Vitamin C, From Supplement to Treatment: A Re-Emerging Adjunct for Cancer Immunotherapy?

Vitamin C (VitC), in addition to its role as a general antioxidant, has long been considered to possess direct anti-cancer activity at high doses. VitC acts through oxidant and epigenetic mechanisms, which at high doses can exert direct killing of tumor cells in vitro and delay tumor growth in vivo. Recently, it has also been shown that pharmacologic-dose VitC can contribute to control of tumors by modulating the immune system, and studies have been done interrogating the role of physiologic-dose VitC on novel adoptive cellular therapies (ACTs). In this review, we discuss the effects of VitC on anti-tumor immune cells, as well as the mechanisms underlying those effects. We address important unanswered questions concerning both VitC and ACTs, and outline challenges and opportunities facing the use of VitC in the clinical setting as an adjunct to immune-based anti-cancer therapies

Systemic immunity shapes the oral microbiome and susceptibility to bisphosphonate‑associated osteonecrosis of the jaw

Background Osteonecrosis of the jaw (ONJ) is a rare but serious adverse drug effect linked to long-term and/or high-dose exposure to nitrogen-bisphosphonates (N-BP), the standard of care for the treatment of bone fragility disorders. The mechanism leading to bisphosphonate-associated ONJ (BAONJ) is unclear and optimal treatment strategies are lacking. Recent evidence suggests that BAONJ may be linked to drug-induced immune dysfunction, possibly associated with increased susceptibility to infections in the oral cavity. The objective of this investigation was to comprehensively assess the relationship linking immune function, N-BP exposure, the oral microbiome and ONJ susceptibility. Methods Leukocyte gene expression of factors important for immunity, wound healing and barrier function were assessed by real-time quantitative PCR and the oral microbiome was characterized by 454 pyrosequencing of the 16S rRNA gene in 93 subjects stratified by N-BP exposure and a history of ONJ. Results There were marked differences in the systemic expression of genes regulating immune and barrier functions including RANK (p = 0.007), aryl hydrocarbon receptor (AHR, p < 0.001), and FGF9 (p < 0.001), which were collectively up-regulated in individuals exposed to N-BP without ONJ relative to treatment controls. In contrast, the expression levels of these same genes were significantly down-regulated in those who had experienced BAONJ. Surprisingly, the oral microbiome composition was not directly linked to either BAONJ or N-BP exposure, rather the systemic leukocyte expression levels of RANK, TNFA and AHR each explained 9% (p = 0.04), 12% (p = 0.01), and 7% (p = 0.03) of the oral bacterial beta diversity. Conclusions The oral microbiome is unlikely causative of ONJ, rather individuals with BAONJ lacked immune resiliency which impaired their capacity to respond adequately to the immunological stress of N-BP treatment. This may be the common factor linking N-BP and anti-RANK agents to ONJ in at-risk individuals. Preventive and/or therapeutic strategies should target the wound healing deficits present in those with ONJ

Pitfalls in the characterization of circulating and tissue-resident human γδ T cells

Dissection of the role and function of human γδ T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire γδ T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human γδ T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles

Inhibition of Proliferation and Induction of Apoptosis in Multiple Myeloma Cell Lines by CD137 Ligand Signaling

BACKGROUND: Multiple myeloma (MM) is a malignancy of terminally-differentiated plasma cells, and the second most prevalent blood cancer. At present there is no cure for MM, and the average prognosis is only three to five years. Current treatments such as chemotherapy are able to prolong a patient's life but rarely prevent relapse of the disease. Even hematopoietic stem cell transplants and novel drug combinations are often not curative, underscoring the need for a continued search for novel therapeutics. CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively. Since CD137 ligand cross-linking enhances proliferation and survival of healthy B cells we hypothesized that it would also act as a growth stimulus for B cell cancers. METHODOLOGY/PRINCIPAL FINDINGS: Proliferation and survival of B cell lymphoma cell lines were not affected or slightly enhanced by CD137 ligand agonists in vitro. But surprisingly, they had the opposite effects on MM cells, where CD137 ligand signals inhibited proliferation and induced cell death by apoptosis. Furthermore, secretion of the pro-inflammatory cytokines, IL-6 and IL-8 were also enhanced in MM but not in non-MM cell lines in response to CD137 ligand agonists. The secretion of these cytokines in response to CD137 ligand signaling was consistent with the observed activation of the classical NF-kappaB pathway. We hypothesize that the induction of this pathway results in activation-induced cell death, and that this is the underlying mechanism of CD137-induced MM cell death and growth arrest. CONCLUSIONS/SIGNIFICANCE: These data point to a hitherto unrecognized role of CD137 and CD137 ligand in MM cell biology. The selective inhibition of proliferation and induction of cell death in MM cells by CD137 ligand agonists may also warrant a closer evaluation of their therapeutic potential

Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ T-Cells

BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials

γδ T lymphocytes from cystic fibrosis patients and healthy donors are high TNF-α and IFN-γ-producers in response to Pseudomonas aeruginosa

BACKGROUND: γδ T cells have an important immunoregulatory and effector function through cytokine release. They are involved in the responses to Gram-negative bacterium and in protection of lung epithelium integrity. On the other hand, they have been implicated in airway inflammation. METHODS: The aim of the present work was to study intracytoplasmic IL-2, IL-4, IFN-γ and TNF-α production by γδ and αβ T lymphocytes from cystic fibrosis patients and healthy donors in response to Pseudomonas aeruginosa (PA). Flow cytometric detection was performed after peripheral blood mononuclear cells (PBMC) culture with a cytosolic extract from PA and restimulation with phorbol ester plus ionomycine. Proliferative responses, activation markers and receptor usage of γδ T cells were also evaluated. RESULTS: The highest production of cytokine was of TNF-α and IFN-γ, γδ being better producers than αβ. No differences were found between patients and controls. The Vγ9δ2 subset of γδ T cells was preferentially expanded. CD25 and CD45RO expression by the αβ T subset and PBMC proliferative response to PA were defective in cystic fibrosis lymphocytes. CONCLUSION: Our results support the hypothesis that γδ T lymphocytes play an important role in the immune response to PA and in the chronic inflammatory lung reaction in cystic fibrosis patients. They do not confirm the involvement of a supressed Th1 cytokine response in the pathogenesis of this disease

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular “exhaustion” described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages

CCL25-CCR9 interaction modulates ovarian cancer cell migration, metalloproteinase expression, and invasion

<p>Abstract</p> <p>Background</p> <p>Ovarian carcinoma (OvCa) is the most lethal gynecological malignancy among women and its poor prognosis is mainly due to metastasis. Chemokine receptor CCR9 is primarily expressed by a small subset of immune cells and its only natural ligand, CCL25, is largely expressed in the thymus, which involutes with age. Other than the thymus, CCL25 is expressed by the small bowel. Interactions between CCL25 and CCR9 have been implicated in leukocyte trafficking to the small bowel, a frequent metastatic site for OvCa cells. The current study shows OvCa tissue and cells significantly express CCR9, which interacts with CCL25 to support carcinoma cell migration and invasion.</p> <p>Methods</p> <p>RT-PCR and flow cytometry techniques were used to quantify the expression CCR9 by OvCa cells. OvCa tissue microarrays (TMA) was used to confirm CCR9 expression in clinical samples. The Aperio ScanScope scanning system was used to quantify immunohistochemical staining. Cell invasion and migration assays were performed using cell migration and matrigel invasion chambers. Matrix metalloproteinase (MMP) mRNAs were quantified by RT-PCR and active MMPs were quantified by ELISA.</p> <p>Results</p> <p>Our results show significantly (<it>p </it>< 0.001) higher expression of CCR9 by mucinous adenocarcinoma, papillary serous carcinoma, and endometriod ovarian carcinoma cases, than compared to non-neoplastic ovarian tissue. Furthermore, CCR9 expression was significantly elevated in OvCa cell lines (OVCAR-3 and CAOV-3) in comparison to normal adult ovarian epithelial cell mRNA. OvCa cells showed higher migratory and invasive potential towards chemotactic gradients of CCL25, which was inhibited by anti-CCR9 antibodies. Expression of collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -9), and stromelysins (MMP-3, -10, and -11) by OvCa cells were modulated by CCL25 in a CCR9-dependent fashion.</p> <p>Conclusions</p> <p>These results demonstrate both biological significance and clinical relevance of CCL25 and CCR9 interactions in OvCa cell metastasis.</p

Genotype-Dependent Tumor Regression in Marek’s Disease Mediated at the Level of Tumor Immunity

Marek’s disease (MD) of chickens is a unique natural model of Hodgkin’s and Non Hodgkin’s lymphomas in which the neoplastically-transformed cells over-express CD30 (CD30hi) antigen. All chicken genotypes can be infected with MD virus and develop microscopic lymphomas. From 21 days post infection (dpi) microscopic lymphomas regress in resistant chickens but, in contrast, they progress to gross lymphomas in susceptible chickens. Here we test our hypothesis that in resistant chickens at 21 dpi the tissue microenvironment is pro T-helper (Th)-1 and compatible with cytotoxic T lymphocyte (CTL) immunity but in susceptible lines it is pro Th-2 or pro T-regulatory (T-reg) and antagonistic to CTL immunity. We used the B2, non-MHC-associated, MD resistance/susceptibility system (line [L]61/line [L]72) and quantified the levels of key mRNAs that can be used to define Th-1 (IL-2, IL-12, IL-18, IFNγ), Th-2 (IL-4, IL-10) and T-reg (TGFβ, GPR-83, CTLA-4, SMAD-7) lymphocyte phenotypes. We measured gene expression in both whole tissues (represents tissue microenvironment and tumor microenvironment) and in the lymphoma lesions (tumor microenvironment) themselves. Gene ontology-based modeling of our results shows that the dominant phenotype in whole tissue as well as in microscopic lymphoma lesions, is pro T-reg in both L61 and L72 but a minor pro Th-1 and anti Th-2 tissue microenvironment exists in L61 whereas there is an anti Th-1 and pro Th-2 tissue microenvironment in L72. The tumor microenvironment per se is pro T-reg, anti Th-1 and pro Th-2 in both L61 and L72. Together our data suggests that the neoplastic transformation is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity in the tissues