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

BackgroundRelatlimab 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.MethodsWe 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+TCRαß-Seq) combined with other multiomics profiling.ResultsThe 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.ConclusionAnti-LAG-3+anti-PD-1 therapy has profound effects on NK cells and Tregs in addition to CD8+ T cells.Trial registrationClinicalTrials.gov (NCT01968109)FundingCancer 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.

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 + TCRαß-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.

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 CD8+ 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.

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-naïve 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 disease ≤ 6 months, N = 6). During therapy, the PB natural killer T (NKT) cell frequency, expression of CD25 and CD45RO on cytotoxic natural killer (NK) cells, and serum CXC chemokine levels were significantly increased in responders. Furthermore, higher age together with age-associated characteristics from PB, lower frequency of PB-naïve CD8+ T cells, and elevated levels of serum MCP-4 and OPG were discovered as baseline predictors of treatment response. We therefore propose that in addition to T cells, anti-PD1 treatment is associated with NK- and NKT-cell population dynamics, and that the age-associated characteristics from PB together with older age may contribute to prolonged PFS in anti-PD1-treated melanoma patients.

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.