Notes and Questions for Anthony Leonardi

Transcript of Interview for Dr. Anthony Leonardi

Interview Date: January 8, 2022

(0:00:45) What are your qualifications?

John’s Hopkins University undergrad and masters
Worked in surgery branch for National Cancer Institute that was using T-cells immunotherapy to treat cancers like melanoma
Research background with cancer immunotherapy and melanoma.

Studied T-cells behaving strangely but he discovered why working as a fellow for 4 yrs
Issue: T-cell differentiation interaction and cytotoxic function, i.e. how T-cells grow up and work and communicate with each other based on this work he did PhD with National Cancer Institute and Kingston University of London;

Went to medical school for 2 yrs at the same time
He is a final year medical student but has taken off right now and might focus on public health rather than just medicine
4 yrs of cancer immunotherapy, culminating in PhD and final yr medical student

(0:03:35) We’ve seen lots of virologists speaking publicly, like Vincent Racaniello, Angie Rasmussem, Peter Hotez, but not many immunologists like yourself or Akiko Iwasaki. What do you think is missing?

Issue is the dynamic of immune system
From virology side: SARS is a one and done = big assumption; when this was proved wrong now we have dynamic of immune system aging which isn’t good news; many immunologists are quiet right now because they don’t want to discuss the implications of being constantly reinfected with this virus, especially when considered in the physiology, the autoantibodies, migration of T-cells into places where they shouldn’t be; overall bad news and nobody wants to mention it on the news.
Another issue: Everyone speaking in terms of averages; but older population and those with immune problems and deficiencies will be marginalized; subsequent infections might not be so severe but for the 60+ there are reasons why a second infection could be more severe; e.g. similarly with the flu, when you are 80+ you would have been exposed to it before and have cross-reactive T-cell immunity, problem is that you will still have a level of severe disease when infected with the flu and not necessarily a mild course
Given the amount of immune aging and pathophysiology that SARS-CoV-2 does, may see similar things but more accelerated

(0:06:11) Akt-fas paper - T-cells - CD4, CD8 cells activity is deranged and causes damage. Can you explain?

T-cells encounter specific peptides they recognize; all T-cells are born with specificity; they have a T-cell receptor (TCR) that recognizes a specific peptide seq that is presented by either MHC class 1 or MHC class 2 (class 1 = CD8 T-cell, class 2 = CD4 T-cell); professional antigen presenting cells (APCs) will present protein fragments (peptides) to cells in their periphery/surface of cell (e.g., MHC1 inside the APC, MHC2 picked up from outside somewhere)
CD8 T-cells that recognize MHC1: react to infections directly; infected cell presents MHC on MHC1 peptides what is inside of it (i.e. get peptide sequence specific to virus and CD8 T-cell react to kill infected cell);
My paper: naïve T-cells were strongly correlated to mild disease (causal); naïve T-cells prevent exuberant function of highly differentiated T-cells;
Differentiated T-cell lifecycle (more like a one-way street than lifecycle): T-cells go from naïve state (never encountered specific peptide that it’s specific to) …

Rafi Ahmed scientist: says T-cells can go back from effector to memory

(0:09:16) Kashif: naïve cells born with them and grow in thymus and thymus disappears at age 2 yrs; fixed supply
As you age, thymus involutes (shrinks) and stop educating/making T-cells that are specific to viruses/cancers; T-cells can grow in number and contract in population size but when older you stop this production of T-cells; when thymus involutes this is hampered, i.e. built in obsolescence, our immune sys stops producing T-cells; all cell types end up aging, even T-cell stem cells that are self-renewing can be pushed to age
My research: how stem cells go to ageing and I found a way to prevent that
Publication shows that naïve T-cells are preventing exuberant function of highly differentiated and cytotoxic CD8 T-cells; they were correlated with mild disease in SARS-CoV-2 and discovered theoretical mechanism of why this is occurring
Publication title = pun to “act fast” because 2 steps in pathway of differentiation to highly achieving cytotoxin function; on the surface of cell there is a CD95 marker/receptor called fas; when cell is not a terminally differentiating cell, engagement of CD95 receptor sends an aging signal into the cell for it to differentiate, then this differentiating signal is specifically carried through akt (signalling protein that carries signal)
Rationale for paper: get out a warning about what serial reinfection could do to age the T-cell repertoire; also include the mechanism of this aging of CD95 mediated differentiation,
A one and done of SARS-CoV-2 infection, physiologically it ages our immune sys, pushes differentiation

MBPI article on telomeres in aging after infection and also data on pushed differentiation of T-cells after infection

Given reinfections we will continuously be pushing this system of differentiation in T-cells in our system don’t know why

One paper (Rivas et al., 2021) found a superantigen on spike because it was pushing T-cells; when you can load a lot of superantigen then you can stimulate T-cells hard
New paper, not peer reviewed: claiming that ORF8 is also superantigen, but evidence is low
Paper in Germany: compliment is pushing T-cells into high cytotoxic function;

Akt-fas paper: you could either block CD95 or inhibit the T-cell affector differentiation with an akt inhibitor; but after infection there is reduction in naïve T-cells; these numbers may normalize but an old person may have a more difficult time reconstituting the level/proportion of naïve T-cells
(0:15:49) Kashif: what is the definition of old?
Not totally linear with age, but 50s-60s and up, but depends on the person;

One paper from Moderbocher: found that older population had less naïve T-cells and this corresponds to more severe disease

Kashif: does it correlate with physical health
In my research, physical health of donors was not known, but presumably they were healthy; but cancer patients have T-cells that are aged/differentiated significant enough to tell by looking at T-cells; will be a problem for those who have had chemotherapy and other major illness

(0:17:45) Does severity of disease matter?

I believe that a lot of the pulmonary damage/severe disease is a manifestation of highly cytotoxic state of T-cells;
One publication claimed that mild illness in children was well correlated with less cytotoxic T-cells function in infections
A lot of damage is mediated by immune system; to become very cytotoxic and cause that damage, T-cells will need a lot of differentiation and exuberant function; will be a proxy for someone who has had a highly pushed T-cell differentiation state

(0:19:29) So the immune system is multilayered. Our vaccines stopped variants mostly up until Delta, and then something shifted, Omicron sails past our first barrier which is antibodies. Severe disease is being held in check mostly, and the second layer of defence, your T-cells, are being touted as our saviour.

You’ve written that there is a big problem with this kind of thinking, that T-cells are actually damaged

T-cells = double edged sword
When things go well, T-cells did their function
When they go bad, T-cells were acting too exuberantly and caused a lot of nonspecific damage or damage to the infected cells
T-cells not always protective and some publications that have highlighted this recently; T-cells are exerting harm in some cases
Not only the immunopathology, but seeing all the blood clots happening and tissue damage from infection and things related to T-cell activity, this mechanism is still unknown

(0:21:27) Superantigens and the Furin Cleavage Site

Superantigen activates T-cells nonspecifically; a T-cell not specific to the virus if it is in proximity to viral antigen, i.e. superantigen, it can interact; the superantigen will send a ligation signal to the T-cell receptor on the CD8 T-cell; this will make the CD8 T-cell think that it’s seen the viral/cancer sequence, so the T-cell will act; for a CD8 T-cell acting means killing, it will be nonspecific activation (i.e. a lot of damage); e.g. exhibited by MISC, delayed syndrome but this delay may be because antigen is in the gut and relatively shielded so it takes some time for immune system to recognize it; but when superantigen (or RF8) is in the lungs you will get more nonspecific T-cell activation

The German paper also shows that compliment is related to nonspecific T-cell activating

In infection, looking at whole blood not just T-cells specific to virus, can see that so many T-cells are stimulated (nonspecific activation); predominately it is superantigen stimulation, but compliment is also doing this big finding; the new German paper is very compelling and helps with understanding the immunopathology of the infection
(0:24:29) Kashif: superantigen can activate large portion of T-cells (20-30%) compared to natural infection (0.1% or 1%), is this right?
Anti CD3 is an antibody you can use, it’s a superantigen and will activate all your T-cells; can activate a high amount of T-cells with superantigen; there is a lot of nonspecific activation which leads to a lot of cell killing mediated by the T-cells; target this during treatment, especially for person with highly differentiated repertoire and low amount of naïve T-cells because naïve T-cells will soak up some of the signals (0:26:10) cytokine sinks = certain T-cells /immune cells will act as sinks for other cytokines and signals; CD95 ligands are not cytokines per se but naïve T-cells will act like sinks for the CD178 fas ligand; naïve T-cells will take up that differentiation signal so this isn’t happening to the T-cells that are differentiated enough to becoming highly cytotoxic; the physiological system effector acquisition is dampened by the CD95 expressing younger T-cells cells; naïve T-cells don’t express CD95, this is called T-cells stem memory, and those will start expressing CD95, soak up signal, like a cytokine sink, and prevent high amount of effector differentiation (illustrated in the Akt-fas paper)
(0:27:58) Kashif: like carbon in a nuclear reactor, absorbs what might cause a runaway reaction = analogy; if older, have more differentiated T-cells because fought off more diseases, and less ability to absorb and survive the massive activation of T-cells caused by SARS-CoV-2
Can explain in part why older people get more severe subsequent infections, it is theoretical, shown in vitro, but not applied specifically to infectious disease; I will work on this to publish another paper but wanted to help out the frontline healthcare workers
(0:29:13) Kashif: can naïve T-cells regenerate?
Yes, if you are younger and your thymus is still working, you will still produce naïve T-cells; some publications stating that naïve T-cells are still coming out of tonsils or other lymphoid organs but it’s unusual and haven’t looked closely at these; as we age, T-cell repertoire is not as good, there is a sloughing that occurs, skin wrinkles, etc.; paradoxically when older, your T-cells are in the phenotype and closer to the effector function, that killing we mentioned; may also have a more exhaustive phenotype as well, but regardless, are in the memory stage predominantly, with less naïve T-cells, and more poised for more cytotoxic activity, paradoxical but can reconcile it with the knowledge of how T-cell differentiates and functions

(0:30:58) Immunosenescence and what that means long term for people. Does this happen in minor cases?

Does this happen with other viruses like flu?

From my own reading because I’m not a virologist, I’m more of a cancer T-cell immunologist;
John Wary publishing from University of Pennsylvania: there is a gene signature in severe cases of COVID-19 when looking at the T-cells and it’s similar to Ebola infection; in Ebola infection there is also superantigenic stimulation; a loose association I have in Ebola infection the T-cells inside the blood, there is a high degree of chaos and a lot of interferon secretion, lympho-collapse and death, and a lot is mediate by CD95 and CD95 ligand; Ebola infection is one the most dramatic collapse of T-cells you can have, paired with extreme amount of death and maybe cytotoxin function; Ebola survivors have a competent and coordinated T-cell response that is earlier which prevents them from dying;
John Wary publication: sees a similar genetic signature as Ebola
Not aware of differentiation states with flu/influenza

(0:33:19) Invasion of other tissues, especially the brain

When doing comparisons between influenza and SARS-CoV-2, (Nature paper): studied people who died of lethal influenza or SARS-CoV-2 and observed T-cell infiltrates in the SAR-CoV-2 group but not in the influenza group; autopsies showed 70-80% of deaths had infiltrating T-cells; this is unusual and worried about this because it’s a superantigenic infection and so close to the brain;
Lots of debate whether virus can enter cranium and be inside the brain/nerves or infecting cells that support the nerves;
(0:34:22) When you have T-cell infiltrating brain this is bad because it will exert a lot of killing, the CD8 T-cells are stimulated and killing things which doesn’t happen in the flu; comparison to flu isn’t valid
Some pedants have stated there is tissue residents in the brain; with SARS-CoV-2 we are talking about infiltrating T-cells not these tissue residents that may have already been there; can look at what T-cells express on the surface to determine if they are resident or not some people are downplaying the findings
In a previous interview I was asked what diseases are caused by viruses vs not; Diabetes and heart disease = caused by western diet; more specifically, type 1 diabetes that is immune mediated, this is from SARS-CoV-2 infection in my opinion; SARS-CoV-2 is activating the immune system so much and causing auto immunity and affecting T-cells responsible for quelling auto-immunity, CD4 T-cells; when these organs (e.g. pancreas) get infiltrated by T-cells or have some response to circulating cytokines or auto-antibodies, the cells that are producing insulin will start dying; I believe SARS-CoV-2 is causing Type 1 diabetes and could be causing cardiac injury
(0:37:01) Kashif: my colleagues have seen uptake in Graves’ disease and it may be the same mechanism along with kids with diabetes
It looks like global autoimmunity and it’s unknown what syndrome it will cause; I guess Graves’ disease, type 1 diabetes are manifesting; but when you have some many auto-antibodies and you turn the T-cells responsible for stopping autoimmunity into T-cells that cause autoimmunity, then you can pick your clinical syndrome; Epstein bar is seeing multiple sclerosis; related to MS recent finding on the sequences to EBV in the brain; no idea what SARScov2 will do

(0:38:19) What do you think it will take to stop Covid-19 for good?

Kashif: You previously mentioned the Manhattan Project; a hack the Israelis are using is boost everyone every 4 months; is there a better solution?
Hate to fall back on to cancer but I see it as a cancer problem; if someone is diagnosed with cancer you want them to live longer with better quality of life maybe in the future there will be better options; [with boosters] it’s great to have the extra 6 months or so and protect the older population and give more time to find a solution; we need to use these even if we have to boost like Israel, should be done for the populations because our technologies will be better in the future;
Disappointed that there is no global Manhattan project for SARS-CoV-2; we need to find something that we can disseminate, especially to the low-income countries and socio-economically disadvantaged countries; we need an infrastructure to be able to deliver the solution and need a solution for it, or something better; now we have vaccines which are great, but we are not disseminating them quick enough;
(0:40:14) Dream scenario: we would have had ample supply of mRNA vaccine to Wuhan 1.0, and it still would have been Wuhan 1.0 because we would have coordinated global efforts (rich countries help poor countries) and all would have scaled up and attack spread in the head on and prevent the spread and immune escape;
(0:40:51) Ideal world: we would have a global supply chain and global manufacturing to disseminate whatever solution that works on all the variants (e.g. the pan-corona virus vaccine)
Without a Manhattan type project it’s going to keep coming and all these autoimmune issues will not go away; even if you’ve compensated to a degree, cranking out antibodies and effector/memory T-cells poised to go, but this system is not totally self-renewing without aging; every 6 months you’re infected by another variant of SARS-CoV-2, there will be decompensation and we are not ready for that; our global population will have a great decrease in life expectancy and some people are seeing that
A life insurance company published that excess death is up from age 30 to 60 yrs
(0:42:55) Kashif: this points to if there is no global Manhattan project, this is the dark future; we all get infected with SARS-CoV-2, our immune systems keep taking hits and we all prematurely age
My fear is that a high proportion of us will have long-covid; people with long-covid are trying to warn us “you don’t want this,” we need to do something because this will disable many people