Lecture 9: "Immunology: T cells"

Hello, I'm richard young, your host with facundo batista and lennon feyen for the mit course on covin 19 sars go v2 and the pandemic. now we have heard experts discuss the pandemic, the coronavirus and what happens to the covid patient. We have also heard an overview of the immune response, but we have not fully explored the critical functions of the key cell type that regulates the immune response, the T cell. Today we have the pleasure of hearing from one of the world's experts on T

cells

. , Dr. Arlene Sharp. Dr. Sharp is. george fabian professor of comparative pathology at harvard medical school also chair of the department of

immunology

at harvard member of the department of pathology at brigham and women's hospital member of the broad institute at mit and harvard leader of cancer

immunology

program at the cancer center Dana Farber of Harvard Co-Director of the Evergren Center for Immune Diseases at Harvard Medical School and Brigham Women's Hospital Dr.

Sharp has been elected to the National Academy of Sciences and the National Academy of Medicine and is Co-Director of the Consortium of Massachusetts on Pathogen Preparedness established this march by Harvard Medical School to respond to the sars cov2 pandemic and prepare for the emerging pathogens of the future arlene thank you for joining us today well thank you it's a real pleasure for me thank you for inviting me to Participate in this wonderful course to preserve my bandwidth. I'm going to pause my video for this presentation and let me see. I'm going to share my screen here.

Can you see my slides and put them in slide mode here? Perfect, okay? alright, well hello everyone, today I'm going to talk to you about T

cells

and in my

lecture

today I'm first going to talk about the regulation of T cell responses, essentially, what turns T cells on and off and why that is important. and then we'll talk a little bit about the roles of T cells in defending against disease-causing pathogenic microbes and then we'll focus on what we're learning about T cell responses to covid19, which is very much a work in progress , so I know you have had

lecture

s on many topics and had an overview of the immune system, but many of you have a variety of backgrounds, so first I want to remind you of some terms: An individual's immune system is truly amazing.

It has the ability to recognize between 10 million and one billion different substances that are called antigens in the language of immunology. Now these antigens are recognized by lymphocytes and lymphocytes have receptors on their surfaces to recognize these antigens. B cells have b cell receptors t cells have t cell receptors there are several classes of lymphocytes which are summarized on this slide there are b cells which are cells that become antibody producing cells and today we are going to have a whole lecture on b cells We will focus on T cells and these T cells come in several types.

There are so-called helper T cells and these cells produce cytokines, small proteins that can help other types of immune cells activate and are important in host defense. Cytolytic T lymphocytes. These lymphocytes are able to recognize infected cells that express microbial antigens and then kill these infected cells. There are also regulatory T cells that suppress immune responses, so I'd like to start by talking a little bit about the regulation of responses and why. Do we need regulation? So to talk a little bit more about these types of T cells that kill infected cells, we have a very limited number of T cells that could recognize a specific antigen, so when we encounter a pathogen and you have a T cell of specific antigen.

This cell needs to be activated and can then expand many times. Studies have shown that a T cell can expand more than fifty thousand times in just one week. Such a rapid response is needed to defend ourselves against infections, but it also means that this response must be carefully regulated so that the appropriate activation of the immune response in response to a virus is obtained at the right time and not in an indeterminate manner. When we think about an immune response, this lymph is activated, these antigen-specific lymphocytes must be activated, then these cells proliferate. and they can differentiate into cells that carry out effector functions, for example, for T cells, these killer T cells, once the pathogen is eliminated, a decrease occurs known as homeostasis and then one of the characteristics that is very special of the immune system is that it remembers this encounter with the pathogen, so the next time it encounters, for example, a virus, it will be able to respond more quickly because it has these memory T cells, so there are passive mechanisms that control these phases, once the microbe lights up, the infection is over and the stimulus is gone, but there are also active mechanisms and we're going to spend some time talking about these active mechanisms, so why are these mechanisms important?

We need to have control of T cell responses to control the activation of these T cells for the cells to function properly. activated, but you also want to control the duration of the response and have adequate resolution of the response, otherwise there may be tissue damage. Additionally, there are certain situations, for example certain chronic viral infections such as HIV, where antigens can persist for a long period of time. Furthermore, one of the most surprising aspects of the immune system is that the wallet can defend us against the diversity of the microbial world and the normal immune system does not respond to itself and so we tolerate ourselves and a breakdown of tolerance can lead to autoimmunity.

We need to have these appropriate control mechanisms to not develop immune-mediated diseases. First we are going to talk about costimulation, which is an important regulator of this balance between the activation of T lymphocytes to obtain the appropriate response against pathogens and control so that we do not obtain our own response to the cells of our own body and we obtain a controlled response to the pathogen, so first we'll talk about costimulation and activation of T cells so that the T cells become activated and need to receive. two signals that are delivered by antigen-presenting cells, such as dendritic cells, the first signal confers specificity to an immune response and involves the recognition of antigens, antigenic peptides are presented by mhc molecules expressed on antigen-presenting cells to the receptor T cells for T cells to complete.

Activation of a co-signal: A second signal known as a co-stimulatory signal is needed and when the T cell receives both signals it can activate and differentiate into effector T cells which can then deal with the pathogen and therefore these two signals. are optimal for T cell activation. Much research has been done in this area and our understanding of costimulation has evolved over time, so we now appreciate that there are not only second positive signals that work with the cell receptor There are also second negative signals that attenuate signals through the T cell receptor. We have learned that many of these second negative inhibitory signals are important in mediating tolerance that protect us against T cell activation. autoimmunity.

These costimulatory signals control a variety of T cells at different levels. stages of their life, we have naive T cells that are controlled by costimulatory signals, effector T cells, memory T cells and those regulatory T cells that I mentioned, they inhibit T cell responses and there are many costimulatory and coinhibitory T cell pathways. and today we will focus only on two pathways that are key immunoregulatory pathways. The first is the pathway that involves the b7 molecules b71 and b72 and their receptors cd28 and ctla4, so the b7 molecules also known as cd80 and cd86 are upregulated on the antigen.

Presenting cells as dendritic cells when the immune system detects their danger, for example, microbes or components of microbes, such as components of the bacterial cell wall, such as lipopolysaccharide, can lead to the expression of these b7 molecules in dendritic cells. These two b7 molecules have overlapping functions but differ. in their expression kinetics and these molecules can interact with cd28 which is constitutively expressed on the surface of t lymphocytes and can also interact with ctla-4 now cd28 is constitutively expressed but in contrast ctla4 is positively regulated in t cells after its activation, while cd28 is expressed on the cell surface, ctla4 is mainly expressed in the cytoplasm and transported to the cell surface, so ctla4 is expressed and reaches the surface of activated T cells and studies have shown that ctla 4 is the high affinity receptor for b71 and b72.

So, first we will talk about the function of cd28. cd28 is the main stimulatory receptor that activates naïve T cells when a T cell receives signals through the T cell receptor or through antigen and by binding of b7 molecules through cd28, the T cell receives them. two signals and the signals through cd28 provide growth factors, survival factors and bioenergetics that allow these t cells to divide and proliferate and then differentiate into effector cells, so the signals through cd28 lead to the synthesis of growth factors such as il2 by t cell survival factors. like bcl2 and then glucose metabolism which provides the energy so these cells can divide.

What about ctla4? ctla-4 has structural similarities to cd28 and is adjacent to cd-28 on the chromosome. In the early studies there was a debate about whether ctla4 was a stimulatory molecule like cd28 or if it could be an inhibitory molecule. My laboratory together with the tacmac laboratory created ctla-4 knockout mice and the phenotype of these mice convinced the field that ctla -4 was a key inhibitory molecule these animals die between three and At four weeks of age they develop enormously enlarged spleens known as splenomegaly and in large lymphocytes known as lymphadenopathy, the T cells in these animals activate spontaneously and infiltrate many different tissues and lead to the destruction of tissue, so here on the left we are looking at the heart of one of these animals, all these little purple cells are lymphocytes and when we have inflammation we refer to this as itis, so there is myocarditis, inflammation and destruction of the heart and also on the right here we are looking at a pancreas.

There has been a complete destruction of the eyelids here, which are important for producing insulin, which is why we have pancreatitis, so this phenotype showed that ctla-4 had a critical role in inhibiting T cell responses and negative regulation of T cell activation, so this pathway then, when we think. Regarding this pathway, there is a balance between the activation mediated by the participation of b7 of cd 28 and the inhibition mediated by the participation of b7 of ctla-4. It is not fully understood how this occurs, but the differences in time and kinetics help us understand this, first when we have a resting situation there are very few b7 molecules, but then when dangerous signals are found, such as a bacterial pathogen , there is an upregulation of these b7 molecules, which can then activate cd28, which will lead to T cell activation as a result of T cell activation, ctla4 is upregulated in these.

Ctla4 cells are the high affinity receptor and when what is activated is activated by b7, this can downregulate the T cell response. How does ctla-4 exert its inhibitory functions? CTLA-4 can be expressed in a variety of T cell types and this cartoon illustrates two ways that CTLA4 can inhibit T cell functions. First, CTLA4 has what is known as an intrinsic cell function, meaning that cells that express ctla4 send signals to those cells and therefore, within cells that express ctla4, ctla4 signals can block signals through the T cell receptor and through cd28 and therefore inhibit the activation of T cells.

Regulatory T cells, those suppressor T cells also highly express ctla4 and ctla4 in these regulatory cells can extract b7 molecules from the membrane of antigen-presenting cells, such as dendritic cells, as a result. There are a reduced number of these b7 molecules and they are not present to interact with 28, so there is reduced costimulation through cd 28 and that also negatively regulates T cell function. What is the importance of the role of ctla? -4 and these immune responses? The ctla phenotype for knockout mice resembled aautoimmune disease and led us and others to wonder if ctla-4 had a role in regulating tolerance and autoimmunity, so first of all, when we think about the immune response, we go back to what I mentioned at the principle of In this lecture, turning off immune responses is important for the proper functioning of the immune system.

We need to activate these cells, but once the pathogen is eliminated, we must return the immune system to its basal state, again referred to as homeostasis. to prevent an inappropriate response to self-antigens, known as tolerance, and as you may have heard before, when antigen receptors are generated and T cells are born in the thymus, these receptors are found on developing lymphocytes, the expression of these antigen receptors is random without any specificity. to find out if these T cells can recognize self or foreign substances so that all individuals can produce lymphocytes whose receptors can see self antigens and these self antigens are not hidden from the immune system, so there is a way to deal with them first in the thymus, where T cells are born, some of these lymphocytes that recognize themselves are eliminated, however, some of these lymphocytes reach the periphery and there are also peripheral mechanisms that can eliminate or inactivate these cells to prevent autoimmunity, so just a little bit more terminology here, I've used the term. autoimmunity and what I mean by this are immune responses against self-antigens or autoantigens and, by implication, these responses are what cause diseases and there are diseases that are classified into these inflammatory diseases mediated by the immune system and when we think about autoimmunity, This can be systemic throughout the body like systemic lupus erythematosus or specific organs like type 1 diabetes and autoimmune disease that occurs in the pancreas, so people have studied the function of ctla4 and autoimmunity and we've learned that, In fact, ctla-4 regulates tolerance and autoimmunity, ctla-4 can control the function of suppressive regulatory T cells and also limits the function of autoreactive effector cells long before we had the entire genome sequenced. , when we had genome-wide association studies, the work of John Todd and Linda Wicker identified polymorphisms in the ctla-4 gene. and we associate them with human autoimmune diseases like type 1 diabetes, we have also learned that there are naturally occurring but rare mutations in ctla-4, like the ones listed here, worked on by mike leonardo and gulbu uzel at the nih called chai and latte. which are mutations, the first is a mutation in ctla4 which is associated with autoimmunity and the second is a mutation in the protein that transports ctla-4 to the cell surface lrba and these individuals will develop characteristics of autoimmunity as well as this understanding of ctla -4 as an inhibitory molecule has been translated into the therapeutic work of jim allison, who was the winner of the nobel prize last year, appreciated that since ctla 4 inhibits t cell activation and blocking ctla4 can improve t cell responses. t cells, it might be possible to block ctla4 and improve t cellular responses against tumors and this is illustrated in this cartoon there are therapeutic antibodies to see tla4 that have been developed these antibodies bind to ctla4 so the b7 molecules can no longer interact with ctla4 and therefore there is unopposed stimulation of b7 molecules that interact with cd28 and this can promote immune system responses and this has actually translated to anti-ctla 4 therapy for cancer, there are many co-stimulatory and co-inhibitory pathways and I would like to talk just about another pathway that is also a key inhibitory pathway, the pathway involving pd1 and I will use this cartoon to introduce the pd1 pathway, so that the pd1 receptor is upregulated in t cells upon activation in a similar manner to ctla4 and when pd-1 is activated by either of its ligands pdl1 or pdl2, it becomes phosphorylated on tyrosine motifs in its cytoplasmic domain and this can lead to the association of protein tyrosine phosphatases such as ship 2, which can then dephosphorylate the kinases downstream of the T cell receptor or cd28, as a result, signaling through the T cell receptor on cd28 is reduced and T cell responses are reduced. reduced cytokine production by T cells and reduced of death by cytolytic T cells.

Now one of the fascinating aspects of this pathway lies in the expression pattern of its ligands and, in particular, the expression of pdl1 really caught our attention when we first discovered that pdl1 and pdl2 were binding partners for pd1 and that these molecules can be expressed on a variety of hematopoietic cell types, such as antigen-presenting cells, but pdl1 in particular can be expressed on several non-hematopoietic cell types, such as blood vessels, a variety of epithelial cell types, muscle cells, liver cells, and liver cells. of the eyelids in the pancreas cells in the placenta and the eye this pattern of tissue expression suggested to us that this pathway could control T cell responses locally within the tissues and, indeed, that is the case, we have learned that the Interferons are potent stimuli for regulating pdl1 and pdl2, so this is one way that the immune system can naturally downregulate immune responses once T cells are activated, these cytokines are made and part of the counterpoint and the counterbalance here is that these cytokines will regulate these ligands, which can then also serve to attenuate immune responses when I think about the pd-1 pathway, it's really an example of why we have somewhat inhibitory signals in the immune system, this pathway is a counterweight to positive signals. through the T cell receptor and cd28 pd1 such as ctla4, these inhibitory signals are also important in mediating tolerance and, in addition, the expression of pdl1 in tissues has an important role because it allows this pathway to play an important role in resolution of pdl1 inflammation in nonhematopoietic tissue. cells, so cells in tissues express pdl-1 and this can control the resolution of inflammation and also protect tissues from autoimmune attack.

Furthermore, it is the smartest immunologists, microbes and tumors, who have time and again exploited this pathway to evade eradication by the immune system. system, this pathway contributes to T cell dysfunction, a process known as exhaustion that can develop during chronic viral infection and also cancer. What is T cell exhaustion? This is a dysfunctional state that is defined by poor function of effector T cells, so the cells do not kill. Also they do not produce cytokines, furthermore these cells express high levels of pd1 and other inhibitory receptors and this is a unique transcriptional and epigenetic state, just to compare acute and chronic infection, here we have a viral infection for example like the virus of influenza, the virus is found. by the immune system and the naïve T cells are activated as we have discussed, these cells can then differentiate into effector cells which are cells that can kill the infected cells and then the virus is eliminated and then we get a memory response, in contrast, there are some infections like hiv, hepatitis b, hepatitis c, a mouse model that is widely used as lymphocytic choreocytic meningitis virus known as lcmv and the situation in cancer where you have tumor antigens that are chronic is quite similar in this situation where you have persistent anagen, these chronically stimulated cells go as they progressively sink into states of T cell exhaustion, first the cells lose the ability to produce IL-2, then they lose the ability to produce certain cytokines and become They become poorer killers and these cells, some of them can disappear, we are learning now. that there are distinct populations of these cells, this understanding of pd-1 and t cell depletion was first described in this lcmv infection model and here what we are seeing is that there is a form of lcmv that causes an acute infection like influenza infection. occurs but then it is cleared and here if we look at the black circles what we can see is that during acute infection the expression of pd-1 increases but then as the virus is cleared the expression of pd-1 will return to decrease during chronic infection pd-1 goes up but stays high and what we have learned is that these cells that highly express pd1 are exhausted and less functional and the work of rafael john weary dan barber and gordon freeman showed that blocking This pathway could revive the function of these exhausted cells, so chronic infection could reduce the viral load by many registers and what I want you to understand from this is that both activated T cells and exhausted cells can express pd1, so you cannot tell if a cell was recently activated or if it is exhausted.

Only with the expression of pd-1 is it necessary to observe these cells and study them more deeply. This part of the pathway has also been translated into therapy. If this inhibitory signal is blocked, this can promote anti-tumor immunity, thus affecting the tumor cells and tissue themselves. The cells can express pdl1 and antibody drugs against pd-1 or pdl-1 have been developed that block this interaction and this can trigger a potent anti-tumor response, so these ctl, these cd8 t cells, can now kill and make cytokines one more time. and this has also translated to therapy in that pd1 blockers have been approved for 22 different types or for 22 different types of cancer.

I would now like to move on to a discussion of the roles of T cells in defense against infection. When we think about the immune response to an acute viral infection, we can think of this in phases, so we have the infection and then as a result of the stimulation, the immune cells activate, the T cells activate, they expand, they differentiate and then we can deal with the virus so that the viral load decreases and the virus is eliminated, then we have what is known as a contraction phase and a transition to memory and therefore we are looking at the time here, this can take a period of typically weeks and These memory cells can last for years, so when we think about the sequence events for the development of these effector T cell responses, we have antigen recognition as we have talked about and these T or B cells Naive cells can be activated and these cells then proliferate and then differentiate into effector cells and two memory cells, so first we're going to talk about the cd4 effector cells and what they can do and then we'll touch on the cd8 cells, which are the cells that they become killer cells and what they are. able to do so first, these naïve cd4 cells are able to differentiate into different subsets and these subsets can recruit and activate different types of cells that they can collaborate with to fight different types of infections, so as shown in this slide, there are effector cells. that they are the th1 cells that produce the cytokine, interferon is the cytokine that defines these cells, that the main target of gamma interferon is macrophages and, as a result of interferon, these cells, macrophages will have a receptor to be able to bind to interferon gamma and then there are signals. that get into these macrophages that activate these macrophages so that they can then deal with the intracellular microbes and kill them.

Th2 cells produce different cytokines il4 il5 and il13 and these cytokines can activate different types of immune cells, eosinophils, mast cells and certain types of macrophages and this type of immune defense is important in particular for certain types of parasites and worms these helmets we also have th17 cells that produce il-17 and il-22 and these can recruit neutrophils and activate them and these neutrophils can deal with extracellular bacteria and fungi, finally, there are follicular auxiliary cells, abbreviated as tfh, these cells can produce cytokines that include il- 21 and they can also produce interferon or il-4 and these target b cells and tfh cells work together with b cells and can lead to the production of antibodies and antibodies are very important in dealing with extracellular pathogens or extracellular forms of, for example, a virus, so antibodies are important to deal with the viral particles and prevent the antibodies can prevent the virus from binding and infecting cell and I think in your next lecture we will talk a lot more about the properties of the antibodies, but we also have forms in the ctl that we'll talk about next, which are important for killing cells that become infected, but first.

We'll talk a little more. I will focus here on the fact that time only allows me to talk in depth about a set of these cells and I am going to talk about the T follicular helper cells because the interactions between the helper cells and theHelper T cells and B cells are important for antibody defense and then we will move on to talk about cytotoxic T cells, so in this cartoon what we are doing is describing the steps and stages for helper T cell interactions. cd4 with the B cells that are necessary for the production of antibodies, so initially we have the activation of these helper T cells with antigen and costimulation, as we have already mentioned, which allows these cells to differentiate into T cells and this initial activation occurs in the T cell zone, so you get antigen recognition and costimulation at the same time in the follicles, the antigen is carried to the same antigen there and these b cells become activated as a result of the activation of the t cells in the b cells that are able to migrate with each other and interact, so when these cells interact, it is the interactions between these cells and the signals between them that allow the initial signals that initiate the antibody response.

As a result, outside the follicle we have these short-lived plasma cells that develop, but then some of these cells migrate back to the follicle where they form the germinal center and these specialized follicular helper t cells can interact with the b cells there and allow these b cells become long-lived plasma cells and memory b cells, i know, michel nuisance. I talked in depth about these cells, but I just want to focus on one aspect of these cells and talk about collaborating against tuberculosis and helping our cd4-mediated activation of these B cell lymphocytes that when these cells interact as we have discussed, these helpers activated.

The t cells will express a molecule called cd40 ligand which is upregulated on the surface of these activated cells and they also produce cytokines and then the b cells when they interact have receptors where they can receive signals through these cytokines and also cd40 is activated when The T cell ligand binds to cb40 on B cells as a result of these signals. These interactions between cd40 and the cd40 ligand are very important for the proliferation and differentiation of B cells and we know this because there are diseases, there are mutations in B cells. gene for the cd40 or cd40 ligand and this can result in a hyper syndrome igm.

Mutations in the gene for the cd40 ligand result in a disease that is characterized by defects in antibody production where it is not switched to isotypes such as igg and maturation of the immunoglobulin response is not achieved and we also know this from studies in mice that have mutations in cd40 or cd40 ligand, so these interactions between these cd4 cells and b lymphocytes are essential to achieve the maturation and activation of the b cell response. that happens, now I would like to move on to the cd8 cells, these are the cells that can develop to kill infected cells, so first I will give you an overview of these responses, both the cd4 and cd8 t cell responses occur within a lymphoid organ, such as a lymph node, has anagen presenting cells, such as dendritic cells, that will transport the viral antigens to the lymph node, where interactions between the T cell receptor and b7 molecules on these dendritic cells will occur with naive cd8 T cells. lead to their activation, their proliferation and then their differentiation into effector cells and these differentiated effector cells then enter the circulation and can migrate back to the sites of initial infection where these ctls can then kill the infected cells, which are called cells. target, so it is important that you know that these naïve CD8 T cells do not kill the infected cells that these cells need to differentiate into mature effector cells and when this differentiation process occurs, these mature cytolytic T cells acquire the machinery that allows them to lyse and kill infected cells.

Now cell death is antigen specific, it is the same mhc peptide antigen that caused these cd8 cells to differentiate into these killer cells that is required to trigger death by these mature differentiated ctls. Now these cytolytic T cells have two main functions: the first is the direct destruction of infected cells and the second is secretion. Of the inflammatory cytokines, these inflammatory cytokines are important in CTL function, but they can also function to activate macrophages, so there is an association between different types of cells to defend us against microbes. How do these cells perform their functions? And this cartoon shows that when there is antigen recognition of a target cell infected here by a ctl, these cells bind very strongly, there are integrins on the surface of these cells that lead to a stable interaction, like the interactions between lfa 1 and i cam For example, these CTL then become activated and have that which leads to excess exocytosis of the granules in these cells and these granules contain granzymes and perforin forms pores in the target cell membranes of the infected cell and then these granzymes can enter the cell and can lead to death of these cells, so it is a process that leads to cell death death of infected cells by these activated ctls finally I would like to move on to the T cell responses in covid19 this is very much a work in progress the type of work being carried out is to characterize virus-specific cd4 and cd8 cells across the spectrum of the disease in hospitalized patients in patients who recover, both in convalescent and In asymptomatic individuals, work is being done to define the specificities of the T cells, what is it about the virus that cb4 and cd8 T cells see, and work is being done to correlate the phenotypes of these T cells with the spectrum of the disease.

First, there are works that have continued lymphopenia indefinitely. This is an abnormal reduction in the number of lymphocytes that has been associated with serious disease, but this. It resolves when patients recover. Lymphopenia has been reported to affect cd4 t cells, cd8 t cells, b cells, as well as natural killer cells. The mechanisms are still unclear. It may be a direct effect of high levels of cytokines such as tnf, but it could also be due to. to hyperactivation, hyperstimulation of T cells or high levels of expression of molecules that can cause the death of these T cells.

Work from one of the previous conferences. Dr. Schiff Pillai has been studying seriously ill patients who had coven 19 who unfortunately succumbed to the disease and when studying the cells of these individuals, they discovered that, unlike individuals who were not infected, there was a marked decrease in the number of b and t cells in the lymph nodes of these individuals who died, as well as in the spleen, the number of b and t cells was reduced. Another thing that was found and I think Dr. Pillai talked a little bit about this last time from an innate immunity perspective is that in these individuals who died from a severe infection they found that there was a blunting of the response of the center germline and this is probable.

Which led to a decrease in the generation of long-lasting antibodies, what was found is that there was a complete loss of the germinal centers in the lymph nodes and spleen of these individuals with severe covin19 and that there was a block in the development of these follicular t. helper cells that are important for the generation of these germinal center responses in the b cells there was an increase in these th1 cells that produce cytokines and there was also a lot of TNF production and TNF secretion from macrophages that may contribute to this defective response. More work is being done.

It is necessary, but this is certainly illuminating and helps us understand some of the defects that are seen in patients with very serious diseases. Other work is being done with people with less severe illnesses and this illustrates the types of studies where people wonder what cd8 t cells and cd4 t cells c that t cells are taken from patients who, in this case , patients who have recovered from an illness, patients who are asymptomatic, for example, and you can take these t cells and then look at what protein portions of the virus are seen and identified and this type of work that just came online in the last month in immunity, for example, has shown that about 90 of the proteins that are epitopes, that is, portions of anagen that you see, are not the spike protein. but it appears that other proteins, such as the nucleocapsid protein, as well as membrane and other open reading frames, are the targets and they found that cd8 t cells from these individuals did not show cross-reactivity with seasonal coronaviruses, furthermore, a paper recent one that was just published and was online last week in Cell was looking at a variety of patients or individuals who were infected those with very severe disease who died those who recovered those with mild disease those who recovered from mild disease exposed family members some of which were asymptomatic and then they also looked at the blood donors and looked in various ways using flow cytometry to look at the phenotype using ellie spot to look at some of the functions of these cells and correlating this with um types of diseases and what and phenotype of the disease and what they found is that in acute infection there are T cells that can show an activated cytotoxic phenotype and that in convalescent individuals when they study these T cells and stimulate them in vitro they can produce, for example , multiple cytokines and, therefore, have memory markers on their surface.

Interestingly, they have found Cyrus Cov2-specific T cells and people who do not have antibodies, suggesting that antibodies alone may underestimate the extent of infected people, so as I mentioned, this is a work in progress every time. further. Articles are published literally every day. I have two recent reviews here that I think are very thoughtful. One from Corolla Venus that was published in Cell in October. One of John Weary in his laboratory that was also published in Nature Reviews. In summary, we are really at a very early stage of understanding T cell responses to coven 19. We are detecting cd4 and cd8 T cells with memory markers.

They have been detected in one hundred percent of the CD4 cells of people who have recovered and in seventy percent of the CD cells. Uh cd8 cells are observed in these patients and in individuals who recovered memory t cells, um cells have been detected that respond to multiple sars cov2 proteins, not only the spike protein but also, for example, the nucleoprotein and the membrane protein. In different studies, it appears that CD8T cellular responses are primarily to internal proteins rather than the spike protein. What has been observed is a particularly high frequency of cd4 cells that can see the viral spike protein in patients who have recovered, but we need more work to understand whether the presence of how to interpret the presence of these cells is still unclear. these cells are providing protective immunity and that work will take time, also as I mentioned there has been recent work done with virus specific T cells that have been detected in antibodies in negative individuals and these were family members of people who developed infection by sars cov2 and in convalescent people, so more work is needed to understand how to interpret this and finally, as one reads articles, some studies show from the first days that you see many cells expressing high levels of these inhibitory receptors such as pd1 and ctla4 and some people refer to these cells as being exhausted.

In preparation for this lecture, I spoke to one of the experts who has been studying these cells and asked him how he interprets this. these cells, as I mentioned, are recently activated or are dysfunctional and your feeling is that these cells are predominantly highly activated cells, as I mentioned, during activation you can have high levels of these inhibitory receptors that are upregulated, but more is needed. work, he said that as he has done transcriptional profiling on some of these cells and they have some characteristics of the transcriptional profiles of exhausted cells, so it's really a work in progress, but I think this work gives us hope that there may be memory T cells to develop. and we need to understand more about the functions of these cells, so I'm going to stop sharing my screen now and turn my video back on.

I will be happy to answer questions. I think we have a few minutes left. Thank you very much, Dr. Sharp, we have. some questions, as you pointed out, the vaccines that are in development focus on the spike protein and you pointed out that T cells often recognize additional portionsof the virus. Do you think this is a problem? So I think that vaccines are going to be very important to develop protective immunity because, as the work of Shiv Pillai's laboratory has indicated that in severe patients we will not be able to develop a good antibody response, it seems that the majority of the CD4 cells are targeted against the spike proteins, so that's good news, but I think this also informs us that to develop a broader response we may want to think more broadly about developing responses to other proteins.

Do you think T cell responses could contribute to the rogue immune response, the cytokine storm, for example? that is observed in a subset of patients, it is a very good question, it is possible that T cells could contribute. The innate immune system certainly plays an important role in this as well. We know that the cytokines produced by the cells of the innate immune system make a lot of interferons and TNF very early, so it could be that those cells are the ones predominantly driving the cytokine storm, but I think the T cells also have the potential to contribute.

Some patients infected with this virus also suffer from cancer and there is some curiosity. about whether we know anything about the response to viral infection when patients receive therapies that modulate the immune response in cancer, so it is a very good question, very little has been published on it, since the current work that has been published indicates that cancer patients can still respond to immunotherapies in some of the patients that I mentioned, where there are high levels of these inhibitory receptors that have been observed in certain patients, especially with hematological malignancies, suggesting that it may be different in certain types of cancer patients who are perhaps some of the most vulnerable to this infection, so more work is needed as we begin to understand how different types of comorbidities, cancer, age, obesity, other things will affect the immune response to copine 19.

And one more question, you talked about T cell depletion, but why not? Don't pathogens evolve that directly suppress ctla-4 or pd1? Yes, I think this is part of a response that develops because we know that if we don't have ctla4 or pd1, these pathogens can upregulate pdl1 tumor ligands. smart in the sense that they can regulate ligands and defend themselves as well, but if we are missing pd1, the pd1 pathway and the mouse models, when you give the animal an infection with the form of lcmv that causes a chronic infection, they die trying to eliminate the infection so it's balanced pathogenicity so to speak the virus can survive and find another host to replicate so there is limited damage to the host's immune system arlene thank you so much for being with us today really It's a pleasure, thanks again.