Generation of highly activated, antigen-specific tumor-infiltrating CD8+ T cells induced by a novel T cell-targeted immunotherapy.

The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8+ T cells to the tumor. Herein, we analyze the unique phenotype of the CD8+ T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8+ T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8+ T cells (PD-1+TIM-3+CTLA-4+) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8+ T cells (PD-1+TIM-3+CTLA-4-) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumor-recruited CD8+ T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8+ T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.

Mast Cell Responses to Viruses and Pathogen Products.

Mast cells are well accepted as important sentinel cells for host defence against selected pathogens. Their location at mucosal surfaces and ability to mobilize multiple aspects of early immune responses makes them critical contributors to effective immunity in several experimental settings. However, the interactions of mast cells with viruses and pathogen products are complex and can have both detrimental and positive impacts. There is substantial evidence for mast cell mobilization and activation of effector cells and mobilization of dendritic cells following viral challenge. These cells are a major and under-appreciated local source of type I and III interferons following viral challenge. However, mast cells have also been implicated in inappropriate inflammatory responses, long term fibrosis, and vascular leakage associated with viral infections. Progress in combating infection and boosting effective immunity requires a better understanding of mast cell responses to viral infection and the pathogen products and receptors we can employ to modify such responses. In this review, we outline some of the key known responses of mast cells to viral infection and their major responses to pathogen products. We have placed an emphasis on data obtained from human mast cells and aim to provide a framework for considering the complex interactions between mast cells and pathogens with a view to exploiting this knowledge therapeutically. Long-lived resident mast cells and their responses to viruses and pathogen products provide excellent opportunities to modify local immune responses that remain to be fully exploited in cancer immunotherapy, vaccination, and treatment of infectious diseases.

Mast Cells and Natural Killer Cells-A Potentially Critical Interaction.

Natural killer (NK) cells play critical roles in host defense against infectious agents or neoplastic cells. NK cells provide a rapid innate immune response including the killing of target cells without the need for priming. However, activated NK cells can show improved effector functions. Mast cells are also critical for early host defense against a variety of pathogens and are predominately located at mucosal surfaces and close to blood vessels. Our group has recently shown that virus-infected mast cells selectively recruit NK cells and positively modulate their functions through mechanisms dependent on soluble mediators, such as interferons. Here, we review the possible consequences of this interaction in both host defense and pathologies involving NK cell and mast cell activation.

Interferon α2 and interferon γ induce the degranulation independent production of VEGF-A and IL-1 receptor antagonist and other mediators from human mast cells.

BACKGROUND: Mast cells are resident immune effector cells, often studied in the context of allergic disease. Found in substantial numbers at sites of potential infection they are increased at sites of angiogenesis and can be pivotal for the sensing and clearance of a variety of pathogens. Interferons (IFNs) are cytokines that are critical for host defence against intracellular pathogens. Increased levels of IFNs are observed during viral infection and in autoimmune diseases. IFNs are also widely used therapeutically and have been examined in the therapy of severe asthma. OBJECTIVE: To define the selective human mast cell cytokine and chemokine response following activation with type I or type II IFN's. METHODS: The ability of both IFNα2 and IFNγ to induce cytokine production by human cord blood-derived mast cells was examined in vitro. Cytokine and chemokine production at 6 and 24 h was assessed by multiplex protein analysis. Degranulation was assessed by ß-hexosaminidase release. Mast cells were also treated with reovirus or respiratory syncytial virus and their production of CXCL10, IL-1 receptor antagonist (IL-1Ra), and vascular endothelial growth factor (VEGF) examined after 24 h. RESULTS: In addition to increased expression of classical IFN response genes, such as CXCL10, small but significant increases in CCL5 and IL-17 production were observed following IFN activation. Notably, human mast cells produced both VEGF and IL-1Ra in a dose dependent manner. These responses occurred in the absence of mast cell degranulation by a mechanism consistent with classical IFN signaling. Both reovirus and respiratory syncytial virus infection of mast cells, were also associated with IFN-dependent IL-1Ra expression. CONCLUSION AND CLINICAL RELEVANCE: Our findings demonstrate that IFNs have profound impact on cytokine and chemokine expression by human mast cells, alone or in the context of viral infection. Mast cell VEGF and IL-1Ra responses to IFNs could impact the regulation of local inflammatory responses and subsequent tissue remodeling.

Virus-Infected Human Mast Cells Enhance Natural Killer Cell Functions.

Mucosal surfaces are protected from infection by both structural and sentinel cells, such as mast cells. The mast cell's role in antiviral responses is poorly understood; however, they selectively recruit natural killer (NK) cells following infection. Here, the ability of virus-infected mast cells to enhance NK cell functions was examined. Cord blood-derived human mast cells infected with reovirus (Reo-CBMC) and subsequent mast cell products were used for the stimulation of human NK cells. NK cells upregulated the CD69 molecule and cytotoxicity-related genes, and demonstrated increased cytotoxic activity in response to Reo-CBMC soluble products. NK cell interferon (IFN)-γ production was also promoted in the presence of interleukin (IL)-18. In vivo, SCID mice injected with Reo-CBMC in a subcutaneous Matrigel model, could recruit and activate murine NK cells, a property not shared by normal human fibroblasts. Soluble products of Reo-CBMC included IL-10, TNF, type I and type III IFNs. Blockade of the type I IFN receptor abrogated NK cell activation. Furthermore, reovirus-infected mast cells expressed multiple IFN-α subtypes not observed in reovirus-infected fibroblasts or epithelial cells. Our data define an important mast cell IFN response, not shared by structural cells, and a subsequent novel mast cell-NK cell immune axis in human antiviral host defense.

Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets.

Regulatory T cells that express CD39 (CD39+ Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39+ Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39+ Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25+ CD39+ T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39+ Treg cells and higher levels of ART1 expression in CD4+ CD39+ T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39+ Treg or CD39- Treg cells was observed by 1mM ATP or 60µM NAD. In contrast, P2Xs receptor-dependent proliferation with 300µM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39+ Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD.

The His155Tyr (489C>T) single nucleotide polymorphism of P2RX7 gene confers an enhanced function of P2X7 receptor in immune cells from patients with rheumatoid arthritis.

We assessed the possible association between several single nucleotide polymorphisms (SNP) of P2RX7 gene with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We determined the function of P2X7 receptor and the frequency of the 489C>T, 1096C>G, and 1513A>C SNP of P2RX7 gene in 111 and 122 patients with SLE and RA, and 98 healthy subjects. We found no significant association between the SNPs studied and SLE or RA. We also detected that lymphocytes from SLE and RA patients with the 489C>T SNP showed a higher ethidium bromide uptake in response to ATP than wild type or 1096C>G/1513A>C subjects. In addition, cells from RA patients and the 489C>T genotype, showed higher [Ca(2+)]i responses to ATP. Our data indicate that the 489C>T SNP of P2RX7 gene confers an enhanced function of this receptor in patients with RA, which may contribute to the pathogenesis of this condition.

Expression and function of P2X(7) receptor and CD39/Entpd1 in patients with type 2 diabetes and their association with biochemical parameters.

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X(7) receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X(7)(+) cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14(+), CD4(+) and CD19(+) subpopulations of P2X(7)(+) cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X(7)(+)CD14(+) cells and blood concentration of LDL-c. In addition, the percentages of CD39(+) cells and CD39(+)CD19(+) cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X(7)(+) cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39(+) cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.

Expression and function of the P2X(7) purinergic receptor in patients with systemic lupus erythematosus and rheumatoid arthritis.

Because the synthesis of pro-inflammatory cytokines and apoptosis of lymphoid cells can be induced through P2X(7), we decided to study its expression, function (apoptosis, shedding of CD62L and synthesis of IL-1beta induced by ATP) and genetic polymorphisms (1513 AC and -762 T/C) in peripheral blood mononuclear cells from 101 patients with systemic lupus erythematosus (SLE), 122 with rheumatoid arthritis (RA) and 90 healthy controls. We found no significant differences in the distribution of 1513 and -762 genotypes of P2X(7) gene in SLE or RA patients compared with healthy controls. However, a diminished induction of apoptosis of CD4(+) T lymphocytes and monocytes was observed in SLE patients with the 1513 AC genotype, and the release of IL-1beta upon stimulation with ATP was significantly decreased in SLE patients. In contrast, in RA patients we detected that the release of IL-1beta was increased. In addition, in patients with SLE and RA the SNPs 1513 AC was associated with a low expression of P2X(7). These results suggest a possible involvement of P2X(7) in the pathogenesis of inflammatory autoimmune diseases.