Panayotis Verginis

Aims: Trained immunity, defined as long-term functional reprogramming of innate immune cells, has been implicated in the heterologous protective effects of certain vaccines against infection and cancer. AS01-adjuvanted vaccines have been suggested to provide such heterologous protective effects. We therefore aimed to investigate the capacity of AS01 to induce trained immunity and evaluated its potential as an antitumoral therapeutic. Methods: Acute cytokine production was assessed in human monocytes after AS01 stimulation. Trained immunity was subsequently evaluated by interleukin-6 (IL-6) and tumor necrosis factor (TNF) quantification following heterologous restimulation. Antitumor efficacy of AS01 was assessed in mice bearing B16-F10 melanoma, MB49 bladder cancer, or Lewis lung carcinoma by monitoring tumor growth. Immune phenotyping of B16-F10 and MB49 tumor microenvironments was determined with flow cytometry. Results: Although AS01 did not stimulate acute inflammation, it induced trained immunity, marked by increased responsiveness upon secondary stimulation in human monocytes. Treatment of mice bearing B16-F10 melanoma or MB49 bladder cancer with AS01 strongly diminished tumor growth, whereas no effect was observed in the Lewis lung carcinoma model. In these responsive models, the antitumor effects of AS01 were associated with an altered tumor microenvironment, characterized by increased infiltration of CD8+ T cells, regulatory T cells, neutrophils, and monocytes, along with a decrease in tumor-associated macrophages. AS01 also decreased expression of immune checkpoint protein PD-L1 in monocytes and neutrophils, suggesting enhanced antitumor immune responsiveness. Conclusion: Our findings show the capacity of AS01 to induce trained immunity and support its potential to be repurposed as an immunotherapeutic approach for cancer.

Introduction: Immune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit.Methods: In this study, we treated tumour-bearing mice with PD-L1 blockage antibody (aPD-L1) immunotherapy, to investigate its effects on cancer-induced emergency myelopoiesis, focusing on bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). We examined the impact of aPD-L1 treatment on HSPC quiescence, proliferation, transcriptomic profile, and functionality.Results: Herein, we reveal that aPD-L1 in tumour-bearing mice targets the HSPCs in the BM, mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, observed in both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice, shifting towards an inflammatory state. Furthermore, HSPCs from aPDL1-treated mice demonstrated resistance to cancer-induced emergency myelopoiesis, evidenced by a lower generation of MDSCs compared to control-treated mice.Discussion: Our findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of cancer-induced emergency myelopoiesis.

Immune checkpoint inhibitor immunotherapy has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite the immense success, still a significant proportion of patients do not develop durable responses, allowing disease progression accompanied by high mortality rates. Therefore, there is an imperative need for identification of reliable non-invasive predictive biomarkers to guide therapeutic decisions. Herein, we constructed a blood immunomap in NSCLC patients with metastatic disease, using a high-dimensional mass cytometry approach. Assessment of clinical responses to aPD1 immunotherapy revealed, among others, a significant expansion of CD8+PD-L1+ T cells in individuals not responding to immunotherapy. Of interest, CD8+PD-L1+ T cells were enriched in tumor biopsies and bronchoalveolar lavage of NSCLC individuals at early stages of disease as well as in pleural infusions of individuals with thoracic malignancies. Transcriptomic analysis revealed that CD8+PD-L1+ T cells exhibited a regulatory/exhausted phenotype, while various transcripts associated with the overall survival of NSCLC individuals, were mapped. Overall, our findings define an immunomap in the early stage and advanced NSCLC patients and identify immune-related events which may benefit the quest for identification of predictive biomarkers of immunotherapy responses.

Immune checkpoint inhibitor immunotherapy has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite the immense success, still a significant proportion of patients do not develop durable responses, allowing disease progression accompanied by high mortality rates. Therefore, there is an imperative need for identification of reliable non-invasive predictive biomarkers to guide therapeutic decisions. Herein, we constructed a blood immunomap in NSCLC patients with metastatic disease, using a high-dimensional mass cytometry approach. Assessment of clinical responses to aPD1 immunotherapy revealed, among others, a significant expansion of CD8 ⁺ PD-L1 ⁺ T cells in individuals not responding to immunotherapy. Of interest, CD8 ⁺ PD-L1 ⁺ T cells were enriched in tumor biopsies and bronchoalveolar lavage of NSCLC individuals at early stages of disease as well as in pleural infusions of individuals with thoracic malignancies. Transcriptomic analysis revealed that CD8 ⁺ PD-L1 ⁺ T cells exhibited a regulatory/exhausted phenotype, while various transcripts associated with the overall survival of NSCLC individuals, were mapped. Overall, our findings define an immunomap in the early stage and advanced NSCLC patients and identify immune-related events which may benefit the quest for identification of predictive biomarkers of immunotherapy responses.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA⁺ CAFs can form immunological synapses with Foxp3⁺ regulatory T cells (Tregs) in tumours. Notably, α-SMA⁺ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA⁺ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA⁺ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA⁺ CAFs and Tregs in an autophagy-dependent manner.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumor microenvironment, serving diverse functions in tumor progression, invasion, matrix remodeling and resistance to therapy; yet, the precise mechanisms via which CAFs imprint on the anti-tumor immunity remain poorly understood. Extensive molecular characterization revealed an increased heterogeneity in the CAF compartment and proposed an interaction between CAFs and tumor-infiltrating immune cells, which may shape tumor immune evasion. Herein, we describe a synapse formation between α-SMA ⁺ CAFs and regulatory T cells (Tregs) in the TME. Foxp3 ⁺ Tregs were localized close to α-SMA ⁺ CAFs in diverse types of tumor models as well as biopsies from melanoma and colorectal cancer patients. Notably, phenotypically tolerogenic α-SMA ⁺ CAFs demonstrated the ability to phagocytose and process tumor antigens, instructing Treg movement arrest, activation and proliferation, in an antigen-specific manner. Of interest, α-SMA ⁺ CAFs were characterized by the presence of double-membrane structures, resembling autophagosomes, in their cytoplasm, while analysis of single-cell transcriptomic data pointed autophagy and antigen processing/presentation pathways to be enriched in α-SMA-expressing CAF clusters. In a mechanistic view, conditional knockout of the autophagy pathway in α-SMA ⁺ CAFs promoted an inflammatory re-programming of CAFs, reduced Treg infiltration, attenuated tumor development, and potentiated the efficacy of immune checkpoint inhibitor immunotherapy. Overall, our findings reveal an immunosuppressive mechanism operating in the TME, which entails the formation of synapses between α-SMA ⁺ CAFs and Tregs in an autophagy-dependent fashion and raises the potential for the development of CAF-targeted therapies in cancer. One Sentence Summary: α-SMA ⁺ Cancer Associated Fibroblasts process and present tumor antigens via autophagy to form immunological synapses with Foxp3 ⁺ T regulatory cells in the tumor microenvironment, promoting tumor development.

Immune checkpoint blockade (ICB) immunotherapy has revolutionized cancer treatment, demonstrating exceptional clinical responses in a wide range of cancers. Despite the success, a significant proportion of patients still fail to respond, highlighting the existence of unappreciated mechanisms of immunotherapy resistance. Delineating such mechanisms is paramount to minimize immunotherapy failures and optimize the clinical benefit. Herein, we reveal that immunotherapy with PD-L1 blockage antibody (αPDL1) in tumour-bearing mice targets the hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (ΒΜ), mediating their exit from quiescence and promoting their proliferation. Notably, disruption of the PDL1/PD1 axis induces transcriptomic reprogramming in HSPCs, from both individuals with Hodgkin lymphoma (HL) and tumour-bearing mice shifting towards an inflammatory state. Functionally, transplantation of HSPCs isolated from αPDL1-treated tumor-bearing mice exhibited resistance to cancer-associated myelopoiesis as evident by the generation of reduced frequencies of myeloid-derived suppressor cells (MDSCs) compared to cells from control-treated mice. Our findings shed light on unrecognized mechanisms of action of ICB immunotherapy in cancer, which involves targeting of BM-driven HSPCs and reprogramming of emergency myelopoiesis.

Abatacept (CTLA4-Ig)—a monoclonal antibody which restricts T cell activation—is an effective treatment for rheumatoid arthritis (RA). Nevertheless, only 50% of RA patients attain clinical responses, while predictors of response are rather limited. Herein, we aimed to investigate for early biomarkers of response to abatacept, based on a detailed immunological profiling of peripheral blood (PB) cells and serum proteins. We applied flow cytometry and proteomics analysis on PB immune cells and serum respectively, of RA patients starting abatacept as the first biologic agent. After 6 months of treatment, 34.5% of patients attained response. At baseline, Th1 and FoxP3+ T cell populations were positively correlated with tender joint counts (p-value = 0.047 and p-value = 0.022, respectively). Upon treatment, CTLA4-Ig effectively reduced the percentages of Th1 and Th17 only in responders (p-value = 0.0277 and p-value = 0.0042, respectively). Notably, baseline levels of Th1 and myeloid cell populations were significantly increased in PB of responders compared to non-responders (p-value = 0.009 and p-value = 0.03, respectively). Proteomics analysis revealed that several inflammatory mediators were present in serum of responders before therapy initiation and strikingly 10 amongst 303 serum proteins were associated with clinical responses. Finally, a composite index based on selected baseline cellular and proteomics’ analysis could predict response to abatacept with a high sensitivity (90%) and specificity (88.24%).