Periodic Reporting for period 4 - ToCCaTa (Tailoring the functional Capacity of Cytotoxic T cells for future Therapies)
Reporting period: 2023-04-01 to 2023-09-30
What was the problem/issue being addressed?
CD8+ T cells have great potential for prophylactic and therapeutic interventions against infections and tumors. A major obstacle to their effective therapeutic use is our limited understanding of the molecular basis that enables CD8+ T cells to perform their diverse functions. Solutions are needed to induce large numbers of pathogen-specific T cells and to adjust their functional capacity to different diseases. For example, large numbers of activated effector T cells are needed for cancer treatment, while resting memory T cells with high proliferative potential and tissue-resident T cells are crucial for limiting viral infection severity. Hypo-functional "exhausted" T cells limit immune responses in chronic infections and cancer, but less aggressive T cells with low tissue-damaging potential could be beneficial for infections in vital organs like the lungs and liver. Thus, adjusting T cell function is a significant challenge. With ERC Consolidator Grant support, we discovered key mechanisms and new insights significantly improving our understanding of CD8+ T cell biology.
What were the overall objectives?
Establishing defined functional profiles of T cells requires detailed knowledge of the molecular mechanisms and networks guiding T cell differentiation and strategies to manipulate them. Using i) experimental systems to mimic disease-related T cell phenotypes, ii) single-cell gene expression assessments, iii) epigenetic characterizations, and iv) high-throughput gene expression alteration strategies, we aimed to establish in vivo verified molecular dependencies and functional profiles. These studies were complemented by hypothesis-based functional assessments of previously identified candidate genes. Our goals were to discover new molecular targets, verify them in patient samples, and evaluate their therapeutic potential in disease-related large animal models. Specifically, we aimed to: Aim 1 - Identify new molecular targets determining T cell effector capacity; Aim 2 - Dissect branching points and molecular networks controlling T cell expansion and diversification; Aim 3 - Uncover new mechanisms controlling effector and memory T cell differentiation.
Why is it important for society?
The SARS-CoV-2 pandemic highlighted the importance of understanding the mechanisms that protect us from viral infections. T cell-mediated immunity played a crucial role in primary pathogen clearance, while antibodies protected against re-infection and after vaccination. Although T cells primarily eliminate infected cells in tissues, excessive or overly strong T cell responses can lead to severe tissue damage, known as immunopathology, which was a major factor in severe COVID-19 cases. Our project investigated the mechanisms controlling the extent and potential of immunopathology, focusing on key mechanisms critical during severe infections, including COVID-19. We aimed to uncover potential therapeutic targets that could be useful in future pandemics.
CD8+ T cells have great potential for prophylactic and therapeutic interventions against infections and tumors. A major obstacle to their effective therapeutic use is our limited understanding of the molecular basis that enables CD8+ T cells to perform their diverse functions. Solutions are needed to induce large numbers of pathogen-specific T cells and to adjust their functional capacity to different diseases. For example, large numbers of activated effector T cells are needed for cancer treatment, while resting memory T cells with high proliferative potential and tissue-resident T cells are crucial for limiting viral infection severity. Hypo-functional "exhausted" T cells limit immune responses in chronic infections and cancer, but less aggressive T cells with low tissue-damaging potential could be beneficial for infections in vital organs like the lungs and liver. Thus, adjusting T cell function is a significant challenge. With ERC Consolidator Grant support, we discovered key mechanisms and new insights significantly improving our understanding of CD8+ T cell biology.
What were the overall objectives?
Establishing defined functional profiles of T cells requires detailed knowledge of the molecular mechanisms and networks guiding T cell differentiation and strategies to manipulate them. Using i) experimental systems to mimic disease-related T cell phenotypes, ii) single-cell gene expression assessments, iii) epigenetic characterizations, and iv) high-throughput gene expression alteration strategies, we aimed to establish in vivo verified molecular dependencies and functional profiles. These studies were complemented by hypothesis-based functional assessments of previously identified candidate genes. Our goals were to discover new molecular targets, verify them in patient samples, and evaluate their therapeutic potential in disease-related large animal models. Specifically, we aimed to: Aim 1 - Identify new molecular targets determining T cell effector capacity; Aim 2 - Dissect branching points and molecular networks controlling T cell expansion and diversification; Aim 3 - Uncover new mechanisms controlling effector and memory T cell differentiation.
Why is it important for society?
The SARS-CoV-2 pandemic highlighted the importance of understanding the mechanisms that protect us from viral infections. T cell-mediated immunity played a crucial role in primary pathogen clearance, while antibodies protected against re-infection and after vaccination. Although T cells primarily eliminate infected cells in tissues, excessive or overly strong T cell responses can lead to severe tissue damage, known as immunopathology, which was a major factor in severe COVID-19 cases. Our project investigated the mechanisms controlling the extent and potential of immunopathology, focusing on key mechanisms critical during severe infections, including COVID-19. We aimed to uncover potential therapeutic targets that could be useful in future pandemics.
Below is a selection of the most significant results obtained with the support of the ERC grant.
a) Identification of Tox as a key determinant (transcription factor) that attenuates T cell responses in chronic infections and tumors:
Chronic infections and malignant tumors are known to attenuate the functional capacity of T cells, leading to T-cell exhaustion. Immunotherapies aim to prevent or overcome this exhaustion to enhance immune responses. We discovered that the transcription factor Tox is stably expressed in exhausted T cells. Knocking out Tox prevented T cell exhaustion in chronic infections, indicating its critical role. However, Tox-deficient T cells declined rapidly, highlighting its importance for maintaining stem-like progenitor cells (Tcf1+ progenitors). This dual role presents challenges for therapeutic targeting. The results were published in Nature in 2019.
b) Observation that exhausted effector T cells, but not stem-like progenitors, require the support of CD4+ helper T cells in chronic infection:
Using single-cell RNA sequencing, we showed that depletion of CD4+ T cells reduces exhausted effector cells but not stem-like progenitors. Restoring CD4+ T cells in chronic infection fully recovers the population. These findings emphasize the need to target both CD8 and CD4 T cells in immunotherapy. The results were published in PNAS in 2019.
c) Discoveries concerning the origin of exhausted T cells:
We have made very interesting discoveries concerning the origin of exhausted T cells, which were previously thought to be primarily involved in chronic infections. We have found that these cells are also constitutively produced in the early phase of an acute infection together with normal memory precursors. We then showed that the type of infection determines whether exhausted or non-exhausted cells are more abundant. This means that in an acute infection, the growth of non-exhausted cells is favored, while in a chronic infection, exhausted cells dominate. However, the crucial point is that both types of infections start with a similarly diversified precursor population in the early phase of infection. Related to this work, we have recently submitted a manuscript, for which we have now received the first set of reviews. They look very promising, and we are confident that we can further progress with this publication.
d) Sensitivity of effector T cells to pyrimidine deficiency:
Using pharmacological inhibitors to block pyrimidine synthesis, we discovered that effector T cells are sensitive to pyrimidine deficiency, while memory T cell progenitors are resistant. This blockade occurs during early T cell expansion when effector T cells have a higher proliferation rate and lower pyrimidine synthesis capacity. This differential susceptibility is a druggable checkpoint for influencing effector T cell responses in acute viral infections. The results were published in Nature Immunology in March 2023, and I am the corresponding senior author.
e) Dynamics and turnover of gut resident T cells:
We have investigated the dynamics and turnover of gut resident T cells. Here we developed a novel CD103 ERT2-Cre RosaLoxP(Stop)ZSgreen reporter mouse that allows time-resolved mapping of resident T cell fate. We found that primary CD103+ resident memory T cells lack secondary expansion potential and require CD103 precursors for their long-term maintenance. We have also developed a novel diphtheria toxin-based depletion system to selectively remove circulating but not resident T cells. With these mice, we demonstrated that resident cells are not competing for resources with circulating cells. Together, our observations improved our so far limited understanding of the kinetics, stability, and turnover of tissue-resident cells during reinfection. The work was published in Science Immunology in 2022, and we received the journal cover for this work.
a) Identification of Tox as a key determinant (transcription factor) that attenuates T cell responses in chronic infections and tumors:
Chronic infections and malignant tumors are known to attenuate the functional capacity of T cells, leading to T-cell exhaustion. Immunotherapies aim to prevent or overcome this exhaustion to enhance immune responses. We discovered that the transcription factor Tox is stably expressed in exhausted T cells. Knocking out Tox prevented T cell exhaustion in chronic infections, indicating its critical role. However, Tox-deficient T cells declined rapidly, highlighting its importance for maintaining stem-like progenitor cells (Tcf1+ progenitors). This dual role presents challenges for therapeutic targeting. The results were published in Nature in 2019.
b) Observation that exhausted effector T cells, but not stem-like progenitors, require the support of CD4+ helper T cells in chronic infection:
Using single-cell RNA sequencing, we showed that depletion of CD4+ T cells reduces exhausted effector cells but not stem-like progenitors. Restoring CD4+ T cells in chronic infection fully recovers the population. These findings emphasize the need to target both CD8 and CD4 T cells in immunotherapy. The results were published in PNAS in 2019.
c) Discoveries concerning the origin of exhausted T cells:
We have made very interesting discoveries concerning the origin of exhausted T cells, which were previously thought to be primarily involved in chronic infections. We have found that these cells are also constitutively produced in the early phase of an acute infection together with normal memory precursors. We then showed that the type of infection determines whether exhausted or non-exhausted cells are more abundant. This means that in an acute infection, the growth of non-exhausted cells is favored, while in a chronic infection, exhausted cells dominate. However, the crucial point is that both types of infections start with a similarly diversified precursor population in the early phase of infection. Related to this work, we have recently submitted a manuscript, for which we have now received the first set of reviews. They look very promising, and we are confident that we can further progress with this publication.
d) Sensitivity of effector T cells to pyrimidine deficiency:
Using pharmacological inhibitors to block pyrimidine synthesis, we discovered that effector T cells are sensitive to pyrimidine deficiency, while memory T cell progenitors are resistant. This blockade occurs during early T cell expansion when effector T cells have a higher proliferation rate and lower pyrimidine synthesis capacity. This differential susceptibility is a druggable checkpoint for influencing effector T cell responses in acute viral infections. The results were published in Nature Immunology in March 2023, and I am the corresponding senior author.
e) Dynamics and turnover of gut resident T cells:
We have investigated the dynamics and turnover of gut resident T cells. Here we developed a novel CD103 ERT2-Cre RosaLoxP(Stop)ZSgreen reporter mouse that allows time-resolved mapping of resident T cell fate. We found that primary CD103+ resident memory T cells lack secondary expansion potential and require CD103 precursors for their long-term maintenance. We have also developed a novel diphtheria toxin-based depletion system to selectively remove circulating but not resident T cells. With these mice, we demonstrated that resident cells are not competing for resources with circulating cells. Together, our observations improved our so far limited understanding of the kinetics, stability, and turnover of tissue-resident cells during reinfection. The work was published in Science Immunology in 2022, and we received the journal cover for this work.
We have published a large number of articles in the most prestigious journals in the field of immunology. These include an article in Nature, two articles in Nature immunology, an article in Science immunology and in PNAS. In addition, there are review articles in Nature immunolgy and Nature reviews immunology.