Periodic Reporting for period 1 - TLSaRNA (Artificial induction of tertiary lymphoid structures (TLS) in tumors using intratumoral mRNAs to evaluate its synergy with immune checkpoint inhibitors)
Berichtszeitraum: 2024-04-01 bis 2026-03-31
Cancer immunotherapy exploits the ability of the host immune system to control the development and progression of tumours. Even though the success of immune checkpoint inhibitors in the treatment of many different tumours, still only a fraction of patients can benefit of them, possibly because the low numbers of intratumoural immune cells in the no-responders and their unresponsiveness state. Therefore, the generation of new therapies aiming to potentiate the immune infiltration can be crucial to improve current treatments. Not only the infiltration of the immune system is important for generating anti-tumoral responses, also their intratumoural organisation. Under situations of chronic inflammation such as cancer, ectopically generated immune structures called tertiary lymphoid structures (TLS) may emerge. TLS have a similar level of organization than the secondary lymphoid organs, characterised by having a B-cell zone adjacent to a T-cell zone, and interestingly their presence has been statistically correlated to a better cancer prognosis in different types of tumours and improved response to immune checkpoint inhibitors (ICB). However, not all the patients develop TLS and the cellular composition and organisation within the structures can vary. Specifically, the role of B cells in the tumour microenvironment is controversial, while some studies have reported a pro-tumoral effect, possibly through differentiation of regulatory B cells, metadata shows a strong association between the presence of a transcriptional B cell profile on the tumours and the improved patient outcome. Moreover, the presence of B cells in the tissue microenvironment is also positively associated with an improved response to ICB. However, even though these strong statistical associations and the relevance of TLS in the cancer prognosis, still little is known about the mechanisms underlying their function and the specific role of B cells in the anti-tumoral response elicited. In this proposal, we will (i) study novel therapies for artificial TLS induction; (ii) characterise their combination with ICBs; and (iii) decipher the function of B cells on the tumour microenvironment. Ultimately, the knowledge generated with this proposal will provide relevant information to guide future immunotherapy treatments.
1- Design and generation of mRNAs encoding for therapeutic cytokines. I generated the mRNAS encoding for the cytokines of interest and tested their expression and functionality in vitro.
2- Optimisation of flow cytometry and tumor clarification protocols. I optimised the flow cytometry and immunofluorescence protocol to analyse TLS.
3- Identification of the effect of intratumoral mRNAs injection in the tumor growth and TLS generation. Local delivery (intratumoral) of two of the cytokines tested delayed tumor growth in mice bearing subcutaneous tumors. We then tested their combination, which enhanced the anti-tumoral effect. Both mRNA-based cytokine therapies were selected for combinatory treatments.
4- Identification of tumor outcome in response to immunotherapy treatments in combination with TLS-induced therapy. We observed an additive effect of immune checkpoint blockade (ICB) when combined with the mRNA-based therapy in controlling tumor growth.
5- Report of the cellular and molecular changes induced in TLS upon ICB treatment. We observed an increased intratumoral T-cell infiltrate along with activation of the intratumoral endothelial cell profile. However, preliminary data did not show an organized immune infiltrate with the formation of mature tertiary lymphoid structures (TLS).
6- Analysis of the anti-tumoral mechanism of action elicited by the mRNA-based therapy. By using depleting anti-CD8 antibodies, I could determine that the anti-tumoral effect exerted by the mRNA-based therapy was partially CD8-dependent.
2- Optimisation of flow cytometry and tumor clarification protocols. I optimised the flow cytometry and immunofluorescence protocol to analyse TLS.
3- Identification of the effect of intratumoral mRNAs injection in the tumor growth and TLS generation. Local delivery (intratumoral) of two of the cytokines tested delayed tumor growth in mice bearing subcutaneous tumors. We then tested their combination, which enhanced the anti-tumoral effect. Both mRNA-based cytokine therapies were selected for combinatory treatments.
4- Identification of tumor outcome in response to immunotherapy treatments in combination with TLS-induced therapy. We observed an additive effect of immune checkpoint blockade (ICB) when combined with the mRNA-based therapy in controlling tumor growth.
5- Report of the cellular and molecular changes induced in TLS upon ICB treatment. We observed an increased intratumoral T-cell infiltrate along with activation of the intratumoral endothelial cell profile. However, preliminary data did not show an organized immune infiltrate with the formation of mature tertiary lymphoid structures (TLS).
6- Analysis of the anti-tumoral mechanism of action elicited by the mRNA-based therapy. By using depleting anti-CD8 antibodies, I could determine that the anti-tumoral effect exerted by the mRNA-based therapy was partially CD8-dependent.
The results obtained during the MSCA fellowship demonstrate:
(i) the potential of local intratumoural therapeutic strategies to enhance local immune responses while avoiding systemic immune-related side effects;
(ii) the feasibility of applying mRNA-based therapy to potentiate local anti-tumoral immune responses;
(iii) that overexpression of specific cytokines facilitates tumour-associated endothelial activation and increases intratumoural CD8⁺ T-cell infiltration;
(iv) that the development of tertiary lymphoid structures (TLS) requires sustained signalling and cannot be easily recapitulated therapeutically at specific locations.
These findings provide valuable insights into which immune modulators may be most promising for therapeutic targeting in solid tumours, supporting the development of novel therapeutic strategies specifically aimed at reshaping the tumour microenvironment. Moreover, while the main objective of the proposal was to therapeutically induce TLS and investigate their anti-tumoral mechanisms of action, the approaches explored so far have successfully increased CD8⁺ T-cell infiltration. However, we have not yet succeeded in generating organized immune structures containing activated B cells, indicating that additional microenvironmental changes are required to facilitate TLS formation.
(i) the potential of local intratumoural therapeutic strategies to enhance local immune responses while avoiding systemic immune-related side effects;
(ii) the feasibility of applying mRNA-based therapy to potentiate local anti-tumoral immune responses;
(iii) that overexpression of specific cytokines facilitates tumour-associated endothelial activation and increases intratumoural CD8⁺ T-cell infiltration;
(iv) that the development of tertiary lymphoid structures (TLS) requires sustained signalling and cannot be easily recapitulated therapeutically at specific locations.
These findings provide valuable insights into which immune modulators may be most promising for therapeutic targeting in solid tumours, supporting the development of novel therapeutic strategies specifically aimed at reshaping the tumour microenvironment. Moreover, while the main objective of the proposal was to therapeutically induce TLS and investigate their anti-tumoral mechanisms of action, the approaches explored so far have successfully increased CD8⁺ T-cell infiltration. However, we have not yet succeeded in generating organized immune structures containing activated B cells, indicating that additional microenvironmental changes are required to facilitate TLS formation.