January 10 2024 - The Immune System of Infants: Not Just Miniature Adults

Name: January 10 2024 - The Immune System of Infants: Not Just Miniature Adults
Uploaded: 2024-02-02T15:35:28.2166667Z
Duration: 55 min 47 s

February 02, 2024

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00:02I'm going to. So Lisa is able
00:05to log in, but not as host.
00:07So what we need to do is open the webinar.
00:11Once the webinar is open,
00:13I will make her a host From there.
00:14It is open. It's open. Oh, OK.
00:17Sorry about that. It's OK Then
00:20let me share my. There she is. There she is.
00:25OK. Thank you. Lisa, you're on.
00:28You're perfect. Oh, my God. Just
00:32relax. Don't worry.
00:36Hi, everybody. We we're
00:38just getting struck. We'll
00:39get started in just a minute.
00:40Allow people to arrive.
00:43We're having some initial
00:47technical difficulties,
00:47but I think we've resolved them.
00:49And I'll start with announcements
00:51in about one minute,
01:14right. Looks like a lot
01:17of people have arrived.
01:17Welcome to Grand Rounds.
01:20And I have a few announcements
01:22before our speaker introduction.
01:26So our upcoming grand rounds next week for
01:29Zana Pashankar who is in our own section
01:32of paediatric hematology and oncology,
01:35we'll be talking about germ cell tumors.
01:38And then on January 24th,
01:41we will have another installment
01:43of our Marjorie S Rosenthal
01:46stories in Pediatrics in 2024.
01:49So that's always a tremendous experience.
01:52I encourage you to join
01:54us. There is no commercial
01:57support for this Grand Rounds
01:58and no conflicts of interest.
02:03And as usual, the Grand
02:05Rounds is accredited for CME.
02:07There's the number to text to the
02:13CME office on your mobile device
02:16and you'll get credit as long
02:18as you're registered with
02:20that office for CME.
02:23And then please use the Q&amp;A or chat or
02:26any questions you might have at the end.
02:29And with that, I'm going to turn
02:30it over to Mark Mercurio who's
02:32going to introduce today's speaker.
02:39Good afternoon.
02:40Our Chief, the neonatology. Chief,
02:42Senator Taylor is is off this meeting.
02:44And so I get the honor of introducing
02:46today's grand House Speaker who
02:48is Doctor Lisa Connaco. Dr.
02:49Connaco is an associate professor
02:51with a prime appointment, of course,
02:53in our department in Pediatrics,
02:54but also secondary appointments at
02:56OBGYN as well as in immunobiology.
03:00She also serves Yale through
03:01work as a member of the Human
03:04Translational Immunology Program,
03:05the Program in Translational Biomedicine,
03:08and the Center for Systems
03:10and Engineering Immunology.
03:11Lisa joined Yale in 2020 in the
03:14midst of the pandemic fund.
03:16She moved her lab and her family
03:18to New Haven and she came to Yale
03:20from the University of Pittsburgh.
03:22She was brought here because
03:23of a tremendous background,
03:23already wonderful accomplishments
03:25in the field.
03:26She went to college at Brandeis,
03:29got an MD and her PhD from
03:31Tuff School of Medicine,
03:32completed Pediatric and Neonatology
03:34fellowship at Boston Children's and
03:36a post doctoral fellowship in Mucosal
03:39immunity at Boston Children's Hospital.
03:42She was also a fantastic candidate
03:44to bring here because achievements
03:46elucidating in the immune system developed.
03:50Now her work has been integral to
03:51our realization that while the
03:53early immune system is maturing,
03:54it's already highly functional
03:56and this functional is critical
03:58to short and long term health.
04:00And Yale.
04:01She's developing A rigorous program in
04:03developmental immunology with federal
04:05and foundation funding and a wide
04:08range of innovative collaborations
04:10both local and international.
04:12If that's not enough,
04:13but Doctor Kanikova also is working
04:15in developing the next generation
04:17of scientists in her lab,
04:18in the classroom and of course,
04:20in the newborn intense security.
04:22We are delighted and honored to have
04:24Lisa as a member of our session.
04:27And she's got a long list of
04:29words and accomplishments.
04:30I'll just let you know of one award,
04:32which is in 2023,
04:33she she was given the New England
04:35Perinatal Society Mentor of the Year.
04:38That's one of just many.
04:39So today,
04:40Doctor Konnikov is gonna speak
04:41about the immune system of infants,
04:43not just miniature adults.
04:45And with that,
04:46I will turn it over to Doctor Lisa Konnikov.
04:48Welcome, Lisa.
04:50Thanks so much, Mark.
04:53It's really an honor to present here.
04:57And I guess the pandemic is
04:59continuing with some COVID,
05:00but I will do my best to make
05:05it through the grand rounds.
05:07Is it in this correct view?
05:10I can't really tell.
05:11You need to put it in presenter mode.
05:14That is that good?
05:15There you go.
05:16That's good.
05:17Perfect.
05:19So I have no conflicts of interest to
05:22disclose fortunately or unfortunately.
05:23And I thought I would start with
05:26some quotes to sort of see how far
05:28we've gone in the past 20 years.
05:30So the first one is from neonatal
05:34immunology by Sri Lanka and Infante
05:37from seminars with perinatology.
05:40And the neonate,
05:41whether premature or normal gestational age,
05:44is a unique host from an
05:46immunologic perspective.
05:47Many components of the immune
05:49system function less well in
05:51neonates compared with adults,
05:52giving rise to the concept of what
05:55we sort of commonly referred to
05:57as immunodeficiency of immaturity.
06:00Now it's this beautiful issue about 2 1/2
06:04three years ago in science that really
06:07started to reshaping this conversation.
06:09And so one of the quotes from this issue,
06:11and that's sort of the illustration here,
06:14is although the precise rules of
06:16specific immune cells during development
06:18require further investigation,
06:19the system as a whole displays
06:22malleable and responsive properties
06:24according to the developmental
06:26needs and environmental challenges.
06:28And another quote from the same
06:30issues suggests evidence is also
06:32mounting that the immune system
06:33programming that starts early in
06:35life may influence the risk of
06:37developing conditions such as allergic,
06:39autoimmune,
06:40reproductive and neuropsychiatric
06:41disorders later in life.
06:43Sort of all suggesting that maybe it's not
06:46as immature as we had previously thought.
06:48And maybe it's actually critically
06:50important not only to the babies
06:53that Mark and I take care of,
06:55but also to adults.
06:58And so I thought I would first start
07:00out with some observations about
07:02infants and their immune system,
07:04then sort of focus on what we thought it was.
07:07But it really isn't.
07:08And then offer some novel insights
07:10from our group and others on what
07:13it might be and then finish up with
07:15a couple of thoughts or ways to
07:18optimize neonatal and lifelong health.
07:20So the first thing that I thought
07:21I would start with is that young
07:23children are at high risk of
07:24severe infection and that's sort
07:26of the common prevailing thought.
07:27And I think that's incredibly true.
07:30This is a map of where sepsis and
07:33neonatal deaths are incredibly common.
07:35And you can see that sort of pretty much
07:38covers almost the entirety of the world.
07:40If you look at neonatal deaths,
07:43almost a third of them,
07:44if not a little bit more are actually
07:47due to infection disease causes.
07:50That's also true if you look at
07:52children under 5 S infection.
07:53Disease related deaths are incredibly
07:56common even in children under 5.
07:59If you look at particular hospitalizations,
08:01this is focusing on the US You can
08:05look at respiratory syncytial virus or
08:07influenza and this is incredibly recent data.
08:10You can see that the peaks are very high,
08:12particularly in the young.
08:14And so for sure for RSV that peak is
08:18highest for zero to four age group.
08:21But even for influenza though,
08:22it's pretty high.
08:23In the elderly population,
08:25the second highest peak is in the young.
08:29If you look at
08:32mortality rates in children overall,
08:36again both RSV and flu have a
08:38very high odds ratio in the those
08:41less than one years of age.
08:43But that's not true for all diseases and
08:46particularly for the COVID-19 one that
08:49we're ultimately familiar with recently.
08:51And I think I fall into this age group,
08:55but you can look and see that
08:57the in the zero to 4H group,
08:58it's actually by far not the
09:01highest and it's incredibly rare
09:03in children to be hospitalized.
09:06And even if you focus and you say
09:07well that's a pretty wide gap,
09:09right, zero to four,
09:09but even if you focus on zero to six months,
09:12so in the very young that again
09:15is still pretty low.
09:17So not all infections are common
09:20in in young children.
09:22The second sort of prevailing
09:23thought is that young children
09:25don't mount good vaccine responses,
09:27and that's certainly true.
09:29So this is a pretty old study,
09:31but it's a it's a review of a lot of
09:34studies looking at the measles vaccine.
09:37And you can see that overall if
09:40you divide kids into those younger
09:43than the nine months and those
09:44that are older than nine months,
09:46overall kids don't mount as good of a
09:50vaccine response than sort of older
09:52than one year old or nine months infants.
09:56But that's not true for all vaccines.
09:58There are some vaccines that
09:59actually work beautifully.
10:00So for example this is a pertussis vaccine
10:02where influence were immunized and then
10:04you re stimulate it with PTX or pertussis.
10:06You can see that they actually
10:08mound a beautiful response.
10:10The same is true for hepatitis
10:12B and actually surprisingly,
10:14infants mound a much better
10:15hepatitis B response than adults.
10:17And you can see sort of after three doses,
10:20the crossbars here are the infants,
10:22the clear bars are adults
10:24that although the you know,
10:26the range is pretty wide,
10:27infants mount a much better response to
10:30hepatitis B immunization than do adults.
10:33And that's true after a pose booster as well.
10:39Another interesting sort of observation is
10:41that not only do infants mount a better
10:45response to some vaccines than adults,
10:48infants in particular,
10:49when certain vaccines are given,
10:52have a reduction in the overall mortality.
10:56So one of these vaccines is BCG.
10:58So a number of studies have shown
11:01that if you give BCG early in life,
11:04there is a sort of a ranging
11:06from 6 to 72% but some degree of
11:10decreased mortality in infants.
11:12And this is also true
11:14for the measles vaccine,
11:16although not as strong as the BCG vaccine.
11:19You can look here that
11:20even in premature infants.
11:21So this is a study from Denmark that
11:24looked at infants less than 2 1/2 kilos,
11:28so premature but not severely
11:31premature infants.
11:32You can see that the overall,
11:34if you gave BCG early,
11:36this is the sort of the dashed
11:38line here both neonatal and
11:42infant mortality decreased.
11:44And this is a concept that we
11:46know now as trained immunity where
11:48there's some primary stimulation
11:50being BCG vaccine or some other
11:53vaccine that resets how the immune
11:55system responds and then responds
11:58much better to secondary stimuli.
12:00So something else that might have
12:03caused death in those kids because
12:05they had early stimulation with BCG
12:07vaccine no longer is fatal to those.
12:12So this sort of concept suggests that
12:15stimulation with the right adjuvant at
12:17the right time can improve immunity.
12:19And this is sort of the concept
12:21of window of opportunity that I
12:23think infants really belong in.
12:26And so of course,
12:27this together brings what I call sort
12:29of the paradox of neonatal immunity, right.
12:32I think it's much more complicated
12:35than just immature, right.
12:37Of course, I showed you data that there's
12:39sepsis and poor vaccine responses in infants,
12:42but by far vast majority of
12:44instruments actually quite healthy
12:45and there's some vaccine responses
12:47that they beautifully mound.
12:49And so that starts a lot of questions.
12:52Is neonatal immunity really mature?
12:54Is it just not adult immunity? If so,
12:58maybe it just serves a different role, right?
13:00And is it site specific, right?
13:02Is your immunity in sort of your
13:05circulation or your blood different
13:07from that that mucosal sites?
13:09And of course, if all of those are true,
13:11when does it first start to develop?
13:14And if you think about what do we ask
13:16of neonatal immunity or fetal immunity?
13:19The requirements are actually drastically
13:21different from what we ask for in adults,
13:23right?
13:23So in the fetus and the neonate,
13:25by far the most predominant function
13:28of the immune system is tolerance,
13:30right?
13:30We really don't want that fetus
13:32to reject the bomb because then
13:34the pregnancy won't continue.
13:35And even early of life it's still
13:38tolerance because the as the baby is born,
13:40it's exposed to myriads of bugs,
13:42right?
13:42And you really don't want the immune
13:44system to respond to all those bugs
13:46because then you would have caused
13:48an overwhelming cytokine storm.
13:49Whereas in adults by far what is really
13:52important is the protection from pathogens,
13:55right.
13:55And so they're the immune systems of the
13:58two are quite conflicting with one another.
14:01Just a brief primer on the immune system,
14:04the immune system in large is
14:06divided into two big farms.
14:07The innate immunity which is the fast
14:10acting immunity that's made-up of a
14:12lot of cells but includes neutrophils,
14:14monocytes, macrophages,
14:15dendritic cells and that is really
14:19the primary first response to any
14:22infection or any, any antigen.
14:24And then there's an adoptive immune
14:26response which is divided into both
14:27B cell which I won't talk that
14:29much about and T cell immunity that
14:30I will talk much more about.
14:32In general.
14:33It's thought to be slow
14:35and it's certainly slower,
14:36but there's some response that that T
14:38cells can mount actually quite quickly.
14:40And then there's a sort of this
14:42grey zone in between where they
14:44are more adaptive like cells,
14:47but there have innate like properties
14:50and particularly in the neonate,
14:51this is quite important.
14:55The T cells that I will talk a little
14:57bit more about have a lot of different
14:59subtypes and I don't think you need
15:00to really remember any of this.
15:01But the main ones that people think about
15:05are TH1TH2 and TH17 and regulatory T cells,
15:09they're all defined by their
15:11cytokines that they make.
15:12So TH ONE cells make a cytokine
15:15known as interfering gamma and
15:17those are really responsible for
15:20against intracellular pathogens.
15:22TH2 once make an interferon,
15:25Illinois four and Illinois 5 and
15:28those are extracellular pathogens and
15:30it's also very related to atrophy
15:32like asthma and allergies etcetera.
15:34And then TH 17 cells are really
15:37associated with autoimmunity.
15:38They make Illinois 17 and they're
15:41responsible for extracellular and fungi.
15:43And then finally regulatory T cells
15:46are make Illinois 10 and they're
15:48really there to dampen the immunity.
15:51So that's really to put the brake on all
15:53the other pro inflammatory immune cells.
15:57Alright,
15:57So what do we really know about infant
16:01immunity and how do we really study this?
16:03So a lot of studies have been
16:04done in mice and we've learned a
16:06lot of very interesting things.
16:08But the problems with mice is that
16:10there's a really very limited
16:12adoptive immune cells in circulation
16:15and for sure almost none at mucosal
16:17sites when mice are born.
16:19But we did for mouse studies,
16:20they suggest that there's sort of Ath
16:232 predominance in the in the T cells.
16:27There's also regulatory T cell skewing.
16:29So they have much more are T
16:32cells than a diet mice,
16:34but of course mice are very inbred
16:35and they have very limited exposure.
16:37So how really reflective is this
16:39of the human immune system?
16:41Their gestation period is
16:42also incredibly short,
16:43right,
16:44so about 20 days versus 280 days and a lot
16:47of things can happen in that time frame.
16:50The other sort of core substitute
16:52that we've used is cord blood.
16:55And what we've learned from cord blood
16:57is that for sure the T&amp;B cells that
16:59are there are almost exclusively naive.
17:02But this is studying term cord blood.
17:05When people actually looked
17:07at preterm cord blood,
17:09sort of a different picture started
17:10to appear and you can see this data
17:12from Tibi Mckenzie's group that
17:14suggests that CXCR 3 positive cells,
17:17these are TH one like cells in central
17:21memory. There's two types of memory.
17:23This is just one of them.
17:26If you look at the preterm cord
17:28blood versus term cord blood,
17:30there's actually a significant
17:32population of of the T cells.
17:34There's a significant population of
17:36memory T cells present in preterm cord blood,
17:38suggesting that fetuses can make
17:41memory T cells.
17:44Interestingly,
17:44sort of more recent work has
17:47suggested that cord blood is really
17:49an independent organ and you can
17:51see this sort of a trajectory of
17:53peripheral blood and cord blood
17:54and preterm and term infants that
17:56sort of converges.
17:57But you can see that cord blood
18:00is completely independent.
18:01And so I think studies with cord
18:03blood are incredibly
18:03interesting, but can't just be
18:06extrapolated to peripheral blood.
18:09So we really need to investigate
18:11appropriate human tissue directly
18:12based on what questions we have.
18:14So what do we know about circulating fetal
18:17and needle immune cells directly in humans?
18:20Well, T cells appear incredibly early on in
18:23human gestation at 10 weeks of life, right?
18:25So barely that you know you're pregnant.
18:27Your fetus already has some T cells.
18:31They are people that have looked at
18:36these very early gestational T cells
18:38have suggested that similar to mice,
18:40fetal cells are more prone to differentiate
18:43into regulatory T cells and very
18:45interestingly are there regulatory T cells.
18:47So usually T cells are against your antigens,
18:51right?
18:51So you so you don't react to yourself.
18:54Fetal T cells are not only against
18:57they're sort of fetal antigens.
18:58There are also a lot of regulatory T cells
19:01against non inherited maternal antigens
19:03that are constantly crossing the plutent
19:06and this is part of the reason that the
19:08fetus doesn't reject the mom as well.
19:11And then a lot of studies have
19:13suggested there's quite unique
19:14features of fetal T cells.
19:16And most of these suggest that
19:18there's specific cell types that
19:20are very abundant in that fetus.
19:22And early neonate infants that are
19:24really meant to suppress the immune
19:27system needs include CD 71 erythrocytes,
19:30myeloid derived suppressive cells,
19:32as well as many others.
19:35But there's very few studies that
19:37investigate directly immune development
19:39in human infants and there's really
19:41almost none in premature infants.
19:43And So what our group has asked is,
19:44can we define immune trajectories
19:46across the first year of life
19:48in term and preterm infants?
19:50And this is work that's
19:51really been led by Booney,
19:52who has done amazing work here.
19:56And what she first asked is, well,
19:58how do we really do these studies,
19:59particularly in premature infants, right?
20:02If you think of a premature infant,
20:03that's 1K,
20:04which is actually pretty big
20:06for a premature infant, right?
20:08There's only 100.
20:09Millilitres of blood that that infant has.
20:12And so really we can't take the
20:14same amount of blood that we take
20:16in adults to do these studies.
20:18And you have to really optimize what you
20:21can do with very limited amount of samples.
20:23And so Buni worked out a protocol
20:25where you can take just two drops
20:28of blood and analyze those drops
20:29and get a lot of information out.
20:32Originally we did something called Cytof,
20:33which is fancy facts where the
20:36antibodies are chelated to heavy metals
20:38instead of conjugated to fluorophores.
20:41And that allows you to combine lots
20:43of different antibodies together.
20:44And so from 1:00 and 2:00 or
20:47from 2 drops of blood,
20:48you can see a lot of different
20:51immune cell types. Here.
20:52Each dot represents an immune cell.
20:54So you can see there's lots
20:55of immune cells there.
20:56And interestingly,
20:57neutrophils are very hard
20:59to detect after freezing.
21:01But even with this protocol,
21:02we were able to see some neutrophils.
21:07And So what we've done is set up a study,
21:11a prospective study of really trying
21:13to analyze how does the immune
21:16system develop in premature infants.
21:18And this has really been work in
21:20collaboration with Sarah Taylor's
21:22Nourished team, who have been really
21:24instrumental in enrolling all of
21:26these patients and collecting,
21:28seeing all of the samples And then
21:30a large team from our group that
21:32have been analysing these samples.
21:34And this is really team science,
21:36what I call because every single
21:38person in our group and even people
21:41in Dean's group have helped out and
21:43process these samples and there's always
21:45somebody on call to process these 24/7.
21:47So this couldn't have been
21:50done without a lot of people.
21:52We've enrolled over 120 babies at
21:55this point and we are continuing
21:59to not only enroll in the NICU,
22:01but we're hoping to expand to the
22:04grad clinic and potentially to
22:05start enrolling a full term cohort.
22:08But in order to validate that all
22:10of this is going to work right,
22:12we first did what my group likes to
22:15call a pilot experiment, 40 samples.
22:18What we took 10 a very preterm
22:22infants ranging from 25 to 30 weeks.
22:24They're samples at one week,
22:26one month and two months of age
22:28at time point T1T2 and T3.
22:31Then we also took five preterm core bloods,
22:35eight term core bloods and then we
22:38also compared them to healthy adults.
22:40We processed all of this according
22:42to the protocol that we've created
22:44where we just try and preserve all
22:46of these cells and then when we need
22:49to analyse them we batch saw them.
22:51And for analysis we take the same
22:55sample and then we split it into three
22:57different ways of analysing just so
23:00that we can optimize what we really
23:03can get from just 100 microliters of blood.
23:07Some of these cells underwent the same
23:09cytof analysis that I showed you earlier
23:11where you were looking at protein markers.
23:14We can phenotype cells.
23:16We also looked at a lot of
23:18signaling molecules with this.
23:19Some of these cells underwent a single
23:21cell R&amp;E SEC analysis where we can look
23:24at transcriptomics of each individual
23:26cell and that was also paired with
23:28looking at T cell receptor and B cell
23:31receptor sequencing that can allow us
23:33to know what particular TCRS and BCRS
23:36were present on the cells that are there.
23:39We also at the same time simultaneously
23:42collect blood spots and these blood spots
23:45can then be processed for proteomics.
23:47And we've used all in proteomics to do this.
23:51So can, sorry, this is just that
23:53table of the initial cohort.
23:55This is the 1st 10 infants
23:57that we've studied.
23:58As I already said, they were quite young.
24:00Their average gestational age
24:02is 28 weeks ranging from 25 to
24:0430 and they all weigh about 1K.
24:06So they all had about 100
24:09milliliters of blood.
24:10They had sort of typical complications
24:12of prematurity seen in this each
24:14group there were two babies with neck
24:16and one baby with sepsis.
24:20And so of course then we asked,
24:21can we really do this, right?
24:22So we took all of this blood and coordinated
24:26thawing all of these samples all together,
24:28splitting them and running
24:30all these separate assays.
24:32And we were incredibly excited to
24:34see that it actually didn't work.
24:36So this is cumulative data on the 30
24:40samples from the preterm peripheral blood,
24:43cord blood and adult samples.
24:45All together, the top is the site of,
24:48so we're looking at the proteins here.
24:50At the bottom is the single cell seek.
24:52We're looking at transcriptomics
24:54and this is just abundance of
24:56the various populations across
24:59longitudinally and preterm core blood,
25:02term core blood in adults.
25:04You can see that overall there's
25:06actually very similar representation in
25:08both transcriptomic part and protein
25:10part of the various populations.
25:12Things that really jump out right away
25:14is that the adult blood is definitely
25:17different from all of the neonatal blood.
25:20There's a much bigger population.
25:22These are memory CDAT cells
25:23and purple in both places.
25:25There's a much bigger
25:27memory population there,
25:29but the sort of the preterm cord blood,
25:32at least from the big picture,
25:33looks pretty similar to term cord
25:35blood and actually fairly similar
25:37to all the longitudinal samples.
25:39What if we zoom in on all of
25:41the individual populations,
25:43right?
25:43This is single cell data looking
25:45at just the monocytes and this
25:47is part of the innate immunity.
25:49You can see majority of the cells
25:51in the blood are either monocytes
25:53or NC is non classical monocytes.
25:55There's a little bit of macrophages,
25:58there's a little bit of DCS,
26:00both that's sort of the classical
26:02DCS and the plasma cytoid DCS.
26:04And then there's this population
26:06that I briefly mentioned before,
26:07which is myeloid deprive suppressive
26:09cells and that's a unique population
26:11that's really much more abundant in
26:14the units that's really thought to
26:17inhibit how the immune system responds.
26:20We can then, sorry,
26:21I'm missing all the legends.
26:23I'm going to do this.
26:24We can then look to see what is
26:26the progression of these cells from
26:28sort of pre term from cord blood
26:30all the way to two months of age.
26:32Those are the samples that we first used.
26:34You can see that overall there's a
26:36little bit of a peak at about one
26:38week of the monocyte populations,
26:40but there's two populations in
26:42particular that really change across
26:45the first two months of life.
26:47One of those is the dendritic cells.
26:49The dendritic cells are really key
26:51it to priming T cells and I'll show
26:54you in a couple of slides T cell
26:56data that this increase in dendritic
26:59cells goes beautifully with.
27:00And the other part that I thought was
27:03incredibly interesting is that for
27:05sure you can see that adult blood has no MDS,
27:07CS or myeloid derived suppressor
27:09cells which is sort of known term.
27:11Cord blood which has been previously shown
27:14certainly has much more of these cells.
27:16But if you look at pre terminal cord blood,
27:17it's actually almost non existent in cord
27:20blood and perhaps This is why there's so
27:24much more inflammation in the preterm infant.
27:26It does go up with age. It peaks at about
27:30one month of age in these preemies,
27:32but it actually never makes it to
27:33the same level as in the term cord.
27:35Look, what about some other populations?
27:40NK cells are one of those population that
27:42sort of lives in between the innate and the
27:45adaptive world that I pointed out earlier.
27:47There's various subtypes of NK populations.
27:49I don't think that matters here,
27:51but I think what's important here is
27:52you can see that this is adult blood,
27:54which is sort of this dot in black.
27:57They're pretty low in the blood in adults.
27:59They are much,
28:00much higher in the blood of both in the
28:03cord blood in particular in both term
28:05and pre term infants and they rapidly
28:07decline over the first two months of
28:09age where they sort of by two months.
28:11They're pretty much equal to what
28:12is seen in adults.
28:14And this population is probably
28:16performing a lot of the functions that
28:19classically CD8T cells would do if
28:22because we had also transcriptional data,
28:25we can look to see what signatures
28:28are enriched for in the preterm cord
28:30blood compared to term cord blood.
28:32And all of these boxes that I
28:35have outlined in red like CD69,
28:37XCL one, etcetera,
28:39they're all associated
28:41with function of NK cells.
28:44And it looks like the preterm cord blood
28:47and K cells are much more activated
28:49and much more functional than term
28:52cord bladder or even adult cells.
28:56We can also look,
28:57because we have information on
28:58so many different cell types,
29:00we can also look to see which cells
29:03interact with which ones and how
29:05is that different between preemies
29:07and adult samples or preterm cord
29:09blood and term cord blood.
29:12And what you can see here,
29:13particularly for NK cells,
29:14there's much more interaction of NK
29:17cells with a lot of other cell types,
29:19particularly early on in life
29:22than compared to adults.
29:24You can see NK cells and preemies interact
29:26with all other cell types much more.
29:28So something that's red is increased
29:31interaction much more than in adults.
29:33The graph here is showing the
29:35the number of interactions.
29:37The graphs here is showing
29:38the strength of interactions.
29:39And so there's an increase of NK
29:41interaction both in number and in strength.
29:44Well, what about the adaptive immune cells,
29:47right.
29:47And so early on it suggested
29:50that maybe at least in mice,
29:52there's very few memory adoptive
29:56cells present.
29:57If you look at blood,
29:58this is single cell data,
30:01certainly in term CRO blood,
30:03almost all of it is and those
30:06are naive cells.
30:07So similarly to mice term CRO blood
30:09stems will actually have very few
30:15memory cells in the memory cells
30:17here in this green population.
30:19But if you sort of focus on this,
30:21this preterm for blood actually has
30:23a pretty good amount of both B1 and
30:25those are specialized B cells that
30:27actually secrete antigen independent
30:30antibodies and memory B cells.
30:35If we zoom in and look at
30:37particular populations to see
30:38how this is changing overall,
30:39these cell numbers are actually
30:41very stable both in in cord blood,
30:44preterm cord blood and longitudinal
30:46sample that seems to be sort of pretty
30:49much defined between 5 and 10% regardless
30:51of what sample you're looking at.
30:53Plasma cells,
30:54those are the antibody producing
30:56cells similar to what we had
30:58thought are incredibly low at birth,
31:00much lower than adults.
31:01But then interestingly they actually go
31:04up pretty quickly and by one month of
31:06age they're fairly similar to adult levels.
31:09And then we found this interesting
31:11population that we're calling,
31:12sorry it's misspelled,
31:13we're calling immature B cells.
31:15These are B cell precursors.
31:17They are very high in term coral
31:20blood and they're much lower in
31:23peripheral preemie samples and
31:24when Boomi looked to see if they're
31:26correlate with gestational age,
31:27you can see this beautiful correlation
31:30that these cells go up with gestation.
31:33We don't know what the function
31:34of these cells are,
31:34but they seem to be at least
31:37gestationally regulated.
31:40If we then look at to see is
31:42there some increased clonality
31:44suggesting that there is some memory
31:48formation and various samples,
31:51it does look like there is at least
31:54some degree of memory formation and
31:56early on this is looking at the various
31:59cell types and how many different
32:01B cell clones have an expansion.
32:03The red here is more than 4B cells
32:05found with the exact same clone,
32:07suggesting they all come from
32:09the same mother cell.
32:10You can see that's highest than B1 cells.
32:14But even if we look at various samples,
32:16even the charcoal blood,
32:17the pre charcoal blood and one week samples
32:20all have some degree of clonal expansion.
32:23What about T cells?
32:26So it is, if you remember when
32:28I talked about the naive cells,
32:30there was a peak of D CS right
32:32around one month of age.
32:33What we see here,
32:35these are proliferating T cells.
32:36Again,
32:37there's a huge proliferation
32:38at about one month of age.
32:41So both the Apcs that are going to
32:43prime the T cells and the T cells
32:45themselves are sort of increasing in
32:47their number right around the same age.
32:49This is just looking at the
32:52various populations across.
32:53You can see that this is quite
32:55different from the B cells in the
32:57sense that of course there is more
32:59naive T cells present early on in life.
33:02But there's already memory formation
33:04both in the CD 8 and the CD4 pool,
33:08very early on in life,
33:09in the preterm cordblad
33:11and longitudinal samples.
33:12And really by about two-month of
33:14eight you have a pretty large
33:16expansion of your memory cell pool.
33:18Sorry, this is showing up blurry.
33:21But you can see sort of correlating
33:23with this expansion at one month of
33:25age of overall T cell proliferation,
33:28a number of different T cell subtypes go up.
33:31So regulatory T cells certainly go up
33:35and that's sort of been the predominant
33:36thought in the nudial immunity.
33:38But if you look to see their proportion is
33:40actually significantly higher than adults,
33:42right, about 6% versus 3%.
33:45But overall they're still quite rare.
33:49Majority of these are naive
33:51CD4T cells or effector cells,
33:53and those again peak at around
33:55one month of age.
33:56But if you look at memory cells,
33:58these are activated effector
34:00memory or essential memory.
34:01They also both go up at
34:03about one month of age.
34:05These are the same central memory
34:07cells that are or at least similar
34:10to what Tippy Mckenzie's group had
34:12identified in preterm cord blood.
34:14And you can see although our numbers
34:15are much smaller than cohort,
34:17this isn't significant.
34:18We see the same trend that preterm cord
34:21blood has almost twice as many central
34:23memory T cells than term cord blood.
34:27What about clonal expansion?
34:29We can see that by two-month of age, right?
34:33There's a this blue group here is
34:36the effect of memory CD8T cells.
34:38There's quite a lot of them.
34:40But if we separate out the
34:42preterm cord blood samples,
34:44there's different responses and different
34:46infants and some of them have a lot
34:49of memory T cells present at birth.
34:51And this particular infant here
34:53was exposed to chorioneonitis,
34:55suggesting that maybe the uterine infection
34:58is really inducing memory T cells.
35:03And then finally we look to see
35:06is sort of more of a concept of
35:08trained immunity that I introduced.
35:10Are there alterations in immune responses
35:13in kids that had been exposed to
35:16chorionitis versus those that have not?
35:19And so Buni took all of the features that
35:21we were able to identify and did APCA plot.
35:24And you can see that the ones in
35:26blue had no chorionitis exposure.
35:28In the ones in green really had Choria,
35:31the Munitis exposure,
35:32they're separating beautifully
35:34and that if we look at the immune
35:37subtypes in those samples,
35:41certainly starting at one at one week
35:44but persisting pretty is at least by one
35:46month age if not by two months of age.
35:48Their immune systems are actually quite
35:50different if they had initial exposure.
35:53And then she could look to see what's
35:56correlated best with coriomonitis exposure.
35:59And the thing that came up on top is the
36:01central membrane CD4 activated T cells.
36:03Again the the cells that Tippy
36:06McKenzie had first identified as
36:08potentially important to preterm labor.
36:10And if we look at different cell
36:12types separated by different cell
36:14types at different ages,
36:15you can see that there this sort
36:18of increase in the number of cell
36:20types including central memory cells
36:22is present at one week of age but
36:24persistent throughout at least the two
36:26months time point that we looked at.
36:29So in summary,
36:31I hope I've convinced you that immune
36:33dynamics are the first two months of
36:35life and preterm babies are highly variable.
36:37Myeloid cells are high in
36:39preterm peripheral blood,
36:39then poor blood with a huge
36:41expansion in DCS and myeloid derived
36:43suppressor cells at one month time
36:46point and K cells are abundant with
36:48increased functional signature.
36:50But although B&amp;T cells
36:51are predominantly naive,
36:53there are particular memory T cells
36:56that can be found and those really
36:59are associated with infants that
37:01were exposed to some infection.
37:03So doing utero exposures really
37:06matter functionally, right.
37:07This is just looking at abundance data.
37:10And so if you take the concept of
37:13trained immunity that I introduced
37:14earlier in in sort of the first
37:17couple of months of life,
37:18it's probably true in utero as well.
37:21And this is a study where in of moms
37:26received hepatitis BCG vaccine and you
37:29can see that in the this this is done
37:32in mice actually but in offsprings
37:35that were exposed to BCG vaccine there
37:39was significantly more response later on.
37:44This has also been shown in mice by
37:47beautiful paper from Yasmin Bell Gates
37:50group that suggested that mice that
37:52have a low level of infection in utero
37:55actually change their offspring's
37:57immune system in a way that if you
38:01then challenge those offsprings with
38:03a different infection they do better.
38:06So in uterus exposures do matter
38:09and not only abundance differences
38:11but also functional, right?
38:14So is this true just in circulation
38:16or can we define immune trajectory
38:19at barrier sites as well?
38:21And what we did is we actually
38:23took intestinal,
38:24small intestinal samples
38:25from early 2nd trimester,
38:28late 2nd trimester therapeutic terminations,
38:31an infant and subjected them to
38:32the same site of analysis that
38:34I was just showing you guys.
38:36And what's really drastic here
38:38is that about 50% of these cells
38:42are of the adoptive phenotype.
38:45And unlike the cord blood that I was
38:47showing you where we can see memory cells,
38:48but they're quite rare.
38:50The memory cells in the intestine
38:53by the time you get to about 22
38:55to 23 weeks make up the majority
38:57by far of the cells there.
39:00So in summary,
39:01maturation of the human Tesla immune
39:03system occurs early in fetal development.
39:05Memory cells exist prenatally and
39:07immune system is compartmentalized,
39:09right.
39:09The cord blood is very different
39:12from your mucosal sites.
39:15This is sort of continuing work
39:17that Weihong do,
39:18Weihong Gu is doing in our group
39:20where she has put together this
39:22beautiful Atlas from really first
39:24trimester all the way to adults to
39:26look at how these cells are changing.
39:29And she's defined A functional signature
39:31of the fetal immune cells that are
39:33drastically different from neonatal,
39:35pediatric and adult cells.
39:37And she can actually track this
39:40both fetal like T cells and adult
39:43like T cells across the samples.
39:45And interestingly,
39:46sort of of course,
39:47they're very high and the fetus and
39:48a little bit lower and then you need,
39:50but even in adults we can find
39:52some fetal like T cells,
39:55suggesting they're quite long lived.
39:57Is this true in other surfaces?
39:59So work that was done by Jessica
40:01Tutuka in my group suggested that yes,
40:04that is actually exactly true.
40:06So she looked at the placenta,
40:07and this is data suggesting
40:10that in the placenta,
40:11T cells that are there are
40:13actually almost exclusively memory,
40:14so that she couldn't find any naive
40:17cells that were present in the placenta.
40:21And then Anthony Vallagalla,
40:22who's a NICU fellow in our group
40:24is following up on this work.
40:26And she put together a cohort
40:28of cases both in the mid second
40:30trimester and term to see how's
40:33the immune system changing.
40:34And she's done a lot of work that
40:36I don't have time to go into.
40:38But the immune system of the
40:40placenta is very different than
40:41the mid gestation and term.
40:43But interestingly,
40:44she is again able to detect memory
40:46T cells and these memory T cells,
40:49if we look at their markers or
40:52activation or their phosphorus
40:53signaling are significantly activated.
40:56And this is just a summary of
40:58everything that's been done
41:00in this field in the past,
41:02I'd say five years,
41:04suggesting that the diversity and
41:06sort of complexity of the fuel immune
41:10system in humans is incredibly high.
41:14Of course,
41:14that sort of begs the questions
41:16are what are the antigens priming
41:18early immune cells work from?
41:21Really pioneering work from
41:26Ganal, Evan, Grohd and all from Mcpherson's
41:30group suggested that it might be the bugs.
41:32They fed radioactive labeled
41:34bugs to the pregnant mice,
41:37and then they were able to detect
41:39bacterial metabolites in the offsprings,
41:42and the immune system of the offsprings
41:45was different from those that weren't fed.
41:48We have similarly done work in infant rate,
41:51and this is impossible to do in humans.
41:53But what we did is we looked at
41:55metabolites and the fetus and the
41:57infant and then adults and they
41:59cluster differently from one another
42:00with the infants being in the middle.
42:02But again, what's driving the
42:05difference predominantly are these
42:07xenobiotics and bacterial metabolites
42:09would fall into that category.
42:12That doesn't really prove that it's the bugs.
42:14So what is it really driving it?
42:18Potentially it's maternal antigen.
42:20And so Jess had looked at maternal antigens
42:22and when she stimulates cells with sort of
42:25ground up Decidua that has maternal antigens,
42:28they're certainly increased
42:29in T cells stimulation,
42:31although it's unlikely to
42:35be explaining everything.
42:37So we still think it's likely
42:39the bugs we have.
42:42A number of us in the field have put
42:44together a paper suggesting that we
42:45don't think there's a live microbiome.
42:48But what we do think is that there
42:49are bacterial components that
42:51are crossing the placenta.
42:52And when we looked at particular bacterial
42:55metabolites and there are metabolomics data,
42:57you can see both hippurate
42:59and P crasal sulfate,
43:00both that have been associated
43:01with sort of a healthy microbiome
43:04but are produced by the bugs.
43:07You can see they're significantly
43:09enriched in the four in the fetus.
43:12That doesn't directly prove
43:13that the bugs are priming,
43:14but at least it suggests
43:16that this is possible.
43:17And so work that's been ongoing and
43:20done by Tyler Rice and our group in
43:23collaboration with Sukhas Kalapur
43:24from UCLA using a non human primate
43:28model of intramniotic infection,
43:30they actually intramniotically
43:32infect these monkeys with E coli.
43:35We have then gotten the samples and
43:38so we can extract T cells from the
43:40fetus that have been exposed to these
43:43E Coli's and reprime them with E coli.
43:48And this is just these big
43:50round cells are Apcs.
43:52And then the little dots here are
43:54the T cells and what we if there is
43:57sort of if these cells are memory
43:59and it's formed memory to the coli,
44:01they should undergo blasting
44:03where colonial expansion.
44:04And you can see this beautiful clonal
44:06expansion on these pictures here.
44:08And Tyler has tracked this where you can
44:11see that there's really minimal clonal
44:13expansion without E coli stimulation
44:15and that really drastically increases.
44:17He can then send these sequences,
44:20these T cells for sequencing.
44:22You can see there's sort of
44:24enhancement in a number of different
44:27sequences in the stimulated group,
44:28and actually interestingly,
44:29a number of them have the same
44:32expansion of the same clones.
44:36This begs a lot more questions, right?
44:38Does the source of these bugs matter?
44:41Do particular Do particular bugs
44:43actually matter in the sense
44:46that are there bugs that stimulate
44:49the memory CD4 cells that are
44:51associated with preterm labour,
44:52and are those different from the ones
44:54that are associated with term labour?
44:55Does the timing matter?
44:57And how do these responses
44:59project future health?
45:01And then I will just finish with two last
45:04thoughts of where do we go from here?
45:06I hope I've convinced you that
45:07new Natal immunity is complex,
45:09dynamic and highly adoptable,
45:11but it's optimized to perform its function,
45:14and the function being protect the
45:17host and prevent overactivation.
45:19And so all of these factors
45:21actually make a huge difference in
45:23how the immune system is set up.
45:25And of course,
45:26this is making a huge difference
45:28into what diseases we're at risk for.
45:30And I think there's a huge need for
45:33large longitudinal studies to study
45:36how various factors prenatally and
45:39in early life dictate to reset the
45:42immune system and sort of put us at
45:44risk for a number of different diseases.
45:47And finally,
45:48I think we need to take all of
45:50this into account in how we design,
45:53improve vaccine strategies.
45:54And this was a beautiful illustration
45:56from the original science issue that
45:58I showed you guys from Sing Sing Wei
46:01and Commons group where if you look
46:03at this sort of infant early on,
46:05there is some pathogen specific antibodies,
46:07but they're quite low.
46:09If we immunize the mom right,
46:11we can sort of have mom's antibody cross
46:15the placenta and protect the fetus.
46:18If we sort of immunize the baby
46:19and the mom right,
46:21you get mom's humoral immunity,
46:23you get trained immunity which is sort
46:25of pathogen agnostic immunity and
46:27you get pathogen specific immunity.
46:29But what if we sort of combine the two,
46:32right?
46:32And maybe we need to find a window
46:34during pregnancy where we immunize the mom,
46:37but that also benefits the developing fetus.
46:40And maybe the fetus themselves can
46:43then mount AT cell response to the
46:47vaccine and then have double protection.
46:50I think I've mentioned all the
46:51people all along,
46:52but these are the people in our group
46:54currently that are doing all this work.
46:56And these are just all of our
46:58collaborators that I think I've mentioned.
47:00But really none of this work would
47:02have been done without Sarah's
47:04group and our partners in crime
47:06that you know my group.
47:07And I'll take any questions.
47:17Thank you so much, Lisa.
47:18Wow, that was quite a tour
47:22of of a complex system.
47:24I'm going to invite folks to send in
47:26questions through the chat and I'll
47:28read it to Lisa for her response.
47:30What was occurring to me as you were
47:32going through this whole thing aside
47:34from the incredible complexity and how
47:36much has been done to describe this
47:38Lisa was the it's it's very appealing.
47:42The notion of intervention
47:44during pregnancy actually.
47:45I mean as you started to describe
47:47all this this complex system the
47:50interventions during pregnancy that
47:51can influence the the immune system.
47:53But I I suppose that's a double
47:55edged sword too because our immune
47:57system can be our best friend
47:58or our worst enemy depending on
48:00what the particular problem is.
48:01And so you know at at at first look I
48:04think what this would be wonderful.
48:06I'd like the hepatitis B vaccine which
48:07you pointed out this is something
48:09that works really well really early.
48:10This works in the baby.
48:11So we can respond to some vaccines.
48:13So presumably there's vaccines you can
48:15give during pregnancy or other things
48:17you can give during pregnancy that
48:19are going to augment the immune system,
48:21if you will,
48:21the immune response in the
48:23fetus and the newborn, right.
48:24This I think you you you've shown us that.
48:28Then I guess what becomes a tone of
48:31caution is these interventions also are
48:32we going to find out sometime later
48:34these kids are more appropriate some
48:36intervention to reduce infections in
48:39the newborn period could ultimately
48:41to increased autoimmune disease that
48:43we hadn't only anticipated with those
48:45interventions And can you talk about
48:47that for a bit or am I off based on
48:49that? I mean I think you hit the
48:50nail on the head mark I think right.
48:53I think we don't understand enough to
48:56really know what is the best window and
48:59what kind of responses should we be looking
49:02for right early on versus hoping to not
49:06induce any adverse reactions later on.
49:09It's clear that sort of very early,
49:10early on in pregnancy,
49:12the immune system is really
49:14designed to be tolerant of the mom.
49:15And so I think interventions very
49:19early on in pregnancy aren't
49:21necessarily going to help prevent
49:23any of the infections later on.
49:25And so I think we have to find that
49:27window sort of later on in pregnancy
49:29where we can intervene and perhaps
49:31sort of reset the OR develop some
49:35memory cells that will be beneficial
49:38in the new Natal infant period.
49:40But I think that's sort of why I meant
49:42we need long longitudinal cohorts
49:44because we don't understand how,
49:46we don't understand how that's
49:47changing even in the new Natal period.
49:49And we certainly don't understand
49:51how that's going to affect children
49:53and adults later on.
49:55Thank
49:56you. Thank you.
49:56There's a few questions that
49:57have come in here with the Q&amp;A.
49:59So let me read one to you.
50:01Very interesting and important work.
50:03If you looked at these immune
50:04markers in relation to feeding type,
50:06IE formula fed,
50:07mixed fed or breast milk fed, yes,
50:11well, Sarah and I have applied for
50:13a grant five times, but hopefully
50:16next time we will get it funded.
50:18I think that's incredibly important,
50:21but there's just not enough data out there.
50:24And I I think for sure nutrition is changing
50:28how the immune system is responding.
50:30And there's clear data, right,
50:31that breast milk is protective,
50:33whether the donor human milk is
50:35just as protective as unclear.
50:37And certainly potentially that
50:39formula is a little bit detrimental,
50:42but more work really needs to be done.
50:44But I think it's an incredibly
50:45important area.
50:47Thank you. Next question, ILC.
50:50Ilcs, excuse me, seem to play an
50:53important role connecting mediate
50:54and adaptive immune systems.
50:56What is their role Ilcs
50:59in these early stages?
51:01Another great question.
51:02So I I just didn't have time to go into
51:04all of the different immune components,
51:05but ILC that incredibly rare in the blood,
51:08so I don't know what role
51:10they would play early on.
51:11They're also incredibly rare in the
51:13blood of immunes at the mucosa side.
51:15I didn't go into it,
51:16but the ILC is actually incredibly
51:18abundant in the fetal intestine in humans.
51:21And we think they're really
51:23actually setting up the immune
51:26niche in the mucosa and they then
51:28drastically go down with gestation.
51:31So I think they're one is that
51:34they're needed there to set up
51:36how the immune system is going
51:38to work and they probably also do
51:40provide some sort of protection
51:44while the adaptive part of the
51:46immune system is being set up.
51:49Thank you. The prevention of
51:51neonatal tetanus relies in part on
51:53the immunization of the mother.
51:55Is this along the lines of
51:56what you were speaking?
51:58Again great question.
52:00So you know part of the protection
52:03that giving vaccines in pregnancy
52:05is that you get humoral immunity,
52:07right, Like antibody media immunity
52:09that gets transferred to the
52:11fetus and thereby neonates are
52:13born with protective antibodies.
52:15I think there is a little bit of data,
52:18I don't, I'm not familiar with the
52:20tetanus data that there's a paper
52:22published on the flu vaccine that if you
52:25give the flu vaccine during pregnancy
52:26you can actually find flu specific
52:29fetal T cells in the fetal blood.
52:31That's sort of what I was suggesting.
52:32Is there is there a window
52:33where we can immunize that?
52:35Not only do you get humoral immunity and
52:37get a bump on your humoral immunity,
52:39you also get a bump in your
52:41T cell memory, in
52:43which case the possibilities
52:44are endless in terms of
52:46infection protection if you can,
52:47if you can do that exactly.
52:49Steroids are often given in
52:52mothers undergoing preterm labor.
52:54What effects do they have when they're born?
52:58Great question, Eduardo.
53:00Yes, I'm sure they do, right?
53:03Steroids drastically
53:04regulate the immune system.
53:08It's incredibly challenging to study
53:10right because almost we at least in our
53:13unit and especially in this cohort,
53:15they universally received steroids.
53:17And so I I don't know what effect
53:20they have because all of the samples
53:22that have been predisposed to them.
53:23But certainly a very important question.
53:28I do over the cohorts of children
53:31born to mothers with CBID or
53:33other immunity deficiencies.
53:35Yeah. So I I am not, but we have
53:39been looking for some, I think
53:44there are some at the NIH that
53:46Carrie and I have been thinking
53:47about because I think that would
53:49be super interesting. I agree. Have
53:52you found any specific differences
53:55in the functional abilities
53:56of these adaptive immune cells
53:58through through through the period?
54:01Are there transitional genetic or
54:04medicine that markers present?
54:08Sorry Mike, you were breaking up.
54:10I'm sorry. I'll I'll reach this again
54:12give you a second to consider it.
54:14Have you found any specific
54:16differences in the functional
54:18abilities of these adaptive immune
54:20cells through the perinatal period?
54:22Are there transitional markers present,
54:24genetic or otherwise?
54:25Yeah. So that's sort of the
54:27I-1 slide that I had up there.
54:28That's what Wei Hong is doing.
54:30She's looking at specific
54:33markers of fetal mucosal T cells,
54:36neonatal mucosal T cells,
54:38and adult mucosal T cells.
54:40The short answer is yes,
54:41they're certainly different.
54:43They have different functional potentials.
54:46So they proliferate.
54:47Neonatal and fetal cells proliferate
54:49drastically more than adult cells.
54:52But that work is ongoing.
54:56This has been quite a quite an hour,
55:00please. Thank you so much.
55:02I think among other things,
55:04science and teaching us all
55:05a lot of community biology.
55:06You've also shown everybody just how cool
55:09the section of this medicine really is.
55:13This is tradition for and we very
55:16much appreciate your sharing and
55:17appreciate the fact that you're
55:19also getting our department in our
55:21division more involved with others
55:23outside of muting this work as well.
55:25So thank you. Thanks to everybody.
55:27Cliff, is there anything else you
55:29need to say before we sign off?
55:33No, that was a Tour de
55:36force. Thank you. Thanks so much, everyone.
55:40Beautiful. Bye. Bye everybody.