Mark Davis and the search for the universal immune test

A traslation of this blog post to Spanish can be downloaded here. I would like to thank Humbert.Cat for the translation.

1. Introduction

These are some notes about the talk that Mark Davis gave during the Community Symposium held in August at Stanford (video). I will introduce some basic notions about T cell receptors (TCR) in paragraphs 2, 3, 4, and 5. Paragraphs 6 is a description of an innovative technology developed by Mark Davis and his colleagues, based on information gathered from the video itself and three research papers published by Davis and others in the last 4 years. This background should be hopefully enough to allow a good understanding of the exciting pilot data presented by Mark Davis on T cell activity in ME/CFS (paragraph 7), and in chronic Lyme (paragraph 8), and to realize why this technology promises to be some sort of universal test for any kind of infectious and autoimmune diseases, known or unknown.

2. T cells

T cells are a type of leukocytes (also known as white blood cells), the cellular component of our immune system. Most of our circulating T cells are represented by T helper cells (Th cells) and cytotoxic T lymphocytes (CTL). While the function of Th cells is to regulate the activity of other leukocytes through the production of a wide range of chemicals (cytokines), CTLs are directly involved in the killing of host cells infected by pathogens. T cells belong to the adaptive arm of the immune system, along with B cells (the factories of antibodies), and as such, they are meant to provide a defence tailored to specific pathogens: our immune system can provide not only antibodies specific for a given pathogen but also specific T cells (both Th cells and CTLs). The innate arm of the immune system (which includes natural killer cells, macrophages, dendritic cells, mast cells…) on the other hand can provide only a one-fits-all type of defense, which represents the first line of immune response, during an infection.

3. T cell receptor

T cells search for their specific pathogens thanks to a molecule expressed on their surface, called T cell receptor (TCR). In figure 1 you can see a schematic representation of the TCR and of the mechanism by which T cells recognize their targets. Antigens (proteins) from pathogens are presented to T cells by other cells of our body: they are displayed on molecules called major histocompatibility complex (MHC), expressed on the outer membrane; if the antigen fits the TCR of a specific T cell, then this T cell is activated and proliferates (clonal expansion). The two chains (α and β) are assembled using the transcription of gene segments with several copies each: in other words, TCRs are assembled with peptides chosen randomly within a set of several possible choices. This leads to a repertoire of 10^15 possible different TCRs (Mason DA 1998). Each T cell displays only one type of TCR.

Figure 1. Upper half. Th cells and CTLs share the same TCR: in both cases this molecule is the assembly of two peptides (chain α and chain β), but while the TCR of Th cells (on the right) is expressed next to the molecule CD4 (which binds to class II MHC), the TCR of CTL is associated with the molecule CD8 (on the left), which is specific for MHC I. Black bars represent four chains (a complex called CD3) that are involved in the signaling of the TCR with the nucleus of the cell (by Paolo Maccallini). Lower half. A beautiful structural representation of the TCR, bound to the peptide-MHC complex (pMHC), from (Gonzàlez PA et al. 2013). In green the peptide, in blue the β chain, in dark green the α chain. CDRs (complementarity determining regions, orange) are composed of those residues of the α and β chains that directly bind the pMHC.

4. T helper cells

Th cells can recognize only antigens presented by class II MHC: this class of MHC is expressed on the outer membrane of some leukocytes, mainly dendritic cells, B cells, and macrophages (referred to as antigen presenting cells, APCs). MHC II engages the TCR of Th cells thanks to peptide CD4 (expressed exclusively by Th cells). The antigen presented by MHC II is a peptide with a length of 13-17 amino acids (Rudensky, et al., 1991) (figure 2).

Figure 2. The TCR expressed by a Th cell binds an epitope presented by a class II MHC expressed on the plasma membrane of an APC. Chains α and β of MHC II are also represented (by Paolo Maccallini).

5. Cytotoxic T lymphocytes

TCRs expressed by CTLs can bind only antigens displayed by class I MHC, which can be found on the outer membrane of any cell of our body. CD8 is the molecule that makes the TCR expressed by CTLs specific for MHC I. While antigens presented by APCs belongs to pathogens that have been collected on the battlefield of the infection, peptides displayed by class I MHC of a specific cell belong to pathogens that have entered the cell itself, therefore they are the proof of an ongoing intracellular infection (figure 3). When a CTL recognizes an antigen that fits its TCR, then the CTL induces apoptosis (programmed death) of the cell that displays it. Antigens presented by MHC I are peptides in the range of 8 to 10 amino acids (Stern, et al., 1994).

Figure 3. An infected cell displays a viral antigen on its MHC I. The TCR of a CTL binds this peptide and send a signal to the nucleus of the CTL itself, that responds with the induction of apoptosis (releasing granzymes, for instance) of the infected cell (by Paolo Maccallini).  

6. The universal immune testing

In his talk, Mark Davis presents an overview of some basic concepts about the immune system, before introducing his exciting new data about ME/CFS and post-treatment Lyme disease syndrome (PTLDS, also known as chronic Lyme). But he also describes a few details of a complex new assay that is theoretically able to read all the information packed in the repertoire of TCRs present – in a given moment – in the blood of a human being. As such, this test – that I have named the universal immune testing – seems to have the potential to determine if a given patient has an ongoing infection (and the exact pathogen) or an autoimmune disease (and the exact autoantigen, i.e. the tissue attached by the immune system). To my understanding, this assay requires three steps, described in the following sections.

6.1. First step: TCR sequencing

As said in paragraph 3, when T cells encounter their specific peptide presented by MHC, they proliferate so that in blood of patients with an ongoing infection (or with a reaction against self, i.e. autoimmunity) we can find several copies of T cells expressing the same TCR: while in healthy controls about 10% of total CD8 T cells is represented by clones of a few different T cells (figure 4, first line), in early Lyme disease – an example of active infection – and in multiple sclerosis (MS) – an example of autoimmune disease – we have a massive clonation of a few lines of CTLs (figure 5, second and third line, respectively). The first step of the universal immune testing is represented by the identification of the exact sequence of TCRs expressed by T cells in blood, as reported in (Han A et al. 2014) where it is described how to sequence genes for the α and the β chain of a given T cell. This approach allows to build graphs of the kind in figure 4, and therefore to determine whether the patient has an abnormal ongoing T cell activity or not. If a clonal expansion is found, then we can speculate that either an active infection is present or some sort of autoimmune condition.

6.2. Second step: TCR clustering

Mark Davis and his group have been able to code a software that allows to identify TCRs that share the same antigen, either within an individual or across different donors. This algorithm has been termed GLIPH (grouping of lymphocyte interaction by paratope hotspots) and has been found capable – for instance – to cluster T CD4 cell receptors from 22 subjects with latent M. tuberculosis infection into 16 distinct groups, each of which comprises TCRs from at least 3 different donors (Glanville J et al. 2017). Five of these groups are reported in figure 5. The idea here is that TCRs that belong to the same cluster, react to the same peptide-MHC complex (pMHC).

6.3. Third step: quest for the epitope(s)

As we have seen, this new technology allows to recognize if T cell clonal expansion is an issue in a given patient, by sequencing TCRs from his peripheral blood. It also allows to cluster TCRs either within an individual or across different patients. The next step is to identify what kind of antigen(s) each cluster of TCRs reacts to. In fact, if we could recognize these antigens in a group of patients with similar symptoms, with T cell clonal expansion and similar TCRs, we would be able to understand whether their immune system is fighting a pathogen (and to identify the pathogen) or if it is attacking host tissues and, if that was the case, to identify what tissue. As mentioned, the number of possible TCR gene rearrangement is supposed to be about 10^15, but the number of possible Th cell epitope is about 20^15 which is more than 10^19. This implies that TCRs have to be cross-reactive to some extent, in order to recognize all possible peptides presented by MHCs (Mason DA 1998). The exact extent of this cross-reactivity and the mechanism by which it is obtained has been elucidated by Mark Davis and his colleagues in a recent paper (Birnbaum ME et al. 2014) that gives us the third step of the universal immune testing. The aim of this phase is to take a given TCR and to find the repertoire of his specific antigens (as said, one TCR reacts to several antigens). In order to understand how this is possible let’s consider one of the experiments described in the paper mentioned above. The researchers considered two well-defined TCRs (named Ob.1A12 and Ob.2F3), cloned from a patient with MS and known to recognize peptide 85-99 (figure 6) of myelin basic protein (MBP) presented by HLA-DR15. They then prepared a set of yeast cells expressing HLA-DR15 molecules, each presenting a different peptide of 14 amino acids, with fixed residues only at position 1 and 4, where the peptide is anchored to MHC (figure 6, left). When copies of Ob.1A12 are added to this culture of yeast cells expressing pMHC complexes, they bind only some of them, and as you can see in the right half of figure 6, for each position of the epitopes bound by Ob.1A12, there is an amino acid that is more likely: for instance, the typical epitope of Ob.1A12 preferentially has alanine (A) at position -4, histidine (H) at position -3, arginine (R) at position -2, and so forth. As you can see, histidine (H) at position 2 and phenylalanine (F) at position 3 are obligate amino acids for a Ob.1A12 epitope.

The table on the right side of figure 6 is, in fact, a substitution matrix with dimension 14×20, a tool that can be used to scan the peptide database in order to find, among all the known peptides expressed by living creatures, all the possible Ob.1A12 specific epitopes. Substitution matrices are commonly used for the so-called peptide alignment, a technique that aims at the identification of similarities between peptides. These matrices are based on evolutionary considerations (Dayhoff, et al., 1978) or on the study of conserved regions in proteins (Henikoff, et al., 1992). But the search for specific epitopes of a given TCR requires (as we have seen here for Ob.1A12) a substitution matrix built ad hoc for that TCR: each TCR requires its own substitution matrix that is obtained adding clones of that TCR on a culture of yeast cells presenting a huge peptide library on their MHCs, and analyzing data from this experiment. So, quite a complex process! In the case of Ob.1A12, this process led to 2330 peptides (including MBP), while the Ob.2F3 specific substitution matrix found 4824 epitopes within the whole peptide database. These peptides included both non-human proteins (bacterial, viral…) and human peptides. For 33 of them (26 non human and 7 human proteins), this group of researchers performed a test in order to directly verify the prediction: 25/26 of environmental peptides and 6/7 of the human peptides induced proliferation of T cells expressing Ob.1A12 and/or Ob.2F3, and this is a huge proof of the validity of this analysis! These 33 peptides are reported in figure 7. This is the last step of the universal immune testing, the one that from the TCR leads to the epitopes. As you have seen, a huge set of different peptides from different sources is linked to each single TCR, in other words, crossreactivity is an intrinsic property of TCR. This also means that lymphocyte transformation tests (LTTs), widely used in Europe for the detection of infections like Borrelia burgdorferi and others, bear a high risk of false-positive results and require a process of experimental and theoretical validation, that is currently lacking (see also this post on this issue).

We are now ready to fully appreciate the pilot data that Mark Davis presented at the Symposium on CD8 T cell clonal expansion in ME/CFS and in chronic Lyme.

7. We have a hit!

Mark Davis, along with Jacob Glanville and José Montoya, has sequenced TCRs from the peripheral blood of 50 ME/CFS patients and 49 controls (first step of the universal immune testing, remember?), then they have clustered them using the GLIPH algorithm (second step). They have found 28 clusters with more than 2500 similar sequences each, where each cluster collects multiple sequences from the same individual as well as (which is perhaps more important) sequences from different patients (figure 8). The cluster that I have circled in red, for instance, is a collection of more than 3000 similar TCRs. The presence of this wide clusters in ME/CFS patients, compared to healthy controls, represents an indirect proof of a specific T cell response to some common trigger in this group of patients, which might be a pathogen or a tissue of the body (or both).

Among these 50 ME/CFS patients, Davis and colleagues selected 6 patients with similar HLA genes (figure 9, left), 5 females among them, and studied their TCRs deeper. In the right half of figure 9, you can see for each patient the degree of CTL clonal expansion. Remember that in healthy controls only about 10% of CTLs is composed by clones of a few cells (figure 4, first raw), while here we see that about 50% of all CTLs is composed by clones. So, a “marked clonal expansion” of CD8 T cells, as Davis said.

Sequences of α and β chains of TCRs from three of the six patients (patients L4-02, L4-10, and L3-20) are reported in figure 10 (I have verified that in fact these are sequences of α and β chains of human TCRs using them as query sequences in standard protein BLAST).

So, we have seen so far the first two steps of the universal immune testing in ME. What about the third step? In his talk, Mark Davis didn’t present any particular epitope, he just showed a slide with what likely is the selection of the epitopes from the peptide library (see paragraph 6.3) by one of the TCRs reported in figure 10. This selection is reported in figure 11, but from that picture, it is not possible to gather any information about the identity of these epitopes. As you probably remember from paragraph 6.3, the analysis of the peptides selected by a TCR among the peptide library allows the identification of a substitution matrix that can be used to select all the possible epitopes of that specific TCR, from the peptide database. This last crucial step has to be performed yet, or it has been already performed, but Davis has not communicated the preliminary results during his talk. Recently new resources have been made available by Open Medicine Foundation, for this promising research to be further pursued, among other projects (R). The aim here, as already said, is to find the antigen that triggers this T cell response. As Mark Davis said, it might be an antigen from a specific pathogen (perhaps a common pathogen that comes and goes) that elicits an abnormal immune response which ends targeting some host tissue (microglia, for instance), thus leading to the kind of immune activation that has been recently reported by Mark Davis himself and others in ME/CFS (Montoya JG et al. 2017). The idea of a common pathogen triggering a pathologic immune response is not new in medicine, and rheumatic fever (RF) is an example of such a disease: RF is an autoimmune disease that attacks heart, brain and joints and is generally triggered by a streptococcal throat infection (Marijon E et al. 2012). The other possible avenue is, of course, that of an ongoing infection of some kind, that has yet to be detected. As said (see par. 6.1), CD8 T cell clonal expansion is present in both acute infections (like early Lyme disease) and autoimmune diseases (like MS) (figure 4), so we have to wait for the antigen identification if we want to understand if the CTLs activity is against a pathogen and/or against a host tissue.

8. Chronic Lyme does exist

It has probably been overlooked that in his talk, Mark Davis reported also very interesting data on post-treatment Lyme disease syndrome (PTLDS, also known as chronic Lyme disease). In particular, he found a marked clonal expansion in CD8 T cells of 4 PTLDS patients (about 40% of total CTLs) as reported in figure 12: consider that in this case, blue slices represent unique T cells, while all the other slices represent clones! All that has been said about CD8 clonal expansion in ME/CFS does apply in this case too: it might be the proof of an ongoing infection – perhaps the same B. burgdorferi, as suggested by several animal models (Embers ME et al. 2017), (Embers ME et al. 2012), (Hodzic E et al. 2008), (Yrjänäinen H et al. 2010) – or a coinfection (a virus?) or it could be the expression of an autoimmune reaction triggered by the initial infection. This has still to be discovered, running the complete universal immune testing, but what is already clear from figure 12 is that PTLDS is a real condition, with something really wrong going on within the immune response: chronic Lyme does exist.

9. Conclusions

Mark Davis and other researchers have developed a complex assay that is able to sequence TCRs from patients, cluster them into groups of TCRs that react to the same antigens, and discover the antigens that triggered that particular T cell response. This assay is a kind of universal immune testing that is theoretically able to recognize if a person (or a group of patients) presents an immune response against a pathogen or against one of his own tissues (or both). This approach has already given pilot data on an ongoing CD8 T cell activity in ME/CFS patients and in chronic Lyme patients and will hopefully identify the trigger of this immune response in the near future. Whether ME/CFS is an ongoing infection, an autoimmune disease or both, the universal immune testing might be able to tell us. This new technology is for immunology, what whole genome sequencing is for genetics, or metabolomics is for molecular diseases: it doesn’t search for a particular pathogen or a particular autoimmune disease. No, it searches for all possible infections and immune disorders, even those that have yet to be discovered.

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