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Pathology Grand Rounds: January 12, 2023

January 13, 2023

The Pathophysiology of Multiple Sclerosis, by David Hafler, MD, FANA

ID
9372

Transcript

  • 00:00At all to this nice, auspicious start
  • 00:03of the new Grand Round series for 2023.
  • 00:07And I'm happy to have Doctor David
  • 00:10Hafler here is our speaker today.
  • 00:13So since 2009, Doctor Heffler has
  • 00:16been the William S and Lower Styles
  • 00:18actually professor and chairman
  • 00:19of the Department of Neurology,
  • 00:21Professor of Immunology, Immunobiology
  • 00:23here at Yale and his neurologist
  • 00:25and chief of the hospital, David.
  • 00:28He's a he's a clinical research scientist
  • 00:30with an interest in understanding the
  • 00:32path of pathogenesis of inflammatory
  • 00:34CNS diseases by studying
  • 00:36both basic properties of.
  • 00:38Due to regulatory pathways in humans
  • 00:40and they run this function in patients.
  • 00:42But as went on, I won't show you all
  • 00:44the ruminations over the 20 odd years,
  • 00:46but with 47,000 patients,
  • 00:4968,000 controls by identified,
  • 00:51233 genetic variants,
  • 00:53all these have been replicated.
  • 00:55All the original ones are
  • 00:57similarly replicated,
  • 00:57which counts for about half the
  • 01:00estimated heritability for Ms.
  • 01:02So that's great.
  • 01:04You can show these wonderful little figures,
  • 01:06but how did the variants cause the disease?
  • 01:09And I think this remains one of
  • 01:11the major challenges for you,
  • 01:12not modern medicine as it's relatively
  • 01:15easy with these technologies.
  • 01:17It wasn't so easy in 2000 where we are
  • 01:20now with different aluminum athlete
  • 01:22technologies to identify genetic
  • 01:24variants with big patient cohorts.
  • 01:27I think the challenge is how to
  • 01:29go from variance to disease.
  • 01:31So this is an effort.
  • 01:32Collaborate effort with Alex Morrison,
  • 01:34Kyle Farr and Brad Bernstein.
  • 01:36We together did genetic and
  • 01:38epigenetic fine mapping of autoimmune
  • 01:40disease variants and these data up.
  • 01:42They'll publish about 5-6 years ago.
  • 01:45I think it's still hand up, pulled up.
  • 01:48So what we did we turns off for a second.
  • 01:50So you all know DNA goes to RNA
  • 01:53goes to protein, you'll learn that.
  • 01:56And so to go from DNA to RNA,
  • 01:58DNA has done wine very well and
  • 02:00there has to be ways so that poll
  • 02:03two another enzymes can get to the
  • 02:05DNA so you can have transcription.
  • 02:08Well you can then use things like
  • 02:10K27 installation,
  • 02:11K4 methylation maps to identify where
  • 02:14this open chromatid on different cell types.
  • 02:17One can then take those data and
  • 02:19overlay them with genetic variants,
  • 02:21arguing that if a genetic variant,
  • 02:24it's a region where there is
  • 02:26no open chromatin,
  • 02:27that variance is not going to be
  • 02:28playing a role that's help with
  • 02:30every place there's a genetic
  • 02:31variant for chromance,
  • 02:32it's open,
  • 02:33then it's likely to be an influencing
  • 02:35that cell type.
  • 02:36That's what we did as part of the
  • 02:38ENCODE project with Brad Bernstein.
  • 02:40Our lab participated in generating the K27K4
  • 02:44methylation maps of human immune cells and.
  • 02:47We have different diseases.
  • 02:49We have neurologic diseases over here.
  • 02:51Here is urate levels,
  • 02:53renal function, kidney disease,
  • 02:55cholesterol and here the autoimmune diseases.
  • 02:58We'll concentrate on Ms.
  • 02:59If you look at Ms.
  • 03:01We found the genetic variance,
  • 03:04the P values of less than 10 to the minus 30.
  • 03:07We're hitting immune cells.
  • 03:09That wasn't surprising T cells.
  • 03:11Macrophages T regs.
  • 03:13But also what was a bit surprising
  • 03:15is there were hitting B cells,
  • 03:17and more so in Ms.
  • 03:19most any other diseases.
  • 03:20If you look at other autoimmune diseases,
  • 03:23it wasn't the case,
  • 03:25say for lupus and primary biliary serositis,
  • 03:29suggesting that B cells play a
  • 03:32critical role in the disease.
  • 03:33Somewhat unfortunately,
  • 03:34around the same time my dear friend
  • 03:37and colleague Steve Hauser made the
  • 03:39observation paper published New England.
  • 03:41Channel,
  • 03:42if you perform B cell depletion,
  • 03:45this is the two different
  • 03:47studies offer one opera 2
  • 03:48compared to the standard treatment
  • 03:50that time barred interferon.
  • 03:52The 9897% decrease in new lesions dramatic
  • 03:55effect and I would just say clinically
  • 03:57will we see a patient we start them on
  • 04:00B cell depletion when we have a patient
  • 04:03who doesn't respond usually isn't Ms.
  • 04:05that's how good that drug is right now.
  • 04:08So these data fit in very
  • 04:10nicely with our observation.
  • 04:12All of these cells in the disease,
  • 04:14but those are those in your
  • 04:16biologist and neuropathologist.
  • 04:17I apologize,
  • 04:18we did not get hits in the brain.
  • 04:22Now I will say, and I won't share Tom
  • 04:24and show the data today with the paper
  • 04:27that is going to be coming out in
  • 04:29nature from our from our consortium,
  • 04:31identifying 2 haplotypes associated
  • 04:33not with the risk of developing Ms.
  • 04:37but with progression.
  • 04:38And these are helpful types which
  • 04:40are found in neuronal cells.
  • 04:42So it's a separate question
  • 04:43of what causes the disease,
  • 04:45what leads to disease progression.
  • 04:46If you have the risk capital type,
  • 04:48your likelihood of progressing
  • 04:51is significantly increased.
  • 04:53So how do you go from Snips
  • 04:56to to functionality?
  • 04:57So we found hits the enough Capital Region.
  • 05:01There are part of the far paper we
  • 05:04found that steps on the NF Kappa B
  • 05:06binding sites across the genome.
  • 05:08And there's snips happen in the
  • 05:10haplotype social NF Kappa B.
  • 05:12Now it's about 10 to the minus 12,
  • 05:14so they're genetic variance here.
  • 05:16However, the odds ratio is about 1.1,
  • 05:19so not a big effect.
  • 05:21So I want to show you.
  • 05:23Just even though the odds ratio is low.
  • 05:26It has a major biologic fact.
  • 05:28In fact, about 1819% of eugenic kits Ms.
  • 05:32are in the NF Capital B and the
  • 05:35TNF NF Capital B signaling region.
  • 05:38So this is published now 7-8 years
  • 05:41ago by will housing our laboratory.
  • 05:43But basically about 20% of these
  • 05:46healthy subjects.
  • 05:47About 20% of you here are GG's.
  • 05:51Do you know enough to know who you are?
  • 05:53Is your homozygote for
  • 05:55this particular variant?
  • 05:57About 20% of you were A and the
  • 05:59rest of you are had to reside in.
  • 06:01If you are G,
  • 06:03you have about a 20 fold increase
  • 06:06in P50 NF Kappa B.
  • 06:08Activity where if you're a it's
  • 06:10significantly less and we every time
  • 06:12we've looked at a genetic variant,
  • 06:14this is what we find that the biology
  • 06:16of these are quite striking and even
  • 06:19though maybe 1.11 point one to the 233
  • 06:23power becomes a very major effect.
  • 06:29So in summary of the genetics big picture,
  • 06:32the genetics of autoimmune
  • 06:34disease dictates lower activation
  • 06:36threshold of different cell types,
  • 06:37including TH 17 cell B cells and T regs
  • 06:41in the dozen or so genes we've looked at.
  • 06:45I also say we've just started a
  • 06:47collaboration with Steve Robbins just
  • 06:49recruiting here from the Broad Institute.
  • 06:51We had these wonderful techniques
  • 06:53of looking at genetic variants
  • 06:54and different cell types look
  • 06:56into effect on motor function.
  • 06:57So there are tools emerging which allows
  • 07:00us to take a whole genome approaches.
  • 07:02So getting back to the question
  • 07:03I started with,
  • 07:04you know the cause of multiple sclerosis,
  • 07:07here's our working model.
  • 07:10That's the genetically,
  • 07:11as I said earlier, it's a genetically
  • 07:13mediated autoimmune disease.
  • 07:15It's initiating the periphery by T
  • 07:17cells and macrophages that traffic
  • 07:19into the central nervous system.
  • 07:22So the the idea is that there are
  • 07:24microbial antigens likely cross reactive
  • 07:27with myelin that something happens to
  • 07:29activate these antigen presenting cells.
  • 07:32Probably be sales, probably EB though
  • 07:35there's no biology behind that yet.
  • 07:37There's expression of
  • 07:39costimulatory molecules.
  • 07:40We have activation of viral
  • 07:42reactive T cells now we all have
  • 07:44autoreactive T cells in our blood.
  • 07:46I could clone mold reactive T
  • 07:48cells from menu in this room.
  • 07:50And a number of years ago,
  • 07:51back in in the late 80s,
  • 07:53we developed technologies for looking
  • 07:55at autoreactive T cells were able to
  • 07:57show for the first time that there are
  • 08:00in fact autoreactive T cells in humans.
  • 08:02Highly robust response and we identified
  • 08:05a dominant epitope Amal and basic
  • 08:08protein are recognized as 84102 region
  • 08:10which went on to to find how it
  • 08:13bounded MHC in a form post doc in the lab,
  • 08:16kyouka fennick with Don Wiley went
  • 08:19down to crystallize this these clones
  • 08:22recognizing this epitope with the T
  • 08:25cell receptor and MHC but was more.
  • 08:28Interesting to me was the fact that we
  • 08:31also found reactivity in healthy individuals.
  • 08:34Significant reactivity
  • 08:34led to decades of work.
  • 08:37Why do we have autoreactive
  • 08:38T cells in their circulation?
  • 08:40This is work done by will count here,
  • 08:42published in STM about seven years ago.
  • 08:46This is principal component analysis
  • 08:48looking at T cell reactivity using a T
  • 08:51cell library approach against published.
  • 08:53Just point out that the paper no peptide
  • 08:55control anger can the myelin peptides.
  • 08:58You can see that the red is Ms.
  • 09:00patients that they tend to go off to GMCSF
  • 09:04gamma in 17 whereas healthy individuals.
  • 09:08The main reactive T cells need
  • 09:10aisle 10 suppressive soda comma,
  • 09:12which makes terrific sense.
  • 09:14And if you do single cell cloning
  • 09:16rather than the library approach,
  • 09:18you can see that and help the individuals.
  • 09:21They tend to make aisle 10 with single
  • 09:24cell they tend they make all ten,
  • 09:27that is Ms.
  • 09:28patients making 17 GMCSF less gamma.
  • 09:31So suggest that these aisle 10 secreting
  • 09:33cells and all of us may play a role.
  • 09:36For example one has damage.
  • 09:38The brain stroke other factors that
  • 09:41these cells may circulate into the
  • 09:43nervous system involving scar formation.
  • 09:46So.
  • 09:48What we find is,
  • 09:49so we have this situation,
  • 09:51we have aisle 10 secreting cells
  • 09:53in healthy individuals,
  • 09:54but you also have regulatory T cells.
  • 09:56Look to both TR1 and Fox V3 cells which
  • 09:59are preventing this from happening.
  • 10:02But multiple sclerosis is a loss of
  • 10:04these Fox P3 regulatory T cells.
  • 10:06I'll show you some recent unpublished
  • 10:09data related to PRDM one.
  • 10:12So the hypothesis is that
  • 10:14in healthy individuals,
  • 10:15these T regs prevent activation of
  • 10:18autoreactive T cells where's Ms.
  • 10:20patients are defective.
  • 10:21This is work done by Dizzy Begleiten,
  • 10:23Claire Batch Allen,
  • 10:25published now almost 20 years ago,
  • 10:28which was the first demonstration
  • 10:29of T Reg dysfunction in the human.
  • 10:31Autoimmune disease,
  • 10:32these are all new ones that untreated Ms.
  • 10:35patients or healthy donors and
  • 10:37this is the presence of oppression
  • 10:39perforation different ratios of T
  • 10:42regs and you can see this market
  • 10:45demolition diminish chip of Reg
  • 10:47function in vitro in patients with Ms.
  • 10:50and the same thing been found in type one
  • 10:53diabetes everyone to arthritis went on
  • 10:55to show that the T regs and patient Ms.
  • 10:57work done by Margaret Dominguez
  • 10:59Pierre is that these T regs and MSN.
  • 11:02Making games that Fearon.
  • 11:03Here we took T Reg,
  • 11:05stimulated for four hours of PNA on a
  • 11:07mycin and measured gamma secretion,
  • 11:09purified populations and using
  • 11:12sample control,
  • 11:13aisle 17 versus gaming can see this gamma.
  • 11:16They all express.
  • 11:17Foxp 3 is a summary of these data
  • 11:20that these T regs were making.
  • 11:22Gamma,
  • 11:22and I'll just point out I'll show
  • 11:24a little bit in a few minutes
  • 11:26that dysfunctional T Rex.
  • 11:28What's happening is they go from
  • 11:30suppressor cells to effector cells.
  • 11:32And start making game interferon.
  • 11:34So an Ms.
  • 11:35is not just bad genes, not bad environment,
  • 11:37but the bad interaction between
  • 11:39genes and the environment.
  • 11:40It's very interested again
  • 11:43in environmental influences.
  • 11:44The instance of Ms. stops at 2000,
  • 11:47but it's continued to increase.
  • 11:49Ms.
  • 11:49Crohn's disease, type one diabetes,
  • 11:51continues to increase.
  • 11:52And of course that can't be genetics.
  • 11:55So the pathophysiology of Ms.
  • 11:58will involve genetic environmental
  • 12:00factors which lead to the immune response.
  • 12:05So well just give background on this about.
  • 12:09We started looking at microbiome
  • 12:11versus TH 17 cells in the blood and
  • 12:15we started looking at dietary history
  • 12:17and we found that if you ate at a
  • 12:20fast food restaurant more than twice
  • 12:22a week you had increased dial 17 cells.
  • 12:25Statistically significant.
  • 12:27We said really and said,
  • 12:29well wasn't the golden
  • 12:31arches provide that and salt.
  • 12:33So we did an incredibly simple experiment.
  • 12:36This work done by Marcus Kletzel,
  • 12:38we added salt to the culture.
  • 12:40And the same time,
  • 12:42my dear friend and colleague Vijay Kutru
  • 12:44was looking at TH 17 cell induction,
  • 12:47identified SGK one as critical
  • 12:49inducing TH 17 cells.
  • 12:51We are very we do lab things together.
  • 12:54We're very closely he told that SGK one,
  • 12:57I told him that salt and we
  • 12:59did the papers in parallel.
  • 13:00I'll just do a little just on terms
  • 13:02of we couldn't have started competing
  • 13:04who could get this out first?
  • 13:06But we're much more clever than that.
  • 13:08We worked in parallel, sent the papers.
  • 13:10Back-to-back to a weekly journal.
  • 13:14And when the editor said, well,
  • 13:16did you know about each other's work,
  • 13:17we said no, not really,
  • 13:20but no one would have believed
  • 13:22that salt did this,
  • 13:23but having two laboratories
  • 13:24showing the same thing at a much
  • 13:27more credence to the work.
  • 13:28So it was and we've gone on to
  • 13:31look at the effect of of salt and
  • 13:33other factors in terms of fat
  • 13:35in terms of T Reg function.
  • 13:37So I'll just show you some of these data.
  • 13:39They're really quite remarkable.
  • 13:40If you add 40 milliequivalents
  • 13:43of salt to a culture,
  • 13:44you have logarithmic increases,
  • 13:46dial 17 and M RNA and and secretion.
  • 13:50They may be saying yourself,
  • 13:51this is artificial right concentration.
  • 13:54Salt and blood is about 150,
  • 13:56which is what sea water is, turns out
  • 13:59the concentration of salt in tissues.
  • 14:02Is higher, it's about 18190 and
  • 14:05blood is a suppressive condition.
  • 14:08You don't want T cells being
  • 14:10activated in the peripheral blood.
  • 14:11So when T cells traffic into the tissue,
  • 14:14they're in the condition of leading to more
  • 14:17activation which is what we were seeing.
  • 14:19I also say that a recent paper by
  • 14:22another group in Germany showed
  • 14:24increased salt concentration using MRI
  • 14:26magnets in tissue skin tissue of Ms.
  • 14:29patients compared to the controls.
  • 14:31So we also this work done.
  • 14:33By the talented graduate student
  • 14:34of the NADPH Mandatory Hernandez.
  • 14:36And she showed that if you add
  • 14:38salt to T regs.
  • 14:39So here we have T Reg effector cells,
  • 14:41we load them with a green dot at the very
  • 14:43green that stimulates them not dead.
  • 14:44With the dye at T regs to go
  • 14:46from here to here,
  • 14:47they suppress entering the cell,
  • 14:49cycle through the same thing
  • 14:50with sodium chloride,
  • 14:51they lose functionality.
  • 14:55And the mechanism, if you look at SGK
  • 14:58one would solve it goes up in the T regs.
  • 15:01You can knock down the SGK one with the
  • 15:04short hairpin RNA and then if you look
  • 15:07at function here's effective function.
  • 15:10If you knockout SGK one you go from
  • 15:12control to here and restore function.
  • 15:15So it was happening so also inducing
  • 15:17SGK one with gamma difference secretion
  • 15:20and T regs leading lots of function.
  • 15:22So I'm presenting to you the importance
  • 15:25of SGK one as a central factor.
  • 15:28And loss of T Rex function.
  • 15:31So come back to that in a moment,
  • 15:32one of the great surprises in my life.
  • 15:35So then the question what's the
  • 15:37transcriptional circuit driving
  • 15:38dysfunctional foxies through positive
  • 15:40regulatory T cells in autoimmunity?
  • 15:42That's can we identify a master
  • 15:44regulator of T cell dysfunction.
  • 15:47Let me just say this is work
  • 15:49done by Thomas Sabita,
  • 15:50who started as a postdoc is now in the system
  • 15:52professor and there's really represents his,
  • 15:55his original work.
  • 15:56So what we basically did was performed a
  • 15:59comprehensive transcriptomic and epigenomic.
  • 16:01Profiling doing bulk and
  • 16:03signals of RNA seek attack.
  • 16:06Seek for epigenetic regulation genome
  • 16:08wide for chromatid accessibility.
  • 16:11He did transcription factor footprint
  • 16:13analysis and accessible chromatin regions and
  • 16:16look to the E QTL effects of Automeris Lucci.
  • 16:18I'll just just show data on the 1st 2:00.
  • 16:21This is a whole one hour talk in itself.
  • 16:24And then we did a CRISPER activation
  • 16:26based validation of this.
  • 16:28It's regulatory elements getting
  • 16:29at the molecular mechanism.
  • 16:31So it's amazing what we can
  • 16:32do in human biology now.
  • 16:34It's unimaginable years ago.
  • 16:35So first of all let me show
  • 16:37you what we found.
  • 16:39Found increases in PRDM 1.
  • 16:42Now for those of you who are mouse
  • 16:44people who say this doesn't make sense.
  • 16:47It's it's well known that PRD one
  • 16:49increases T Reg function announce
  • 16:51T cells and I'll show you what
  • 16:54what it was and that it we found
  • 16:56that it drives a dysfunctional
  • 16:58sheets something like program.
  • 17:00But what's happened is not the
  • 17:02long form that's increased it's
  • 17:03a short form which inhibits the
  • 17:05long form and here's the kicker,
  • 17:07it drives SGK one of all the kinases and
  • 17:11proteins that could have been induced by
  • 17:14the shore former PhD one it was the SGK 1.
  • 17:17So let me now I showed you the results,
  • 17:19let me show you the data.
  • 17:20See here we looked at T Reg,
  • 17:22this is doing vulgar in DC looking at
  • 17:26overlapping differential expressed genes
  • 17:28between memory T regs and seating for itself.
  • 17:31So you can see this market
  • 17:34increase in PRDM one,
  • 17:35BCL 3 pin three will also regulated.
  • 17:38These are all induced by PRDM one and genes
  • 17:41are downregulated by PRDM one like ID 3,
  • 17:44LBH were down regulated.
  • 17:45So we had this increase in.
  • 17:47PRDM one did a replication of the set of
  • 17:51patients and showed here that the PRDM
  • 17:54one is upregulated in patient with Ms.
  • 17:57but again confusing because
  • 17:59of the mouse data.
  • 18:01It's all about mice of course,
  • 18:03but then we learned that there are two
  • 18:06isoforms in humans that don't exist in mice.
  • 18:09Dry nosed mammals do not express a
  • 18:12short form of PRDM one and so the
  • 18:16short form the dominant negative.
  • 18:18When we looked in normal cell types,
  • 18:20we found that the short form is what's
  • 18:23expression memory cells and T regs,
  • 18:24but not in B cells.
  • 18:27So then we looked at the short
  • 18:29former period you want by PCR,
  • 18:30and indeed it's this short
  • 18:32form that's increased NS,
  • 18:33not the long form.
  • 18:36And we rapidly perform Western
  • 18:38blotting to show that indeed the
  • 18:41short form is what's induced.
  • 18:43We then looked at the data sets in
  • 18:46particular set by oted all that's
  • 18:48published in cell and we found that
  • 18:51the PRD one isoform PRD one is also
  • 18:54increased in most autoimmune diseases.
  • 18:56So appears to be a common regulator of in
  • 18:59T regs among different autoimmune diseases.
  • 19:02And of course PRD one drives the
  • 19:04blimp one and we looked at blimp
  • 19:06one expression and MST regs it was
  • 19:08in and it was it was increased.
  • 19:10So it wasn't memory T Rex and memory T Rex.
  • 19:13By flow. By both PCR and flow.
  • 19:18So then here's the experiment
  • 19:20where we transfected PRD one into
  • 19:22T regs and integer cat cells.
  • 19:24We have the induction of SGK one and here's
  • 19:27measuring SGK one and primary union T Rex.
  • 19:30See this? When we overexpressed
  • 19:31the short form of T regs,
  • 19:34you had an increase in SGK one expression.
  • 19:39And then if you go in and do all the
  • 19:40other experiments, not get SGK one,
  • 19:42you lose the loss of T rate function but the
  • 19:45short form in T Rex become dysfunctional.
  • 19:48The whole story came together.
  • 19:50So this suggests to us that the short form
  • 19:52of PD one may be critical in different
  • 19:55autoimmune diseases and driving dysfunction.
  • 19:58And again we believe it's related to
  • 20:00salt and to genetic factors that will
  • 20:03show the data particular CD toward the
  • 20:05genetic variant in CD28 who drives a P1.
  • 20:10Now switch gears and show some public,
  • 20:13recently published work looking at
  • 20:15T cell traffic between blood and
  • 20:17the CNS and the single cell work.
  • 20:19So.
  • 20:20T cell traffic into the CNS is very tightly
  • 20:23regulated and CXCR 3 positive cells
  • 20:25are the ones that get into the brain,
  • 20:28crossing the correct plexus
  • 20:30near great interest,
  • 20:32and they get into the brain.
  • 20:34And what I'll show you is a T cells
  • 20:36in the central nervous system are
  • 20:38CXCR 3 positive and express Tibet
  • 20:41and make gamma interferon.
  • 20:43We believe that this relates to the
  • 20:45fundamental observation by the late
  • 20:47Ben Barris that astrocytes are driving
  • 20:49homeostatic communication would not.
  • 20:51This is Michael Glee up but also are
  • 20:54driving through secretion of cholesterol
  • 20:56and TGF beta are driving this T bet
  • 20:59induction that isn't the T cells go
  • 21:02into the nervous system then they'll
  • 21:04you in the brain drives this function.
  • 21:07So give them on the knowledge of
  • 21:09particular genre pop or Lotto submitter.
  • 21:11Our Krishnaswamy and David Vandyke and Lee
  • 21:14sang together did this worker with us.
  • 21:17And basically we took spinal
  • 21:20fluid group of patients,
  • 21:21isolated the spinal fluid homonuclear cells,
  • 21:25perform 10X the usual way that
  • 21:28most you're familiar with now.
  • 21:30And then we perform this on 6 healthy donors,
  • 21:34get the bed in the moment, 5 patient with Ms.
  • 21:36And looked at over 100,000 cells
  • 21:39into 50,000 T cell receptor.
  • 21:41Now does show high level summary
  • 21:43of the of this work.
  • 21:46So first in terms of blood
  • 21:48versus spinal fluid,
  • 21:49what we found wasn't surprising that the
  • 21:51majority of cells in the spinal fluid
  • 21:54or T cells what we observed before.
  • 21:56So we started first bikes adding a
  • 21:58spinal fluid from patients with Ms.
  • 22:00right blood spinal fluid.
  • 22:02We found the spinal fluid was very inflamed.
  • 22:05I love this picture and we're sitting
  • 22:07at the immunology repeat this change
  • 22:09that we need to do controls goes that
  • 22:12means we have to do spinal taps on age
  • 22:14match 20 something year old students
  • 22:17to do that. So Full disclosure.
  • 22:20I do not know who would spinal taps, I said.
  • 22:25I do not want to know because I
  • 22:27do not want to be accused
  • 22:29of coercion, collusion.
  • 22:30So only Jenna knows who volunteered.
  • 22:33They're all de identified.
  • 22:35But at the I acknowledge all these wonderful
  • 22:37students who had Spinal Tap stuff.
  • 22:40So these cells acquire an
  • 22:42inflammatory signature.
  • 22:43So just quickly the menu now fate
  • 22:46where the cell progression of
  • 22:48blood and CSF release fate maps.
  • 22:51This is a fading out.
  • 22:52The red is blood, the blue is CSF
  • 22:55via very different characteristics.
  • 22:58They sense potential of heat diffusion,
  • 23:00if any based transition embedding.
  • 23:03I love the words they come up with and
  • 23:05that world, but it's a way of looking at it.
  • 23:08Unsupervised visualization that looks
  • 23:10at geometric distance between data points.
  • 23:14So here's the original tissue
  • 23:15on the fate map.
  • 23:16We can define the CD.
  • 23:188 cells are here,
  • 23:20CD4 cells are here.
  • 23:22And we were able to describe we took
  • 23:24the top 10 differential expressed
  • 23:26genes in each cluster and to find
  • 23:29different populations naive cells,
  • 23:31naive CD4,
  • 23:32CD 8 and really three different
  • 23:34populations CSF cell we called CSF
  • 23:371/2 and three in memory CD8 cells.
  • 23:41So one could do something very interesting,
  • 23:44which is looking at the continuum
  • 23:46between blood and spinal fluid,
  • 23:48getting back to the point of how T
  • 23:51cells change function going to tissue.
  • 23:53So we merge the fate to fusion
  • 23:56operator with the original identity
  • 23:57of itself come up with a tissue score
  • 24:00which is basically the probability
  • 24:02of transitioning from one form to
  • 24:05another in a random walk and you
  • 24:07can see the Tisha score that their
  • 24:09cells are very blood like over
  • 24:11here CD4CD8 cells in transition
  • 24:13and cells is very CSF like.
  • 24:18And so I did have a reality test.
  • 24:21Ensuring that tissue
  • 24:22score captures no biology.
  • 24:23ITG Force is the LA4CD49D and
  • 24:27requires this T cell traffic
  • 24:28into the into nervous system.
  • 24:30It's a treatment for Ms.
  • 24:32blocking T cell traffic and you can see
  • 24:35that it's expressed predominantly in the T
  • 24:38cells and transition CSL against the XR3,
  • 24:41KEMA concord and fatty cell
  • 24:43entry predominant expressed.
  • 24:45It's expressed only really
  • 24:47in PCNSL CR7 Express 9 T.
  • 24:51So she excluded from the brain
  • 24:53and you can see that it's almost
  • 24:55exclusively in the peripheral blood.
  • 24:58So we've found nine clusters
  • 24:59of T cells and spinal fluid.
  • 25:01We use the gene expressions imputed
  • 25:04with something called magic because
  • 25:06basically looked at gene expression
  • 25:09across the gene expression patterns.
  • 25:11You see different patterns
  • 25:13across different tissue scores.
  • 25:14Again,
  • 25:15all the details in the paper have
  • 25:16the 9 clusters and I want to start
  • 25:18with the teach one cluster and
  • 25:20think about single cell data.
  • 25:22There's so many things you
  • 25:23can explore with it.
  • 25:25So to me,
  • 25:26I find a few interesting stories,
  • 25:27validate them biologically,
  • 25:29get the data out in the literature,
  • 25:31and I've been so pleased by how many
  • 25:33others have taken our data and use
  • 25:35it for really important experiment.
  • 25:37That's what we're trying to do.
  • 25:38So even treat bothers TH1 cluster.
  • 25:41So if you look at the TH with the CD
  • 25:44four cells to different populations,
  • 25:47the CSF 3 populations particular
  • 25:49and high amounts of gabito Ferrum
  • 25:52tibets CXCR 3 run 3 stat.
  • 25:544, so an aisle 12 receptor,
  • 25:57another CD4 population of tissue
  • 26:00resident markers like lag 3CD-69
  • 26:03and PRDM 1 interestingly enough
  • 26:06and markets have soda toxicity,
  • 26:08a grandson and grandson K,
  • 26:10which is interesting.
  • 26:12Colleague Michael Brenner at Harvard
  • 26:14recently identified grandson Kay
  • 26:16as being involved in complement
  • 26:18deposition and finally CDA population
  • 26:20markets at tissue residence and,
  • 26:22not surprising cytotoxicity.
  • 26:26So to summarize a lot of data,
  • 26:28what do we find in the blood cells
  • 26:31excluded from entering the CSF and
  • 26:33we found cells that are rich for
  • 26:36for traits necessary for entry
  • 26:38and then finally markers for CSF
  • 26:41entry and tissue dependent changes.
  • 26:44So in CSF we found gamma interferon
  • 26:47signature rest in T cells,
  • 26:49cholesterol homeostasis,
  • 26:50TGIF beta pathway and these
  • 26:53cohabitated receptors will get
  • 26:55to that in just a moment.
  • 26:57So then the question is,
  • 26:58do we actually see gamma difference
  • 27:00creating T cells from spinal fluid?
  • 27:03So first we looked at PPD one.
  • 27:05So this is from another three healthy
  • 27:08subjects looking at blood versus CSF.
  • 27:10This is no stimulation,
  • 27:114 hours of stimulation with PM out of
  • 27:14mice and see this market expression of PD1.
  • 27:17And see itself compared to blood
  • 27:20with stimulation goes even higher.
  • 27:22So there is very high expression of this
  • 27:25Co inhibitory stepter in spinal fluid cells.
  • 27:28We then looked at that together,
  • 27:29interferon response with blood and
  • 27:31you can see this major gamma signature
  • 27:34recapitulating what we found with
  • 27:37the RNC data and compared to blood.
  • 27:40So this is major gamma signature
  • 27:42and T cells from spinal fluid.
  • 27:44I'll show you another experiment
  • 27:46which I found interesting.
  • 27:47This is work we've done.
  • 27:48We did looking at PD1 glioblastoma
  • 27:51and basically we took the PD1 high,
  • 27:54PD1 intermediate,
  • 27:55PD one negative and total T cells
  • 27:58stimulated them and not surprisingly
  • 28:00PD1 high cells do not enter cell cycle.
  • 28:04But they did make gamuts to pharon.
  • 28:073% to 50 to over 50%.
  • 28:10So it suggests to us it's phenocopies,
  • 28:13what we see in the brain,
  • 28:14the cells in the brain,
  • 28:15they're condition in the brain have
  • 28:18high amounts of combinatory molecules
  • 28:20that make gaming jefferon and we wonder
  • 28:23is this what immune privilege is?
  • 28:25Is that what if you privilege
  • 28:27the high expression?
  • 28:28Comunitar molecules can enter the cell cycle,
  • 28:31but they are functional.
  • 28:33So now we know normal spot on the floor.
  • 28:35What about multiple sclerosis?
  • 28:36Did the single cell analysis
  • 28:39say looking at the populations
  • 28:41between healthy blood and Ms.
  • 28:43or no difference?
  • 28:44Not surprising.
  • 28:45We never found any differences before.
  • 28:48But if we look at log fold changes,
  • 28:49I'll just highlight some of them
  • 28:51were just beginning to work out
  • 28:53what these different factors being.
  • 28:54We're intrigued by mallet.
  • 28:56Mallet one which is involved in
  • 28:59gene expression and epigenetic
  • 29:01modulation of gene expression.
  • 29:03We found I 32 it's a pro
  • 29:07inflammatory cytokine induces
  • 29:08TNF alpha associated with Ms.
  • 29:11And we found June a part of the AP1 bonding.
  • 29:16I won't show all these
  • 29:17dates to talk in itself,
  • 29:18but we looked at healthy Ms.
  • 29:20versus non expanded versus expanded itself.
  • 29:24It's tremendous clonal expansion
  • 29:26these cells and again looking at
  • 29:29the different populations within
  • 29:32the aisle 32AP1 and there was
  • 29:34more distinct in the clone expand
  • 29:37itself both in CD4 and CD8 cells.
  • 29:39I think the next decade will
  • 29:41be taking these various.
  • 29:42A fact is we found replicating
  • 29:44them by protein and then seeing
  • 29:46how they involved in Ms.
  • 29:48induction.
  • 29:49But this is really a road map as genetics
  • 29:52work road map for what drives and drives
  • 29:55autoimmunity in the nervous system.
  • 29:57And of course,
  • 29:57what about the brain?
  • 29:58I couldn't resist being a pathology group.
  • 30:02So here we did characterize T cells
  • 30:05in normal human brain prank them up
  • 30:08here different cluster patients.
  • 30:09Some of these involve fresh RNC from
  • 30:13brain that provided by Jack Intel
  • 30:16doing epilepsy surgery and here
  • 30:19are the T cells over here at the
  • 30:21very end and different populations.
  • 30:23While summarizing here we're
  • 30:25looking at the tissue residence and
  • 30:27functional gene expression and T cell
  • 30:30with normal brain prank comma and.
  • 30:32You know,
  • 30:33I mentioned we saw this game
  • 30:35interferon signature.
  • 30:36In the spinal fluid and we see here
  • 30:39in the brain this is RNA seek up
  • 30:42T cells right out of the brain.
  • 30:45Here is with Duke seek his major
  • 30:47gaming different signature.
  • 30:48So these data indicate that the gamma
  • 30:51different signature is there in the brain.
  • 30:53Speculate at the end what that
  • 30:56might be doing also Joe work from
  • 30:58Tomo samita done with Andrew Wang
  • 31:01out loud that's predominant humans,
  • 31:03we do mice, we work with vice people.
  • 31:06And this is data from a few months ago and
  • 31:08she's getting barrage of write her thesis.
  • 31:11But basically we wanted to look at T cells
  • 31:13isolated directly from the house brain.
  • 31:16So we saw this in humans.
  • 31:17The question is to see it in mouse brain.
  • 31:20And so here we're looking at T cells
  • 31:23isolated directly from parenchymal tissue.
  • 31:25We wash out the vessels
  • 31:27and work with HomeGoods.
  • 31:28Lander suggests he T cells
  • 31:29are in the prank him up.
  • 31:31He's gamma to Fearon on the X axis.
  • 31:36Case in the CD three you can see this
  • 31:38prominent cabinet different signature in
  • 31:41CD4CD8 cells as we saw in the brain with
  • 31:4440% that cells are making game interferon.
  • 31:47You can see this here,
  • 31:48but you don't see it,
  • 31:50you don't see it in the
  • 31:51in the peripheral blood,
  • 31:53you only see it in the nervous system.
  • 31:55So it suggests that these Gavin
  • 31:57difference between T cells are
  • 31:59physiologic and won't show that the data.
  • 32:02But if you put if you do this
  • 32:04experiments and germ free.
  • 32:06Animals work done with Noah Palm,
  • 32:08they don't make these cells.
  • 32:10These gamma different secreting T cells
  • 32:12are being driven by gut microbiome
  • 32:15and if you label these T cells.
  • 32:17In the gut with the dye that turns
  • 32:20color with the fluorescent probe,
  • 32:22you can show that all these cells in
  • 32:24the brain are coming from the gut,
  • 32:26similar to what BJ's Country did.
  • 32:29And E model.
  • 32:30But this is normal Physiology,
  • 32:33so we can speculate why is it that T
  • 32:36cells in normal central nervous system?
  • 32:39Our country from the cotton
  • 32:40what are they doing there?
  • 32:41Nature doesn't do this for no reason.
  • 32:44I'm sort of speculate that maybe at
  • 32:46night when you have the lymphatics and
  • 32:48you clean your brain out these T cells
  • 32:51that surfing along secreting gamma
  • 32:53influencing the microglia experiments
  • 32:54were battery to begin it's to look
  • 32:57at Tibet gamma knockouts to see what
  • 33:00happens to synaptic pruning and what it
  • 33:03does to the microglia influencing the
  • 33:06neuronal excellent interactions and by.
  • 33:08In summary,
  • 33:09so is there.
  • 33:11Is there glial tea sub
  • 33:13communications circuits?
  • 33:14This is again from light
  • 33:15Ben Barris sowing TGF.
  • 33:17Beta and cholesterol drove these together.
  • 33:20And after I drive TJF,
  • 33:22painting classes are required for my
  • 33:24survival and these sizeable factors are
  • 33:26produced by astrocytes including cholesterol,
  • 33:28lipoprotein which are found in CSF.
  • 33:31I won't show the data but if you take
  • 33:33spinal fluid or cholesterol and TGF beta,
  • 33:36it drives gamma interferon
  • 33:37almost as much as Isle 12.
  • 33:39So we think the cholesterol in the
  • 33:42brain brains what mainly cholesterol
  • 33:44is driving this gamma driving this
  • 33:47gamma and what it's unknown function.
  • 33:49Gamma interferon in the CNS.
  • 33:52It's known to involve in chemical
  • 33:55production record Plexus.
  • 33:56Gamma has known neuroprotective
  • 33:58function like glutamate clearance,
  • 34:00neuronal survival.
  • 34:01We speculate synaptic pruning
  • 34:04and work done by Yoni Kipness
  • 34:06published a few years ago in nature.
  • 34:09So if you knock out gamma to Ferron in mice,
  • 34:12they developed depression.
  • 34:13How do you measure depression mice?
  • 34:14I don't know, but they clearly had changes
  • 34:17in behavior until they meet them working.
  • 34:20That with a number of individuals
  • 34:22and psychiatry department I can
  • 34:23tell you that if you put animals the
  • 34:25germ free environment and get rid
  • 34:27of these games to creating cells
  • 34:29that market changes in behavior.
  • 34:31So it is psychiatrist one field,
  • 34:33neurologist another.
  • 34:34But what's beginning to happen feels
  • 34:37are colliding of course centered
  • 34:39around their inflammation but to me
  • 34:41the normal Physiology the discovery
  • 34:43of gamma difference between T cells
  • 34:45that are normal Physiology which arose
  • 34:48from studying disease is actually.
  • 34:50More interestingly enough,
  • 34:51observations please to how how things work.
  • 34:56And one other experiment again which
  • 34:58I didn't show the data for is you can
  • 35:01ask the question well what if he's
  • 35:02teased cell receptor as a barcode?
  • 35:04To look at this identical T cell
  • 35:07in spinal fluid versus the blood.
  • 35:10Does it change or is there
  • 35:12a selective migration?
  • 35:13The answer is it changes.
  • 35:15The T cells in the blood that share
  • 35:18the same T cell receptor sequence with
  • 35:21cells in the CSF have very different
  • 35:24characteristics the CSF cells.
  • 35:26Have the gamma signature and other PD.
  • 35:29One signature with the identical
  • 35:31T cell in the blood does not
  • 35:33have markets knife cells,
  • 35:35so this provides strong evidence that
  • 35:37what's happening as the T cells migrate
  • 35:39into the central nervous system,
  • 35:41they're acquiring these phenotypes.
  • 35:44So in summary,
  • 35:45all immune disorders are complex
  • 35:48complex genetic diseases where genetic
  • 35:50variants mapped to the immune system
  • 35:52in MSB cells drive the inflamed
  • 35:55Mon reactive CD4 cells instead.
  • 35:57Let me just comment on EB for a moment.
  • 36:00May have heard that beautiful paper
  • 36:02by Alberto Mascaro clearly showing
  • 36:05that if you are a he looked at
  • 36:081,000,000 recruits in the army.
  • 36:10Identified individuals are EB
  • 36:12negative and follow.
  • 36:14Deerfield their light chains come CPK
  • 36:16the brain shows brain damage and he
  • 36:20showed that the on the serial samples
  • 36:22they collected that when NFL went up
  • 36:25in the serum followed by diagnosis of Ms.
  • 36:28it was 49 and 50 ton
  • 36:31preceded by EB infection.
  • 36:32You have to look at a million
  • 36:34people to find that.
  • 36:35But incredibly provocative
  • 36:37data that's EV trigger Ms.
  • 36:40what's the experiment we need to do.
  • 36:42There's one key experiment.
  • 36:44Which we're working on,
  • 36:46which is to vaccinate patients at risk.
  • 36:49So there's no EV vaccine out there now?
  • 36:53Ohh Danner GSK have one and I'm on a
  • 36:55group of devices trying to convince
  • 36:57GSK to do a subset with the clinical
  • 36:59trial patients at risk that we
  • 37:01can vaccinate and prevent disease
  • 37:03as we prevented SP with measles
  • 37:05vaccination that be the defendant
  • 37:08rather definitive evidence we've
  • 37:10not been able to find any biology
  • 37:12B we've looked in the brain of Ms.
  • 37:15patient just interviewing our graduate
  • 37:17student he said you haven't published
  • 37:19much on EV why haven't you go we've
  • 37:21been looking we haven't found any.
  • 37:23In your own publishing that data right.
  • 37:25But what we have your IRB approval
  • 37:28to do now and start shortly it's
  • 37:31a do tonsil aspirates it,
  • 37:33wants it in this patience.
  • 37:34EB lives in the nasal pharynx.
  • 37:37Then you have any ideas how to
  • 37:39do this but we do singles RDC can
  • 37:41do CV expression so we can fund
  • 37:43the EB signature we once and Ms.
  • 37:45patients but of course there may
  • 37:47be gone by the time we do it.
  • 37:49So once again summary of the
  • 37:52talk autoimmune disorders.
  • 37:53Particular mass or complex genetic diseases,
  • 37:56genetic variance mapped to the
  • 37:58immune system and MSB cells.
  • 38:00We believe Dr.
  • 38:01Inflame months specific CD4 cells in the CNS.
  • 38:05Also mentioned that we're doing
  • 38:07single cell RNA sequencing pre
  • 38:08post treatment THC stated about
  • 38:10two weeks all we've been spending
  • 38:12now year analyzing the data but
  • 38:14the most promising we're seeing
  • 38:16would be cell depletion myeloid,
  • 38:19express myeloid, induction of TNF.
  • 38:22You might say, are you kidding me?
  • 38:24It's working by inducing
  • 38:27inflammatory cytokine.
  • 38:28But go back to the clinical trial,
  • 38:30anti TNF makes Ms.
  • 38:32work works great in IBD and RA.
  • 38:35So the data is now suggesting that
  • 38:37TNF induced by the B cell depletion
  • 38:40is leading to TNF secretion which
  • 38:43then induces increased T Reg
  • 38:45function with TNFR 2 receptor
  • 38:47doing the single cell analysis.
  • 38:49So we'll see where that goes.
  • 38:51Yeah I showed you the T cell
  • 38:53traffic between blood and spinal
  • 38:54fluid and brain tightly regulated
  • 38:56showed the TH one signature and
  • 38:58that the blood and sees that for
  • 38:59functionally different indicating
  • 39:01the CNS shapes homeostatic T cell
  • 39:03states that happens all the tissues
  • 39:05at T cell center in the tissues.
  • 39:08This is phenotypic difference
  • 39:09in healthy and masks and control
  • 39:11particularly more expanded cells and
  • 39:13we're beginning to explore what these
  • 39:15are what they mean and this help us
  • 39:17teach one signature seen healthy
  • 39:19brain and of course with the PRT.
  • 39:21And positive teabags think we may have
  • 39:23identified a major transcriptional
  • 39:25factor driving autoimmunity.
  • 39:28So let me end by thanking I I put
  • 39:31here the members of the lab who made
  • 39:35major contributions work Jenna Pappalardo,
  • 39:37who is now out in West Coast,
  • 39:40an industry.
  • 39:41Tomo, who's assistant professor Tomia,
  • 39:43graduate student,
  • 39:44please thank assistant professor
  • 39:46in our department.
  • 39:47Others in the lab now is the former lab
  • 39:49members who contributed the work I discussed.
  • 39:52Today,
  • 39:52Matt Lincoln and the PRD one projects
  • 39:55computational Margot who did the
  • 39:57work with the TH1 T Rex phone
  • 39:59contact and the Center colleagues
  • 40:01of the Broad Institute.
  • 40:02In particular Brad Bernstein
  • 40:04analyst Kellison,
  • 40:05Chuck Epstein who worked with us,
  • 40:07the PRD one project collaborators
  • 40:09at the MI Year in Yale Genetics and
  • 40:12Marcelo and Yang who we feel are
  • 40:14part of our Neuro inflammation group.
  • 40:17So thank you for your time and just
  • 40:19say here's my e-mail and here's
  • 40:21our last picture of our.
  • 40:22Ms.
  • 40:22Group if we have a lot of meetings
  • 40:25every week, all winter, not really.
  • 40:27But anyway,
  • 40:28thank you for giving me the
  • 40:29opportunity to talk to.
  • 40:30I really appreciate it.
  • 40:43So do we know who's in the chat?
  • 40:46That is OK. Good story here.
  • 40:55OK, question.
  • 40:58Thank you for a great thank you.
  • 41:01What about this are those? Who
  • 41:05are they?
  • 41:11You should ask.
  • 41:13Unable to defrag the hard problem.
  • 41:18Here's how to approach.
  • 41:19Do you have a collaboration?
  • 41:23Change. And what we're doing is we're
  • 41:27taking the T cell receptor, thousands of
  • 41:29T cell receptors, popping them into
  • 41:31reporter cell lines and then using
  • 41:33antigen libraries see what they react to.
  • 41:36We're also have tetramers loaded with
  • 41:39different peptide libraries barcoded.
  • 41:42We do single cell and pull them
  • 41:43out and see what they're reacting
  • 41:45with so far, guess what?
  • 41:46Answer we're finding the spinal fluid
  • 41:48across different patient, anything else?
  • 41:52Beebe. Whether it's primarily I
  • 41:56don't know but we also see that the
  • 41:58activity and what we're doing you
  • 42:00know we can I can tell you that
  • 42:02team cell rachamim based approach
  • 42:04that's and tells me one thing right.
  • 42:06So you'd like non hypothetical need
  • 42:08approaches so we're halfway in the
  • 42:10project and hopefully hear so the
  • 42:12better idea to do the same thing
  • 42:13with cancer antigens or they'll know
  • 42:15and guess what they're recognizing.
  • 42:18Don't just rent apart.
  • 42:24The train.
  • 42:31Yeah, the traffic.
  • 42:36Of the possible.
  • 42:41Requires.
  • 42:48Or.
  • 42:50Stop it.
  • 42:56Sure.
  • 43:07What happens with the message? All Star.
  • 43:19K1 did not qualify. In the South. So.
  • 43:27But since it's increased solid,
  • 43:30expect to see it. But you know.
  • 43:35And then?
  • 43:38And yes, it's laughing.
  • 43:43Disease.
  • 43:45That's where we had she is thought
  • 43:48to subtractive stuff because
  • 43:50these papers the post office.
  • 43:54How much is that?
  • 43:57That there's whereas I think
  • 43:59we have a good working model
  • 44:02for relaxing the EMS not yet to
  • 44:04discover but a good model I have
  • 44:06no idea where cost of progressive.
  • 44:10Before we had trees
  • 44:11that will be 50%.
  • 44:15Now the most important question
  • 44:18here is that we have a plan.
  • 44:23Question.
  • 44:26The station.
  • 44:29Progressive disease.
  • 44:31But that's a major question by suspected
  • 44:34to be progressive disease cells stay
  • 44:36there trapped in the back and forth
  • 44:39that we might see on the sausage.
  • 44:42Is it really correct?
  • 44:43That's terrific questions and
  • 44:45we'll go back and then you have to.
  • 44:48The second version.
  • 44:51This is really interesting people,
  • 44:53normal people have T cells are going.
  • 44:56They're making their gambling responsibly.
  • 45:02Yeah, So what are the time scales involved?
  • 45:07T cell recognizes something
  • 45:09that wasn't changed.
  • 45:10You might not know, obviously, but you.
  • 45:12Visioning that it's terms of making
  • 45:15a change there and then having
  • 45:16some response back afterwards,
  • 45:19well, I I could do it my side.
  • 45:24New brand. I can get
  • 45:26spinal fluid but drain it.
  • 45:29But now that it's very
  • 45:30radically so, it happens. If
  • 45:31we so pre, we don't see them at
  • 45:34the time of reading when you start
  • 45:37acquiring the microphone and
  • 45:39that's when we started seeing it.
  • 45:40That happens very quickly.
  • 45:42So we label them,
  • 45:43I can answer that question.
  • 45:44We might label them in
  • 45:47the gut within 2-3 days.
  • 45:49So, so the experiment I did as
  • 45:51as a postdoc.
  • 45:53The terrific experiment.
  • 45:54There's some new thing called
  • 45:57monoclonal antibodies and we
  • 45:58want to put them into people.
  • 45:59No one has really done
  • 46:00that yet. So we're kind of cowboy.
  • 46:03Please stop the recording now. So I've got 5.
  • 46:09So what we did was
  • 46:10we had invited.
  • 46:15Two and I did it with my 5.
  • 46:21Make these. And we said, Gee,
  • 46:24if we don't find material across state lines,
  • 46:27that would be Massachusetts.
  • 46:29You don't need that TA approval.
  • 46:32That now, but you know IRB approval.
  • 46:35So we're very careful what we did,
  • 46:37we had RV approval and what
  • 46:39we do and so we injected.
  • 46:43And to our patients and we showed
  • 46:46that major biological effects
  • 46:48but they're now sanctified
  • 46:50after one child human anti mouse
  • 46:52antibody would deactivate them. So
  • 46:55we only had to make.
  • 46:58But 11 experiments that anti CD 2
  • 47:02N IG23 coded all the T cell but
  • 47:04did not cross into the central
  • 47:06nervous system showed that.
  • 47:08So I would do this thing.
  • 47:10That's a new call, Microsoft you guys.
  • 47:13There was one solicitation to
  • 47:15Harvard Medical School at the
  • 47:16time and I did the first thing
  • 47:19pretreatment did with respondent.
  • 47:20Few days respond that did the same thing.
  • 47:23We got anti mouse and everything but.
  • 47:25Then we did this that it's standing
  • 47:28after the treatment and majority of
  • 47:30cells lit off the coat anti mass and
  • 47:32said what's going on here? Let's just
  • 47:33screw it up again.
  • 47:37Oh my God they're covered with
  • 47:39antibody with mouse antibody.
  • 47:40So we use this way of labeling all
  • 47:43the peripheral blood T cell and that's
  • 47:46being traffic into the CNS policy.
  • 47:48And because everyone looked
  • 47:50at the blood brain barrier,
  • 47:5180% of the cells traffic within three days.
  • 47:56How did they do it?
  • 47:59Found the entry right before crossing.
  • 48:01Well, because when we took
  • 48:03that we couldn't find the party even though.
  • 48:10No.
  • 48:13Now that doesn't listen all
  • 48:16the circulating T cells.
  • 48:23Invite them. So it's suggested
  • 48:26you can follow the connection.
  • 48:30So it's suggested and that nice people
  • 48:33have gone to replicate that more output.
  • 48:36So the traffic of T cells from the blood
  • 48:39to the nervous system side is very fast.
  • 48:42And I think it's continuing this intro data,
  • 48:43but there are three different
  • 48:45scenes of population in CSF.
  • 48:47One and three are about basically residents.
  • 48:50LCF 2 looks like sales and traffic,
  • 48:54so one opposition doesn't have CD-69
  • 48:57was like a population is garden.
  • 48:59We have a question in chat. Yes, yeah.
  • 49:03What's the concordance of Ms.
  • 49:05and identical twins?
  • 49:06That might be a good group in
  • 49:07which to consider prophylaxis.
  • 49:09And a second related question,
  • 49:10is there any handle on what
  • 49:11causes spontaneous remission?
  • 49:13And by these days goes untreated,
  • 49:15Nope, no one goes and I want to
  • 49:17comes to Yelp goes untreated.
  • 49:18Well occasionally they don't
  • 49:19want to do a patient want.
  • 49:21So the answer Jeff is about
  • 49:233040% and yes that would be a
  • 49:25great group to consider for
  • 49:27prophylaxis but it's a small number,
  • 49:30it's two small numbers.
  • 49:31So what we're doing in the Rs
  • 49:33study is developing the tools
  • 49:35to develop to identify average
  • 49:37patients of intake first.
  • 49:38So the question is what is the incident Ms.
  • 49:41in daughters of patients with Ms.
  • 49:43about one in.
  • 49:4430 It's quite high and we can do
  • 49:47polygenic risk score and increase
  • 49:48it even more so and then we can
  • 49:51use NFL we were fully light chains
  • 49:53to follow them so that's why I'm
  • 49:56proposing GSK first as a subset take
  • 49:595000 and first degree relatives
  • 50:01children at risk before they become
  • 50:03EB positive seriously follow them
  • 50:05with NFL NFL go up MRI them I think
  • 50:09that's attractable study to do.
  • 50:10That's how we're going to try to
  • 50:12do it will cause spontaneous.
  • 50:14So what So what relapse,
  • 50:15it's a really good question.
  • 50:17What happens I think in Ms.
  • 50:19is that it's there's an acute
  • 50:21event there's the attacks occur
  • 50:23very quickly within a day within
  • 50:26forty 2448 hours they come on,
  • 50:27it's T cell trafficking into the CNS.
  • 50:30There is Dima that breakdown,
  • 50:32the barbarian barrier,
  • 50:34gadolinium enhancement.
  • 50:35I think it's the edema that's
  • 50:37causing neurologic symptoms with
  • 50:39time there is retraction,
  • 50:41edema goes away,
  • 50:41the blood brain barrier is closed
  • 50:43and rather than having a big lesion.
  • 50:45With a tiny scar,
  • 50:46like it's just just normal Physiology
  • 50:49of of the lesions resolving as
  • 50:51edema goes away and steroids
  • 50:52makes that happen faster.
  • 50:54I think that's what's happening.
  • 50:58That one. Alright.
  • 51:02Well, thank you very much to David.