A mixture-like model for tumor-immune system interactions.

We introduce a mathematical model based on mixture theory intended to describe the tumor-immune system interactions within the tumor microenvironment. The equations account for the geometry of the tumor expansion, and the displacement of the immune cells, driven by diffusion and chemotactic mechanisms. They also take into account the constraints in terms of nutrient and oxygen supply. The numerical investigations analyze the impact of the different modeling assumptions and parameters. Depending on the parameters, the model can reproduce elimination, equilibrium or escape phases and it identifies a critical role of oxygen/nutrient supply in shaping the tumor growth. In addition, antitumor immune cells are key factors in controlling tumor growth, maintaining an equilibrium while protumor cells favor escape and tumor expansion.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

Analysis of the Equilibrium Phase in Immune-Controlled Tumors Provides Hints for Designing Better Strategies for Cancer Treatment.

When it comes to improving cancer therapies, one challenge is to identify key biological parameters that prevent immune escape and maintain an equilibrium state characterized by a stable subclinical tumor mass, controlled by the immune cells. Based on a space and size structured partial differential equation model, we developed numerical methods that allow us to predict the shape of the equilibrium at low cost, without running simulations of the initial-boundary value problem. In turn, the computation of the equilibrium state allowed us to apply global sensitivity analysis methods that assess which and how parameters influence the residual tumor mass. This analysis reveals that the elimination rate of tumor cells by immune cells far exceeds the influence of the other parameters on the equilibrium size of the tumor. Moreover, combining parameters that sustain and strengthen the antitumor immune response also proves more efficient at maintaining the tumor in a long-lasting equilibrium state. Applied to the biological parameters that define each type of cancer, such numerical investigations can provide hints for the design and optimization of cancer treatments.

LLT1-CD161 Interaction in Cancer: Promises and Challenges.

The success of immune checkpoint therapy in cancer has changed our way of thinking, promoting the design of future cancer treatments that places the immune system at the center stage. The knowledge gained on immune regulation and tolerance helped the identification of promising new clinical immune targets. Among them, the lectin-like transcript 1 (LLT1) is the ligand of CD161 (NKR-P1A) receptor expressed on natural killer cells and T cells. LLT1/CD161 interaction modulates immune responses but the exact nature of the signals delivered is still partially resolved. Investigation on the role of LLT1/CD161 interaction has been hampered by the lack of functional homologues in animal models. Also, some studies have been misled by the use of non-specific reagents. Recent studies and meta-analyses of single cell data are bringing new insights into the function of LLT1 and CD161 in human pathology and notably in cancer. The advances made on the characterization of the tumor microenvironment prompt us to integrate LLT1/CD161 interaction into the equation. This review recapitulates the key findings on the expression profile of LLT1 and CD161, their regulation, the role of their interaction in cancer development, and the relevance of targeting LLT1/CD161 interaction.

CD161 expression and regulation defines rapidly responding effector CD4+ T cells associated with improved survival in HPV16-associated tumors.

BACKGROUND: Expression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown. METHODS: We examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies. RESULTS: Central and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealed KLRB1 expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivator SOX4, known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1 and SOX4 was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)ß1. CONCLUSION: High levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells.

A size and space structured model of tumor growth describes a key role for protumor immune cells in breaking equilibrium states in tumorigenesis.

Switching from the healthy stage to the uncontrolled development of tumors relies on complicated mechanisms and the activation of antagonistic immune responses, that can ultimately favor the tumor growth. We introduce here a mathematical model intended to describe the interactions between the immune system and tumors. The model is based on partial differential equations, describing the displacement of immune cells subjected to both diffusion and chemotactic mechanisms, the strength of which is driven by the development of the tumors. The model takes into account the dual nature of the immune response, with the activation of both antitumor and protumor mechanisms. The competition between these antagonistic effects leads to either equilibrium or escape phases, which reproduces features of tumor development observed in experimental and clinical settings. Next, we consider on numerical grounds the efficacy of treatments: the numerical study brings out interesting hints on immunotherapy strategies, concerning the role of the administered dose, the role of the administration time and the interest in combining treatments acting on different aspects of the immune response. Such mathematical model can shed light on the conditions where the tumor can be maintained in a viable state and also provide useful hints for personalized, efficient, therapeutic strategies, boosting the antitumor immune response, and reducing the protumor actions.

High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture.

The integrative analysis of tumor immune microenvironment (TiME) components, their interactions and their microanatomical distribution is mandatory to better understand tumor progression. Imaging Mass Cytometry (IMC) is a high dimensional tissue imaging system which allows the comprehensive and multiparametric in situ exploration of tumor microenvironments at a single cell level. We describe here the design of a 39-antibody IMC panel for the staining of formalin-fixed paraffin-embedded human tumor sections. We also provide an optimized staining procedure and details of the experimental workflow. This panel deciphers the nature of immune cells, their functions and their interactions with tumor cells and cancer-associated fibroblasts as well as with other TiME structural components known to be associated with tumor progression like nerve fibers and tumor extracellular matrix proteins. This panel represents a valuable innovative and powerful tool for fundamental and clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies.

Cutaneous Squamous Cell Carcinoma Development Is Associated with a Temporal Infiltration of ILC1 and NK Cells with Immune Dysfunctions.

NK cells and tissue-resident innate lymphoid cells (ILCs) are innate effectors found in the skin. To investigate their temporal dynamics and specific functions throughout the development of cutaneous squamous cell carcinoma (cSCC), we combined transcriptomic and immunophenotyping analyses in mouse and human cSCCs. We identified an infiltration of NK cells and ILC1s as well as the presence of a few ILC3s. Adoptive transfer of NK cells in NK cell‒ and ILC-deficient Nfil3-/- mice revealed a role for NK cells in early control of cSCC. During tumor progression, we identified a population skewing with the infiltration of atypical ILC1 secreting inflammatory cytokines but reduced levels of IFN-γ at the papilloma stage. NK cells and ILC1s were functionally impaired, with reduced cytotoxicity and IFN-γ secretion associated with the downregulation of activating receptors. They also showed a high degree of heterogeneity in mouse and human cSCCs with the expression of several markers of exhaustion, including TIGIT on NK cells and PD-1 and TIM-3 on ILC1s. Our data show an enrichment in inflammatory ILC1 at the precancerous stage together with impaired antitumor functions in NK cells and ILC1 that could contribute to the development of cSCC and thus suggest that future immunotherapies should take both ILC populations into account.

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development.

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC.

NK Cell and Fibroblast-Mediated Regulation of Skin Squamous Cell Carcinoma Invasion by CLEC2A Is Compromised in Xeroderma Pigmentosum.

The ability of cancer cells to invade and disseminate can be affected by components of the surrounding microenvironment. To identify dermal components that regulate the growth of epidermal carcinomas, we studied the genetic disease called xeroderma pigmentosum that bears mutations in genes involved in the nucleotide excision repair of DNA. Patients with xeroderma pigmentosum are more prone to develop cutaneous tumors than the general population and their dermal fibroblasts display the features of dermal cancer-associated fibroblasts, which promote the invasion of keratinocytes. Here, we report that 3-dimensional dermal cultures of fibroblasts from healthy donors but not from patients with xeroderma pigmentosum complementation group C express CLEC2A, which is the ligand of the activating NK cell receptor NKp65. A similar loss of CLEC2A was observed in sporadic dermal cancer-associated fibroblasts and upon the culture of fibroblasts with cutaneous squamous cell carcinoma-conditioned medium. Using an innovative 3-dimensional organotypic skin culture model that contain NK cells in addition to fibroblasts and squamous cell carcinoma cells, we unveiled a key role of CLEC2A that orchestrates a crosstalk between fibroblasts and NK cells, thereby leading to the control of squamous cell carcinoma invasion. These findings indicate that CLEC2A-expressing dermal fibroblasts play a major role in immune surveillance of the skin.

A size and space structured model describing interactions of tumor cells with immune cells reveals cancer persistent equilibrium states in tumorigenesis.

The recent success of immunotherapies for the treatment of cancer has highlighted the importance of the interactions between tumor and immune cells. Mathematical models of tumor growth are needed to faithfully reproduce and predict the spatiotemporal dynamics of tumor growth. We introduce a mathematical model intended to describe by means of a system of partial differential equations the early stages of the interactions between effector immune cells and tumor cells. The model is structured in size and space, and it takes into account the migration of the tumor antigen-specific cytotoxic effector cells towards the tumor micro-environment by a chemotactic mechanism. We investigate on numerical grounds the role of the key parameters of the model such as the division and growth rates of the tumor cells, and the conversion and death rates of the immune cells. Our main findings are two-fold. Firstly, the model exhibits a possible control of the tumor growth by the immune response; nevertheless, the control is not complete in the sense that the asymptotic equilibrium states keep residual tumors and activated immune cells. Secondly, space heterogeneities of the source of immune cells can significantly reduce the efficiency of the control dynamics, making patterns of remission-recurrence appear.

Expression of LLT1 and its receptor CD161 in lung cancer is associated with better clinical outcome.

Co-stimulatory and inhibitory receptors expressed by immune cells in the tumor microenvironment modulate the immune response and cancer progression. Their expression and regulation are still not fully characterized and a better understanding of these mechanisms is needed to improve current immunotherapies. Our previous work has identified a novel ligand/receptor pair, LLT1/CD161, that modulates immune responses. Here, we extensively characterize its expression in non-small cell lung cancer (NSCLC). We show that LLT1 expression is restricted to germinal center (GC) B cells within tertiary lymphoid structures (TLS), representing a new hallmark of the presence of active TLS in the tumor microenvironment. CD161-expressing immune cells are found at the vicinity of these structures, with a global enrichment of NSCLC tumors in CD161+ CD4+ and CD8+ T cells as compared to normal distant lung and peripheral blood. CD161+ CD4+ T cells are more activated and produce Th1-cytokines at a higher frequency than their matched CD161-negative counterparts. Interestingly, CD161+ CD4+ T cells highly express OX40 co-stimulatory receptor, less frequently 4-1BB, and display an activated but not completely exhausted PD-1-positive Tim-3-negative phenotype. Finally, a meta-analysis revealed a positive association of CLEC2D (coding for LLT1) and KLRB1 (coding for CD161) gene expression with favorable outcome in NSCLC, independently of the size of T and B cell infiltrates. These data are consistent with a positive impact of LLT1/CD161 on NSCLC patient survival, and make CD161-expressing CD4+ T cells ideal candidates for efficient anti-tumor recall responses.

A Real-Time Cytotoxicity Assay as an Alternative to the Standard Chromium-51 Release Assay for Measurement of Human NK and T Cell Cytotoxic Activity.

This unit describes the monitoring and quantification of cellular cytotoxicity using a non-radioactive and real-time cytotoxic assay. The extent of target-cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell-imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO2 . The basic protocol describes natural killer cell-mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody-dependent cell-mediated cytotoxicity (ADCC). The assay is also suitable for antigen specific T-cell cytotoxic assays. Until now, the standard chromium 51 (51 Cr)-release assay has remained the sole sensitive assay but its major drawbacks include cost and hazard of handling radioactivity. The real-time cytotoxic assay is therefore an effective alternative providing a robust and sensitive assay that accurately monitors lysis of target cells over time. © 2017 by John Wiley & Sons, Inc.

Sublingual Priming with a HIV gp41-Based Subunit Vaccine Elicits Mucosal Antibodies and Persistent B Memory Responses in Non-Human Primates.

Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.

A real-time digital bio-imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium-51 release assay.

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard 51 Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to 51 Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

Lectin-like transcript 1 is a marker of germinal center-derived B-cell non-Hodgkin's lymphomas dampening natural killer cell functions.

Non-Hodgkin's lymphomas (NHLs) are malignant neoplasms which are clinically and biologically diverse. Their incidence is constantly increasing and despite treatment advances, there is a need for novel targeted therapies. Here, we identified Lectin-like transcript 1 (LLT1) as a biomarker of germinal center (GC)-derived B-cell NHLs. LLT1 identifies GC B cells in reactive tonsils and lymph nodes and its expression is maintained in B-cell NHLs which derive from GC, including Burkitt lymphoma (BL), follicular lymphoma (FL), and GC-derived diffuse large B-cell lymphoma (DLBCL). We further show that LLT1 expression by tumors dampens natural killer (NK) cell functions following interaction with its receptor CD161, uncovering a potential immune escape mechanism. Our results pinpoint LLT1 as a novel biomarker of GC-derived B-cell NHLs and as a candidate target for innovative immunotherapies.

Induction of lectin-like transcript 1 (LLT1) protein cell surface expression by pathogens and interferon-γ contributes to modulate immune responses.

CD161 is a C-type lectin-like receptor expressed on human natural killer (NK) cells and subsets of T cells. CD161 has been described as an inhibitory receptor that regulates NK cell-mediated cytotoxicity and IFN-γ production. Its role on T cells has remained unclear. Studies have shown that triggering of CD161 enhances NK T cell proliferation and T cell-IFN-γ production while inhibiting TNF-α production by CD8(+) T cells. Lectin-like transcript 1 (LLT1), the ligand of CD161, was found to be expressed on Toll-like receptor (TLR)-activated plasmacytoid and monocyte-derived dendritic cells (DC) and on activated B cells. Using newly developed anti-LLT1 mAbs, we show that LLT1 is not expressed on the surface of circulating B and T lymphocytes, NK cells, monocytes, and dendritic cells but that LLT1 is up-regulated upon activation. Not only TLR-stimulated dendritic cells and B cells but also T cell receptor-activated T cells and activated NK cells up-regulate LLT1. Interestingly, IFN-γ increases LLT1 expression level on antigen-presenting cells. LLT1 is also induced on B cells upon viral infection such as Epstein-Barr virus or HIV infection and in inflamed tonsils. Finally, expression of LLT1 on B cells inhibits NK cell function but costimulates T cell proliferation or IFN-γ production, and coengagement of CD161 with CD3 increases IL-17 secretion. Altogether, our results point toward a role for LLT1/CD161 in modulating immune responses to pathogens.

Characterization of alternatively spliced transcript variants of CLEC2D gene.

Lectin-like transcript 1 (LLT1) encoded by CLEC2D gene is a C-type lectin-like molecule interacting with human CD161 (NKR-P1A) receptor expressed by natural killer cells and subsets of T cells. Using RT-PCR and sequencing, we identified several CLEC2D alternatively spliced transcript variants generated by exon skipping. In addition to the reported transcript variants 1 (LLT1) and 2, we identified a novel splice variant 4 and transcripts coding for putative soluble proteins. CLEC2D transcripts were detected primarily in hematopoietic cell lines and were found to be co-induced by the same activation signals. Although very low amounts of putative soluble CLEC2D protein isoforms could be produced by transfectants, CLEC2D isoforms 2 and 4 were efficiently expressed. By contrast to LLT1, which was detected on the cell surface, isoform 2 and 4 remained in the endoplasmic reticulum where they formed homodimers or heterodimers with LLT1. They failed to interact with CD161, leaving LLT1 as the sole ligand for this receptor. CLEC2D therefore uses gene splicing to generate protein isoforms that are structurally distinct and that have different biological activities.

Cutting edge: lectin-like transcript 1 is a ligand for the CD161 receptor.

Human NK cells and subsets of T cells or NKT cells express the orphan C-type lectin receptor CD161 (NKR-P1A) of unknown function. In contrast to rodents that possess several NKR-P1 genes coding for either activating or inhibitory receptors, the nature of signals delivered by the single human NKR-P1A receptor is still to be clarified. In this article, we show that the lectin-like transcript 1 (LLT1) molecule is a ligand for the CD161 receptor. Engagement of CD161 on NK cells with LLT1 expressed on target cells inhibited NK cell-mediated cytotoxicity and IFN-gamma secretion. Conversely, LLT1/CD161 interaction in the presence of a TCR signal enhanced IFN-gamma production by T cells. These findings identify a novel ligand/receptor pair that differentially regulate NK and T cell functions.

Requirement of the proteasome for the trimming of signal peptide-derived epitopes presented by the nonclassical major histocompatibility complex class I molecule HLA-E.

The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.