Tertiary lymphoid structures in anticancer immunity.

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates where adaptive antitumour cellular and humoral responses can be elaborated. Initially described in non-small cell lung cancer as functional immune lymphoid structures associated with better clinical outcome, TLS have also been found in many other carcinomas, as well as melanomas and sarcomas, and associated with improved response to immunotherapy. The manipulation of TLS as a therapeutic strategy is now coming of age owing to the likely role of TLS in the improved survival of patients with cancer receiving immune checkpoint inhibitor treatment. TLS have also garnered considerable interest as a predictive biomarker of the response to antitumour therapies, including immune checkpoint blockade and, possibly, chemotherapy. However, several important questions still remain regarding the definition of TLS in terms of both their cellular composition and functions. Here, we summarize the current views on the composition of TLS at different stages of their development. We also discuss the role of B cells and T cells associated with TLS and their dialogue in mounting antibody and cellular antitumour responses, as well as some of the various mechanisms that negatively regulate antitumour activity of TLS. The prognostic value of TLS to the clinical outcome of patients with cancer and the relationship between TLS and the response to therapy are then addressed. Finally, we present some preclinical evidence that favours the idea that manipulating the formation and function of TLS could lead to a potent next-generation cancer immunotherapy.

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.

[The Nobel Prize in Physiology or Medicine 2023: Katalin Karikó and Drew Weissman - A vaccine revolution driven by fundamental research in immunology and molecular biology]. / Prix Nobel de physiologie ou médecine 2023 : Katalin Karikó et Drew Weissman - Une révolution vaccinale portée par la recherche fondamentale en immunologie et en biologie moléculaire.

Title: Prix Nobel de physiologie ou médecine 2023 : Katalin Karikó et Drew Weissman - Une révolution vaccinale portée par la recherche fondamentale en immunologie et en biologie moléculaire. Abstract: Le 2 octobre 2023, le prix Nobel de physiologie ou médecine a été décerné à Katalin Karikó et Drew Weissman, tous deux professeurs à l'université de Pennsylvanie, pour leur « découverte concernant les modifications des nucléosides qui ont permis le développement de vaccins ARN efficaces contre le COVID-19 ¼. Le communiqué du comité Nobel indique que « grâce à leurs découvertes exceptionnelles qui ont changé radicalement notre compréhension des mécanismes par lesquels l'ARN messager interagit avec notre système immunitaire, ces deux lauréats ont contribué au développement, avec une rapidité sans précédent, d'un vaccin contre l'une des plus grandes menaces des temps modernes affectant la santé humaine ¼.

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.

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.

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.

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.

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.

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.

[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.

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.

A rationally-engineered IL-2 improves the antitumor effect of anti-CD20 therapy.

Anti-CD20 treatment represents a therapeutic benefit for patients with B-cell lymphomas, although more efficient therapies are needed for refractory or relapsing patients. Among them, the combination of anti-CD20 and IL-2 that induces T cell response has been hampered by the expansion of FoxP3+ Tregs that strongly express the high affinity IL-2 receptor (IL-2R αßγ). We explore here the anti-tumor effect of an anti-CD20 antibody combined with a mutated IL-2 (no-alpha mutein) which has a disrupted affinity for the IL-2R αßγ. We demonstrate that anti-CD20/no-alpha mutein combination significantly augments the survival rate of mice challenged with huCD20+ cells as compared to animals treated with anti-CD20 ± IL-2. Moreover, the combination with no-alpha mutein but not IL-2 provokes an increase of granzyme B and perforin in splenic NK and CD8+ T cells, a reduction of Tregs and an increase in activated macrophages. The former combination also induces a T helper profile different from that obtained with IL-2, with an earlier polarization to Th1 and no increase in Th17. The therapeutic effect of anti-CD20/no-alpha mutein was accompanied by an expansion of peripheral central (TCM) and effector (TEM) memory CD8+ T cell compartments. Last, as opposed to IL-2, no-alpha mutein administered at the beginning of anti-CD20 treatment did not dampen the long-term protection of surviving mice after tumor rechallenge. Thus, this study shows that the combination of anti-tumor antibodies and no-alpha mutein is a promising approach to improve the therapeutic effect of these antibodies by potentiating NK/macrophage-mediated innate immunity and the adaptive T-cell response.