Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming

Natural Killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human Cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C+Fc∝RI≥- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterised yet. Here we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells, and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansion. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting, and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1–4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection

Characterization of human memory NK cells ex vivo and study of therapeutic monoclonal antibody capability to promote their expansion in vitro

Le cellule Natural Killer (NK) “memory” mostrano caratteristiche distintive (longevita’ e iperresponsivita’ alla stimolazione attraverso il CD16) che le rendono potenzialmente interessanti nell’ambito dell’immunosorveglianza contro i tumori. Abbiamo studiato alcuni dei meccanismi che permettono l’espansione e il mantenimento delle cellule NK memory in vivo. Abbiamo ottimizzato un sistema in cui la stimolazione con una linea tumorale B opsonizzata con anticorpi terapeutici anti-CD20 induce l’espansione selettiva delle memory NK in vitro. Questo sistema ci ha permesso di analizzare le loro caratteristiche fenotipiche e funzionali, di confrontarle con la controparte ex vivo e di esplorare alcuni dei requisiti molecolari alla base della loro proliferazione. La conoscenza delle capacità funzionali e della dinamica delle NK memory in vivo ed in vitro potrà essere rilevante nella progettazione di schemi immunoterapeutici innovativi, tesi al potenziamento delle loro funzioni anti-tumorali

Regulation of DNAM-1 family receptors and their ligands in T lymphocytes and intestinal epithelial cells

Purpose: DNAM-1 family co-receptors are expressed on T lymphocyte subsets and provide activating (DNAM-1) or inhibitory (TIGIT) signals that regulate T cell functions and proliferation. We previously found that the expression pattern of these co-receptors strikingly differs between circulating and mucosal T cell populations. Moreover, perturbed expression of DNAM-1/ligand system distinctly characterizes infiltrate and epithelial counterpart in the inflamed mucosa microenvironment of active Inflammatory Bowel Disease (IBD) pediatric patients. Here we analyzed the capability of polyclonal TCR-dependent stimulation or selected cytokines to modulate the expression of DNAM-1 family co-receptors and shared ligands (PVR and Nectin-2) on peripheral blood (PB) T cell subsets and HT-29 colon carcinoma-derived cell line. Results: IL-2 family cytokines or TCR/CD28 stimulation increases the frequency of TIGIT+ T cells, suggesting that such stimuli may partially explain the higher frequency of TIGIT+ mucosal T cells, as compared to PB counterpart. Differently, DNAM-1 levels are increased by TGF-b, and decreased by IL-17A. The dysregulated abundance of these two cytokines in inflamed mucosa microenvironment could underlie the downregulated DNAM-1 expression on mucosal T cells from active IBD patients. Moreover, Nectin-2 expression on HT-29 cells was decreased by TGF-b and IL-10 anti-inflammatory cytokines, suggesting that the reduced amount of these factors may lead to the increased frequency of Nectin-2+ gut epithelial cells recorded in active IBD lesions. Conclusion: Our data suggest that mucosal microenvironment factors shape the physiological expression pattern of DNAM-1 family co-receptor/ligand system and contribute to its alteration in IBD

Shigella modulation of polyamines during the invasion of host cells

Polyamines are small molecules found in all cells and associated with a wide variety of physiological processes. In bacterial pathogens the modulation of polyamine content could represent a strategy to optimize bacterial fitness within the host. Shigella, the etiological agent of bacillary dysentery, in contrast to its innocuous ancestor Escherichia coli, has a polyamine profile characterized by high level of intracellular spermidine. To understand the role of polyamines in the Shigella-host interaction, we analysed the expression of host cell genes involved in the biogenesis and back-conversion of polyamines during Shigella infection. Preliminary data indicate that the acetyl polyamine oxidase (APAO) is induced at early stages of Shigella infection, while induction of the spermine/spermidine acetyltransferase and of the spermine oxidase is detected later during the infection. These observations lead us to speculate that the early release of H2O2 as secondary product of APAO activity might represent a signal for Shigella to activate the expression of mdtJI efflux pump operon in order to export bacterial polyamines which have a scavenger function during oxidative stress conditions

The interplay between anti-CD20 therapeutic antibodies and "memory" Natural Killer cells

Purpose: Our study focuses on the recently described long-lived and highly functional NK cell populations (dubbed memory NK cells), defined by the lack of expression of CD16-associatedFceRIg chain and the ability to produce high amounts of IFNg upon CD16 re-stimulation (1). Particularly relevant are our recent observations demonstrating that the sustained stimulation of NK cells with obinutuzumab (anti-CD20 mAb)-opsonised tumor cells leads to the selective down-regulation of FceRIg chain, along with the priming for enhanced IFNg production (2). Here we want to study the capability of anti-CD20 mAbs to support memory NK cell expansion. Methods: CD56+CD16+CD3-g- (memory) and CD56+CD16+CD3-g+(conventional) NK cells from healthy donors were quantified ex vivo and after 10 day co-culture with anti-CD20 mAb-opsonised CD20+ Raji cells in the presence of IL-2. Two different antiCD20 mAbs, currently employed in the treatment of B cell malignancies were chosen: first generation, reference molecule, rituximab, and next generation, Fc-engineered, obinutuzumab, which shows increased binding affinity to CD16. Results: Almost 55% of healthy donors exhibit a population of memory NK cells, accounting for 5%-70% of total peripheral blood NK cells. We observed that CD56+CD16+CD3- g- (memory) NK cells selectively undergo 2- to 12-fold expansion, upon co-culture with anti-CD20 opsonised targets, with no major differences between different anti-CD20 mAbs; on the opposite, CD56+CD16+CD3-g+ (conventional) NK cell proliferation is not affected by CD16 stimulation. The phenotypic and functional characterization of anti-CD20 mAb-expanded memory NK cells is under investigation. Conclusions: Our data highlight a new aspect of the interplay between therapeutic mAbs and NK cell plasticity, suggesting a potential tool for the clinical exploitment of NK cell effector functions

Peripheral blood T cell alterations in newly diagnosed diffuse large B cell lymphoma patients and their long-term dynamics upon rituximab-based chemoimmunotherapy

The importance of T cell-dependent immune responses in achieving long-term cure of chemoimmunotherapy-treated cancer patients is underscored by the recently described "vaccinal effect" exerted by therapeutic mAbs. In accordance, pre- and post-therapy peripheral blood lymphopenia represents a well-established negative prognostic factor in DLBCL. We analyzed the phenotypic and functional (IFNγ production, and Granzyme B (GrzB) cytotoxic granule marker expression) profile of peripheral blood T lymphocyte subsets ("conventional" CD4(+) and CD8(+), FOXP3(+)CD25(bright) Treg, and "innate-like" CD56(+)) in DLBCL patients at diagnosis, and assessed the long-term impact of R-CHOP chemoimmunotherapy, in a prospective study. At diagnosis, DLBCL patients showed lower lymphocyte counts, due to selective decrement of CD4(+) T (including Treg) and B lymphocytes. While all T cell subsets transiently decreased during therapy, CD4(+) T cell and Treg remained significantly lower than controls, up to 1 year after R-CHOP. Phenotypically skewed profile of CD4(+) and CD8(+) T cell subsets associated with higher frequencies of IFNγ(+) and GrzB(+) cells at diagnosis, that transiently decreased during therapy, and re-attained persistently elevated levels, till up to 1 year after therapy. Differently, the pre-therapy elevated levels of circulating monocytes, and of plasma IL-6 and IL-10 rapidly normalized upon R-CHOP. In sum, we describe a quantitatively and functionally altered status of the peripheral blood T cell compartment in DLBCL patients at diagnosis, that persists long-term after tumor eradication, and it is only transiently perturbed by R-CHOP chemoimmunotherapy. Moreover, data suggest the association of selected T cell functional features with DLBCL phenotype, and with therapy outcome

image_1_Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.PDF

<p>Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C⁺FcεRIγ^- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor’ HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.</p

The role of CD101-expressing CD4 T cells in HIV/SIV pathogenesis and persistence.

Despite the advent of effective antiretroviral therapy (ART), human immunodeficiency virus (HIV) continues to pose major challenges, with extensive pathogenesis during acute and chronic infection prior to ART initiation and continued persistence in a reservoir of infected CD4 T cells during long-term ART. CD101 has recently been characterized to play an important role in CD4 Treg potency. Using the simian immunodeficiency virus (SIV) model of HIV infection in rhesus macaques, we characterized the role and kinetics of CD101+ CD4 T cells in longitudinal SIV infection. Phenotypic analyses and single-cell RNAseq profiling revealed that CD101 marked CD4 Tregs with high immunosuppressive potential, distinct from CD101- Tregs, and these cells also were ideal target cells for HIV/SIV infection, with higher expression of CCR5 and α4β7 in the gut mucosa. Notably, during acute SIV infection, CD101+ CD4 T cells were preferentially depleted across all CD4 subsets when compared with their CD101- counterpart, with a pronounced reduction within the Treg compartment, as well as significant depletion in mucosal tissue. Depletion of CD101+ CD4 was associated with increased viral burden in plasma and gut and elevated levels of inflammatory cytokines. While restored during long-term ART, the reconstituted CD101+ CD4 T cells display a phenotypic profile with high expression of inhibitory receptors (including PD-1 and CTLA-4), immunsuppressive cytokine production, and high levels of Ki-67, consistent with potential for homeostatic proliferation. Both the depletion of CD101+ cells and phenotypic profile of these cells found in the SIV model were confirmed in people with HIV on ART. Overall, these data suggest an important role for CD101-expressing CD4 T cells at all stages of HIV/SIV infection and a potential rationale for targeting CD101 to limit HIV pathogenesis and persistence, particularly at mucosal sites