Final Report Summary - PROTECTC (Identify novel pathways to enhance the induction of protective CD8+ T cell responses) During the funding period, we have made the following discoveries:1) We established the concept that T cells undergo stable differentiation in chronic infection: At the beginning of the project, it was well known that chronic viral infections and malignant tumors induce T cells that have a reduced ability to secrete effector cytokines and have upregulated expression of the inhibitory receptor PD1 (programmed cell death protein 1). These features were so far considered to mark terminally differentiated 'exhausted' T cells. However, along with work that we had published before the starting of the project, there were several clinical and experimental observations which indicated that phenotypically exhausted T cells can still mediate a crucial level of pathogen or tumor control. We therefore proposed before the starting of the project that the exhausted phenotype results from a differentiation process in which T cells stably adjust their effector capacity to the needs of chronic infection. We argued that this phenotype is optimized to cause minimal tissue damage while still mediating a critical level of pathogen control. At the beginning of funding, this view strongly contrasted to the commonly held view of functional exhaustion. With the ERC funding, we managed to establish and verify many of the concepts that we had proposed. 1a) Demonstration that exhausted and non-exhausted T cells show in a similar environment similar expansion and survival kinetics (Utzschneider et al., Jourmal of Experimental Medicine, 2016). We found that the key trigger for inducing T-cell exhausting is high antigen exposure; an environment of chronic infection with low antigen exposure is not sufficient. We demonstrated this using an experimental system in which we can selectively reduce the level of expression of a particular antigen, while we left the chronic infection as such unaffected. When T cells are exposed to such low level antigen-stimulation in chronic infection, they retain over in-definitive periods a normal effector phenotype without showing signs of transitioning into memory T cells. Surprisingly, T cells with a chronic phenotype exposed to high antigen levels underwent similar expansion and survival rates as T cells with an acute phenotype exposed to low antigen levels. This underscores that phenotype and expansion/survival kinetics are uncoupled. Moreover, our system allows highly specific side-by-side comparisons of cells with an acute or chronic phenotype. This sustained a paradigm shift that T cells with or without an exhausted/chronic phenotype undergo similar expansion in chronic infection.1b) Demonstrated that a population of proliferation competent T cells is needed to maintain T cell responses in chronic infection (Utzschneider et al., Immunity, 2016). This is arguably the most significant of the findings published so far that emerged from the ERC grant. Before the project we had published that populations of exhausted T cells still contain a minor subset that retained proliferative capacity and we had proposed that this population could be critical for long-term maintenance of T cell responses. Within the ERC project, we were therefore interested in identifying the molecular foundation of this population. Anticipating that Tcf-1 may mark cells which retain proliferative potential in chronic infection, we observed in a collaboration with Werner Held (U. of Lausanne, CH) that Tcf-1 deficient mice show similar early T cell expansion but later have major deficiencies in maintaining the T cell response. Using GFP reporter mice, we confirmed that proliferative capacity in chronic infection is restricted to Tcf-1 expressing cells and that absence of these cells results in impaired T cell maintenance. Moreover, we demonstrated that increases in T cell numbers after blocking PD1 signaling also requires this memory-like population. This work resulted in a co-senior author publication in Immunity. We consider the Tcf-1+ population as key for successful immunotherapies. Moreover, a number of publications were subsequently released which confirmed our findings in other modes and in human chronic infection and for human tumor-specific T cell.1c) We followed up on this observation of the significant of the Tcf-1 population (manuscript submitted). We performed extensive molecular network analysis using single cell RNA sequencing. The results of these data are pending publication. Moreover, we tested the CD4 dependence of this population. The resulted in very surprising outcomes and underlined that Tcf-1+ population in chronic infection are very different from conventional memory T cells.1d) Using the system described under 1b, we made extensive side by side composition of exhausted and not-exhausted T cells. We therefore identify a transcription factor that was associated with chronic infection (manuscript submitted). Strikingly, the elimination of this factor prevented T-cell from becoming exhausted. Alongside with a number of tests in mice and humans, we think we have identified the transcription factor that serves as master regulator for T cell exhaustion. We have submitted these findings to a high profile journal and we recently submitted a revised version of this manuscript. Altogether, we think we can claim that our findings had a significant impact onto the field. Our findings were meanwhile complemented and confirmed by several other publications. Moreover, the significant of targeting the Tcf-1+ population in clinical setting against cancer is presently investigated. 2) Identification of an early commitment of naïve T-cells into precursors memory T cells and identification of a new molecular pathways that controls differentiation of effector and memory T cells (Manuscript in final stage of preparation). We have identified a metabolic modulator that alters T cell differentiation on acute infections in such a way that it almost completely blocks effector T cell formation without impacting memory generation. We apologize for not being able to reveal the entity of this factors in this format. The corresponding publication is under preparation.3) A minimum epitope overlap focuses the T cell repertoire in secondary infections (Oberle, Cell reports, 2016): Following observations that primary infections recruit very broad T cell repertoires (Zehn et al., Nature 2009), we investigated the TCR stimulation requirement in situations where an individual bears pathogen-specific memory T cells at the time of infection. We found that even the presence of a single epitope recognized by memory T cells raises strongly the T cell stimulation threshold, such that only very high affinity T cells become activated. Interestingly, this threshold increase appears to be predominantly mediated by resident memory T cells . Subsequently, we are investigating the cellular mechanisms that cause resident memory T cells to interfere with antigen presentation and low affinity T cell stimulation. We pursued this with the help of whole organ conventional histological imaging. Presently, we are in the final stages of finishing this project through institutional support.
