaded in tumor bearing C57Bl/6J mice.

I suggest you 2 paper in which they highlighted many differences in TCR repertoire between Balb/c and tumor bearing Balb-neuT mice after immunization.

Hope this help.

Best

Sergio

There are many resources, where you can inform quite well regarding different surface stains using different protocols and different clones (e.g. CD4); I personally like cytobank a lot:

Or ebioscience to have a quick look...

Or BD...

Good luck and cheers

I agree with Isabella. You might also try Sigma for the LPS. As for measuring cytokine production using the supernatants for quantitating cytokine production by ELISA, multiplex, or other is good. Using intracellular staining/flow cytometry to look at single cell production is not quantitative for cytokine produced, but provides the frequency of cytokine producing cells. They are different data points and both may be useful depending on the context of your experiments or model.

You can try GP-62C from Progen.

By removing the beads magnetically you mean just to put them again on the magnet for 1 minute? Then pipet the cells into any other eppi? So following your suggestions  I don't loose activated T cells as the beads anyway do not stick any longer to the target (e.g. after 24hrs). Thanks in advance! 

active motif antibody for ChIP is very good.But  I am not sure if they have this product, you can go to their website and check.

Can't think of an exact place off hand.  Might want to check ATCC. 

Read through papers in your field.  Many researchers may have cells that would work for your purpose.    Additionally, many will just give you the cells so long as they are acknowledged and your lab just pays shipping.  It can be much cheaper.    And ask people around you at your institution.

Thanks Rainer! Yes, i found someone in Spain willing to collaborate on this, so all good. But thanks anyway for your kind reply!

CD34 for breast cancer

Also

This thread is old, but I have a question to Bedrich.

Step 4 says dilute sample to 20ml with DMEM (1%BSA), is this when the tumor is small and digested in an enzymatic mix (serum-free DMEM plus collagenase) of less than 20ml? If that is the case, how about if the tumor is large (flank tumor ~1000mg) and the enzyme mix volume is already 20ml at 3.

Another point is regarding the specifications serum-free DMEM at 1 and DMEM/BSA at 4, what would the consequence be if one type (either serum -free DMEM or DMEM/BSA) is used through out? 

Still one more point (one before the last), I do not see DNAse in your protocol which I usually use to digest DNA released from dead cells and reduce sample stickiness; any explanation?

LAST POINT: I know of collagenase I and IV and that collagenase I is good when downstream analysis involves lymphocytes while IV is preferred when downstream analysis is on myeloid cells, but here you mentioned collaginase A and I would appreciate it if you elaborate on its use in terms of the points mentioned for collagenase I and IV.

I am glad to hear that :)  

Chung Sub 

Search here

Hi Laura

I have attached the typical PGE2 standard curve given by R&D Systems on their website (assuming this is the same kit you are using). On a log scale the working range is still slightly sigmoidal in shape. I assume you are fitting a polynomial curve of some description to give the best fit to your calibration standards? If you are fitting a linear regression line to these standards it would explain a low R^2 value.

However, even if you are correctly fitting a polynomial curve to your standards, I would still be happy with R^2 being consistently above 0.9. You have proven by repeated trial runs that the kit is reasonably reproducible with respect to R^2 - this consistency is more important than the accuracy of your fit. While we have not used PGE2 kits, in our experience R^2>0.9 for an immunoassay is perfectly acceptable. It is more important to assess the accuracy of the kit in quantifying concentrations in your control samples.

Since this is a kit with pre-prepared reagents, there is little troubleshooting you can do other than proper storage, ensuring your plate washing is consistent and thorough and, as mentioned by Charles, that your pipetting technique is as good as possible.

Best of luck.

I extend my gratitude Dr. Sihem for taking time to look for relevant articles, answering my question and for sharing really insightful articles for my help.

I failed to fetch any scoring system after going through few of the articles and hence was perplexed which way to design my small study of cxcl14 expression in different tumors. I wish to express more..ours is a limited facility department with no molecular facility. However it is there in other departments which can be collaborated with if need ed. At the most, we can do IHC here. I have purchased CXCL14 antibody and have just started applying on some paraffin sections.

Can you please elaborate on cohort of stained tissues please.

At last I thank you again.

Besides using the above stated reagents to permeabilize cells' membrane, you could use an another well known reagent ,saponin.

Miss Nicole, The principal of this reaction is based on NAD/NADH2 and 340 is the wavelength. I hope i may not get visible color reaction.

Why not use T cell isolated from volunteers.  Just like mixed lymphocyte reaction. The cells line are convenient but may not show the truth. Good luck.

Even if the status of p53 is known in the cell line, one may require to optimize the concentration of hydrogen peroxide for a given time. Additionally, culture conditions (medium and growth factors) may influence the outcome. 

We use cimetidine 400 mg 2 x/day to reverse the MDSC. With patients.

Both ProPred and netMHCPan are algorithms that predict the likelihood that a peptide will be bound by a MHC molecule.  This is important because peptides that are not bound by MHC will not be presented to T cells and thus cannot be targeted in T cell mediated immune responses.  It is also important to note that a peptide can be bound by MHC and presented to T cells and still not be immunogenic if the organism does not have any T cells bearing T cell receptors that recognize that peptide.

I am not familiar with the ProPred output, however I find the most relevant number to review in the netMHCPan output is the predicted "Score Aff(nM)" value (i.e. the predicted binding affinity for MHC).  In the tumor neoantigen literature, the standard convention (for now) seems to be that peptides with affinities < 500nM are considered MHC binders.  

In one study from our group, most peptides that were predicted to bind at 50nM or less were confirmed by binding affinity measurements, whereas about half of peptides predicted at 50-500nM were confirmed.

Hope this helps.

Benjamin

You are most welcome, please let me know how it goes, and i might be able to help you with one of the Books (2010).   Best wishes.

Ghanim.

tanks for your tips, just few question, you permeabilized with port K before the fixation with PFA 4 % or after ?

And what il the concentration of the proteinase K ?

Thanks again

Tom

I presume this is because you suggest that the majority of the cells you isolate are leukaemic? It would be still useful to use a pan-marker to delineate a leukaemic population.

My group works on CLL, and the level of heterogeneity of spontaneous apoptosis is high. I would worry that all cells are PI+ without going through the double-positive stage first, particularly after 24h.

It could well be that you observe a toxic effect, rather than apoptosis.

Why not to stain for Ann+PI+  the cells straight ex vivo, then after shorter periods of time post-treatment with your drug, such as 1h, 2h, 4h. You should see no apoptosis as early as 1-2h post-treatment, so if the cells are dead, it would indicate toxicity. Your untreated controls should be the same as ex vivo. Likewise, if you use leukaemic marker, and your drug is specific for leukaemic cells you should be able to see if all PBMCs are dead (toxicity), or the leukaemic cells exclusively.

Hello Maryam,

Yes cell death definitely affects the exosomes...its like, entire profile change after cell death and there is also release of certain toxic cytokines into systems,..also if cells will be dead, then no healthy exosomes will be secreted as normal cellular processes will come to a halt...so, care is always taken that once you isolate exosomes, you always characterize the residual cells for cell viability and health status by Acridine orange staining or Annexin-pI apoptosis assay.That is must to confirm the viability of cells after exosome harvesting..So first, make sure that all your cells are healthy and viable and then only proceed with any of exosome related experiments...

Hope it helps !

... ni idea...

Thank You for the suggestions

I direct you to this interesting article from the Daily Dongle, a blog about flow cytometry from the same team that works on FlowJo flow cytometry analysis software.

The following protocol we use for human cells: coat plate with aCD3 OKT3 (1 µg/ml in PBS, although 0,1/0,01 also works) incubate 4 hours at roomtemperature. Wash three times with PBS and add the cells to your plate.If we do a costimulation with CD28 (15E8, 5 µg/ml) in a pre incubation so cells in a small volume (to save antibody) and then adjust cellconcentration to the wanted concentration. Depending on what you want to analyze, activation molecules/cytokines you incubate for 4-72 hrs for cell proliferatiopn >4 days. After 3-4 days you should start to see clumbs. If it's pure T-cells you use, coating with aCD3 is essential, but also when you use PBMC coating is more optimal than soluble. Good luck

I agree with Ellen. I'd like to add another point concerning B cell depletion therapies. Some chronic lymphocytic leukemias have relatively low levels of CD20 but express high levels of CD37. Thus, targeting a non-CD20 surface receptor may make sense, and not only that, the combined binding of CD20 and CD37 (which signals differently than CD20) may lead to more cell death as well as ADCC.

Thank you Stefan Koch. I wanted to use it for RT-PCR. I also wanted to check by flow if possible so thank you for your suggestion.

Dear Goodwin,

Your article (the one you attached in your previous response) is actually gorgious!!

I learned a lot and I encounrage RG members interested by "my question" to have an in-depth reading of your article!

Take care!

Robert

NK cells originally described as large granular lymphocytes, exhibited natural cytotoxicity against certain tumor cells in the absence of preimmunization or stimulation.

CD56 dim NK cells, which make up the majority of circulating cells, are the most potent cytotoxic NK cells against tumor cells.

(i) by releasing cytoplasmic granules containing perforin and granzymes that leads to tumor-cell apoptosis by caspase-dependent and -independent pathways

(ii) by death receptor-mediated apoptosis.   Some NK cells express tumor-necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL)

(iii) by secreting various effector molecules, such as IFN-c, that exert antitumor functions.

(iv) through antibodydependent cellular cytotoxicity (ADCC) by expressing CD16 to

destroy tumor cells.

Cheng, Min, et al. "NK cell-based immunotherapy for malignant diseases." Cellular & molecular immunology 10.3 (2013): 230-252.‏

IF YOU COAT THE WELLS BY AGAROSE GEL YOU CAN SEE NEUTROPHİL MOVEMENTS UNDER AGAROSE.  THE END OF STUDY YOU WİLL GET OUT AGAROSE GEL AND STAIN WELLS TO SEE MOVEMENT TRACES ON THE WELLS.

Thank you for both answers. Yes I am familiar with ILTs. However,  at the moment I am wondering whether HLA disparity could be theoretically recognized between allogeneic antigen-presenting cells (macrophages, DCs) in the absence of lymphocytes. This has not been addressed anywhere and in my opinion most likely does not exist in nature. But also true... nobody has addressed this so far...

We use 10ml Liberase TL (307ul of 5mg/ml in HBBS) with 25ug/ml DNAse II. Our protocol is to add the liberase/DNAse mix, mince the skin into small (<2mm) pieces using scissors, add 25ug/ul dispase and incubate 37degC for 2 hours in a shaker.

Take it out every 30mins, then pipette up and down using a serological pipette to separate the clumps. After the 2 hours, add an equal amount of media (we use fibroblast), filter through a 70um strainer, pellet the cells at 400g 5mins, tip off supernatant.

This has given us the best yield vs. viability from any previous skin dissociation protocol.

Removal of unbound crystals from the surface of macrophages is quite straightforward with three washes with ice cold PBS supplemented with 5mM EDTA (remember that EDTA needs to be made up as a 0.5M stock solution first with warming and pH adjustment to ph 8.0 so it goes into solution). The washing protocol is described in our paper describing macrophage phagocytosis of MSU crystals in 2000 (Yagnik DR, Hillyer P, Marshall D, Smythe CD, Krausz T, Haskard DO, Landis RC. Arthritis Rheum. 2000 Aug;43(8):1779-89). Good luck with your experiments.

When you use cells for compensation antigens may indeed have an influence. Are you using the brightest condition for each fluorescence during compensation? I mean if you are working with a marker that is increased upon stimulation, than your stimulated cell should be the sample used for compensation. But that shouldn't be a problem when you use beads.

I've never used eFluor780 but I read that it's excited by the red laser and detected by the 780/60 band filter. So I believe it might have some spill on APC (and vice versa). The same as for FITC/PE (but considering blue laser excitation).

What comes to mind is:

Dear Akshaya 

Please follw my collegue Ahmad Darwish , he is really expert in this area , his profile contains alot of his papers .

Good luck. 

This is a major problem ocurring more and more often in labs.

Here attached you have two comments about this problem and also people who you could contact to bring you a precise response.

Best regards

Robert

thnx Baskaran

Dear Beatrice, Thanks a lot, that is very helpful!

Anne

I realise this is an old thread - I'm having trouble getting hold of anti-mouse PD-1 (clone G4 ideally), would any one have access to this (and be willing to ship to Belfast, expenses paid!) or recommend a commercial alternative?

Thanks in advance!