Expanded Human Blood-Derived γδT Cells Display Potent Antigen-Presentation Functions

Cell-based immunotherapy strategies target tumors directly (via cytolytic effector cells) or aim at mobilizing endogenous anti-tumor immunity. The latter approach includes dendritic cells (DC) most frequently in the form of in vitro cultured peripheral blood monocytes-derived DC. Human blood γδT cells are selective for a single class of non-peptide agonists (“phosphoantigens”) and develop into potent antigen-presenting cells (APC), termed γδT-APC within 1–3 days of in vitro culture. Availability of large numbers of γδT-APC would be advantageous for use as a novel cellular vaccine. We here report optimal γδT cell expansion (>10(7) cells/ml blood) when peripheral blood mononuclear cells (PBMC) from healthy individuals and melanoma patients were stimulated with zoledronate and then cultured for 14 days in the presence of IL-2 and IL-15, yielding γδT cell cultures of variable purity (77 ± 21 and 56 ± 26%, respectively). They resembled effector memory αβT (T(EM)) cells and retained full functionality as assessed by in vitro tumor cell killing as well as secretion of pro-inflammatory cytokines (IFNγ, TNFα) and cell proliferation in response to stimulation with phosphoantigens. Importantly, day 14 γδT cells expressed numerous APC-related cell surface markers and, in agreement, displayed potent in vitro APC functions. Day 14 γδT cells from PBMC of patients with cancer were equally effective as their counterparts derived from blood of healthy individuals and triggered potent CD8(+) αβT cell responses following processing and cross-presentation of simple (influenza M1) and complex (tuberculin purified protein derivative) protein antigens. Of note, and in clear contrast to peripheral blood γδT cells, the ability of day 14 γδT cells to trigger antigen-specific αβT cell responses did not depend on re-stimulation. We conclude that day 14 γδT cell cultures provide a convenient source of autologous APC for use in immunotherapy of patients with various cancers

Different features of tumor-associated NK cells in patients with low-grade or high-grade peritoneal carcinomatosis

Peritoneal carcinomatosis (PC) is a rare disease defined as diffused implantation of neoplastic cells in the peritoneal cavity. This clinical picture occurs during the evolution of peritoneal tumors, and it is the main cause of morbidity and mortality of patients affected by these pathologies, though cytoreductive surgery with heated intra-peritoneal chemotherapy (CRS/HIPEC) is yielding promising results. In the present study, we evaluated whether the tumor microenvironment of low-grade and high-grade PC could affect the phenotypic and functional features and thus the anti-tumor potential of NK cells. We show that while in the peritoneal fluid (PF) of low-grade PC most CD56dim NK cells show a relatively immature phenotype (NKG2A+KIR\u2013CD57\u2013CD16dim), in the PF of high-grade PC NK cells are, in large majority, mature (CD56dimKIR+CD57+CD16bright). Furthermore, in low-grade PC, PF-NK cells are characterized by a sharp down-regulation of some activating receptors, primarily NKp30 and DNAM-1, while, in high-grade PC, PF-NK cells display a higher expression of the PD-1 inhibitory checkpoint. The compromised phenotype observed in low-grade PC patients corresponds to a functional impairment. On the other hand, in the high-grade PC patients PF-NK cells show much more important defects that only partially reflect the compromised phenotype detected. These data suggest that the PC microenvironment may contribute to tumor escape from immune surveillance by inducing different NK cell impaired features leading to altered anti-tumor activity. Notably, after CRS/HIPEC treatment, the altered NK cell phenotype of a patient with a low-grade disease and favorable prognosis was reverted to a normal one. Our present data offer a clue for the development of new immunotherapeutic strategies capable of restoring the NK-mediated anti-tumor responses in association with the CRS/HIPEC treatment to increase the effectiveness of the current therapy

Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cell

The T cell compartment is phenotypically and functionally heterogeneous; subsets of naive and memory cells have different functional properties, and also differ with respect to homeostatic potential and the ability to persist in vivo. Human stem cell memory T (TSCM) cells, which possess superior immune reconstitution and antitumor response capabilities, can be identified by polychromatic flow cytometry on the basis of the simultaneous expression of several naive markers together with the memory marker CD95. We describe here a protocol based on the minimum set of markers required for optimal identification of human and nonhuman primate (NHP) TSCM cells with commonly available flow cytometers. By using flow sorters, TSCM cells can thereby be isolated efficiently at high yield and purity. With the use of the 5.5-h isolation procedure, depending on the number of cells needed, the sorting procedure can last for 2-15 h. We also indicate multiple strategies for their efficient expansion in vitro at consistent numbers for functional characterization or adoptive transfer experiments

Dendritic Cells/Natural Killer Cross-Talk: A Novel Target for Human Immunodeficiency Virus Type-1 Protease Inhibitors

BACKGROUND: HIV-1 Protease Inhibitors, namely PIs, originally designed to inhibit HIV-1 aspartic protease, can modulate the immune response by mechanisms largely unknown, and independent from their activity on viral replication. Here, we analyzed the ability of PIs to interfere with differentiation program of monocytes toward dendritic cell (DCs) lineage, a key process in the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors were isolated and induced to differentiate in vitro in the presence or absence of saquinavir, ritonavir, nelfinavir, indinavir or amprenavir (sqv, rtv, nlfv, idv, apv, respectively). These drugs demonstrated a differential ability to sustain the generation of immature DCs (iDCs) with an altered phenotype, including low levels of CD1a, CD86, CD36 and CD209. DCs generated in the presence of rtv also failed to acquire the typical phenotype of mature DCs (mDCs), and secreted lower amounts of IL-12 and IL-15. Accordingly, these aberrant mDCs failed to support activation of autologous Natural Killer (NK) cells, and resulted highly susceptible to NK cell-mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: Our findings uncover novel functional properties of PIs within the DC-NK cell cross-talk, unveiling the heterogeneous ability of members of this class drugs to drive the generation of atypical monocyte-derived DCs (MDDCs) showing an aberrant phenotype, a failure to respond appropriately to bacterial endotoxin, a weak ability to prime autologous NK cells, and a high susceptibility to NK cell killing. These unexpected properties might contribute to limit inflammation and viral spreading in HIV-1 infected patients under PIs treatment, and open novel therapeutical perspectives for this class drugs as immunomodulators in autoimmunity and cancer

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24 pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-Ineg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56neg/CD16pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24pos blasts derived from primary T cells

Identification of Tissue-Resident Natural Killer and T Lymphocytes with Anti-Tumor Properties in Ascites of Ovarian Cancer Patients

Women with ovarian cancer have limited therapy options, with immunotherapy being unsatisfactory for a large group of patients. Tumor cells spread from the ovary or the fallopian tube into the abdominal cavity, which is commonly accompanied with massive ascites production. The ascites represents a unique peritoneal liquid tumor microenvironment with the presence of both tumor and immune cells, including cytotoxic lymphocytes. We characterized lymphocytes in ascites from patients with high-grade serous ovarian cancer. Our data reveal the presence of NK and CD8+ T lymphocytes expressing CD103 and CD49a, which are markers of tissue residency. Moreover, these cells express high levels of the inhibitory NKG2A receptor, with the highest expression level detected on tissue-resident NK cells. Lymphocytes with these features were also present at the primary tumor site. Functional assays showed that tissue-resident NK cells in ascites are highly responsive towards ovarian tumor cells. Similar results were observed in an in vivo mouse model, in which tissue-resident NK and CD8+ T cells were detected in the peritoneal fluid upon tumor growth. Together, our data reveal the presence of highly functional lymphocyte populations that may be targeted to improve immunotherapy for patients with ovarian cancer.This research was funded by a Research Award from the Wellbeing of Women (RG2331; F.C., E.B., J.B.), a Wellcome Investigator Award (094073/Z/10/Z; F.C.), the Cambridge NIHR BRC Cell Phenotyping Hub (F.C.), the Swedish cancer foundation society (20 0226 P 01 H; E.B. and CAN 2018/384; K.S.), the Swedish research council (2019-06328; E.B.), the Swedish Society for Medical Research (E.B.), the Swedish Society for Medicine (SLS-881711; E.B.), the Assar Gabrielsson founda- tion (BRG20-06; E.B.), Swedish state under the agreement between the Swedish government and the county council, the ALF agreement (ALFGBG-965552; K.S.), Italian Ministry of Health (Bando Ri- cerca Finalizzata PE-2016-02363915, D.M.), Associazione Italiana per la Ricerca sul Cancro (IG 14687; D.M.), Intramural research and clinical funding programs of Humanitas Research Hospital (5 X 1000; D.M.) and University of Milan (D.M.). O.H. was supported by a AstraZeneca-MedImmune- Cambridge PhD fellowship. V.C. is a recipient of a competitive fellowship awarded from the Ph.D. program of Experimental Medicine at the University of Milan

Identification of Tissue-Resident Natural Killer and T Lymphocytes with Anti-Tumor Properties in Ascites of Ovarian Cancer Patients.

Peer reviewed: TrueFunder: Cambridge NIHR BRC Cell Phenotyping HubFunder: Swedish Society for Medical ResearchFunder: University of MilanFunder: AstraZeneca-MedImmune-Cambridge PhD fellowshipFunder: Experimental Medicine at the University of MilanFunder: Cancer Research UKFunder: Cancer Research UK Cambridge CentreFunder: Mark Foundation Institute for Integrated Cancer MedicineWomen with ovarian cancer have limited therapy options, with immunotherapy being unsatisfactory for a large group of patients. Tumor cells spread from the ovary or the fallopian tube into the abdominal cavity, which is commonly accompanied with massive ascites production. The ascites represents a unique peritoneal liquid tumor microenvironment with the presence of both tumor and immune cells, including cytotoxic lymphocytes. We characterized lymphocytes in ascites from patients with high-grade serous ovarian cancer. Our data reveal the presence of NK and CD8+ T lymphocytes expressing CD103 and CD49a, which are markers of tissue residency. Moreover, these cells express high levels of the inhibitory NKG2A receptor, with the highest expression level detected on tissue-resident NK cells. Lymphocytes with these features were also present at the primary tumor site. Functional assays showed that tissue-resident NK cells in ascites are highly responsive towards ovarian tumor cells. Similar results were observed in an in vivo mouse model, in which tissue-resident NK and CD8+ T cells were detected in the peritoneal fluid upon tumor growth. Together, our data reveal the presence of highly functional lymphocyte populations that may be targeted to improve immunotherapy for patients with ovarian cancer.This research was funded by a Research Award from the Wellbeing of Women (RG2331; F.C., E.B., J.B.), a Wellcome Investigator Award (094073/Z/10/Z; F.C.), the Cambridge NIHR BRC Cell Phenotyping Hub (F.C.), the Swedish cancer foundation society (20 0226 P 01 H; E.B. and CAN 2018/384; K.S.), the Swedish research council (2019-06328; E.B.), the Swedish Society for Medical Research (E.B.), the Swedish Society for Medicine (SLS-881711; E.B.), the Assar Gabrielsson founda- tion (BRG20-06; E.B.), Swedish state under the agreement between the Swedish government and the county council, the ALF agreement (ALFGBG-965552; K.S.), Italian Ministry of Health (Bando Ri- cerca Finalizzata PE-2016-02363915, D.M.), Associazione Italiana per la Ricerca sul Cancro (IG 14687; D.M.), Intramural research and clinical funding programs of Humanitas Research Hospital (5 X 1000; D.M.) and University of Milan (D.M.). O.H. was supported by a AstraZeneca-MedImmune- Cambridge PhD fellowship. V.C. is a recipient of a competitive fellowship awarded from the Ph.D. program of Experimental Medicine at the University of Milan

Immune Evasion and Recognition of the Syphilis Spirochete in Blood and Skin of Secondary Syphilis Patients: Two Immunologically Distinct Compartments

<div><h3>Background</h3><p>The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of <em>Treponema pallidum</em> (<em>Tp</em>) to escape immune recognition while simultaneously inducing inflammation.</p> <h3>Methods</h3><p>To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. <em>Ex vivo</em> opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions <em>in vivo</em>.</p> <h3>Results</h3><p>Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56−/CD16+ NK-cell subset in blood. Skin lesions, which had visible <em>Tp</em> by IHC and substantial amounts of <em>Tp</em>-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-β and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of <em>Tp</em> in conjunction with monocyte activation, most spirochetes were not internalized.</p> <h3>Conclusions</h3><p>Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of <em>Tp</em> subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.</p> </div