Neuronal network inference and membrane potential model using multivariate Hawkes processes.

BACKGROUND: In this work, we propose to catch the complexity of the membrane potential's dynamic of a motoneuron between its spikes, taking into account the spikes from other neurons around. Our approach relies on two types of data: extracellular recordings of multiple spikes trains and intracellular recordings of the membrane potential of a central neuron. NEW METHOD: We provide a unified framework and a complete pipeline to analyze neuronal activity from data extraction to statistical inference. To the best of our knowledge, this is the first time that a Hawkes-diffusion model is investigated on such complex data. The first step of the proposed procedure is to select a subnetwork of neurons impacting the central neuron using a multivariate Hawkes process. Then we infer a jump-diffusion dynamic in which jumps are driven from a Hawkes process, the occurrences of which correspond to the spike trains of the aforementioned subset of neurons that interact with the central neuron. RESULTS: From the Hawkes estimation step we recover a small connectivity graph which contains the central neuron, and we show that taking into account this information improves the inference of membrane potential through the proposed jump-diffusion model. A goodness of fit test is applied to validate the relevance of the Hawkes model in such context. COMPARISON WITH EXISTING METHODS: We compare an empirical inference method and two sparse estimation procedures based on the Hawkes assumption for the reconstruction of the connectivity graph using the spike-trains. Then, the Hawkes-diffusion model is competed with the simple diffusion in terms of best fit to describe the behavior of the membrane potential of a central neuron surrounded by a network. CONCLUSIONS: The present method takes advantage of both spike trains and membrane potential to understand the behavior of a fixed neuron. The entire code has been developed and is freely available on GitHub.

Clinical and Prognostic Value of Immunogenetic Characteristics in Anti-LGI1 Encephalitis.

OBJECTIVE: Antibodies against leucine-rich glioma-inactivated 1 (LGI1-Abs) characterize a limbic encephalitis (LE) strongly associated with HLA-DRB1*07:01, although some patients lack LGI1-Abs in CSF or do not carry this allele. Whether they represent a different subtype of disease or have different prognoses is unclear. METHODS: Retrospective analysis of clinical features, IgG isotypes, and outcome according to LGI1-Ab CSF positivity and DRB1*07:01 in a cohort of anti-LGI1 LE patients. RESULTS: Patients with LGI1-Abs detected in both CSF and serum (105/134, 78%) were compared with those who were CSF negative (29/134, 22%). Both groups had similar clinical features and serum levels, but CSF-positive patients had shorter diagnostic delay, more frequently hyponatremia, inflammatory CSF, and abnormal MRI (p < 0.05). Human leukocyte antigen (HLA) genotyping was performed in 72/134 (54%) patients and 63/72 (88%) carried DRB1*07:01. Noncarriers (9/72, 12%) were younger, more commonly women, and had less frequently psychiatric and frontal symptoms (p < 0.05). No difference in IgG isotypes according to CSF positivity or HLA was found (p > 0.05). HLA and IgG isotypes were not associated with poor outcome (mRS >2 at last follow-up) in univariate analyses; CSF positivity was only identified as a poor outcome predictor in the multivariate analysis including the complete follow-up, whereas age and female sex also remained when just the first year was considered. CONCLUSIONS: LE without CSF LGI1-Abs is clinically indistinguishable and likely reflects just a lesser LGI1-Ab production. HLA association is sex and age biased and presents clinical particularities, suggesting subtle differences in the immune response. Long-term outcome depends mostly on demographic characteristics and the intensity of the intrathecal synthesis.

CXCR6 deficiency impairs cancer vaccine efficacy and CD8+ resident memory T-cell recruitment in head and neck and lung tumors.

BACKGROUND: Resident memory T lymphocytes (TRM) are located in tissues and play an important role in immunosurveillance against tumors. The presence of TRM prior to treatment or their induction is associated to the response to anti-Programmed cell death protein 1 (PD-1)/Programmed death-ligand 1 (PD-L1) immunotherapy and the efficacy of cancer vaccines. Previous work by our group and others has shown that the intranasal route of vaccination allows more efficient induction of these cells in head and neck and lung mucosa, resulting in better tumor protection. The mechanisms of in vivo migration of these cells remains largely unknown, apart from the fact that they express the chemokine receptor CXCR6. METHODS: We used CXCR6-deficient mice and an intranasal tumor vaccination model targeting the Human Papillomavirus (HPV) E7 protein expressed by the TC-1 lung cancer epithelial cell line. The role of CXCR6 and its ligand, CXCL16, was analyzed using multiparametric cytometric techniques and Luminex assays.Human biopsies obtained from patients with lung cancer were also included in this study. RESULTS: We showed that CXCR6 was preferentially expressed by CD8+ TRM after vaccination in mice and also on intratumoral CD8+ TRM derived from human lung cancer. We also demonstrate that vaccination of Cxcr6-deficient mice induces a defect in the lung recruitment of antigen-specific CD8+ T cells, preferentially in the TRM subsets. In addition, we found that intranasal vaccination with a cancer vaccine is less effective in these Cxcr6-deficient mice compared with wild-type mice, and this loss of efficacy is associated with decreased recruitment of local antitumor CD8+ TRM. Interestingly, intranasal, but not intramuscular vaccination induced higher and more sustained concentrations of CXCL16, compared with other chemokines, in the bronchoalveolar lavage fluid and pulmonary parenchyma. CONCLUSIONS: This work demonstrates the in vivo role of CXCR6-CXCL16 axis in the migration of CD8+ resident memory T cells in lung mucosa after vaccination, resulting in the control of tumor growth. This work reinforces and explains why the intranasal route of vaccination is the most appropriate strategy for inducing these cells in the head and neck and pulmonary mucosa, which remains a major objective to overcome resistance to anti-PD-1/PD-L1, especially in cold tumors.

Resident memory T cells, critical components in tumor immunology.

CD8+ T lymphocytes are the major anti-tumor effector cells. Most cancer immunotherapeutic approaches seek to amplify cytotoxic T lymphocytes (CTL) specific to malignant cells. A recently identified subpopulation of memory CD8+ T cells, named tissue-resident memory T (TRM) cells, persists in peripheral tissues and does not recirculate. This T-cell subset is considered an independent memory T-cell lineage with a specific profile of transcription factors, including Runx3+, Notch+, Hobit+, Blimp1+, BATF+, AHR+, EOMESneg and Tbetlow. It is defined by expression of CD103 (αE(CD103)ß7) and CD49a (VLA-1 or α1ß1) integrins and C-type lectin CD69, which are most likely involved in retention of TRM cells in non-lymphoid tissues, including solid tumors. CD103 binds to the epithelial cell marker E-cadherin, thereby favoring the location and retention of TRM in epithelial tumor regions in close contact with malignant cells. The CD103-E-cadherin interaction is required for polarized exocytosis of lytic granules, in particular, when ICAM-1 expression on cancer cells is missing, leading to target cell death. TRM cells also express high levels of granzyme B, IFNγ and TNFα, supporting their cytotoxic features. Moreover, the local route of immunization targeting tissue dendritic cells (DC), and the presence of environmental factors (i.e. TGF-ß, IL-33 and IL-15), promote differentiation of this T-cell subtype. In both spontaneous tumor models and engrafted tumors, natural TRM cells or cancer-vaccine-induced TRM directly control tumor growth. In line with these results, TRM infiltration into various human cancers, including lung cancer, are correlated with better clinical outcome in both univariate and multivariate analyses independently of CD8+ T cells. TRM cells also predominantly express checkpoint receptors such as PD-1, CTLA-4 and Tim-3. Blockade of PD-1 with neutralizing antibodies on TRM cells isolated from human lung cancer promotes cytolytic activity toward autologous tumor cells. Thus, TRM cells appear to represent important components in tumor immune surveillance. Their induction by cancer vaccines or other immunotherapeutic approaches may be critical for the success of these treatments. Several arguments, such as their close contact with tumor cells, dominant expression of checkpoint receptors and their recognition of cancer cells, strongly suggest that they may be involved in the success of immune checkpoint inhibitors in various cancers.

Targeting Resident Memory T Cells for Cancer Immunotherapy.

A novel population of memory CD8+ T cells called resident memory T cells (TRM) has been identified based on their phenotype (CD103, CD69) and on their local tissue residency without recirculating in the blood. These cells have been implicated in protective immune response against pathogens in both animal models and humans. Their role in cancer is just emerging as a key player in tumor immunosurveillance. Many properties of these cells suggest that they could control tumor growth: (i) they respond much faster to reexposure to cognate antigen than circulating memory cells, (ii) they express high levels of cytotoxic molecules, and (iii) they are enriched in tumor-specific T cells in close contact with tumor cells. TRM are present in many human cancers and are associated with a good clinical outcome independently of the infiltration of CD8+ T cells. It has been recently shown that the efficacy of cancer vaccines depends on their ability to elicit TRM. In adoptive cell therapy, the transfer of cells with the ability to establish TRM at the tumor site correlates with the potency of this approach. Interestingly, TRM express immune checkpoint molecules and preliminary data showed that they could expand early during anti-PD-1 treatment, and thus be considered as a surrogate marker of response to immunotherapy. Some cues to better expand these cells in vivo and improve the success of cancer immunotherapy include using mucosal routes of immunization, targeting subpopulations of dendritic cells as well as local signal at the mucosal site to recruit them in mucosal tissue.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

[Tim-3: a novel biomarker and therapeutic target in oncology]. / Tim-3 - Biomarqueur et cible thérapeutique en cancérologie.

T cells harboring multiple co-inhibitory molecules lose their anti-tumoral functionality. PD-1 is a clinically approved target in cancer therapy, but its expression alone does not mean dysfunctionality. The expression of Tim-3 on numerous cell types (T cell, Treg, dendritic cell, myeloid cells) favors tumor escape to immune cells. Within many tumors, PD-1/Tim-3 coexpressing CD8-T cells lose their ability to secrete cytokines (IFNγ, IL-2, TNFα) and their intratumoral infiltration correlates with a bad prognosis. Tim-3 recently appeared as a potential biomarker of anti-PD-1 resistance. Combined blockade of PD-1 and Tim-3 axis demonstrated potent clinical efficacy in preclinical models and reinforced the rationale of using an anti-Tim-3 to override tumor resistance.

Mechanisms of action and rationale for the use of checkpoint inhibitors in cancer.

The large family of costimulatory molecules plays a crucial role in regulation of the immune response. These molecules modulate TCR signalling via phosphorylation cascades. Some of the coinhibitory members of this family, such as PD-1 and CTLA-4, already constitute approved targets in cancer therapy and, since 2011, have opened a new area of antitumour immunotherapy. Many antibodies targeting other inhibitory receptors (Tim-3, VISTA, Lag-3 and so on) or activating costimulatory molecules (OX40, GITR and so on) are under evaluation. These antibodies have multiple mechanisms of action. At the cellular level, these antibodies restore the activation signalling pathway and reprogram T cell metabolism. Tumour cells become resistant to apoptosis when an intracellular PD-L1 signalling is blocked. CD8+ T cells are considered to be the main effectors of the blockade of inhibitory receptors. Certain CD8+ T cell subsets, such as non-hyperexhausted (CD28+, T-bethigh, PD-1int), follicular-like (CXCR-5+) or resident memory CD8+ T cells, are more prone to be reactivated by anti-PD-1/PD-L1 monoclonal antibody (mAb). In the future, the challenge will be to rationally combine drugs able to make the tumour microenvironment more permissive to immunotherapy in order to potentiate its clinical activity.

Induction of resident memory T cells enhances the efficacy of cancer vaccine.

Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in tissue and do not recirculate. Although they are considered as early immune effectors in infectious diseases, their role in cancer immunosurveillance remains unknown. In a preclinical model of head and neck cancer, we show that intranasal vaccination with a mucosal vector, the B subunit of Shiga toxin, induces local Trm and inhibits tumour growth. As Trm do not recirculate, we demonstrate their crucial role in the efficacy of cancer vaccine with parabiosis experiments. Blockade of TFGß decreases the induction of Trm after mucosal vaccine immunization, resulting in the lower efficacy of cancer vaccine. In order to extrapolate this role of Trm in humans, we show that the number of Trm correlates with a better overall survival in lung cancer in multivariate analysis. The induction of Trm may represent a new surrogate biomarker for the efficacy of cancer vaccine. This study also argues for the development of vaccine strategies designed to elicit them.

Is There Still Room for Cancer Vaccines at the Era of Checkpoint Inhibitors.

Checkpoint inhibitor (CPI) blockade is considered to be a revolution in cancer therapy, although most patients (70%-80%) remain resistant to this therapy. It has been hypothesized that only tumors with high mutation rates generate a natural antitumor T cell response, which could be revigorated by this therapy. In patients with no pre-existing antitumor T cells, a vaccine-induced T cell response is a rational option to counteract clinical resistance. This hypothesis has been validated in preclinical models using various cancer vaccines combined with inhibitory pathway blockade (PD-1-PDL1-2, CTLA-4-CD80-CD86). Enhanced T cell infiltration of various tumors has been demonstrated following this combination therapy. The timing of this combination appears to be critical to the success of this therapy and multiple combinations of immunomodulating antibodies (CPI antagonists or costimulatory pathway agonists) have reinforced the synergy with cancer vaccines. Only limited results are available in humans and this combined approach has yet to be validated. Comprehensive monitoring of the regulation of CPI and costimulatory molecules after administration of immunomodulatory antibodies (anti-PD1/PD-L1, anti-CTLA-4, anti-OX40, etc.) and cancer vaccines should help to guide the selection of the best combination and timing of this therapy.

CSF IgA NMDAR antibodies are potential biomarkers for teratomas in anti-NMDAR encephalitis.

OBJECTIVE: To evaluate the presence of immunoglobulin A (IgA) subtype of anti-NMDA receptor (NMDAR) antibodies (IgA-NMDAR-Abs) in the CSF of patients with immunoglobulin G (IgG)-NMDAR-Ab encephalitis and to describe the potential association with a specific clinical pattern. METHODS: The retrospective analysis for the presence of IgA-NMDAR-Abs in 94 CSF samples from patients with anti-NMDAR encephalitis diagnosed between October 2007 and February 2014 was conducted at the French Reference Centre on Paraneoplastic Neurological Syndrome. This observational study compared 39 patients with both IgA- and IgG-NMDAR-Abs to 55 patients with only IgG-NMDAR-Abs. RESULTS: In the retrospective cohort, 41% of the patients with NMDAR-Ab encephalitis had both CSF IgG- and IgA-NMDAR-Abs. Approximately half of the IgA-NMDAR-Ab-positive patients (18/38, 49%) definitively possessed associated tumors, primarily ovarian teratomas (17/18, 94%), compared with only 5% (3/55) of the patients in the IgA-NMDAR-Ab-negative group (p < 0.001). In the adult female population at risk for ovarian teratoma, the detection of CSF IgA-NMDAR-Ab positivity showed 85% sensitivity, 70% specificity, a 57% positive predictive value, and a 90% negative predictive value for the diagnosis of ovarian teratoma. No other specific clinical features or clinical outcome were associated with CSF IgA-NMDAR-Ab positivity. CONCLUSION: These results suggest that in patients with IgG-NMDAR-Ab encephalitis, CSF IgA-NMDAR-Abs could be used as a biological marker for the presence of an ovarian teratoma.