Activation of B cells in Tertiary Lymphoid Structures in cancer: Anti-tumor or anti-self?

Whereas T cells in the tumor microenvironment have been the main focus as cancer controlling cells and targets of immunotherapies, B cells have recently gained strong attention. Being associated to Tertiary Lymphoid Structures (TLS) located at the vicinity of tumor nests, the fate of B cell depends on TLS maturity. In immature TLS they may evolve as regulatory B cells producing immunosuppressive cytokines and promote tumor growth. In mature TLS with a germinal center, B cells are selected, amplified, undergo affinity maturation and isotypic switching, resulting in plasma cell generation and production of anti-tumor antibodies. In that case, they are associated with longer patient's survival and therapeutic response to immunotherapy. Identification of tumor specific, or tumor overexpressed, antigens recognized by "in situ" produced antibodies and their discrimination from self-antigens induced by ICI treatments is a major challenge to develop novel antibody-based immunotherapies.

Effective control of Staphylococcus aureus lung infection despite tertiary lymphoid structure disorganisation.

BACKGROUND: Tertiary lymphoid structures (TLS) are triggered by persistent bronchopulmonary infection with Staphylococcus aureus, but their roles remain elusive. The present study sought to examine the effects of B- and/or T-cell depletion on S. aureus infection and TLS development (lymphoid neogenesis) in mice. METHODS: C57Bl/6 mice were pre-treated with 1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or 2) an anti-CD4 and/or an anti-CD8 mAb (T-cell depletion) or 3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion) or 4) isotype control mAbs. After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing S. aureus (106 CFU per mouse). 14 days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures and immunohistochemistry, respectively. RESULTS: 14 days after S. aureus-bead instillation, lung bacterial load was comparable between control and lymphocyte-depleted mice. While TLS were observed in the lungs of infected mice pre-treated with control mAbs, these structures were disorganised or abolished in the lungs of lymphocyte-depleted mice. The absence of CD20+ B-lymphocytes had no effect on CD3+ T-lymphocyte infiltration, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell infiltration. Depletion of CD4+ or CD8+ T-cells separately had limited effect on B-cell infiltration, but led to the absence of germinal centres. CONCLUSION: TLS disorganisation is not associated with loss of infection control in mice persistently infected with S. aureus.

Tight Interplay Between Therapeutic Monoclonal Antibodies and the Tumour Microenvironment in Cancer Therapy.

Therapeutic monoclonal antibodies (mAb) have changed the landscape of cancer therapy. With advances in the understanding of tumour biology and its microenvironment, different categories of mAbs have been developed; a first category is directed against tumour cells themselves, a second one comprises antibodies blocking the formation of neo-vasculature that accompanies tumour development, and, during the last decades, a third new category of immunomodulatory antibodies that target immune cells in the tumour microenvironment rather than cancer cells has emerged. In this chapter, we outline the main mechanisms of action of the different anti-tumour antibodies. We discuss the notion that, rather than passive immunotherapy that solely induces tumour cell killing, mAbs have multifaceted effects on the tumour microenvironment and could, qualitatively and quantitatively, reshape the immune infiltrate. We also discuss bystander effects of mAbs on the tumour microenvironment that should be carefully considered for the design of new therapeutic strategies.

Presence of T cells directed against CD20-derived peptides in healthy individuals and lymphoma patients.

Preclinical and clinical studies have suggested that cancer treatment with antitumor antibodies induces a specific adaptive T cell response. A central role in this process has been attributed to CD4+ T cells, but the relevant T cell epitopes, mostly derived from non-mutated self-antigens, are largely unknown. In this study, we have characterized human CD20-derived epitopes restricted by HLA-DR1, HLA-DR3, HLA-DR4, and HLA-DR7, and investigated whether T cell responses directed against CD20-derived peptides can be elicited in human HLA-DR-transgenic mice and human samples. Based on in vitro binding assays to recombinant human MHC II molecules and on in vivo immunization assays in H-2 KO/HLA-A2+-DR1+ transgenic mice, we have identified 21 MHC II-restricted long peptides derived from intracellular, membrane, or extracellular domains of the human non-mutated CD20 protein that trigger in vitro IFN-γ production by PBMCs and splenocytes from healthy individuals and by PBMCs from follicular lymphoma patients. These CD20-derived MHC II-restricted peptides could serve as a therapeutic tool for improving and/or monitoring anti-CD20 T cell activity in patients treated with rituximab or other anti-CD20 antibodies.

Human iNKT and MAIT cells exhibit a PLZF-dependent proapoptotic propensity that is counterbalanced by XIAP.

Invariant natural killer (iNKT) T cells and mucosal-associated invariant T (MAIT) cells represent peculiar T-lymphocyte subpopulations with innate-like properties that differ from conventional T cells. iNKT are reduced in the primary immunodeficiency caused by mutations in the X-linked inhibitor of apoptosis (XIAP). By studying the mechanism of this depletion, we herein report that iNKT cells exhibit a high susceptibility to apoptosis that is not observed with conventional T cells. Elevated expression of caspases 3 and 7 accounts for the proapoptotic phenotype of iNKT cells, which is inhibited by XIAP although it exerts a moderate effect in conventional T cells. Similarly, MAIT cells exhibit a proapoptotic propensity with elevated expression of activated caspases and are decreased in XIAP-deficient individuals. Knockdown of the transcription factor PLZF/ZBTB-16, which is involved in the effector program of iNKT cells, diminishes their proapoptotic phenotype. Conversely, overexpression of PLZF/ZBTB-16 in conventional T cells leads to a proapoptotic phenotype. Our findings identify a previously unknown pathway of regulation of innate-like T-cell homeostasis depending on XIAP and PLZF. The proapoptotic feature of iNKT cells also gives a reliable explanation of their exhaustion observed in different human conditions including the XIAP immunodeficiency.

Longitudinal and integrative biomodeling of effector and memory immune compartments after inactivated influenza vaccination.

Most vaccines, including those against influenza, were developed by focusing solely on humoral response for protection. However, vaccination activates different adaptive compartments that might play a role in protection. We took advantage of the pandemic 2009 A(H1N1) influenza vaccination to conduct a longitudinal integrative multiparametric analysis of seven immune parameters in vaccinated subjects. A global analysis underlined the predominance of induction of humoral and CD4 T cell responses, whereas pandemic 2009 A(H1N1)-specific CD8 responses did not improve after vaccination. A principal component analysis and hierarchical clustering of individuals showed a differential upregulation of influenza vaccine-specific immunity including hemagglutination inhibition titers, IgA(+) and IgG(+) Ab-secreting cells, effector CD4 or CD8 T cell frequencies at day 21 among individuals, suggesting a fine-tuning of the immune parameters after vaccination. This is related to individual factors including the magnitude and quality of influenza-specific immune responses before vaccination. We propose a graphical delineation of immune determinants that would be essential for a better understanding of vaccine-induced immunity in vaccination strategies.

Unraveling the complex interplay between anti-tumor immune response and autoimmunity mediated by B cells and autoantibodies in the era of anti-checkpoint monoclonal antibody therapies.

The intricate relationship between anti-tumor immunity and autoimmunity is a complex yet crucial aspect of cancer biology. Tumor microenvironment often exhibits autoimmune features, a phenomenon that involves natural autoimmunity and the induction of humoral responses against self-antigens during tumorigenesis. This induction is facilitated by the orchestration of anti-tumor immunity, particularly within organized structures like tertiary lymphoid structures (TLS). Paradoxically, a significant number of cancer patients do not manifest autoimmune features during the course of their illness, with rare instances of paraneoplastic syndromes. This discrepancy can be attributed to various immune-mediated locks, including regulatory or suppressive immune cells, anergic autoreactive lymphocytes, or induction of effector cells exhaustion due to chronic stimulation. Overcoming these locks holds the risk to induce autoimmune mechanisms during cancer progression, a phenomenon notably observed with anti-immune checkpoint therapies, in contrast to more conventional treatments like chemotherapy or radiotherapy. Therefore, the challenge arises in managing immune-related adverse events (irAEs) induced by immune checkpoint inhibitors treatment, as decoupling them from the anti-tumor activity poses a significant clinical dilemma. This review summarizes recent advances in understanding the link between B-cell driven anti-tumor responses and autoimmune reactions in cancer patients, and discusses the clinical implications of this relationship.

Human X-linked variable immunodeficiency caused by a hypomorphic mutation in XIAP in association with a rare polymorphism in CD40LG.

The present study focuses on a large family with an X-linked immunodeficiency in which there are variable clinical and laboratory phenotypes, including recurrent viral and bacterial infections, hypogammaglobulinemia, Epstein-Barr virus-driven lymphoproliferation, splenomegaly, colitis, and liver disease. Molecular and genetic analyses revealed that affected males were carriers of a hypomorphic hemizygous mutation in XIAP (XIAP(G466X)) that cosegregated with a rare polymorphism in CD40LG (CD40 ligand(G219R)). These genes are involved in the X-linked lymphoproliferative syndrome 2 and the X-linked hyper-IgM syndrome, respectively. Single expression of XIAP(G466X) or CD40L(G219R) had no or minimal effect in vivo, although in vitro, they lead to altered functional activities of their gene products, which suggests that the combination of XIAP and CD40LG mutations contributed to the expression of clinical manifestations observed in affected individuals. Our report of a primary X-linked immunodeficiency of oligogenic origin emphasizes that primary immunodeficiencies are not caused by a single defective gene, which leads to restricted manifestations, but are likely to be the result of an interplay between several genetic determinants, which leads to more variable clinical phenotypes.

Acute Influenza Infection Promotes Lung Tumor Growth by Reprogramming the Tumor Microenvironment.

One billion people worldwide get flu every year, including patients with non-small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.

Autophagy-Related Gene Signature Highlights Metabolic and Immunogenic Status of Malignant Cells in Non-Small Cell Lung Cancer Adenocarcinoma.

Autophagy is a self-degradative mechanism involved in many biological processes, including cell death, survival, proliferation or migration. In tumors, autophagy plays an important role in tumorigenesis as well as cancer progression and resistance to therapies. Usually, a high level of autophagy in malignant cells has been associated with tumor progression and poor prognostic for patients. However, the investigation of autophagy levels in patients remains difficult, especially because quantification of autophagy proteins is challenging in the tumor microenvironment. In this study, we analyzed the expression of autophagy genes in non-small cell lung (NSCLC) cancer patients using public datasets and revealed an autophagy gene signature for proliferative and immune-checkpoint-expressed malignant cells in lung adenocarcinoma (LUAD). Analysis of autophagy-related gene expression profiles in tumor and adjacent tissues revealed differential signatures, namely signature A (23 genes) and signature B (12 genes). Signature B correlated with a bad prognosis and poor overall and disease-specific survival. Univariate and multivariate analyses revealed that this signature was an independent factor for prognosis. Moreover, patients with high expression of signature B exhibited more genes related to proliferation and fewer genes related to immune cells or immune response. The analysis of datasets from sorted fresh tumor cells or single cells revealed that signature B is predominantly represented in malignant cells, with poor expression in pan-immune population or in fibroblast or endothelial cells. Interestingly, autophagy was increased in malignant cells exhibiting high levels of signature B, which correlated with an elevated expression of genes involved in cell proliferation and immune checkpoint signaling. Taken together, our analysis reveals a novel autophagy-based signature to define the metabolic and immunogenic status of malignant cells in LUAD.

[Autophagy modulation by viruses: An important role in tumor progression]. / L'autophagie modulée par les virus - Un rôle dans la progression tumorale.

Autophagy is an important process for cellular homeostasis at critical steps of development or in response to environmental stress. In the context of cancers, autophagy has a significant impact on tumor occurrence and tumor cell growth. On the one hand, autophagy limits the transformation of precancerous cells into cancer cells at an early stage. However, on the other hand, it promotes cell survival, cell proliferation, metastasis and resistance to anti-tumor therapies in more advanced tumors. Autophagy can be induced by a variety of extracellular and intracellular stimulus. Viral infections have often been associated with a modulation of autophagy, with variable impacts on viral replication and on the survival of infected cells depending on the model studied. In a tumor context, the modulation of autophagy induced by the viral infection of tumor cells seems to have a significant impact on tumor progression. The aim of this review article is to present recent findings regarding the consequences of autophagy disturbance by viral infections on tumor behavior.

TITLE: L'autophagie modulée par les virus - Un rôle dans la progression tumorale. ABSTRACT: L'autophagie est un processus métabolique important pour maintenir l'homéostasie cellulaire à des moments critiques du développement et/ou en réponse à un stress environnemental. Cela est particulièrement pertinent dans le cas des cancers, pour lesquels il a été montré que l'autophagie a un impact important sur leur survenue et sur la croissance tumorale. D'une part, elle limite la transformation cancéreuse des cellules précancéreuses à un stade précoce, mais, d'autre part, elle favorise la survie et la prolifération cellulaires, les métastases et la résistance aux thérapies anti-tumorales dans les tumeurs plus avancées. L'autophagie peut être induite par une grande variété de stimulus extracellulaires et intracellulaires. Les infections virales ont souvent été associées à une modulation de l'autophagie, dont l'impact sur la réplication virale ou la survie des cellules infectées diffère selon le modèle étudié. Dans un contexte tumoral, certains mécanismes moléculaires complexes par lesquels la modulation de l'autophagie par les virus peut influencer le développement des cellules précancéreuses ou cancéreuses ont été révélés. Cette revue présente les découvertes récentes concernant les répercussions d'une perturbation de l'autophagie par l'infection virale sur la survenue et la progression des tumeurs cancéreuses.

Intravenous immunoglobulin induces proliferation and immunoglobulin synthesis from B cells of patients with common variable immunodeficiency: a mechanism underlying the beneficial effect of IVIg in primary immunodeficiencies.

Common variable immunodeficiency (CVID) is associated with low serum immunoglobulin concentrations and an increased susceptibility to infections and autoimmune diseases. The treatment of choice for CVID patients is replacement intravenous immunoglobulin (IVIg) therapy. IVIg has been beneficial in preventing or alleviating the severity of infections and autoimmune and inflammatory process in majority of CVID patients. Although the mechanisms of action of IVIg given as 'therapeutic high dose' in patients with autoimmune diseases are well studied, the underlying mechanisms of beneficial effects of IVIg in primary immunodeficiencies are not completely understood. Therefore we investigated the effect of 'replacement dose' of IVIg by probing its action on B cells from CVID patients. We demonstrate that IVIg at low doses induces proliferation and immunoglobulin synthesis from B cells of CVID patients. Interestingly, B cell stimulation by IVIg is not associated with induction of B cell effector cytokine IFN-γ and of transcription factor T-bet. Together, our results indicate that in some CVID patients, IVIg rectifies the defective signaling of B cells normally provided by T cells and delivers T-independent signaling for B cells to proliferate. IVIg 'replacement therapy' in primary immunodeficiencies is therefore not a merepassive transfer of antibodies to prevent exclusively the recurrent infections; rather it has an active role in regulating autoimmune and inflammatory responses through modulating B cell functions and thus imposing dynamic equilibrium of the immune system.

Autophagy Modulation by Viral Infections Influences Tumor Development.

Autophagy is a self-degradative process important for balancing cellular homeostasis at critical times in development and/or in response to nutrient stress. This is particularly relevant in tumor model in which autophagy has been demonstrated to have an important impact on tumor behavior. In one hand, autophagy limits tumor transformation of precancerous cells in early stage, and in the other hand, it favors the survival, proliferation, metastasis, and resistance to antitumor therapies in more advanced tumors. This catabolic machinery can be induced by an important variety of extra- and intracellular stimuli. For instance, viral infection has often been associated to autophagic modulation, and the role of autophagy in virus replication differs according to the virus studied. In the context of tumor development, virus-modulated autophagy can have an important impact on tumor cells' fate. Extensive analyses have shed light on the molecular and/or functional complex mechanisms by which virus-modulated autophagy influences precancerous or tumor cell development. This review includes an overview of discoveries describing the repercussions of an autophagy perturbation during viral infections on tumor behavior.

Activation of human peripheral IgM+ B cells is transiently inhibited by BCR-independent aggregation of Fc gammaRIIB.

Immune complexes can trigger a SHIP-1-independent proapoptotic signal in mouse class-switched IgG(+) B cells and plasma cells by binding to Fc gammaRIIB, in the absence of concomitant coaggregation with BCR, hence regulating plasma cell survival and participating in the selection of B cells producing high affinity Abs during secondary Ab responses. By contrast, we demonstrate in the present study that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells does not induce apoptosis but transiently inhibits B cell proliferation and calcium influx triggered by BCR cross-linking. Using human peripheral B cells and IIA1.6 lymphoma B cells expressing wild-type human Fc gammaRIIB (IIA1.6-Fc gammaRIIB), we also show that the unique aggregation of human Fc gammaRIIB induces ITIM phosphorylation. This aggregation provokes the recruitment of phosphorylated SHIP-1 by Fc gammaRIIB and inhibits the constitutive phosphorylation of Akt in human IIA1.6-Fc gammaRIIB cells. This inhibitory signaling pathway is abrogated in IIA1.6 cells expressing ITIM-mutated Fc gammaRIIB (Fc gammaRIIB(Y292G)), suggesting that ITIM phosphorylation is necessary for Fc gammaRIIB-induced B cell blockade. Overall, we demonstrate that the unique aggregation of Fc gammaRIIB on human peripheral IgM(+) B cells is sufficient to transiently down-regulate their activation without inducing apoptosis. Our results suggest that Fc gammaRIIB could negatively regulate IgM(+) B cells before class-switch occurrence and that its unique engagement by immune complexes represents a reversible checkpoint for peripheral IgM(+) B cells.

Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells.

FcgammaRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcgammaRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgammaRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgammaRIIIB extra-cellular domains. These sdAbs bind FcgammaRIIIA+ NK cells and FcgammaRIIIB+ polymorphonuclear cells, but not FcgammaRI+ or FcgammaRII+ cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgammaRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgammaRIII with a K(D) in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgammaRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-gamma production, respectively. These anti-FcgammaRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgammaRIII killer cells to target and destroy tumor cells.

Exploring the Role of Tertiary Lymphoid Structures Using a Mouse Model of Bacteria-Infected Lungs.

Animal models can be helpful tools for deciphering the generation, maintenance, and role of tertiary lymphoid structures (TLS) during infections or tumor development. We describe here the establishment of a persistent lung infection in immune-competent mice by intratracheal instillation of agarose beads containing Pseudomonas aeruginosa or Staphylococcus aureus bacteria. After instillation, animals develop a chronic pulmonary infection, marked by the presence of TLS. This experimental setting allows the study of the function of TLS induced by bacteria encountered in patients with cystic fibrosis (CF) as P. aeruginosa and S. aureus are the two main bacterial strains that infect bronchi of adult CF patients. Additionally, we describe also how to manipulate the immune response in these infected animals by targeting immune cells involved in TLS function. Overall, this approach makes it possible to explore the role of chronic inflammation in the induction and maintenance of TLS in infected tissues.

Rescuing CD4+CD25+ regulatory T-cell functions in rheumatoid arthritis by cytokine-targeted monoclonal antibody therapy.

CD4+CD25+ regulatory T cells (Tregs) play a crucial role in controlling the development of autoimmune diseases such as rheumatoid arthritis (RA). However, despite an increased number of Tregs, the persistence of inflammation in the rheumatoid joints suggests that Tregs are unable to suppress ongoing disease, perhaps due to an inhibition of their functions by pro-inflammatory cytokines. Treatment of RA patients with anti-TNF-alpha monoclonal antibodies such as infliximab and adalimumab has been found to induce and restore the functions of Tregs. Thus, manipulation of the pro-inflammatory environment in the inflamed synovia via neutralization of inflammatory cytokines by monoclonal antibodies could represent a novel therapeutic strategy for restoring the suppressive functions of Tregs and induction and/or expansion of Tregs in order to reinforce tolerance mechanisms.

FcgammaR: The key to optimize therapeutic antibodies?

The binding of IgG to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and the control of effector immune functions. Activating FcgammaR induce antibody-dependent cell cytotoxicity (ADCC), endocytosis of immune complexes followed by antigen presentation, phagocytosis, and release of cytokines or pro-inflammatory mediators. By contrast, inhibitory FcgammaR regulate immune responses by inhibiting the activation of B lymphocytes, monocytes, mast cells and basophils, induced through activating receptors. Studies with FcgammaR-deficient mice support the critical role of the different FcgammaR in the in vivo functional effects of therapeutic monoclonal antibodies. Structural studies have provided detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions. The importance of the sugar components linked to asparagine 297 in the function of IgG has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.