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Again we want to thank the reviewers for their comments. Indeed, looking more critically at our data we noticed that for the velocity we took into account all the cells, including the ones that were not actually migrating but “jingling around” without really displacing on the substrate. This increased artificially the mean instantaneous velocity of the whole cell population. To avoid this problem, we followed the tracks of individual cells and obtained their mean instantaneous velocities and maximum distance travelled on the gels. We considered cells as either arrested, if their maximum displacement, i.e. the maximum distance travelled, was lower than 10 μm in the monitored 5 min, or migrating. Mean instantaneous velocities are now given only for migrating T cells. We counted the number of arrested T lymphoblasts and added this parameter to the results in the new figure that shows the percentage of arrested T lymphoblasts on the anti-CD3+aCD28+ICAM-1 coated PA-gels for 2 donors ( Figure 1C ). As stated now in the new version of the Results, more T cells arrested on the stiffest (100 kPa) substrate than on the softer ones (0.5 and 6.4 kPa). Yet, the percentage of arrested cells never attained the value found on anti-CD3+aCD28+ICAM-1 coated glass slides, even when tested after 7h of culture of T lymphoblasts on the different PA-gels. These results thus suggest that at low density of TCR ligands (condition of this study) and in the range of physiological APC rigidities (Bufi et al., 2015), most of the T lymphocytes do not stop to form a synapse but rather form kinapses (Dustin, 2008). Yet, in more extreme conditions of rigidities (extracellular matrix, pathological tissues) more cells stop.

Concerning the scanning electron microscopy images shown (previously Figure 4 , currently Figure 1D ), the reviewers are right. Because of the multiple treatments of the slides and washing conditions, we probably imaged mostly T lymphoblasts interacting strongly with the substrates (arrested cells). Indeed, a handful of images of migrating cells were observed (see Mens Cale Wing Tip Brogues Hush Puppies ipYHQZryc
). This explains why the images are not consistent with the fast motility values reported. We have now clearly stated this bias in the new version of the manuscript.

Author response image 1
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Again we would like to stress that the reviewers’ points were particularly useful to better characterize the co-culture model we used in our study. However, the first thing we would like to mention is that we initially tried to culture HeLa cells on PA-gels but were unable to succeed (and thus used commercial PDMS culture gels only available at 1.5 and 28 kPa). Indeed, fibronectin coating of PA-gel surfaces was probably too low to allow adhesion of HeLa cells that cannot adhere directly to the uncharged polyacrylamide. Following the reviewers’ advice, we tried, without success, to develop PDMS substrates of higher Young moduli and of “good quality” (flat homogeneous surfaces) to compare with the commercial ones we are using. Therefore, in order to address the point mentioned by the reviewers and use one higher stiffness value, we cultured the HeLa cells directly on fibronectin coated glass slides that have a Young modulus in the order of GPa (see below).

Figure 4 with 1 supplement
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Production of () IFNγ (n: 13) and () TNFα (n: 10) on PA-gels of varying stiffness. In the presence of aCD3, the aCD3:aCD28 coating ratio was 1:10. () FACS plot of CD25 staining. A …

Figure 4—figure supplement 1
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() Production of IFNγ on PA-gels of varying stiffness; in the presence of aCD3, the aCD3:aCD28 coating ratio was 1:100 (n: 5).

() Production of IFNγ on PA-gels of varying stiffness for non-biotinylated soluble aCD3+aCD28 antibodies at concentrations of 1+10 μg/mL respectively (n: 4). () Production of IFNγ and TNFα on PA-gels of varying stiffness coated with aCD3+aCD28 only. The aCD3:aCD28 coating ratio was 1:10 (n: 5). () Production of IFNγ and IL-2 and percentage of CD69 cells for memory CD4 T cells cultured on PA-gels of varying stiffness (n: 3). Mean values with standard error are shown. For statistical analysis, paired parametric t-tests were performed: *p-value<0.05.

We then sought to confirm whether stiffness sensing by T cells required physical interaction of the TCR/CD3 complex to the gel substrate. CD4 + T lymphoblasts were cultured on PA-gels of varying stiffness coated with ICAM-1 and soluble aCD3+aCD28 were added. In these conditions, IFNγ production was not affected by gel stiffness ( FOOTWEAR Ballet flats Fendi rBpptOcoS
) showing that mechanosensing indeed requires direct engagement of the TCR/CD3 complex with ligands on substrates of varying stiffness. Moreover, in the absence of ICAM-1 (PA-gels coated solely with biotinylated aCD3+aCD28), production of cytokines still followed substrate stiffness ( Figure 4—figure supplement 1C ) showing that ICAM-1 is not essential for the stiffness-mediated modulation of cytokine production.

These results were confirmed on freshly isolated memory CD4 + T cells, which also showed increased production of cytokines (IFNγ and IL-2) and increased expression of the activation marker CD69 in response to increasing stiffness ( Figure 4—figure supplement 1D ).

Overall, our results show that TCR/CD3-induced cytokine production by effector and memory human CD4 + T cells is increased by the rigidity of substrates bearing TCR ligands and shows exquisite sensitivity within the physiological range of cellular (0.5 and 6.4 kPa) and tissue rigidity (100 kPa).

Gene expression profiles suggested that T cell metabolism was sensitive to the stiffness of PA-gels. This was in line with the fact that T cells dramatically change their metabolic activity, switching from a metabolically quiescent state to aerobic glycolysis upon TCR triggering and co-stimulatory activation ( Pearce et al., 2013 ). To confirm T cell metabolic response to stiffness, we first measured the production of lactate in supernatants of cultures on PA-gels. The presence of aCD3 on PA-gels induced more lactate production by T cells than aCD28+ICAM1 alone ( Womens Drew Salt Stone Jeans Wrangler 7WDMN4iHw
). This increase was measurable 16 hr after activation but was only significant for T cells activated on 100 kPa. After 24 hr, lactate production was significantly higher in the presence of aCD3, similar when cells were activated on 0.5 and 6.4 kPa PA-gels and more pronounced on the stiff 100 kPa PA-gel ( FOOTWEAR Lowtops amp; sneakers Bisgaard fZ7lKQUmna
). These results show that the T cell glycolytic switch induced by TCR triggering was increased by high stiffness values (100 kPa) but not by low ones (hundreds to thousands of Pa).

No. Your life is not over at 59. But you need to find a doctor, or some caregiver, who can explain what’s going on clearly and whom you can trust. Keep shopping around till you find one.


Jan: at 151 you really need B12 injections. Preferably with methylcobalamin if you can convince your doctor to use that (tell him/her studies have shown it is the best absorbed form with the lowest toxicity). Yes, 151 is low and you need to take action to bring the level back up. Another option is sublingual methylcobalamin – but I would highly recommend you do this under a health care practitoner’s care. You need to find out WHY your B12 levels are so low. If you have an absorption problem like pernicious anemia, which is not uncommon in the presence of other autoimmune diseases like MS, you will have to be on injections or at the very least high dose sublingual B12 indefinitely. I’d say go visit whichever of the docs you listed that you have the best relationship.


Chris, I got back the results of the lumbar punch today from my new neurologist and I do not show that I have MS, as the Dr. she replaced had diagnosed said. To complicate this even more, I was shown on MRI in 2006 to have a lesion on my midbrain that had grown from a previous MRI done in 2004. This is what started the ball rolling. When I had breast cancer in 04 I owned a small investment company. I had to close it but I had felt that I was not on top of my game for some time. To reenter the corp work environment at 51 was daunting to say the least so I had a full neurological work up. There was a problem in the brain stem at that time but they did not report it to me. After working for two years and struggling both mentally and emotionally, a Dr, ordered a MRI and the place had grown a bit and that was when I found out it was even there. It sent me to a brain surgeon at the hosp. where I ended up with this MS clinic that I am still seeing. He felt it had not grown but rather the MRI was slicing thinner. And that it was just showing larger. He saw spots on my brain, sent me to the MS doc. He diagnosed MS with the 261 B12 level in front of him and told me that the brain stem issue was totally gone every year since. Had disappeared altogether.

Well, it has not. The neurosurgeon thought it was a hamartoma, something that is a malformation from birth, and not a glioma. However, my spinal fluid was full of protein so now he says it is due to this brain stem “glioma”. So, seems like I have a tumor on my brain stem,, worst place ever, and a B12 deficiency. I am seeing my PCP today at 3. What am I? When this started, my tongue would get thick, I could not speak correctly, like I was on drugs, could not spell, word find, emotionally I was broken totally. Could not train for new things and retain them. Yesterday I collapsed totally, and I can hardly walk. I am in intense pain in my legs at night and my entire body hurts. Am I doomed to this thr rest of my life because I was not treated in time? What stage would you think I am in? My red cells seem ok, there has been a problem or two along the way. After my breast cancer surgery, they were way out of whack and taking iron for 6 mos corrected that. They said I lost too much blood. I am giving myself injections. But with the other B12, the least desired one. I am going to take your recommendations to the dr. today. I also feel that there is some liability on behalf of the first Dr. who ignored the B12, and there are other issues with him. There is a reason he is no longer at the clinic. The new dr. recommended that I may want to go to the Mayo Clinic also.


Thats a fine theory but keep in mind that suboptimal thyroid function could be causing lack of enzymes and weak stomach acids that can’t break down the gluten protein and perhaps there is a progression to more of a leaky gut situation that might have occurred. The whole proteins are making it into the blood stream by passing through an inflamed stomach lining where they are attacked by the immune system causing detectable antibodies.

Thyroid disfunction is notorious for causing low iron and there is often a B vitamin issue as well. This is my situation.

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Hello, this sounds like me, and I also have Rheumatoid Arthritis on top of this, and definitely a leaky gut. I am suppose to go for a Myers Cocktail injection, B injections. I do have the MTHFR factor as well. Any suggestions, for me, I have been taking HCI for my stomach acid and enzymes too. I want to heal my gut and inflammation, and the Natural doctors don’t seem to be helping me fast enough. I eat nothing processed only fresh foods. My thyroid is off again, I have anemia, and malabsorbtion problems and eat organic.

Any help would be great!

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I woluld start taking kelp. It is a natural source of iodine and should help your thyroid and it is amazing when your thyroid gets enough iodine how much better you feel. Your hair gets lustrous and your skin gets better. a majority of people in USA are deficent in magnesium so you might try taking that.


I’m living walking proof…. I hope my story will inspire you! http://www.youtube.com/watch?v=CH-N3ktF25g


I too have eaten meat and had a deficiency though it is worse when I don’t eat meat. But before it was diagnosed I had other health issues for years that doctors didn’t understand or know what to do with. These promptly disappeared when I started methyl b12


Hi Jesse,

Anyone taking Losec or any other antacid (even over the counter ones) will also have issues absorbing B12, also, people who have had stomach surgery that affects production of intrinsic factor, such as a gastric sleeve or gastric bypass, stomach cancer survivors who have lost part of their stomach, also people with bowel disorders such as IBS, Crohns or Coeliac Disease as the foods rich in B12 tend to go right past the small intestine section that absorbs the B12, people who don’t eat much red meat and prefer to eat chicken or fish, people like myself with pernicious anaemia.

B12 is a really safe supplement to take so boosting your intake isn’t going to cause any harm and may end up bringing an overall improvement to energy levels, sleep, mood and in my case, also stops me randomly passing out and falling over like a drunk person.

Toodles, m


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