CD40L-expressing recombinant vaccinia virus (rVV40L) : generation of central memory CD8+ T cells and induction of tumor cell death

Generation of immunological memory represents a key tenet of the adaptive immune system. CD8+ T cell memory compartment encompasses two main subsets, central (TCM) and effector memory (TEM) lymphocytes, displaying a differential expression of homing and chemokine receptors. Furthermore, TCM are characterized by high proliferative potential in response to antigen stimulation, high responsiveness to homeostatic cytokines and the ability to home into secondary lymphoid organs.In contrast, TEM produce higher levels of effector cytokines, preferentially home in non- lymphoid tissues and have limited proliferation capacity. Immune responses conferring protection against infectious agents or efficiently eradicating established tumors critically necessitate the generation of antigen specific TCM. We have generated a recombinant vaccinia virus encoding human CD40 ligand (rVV40L) and used it here, in replication incompetent form, to promote the differentiation of naïve CD8+ T cells into TCM specific for viral and tumor associated antigens. Soluble CD40L recombinant protein (s40L) and wild-type vaccinia virus (VV WT) alone or in combination were used as controls. We have found that, in the absence of CD4+ T cells, a single antigenic stimulation “in vitro” of naïve CD8+ T cells by rVV40L-infected non-professional CD14+ antigen presenting cells (APCs) promotes the rapid generation of viral or tumor associated antigen specific CD8+ T cells displaying TCM phenotypic and functional properties. In particular these cells are able to respond with vigorous proliferation and IL-2 production to “in vitro” antigenic challenge. In addition, we have also evaluated the capacity of rVV40L to mediate cytotoxic effect on tumor cells upon direct infection or by promoting the activation of myeloid cells of monocyte/macrophage lineage. Interestingly, rVV40L infection as compared to s40L treatment resulted in the inhibition of proliferative capacity of CD40 receptor expressing tumor cells. Furthermore, rVV40L-infection of tumor cells resulted also in the acquisition of tumorocidal activity, as indicated by the increased TNF-α gene expression, by CD14+ monocytes. Taken together, these data fully support the use of CD40L-expressing recombinant vaccinia virus for clinical investigation in view of its direct cytotoxic activity on tumor cells and by its ability to to promote the generation of of effective and long-lasting antigen specific CD8+ T cell responses through the modulation of antigen presenting capacity of non-professional CD14+ antigen presenting cells

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8+ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8+ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8+ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8+ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection

MAGE-A Antigens and Cancer Immunotherapy

MAGE-A antigens are expressed in a variety of cancers of diverse histological origin and germinal cells. Due to their relatively high tumor specificity, they represent attractive targets for active specific and adoptive cancer immunotherapies. Here, we (i) review past and ongoing clinical studies targeting these antigens, (ii) analyze advantages and disadvantages of different therapeutic approaches, and (iii) discuss possible improvements in MAGE-A-specific immunotherapies

Hemidesmus indicus induces immunogenic death in human colorectal cancer cells

The ability of anticancer treatments to promote the activation of tumor-reactive adaptive immune responses is emerging as a critical requirement underlying their clinical effectiveness. We investigated the ability of Hemidesmus indicus, a promising anticancer botanical drug, to stimulate immunogenic cell death in a human colorectal cancer cell line (DLD1). Here we show that Hemidesmus treatment induces tumor cell cytotoxicity characterized by surface expression of calreticulin, increased HSP70 expression and release of ATP and HMGB1. Remarkably, the exposure to released ICD-inducer factors from Hemidesmus-treated DLD1 cells caused a modest induction of CD14-derived dendritic cells maturation, as demonstrated by the increased expression of CD83. Moreover, at sub-toxic concentrations, H.i. treatment of monocytes and dendritic cells induced their mild activation, suggesting its additional direct immunostimulatory activity. These data indicate that Hemidesmus indicus induces immunogenic cell death in human tumor cells and suggest its potential relevance in innovative cancer immunotherapy protocols

The Interplay Between Neutrophils and CD8+ T Cells Improves Survival in Human Colorectal Cancer

Purpose: Tumor infiltration by different T lymphocyte subsets is known to be associated with favorable prognosis in colorectal cancer. Still debated is the role of innate immune system. We investigated clinical relevance, phenotypes, and functional features of colorectal cancer-infiltrating CD66b+ neutrophils and their crosstalk with CD8+ T cells.Experimental Design: CD66b+ and CD8+ cell infiltration was analyzed by IHC on a tissue microarray including <650 evaluable colorectal cancer samples. Phenotypic profiles of tissue-infiltrating and peripheral blood CD66b+ cells were evaluated by flow cytometry. CD66b+/CD8+ cells crosstalk was investigated by in vitro experiments.Results: CD66b+ cell infiltration in colorectal cancer is significantly associated with increased survival. Interestingly, neutrophils frequently colocalize with CD8+ T cells in colorectal cancer. Functional studies indicate that although neutrophils are devoid of direct antitumor potential, coculture with peripheral blood or tumor-associated neutrophils (TAN) enhances CD8+ T-cell activation, proliferation, and cytokine release induced by suboptimal concentrations of anti-CD3 mAb. Moreover, under optimal activation conditions, CD8+ cell stimulation in the presence of CD66b+ cells results in increasing numbers of cells expressing CD45RO/CD62L "central memory" phenotype. Importantly, combined tumor infiltration by CD66b+ and CD8+ T lymphocytes is associated with significantly better prognosis, as compared with CD8+ T-cell infiltration alone.Conclusions: Neutrophils enhance the responsiveness of CD8+ T cells to T-cell receptor triggering. Accordingly, infiltration by neutrophils enhances the prognostic significance of colorectal cancer infiltration by CD8+ T cells, suggesting that they might effectively promote antitumor immunity. Clin Cancer Res; 1-12. (c)2017 AACR

Tuning of apoptotic epitope-specific CD8⁺ T cells by PD-1.

<p>(<b>A</b>) Representative experiment in which PBMCs from a patient with acute HCV infection were stained with mAb to CD8 and the pentamer complexed with the indicated apoptotic epitope. Cells were then stimulated with the soluble form of the indicated peptide and anti-CD28 mAb in the presence or absence of a blocking antibody to PD-L1 or an isotype control. After 6 h and 10 d of culture, cells were processed for the detection of the indicated cytokines by ICS assay with the relevant mAbs. (<b>B</b>) Cumulative experiments, performed as described in (a), showing the percentages of cells producing the indicated cytokines in CD8⁺pentamer⁺ cells, evaluated at different time points from the stimulation with the apoptotic peptides in the presence or absence of mAb to PD-L1. NS = not significant.</p

Polyfunctional CD8⁺ T_EM cells specific to apoptotic epitopes distinguish patients experiencing chronic infection.

<p>(<b>A,B</b>) Representative flow cytometry analyses of PBMCs from patients with acute HCV infection experiencing chronic infection (A) or undergoing infection resolution (B) that were stained with mAb to CD8 and pentamers complexed to the indicated apoptotic or viral epitopes. Cells were then stimulated with the relevant soluble peptides plus anti-CD28 mAb and processed for the detection of IL-17, IL-22, IFN-γ, IL-4, and IL-2 by ICS assay with the relevant mAbs. Counterplot analyses are gated on CD8⁺pentamer⁺ cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. The small histograms show ICS analyses of representative cytokine (IL-17, IL-22, IFN-γ, IL-4, or IL-2) production without antigenic stimulation by gated CD8⁺pentamer⁺ cells. (<b>C,D</b>) Peptide dose-response curves of cytokine-producing CD8⁺pentamer⁺ cells from patients with acute HCV infection experiencing chronic infection (C) or undergoing infection resolution (D). Cells were stained with the pentamers expressing the indicated peptides and stimulated for 6 h with the same soluble peptides. They were then processed for the detection of the different cytokines indicated by ICS assay. Values are shown with the background subtracted.</p

Decay kinetics of pentamer staining for CD8⁺ T cells.

<p>(<b>A</b>) Representative decay plot of the natural logarithm of the normalized fluorescence versus time after anti-HLA-A2 mAb addition for fresh PBMCs stained with pentamers that were complexed with the indicated apoptotic epitope. The <i>t</i>_1/2 represents the staining half-times for the pentamers to CD8⁺ T cells. Filled or empty circles represent PBMCs from patients with acute HCV infection experiencing chronic infection or undergoing infection resolution, respectively. (<b>B</b>) The <i>t</i>_1/2 for the pentamers (complexed with apoptotic or viral epitopes) binding CD8⁺ T cells from patients with acute HCV infection experiencing chronic infection (filled symbols) or undergoing infection resolution (empty symbols). Apoptotic epitopes: circle symbols represent the MYH9_478–485 pentamer specificity, square symbols represent MYH9_741–749 pentamer specificity, and triangle symbols represent VIME_78–87 pentamer specificity. Viral epitopes: circle symbols represent HCV-NS3_1073–1081 pentamer specificity, square symbols represent HCV-NS3_1406–1415 pentamer specificity, and triangle symbols represent HCV-Core_132–140 pentamer specificity.</p

Functional flexibility of fresh IL-17–producing CD8⁺ T cells specific to apoptotic epitopes.

<p>(<b>A</b>) PBMCs from three independent patients with acute HCV infection were stained with mAb to CD8 and pentamers complexed with the indicated apoptotic peptide. Cells were stimulated with the relevant soluble peptide plus anti-CD28 mAb for 6 h and then processed for the detection of IL-17 and IFN-γ by ICS assay with the relevant mAbs. Counterplot analyses are gated on CD8⁺pentamer⁺ cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. The small histograms show ICS analyses of representative cytokine (IL-17, or IFN-γ) production without antigenic stimulation by gated CD8⁺pentamer⁺ cells. (<b>B</b>) Representative flow cytometry analysis in which cells were stimulated as previously described and processed for the detection of IL-17, IFN-γ, RORC, and T-bet by ICS assay with the relevant mAbs. Analysis gated on CD8⁺pentamer⁺IL-17⁺ IFN-γ⁺ cells (signed by the star symbol in the panel A) show percentages of RORC- and Tbet-expressing cells. The percentage of cells is reported in each quadrant. (<b>C</b>) Mean percentages of RORC⁺ or T-bet⁺ cells detected in all the timely followed CD8⁺pentamer⁺ cells producing IL-17 or IFN-γ upon antigen stimulation. (<b>D</b>)IL-17–producing CD8⁺ T cells specific to apoptotic epitopes efficiently convert to IFN-γ producing cells <i>in vitro</i>. One representative of three experiments in which PBMCs from patients with acute HCV infection were stained with mAb to CD8 and pentamers complexed with the indicated apoptotic peptide, stimulated with the relevant soluble peptide plus anti-CD28 mAb for 6 h, and processed for the detection of IL-17 and IFN-γ by ICS assay with the relevant mAbs. Counterplot analyses are gated on CD8⁺pentamer⁺ cells and show percentages of cytokine-producing cells. The percentage of cells is reported in each quadrant. Cells producing IL-17 were sorted by using anti-CCR6 and anti-CCR4 mAbs and FACSAria processing (the purity of IL-17⁺ cell population was evaluated by stimulating CCR6⁺CCR4⁺ cells with anti-CD3 and anti-CD28 mAbs). CCR6⁺CCR4⁺ cells were re-stimulated with the same apoptotic epitope and autologous APCs in the presence or absence of either the cytokine mixture <i>type-17</i> (IL-1β, IL-6, IL-23 and TGF-β) polarizing toward the type-17 cell phenotype or the cytokine mixture <i>type</i>-1 (IFN-γ and IL-12) polarizing toward the type-1 cell phenotype. After 10–12 d of culture in IL-2 conditioned medium, cells were stained with pentamers expressing MYH9_741–749 peptide, further antigen-stimulated as previously described, and tested for their capacity to produce IL-17 and IFN-γ by ICS assay.</p