SAR442085, a novel anti-CD38 antibody with enhanced antitumor activity against multiple myeloma.

Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.

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.

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.

Presence of B cells in tertiary lymphoid structures is associated with a protective immunity in patients with lung cancer.

RATIONALE: It is now well established that immune responses can take place outside of primary and secondary lymphoid organs. We previously described the presence of tertiary lymphoid structures (TLS) in patients with non-small cell lung cancer (NSCLC) characterized by clusters of mature dendritic cells (DCs) and T cells surrounded by B-cell follicles. We demonstrated that the density of these mature DCs was associated with favorable clinical outcome. OBJECTIVES: To study the role of follicular B cells in TLS and the potential link with a local humoral immune response in patients with NSCLC. METHODS: The cellular composition of TLS was investigated by immunohistochemistry. Characterization of B-cell subsets was performed by flow cytometry. A retrospective study was conducted in two independent cohorts of patients. Antibody specificity was analyzed by ELISA. MEASUREMENTS AND MAIN RESULTS: Consistent with TLS organization, all stages of B-cell differentiation were detectable in most tumors. Germinal center somatic hypermutation and class switch recombination machineries were activated, associated with the generation of plasma cells. Approximately half of the patients showed antibody reactivity against up to 7 out of the 33 tumor antigens tested. A high density of follicular B cells correlated with long-term survival, both in patients with early-stage NSCLC and with advanced-stage NSCLC treated with chemotherapy. The combination of follicular B cell and mature DC densities allowed the identification of patients with the best clinical outcome. CONCLUSIONS: B-cell density represents a new prognostic biomarker for NSCLC patient survival, and makes the link between TLS and a protective B cell-mediated immunity.

Natural autoantibodies to Fcγ receptors in intravenous immunoglobulins.

A considerable progress has been achieved in the comprehension of the cellular and molecular mechanisms that account for the therapeutic benefit of intravenous immunoglobulin (IVIg) in several autoimmune and inflammatory conditions. However, the precise mechanisms responsible for such a wide range of biological activities have not been proven unambiguously. A wide range of specificities have been identified within IVIg including idiotypes of immunoglobulins, T cell receptor, HLA molecules, several cell surface molecules of immunological importance such as CD4, CD5, Fas, BAFF, cytokines and cytokine receptors, chemokine receptors, CD40 among others. Here we identify and characterize the natural autoantibodies of IgG isotype directed against the human Fc receptors. We show that the F(ab')2 of IVIg recognize the FcγRIII (CD16) and FcγRII (CD32). Interestingly, the immunopurified anti-FcγIII and anti-FcγII antibodies isolated from IVIg bind soluble and membrane-bound FcR and inhibit rosette formation. Altogether, these results along with previous reports provide pointers on the existence of functionally relevant natural autoantibodies towards a wide range of self-motifs that may participate in regulation of the immune response. Their presence in the therapeutic immunoglobulin preparations may explain at least in part, the beneficial effect of IVIg in autoimmune diseases.

Absence of sympathetic innervation hampers the generation of tertiary lymphoid structures upon acute lung inflammation.

Tertiary lymphoid structures (TLS) are lymphoid organs present in inflammatory non-lymphoid tissues. Studies have linked TLS to favorable outcomes for patients with cancers or infectious diseases, but the mechanisms underlying their formation are not fully understood. In particular, secondary lymphoid organs innervation raises the question of sympathetic nerve fibers involvement in TLS organogenesis. We established a model of pulmonary inflammation based on 5 daily intranasal instillations of lipopolysaccharide (LPS) in immunocompetent mice. In this setting, lung lymphoid aggregates formed transiently, evolving toward mature TLS and disappearing when inflammation resolved. Sympathetic nerve fibers were then depleted using 6-hydroxydopamine. TLS quantification by immunohistochemistry showed a decrease in LPS-induced TLS number and surface in denervated mouse lungs. Although a reduction in alveolar space was observed, it did not impair overall pulmonary content of transcripts encoding TNF-α, IL-1ß and IFN-γ inflammation molecules whose expression was induced by LPS instillations. Immunofluorescence analysis of immune infiltrates in lungs of LPS-treated mice showed a drop in the proportion of CD23+ naive cells among CD19+ B220+ B cells in denervated mice whereas the proportion of other cell subsets remained unchanged. These data support the existence of neuroimmune crosstalk impacting lung TLS neogenesis and local naive B cell pool.

High lipid content of irradiated human melanoma cells does not affect cytokine-matured dendritic cell function.

Gamma irradiation is one of the methods used to sterilize melanoma cells prior to coculturing them with monocyte-derived immature dendritic cells in order to develop antitumor vaccines. However, the changes taking place in tumor cells after irradiation and their interaction with dendritic cells have been scarcely analyzed. We demonstrate here for the first time that after irradiation a fraction of tumor cells present large lipid bodies, which mainly contain triglycerides that are several-fold increased as compared to viable cells as determined by staining with Oil Red O and BODIPY 493/503 and by biochemical analysis. Phosphatidyl-choline, phosphatidyl-ethanolamine and sphingomyelin are also increased in the lipid bodies of irradiated cells. Lipid bodies do not contain the melanoma-associated antigen MART-1. After coculturing immature dendritic cells with irradiated melanoma cells, tumor cells tend to form clumps to which dendritic cells adhere. Under such conditions, dendritic cells are unable to act as stimulating cells in a mixed leukocyte reaction. However, when a maturation cocktail composed of TNF-alpha, IL-6, IL-1beta and prostaglandin E2 is added to the coculture, the tumor cells clumps disaggregate, dendritic cells remain free in suspension and their ability to efficiently stimulate allogeneic lymphocytes is restored. These results help to understand the events following melanoma cell irradiation, shed light about interactions between irradiated cells and dendritic cells, and may help to develop optimized dendritic cell vaccines for cancer therapy.

[The Janus face of monoclonal antibodies in oncology: passive immunity and vaccination]. / Le double visage des anticorps monoclonaux en oncologie - Immunité passive et vaccination.

Monoclonal antibodies used in oncology exert direct anti-tumor action leading to cancer cell death. This is due to a variety of mechanisms, ranging from the induction of apoptosis to the recruitment of effector cells from the innate immunity. However, antibodies can also induce long-lasting anti-tumor effects thanks to the induction of an adaptive immunity where CD4(+) and CD8(+) T cells play a central role. Different preclinical experimental models, strengthened by a few clinical observations, have shown that, far from being involved only in passive immunotherapy, monoclonal antibodies used in oncology are also endowed with a "vaccine" effect, inducing immune memory, likely responsible for the long-lasting clinical responses that have been sometimes observed. This capacity of triggering/re-installing tumor immune surveillance could be also reinforced by the use, possibly in combination, of antibodies antagonizing molecules such as CTLA-4 or PD-1 that play a key role in the inhibition of the anti-tumor immune responses. Finally, this novel paradigm of therapeutic anti-tumor antibodies as inducers of anti-tumor adaptive immune responses with long-term memory should lead us to re-examine how antibody treatment, chemotherapy, radiotherapy, and biological response modifiers are combined, in particular both in terms of timing and doses.

Expression and Purification of scFv2H7-P18F3, a Bi-Modular Fusion Protein (BMFP) Targeting Human CD20.

P18F3-based bi-modular fusion proteins (BMFPs), designed to re-direct pre-existing anti-Epstein-Barr virus (EBV) endogenous polyclonal antibodies towards defined target cells, demonstrated efficient biological activity in a mouse tumor model and could potentially represent a universal and versatile platform to develop novel therapeutics against a broad range of diseases. This protocol provides step-by-step instructions for expressing scFv2H7-P18F3, a BMFP targeting human CD20, in Escherichia coli (SHuffle®), and for purifying soluble proteins using a two-step process, namely immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography. This protocol can also be used for expression and purification of other BMFPs with alternative binding specificities.

HEV-associated dendritic cells are observed in metastatic tumor-draining lymph nodes of cutaneous melanoma patients with longer distant metastasis-free survival after adjuvant immunotherapy.

Introduction: Tissue biomarkers that aid in identifying cutaneous melanoma (CM) patients who will benefit from adjuvant immunotherapy are of crucial interest. Metastatic tumor-draining lymph nodes (mTDLN) are the first encounter site between the metastatic CM cells and an organized immune structure. Therefore, their study may reveal mechanisms that could influence patients´ outcomes. Methods: Twenty-nine stage-III CM patients enrolled in clinical trials to study the vaccine VACCIMEL were included in this retrospective study. After radical mTDLN dissection, patients were treated with VACCIMEL (n=22) or IFNα-2b (n=6), unless rapid progression (n=1). Distant Metastasis-Free Survival (DMFS) was selected as an end-point. Two cohorts of patients were selected: one with a good outcome (GO) (n=17; median DMFS 130.0 months), and another with a bad outcome (BO) (n=12; median DMFS 8.5 months). We analyzed by immunohistochemistry and immunofluorescence the expression of relevant biomarkers to tumor-cell biology and immune cells and structures in mTDLN, both in the tumor and peritumoral areas. Results: In BO patients, highly replicating Ki-67+ tumor cells, low tumor HLA-I expression and abundant FoxP3+ lymphocytes were found (p=0.037; p=0.056 and p=0.021). In GO patients, the most favorable biomarkers for prolonged DMFS were the abundance of peri- and intra-tumoral CD11c+ cells (p=0.0002 and p=0.001), peri-tumoral DC-LAMP+ dendritic cells (DCs) (p=0.001), and PNAd+ High Endothelial Venules (HEVs) (p=0.004). Most strikingly, we describe in GO patients a peculiar, heterogeneous structure that we named FAPS (Favoring Antigen-Presenting Structure), a triad composed of DC, HEV and CD62L+ naïve lymphocytes, whose postulated role would be to favor tumor antigen (Ag) priming of incoming naïve lymphocytes. We also found in GO patients a preferential tumor infiltration of CD8+ and CD20+ lymphocytes (p=0.004 and p=0.027), as well as peritumoral CD20+ aggregates, with no CD21+ follicular dendritic cells detected (p=0.023). Heterogeneous infiltration with CD64+CD68-CD163-, CD64+CD68+CD163- and CD64+CD68+CD163+ macrophages were observed in both cohorts. Discussion: The analysis of mTDLN in GO and BO patients revealed marked differences. This work highlights the importance of analyzing resected mTDLN from CM patients and suggests a correlation between tumor and immune characteristics that may be associated with a spontaneous or vaccine-induced long DMFS. These results should be confirmed in prospective studies.

Modulation of tumor immunity by therapeutic monoclonal antibodies.

The surveillance of tumors by the immune system of cancer patients and its impact on disease progression and patient survival have been largely documented over the last years. In parallel, the use of therapeutic monoclonal antibodies (mAbs) in oncology has gained a widespread recognition as it has made it possible to increase patient survival and to ameliorate the quality of life in a number of cancers. However, the clinical responses observed following mAb treatment remain largely heterogeneous and their duration is still highly unpredictable. Recently, the concept that the injection of therapeutic antibodies not only triggers early anti-tumor events such as receptor blockade, cytostasis, apoptosis, complement-dependent cytotoxicity and/or antibody-dependent cytotoxicity but also allows the host immune system to fight tumor cells through the development of a long-lasting adaptive immunity has emerged. In the present review, we will examine the arguments that support this concept by detailing the cellular and molecular events likely to underlie the induction of an efficient anti-tumor adaptive immune response by mAbs. We will also discuss the consequences of this induction on the way therapeutic antibodies can be used and inserted in a more global immunotherapeutic approach aiming at strengthening the adaptive anti-tumor immune response developed by cancer patients.

Long-lasting antitumor protection by anti-CD20 antibody through cellular immune response.

The anti-CD20 monoclonal antibody (mAb) rituximab has been used successfully for lymphoma therapy for more than 10 years. Although several direct mechanisms by which anti-CD20 mAbs act have been characterized in vitro, their specific role in clinical efficacy is still debated. Little is known about the possible antitumor immune response that they may induce in patients, despite clinical data suggesting a "vaccinal" effect. We show here that an initial treatment with anti-CD20 induces protection against human CD20-expressing tumor cells and allows immunocompetent mice to survive tumor challenge. This long-lasting protection requires the presence of the Fc portion of the anti-CD20 mAb and is achieved through the induction of a cellular immune response. Only CD4(+) cells were needed at the beginning of the treatment, but both CD4(+) and CD8(+) cells were required after tumor challenge to achieve protection. Finally, we show that interleukin-2 treatment, given after tumor challenge, improves the overall survival rate, compared with that obtained by anti-CD20 treatment alone. These findings demonstrate that anti-CD20 mAbs exert therapeutic effects through the induction of an adaptive cellular immune response, aside from any direct mechanisms involving effectors from innate immunity.

Intratumoral plasma cells: More than a predictive marker of response to anti-PD-L1 treatment in lung cancer?

In this issue of Cancer Cell, Patil et al. report that increased plasma cell signatures are predictive of an extended overall survival in non-small-cell lung cancer patients treated with a PD-L1 inhibitor and that these cells are associated with the presence of tertiary lymphoid structures.

BMFPs, a versatile therapeutic tool for redirecting a preexisting Epstein-Barr virus antibody response toward defined target cells.

Industrial production of therapeutic monoclonal antibodies is mostly performed in eukaryotic-based systems, allowing posttranslational modifications mandatory for their functional activity. The resulting elevated product cost limits therapy access to some patients. To address this limitation, we conceptualized a novel immunotherapeutic approach to redirect a preexisting polyclonal antibody response against Epstein-Barr virus (EBV) toward defined target cells. We engineered and expressed in bacteria bimodular fusion proteins (BMFPs) comprising an Fc-deficient binding moiety targeting an antigen expressed at the surface of a target cell, fused to the EBV-P18 antigen, which recruits circulating endogenous anti-P18 IgG in EBV+ individuals. Opsonization of BMFP-coated targets efficiently triggered antibody-mediated clearing effector mechanisms. When assessed in a P18-primed mouse tumor model, therapy performed with an anti-huCD20 BMFP significantly led to increased survival and total cancer remission in some animals. These results indicate that BMFPs could represent potent and useful therapeutic molecules to treat a number of diseases.

Defining biomarkers in oral cancer according to smoking and drinking status.

Introduction: Oral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and Methods: Cells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. Results: Normal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4+ T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4+ T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8+ T lymphocytes among CD45+ cells in NSND patients was associated with a better disease-free survival (p=0.004). Conclusion: Granulocytes, monocytes-macrophages, and CD4+ T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4+ T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8+ T cells in NSND patients could help defining the prognostic of OSCC patients.

Potent immunomodulatory and antitumor effect of anti-CD20-IL2no-alpha tri-functional immunocytokine for cancer therapy.

Introduction: The anti-CD20 antibody rituximab (RTX) has substantially improved outcomes of patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing lymphomas. An approach to increase the clinical effectiveness of anti-tumor therapy is the use of antibody-cytokine fusion proteins (immunocytokines (ICKs)) to deliver at the tumor site the antibody effector functions and cytokines that trigger anti-tumor activities. In particular, IL-2-based ICKs have shown significant results in preclinical studies but not in clinical trials due to the toxicity profile associated to high doses IL-2 and the undesired expansion of Tregs. Methods: To improve the efficacy of RTX therapy, we fused a murine (mIgG2a) or a human (hIgG1) version of RTX to a mutated IL-2 (no-alpha mutein), which has a disrupted affinity for the high affinity IL-2 receptor (IL-2R) to prevent the stimulation of Tregs and reduce the binding to endothelial cells expressing CD25, the α chain of high affinity IL-2R. Characterization of anti-CD20-IL2no-alpha ICKs was performed by SDS-PAGE, Western-blotting and SEC-HPLC and also by several functional in vitro techniques like T-cell proliferation assays, apoptosis, CDC and ADCC assays. The in vivo activity was assessed by using murine tumor cells expressing huCD20 in C57/Bl6 mice. Results: Both ICKs exhibited similar in vitro specific activity of their IL2no-alpha mutein moieties and kept CD20-binding capacity. Anti-CD20-IL2no-alpha (hIgG1) retained antibody effector functions as complement-dependent cytotoxicity and enhanced direct apoptosis, NK cell activation and antibody-dependent cellular cytotoxicity relative to RTX. In addition, both ICKs demonstrated a higher antitumor efficacy than parental molecules or their combination in an EL4-huCD20 tumor model in immunocompetent mice. Anti-CD20-IL2no-alpha (hIgG1) strongly expanded NK and CD8+ T cells but not Tregs in tumor-bearing mice. Discussion: These findings suggest that anti-CD20-IL2no-alpha could represent an alternative treatment for B cell lymphoma patients, mainly those refractory to RTX therapy.

[Bispecific antibodies: An old story with a bright future with CAR-T cells!] / Les anticorps bispécifiques : une vieille histoire pleine d'avenir avec les CAR-T !

CAR-T cells originate from two different approaches, cellular immunotherapy based on tumor immunosurveillance by T lymphocytes, combined with molecular engineering of bispecific antibodies and antibody fragments. The latter makes it possible to retarget immune effector cytotoxic cells (such as NK cells and T lymphocytes) to tumor cells through the binding to tumor-associated antigens. We present herein the history of bispecific antibodies, highlighting how such antibodies played a major role in CAR-T cell development. We will first evoke how antibody engineering led to the construction of various bispecific formats, in particular using the single chain Fv fragment (scFv) which has been used as the initial building block to generate chimeric bi-, tri- or multifunctional molecules. We will also describe how bispecific antibodies, either full IgG or as scFv or F(ab')2 format, directed against Fcγ receptors or CD3ɛ and against tumor-associated antigens, induce a potent anti-tumor cytotoxicity following the recruitment and activation of immune effector cells, including CD3+ T lymphocytes. These anti-tumor effects have been translated into the clinics, especially to treat malignant hemopathies. At last, recently generated bispecific CAR-T cells suggest that the embrace between cell therapy and bispecific antibodies is not over and that we are yet to witness further discoveries enabling these cells to be even more efficient.

Tumor-Associated Tertiary Lymphoid Structures: From Basic and Clinical Knowledge to Therapeutic Manipulation.

The tumor microenvironment is a complex ecosystem almost unique to each patient. Most of available therapies target tumor cells according to their molecular characteristics, angiogenesis or immune cells involved in tumor immune-surveillance. Unfortunately, only a limited number of patients benefit in the long-term of these treatments that are often associated with relapses, in spite of the remarkable progress obtained with the advent of immune checkpoint inhibitors (ICP). The presence of "hot" tumors is a determining parameter for selecting therapies targeting the patient immunity, even though some of them still do not respond to treatment. In human studies, an in-depth analysis of the organization and interactions of tumor-infiltrating immune cells has revealed the presence of an ectopic lymphoid organization termed tertiary lymphoid structures (TLS) in a large number of tumors. Their marked similarity to secondary lymphoid organs has suggested that TLS are an "anti-tumor school" and an "antibody factory" to fight malignant cells. They are effectively associated with long-term survival in most solid tumors, and their presence has been recently shown to predict response to ICP inhibitors. This review discusses the relationship between TLS and the molecular characteristics of tumors and the presence of oncogenic viruses, as well as their role when targeted therapies are used. Also, we present some aspects of TLS biology in non-tumor inflammatory diseases and discuss the putative common characteristics that they share with tumor-associated TLS. A detailed overview of the different pre-clinical models available to investigate TLS function and neogenesis is also presented. Finally, new approaches aimed at a better understanding of the role and function of TLS such as the use of spheroids and organoids and of artificial intelligence algorithms, are also discussed. In conclusion, increasing our knowledge on TLS will undoubtedly improve prognostic prediction and treatment selection in cancer patients with key consequences for the next generation immunotherapy.

Tertiary Lymphoid Structure-B Cells Narrow Regulatory T Cells Impact in Lung Cancer Patients.

The presence of tertiary lymphoid structures (TLS) in the tumor microenvironment is associated with better clinical outcome in many cancers. In non-small cell lung cancer (NSCLC), we have previously showed that a high density of B cells within TLS (TLS-B cells) is positively correlated with tumor antigen-specific antibody responses and increased intratumor CD4+ T cell clonality. Here, we investigated the relationship between the presence of TLS-B cells and CD4+ T cell profile in NSCLC patients. The expression of immune-related genes and proteins on B cells and CD4+ T cells was analyzed according to their relationship to TLS-B density in a prospective cohort of 56 NSCLC patients. We observed that tumor-infiltrating T cells showed marked differences according to TLS-B cell presence, with higher percentages of naïve, central-memory, and activated CD4+ T cells and lower percentages of both immune checkpoint (ICP)-expressing CD4+ T cells and regulatory T cells (Tregs) in the TLS-Bhigh tumors. A retrospective study of 538 untreated NSCLC patients showed that high TLS-B cell density was even able to counterbalance the deleterious impact of high Treg density on patient survival, and that TLS-Bhigh Treglow patients had the best clinical outcomes. Overall, the correlation between the density of TLS-Bhigh tumors with early differentiated, activated and non-regulatory CD4+ T cell cells suggest that B cells may play a central role in determining protective T cell responses in NSCLC patients.

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.