Modelling of killer T-cell and cancer cell subpopulation dynamics under immuno- and chemotherapies

Each patient’s cancer has a unique molecular makeup, often comprised of distinct cancer cell subpopulations. Improved understanding of dynamic processes between cancer cell populations is therefore critical for making treatment more effective and personalized. It has been shown that immunotherapy increases the survival of melanoma patients. However, there remain critical open questions, such as timing and duration of immunotherapy and its added benefits when combined with other types of treatments. We introduce a model for the dynamics of active killer T-cells and cancer cell subpopulations. Rather than defining the cancer cell populations based on their genetic makeup alone, we consider also other, non-genetic differences that make the cell populations either sensitive or resistant to a therapy. Using the model, we make predictions of possible outcomes of the various treatment strategies in virtual melanoma patients, providing hypotheses regarding therapeutic efficacy and side-effects. It is shown, for instance, that starting immunotherapy with a denser treatment schedule may enable changing to a sparser schedule later during the treatment. Furthermore, combination of targeted and immunotherapy results in a better treatment effect, compared to mono-immunotherapy, and a stable disease can be reached with a patient-tailored combination. These results offer better understanding of the competition between T-cells and cancer cells, toward personalized immunotherapy regimens.Peer reviewe

Age-associated changes in the immune system may influence the response to anti-PD1 therapy in metastatic melanoma patients

Anti-PD1 treatment has improved the survival of metastatic melanoma patients, yet it is unknown which patients benefit from the treatment. In this exploratory study, we aimed to understand the effects of anti-PD1 therapy on the patients' immune system and discover the characteristics that would result in successful treatment. We collected peripheral blood (PB) samples from 17 immuno-oncology-naive metastatic melanoma patients before and after 1 and 3 months of anti-PD1 therapy. In addition, matching tumor biopsies at the time of diagnosis were collected for tissue microarray. The complete blood counts, PB immunophenotype, serum cytokine profiles, and tumor-infiltrating lymphocytes were analyzed and correlated with the clinical data. Patients were categorized based on their disease control into responders (complete response, partial response, stable disease > 6 months, N = 11) and non-responders (progressive disease, stable diseasePeer reviewe

Evolution and modulation of antigen-specific T cell responses in melanoma patients

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCRαβ-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.</p

Evolution and modulation of antigen-specific T cell responses in melanoma patients

Analyzing antigen-specific T cell responses at scale has been challenging. Here, we analyze three types of T cell receptor (TCR) repertoire data (antigen-specific TCRs, TCR-repertoire, and single-cell RNA + TCR alpha beta-sequencing data) from 515 patients with primary or metastatic melanoma and compare it to 783 healthy controls. Although melanoma-associated antigen (MAA) -specific TCRs are restricted to individuals, they share sequence similarities that allow us to build classifiers for predicting anti-MAA T cells. The frequency of anti-MAA T cells distinguishes melanoma patients from healthy and predicts metastatic recurrence from primary melanoma. Anti-MAA T cells have stem-like properties and frequent interactions with regulatory T cells and tumor cells via Galectin9-TIM3 and PVR-TIGIT -axes, respectively. In the responding patients, the number of expanded anti-MAA clones are higher after the anti-PD1(+anti-CTLA4) therapy and the exhaustion phenotype is rescued. Our systems immunology approach paves the way for understanding antigen-specific responses in human disorders.Peer reviewe

Uusien immuuniaktivaation vapauttajahoitojen vaikutus melanoomapotilaiden immuunijärjestelmään

Checkpoint inhibitor drugs, anti-PD1 and anti-LAG3, aim to reinvigorate the immune response of CD8+ T cells against tumors. However, their effects on other immune cells like natural killer (NK) and NKT cells, as well as the varying patient responses, remain unclear. This doctoral research aimed to study the effects of checkpoint inhibitors on melanoma patients' immune system, while also seeking biomarkers for therapy response. In study I, we analyzed peripheral blood (PB) and tumor samples of metastatic melanoma patients treated with anti-PD1. The results indicated that the frequency of NKT cells, CD25+ and CD45RO+ cytotoxic NK cells, and serum CXC chemokines increased significantly in responders. The baseline predictors of treatment response included age-associated biological characteristics. In study II, we developed a bioinformatic tool to identify tumor peptides resembling viral epitopes and showed their ability to enhance cancer immunotherapy effectiveness in mice. In melanoma patients receiving anti-PD1, high CMV-specific antibody levels correlated with extended progression-free survival, and their immune cells exhibited potential reactivity to both melanoma and CMV peptides. Further, T cell receptor (TCR) sequencing identified the expansion of the same CD8+ T cell clones in response to specific tumor and viral peptides. In study III, we analyzed PB samples from immuno-oncology naïve or refractory metastatic melanoma patients treated with anti-LAG3+anti-PD1 therapy. The results indicated that NK cells, regulatory T cells, and CD8+ T cells had the highest LAG3 expression and significant changes during treatment. Responders had higher baseline TCR clonality, enriched adaptive NK cells that became activated during treatment, and their expanding CD8+ T-cell clones gained a more cytotoxic NK-like phenotype. In study IV, we analyzed melanoma patients' CD56+ lymphocytes from paired tumor and PB samples. The results suggested enrichment in cytokine stimulus and IFN-γ signaling in the tumor infiltrating NK and NKT cells. Also, many T cell clones identified in PB CD56+ lymphocytes were present in tumor samples, with some predicted to target melanoma-associated antigens. In conclusion, this thesis reveals that immune checkpoint inhibitors have profound effects on various immune cells, enhancing the antitumor potential of NK and NKT cells and altering cytokine stimulus for an improved antitumor immune response. Potential biomarkers of favorable therapy response were identified, warranting further investigation in larger clinical trials. Additionally, the findings highlight the influence of cross-reacting T-cells and pre-existing immunity on immunotherapy response.Immuuniaktivaation vapauttajat, anti-PD1 ja anti-LAG3, pyrkivät elvyttämään CD8+ T-solujen immuunivasteen syöpää vastaan. Näiden vaikutus muihin immuunisoluihin, kuten luonnollisiin tappaja (NK) ja NKT-soluihin, sekä vaihtelevien hoitovasteiden syyt, ovat vielä epäselviä. Tämä väitöstutkimus pyrki tutkimaan immuuniaktivaation vapauttajien vaikutuksia melanoomapotilaiden immuunijärjestelmään, etsien samalla biomarkkereita hoitovasteen ennustamiseksi. Osatyössä I tutkimme kokoveri- ja kasvainnäytteitä anti-PD1 hoidetuilla edennyttä melanoomaa sairastavilla potilailla. Tulokset osoittivat NKT-solujen, CD25+ ja CD45RO+ NK-solujen ja seerumin CXC-kemokiinien kasvaneen merkittävästi vasteen saaneilla potilailla. Hoitovastetta ennustaviin tekijöihin lukeutui ikään liittyviä biologisia muuttujia. Osatyössä II kehitimme bioinformatiikan työkalun tunnistamaan virusepitooppeja muistuttavia kasvainpeptidejä, ja osoitimme niiden kyvyn tehostaa syövän immunoterapiaa hiirimalleissa. Anti-PD1 hoidetuilla melanoomapotilailla, korkeat CMV-spesifiset vasta-ainetasot liittyivät pidentyneeseen etenemättömyysaikaan, ja heidän immuunisolut reagoivat sekä melanooma- että CMV-peptidien kanssa osoittaen ristireaktiivista potentiaalia. Lisäksi T-solureseptorien (TCR) sekvensointi osoitti, että kasvain- ja viruspeptideille spesifiset samat CD8+ T-solukloonit ekspandoituivat. Osatyössä III tutkimme kokoverinäytteitä immunoterapia naiiveilla tai refraktorisilla anti-LAG3+anti-PD1-hoidetuilla edennyttä melanoomaa sairastavilla potilailla. Tulokset osoittivat, että NK-solut, regulatoriset T-solut ja CD8+ T-solut ilmensivät eniten LAG3:a ja muuttuivat merkittävästi hoidon aikana. Vasteen saaneilla potilailla oli korkeampi lähtötason TCR-klonaalisuus, enemmän adaptiivisia NK-soluja, jotka aktivoituivat hoidon aikana, ja heidän ekspandoituvat CD8+ T-solukloonit saivat sytotoksisemman NK-solu fenotyypin. Osatyössä IV tutkimme melanoomapotilaiden CD56+ lymfosyyttejä kasvain- ja kokoverinäytteistä. Tulokset viittasivat sytokiiniärsykkeen ja IFN-γ-signaloinnin vahvistumiseen kasvaimen NK- ja NKT-soluissa. Kasvainnäytteissä oli myös monia kokoveren CD56+ lymfosyyteistä tunnistettuja T-soluklooneja, joista osan ennustettiin kohdistuvan melanoomaan liittyviin antigeeneihin. Yhteenvetona tämä väitöskirja osoittaa, että immuuniaktivaation vapauttajilla on vaikutuksia moniin immuunisoluihin, tehostaen NK- ja NKT-solujen syöpävastaista potentiaalia ja muuttaen sytokiinistimulaatiota saaden paremman immuunivasteen syöpää vastaan. Mahdollisia hyvän hoitovasteen biomarkkereita tunnistettiin, ja niitä on syytä tutkia laajemmissa kliinisissä tutkimuksissa. Tulokset myös korostavat ristireagoivien T-solujen ja olemassa olevan immuniteetin vaikutusta immunoterapiavasteeseen

Resistance in hybrid aspen to pathogens

publishedVersio

Single-cell characterization of anti-LAG-3 and anti- PD-1 combination treatment in patients with melanoma

BACKGROUND. Relatlimab plus nivolumab (anti-lymphocyte-activation gene 3 plus anti-programmed death 1 [anti-LAG-3+anti-PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown.METHODS. We evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy-refractory patients with metastatic melanoma treated with anti-LAG-3+anti-PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCR alpha beta-Seq) combined with other multiomics profiling.RESULTS. The highest LAG3 expression was noted in NK cells, Tregs, and CD8+ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3+ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8+ T cell clones gained a more cytotoxic and NK-like phenotype.CONCLUSION. Anti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.TRIAL REGISTRATION. ClinicalTrials.gov (NCT01968109)Peer reviewe

Viral molecular mimicry influences the antitumor immune response in murine and human melanoma

Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8þ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.publishedVersionPeer reviewe

Single-cell characterization of anti–LAG-3 and anti–PD-1 combination treatment in patients with melanoma

Abstract Background: Relatlimab plus nivolumab (anti–lymphocyte-activation gene 3 plus anti–programmed death 1 [anti–LAG-3+anti–PD-1]) has been approved by the FDA as a first-line therapy for stage III/IV melanoma, but its detailed effect on the immune system is unknown. Methods: We evaluated blood samples from 40 immunotherapy-naive or prior immunotherapy–refractory patients with metastatic melanoma treated with anti–LAG-3+anti–PD-1 in a phase I trial using single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-Seq) combined with other multiomics profiling. Results: The highest LAG3 expression was noted in NK cells, Tregs, and CD8⁺ T cells, and these cell populations underwent the most significant changes during the treatment. Adaptive NK cells were enriched in responders and underwent profound transcriptomic changes during the therapy, resulting in an active phenotype. LAG3⁺ Tregs expanded, but based on the transcriptome profile, became metabolically silent during the treatment. Last, higher baseline TCR clonality was observed in responding patients, and their expanding CD8⁺ T cell clones gained a more cytotoxic and NK-like phenotype. Conclusion: Anti–LAG-3+anti–PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8⁺ T cells. Trial registration: ClinicalTrials.gov (NCT01968109) Funding : Cancer Foundation Finland, Sigrid Juselius Foundation, Signe and Ane Gyllenberg Foundation, Relander Foundation, State funding for university-level health research in Finland, a Helsinki Institute of Life Sciences Fellow grant, Academy of Finland (grant numbers 314442, 311081, 335432, and 335436), and an investigator-initiated research grant from BMS