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Background Cervical cancer’s lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes’ immune gene expression profiles and immunoregulation mechanisms have yet to be explored.Methods Our study aimed to elucidate the immune cell populations and their roles in the immune gene expression profile of negative SLNs compared with positive SLNs and non-SLNs using Nanostring RNA seq analysis. We performed a principal component analysis on the log2 normalized expression of 685 endogenous genes in the nCounter PanCancer Immune Profiling Panel, followed by an assessment of the differential expression of genes and immune cell type abundance.Results We found significant variations in gene expression among the groups, with negative SLNs displaying overexpression of genes related to tumor-infiltrating immune cells, specifically innate cell populations. They also demonstrated the upregulation of genes involved in antigen presentation and T-cell priming. In contrast, positive SLNs were enriched in regulatory networks, suggesting their potential role in immune evasion. A comparison of negative SLNs and non-SLNs revealed increased innate and adaptive immune cell types, underscoring the ongoing T cell response to tumor antigens.Conclusion Our findings underscore a specific immunogenetic phenotype profile in negative SLNs, emphasizing their crucial role in the initial anticancer response, immunosurveillance, and the propagation of immune tolerance from the primary cervical tumor. These results highlight the potential of SLNs as a novel target for immunotherapy strategies and underscore the importance of new imaging methods for accurately identifying SLN status without removal. Future investigations are needed to understand further the immunological interplay within SLNs and their influence on cervical cancer progression.

BackgroundWhile much progress has been accomplished in the understanding of radiation-induced immune effects in tumors, little is known regarding the mechanisms involved at the tumor draining lymph node (TDLN) level. The objective of this retrospective study was to assess the immune and biological changes arising in non-involved TDLNs upon node sparing concurrent chemoradiotherapy (CRT) of non-small cell lung cancer (NSCLC) tumors.MethodsPatients with proven localized (cN0M0) NSCLC, treated by radical surgery plus lymph node dissection with (CRT+) or without (CRT-) neoadjuvant chemoradiotherapy, whereby radiotherapy was targeted on the primary tumor with no significant incidental irradiation of the non-involved TDLN station (stations XI), were identified. Bulk RNA sequencing of TDLNs was performed and data were analyzed based on differential gene expression (DGE) and gene sets enrichment.ResultsSixteen patients were included and 25 TDLNs were analyzed: 6 patients in the CRT+ group (12 samples) and 10 patients in the CRT- group (13 samples). Overall, 1001 genes were differentially expressed between the two groups (CRT+ and CRT-). Analysis with g-profiler revealed that gene sets associated with antitumor immune response, inflammatory response, hypoxia, angiogenesis, epithelial mesenchymal transition and extra-cellular matrix remodeling were enriched in the CRT+ group, whereas only gene sets associated with B cells and B-cell receptor signaling were enriched in the CRT- group. Unsupervised dimensionality reduction identified two clusters of TDLNs from CRT+ patients, of which one cluster (cluster 1) exhibited higher expression of pathways identified as enriched in the overall CRT+ group in comparison to the CRT- group. In CRT+ cluster 1, 3 out of 3 patients had pathological complete response (pCR) or major pathological response (MPR) to neoadjuvant CRT, whereas only 1 out of 3 patients in the other CRT+ cluster (cluster 2) experienced MPR and none exhibited pCR.ConclusionNeoadjuvant node sparing concurrent CRT of NSCLC patients is associated with distinct microenvironment and immunological patterns in non-involved TDLNs as compared to non-involved TDLNs from patients with non-irradiated tumors. Our data are in line with studies showing superiority of lymph node sparing irradiation of the primary tumor in the induction of antitumor immunity.

Abstract Immune checkpoint blockers (ICB) have revolutionized cancer therapy. However, complete response is observed in a minority of patients and most patients develop immune-related adverse events (irAEs). These include colitis, which can be treated with anti-tumor necrosis factor (TNF) antibodies such as Infliximab. In a recent issue of the Journal for ImmunoTherapy of Cancer, Badran et al. reported that co-administering Infliximab together with ICB to five cancer patients prevents colitis recurrence, with four of them exhibiting overall disease stability. The basis for this treatment strategy stemmed from our pre-clinical demonstration that TNF contributes to resistance to anti-PD-1 therapy. In agreement with this concept, we have shown that TNF blockers improve the anti-tumor therapeutic activity of ICB in mice and based on these findings we are currently evaluating the combination in melanoma patients enrolled in the TICIMEL clinical trial. Herein, (i) we discuss the scientific rationale for combining anti-TNF and ICB in cancer patients, (ii) comment on the paper published by Badran et al. and (iii) provide the TICIMEL clinical trial design.

Radiation-induced immune effects have been extensively deciphered over the last few years, leading to the concept of the dual immune effect of radiotherapy with both immunostimulatory and immunosuppressive effects. This explains why radiotherapy alone is not able to drive a strong anti-tumor immune response in most cases, hence underlining the rationale for combining both radiotherapy and immunotherapy. This association has generated considerable interest and hundreds of trials are currently ongoing to assess such an association in oncology. However, while some trials have provided unprecedented results or shown much promise, many hopes have been dashed. Questions remain, therefore, as to how to optimize the combination of these treatment modalities. This narrative review aims at revisiting the old, well-established concepts of radiotherapy relating to dose, fractionation, target volumes and organs at risk in the era of immunotherapy. We then propose potential innovative approaches to be further assessed when considering a radio-immunotherapy association, especially in the field of non-small-cell lung cancer (NSCLC). We finally propose a framework to optimize the association, with pragmatic approaches depending on the stage of the disease.

Tumor antigen–specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

γδ T lymphocytes diverge from conventional T CD8 lymphocytes for ontogeny, homing, and antigen specificity, but whether their differentiation in tumors also deviates was unknown. Using innovative analyses of our original and ~150 published single-cell RNA sequencing datasets validated by phenotyping of human tumors and murine models, here we present the first high-resolution view of human γδ T cell differentiation in cancer. While γδ T lymphocytes prominently encompass TCRVγ9 cells more differentiated than T CD8 in healthy donor’s blood, a different scenario is unveiled in tumors. Solid tumors and lymphomas are infiltrated by a majority of TCRVγnon9 γδ T cells which are quantitatively correlated and remarkably aligned with T CD8 for differentiation, exhaustion, gene expression profile, and response to immune checkpoint therapy. This cancer-wide association is critical for developing cancer immunotherapies.

Background Besides the interest of an early detection of ovarian cancer, there is an urgent need for new predictive and prognostic biomarkers of tumor development and cancer treatment. In healthy patients, circulating blood monocytes are typically subdivided into classical (85%), intermediate (5%) and non-classical (10%) populations. Although these circulating monocyte subsets have been suggested as biomarkers in several diseases, few studies have investigate their potential as a predictive signature for tumor immune status,tumor growth and treatment adaptation.Methods In this study, we used a homogeneous cohort of 28 chemotherapy-naïve patients with ovarian cancer to evaluate monocyte subsets as biomarkers of the ascites immunological status. We evaluated the correlations between circulating monocyte subsets and immune cells and tumor burden in peritoneal ascites. Moreover, to validate the use of circulating monocyte subsets tofollow tumor progression and treatment response, we characterized blood monocytes from ovarian cancer patients included in a phase 1 clinical trial at baseline and following murlentamab treatment.Results We demonstrate here a robust expansion of the intermediate blood monocytes (IBMs) in ovarian cancer patients. We establish a significant positive correlation between IBM percentage and tumor–associate macrophages with a CCR2high/CD163high/CD206high/CD86lowprofile. Moreover, IBM expansion is associated with a decreased effector/regulatory T-cell ratio in ascites and with the presence of soluble immunosuppressive mediators. We also establish that IBM proportion positively correlates with the peritoneum tumor burden. Finally, the study of IBMs in patients with ovarian cancer under immunotherapy during the phase clinical trial supports IBMs to follow the evolution of tumor development and the treatment adaptation.Conclusions This study, which links IBM level with immunosuppression and tumor burden in peritoneum, identifies IBMs as apotential predictive signature of ascites immune status and as a biomarker ofovarian cancer development and treatment response.Trial registration number EudraCT: 2015-004252-22 NCT02978755.

Immunotherapies have achieved clinical benefit in many types of cancer but remain limited to a subset of patients in colorectal cancer (CRC). Resistance to immunotherapy can be attributed in part to tissue-specific factors constraining antitumor immunity. Thus, a better understanding of how the colon microenvironment shapes the immune response to CRC is needed to identify mechanisms of resistance to immunotherapies and guide the development of novel therapeutics. In an orthotopic mouse model of MC38-CRC, tumor progression was monitored by bioluminescence imaging and the immune signatures were assessed at a transcriptional level using NanoString and at a cellular level by flow cytometry. Despite initial tumor growth in all mice, only 25% to 35% of mice developed a progressive lethal CRC while the remaining animals spontaneously rejected their solid tumor. No tumor rejection was observed in the absence of adaptive immunity, nor when MC38 cells were injected in non-orthotopic locations, subcutaneously or into the liver. We observed that progressive CRC tumors exhibited a protumor immune response, characterized by a regulatory T-lymphocyte pattern, discernible shortly post-tumor implantation, as well as suppressive myeloid cells. In contrast, tumor-rejecting mice presented an early inflammatory response and an antitumor microenvironment enriched in CD8+ T cells. Taken together, our data demonstrate the role of the colon microenvironment in regulating the balance between anti or protumor immune responses. While emphasizing the relevance of the CRC orthotopic model, they set the basis for exploring the impact of the identified signatures in colon cancer response to immunotherapy.

CD4 T cells secreting IL-10 or IL-17 are frequent at mucosal sites, where their equilibrium is important for simultaneously maintaining tolerance and immunity to the resident microbiota. The mode of action of these cells, however, is as yet incompletely understood. In this study, we have combined ex vivo analysis of CD4 T cells producing IL-10 or/and IL-17 with assessment of clonal populations isolated ex vivo using a cytokine catch assay. We found that circulating CD4 T cells secreting IL-10 or/and IL-17 ex vivo include both conventional FOXP3- CD4 T cells and FOXP3+ Helios- Treg. Upon assessment of clonal populations derived from single ex vivo isolated cytokine secreting cells, we found that IL-10 or/and IL-17 secreting cells prevalently secrete one or the other cytokine depending on the type of stimulation, the time after stimulation and the presence of microbial products. Namely, IL-10 secretion by clonal cells was prevalent at early time points after TCR mediated stimulation, was independent of co-stimulation and was increased in the presence of the microbial fermentation product butyrate. In contrast, IL-17 secretion was higher at later time points after TCR mediated stimulation and in the presence of co-stimulatory signals. Taken together, these results provide insights into the mechanisms that, through modulation of cytokine secretion depending on conditions, allow IL-10 and IL-17 producing CD4 T cells to contribute to maintain tolerance to microbes locally, while retaining the ability to participate in protective immune responses at distant sites.

Generation of tumor-antigen specific CD4+ T-helper (TH) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4+ T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific TH lines. We isolated phenotypically defined CD4+ T-cell subpopulations from circulating lymphocytes of DR52b+ healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO119-143, autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO119-143 tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer+ cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer+ cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific TH lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer+ TH lines from conventional CD4+CD25− naïve and central memory populations, but not from effector memory populations or CD4+CD25+ Treg. In vitro primed TH lines recognized ESO with affinities comparable to ESO-tetramer+ cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal TH lines from non-immune individuals. This is an approach that is of potential interest for adoptive cell therapy of patients bearing ESO-expressing cancers.

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.