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How do killer cells manage to survive?

It is not easy for the body to protect itself once a virus has broken through the body’s lines of defence and caused a chronic infection. This task is made even more difficult when the intruders are viral hepatitis pathogens that attack the liver, the place where immunological tolerance is induced. Jörg Reimann (physician) and Reinhold Schirmbeck (biologist) are working on the development of T-cell-mediated therapeutic vaccination strategies to suppress chronic virus infections in the liver. Their approach takes them into the impassable terrain of basic immunological principles of regulation and dominance.

The two researchers from Ulm are using a transgenic mouse model to obtain detailed insights into the complex regulation of the human defence system. They are looking for ways to use CD8 T-cells to overcome immunological tolerance and induce a response that will promise to deliver permanent protection against hepatitis pathogens. Rare cases involving the spontaneous cure of chronic viral liver infections show that their venture has a chance of success. The two scientists are convinced that such spontaneous remissions do not happen by chance. Uncovering the secret behind these remissions is what motivates the scientists, particularly as evidence shows that they are not chasing ghosts.

The problem of different shapes

In order for T-cells to be able to recognise antigens, the antigens must be specifically altered and presented on MHC (major histocompatibility complex)-encoded class I and class II receptors on the cell surface. These receptors are extremely variable. Using the MHC class I pathway, virally infected and abnormal cells that produce allogeneic proteins are identified and eliminated by the (CD8) T-killer cells. These MHC membrane proteins are of key importance in the specific activation of T-cells.

The extremely varied repertoire of cytotoxic CD8 T-lymphocytes is selected in such a way that the aggressive killer cells do not normally bind with their antigen-specific T-cell receptor to cells that present a peptide originating from the body’s own proteins. This self-tolerance protects the body from autoaggressive attacks from its own immune system. However, when a cell is infected with viruses or affected by mutations, thereby leading to the expression of new, i.e. non-self/unknown antigens, these peptides will be presented as part of the MHC class I complex. This leads to the activation of cytotoxic T-lymphocytes that will then eliminate the abnormal cells.

Very individualistic immune responses

The hepatitis B virus attacks an organ that is highly delicate in immunological terms. © NCI

The two researchers from Ulm hope to find out how the hepatitis viruses manage to frequently outwit the body's immune system and establish chronic infections. They are restimulating in vivo vaccine-induced T-cells under different conditions in a transgenic mouse model (in which selected antigens of hepatotropic viruses or complete, replicating viral genomes are selectively expressed in the liver).

The presentation of peptides on specific MHC molecules leads to the activation of T-cells. Subsequently, a range of antigenic peptides that are able to specifically activate T-cells then need to be determined for each MHC haplotype (e.g. H-2d, H-2b). This makes the characterisation of human T-cells quite difficult because the human immune system reacts in very individual ways to viral attacks. The body's immune defence mechanisms target a specific antigen fragment (epitope), a process that is carried out by so-called HLA (human leukocyte antigens) molecules (every human individual expresses 3 - 6 different HLA molecules) which bind the appropriate epitope and present it on the cell surface, thereby stimulating CD8 T-cells. The binding of epitopes to MHC class I molecules can trigger very individualistic immune responses. "Unfortunately, we are only at the very beginning," said Reinhold Schirmbeck. There are at present only a handful of humanised mouse systems that express human class I molecules such as HLA A2 (50% of the European population possess HLA A2) available.

However, the researchers have already made one important observation. They found that only T-cells that were specific for a small number of the epitopes identified were able to mediate protective, anti-viral T-cell immunity. Whilst the researchers have had no difficulty in transferring tailor-made epitopes into the model animals, they are finding it far more difficult to find an answer to the question as to which epitope guarantees the greatest (immunological) success. In principle, viruses such as HBV are very large, and the number of eptitopes that can be presented on the surface is equally large (up to over 106).

Immununodominance and fishing for effects

Complicated immunodominance: Cells with MHC type A are potentially able to present the epitopes A1 – A5. But not all epitopes are simultaneously active, and not all induced T-cell responses have an anti-viral effect. A more detailed analysis of the anti-viral T-cell response in MHC-A animals shows that normally only one “immunodominant” response against A1 occurs during infection; responses against the “subdominant” epitopes A2 – A5 cannot be detected. Only the deletion of epitope A1 makes the subdominant epitopes A2 and A3 (but not A4 and A5) visible to the T-cells. Epitopes A1, A2 and A3 need to be deleted in order to generate T-cells that are specific for the epitopes A4 and A5. Only a few selected epitopes have the desired “quality”, i.e. the ability to specifically activate anti-viral T-cells. A hierarchy of potentially interesting epitopes becomes visible when the viral antigen is “engineered” (i.e. when immunogenic sequences are selectively deleted). © Profs. Schirmbeck/Reimann/University Hospital Ulm

The Ulm researchers focus on immunodominance, a phenomenon that is not yet clarified in detail. The researchers have found out that the dominant epitope triggers an effective immunological response, but fails to have a biological effect. This is in contrast to epitopes found on a second, subdominant level.

The subdominant, but not the dominant, epitopes potentially trigger the sought-after immunological effect. Therefore, the dominant epitopes must be removed to enable the previously suppressed subdominant epitopes to exert their effect. It is still not clear what makes the epitopes dominant or subdominant and which MHC class I molecules are involved in these regulatory processes. Jörg Reimann and Reinhold Schirmbeck are slowly but surely gaining an understanding of the molecular basis of this phenomenon using mouse models.

And then there is the liver…

Interactions between antigen-presenting liver cells and CD8 T-cells: (1) virus-infected cells present viral epitopes by way of MHC class I molecules on the cell surface. These complexes are specifically recognised by CD8 T-cells. (8) Co-stimulatory and/or (3) co-inhibitory ligand/receptor interactions mediate additional signals that have an effect on the viability and functionality of CD8 T-cells. © Profs. Schirmbeck/Reimann/University Hospital Ulm

As if the interactions between virus and CD8 T-cells were not complicated enough, the immunologically somewhat delicate liver makes the two scientists’ work even more difficult. Once hepatitis viruses have infested the liver, the liver starts to communicate with the T-cells, with an often fatal outcome for the latter. Schirmbeck and Reimann then have to deal with another regulation wave of co-stimulatory and co-inhibitory molecules in the liver cell.

The two researchers are using therapeutic approaches in their attempt to shift the balance towards the T-cells, either by reducing the antigen freight in the liver, or through interferon and anti-viral therapies that give the T-cells permanent advantages and enable them to reach stable immunity.

Regulatory cells render the life difficult for T-cells in the liver

Reimann and Schirmbeck have successfully solved the methods of vaccination (DNA vaccines, recombinant viruses, peptide vaccines). They have also succeeded in defining immunodominant and subdominant epitopes in the animal model and “tuning” T-cells in such a way as to enable them to reach their target organ, increase the local interferon production and hence reduce the replication of the viruses. The researchers have managed to sustain the process for a week. Jörg Reimann assumes that regulatory cells of the innate immune system make life difficult for T-cells in the liver because they contribute to reducing the effect of the specific immune system in just a few days.

Once the two researchers have defined the regulation levels, they will know which are the right questions to ask: how and where can they interfere with the regulation process of T-cell and liver cell recognition, from where or which molecules stems the signal that switches the T-cells off and suppresses their function? Reimann and Schirmbeck hope to be able to characterise this signal using boost immunisation strategies.

In order to decipher these regulatory mechanisms, the scientists need to work on basic biology (Reimann: we need to approach the problem in a reductionist way) using preclinical mouse models. Reimann and Schirmbeck are well aware of the need for substantial preclinical evidence before they can even begin to think of translation. It is also clear for them that the T-cell-mediated induced response (induced by vaccines) would be a far less invasive therapy for millions of HBV-infected and HCV-infected people than a therapy involving antiviral drugs that is associated with numerous undesired side effects.

Riedl, P., Wieland, A. et al.: Elimination of Immunodominant Epitopes from Multispecific DNA-based Vaccines Allows Induction of CD8 T Cells That Have a Striking Antiviral Potential), in: Journal of Immunology, 2009, 183:370-380.

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